WO2022090419A1

WO2022090419A1 - Process for the treatment of non-crosslinked tissue

Info

Publication number: WO2022090419A1
Application number: PCT/EP2021/080039
Authority: WO
Inventors: Alexander Rzany; Daniel NIOPEK; Nina Foh; Bernhard Hensel
Original assignee: Biotronik Ag
Priority date: 2020-10-30
Filing date: 2021-10-28
Publication date: 2022-05-05

Abstract

The present invention relates to a process for the treatment of tissue, preferably native biological tissue or pretreated tissue (e.g., decellularized) biological tissue, preferably pericardial tissue, comprising (chemically and/or biochemically) crosslinkable groups.

Description

Process for the treatment of non-crosslinked tissue

The present invention relates to several aspects for the treatment of tissue, preferably native biological tissue or pretreated tissue (e.g. decellularized) biological tissue, preferably pericardial tissue, comprising (chemically and/or biochemically) crosslinkable groups.

Ultrathin Tissue - crosslinking and/or shaping of tissue

The first aspect of the invention relates to a method for chemically crosslinking and optionally shaping (including thickness reduction) tissue, in particular biological tissue, using a permeable material layer, optionally in combination with a stepless pressure load/compression on the tissue, so as to always allow fluid transport into and out of the tissue to be treated under the chemical crosslinking; along the lines of drainage. According to the first aspect of the invention, the method can be carried out under different pressure loads/compressions, in particular in order to specifically influence the resulting tissue properties, and, for example, to bring about a homogeneous thickness distribution of the tissue and/or to deliberately and specifically place inhomogeneities, if desired. Furthermore, the method enables the introduction/imprinting of a consistent three-dimensional shape into the tissue to be treated, for example inhomogeneities in the thickness of the tissue, in order to exert a sealing function against a vessel lumen with additional use of a pressure compensation layer and at least one rigid counterform in the method, such as for example a foam layer, in particular a polyurethane foam layer.

Methods for reducing the thickness of, for example, bovine pericardium by approx. 50% by means of planar crosslinking between plates are known, for example, from US 7,141,064 B2.

Methods for thickness reduction of e.g. bovine pericardium by layer ablation, e.g. by means of a laser and subsequent crosslinking under compression in porous ceramic carriers, are known e.g. from US 2013/0310929 Al. Furthermore, methods for three-dimensional shaping by means of rigid molded bodies on both sides are known, for example, from US 8,136,218 B2. In the method described therein, the tissue is placed between two rigid shaped bodies and chemically crosslinked in this state.

The crosslinking of pericardium, e.g. on plastic frames, and a subsequent matching of individual leaflet/skirt components according to their thickness distribution, the tissue thickness profile as well as according to their bending behavior in order to enable a uniform opening and closing behavior of a later TAVI/TAVR valve as an implant is also known.

However, the exemplary methods of the prior art named above are subject to disadvantages. This is due to the fact that the thickness reduction is mostly based on chemical crosslinking under pressure, and thus on the principle of displacement of water from the original tissue, and an associated densification of the fibers of the tissue. However, if the tissue is limited from both sides by means of a fixed counterform (as, for example, in the case of the use of two rigid molded bodies), there is no sufficient exchange surface for the water, and a very high pressure is necessary to be able to reduce this in turn.

Accordingly, US 7,141,064 B2 specifies a maximum tissue length of 6 inches and a pressure of at least 250 pounds; which, assuming a square maximum area, corresponds to a minimum pressure of 0.5 kg/cm² However, this high pressing force requires a high technical effort and can lead to damage of the collagen fibers and thus to deteriorated mechanical properties of the resulting tissue. In addition, the thickness reduction that can be achieved with this method is approx. 50%, which, however, does not correspond to the maximum thickness reduction that can be achieved.

The abovementioned removal of layers to reduce the tissue thickness inevitably results in damage to the tissue surface and thus to load-bearing collagen fibers. This in turn entails the risk of inadequate mechanical properties or insufficient long-term performance for the tissue obtained.

The rigid molded bodies mentioned at the beginning are, for example, unable to compensate for the naturally always present inhomogeneity in the tissue thickness. In areas of higher tissue thickness, this also results in pressure peaks which cause partial fiber compaction and the associated stiffening of the tissue. Visually, these pressure points can usually be identified as transparent areas on the tissue surface. Air bubbles trapped between the two moldings also cause this effect. In addition, the rigid mold bodies hinder the access of the crosslinking solution to the tissue to be formed, which in turn results in poorer crosslinking quality of the tissue.

A current typical method for the fixation of biological tissue for use as a heart valve implant (e.g., a TAVI/TAVR valve), provides for planar crosslinking of the original tissue, e.g., on plastic frames, to achieve wrinkle-free results. After chemical fixation, e.g. using glutaraldehyde solution, the individual tissue components for a TAVI/TAVR valve (e.g. one or more leaflets, one or more skirt elements; inner and/or outer skirt) are e.g. laser cut or via other cutting, and subsequently grouped according to their thickness profile and flexibility or assigned as a suitable match. This is a selection method for finding suitable and matching skirt/leaflet combinations. However, the necessary selection method results in high reject rates; in addition, the flexibility of the tissue is reduced due to the pretension on the frame during crosslinking.

With regard to the above-mentioned disadvantages of the prior art, a technical problem is to provide a method for the production of crosslinked and/or shaped tissue, in particular crosslinked and/or shaped biological tissue, in particular for medical applications, which enables targeted adaptation of the tissue properties, such as in particular thickness, shape and/or flexibility, directly during chemical crosslinking. Thus, the method according to the first aspect of the invention is intended to provide, among other things, tissue/tissue components with optimized thickness homogeneity.

The optimized thickness homogeneity is to be understood as significantly better than in conventionally crosslinked tissue. For example, pericardium in conventionally crosslinked tissue shows a variance in thickness homogeneity of e.g. 160 - 200 pm. In contrast, a pericardium that has undergone a thickness-reducing method according to the first aspect of the invention shows a thickness variance around 40 pm, as a reduced thickness by up to a factor of 5 in section. In this case, a tolerance of +/- 5 pm can be specified as the measurement tolerance per measured point.

Due to the controlled and even willingly "adjustable" final thickness of the tissues obtained from the methods disclosed herein, thickness-homogeneous (in particular ultra-compact) tissues/tissue components are provided, which may also allow a more evenly distributed mechanical load absorption. Consequently, a further object of the first aspect of the invention is to provide, in particular, methods for the production of ultra-thin or ultra-compact tissue or to provide corresponding tissue components.

The term "ultrathin tissue” denotes that the treated tissue has a thickness homogeneity characterized by a substantially constant thickness of the tissue of preferably less than 40% of the thickness of the starting tissue (with a tolerance range for a measurement error of ± 5 pm) or that the tissue has a thickness of less than 80 pm, preferably between 80 and 20 pm or between 25 pm and 20 pm.

Normally, the normal remaining thickness of a pericardial tissue is 120% - 40% of the initial thickness. 120%, i.e. even an increase in thickness, for the case of crosslinking performed freely on a mesh (pericardium becomes thicker during free crosslinking with glutaraldehyde). The thickness reduction is already 40% for standard UTT patches. However, anything below this marks extremely thin patches, where 10% of the initial thickness would be an absolute lower limit. The absolute minimum value for porcine pericardial tissue, for example, is 20 pm thickness.

The methods disclosed herein of a deliberately targeted thickness influence/reduction, up to ultra-thin biological tissue, with an optional additional shaping and/or influencing of the flexibility of the tissue, enable new fields of application, in particular new medical fields of application.

The above-mentioned adaptation of the tissue properties, such as in particular the thickness, but at the same time also the shape and/or flexibility, is achieved by chemical crosslinking and optional shaping using a permeable material layer (preferably made of a technical fabric), optionally in combination with a (continuous) pressure load on the tissue and the use of an added pressure compensation layer, such as, for example, a compressible foam that is also permeable, or an elastomer mat/silicone mat that is not permeable. Thus, the pressure compensation layer can be permeable, but it does not have to be. Essential for the access of the crosslinking solution to the tissue and for the drainage of the tissue water is the addition of a permeable material layer, e.g. actually only one, or at least two or even more permeable material layers, preferably made of a technical fabric. The at least one, preferably two, permeable material layer(s) may be an organic polymer layer, preferably a polyester layer. The at least one, preferably two, permeable material layer(s) has a mesh size or pore size enabling the crosslinking solution to pass through the permeable material layer(s). This enables a sufficient contact of the tissue with the crosslinking solution passing through the permeable material layer(s). The at least one, preferably two, permeable material layer(s) can have a mesh size or pore size of less than 60 pm, preferably ranging from 10 pm to 60 pm. Mesh sizes or pore sizes of less than 60 pm lead to even tissue surfaces. Mesh sizes or pore sizes larger than 60 pm lead to uneven tissue surfaces so that an imprinted structure on the surface of the biological tissue may be visible to the naked eye. Nevertheless, mesh sizes or pore sizes larger than 60 pm can be used to imprint structures on the surface of the biological tissue if this is desired, namely, whenever one wishes to imprint a technically functional surface on the tissue to be treated, such as, for example, a roughening of a surface or specific depressions in a surface, etc. The at least one, preferably two, permeable material layer can have a thickness of 40 pm to 70 pm. Preferably the tissue is sandwiched between two permeable material layer(s).

In addition to the at least one, preferably two, material layers at least one pressure compensation layer can be used. The pressure compensation layer, even if it is not permeable, at least supports the adequate distribution of the crosslinking solution by its technical properties. The at least one, preferably two, pressure compensation layer(s) avoid(s) local stress peaks and enable a homogeneous thickness of the obtained tissue. Preferably the tissue is sandwiched between two permeable material layer(s) and the two permeable material layer(s) are sandwiched between two pressure compensation layers and the two pressure compensation layers are sandwiched between two (rigid) counterforms (via which an external pressure is applied). The at least one, preferably two, material layers and/or the at least one pressure compensation layer can be made of a hydrophilic material. Hydrophilic materials enable a better permeability and/or wettability with the crosslinking solution, preferably an aqueous (glutar)aldehyde solution, than hydrophobic materials. The at least one pressure compensation layer can be a solid polymeric foam, preferably a polyurethane foam. The at least one pressure compensation layer can have a compression hardness of 65kPa or less, preferably 60kPa, and/or a density of 45 kg/m³ or less, preferably 45 kg/m³. The higher the compression hardness and bulk density are selected the higher is the pressure built up on the tissue.

That is, in some embodiments of the first aspect of the invention, a method is provided which, on the one hand, equalizes the natural inhomogeneity of the tissue, possibly allowing shaping of the tissue to be treated, and, on the other hand, allows access of the crosslinking solution to the tissue to be crosslinked/ shaped. This is essentially realized for these embodiments by means of a stepless pressure load on the tissue to be treated, preferably between two rigid and possibly perforated counterforms, and by means of one or more pressure compensation layers located in between, such as one or more compressible foams, in addition to the permeable material layer to be used essentially anyway.

In addition, a three-dimensional shape can be introduced/imprinted into the tissue during chemical crosslinking, e.g. by means of glutaraldehyde, by means of a combination of shaping of the foam and the counterform(s) that is arbitrarily selected but suitable for the method.

Alternatively, there is another possibility of 3D molding besides the variant with the molded foam: The basic method remains the same, but the foam can be replaced by a silicone mat, but in this case more emphasis must be placed on perforations on the opposite side, i.e. the counterform. That is, the structure would be as follows from bottom to top: i) porous or perforated solid lower counterform, ii) mesh as a technical fabric and thus an example of a permeable material layer, iii) the tissue to be treated, iv) silicone layer as a pressure compensation layer, v) followed by upper solid counterform. The silicone pressure compensation layer can also be, for example, a balloon of silicone used to pressurize the pericardium pneumatically or hydraulically from the inside out against a perforated outer mold. With this 3D arrangement, it is not only possible to create valve segments, but also, for example, a complete valve with an adhesive seam.

As mentioned above, in one embodiment according to the first aspect of the invention, it is essential for chemical crosslinking and simultaneous molding to use a pressure compensation layer, such as a compressible foam or a firmer silicone mat. For the drainage function with respect to the chemical crosslinking solution and the tissue water, however, the only decisive factor is that a permeable material layer of technical fabric, such as a mesh, is present. This allows fluid exchange between the tissue and the environment. This makes it possible to remove the water contained in the tissue by applying comparatively little force and, at the same time, to reduce the tissue thickness considerably (from, for example, 200 pm initial thickness to up to 20 pm final thickness of the treated tissue, i.e. 10% of the initial thickness), while retaining the mechanical stability of the tissue itself. In addition, the permeable pressure compensation layer ensures good contact between the tissue and the crosslinking solution. The methods of the first aspect of the invention thus enable a specific adaptation of the properties of biological tissue, in particular collagen-containing biological tissue, such as in particular thickness, shape and flexibility (but not limited in this respect), namely this by a specific adaptation of the chemical crosslinking step of the tissue, whereby on the one hand the tissue waste is reduced and on the other hand new fields of application, in particular new medical fields of application, are opened up by the arbitrary adaptation.

The method is used, for example, to produce ultrathin pericardium, which can be reduced in thickness by about 50% even by applying a low force of 0.1 kg/cm² during fixation. Since mechanical stability is maintained despite the substantial thickness reduction, the tissue is suitable for use as a leaflet/skirt tissue component in a TAVI/TAVR valve, for example. Advantage of using such low force application is on the one hand the simple technical feasibility and on the other hand the low stress on the collagen fibers.

Another major advantage of the methods with the crosslinking variants under infinitely variable pressure is the improved thickness homogeneity with individually adjustable final thickness of the tissue to be treated, in particular non-crosslinked tissue such as collagen-containing biological tissue. Depending on the crosslinking variant, this varies between 120% (see embodiment variant 1 - crosslinking without stepless pressure loading; resulting swelling of the tissue) and 40% (see embodiment variant 2 - crosslinking under stepless pressure loading and associated thickness reduction) compared to the initial thickness in the native state.

Due to the reduced variation in the thickness of the tissues obtained according to the first aspect of the invention, the scrap within the production can be significantly reduced. The optional additional use of one or more, possibly permeable, pressure compensation layers (such as a compressible foam) for force transmission guarantees the equalization of the thickness differences naturally occurring in the biological tissue - without causing local stress peaks that can lead to local stiffening of the tissue.

A further advantage is that the same components described above can also be used to produce three-dimensional (3D)-shaped tissue, in particular 3D-shaped, collagen-containing biological tissue (see embodiment variant 3 - crosslinking under stepless compressive loading and associated thickness reduction in the presence of at least one pre-shaped rigid and optionally perforated counterform). Furthermore, according to the first aspect of the invention, the methods disclosed herein can also be used to bring about connect! ons/joints of tissue pieces.

Due to the different variants and implementation possibilities of the methods according to the first aspect of the invention, very wide-ranging areas of application result. For example, the cross-linked and optionally formed tissue can be found in a TAVI/TAVR valve (valve component but also inner and/or outer skirt elements to reduce paravalvular leakage; PVL), as a stent-graft, as a pouch for pacemakers or, for example, as a collagen tube, as a Left Atrial Appendage Closure Device, as all four heart valve prostheses, as esophageal implants, as bile duct implants.

Transcatheter aortic valve implantation ("TAVI"), or transcatheter aortic valve replacement ("TAVR"), or percutaneous aortic valve replacement ("PAVR") is a minimally invasive procedure in which an artificial aortic valve prosthesis is placed and released within the native aortic valve in a collapsed (crimped; compressed) state.

The implant usually consists of individual, manually sutured, collagen-containing tissue components integrated into a suitable self-expanding or mechanically expandable stent (e.g., balloon-expandable) or support structure. Through the typically complex and error-prone suturing method, a complex, three-dimensional tissue geometry is thereby created, which is essential for the functionality of the prosthesis. At the same time, the expert is aware that the numerous surgical nodes/sutures represent mechanical weak points that can potentially lead to failure of the implant, and thus can also sometimes cause severe complications in the patient.

There are basically three different types of prosthetic heart valves, especially aortic valve prostheses: Prostheses with mechanical valves, which are manufactured artificially, mostly from graphite coated with pyrolytic carbon; prostheses with valves made from biological tissue (or partly biological tissue locally reinforced by artificial fibers, if necessary), mostly pericardial tissue typically derived from animal sources (e.g., porcine or bovine); and valves made from artificial materials such as polymers. The heart valve formed from the biological tissue is generally secured in a base body (e.g., a solid plastic scaffold or a self-expanding stent or a balloon-expanding stent) and this is implanted in the position of the natural valve. The first aspect of the invention describes, among other things, a method for sutureless and integral connection/jointing of such tissue for use in a prosthetic aortic valve to be implanted in place of a natural aortic valve.

Usually, the initial tissue must be thoroughly cleaned and prepared prior to implantation. As far as possible, the tissue is modified in such a way that it is not recognized by the body as foreign tissue, has as little calcification as possible, and has as long a service life as possible. Essentially, such a method for preparing tissue comprises several steps:

One possible preparation step is the so-called decellularization of the tissue. In this step, cell membranes, intracellular proteins, cell nuclei and other cellular components are almost completely removed from the tissue to obtain an approximately pure extracellular matrix. Cells and cellular components remaining in the tissue represent in particular a possible cause of undesired calcification of the biological implant material. Decellularization should be carried out so gently that the structure of the extracellular matrix and in particular the collagen fibers in the extracellular matrix remain as unaffected as possible, while on the other hand all cells and cellular components contained therein are removed from the tissue as completely as possible.

Preferably, according to the first aspect of the invention, the biological and/or artificial tissue is subjected to a pretreatment comprising an optional decellularization with a suitable detergent, preferably with a solution containing surfactin and deoxycholic acid. The decellularization can also be carried out in another way, for example by lysis of the cells or by osmotic digestion.

In the context of the invention, the expressions/terms "biological(s) and/or artificial(s) tissue" or similar terminology describe the tissue types suitable for the seamless joining/jointing methods according to the first aspect of the invention. That is, for example, purely biological tissue is tissue of purely natural origin, e.g., porcine pericardium taken from a porcine pericardium. Purely artificial tissue is tissue that has been artificially produced, for example, from one or more different polymer(s) - e.g., by means of suitable 3D printing methods or the like. Biological and artificial tissue refers to mixed forms of e.g. a biological basic substance such as porcine pericardium, but including artificial materials, e.g. for local reinforcement of certain tissue regions, which are exposed to e.g. enormous physiological pressure and/or tensile loads - e.g. leaflets of a TAWTAVR valve. However, in the context of the invention, common to all these tissue types, and essential, is that they comprise crosslinkable groups, e.g. free amino groups, in particular collagen fibers, which are chemically and/or biochemically crosslinkable. It is also essential for the methods according to the first aspect of the invention that the starting tissue/ tissue components are introduced into the methods according to the first aspect of the invention substantially non-crosslinked at least in the overlap region (i.e. the tissue region(s) to be joined/joined, but preferably in its entirety; i.e. that, if possible, no substantial precrosslinking has taken place, for example by means of glutaraldehyde solution. Substantially non-crosslinked tissue throughout the application means that the proportion of crosslinkable groups in the tissue to be treated (compared to non-crosslinkable groups) is greater than 50%, preferably greater than 60%, even more preferably greater than 80%, most preferably greater than 90%. However, this also means that lightly or only slightly pre-crosslinked or partially crosslinked tissue is suitable for the methods of the first aspect of the invention.

The methods according to the first aspect of the invention are thus suitable for seamless joining/joining of tissue, e.g. native tissue, non-crosslinked decellularized tissue or non- crosslinked non-decellularized tissue. Also suitable are natively dried tissues, which optionally have also been previously subjected to decellularization. The prerequisite is always that the tissue to be joined/joined must contain crosslinkable groups, e.g. free amino groups, in particular collagen, e.g. contained in collagen fibers.

After decellularization, as many cellular components as possible are removed from the tissue and the biological material consists exclusively of extracellular matrix. In pericardial tissue, the extracellular matrix is predominantly formed from said collagen fibers. In order to achieve a biological material with the best possible mechanical properties and to prevent defense reactions of the receiving body, in the prior art the collagen fibers are crosslinked by means of a suitable crosslinking agent through the incorporation of chemical bonds.

The crosslinking agent specifically binds to free amino groups of the collagen fibers and forms chemically stable bonds between the collagen fibers. In this way, a long-term stable biological material is formed from the three-dimensionally arranged collagen fibers, which, moreover, is no longer recognized as foreign biological material. The three-dimensional crosslinking or linking of the individual collagen fibers via the crosslinking agent significantly increases the stability and stressability of the tissue. This is particularly crucial when used as the tissue of a heart valve, where the tissue must open and close as a valve every second. According to the prior art, the tissue treated in this way is attached to a basic body (e.g., a hollow cylindrical nitinol stent), far predominantly by suturing using a plurality of surgical knots. The main body or scaffold is implantable by surgical techniques (mostly catheter-based). Frequently, the basic scaffold is self-expanding or mechanically expandable with the aid of a balloon, so that the prosthetic heart valve can be guided to the implantation site in a compressed state by means of a catheter and implanted within the natural valve.

In the prior art, such catheter-implantable prosthetic heart valves are usually stored in a storage solution, correspondingly in a moist state. The storage solution serves to sterilely stabilize the biological tissue. One conceivable storage solution is, for example, glutaraldehyde.

For implantation, the prosthetic heart valve must then be removed from the storage solution in the operating room and mounted on the catheter after several rinsing procedures. This assembly of the prosthetic heart valve only in the operating room is cumbersome and labor-intensive. In addition, the correct performance of the assembly depends on the skills of the particular surgical team.

In the case of various medical implants, the problem arises that after implantation, there is a leakage between the surface of the implant and an anatomical structure of the patient, for example, a vessel wall in which the implant was implanted. In the case of a prosthetic heart valve as a medical implant, for example, paravalvular leakage (PVL) may occur, limiting the performance of the prosthetic heart valve.

For example, a method of manufacturing a prosthetic heart valve that includes processing dried biological material has been disclosed in US 8,105,375. According to the method disclosed therein, the biological tissue is fixed or crosslinked with an aldehyde-containing solution (e.g., glutaraldehyde or formaldehyde solution), and treated with at least one aqueous solution containing at least one biocompatible and non-volatile stabilizer prior to drying. Stabilizers include hydrophilic hydrocarbons with a plurality of hydroxyl groups, and examples include water-soluble sugar alcohols such as glycerol, or ethylene glycol or polyethylene glycol.

Basically, heart valve defects (Latin: vitia, singular: vitium) as medical indications for a prosthetic heart valve can be divided into stenoses and insufficiencies according to their functional disturbance. Of all valve vitias, calcifying aortic valve stenosis is the most common acquired valvular heart disease in Western industrialized nations and thus the most common medical indication for heart valve replacement (TAVI/TAVR/PAVR).

A conventionally manufactured transcatheter aortic valve prosthesis typically consists of up to six individual tissue parts/components that are manually sutured together in a usually extremely time-consuming and cost-intensive method, and then integrated into a stent or other frame structure. This gives the implant a complex, three-dimensional geometry that is essential for the functionality of the prosthesis. The mostly three freely supported, inwardly directed leaflets form semilunar pockets that passively effect valve closure. The additional skirt components (inner and/or outer skirt) attached to the stent/frame structure serve to prevent or seal against paravalvular leakage (PVL).

Thus, the tissue portion of a TAVI/TAVR valve usually consists of a total of six individual tissue components cut from cross-linked tissue patches. The three leaflet parts, which functionally effect the opening and closing of the prosthesis, are called "leaflets". The three so-called inner skirt parts are immovably attached internally to the stent/frame structure in the final product and serve primarily to reduce paravalvular leakage. A shaping method, e.g. laser cutting or punching, is followed by a complex, multi-stage sewing method, which gives the valve implant its characteristic three-dimensional geometry. In some variants of the prior art, an outer skirt is additionally attached to the outside of the TAVI/TAVR valve, which is also mostly made of tissue and addresses PVL.

The entire suturing method of the valve is performed entirely manually under the microscope and is thus extremely time, cost and resource intensive. In total, several hundred individual surgical knots are tied, with about half of the knots being for suturing the above-mentioned tissue parts/components together and the other half for suturing the tissue components into the stent/frame structure. The difficulty here is that if a single knot is placed incorrectly, this immediately leads to rejection of the valve prosthesis and additional costs in the manufacturing method. Furthermore, sutures form mechanical weak points that can potentially lead to failure of the implant - as mentioned at the beginning.

Typically, the manufacturing of a TAVI/TAVR valve starts with the mechanical processing of the tissue (e.g. pericardium), where the required tissue component(s) is/are prepared and cleaned (e.g. from the pericardium). In the subsequent crosslinking method, the tissue is usually placed and/or fixed (e.g., stretched at the edges) on a suitable planar mold (e.g., one or more plates or a plastic frame), and placed in a suitable crosslinking solution (e.g., glutaraldehyde solution comprising glutaraldehyde oligomers) for several days.

Chemical crosslinking by means of glutaraldehyde oligomers leads to inter- and intramolecular crosslinking in the collagen, and this is essential to protect the tissue from enzymatic degradation and thus ensure the long-term stability of the implant. In addition, this step forces the tissue into a planar shape, facilitating the laser cutting or a punch-out that typically follows.

In this regard, it should be mentioned in general, and without attachment to this theory, that crosslinking in solutions comprising glutaraldehyde oligomers typically occurs via a plurality of glutaraldehyde macromolecules present in the solution. Due to the large number of molecular variants present, good crosslinking takes place. The spacing of the binding sites on the collagen fibers involved can therefore vary and yet chemically covalent binding can occur due to the glutaraldehyde oligomers.

The background to the need for chemical crosslinking is that biological tissue, unless it is supplied by cells and endogenous methods in the body, is subject to natural decomposition and denaturation methods. Accordingly, it must be selectively processed for further processing into a functional long-term implant.

Glutaraldehyde, more correctly referred to as glutardialdehyde, was first used for chemical fixation in the early 1960s and has since become the gold standard for crosslinking collagen- containing tissues. Chemical crosslinking of the collagen structure by glutaraldehyde reduces the immune response and prevents enzymatic degradation after implantation - without compromising the anatomical integrity of the tissue and the viscoelastic properties of the collagen. In addition to its crosslinking property, it can also be used as a sterilizing agent, as it has a killing effect against bacteria, viruses and spores. The great success of glutaraldehyde is due to its commercial availability at low cost, as well as its excellent solubility and high reactivity.

As exemplified above for TAVI/TAVR valves, artificial compounds of tissues/components (biological and/or artificial), especially tissues for medical use, are known. However, the compounds of the prior art are predominantly made of surgical materials, in particular surgical sutures comprising one or more surgical knots.

As mentioned, such surgical sutures usually have to be placed manually. This method is very time-consuming, expensive and error-prone - to list just a few of the associated disadvantages. Surgical knots, for example, must be placed individually by personnel in a highly concentrated manner and must always be visually inspected. In addition, each individual knot represents a potential weak point of the medical tissue, since mechanical forces occurring under stress of a medical implant are focused on the knots. Surgical sutures also have a non-negligible space requirement (space requirement), which means that minimum structural sizes of a few millimeters cannot be undercut, especially in the case of medical implants. This noticeably restricts medical implants in their medical fields of application.

The connection of several tissue segments by sutures of surgical material to create a three- dimensional tissue geometry, e.g. of a TAVI/TAVR valve, are known. Furthermore, a method for three-dimensional shaping by means of rigid shaped bodies on both sides is known, for example, from US 8,136,218 B2. In this method, the tissue is placed between two rigid molded bodies and chemically crosslinked in this state so that the geometry of the molded bodies is permanently imprinted in the tissue.

However, there are also disadvantages associated with the prior art methods. For example, surgical sutures usually have to be placed manually. This method is very time-consuming, expensive and error-prone. The knots must be visually inspected individually. In addition, each individual knot represents a potential weak point, since mechanical forces that occur are focused on the knots. Surgical sutures also have a non-negligible space requirement, which means that minimum structural sizes of a few millimeters cannot be undercut for implants.

Furthermore, the rigid molded bodies described above are not capable of compensating for inhomogeneities in tissue thickness that are naturally always present. In areas of higher tissue thickness, this results in pressure peaks that cause partial fiber compaction and the associated stiffening of the tissue. Visually, these pressure points can be identified as transparent areas on the tissue surface. Air bubbles trapped between the two moldings also have this effect. In addition, usually the rigid molded bodies hinder the access of the crosslinking solution to the tissue, resulting in poorer crosslinking quality of the tissue. Furthermore, on the method side, the first aspect of the invention comprises a chemical crosslinking of tissue joining partners comprising crosslinkable groups, such as, for example, free amino groups, by means of a suitable crosslinking agent under static, quasi-static or periodic pulsatile pressure loading in a defined overlap region for seamless, dense and tight tissue closure disclosed - for example, for tissue closure for a one-piece valve component made of pericardial tissue for a TAVI/TAVR valve. Thereby, a seamless, homogeneous, and at the same time mechanically stable connect! on/jointing of tissue/tissue components is achieved.

That is, the invention exploits, among other things, for the first time in a targeted manner, in sufficient quantity and density, the effect that a crosslinking agent such as, for example, glutaraldehyde can also form interfibrillar connect! ons/crosslinks between two joining partners, such as, for example, tissue surfaces for a one-piece valve component, in order to realize a seamless, materially bonded and durable connect! on/joint.

The crosslinking agent is preferably an aldehyde-containing crosslinking agent, more preferably glutaraldehyde. In alternative embodiments of the invention, the crosslinking agent contains carbodiimide, formaldehyde, glutaraldehyde acetals, acyl azides, cyanimide, genepin, tannin, pentagalloyl glucose, phytate, proanthocyanidin, reuterin and/or epoxy compounds.

An exemplary and preferred crosslinking agent is a glutaraldehyde-containing solution consisting of glutaraldehyde at a concentration of 6 g/1 in DPBS without calcium and magnesium.

Glutaraldehyde, e.g. in aqueous solution, is a known crosslinking agent, especially of free amino groups, proteins, enzymes, and e.g. collagen fibers (Isabelle Migneault, Catherine Dartiguenave, Michel J. Bertrand, and Karen C. Waldron: Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking; BioTechniques 37:790-802 (November 2004).

A particular advantage of the methods disclosed herein is that, for example, a glutaraldehyde solution can in principle be used as a crosslinking agent irrespective of concentration. In one embodiment, for example, the tissue/components to be bonded is placed in a glutaraldehyde oligomer-containing solution at pH 7.4 for 48 hours at a temperature of 4°C during the chemical crosslinking step, and subjected to quasi-static or periodic pulsatile pressure loading/compression.

In general, the skilled person is aware that chemical crosslinking, depending on the tissue to be treated and the desired properties of the crosslinked tissue, can also be regulated or controlled by temperature. Crosslinking generally starts at a temperature above 0°C. Preferred temperature ranges for chemical crosslinking in the sense of the invention are 1 - 50°C, preferably 10 - 50°C, more preferably 20 - 50°C, even more preferably 25 - 40°C, most preferably 35 - 40°C, for example at 37°C.

Advantageously, the tissue is rinsed at least once, preferably several times, with a suitable solvent, in particular a buffered salt solution and/or an alcohol solution, before and particularly preferably after the decellularization (if it is decellularized tissue). Buffered sodium chloride solutions and/or an ethanol solution are particularly advantageous.

In one embodiment of the first aspect of the invention, alpha-gal epitopes may additionally be removed from the tissue in a further treatment step, which may be performed after or before the optional decellularization step. Any suitable alpha-galactosidase can be used for such an additional treatment step, e.g., alpha-galactosidase from green coffee bean (GCB) or Cucumis melo.

As mentioned above, on the device side, the task posed is solved, inter alia, by a medical implant comprising the seamlessly and integrally bonded/joined tissue subjected to one of the methods according to the first aspect of the invention.

With the context of the first aspect of the invention, the term "medical implant" or similar terms particularly includes stent-based implants and heart valve prostheses, particularly aortic valve prostheses, which are stent-based. According to the first aspect of the invention, the term "medical implant" also reads to any medical implant for which the suture-free joined/ connected tissue is suitable as a method product, for example, to seal the implant against an anatomical structure. Also included as a medical implant are pockets that can receive and be implanted with, for example, a pacemaker, an implantable leadless pacemaker, or a defibrillator.

Nowadays, stents are particularly frequently used as implants for the treatment of stenoses (narrowing of blood vessels). They have a body in the form of a possibly perforated tubular or hollow cylindrical basic structure, which is open at both longitudinal ends. The basic structure of the stent may be composed of individual meshes formed by zigzag or meander-shaped webs. The tubular basic structure of such an endoprosthesis is inserted into the vessel to be treated and serves to support the vessel.

Stents have become particularly popular for the treatment of vascular diseases. The use of stents can widen constricted areas in the vessels, resulting in a gain in lumen. Although the use of stents or other implants can achieve an optimal vessel cross-section, which is primarily necessary for the success of the therapy, the permanent presence of such a foreign body initiates a cascade of microbiological methods which, for example, promote inflammation of the treated vessel or necrotic vascular changes and which can lead to a gradual overgrowth of the stent through the formation of plaques.

Stent graft(s)" are stents that contain a fleece or other flat covering, such as a film or tissue, on or in their often grid-like basic structure. In this context, the term "nonwoven" is understood to mean a textile tissue formed by individual fibers.

In the context of the first aspect of the invention, the term "nonwoven" also includes the case in which the textile sheet-like structure consists of only a single "continuous" fiber. Such a stent graft is used, for example, to support weak points in arteries, esophagus or bile ducts, for example in the area of an aneurysm or a rupture of the vessel wall (so-called bail-out device), in particular as an emergency stent.

Medical endoprostheses or implants for a wide variety of applications are known in great variety from the prior art and can be combined with the seamless and materially joined tissue of the invention for suitable purposes. Implants in the sense of the first aspect of the invention are in particular endovascular prostheses or other endoprostheses, e.g. stents (vascular stents, bile duct stents, vascular stents, peripheral stents or, e.g., mitral stents), endoprostheses, endoprostheses or endoprostheses, endoprostheses for closing persistent foramen ovale (PFO), pulmonary valve stents, endoprostheses for closing an ASD (atrial septal defect), as well as prostheses in the area of hard and soft tissue. Also possible as an implant is a left atrial appendage closure device (LAAC).

In an alternative, preferably the medical implant is a prosthetic heart valve, more preferably a TAVI/TAVR valve, which comprises an artificial heart valve made of sutureless and materially bonded/joined tissue and/or a seal made of said tissue attached, preferably sutured, to an expandable or self-expanding and catheter implantable base frame, stent, or retaining device.

In all embodiments of the first aspect of the invention, the decellularization method, if performed, is applied to tissue that is not conventionally crosslinked after decellularization; rather, crosslinking occurs exclusively in the methods disclosed herein under quasi-static or periodic pulsatile pressure/compression in one or more selected overlap region(s) of the tissues involved.

Such a tissue could be used, for example, in cases where cellular ingrowth is preferred, such as in the treatment of a wound or bum with a porous matrix or when used as a means of sealing an implant or graft.

After the optional decellularization and crosslinking methods disclosed herein, the tissue/tissue component can undergo a dimensional and structural stabilization step. It has also been shown that stabilization of the tissue can be significantly enhanced by exposure to certain stabilizing agents.

In a preferred stabilization step, the tissue is exposed to at least one solution containing glycerol and/or polyethylene glycol, wherein the tissue is exposed to either one of these solutions or to the two solutions sequentially in any order and composition as first and second solutions or to both solutions or even to multiple solutions with different molecular weights of PEG simultaneously as a mixture of solutions or sequentially in any order. When drying tissue, e.g., for storage or transportation of the tissue, the stabilization method is preferably carried out prior to drying.

As a non-limiting example, the stabilization method may be performed, for example, after decellularization and crosslinking by immersing the tissue in a series of one or more stabilizing solutions of glycerol and/or polyethylene glycol to sufficiently saturate the tissue with stabilizing agents, and ultimately to produce a stable, dry tissue with a seamless joint/joint. Saturation times can vary, but typically take about 5 minutes to 2 hours or 5 minutes to 15 minutes, depending on the properties of the tissue. The stabilized tissue can be dried by placing the tissue, for example, in a suitable environment with constant low relative humidity or, for example, controllable humidity and/or temperature, for example, in a climate chamber or desiccator and reducing the relative humidity. For example, from 95% to 10% over 12 hours at 37°C. It is obvious to the person skilled in the art that, depending on the circumstances, another suitable drying protocol may be applied.

In general, for the entire present disclosure the skilled person can suitably adjust the technical parameters such as times, amounts, concentrations, temperatures and, for example, pressures depending on the type of tissue to be treated and the desired crosslinking/bonding results.

The polyethylene glycol-containing solutions typically contain polyethylene glycol with an average molecular weight between 150 g/mol and 6000 g/mol, or a mixture thereof. As used herein, the term "between" also includes the upper and lower specified values. Thus, an average molecular weight between 150 g/mol and 6000 g/mol is intended to include 150 g/mol and 6000 g/mol.

In some embodiments, at least one polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 150 g/mol and 200 g/mol, between 150 g/mol and 300 g/mol, between 200 g/mol and 300 g/mol, between 200 g/mol and 600 g/mol, between 200 g/mol and 400 g/mol, between 150 g/mol and 400 g/mol, or between 400 g/mol and 600 g/mol. According to a particularly preferred embodiment, the polyethylene glycol-containing solution provided alone or before or after a glycerol solution contains polyethylene glycol at or about 150 g/mol to 300 g/mol or at or about 200 g/mol (e.g., PEG200), and in an even more preferred embodiment, the polyethylene glycol-containing solution contains 40% PEG200 or about 40% PEG200.

The term "about" as used herein is intended to encompass a variation above and below the stated amount that would be expected in normal use, such as a variation of 5% or 10%.

Glycerin may be added to any of the above stabilizing solutions to form a mixture, or it may be provided separately for stabilizing purposes, such as in aqueous solution. In some embodiments, a subsequently applied polyethylene glycol-containing solution contains polyethylene glycol having a higher average molecular weight than a previously applied polyethylene glycol-containing solution. In some embodiments, the subsequently applied polyethylene glycol-containing solution contains polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol, or a mixture thereof. In some embodiments, the subsequently applied polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 300 g/mol and 1500 g/mol, or a mixture thereof.

In some embodiments, the subsequently applied polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 400 g/mol and 1200 g/mol, or a mixture thereof. In some embodiments, the subsequently applied polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 400 g/mol and 800 g/mol, or a mixture thereof. In some embodiments, the subsequently applied polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 400 g/mol and 600 g/mol, or a mixture thereof. In some embodiments, the subsequently applied polyethylene glycol-containing solution contains polyethylene glycol having an average molecular weight of 400 g/mol (PEG400) or about 400 g/mol.

Again, glycerol may be added to any of the above stabilizing solutions to form a mixture, or it may be provided separately as a stabilizing solution.

In this regard, the skilled person is aware that the temperature during the stabilization step can affect the results. For example, too high a temperature (e.g., above about 85°C) will cause denaturation and irreversible damage to the tissue cross-linked, e.g., glutaraldehyde cross-linked, for the purpose of bonding/jointing. Again, however, too low a temperature can lead to a solution that is too viscous. Preferably, exposure to the stabilizing solutions is at 37°C, but temperatures from room temperature up to 60°C should be tolerable.

As mentioned at the outset, the methods described in the first aspect of the invention are suitable for the preparation of substantially non-crosslinked tissue or, for example, decellularized, substantially non-crosslinked tissue - with the proviso that crosslinkable groups, e.g., free amino groups, must be present in the tissue. Optionally, all of the tissues addressed within the scope of the invention may be stabilized as described herein. Optionally, alpha-gal epitopes can be removed from all these tissues by a suitable alpha-galactosidase treatment (preferably originating from GCB or Cucumis melo, see above).

With regard to the implant itself, the aforementioned problem is further solved by an implant containing biological tissue which has been subjected to one of the methods according to the first aspect of the invention and, if necessary, subsequently stabilized and/or dried.

In this case, the drying of the tissue is designed in such a way that a slow and gentle removal of the water in the liquid state from the tissue is ensured. This is advantageously achieved by the controlled reduction of the ambient humidity of the biological tissue in a suitable environment, such as a desiccator or a climatic chamber, with controlled adjustment of the parameters of the ambient atmosphere of the biological tissue.

A core of the method for seamless joining according to the first aspect of the invention lies in the surprising realization that various suitable crosslinking agents, such as for example and preferably glutaraldehyde, not only have the ability to form inter- and intramolecular crosslinks within a collagen fiber (see prior art above), but also interfibrillar crosslinks between individual fibers. Thus, it is possible for the first time to generate seamless and materially cohesive connections/joints in an overlapping area of two tissue joining partners, which comprise crosslinkable groups, such as free amino groups, e.g. containing collagen, by simultaneously applying a static, quasi-static or periodic pulsatile pressure load/compression by means of a suitable device.

The basic requirement for this is that the distance between the collagen fibers is smaller than the length of the crosslinking molecules involved, such as the glutaraldehyde oligomers mentioned above, which form the actual crosslinks. Therefore, in the context of the invention, a pressuregenerating device has been provided to generate a quasi-static or a periodic pulsatile vertical force application (pressure load/compression), with desired repetition cycles and over a desired time period, to a defined tissue region during the crosslinking method. According to the first aspect of the invention, the pressure generating device can be based on the physical principles of pneumatics, mechanics, and, for example, hydraulics, but is not limited in this respect. In the context of the invention, hydraulics is a particularly preferred embodiment for generating the pressure load/compression. The basic requirement for said formation of interfibrillar crosslinks is that the distance between the collagen fibers and microfibrils involved is smaller than the length of the glutaraldehyde oligomers involved (see above), which essentially form the crosslink. Appropriate pressing parameters over suitable time periods to reduce the fiber spacing are thus essential to enable a stable, seamless and cohesive bond using glutaraldehyde oligomers. At the same time, however, a high pressing pressure potentially, and thus not necessarily, results in preventing accessibility of the crosslinking solution to the tissue during force application.

Therefore, according to the first aspect of the invention, in a preferred embodiment, not only a quasi-static pressure on the tissue over a suitable longer period of time during crosslinking is considered (quasi-static refers to a constant pressure over a longer period of time (e.g. 300 seconds), which may be less frequently interrupted by short and suitable pressure pauses (e.g. 1 or 2 second(s)), but a periodic-pulsatile pressure load/compression over suitable shorter periods, but with possibly more frequent repetition of the pressure phases, also interrupted by short pressure pauses (e.g. 30 seconds pressure, 1 or 2 second(s) pressure pause, followed by 30 seconds pressure, 1 or 2 second(s) pause, etc.).

That is, in the pressureless phases (pressure pauses) during the crosslinking method, sufficient contact between the crosslinking agent, e.g. the glutaraldehyde oligomers, and the tissue to be joined/joined is ensured in this way. For this purpose, a suitable device is provided with which both a quasi-static, relatively constant pressure can be realized over longer period cycles, and a dynamic, periodic pulsatile pressure can be generated on the tissue, but over shorter and more frequent period cycles.

In an alternative embodiment, it is possible to achieve the crosslinking according to the first aspect of the invention for seamless and material-locking connect! on/jointing via a static, i.e. permanent pressure, without pauses. The prerequisite for this is to provide a support surface for the tissue to be joined/joined which is perforated, i.e. is open to the crosslinking solution, in order to ensure its access to the tissue to be crosslinked.

The terms "amino group-containing(s)" / "comprising free amino groups" or similar terminology mean, in the context of the present invention, that the tissue(s) to be joined/joined must comprise free amino groups that are chemically crosslinkable by means of a suitable crosslinking agent in order to be seamlessly and cohesively joined/joined via the methods described herein. A preferred embodiment for amino group-containing tissue(s) are collagen-containing tissues such as connective tissue, skin, subcutaneous tissue, ligaments, cartilage, bone, tendons, teeth, and in particular pericardium (porcine and bovine for example), etc. Accordingly, the methods disclosed herein lend themselves particularly to the production of medical implants in the areas of: Skin, wound healing, therapies of bum patients, replacement of ligaments, cartilage, bone, or tendons, and in implantology. It is clear to the skilled person that due to the very broad medical application possibilities of compounds/joints of e.g. collagen-containing biological tissues, the aforementioned listing is by no means to be interpreted as exhaustive.

With this context, the term "collagen-containing(s)" or similar terms used in the context of the invention describes that the tissue(s) to be joined/joined must comprise free collagen fibers in order to be seamlessly and cohesively joined/joined via the methods described herein.

Suitable collagen-containing tissues within the scope of the invention are, for example, native collagen-containing tissues, moist collagen-containing tissues, already processed (but essentially non-crosslinked) collagen-containing tissues, such as, for example, already stabilized collagen- containing tissues, already preserved collagen-containing tissues, already dried (non-crosslinked) collagen-containing tissues, already decellularized tissues, as well as mixed forms of the aforementioned tissues. It is clear to the person skilled in the art that this list of suitable collagen- containing tissue forms is not exhaustive, but that further collagen-containing tissue types may be suitable for the disclosed method.

In accordance with the invention, bonding methods for stabilized, dried (non-crosslinked) tissue in particular have been tested.

In one alternative, even the seamless and material-locking bonding of already fully or partially crosslinked tissue is possible in principle, whereby exceptionally either no crosslinking solution such as, for example, glutaraldehyde solution or only a very low-concentration glutaraldehyde solution (0 - 1% glutaraldehyde) is additionally required.

In a further preferred variant, the medical implant is a vascular valve prosthesis, in particular a heart valve prosthesis. For example, an aortic valve prosthesis, a tricuspid valve prosthesis, a mitral valve prosthesis and a pulmonary valve prosthesis are suitable examples of a heart valve prosthesis. Typically, such prostheses or implants have a stent-like structure that carries a valve assembly inside it to replace a natural vascular or heart valve. In this regard, the seamless and materially bonded/joined tissue may be applied to a surface of the prosthetic heart valve (internal and/or external).

In a further variant, the medical implant is a dry-stored and/or dry-delivered complete system, in particular a dry-stored/dry-delivered heart valve prosthesis, in particular an aortic valve prosthesis.

In a further variant, the heart valve prosthesis, in particular aortic valve prosthesis, comprising one or more of the seamless and integral tissue/joint tissue components, is loaded in a dehydrated state into a so-called catheter delivery system and is delivered in this preloaded state to an operating room.

With the context of the invention, the terms/expressions "quasi-static compressive loading/compression" or similar terms/expressions denote an essentially vertical physical application of force to the tissue to be joined/joined, carried out in such a way that it can be considered exclusively as a sequence of equilibrium states. Thus, the time scale on which a quasi-static method occurs must be much slower than the time period in which equilibrium is reached (the so-called relaxation time). Although a respective state of equilibrium prevails to a large extent at each point in time of the method, it is nevertheless generally an objective of the method to obtain different states or a characteristic curve. This means that the equilibrium state at time tl (pressure load) may well differ considerably from the equilibrium state at time t2 (pressure relief or pressure pause). The above definition is merely intended to exclude the possibility that dynamic or more dynamic methods, e.g. a periodic pulsatile pressure load/compression, have any appreciable influence on the joining/joining behavior of the tissue components to be joined/joined.

Specifically, in the context of the invention, this means that the relationship between "with pressure loading" and "pressure relief/pressure pause" during the chemical crosslinking method in the case of "quasi-static", is more protracted over time for the pressure loading, and with longer periods of time, possibly several times alternating, than in the direct The "periodic- pulsatile" relationship of "pressure load" and "pressure relief/pressure pause" is shorter for the pressure load, which means that the two states "with pressure" / "without pressure" are also shorter over time and, if necessary, are repeated alternately much more often. Conversely, the terms/expressions "periodic-pulsatile pressure loading/compression" or similar terms/expressions denote that the relationship between "pressure loading" and "pressure relief/pressure pause" during the chemical crosslinking method is more short-lived over time, especially for the pressure loading, and thus the states "with pressure"/"without pressure" and with smaller time spans also alternate noticeably more often, in direct comparison to the "quasistatic" conditions described above.

Specifically, in the context of the invention, the terms/expressions "quasi-static pressure load compression" or similar terms/expressions can be used over a ratio of, for example, 300: 1 seconds with respect to "with pressure load" (300 seconds) vs. "pressure release/pressure pause" (for example. 1 or 2 second(s)), and thus differ from the terms/expressions "periodic-pulsatile pressure load/compression" or similar terms/expressions in such a way that in the latter case a ratio of e.g. 30:1 seconds exists with respect to "with pressure load" (e.g. 30 seconds) versus "pressure relief/pressure pause" (e.g. 1 or 2 second(s)).

That is, "quasi-static" includes, for example, a single, constant pressure load/compression on the tissue to be joined/joined of 5 minutes (= 300 seconds) in the presence of a suitable crosslinker solution with, for example, 1 or 2 second(s) pressure relief/pressure pause. Likewise, however, "quasi-static" also describes those cases in which two or more times of constant pressure load/compression with the pressure releases/pressure pauses as described above act on the tissue to be joined/joined. That is, even corresponding multiple cycles of this rather protracted "quasi- static" form of pressure loading and very short pressure pauses in between falls under these terms.

In contrast, this means, for example, that "periodic-pulsatile" includes at least two, but also several, short pressure loads/compressions on the tissue to be joined/joined of, for example, 30 seconds in the presence of a suitable crosslinking solution, but also always with 1 or 2 second(s) pressure relief/pressure pause. This means that even correspondingly multiple cycles of this rather short "periodic-pulsatile" form of pressure loading with short pressure pauses in between fall under these latter terms.

It is obvious to the skilled person that within the scope of the invention he does not have to slavishly adhere to the exact ratios of 300: 1 seconds for "pressure load" (300 seconds) versus "pressure relief/pressure pause" (e.g. 1 second) in terms of "quasi-static", and 30: 1 for "pressure load" (e.g. 30 seconds) versus "pressure relief (e.g. 1 second) in terms of "periodic-pulsatile". 1 second) in terms of "periodic-pulsatile", but rather the relations of the mentioned time spans to each other distinguish the variants "quasi-static" from "periodic-pulsatile" during chemical crosslinking, and the exact values may suitably deviate from the above examples. For example, for "quasi-static" the above-described ratios of 250: 1 seconds or, for example, 350: 1 seconds are also conceivable, and with regard to "periodic-pulsatile", for example, 15:1 seconds or 30:2 seconds are conceivable.

In the pressureless phases, quasi in the pauses of the external pressure load, a sufficient contact between the chemical crosslinking agent (e.g. glutaraldehyde) and the tissue components in the contact area is ensured in this way.

The above-mentioned alternative case of static crosslinking - without pressure pause - but with a perforated/holed counterform for accessibility of the crosslinking solution is appropriately delimited with the above definition of the quasi-static case.

The person skilled in the art is generally aware that the above-mentioned times can vary considerably depending on the tissue to be treated and the crosslinking agent to be used. Too short times are likely to lead to insufficient stability, too long times are likely to end in a waste of time, and the skilled person would optimize the parameters (time, temperature, concentrations, etc.) depending on the material.

At this point, the overlap length of the tissue components, the physical compression type (hydraulic, mechanical, etc.), cylinder force and the crosslinking time itself should be highlighted as other significant factors influencing the methods according to the first aspect of the invention. Thus, despite a lower breaking load, a reduction in the overlap length tends to result in a higher bond strength. In order to ensure the accessibility of the crosslinking agent, e.g. the glutaraldehyde solution, to the overlap area, a quasi-static or periodic pulsatile pressure load/compression is indispensable according to the first aspect of the invention.

The cylinder force has to be chosen appropriately, depending on the compression area, in order to cause significant (collagen) fiber densification. With regard to the crosslinking duration, a total period of static, quasi-static or periodic pulsatile compressive loading/compression of three days is particularly preferred.

The crosslinking of overlapping tissue joining partners according to the first aspect of the invention is a valid concept for the seamless and material -locking joining/joining of tissue, in particular tissue containing collagen. With regard to the application itself, however, the skilled person must always take into account the load limits of the bonded joint in different load cases as well as the effects of the compression method on the properties of the tissue joining partners.

Examples

The method according to the first aspect of the invention is suitable in principle for chemically/biochemically crosslinkable tissue which is itself essentially non-crosslinked, in particular for substantially non-crosslinked tissue containing collagen. That is, the tissue to be crosslinked according to the first aspect of the invention must have crosslinkable chemical/biochemical groups, such as free amino groups.

The following concrete implementation of the invention by way of embodiment examples is explained using pericardial tissue as an example. Suitable pericardium is that of pigs or cattle in the native, native-stabilized dried or decellularized state, but always non-crosslinked. It is obvious to the person skilled in the art that the methods according to the first aspect of the invention can be used in a generalized manner for tissues, in particular biological tissues, which are substantially non-crosslinked but can be chemically and/or biochemically crosslinked.

Common to all embodiments is a processing of the pericardial tissue comprising both an initial mechanical preparation and the necessary step of chemical crosslinking. The tissue obtained according to the first aspect of the invention can then be subjected to further processing steps, such as stabilization and drying from US 10,390,946 B2.

The methods according to the first aspect of the invention can generally be divided into three variants, which are specifically detailed below - with reference to the figures and reference signs where useful. Common to all variants is the use of at least one permeable material layer e.g. of technical fabric, as a direct support for the pericardial tissue with fluid exchange function; partly in the sense of a drainage of tissue water and for the accessibility of the glutaraldehyde. Variant 1 - Planar crosslinking with permeable material layers - without pressure loading

To ensure wrinkle-free crosslinking of pericardial tissue, the pericardium has so far typically been stretched on a plastic frame and then chemically crosslinked, e.g. using a glutaraldehyde solution.

However, since the pericardium is subject to pretension during crosslinking according to this state of the art, the result is a stiffer tissue compared to the initial state. The collagen fibers are therefore no longer present in their original wave form, but are already in a stretched and thus strained state. Various approaches (e.g. spray fixation or fixation on plates) have already been tested, but all these prior art approaches also exhibit the technical disadvantages mentioned at the beginning.

In contrast, the use of at least one permeable material layer disclosed herein, e.g., made of a technical fabric, enables wrinkle-free crosslinking - without biasing/ stressing the collagen fibers, so that a significantly more flexible pericardial tissue (8) can be provided for medical applications in particular.

For this purpose, according to the present embodiment example, pericardial tissue (8) is placed on or between the technical tissue as a permeable material layer after mechanical preparation (e.g. removal of excess tissue, in particular fatty tissue, and cutting) and chemically crosslinked in this state in a 0.5% glutaraldehyde solution. A flexible and wrinkle-free crosslinked pericardium results directly from this method, which, however, still exhibits the original thickness distribution inherent to the biological starting material. In this variant, the technical fabric serves as a permeable material layer, but not primarily for a two-dimensional water drainage option in the sense of drainage, but rather for shape stabilization and thus for wrinkle- free crosslinking with simultaneous accessibility of the crosslinking solution.

Various embodiments of this form of crosslinking according to the first aspect of the invention - without the effect of pressure. Specifically, at least two layers of the technical tissue are formed into a kind of receiving pocket for crosslinking, which can receive the previously processed pericardial tissue. Several permeable material layers, e.g. of technical tissue, e.g. three, four, five, six or more, can also be arranged and connected on top of each other in such a way that two adjacent layers each form a type of receiving pocket for a pericardial tissue. This arrangement of permeable material layers, quasi as a crosslinking unit, is then transferred into a suitable container/receptacle filled with, for example, 0.5% glutaraldehyde solution or alternatively laid out flat/planar in a container/receptacle in such a way that the arrangement of permeable material layers is completely covered with crosslinking solution.

Variant 2 - Planar crosslinking with permeable material layers under stepless pressure loading Essential for these embodiments of the method according to the first aspect of the invention is the modification of the chemical crosslinking step. As can be seen in Figure 6, the crosslinking of the pericardial tissue takes place while it lies in a device or crosslinking unit consisting of two rigid counterforms, a polyurethane foam as a pressure compensation layer and two permeable material layers of technical tissue (in the sense of a drainage). For crosslinking, the entire device is located in a suitable container (e.g. vertical or horizontal) filled with e.g. 0.5% glutaraldehyde solution, so that the device is completely covered by the crosslinking solution.

The use of permeable material layers made of technical fabric is essential for successful implementation. On the one hand, this enables water present in the pericardial tissue to be removed with comparatively low pressure, and on the other hand it ensures sufficient accessibility of the crosslinking solution to the tissue.

The compression of the pressure compensation layer under the effect of pressure between the rigid counterforms, results in a pressure load on the pericardial tissue. The water present in the pericardial tissue escapes via the layers of the technical fabric already during the assembly of the device/crosslinking unit. Since the entire device/crosslinking unit is placed in a container filled with, for example, a 0.5% glutaraldehyde solution directly after assembly, which in particular forms interfibrillar crosslinks, the compacted state of the pericardial tissue is permanently maintained. Depending on the applied pressure load, thickness-reduced to ultrathin tissues can be obtained according to the first aspect of the invention, in particular ultrathin porcine pericardium with a final thickness of up to a maximum of 20 pm.

The individual concrete method steps for producing such a planar, ultra-compact/ultra-thin porcine pericardial tissue can be summarized as follows: i) A pericardium, e.g. from a pig, is freshly collected at the slaughterhouse and stored for 2 h at 4°C in a suitable storage solution, e.g. EDTA/isopropanol or saline;

(ii) The pericardial tissue is rinsed three times for 5 min in saline (0.9%);

(iii) The pericardial tissue is prepared wet in saline (0.9%): Removal of fat/connective tissue and this is followed by gross cutting to approximately 12 cm x 8 cm; (iv) Followed by irrigation in physiological saline with gentle mechanical agitation; v) Provision of a first rigid counterform comprising holes - lower counterform; vi) fitting the first rigid counterform with a suitable connecting means, e.g. screws; vii) Placement of a first permeable material layer (technical fabric) centrally on the first rigid counterform as a support surface without folds; viii) Central wrinkle-free support of the pericardial tissue on the first permeable material layer, followed by a smoothing out of any air bubbles present in the pericardial tissue; ix) Central wrinkle-free overlay of a second permeable material layer of technical tissue on the pericardial tissue; x) central overlay of a pressure compensation layer of polyurethane foam on the second permeable material layer; xi) Precisely fitting and form-fitting support of a second rigid counterform with perforations and comprising holes; xii) connecting the two counterforms via the screws and fixing the counterforms by means of a suitable continuously adjustable retaining means, such as, for example, one or more nuts; xiii) adjustment of a desired pressure load (application of force) between the counterforms via a suitable regulation of the distance between the two plates; for example, via a looser or tighter screwing of both plates. This means that the pressure load is controlled as a function of the plate spacing, which can be implemented mechanically, hydraulically or via a suitable pneumatic system, for example; xiv) placing the device/crosslinking unit in a suitable receptacle/container - e.g. substantially vertically or substantially horizontally; xv)Filling the container/receptacle with a sufficient amount of 0.5% glutaraldehyde solution such that the device/crosslinking unit is completely covered with the crosslinking solution; xvi) Followed by chemical crosslinking for 2 days at a temperature of 37°C; xvii) Storage of the final processed pericardial tissue in glutaraldehyde solution or followed by any further processing.

The use of the polyurethane foam as a pressure compensation layer and to transmit the force of the compressive load ensures compensation of the natural inhomogeneities of the pericardial tissue, avoiding local stress peaks. As a result, the resulting tissue has a particularly advantageous, and if required, extremely thin thickness homogeneity (. Furthermore, the foam also promotes wetting of the pericardial tissue with the crosslinker solution, thus ensuring a high crosslinking quality.

By continuously reducing the plate spacing (e.g., by means of suitable screws and nuts) and the associated compression of the foam, the final thickness of the pericardial tissue can be specifically adjusted to the requirements of a subsequent application, in particular for a medical application.

Furthermore, the flexibility of the pericardial tissue can be modified in addition to the final thickness by adjusting the plate spacing.

Depending on the starting tissue used, different end thicknesses can be achieved. For example, for porcine pericardial tissue, the use of two permeable material layers, e.g. of technical tissue, results in a thickness reduction of 50% already at a pressure load of about 0.1 kg/cm² Depending on the starting tissue, however, a greater thickness reduction can also be achieved by increasing the pressing force), with complete fiber compaction representing a lower limit of the final thickness. In the case of porcine pericardium, for example, this is about 20 pm.

Since the at least one, preferably at least two permeable material layers e.g. made of technical tissue, allow large-area drainage of the stored water from the tissue even at low pressures, the load-bearing collagen fibers remain undamaged, so that the parameters of elongation at break and breaking stress are maintained and the pericardial tissue is thus still suitable, e.g. for use in a TAVI/TAVR valve.

Variant 3 - 3D crosslinking under pressure load

By means of a suitable shaping of a rigid counterform in interaction with the pressure compensation layer, e.g. of a suitable foam, preferably of a polyurethane foam, it is also possible according to the first aspect of the invention to introduce or imprint a three-dimensional shaping into the substantially non-crosslinked starting tissue.

For example, by using the foam as a pressure compensation layer in conjunction with the technical fabric of the permeable material layers, a three-dimensional structure can be introduced into the tissue during the crosslinking step, while at the same time also improving the homogeneity of the tissue thickness and, if necessary, reducing the tissue thickness. The procedure is analogous to the method presented in variant 2 above, with the difference that instead of the two planar, rigid counterforms, a rigid outer mold adapted to the desired geometry (e.g., a negative of a one-piece skirt/leaflet configuration for a TAVI/TAVR valve) and an equally adapted foam are used. Crosslinking is then also performed in a container, e.g. filled with a 0.5% glutaraldehyde solution.

In the case of large shape changes, e.g. leaflet, the technical fabric is also compression molded to achieve a better shape. Outer skirts can be made without shape embossing the mesh. Mesh made of polyester, for example, can be pre-embossed by heat in metal molds.

Influence parameters

Pressure compensation layer (e.g. a foam or a silicone)

In the context of the invention, the pressure compensation layer is characterized by the parameters: Compression hardness, density, material composition, material thickness and, if necessary, permeability. Since in some embodiments the pressure is regulated by a stepless reduction of the distance between two rigid counter-forms, for example by suitable connecting means in combination with steplessly adjustable retaining means, the pressure compensation layer is of particular importance with regard to the transmission of pressure to the tissue to be treated. According to the first aspect of the invention, compression hardness, bulk density, thickness and type of material used directly influence the maximum achievable pressure at a defined and desired distance between the counterforms. The higher the compression hardness and bulk density are selected, and the thicker the material, the higher the pressure built up on the tissue.

Hydrophobic materials therefore appear less suitable for the pressure compensation layer with this context. Nevertheless, hydrophilic pressure compensation layers are preferred according to the first aspect of the invention.

A particularly preferred embodiment of the invention for the pressure compensation layer comprises the following set of parameters, which is very suitable for the formation of thickness- reduced tissue, in particular ultra-compact tissue:

Compression hardness: 60 kPa density: 40 kg/m³ Thickness: 3 cm

Material: polyurethane foam

It is obvious to the expert that, depending on the requirements of the tissue to be treated and for the particular application, different constellations may be preferred for the pressure compensation layer.

Compression pressure/pressure load

The applied compression pressure/pressure load is another important influencing parameter according to the first aspect of the invention. In general, at lower pressures, this results directly in higher tissue thickness. Higher pressures in turn reduce the tissue thickness; but are quasi ineffective after a certain point of thickness reduction, but still cause a stiffening of the tissue, which can be illustrated by bending behavior measurements. The greatest possible flexibility of the tissue obtained results from a mere chemical crosslinking in the presence of at least one, preferably at least two permeable material layers, e.g. of technical fabric - without the application of a pressure (see variant 1 above).

Preferred in the context of the invention is a compression pressure/load in the range of 0.002 kg/cm² to 0.15 kg/cm²

Here, too, it is obvious to the skilled person that, depending on the requirements of the tissue to be treated and for the particular application, different compression pressures/pressure loads may be preferred.

Duration and temperature of pressure crosslinking

The applied duration and temperature under pressure crosslinking is another important influencing parameter according to the first aspect of the invention. A suitable total duration of pressure crosslinking is in the range of 4 h to 5 days, preferably 4 h to 4 days, more preferably 3 days, even more preferably 2 days. Suitable temperatures for pressure crosslinking range from 10°C to 50°C, preferably 25°C to 40°C, more preferably 30°C to 37°C, even more preferably at or around 37°C.

Thus, in some embodiments of the invention, pressure crosslinking for 2 days at 37°C or for 4 days at room temperature as described herein is preferred. In general, a shorter crosslinking time under pressure than, for example, under 24 h appears conceivable, but seems to lead to rather inadequate tissue properties. In particular, the compression molding then appears to be insufficient. On the other hand, a longer crosslinking time, i.e. beyond 5 days, under pressure does not seem to result in any significant advantages with regard to the tissue properties.

The duration of an optional post-crosslinking can be made more flexible in this respect, but should preferably take place over at least 3 days at 37°C or over 5 days at room temperature.

Furthermore, in the context of the invention, an increase in temperature during chemical crosslinking (e.g., under the pressure load disclosed herein) generally leads to an acceleration of the crosslinking method. However, the denaturation temperature of the tissue to be treated should be kept well below this temperature.

Properties/requirements for the permeable material layer(s) - e.g. made of technical fabric

One task of the permeable material layer(s) (7a, 7b, 7ac) of the technical fabric disclosed herein, for example, is to ensure a sufficient surface area for the exchange of liquids, in particular of the tissue water and the crosslinking solution, during crosslinking under pressure. This includes, on the one hand, the drainage of water from the (biological) tissue during compression/pressure loading (drainage function) and, on the other hand, the supply of the crosslinking solution to the tissue to be treated. Characteristic parameters are therefore essentially the surface properties, permeability and stiffness of the permeable material layer(s), e.g. of the technical fabric used here.

Due to the direct contact of the permeable material layer(s) (7a, 7b, 7c) with the tissue to be crosslinked, special requirements must be placed on the surface of the technical tissue, for example, such as the absence of detaching particles or also the structuring of the material layers themselves, since these may be imprinted in the (biological) tissue during crosslinking.

Preferred in the context of the invention are polyester meshes with 100 - 180 threads/cm. The mesh size ranges from 10 pm - 60 pm, with a thickness of the technical fabric of about 40 pm - 70 pm being suitable. Larger mesh sizes also lead to a sufficient exchange of fluids, but the imprinted structure on the surface of the biological tissue may then even be visible to the naked eye.

Nevertheless, in another embodiment these properties of the imprinted surfaces of the permeable material layers (7a, 7b, 7c) can also be used within the scope of the invention in a positive sense. Namely, whenever one wishes to imprint a technically functional surface on the tissue to be treated, such as, for example, a roughening of a surface or specific depressions in a surface, etc.

With respect to the foregoing disclosure for ultrathin tissue, the first aspect of the invention also comprises the following embodiments, numbered in ascending order:

1. Method for the preparation of crosslinked and/or shaped tissue, in particular crosslinked and/or shaped biological tissue, with selective adaptation of the thickness, shape and/or flexibility of the tissue, for medical applications, in particular for use as a component of a medical implant, preferably a vascular implant, more preferably an artificial heart valve or a covered stent, wherein the method comprises at least the following steps: a) providing one or more tissue(s), preferably pericardial tissue, comprising chemically and/or biochemically crosslinkable groups; b) providing at least one, preferably at least two, permeable material layers configured, optionally bonded to each other, to act as at least a support surface covering the tissue and/or as at least a perfectly fitting receiving pocket for the tissue; c) optional cutting of the tissue(s) to be crosslinked according to step a) by means of a suitable cutting instrument and/or a suitable cutting device; d) placing/arranging the tissue according to step a) or c) on, in or between the permeable material layer(s) according to step b); e) providing one or more pressure compensation layers, which are configured to completely cover at least the tissue and the at least one or more permeable material layers as a support surface; f) optionally providing a container or receptacle suitable for chemical crosslinking; g) chemically crosslinking the tissue according to step d) with the addition of a suitable crosslinking agent, optional withinthe container or receptacle according to step f), preferably over a total period of at least 4 hours to a maximum of 5 days in combination with a (continuous) pressure load on the tissue; h) removal of the crosslinked and optionally shaped tissue according to step g); i) optional purely chemical post-crosslinking by means of a suitable crosslinking agent. 2. Method for the preparation of crosslinked and/or shaped tissue, in particular crosslinked and/or shaped biological tissue, with selective adaptation of the thickness, shape and/or flexibility of the tissue, for medical applications, in particular for use as a component of a medical implant, preferably a vascular implant, more preferably an artificial heart valve or a covered stent, wherein the method comprises at least the following steps: a) providing one or more tissues comprising chemically and/or biochemically crosslinkable groups; b) providing at least one, preferably at least two, permeable material layers configured, preferably bonded together, to act as at least a support surface covering the tissue and/or at least a perfectly fitting receiving pocket for the tissue; x) providing a device comprising at least two rigid counterforms, preferably at least one of said counterforms being perforated, and configured to be connected via one or more suitable spaced apart adjustable connecting and/or retaining means, preferably one or more threaded screws with accurately fitting nuts, adjustable in distance to each other, so as to be able to exert a stepless pressure load on the tissue according to step b), and in such a way that the at least one, preferably the at least two permeable material layers provide a fluid exchange between tissue and environment; e) providing one or more pressure compensation layers, preferably one or more foam layers, which are configured to completely cover at least the tissue and at least the one or more permeable material layers as a support surface, and which are optionally placed on one or more of the permeable material layers for an exact fit, and thus come to rest between the at least two rigid counterforms; c) optionally cutting the tissue(s) to be crosslinked according to step a) by means of a suitable cutting instrument and/or a suitable cutting device; d) placing/arranging the tissue according to step a) or c) on/in the permeable material layers according to step b) and optionally thereon in the device according to step c) with an optional placing/addition of one or more of the pressure compensation layers according to step d) and wherein the rigid counterforms are gradually reduced in their distance via the adjustable connecting means/fixing means in such a way that a continuous pressure effect on the tissue is obtained; f) optionally providing a container or receptacle suitable for chemical crosslinking; g) chemically crosslinking the arranged tissue according to step f) with the addition of a suitable crosslinking agent, optionally within the container or receptacle according to step g), over a total period of at least 4 hours up to a maximum of 5 days; or j) chemically crosslinking the arranged tissue according to step d) in said device with the optionally inserted pressure compensation layer(s) with the addition of a suitable crosslinking agent into the container or receptacle according to step g), and optionally further reducing the spacing of the rigid counterforms via the adjustable connecting means/fixing means, over a total period of at least 4 hours to a maximum of 5 days; h) demolding/removal of the tissue crosslinked and molded according to step (g) or step (i); i) optional purely chemical post-crosslinking by means of a suitable crosslinking agent.

3. Method for the preparation of crosslinked and/or shaped tissue, in particular crosslinked and/or shaped biological tissue, with selective adaptation of the thickness, shape and/or flexibility of the tissue, for medical applications, in particular for use as a component of a medical implant, preferably a vascular implant, more preferably an artificial heart valve or a covered stent, wherein the method comprises at least the following steps: a) providing one or more tissue(s) comprising chemically and/or biochemically crosslinkable groups; b) providing at least one, preferably at least two, permeable material layers configured, preferably bonded together, to act as at least a support surface covering the tissue and/or at least a perfectly fitting receiving pocket for the tissue; x) providing a device comprising at least two rigid counterforms, preferably at least one of said counterforms being perforated, and configured to be adjustable in distance to each other via one or more suitable connecting and/or retaining means adjustable in distance to each other, preferably one or more threaded screws with accurately fitting nuts, so as to be able to exert a stepless compressive load on the tissue according to step a), in such a way that the at least one, preferably the at least two permeable material layers provide a fluid exchange between tissue and environment; e) providing one or more permeable pressure compensation layers, preferably one or more foam layers, which are configured to completely cover at least the tissue and at least the one or more permeable material layers as a support surface, and which are optionally placed on one or more of the permeable material layers for a snug fit, and thus come to rest between the at least two rigid counterforms; c) optionally cutting the tissue(s) to be crosslinked according to step a) by means of a suitable cutting instrument and/or a suitable cutting device; d)placing/arranging the tissue according to step a) or e) on/in the permeable material layers according to step b), which have previously been placed in the device according to step c), followed by placing/adding one or more of the permeable pressure compensation layers according to step d) in the device according to step c), and whereupon the rigid counterforms are gradually reduced in their distance via the adjustable connecting means/restraining means in such a way that a continuous pressure effect on the tissue is created; f) providing a container or receptacle suitable for chemical cross-linking; j) chemically crosslinking the arranged tissue according to step f) in said device with the addition of a suitable crosslinking agent into the container or receptacle according to step g), and wherein optionally the rigid counter-forms are further reduced in their distance stepwise via the adjustable connecting means/fixing means, over a total period of at least 4 hours up to a maximum of 5 days; h) demolding/removal of the crosslinked and molded tissue after step (h); i) Optional pure chemical post-crosslinking using a suitable crosslinking agent.

4. The method according to any of the preceding embodiments, wherein the crosslinking agent is an aldehyde-containing solution or is selected from the group consisting of glutaraldehyde, carbodiimide, formaldehyde, glutaraldehyde acetals, acyl azides, cyanimide, genipin, tannin, pentagalloyl glucose, phytate, proanthocyanidin, reuterin, and/or epoxy compounds.

5. The method according to any of the preceding embodiments, wherein the crosslinking agent is glutaraldehyde, preferably a 0.5% glutaraldehyde solution.

6. The method according to any one of the preceding embodiments, wherein the tissue has been subjected to a pretreatment comprising an optional decellularization, preferably with a solution containing surfactin and deoxycholic acid, and optionally a pre-crosslinking, preferably with a solution containing glutaraldehyde.

7. The method according to any of the preceding embodiments, wherein the tissue is rinsed at least once with a suitable solution, in particular a salt solution and/or an alcohol solution, before and/or after the crosslinking, the optional pre-crosslinking and/or the optional post-crosslinking. 8. The method according to any of the preceding embodiments, wherein the method further comprises performing a structural stabilization step on the, optionally decellularized, tissue before or after the crosslinking, the optional pre-crosslinking and/or the optional postcrosslinking.

9. The method according to embodiment 8, wherein the structural stabilization step is performed on the, optionally decellularized, tissue after the crosslinking, after the optional pre-crosslinking, or after the optional post-crosslinking.

10. The method according to any of the preceding embodiments 8 or 9, wherein the structure stabilization step comprises exposing the, optionally decellularized, tissue to at least one solution, but preferably at least two different solutions, wherein one solution comprises glycerol and another solution comprises polyethylene glycol.

11. The method according to embodiment 10, wherein exposure to one or more of the solutions lasts from 5 minutes to 2 hours.

12. The method according to any of the preceding embodiments, further comprising drying the tissue in a suitable environment of constant low relative humidity or in a climate chamber by reducing the relative humidity, optionally from 95% to 10% over 12 hours at 37°C.

13. The method according to any one of embodiments 10 to 12, wherein of the at least two different solutions, a first solution comprises polyethylene glycol having an average molecular weight between 150 g/mol and 300 g/mol; and a second solution is an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol.

14. The method according to any one of embodiments 10 to 13, wherein of the at least two different solutions, a first solution comprises polyethylene glycol having an average molecular weight between 200 g/mol and 600 g/mol; and a second solution is an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol.

15. The method according to any of the preceding embodiments, wherein the method additionally comprises removal of alpha-gal epitopes by use of a suitable alpha-galactosidase. 16. The method according to embodiment 15, wherein the alpha-galactosidase is obtained from green coffee bean (GCB).

17. The method according to embodiment 15, wherein the alpha-galactosidase is derived from Cucumis melo.

18. The method according to any of the preceding embodiments, wherein the chemical crosslinking takes place over a period of time in the range of 4 h to 5 days, preferably 4 h to 4 days, more preferably 3 days, even more preferably over 2 days.

19. The method according to any of the preceding embodiments, wherein the chemical crosslinking takes place at a temperature in the range of 10°C to 50°C, preferably 25°C to 40°C, more preferably 30°C to 37°C, even more preferably at or around 37°C.

20. The method according to any one of the preceding embodiments, wherein the optional postcrosslinking is carried out for at least 3 days at 37°C or for 5 days at room temperature.

21. The method according to any one of the preceding embodiments, wherein the permeable material layers comprise a technical fabric.

22. The method according to any one of the preceding embodiments, wherein the permeable material layers have a mesh size in the range of 10-60 pm.

23. The method according to any one of the preceding embodiments, wherein the permeable material layers have a thickness/height in the range of 40 - 70 pm.

24. The method according to any one of the preceding embodiments, wherein the permeable material layers comprise a polyester mesh having 100 - 180 threads/cm and a mesh size in the range of 10 - 60 pm and a thickness/height in the range of 40 - 70 pm.

25. The method according to any of embodiments 2 to 24, wherein the pressure compensation layer has a compression hardness in the range of 20 - 80 kPa, preferably 30 - 70 kPa, more preferably 40 - 60 kPa, even more preferably 60 kPa. 26. The method according to any one of embodiments 2 to 24, wherein the pressure compensation layer has a Shore hardness A in the range of 5 - 70, preferably 10 - 50, more preferably 15 - 30, still more preferably 20.

27. The method according to any one of embodiments 2 to 26, wherein the pressure compensation layer has a bulk density in the range of 10 - 60 kg/m³, preferably 20 - 50 kg/m³, more preferably 30 - 40 kg/m³, even more preferably 40 kg/m³.

28. The method according to any of embodiments 2 to 27, wherein the pressure compensation layer has a thickness/height in the range of 1 - 5 cm, preferably 2 - 4 cm, more preferably 3 - 4 cm, even more preferably 3 cm.

29. The method obtained according to any one of embodiments 2 to 28, wherein the pressure compensation layer is a polyurethane foam, having a compression hardness of 60 kPA, a density of 40 kg/m³, and a thickness of 3 cm.

30. Tissue obtained according to one of the above methods for medical application, in particular for use in vascular implants, preferably in an artificial heart valve or in a covered stent.

31. Tissue for medical application, in particular for use in vascular implants, preferably in an artificial heart valve or in a covered stent, which has a thickness homogeneity with a variation of the order of 40 pm with a measurement tolerance in thickness per measurement of ± 5 pm.

32. Tissue for medical application, in particular for application in vascular implants, preferably in an artificial heart valve or in a covered stent according to embodiment 31, wherein the ultracompact thickness homogeneity is characterized by a substantially constant thickness of the tissue of below 40%.

33. Medical implant, preferably having a hollow cylindrical base structure, wherein in and/or on a surface of the base structure the tissue according to embodiments 30-32 is arranged, which in the implanted state of the medical implant is intended and arranged to contact an anatomical structure of a patient, in particular a vessel wall, in particular a vessel, to which the medical implant has been implanted. 34. The medical implant according to embodiment 33, wherein the implant is a prosthetic heart valve comprising an artificial heart valve made of said tissue and/or a seal made of said tissue, which is attached, preferably sutured, to an expandable or self-expanding base body implantable by catheter.

35. The medical implant according to one of embodiments 33 or 34, wherein the medical implant is selected from the group consisting of an artificial heart valve, in particular an artificial aortic valve, a coronary or peripheral vascular stent, in particular a covered stent and/or a stent graft.

36. The medical implant according to embodiment 35, wherein the tissue is selected from the group consisting of pericardium, ligaments, tendon, cartilage, bone, skin.

37. Tissue, preferably pericardial tissue, obtained by a method according to one of the embodiments 1 to 29 having a thickness homogeneity with a variation of 40 pm or less, with a measurement tolerance in thickness per measurement of ± 5 pm.

38. Tissue according to embodiment 37 or obtained by a method according to one of the embodiments 1 to 29 having a thickness of less than 80 pm, preferably between 80 and 20 pm or between 25 pm and 20 pm.

3D shaped tissue - three-dimensional shaping of a tissue or tissue component using granulate

The second aspect of the invention relates to a process for three-dimensional shaping of a biological and/or artificial tissue/tissue component, which enables a desired three-dimensional, reproducible and permanent shaping of the tissue/tissue component via crosslinking by means of a suitable crosslinking agent in combination with a rigid shaped body, in particular a single rigid shaped body, and a granulate.

Processes for three-dimensional shaping by means of multiple rigid molded bodies, for example by means of molded bodies that are rigid on both sides, are already known from US 8,136,218 B2. In this process, the tissue to be formed is placed/arranged between two rigid molded bodies and chemically crosslinked in this state, so that the geometries of the molded bodies are permanently "imprinted" into the tissue. However, such rigid molded bodies on both sides are not capable of compensating for inhomogeneities in the tissue thickness that are naturally always present. In areas of thicker tissue, for example, pressure peaks are created which can cause partial fiber compaction and the associated stiffening of the tissue.

Visually, these pressure points can often be identified by the skilled person as transparent areas on the tissue surface (see Figure 1). Air bubbles trapped between the two moldings also have this effect. In addition, the two rigid molded bodies regularly obstruct the access of a crosslinking solution to the tissue to be molded, which results in a poorer crosslinking quality of the tissue as a direct consequence.

It is generally known that chemical crosslinking of, for example, pericardium by means of a suitable crosslinking agent, such as glutaraldehyde, leads to the formation of inter- and intramolecular bonds in the tissue, which restrict the fibers in their freedom of movement. Due to these additional covalent bonds, the bending stiffness of the tissue increases and the tissue loses flexibility. Based on these findings, the following hypothesis can be made: The crosslinks that result from the chemical processing of collagen-containing tissue such as pericardium with, for example, glutaraldehyde, lead to a preservation of the elongation state currently prevailing in the tissue. That is, fibers that are compressed or stretched during the crosslinking process by, for example, external physical forces remain in the compressed or stretched state after crosslinking even when these forces are removed; in effect, they are fixed.

In view of the disadvantages of the prior art summarized above, it is desirable to provide methods for the three-dimensional shaping of tissue, in particular tissue containing collagen, for medical purposes, which also address, among other things, the problem of inhomogeneous tissue thickness distribution.

Against this background, a technical problem of the second aspect of the invention is to provide a process for crosslinking/treatment of a biological and/or artificial tissue/tissue component, which is suitable for reproducible, durable, three-dimensional shaping of the tissue/tissue component with high crosslinking quality and homogeneous surface.

The shaping of biological and/or artificial tissue/tissue component comprising crosslinkable groups, such as collagen fibers, according to the second aspect of the invention is carried out using at least one, in particular a single, rigid molded body in combination with a suitable crosslinking agent, such as glutaraldehyde, and a granulate. The rigid molded body serves as a mere support surface and, if necessary, for shaping the tissue/tissue component, whereas the granulate mechanically fixes the tissue to be shaped to the molded body during the chemical crosslinking process and, at the same time, can be penetrated by the crosslinking solution, such as glutaraldehyde.

Advantageous effects of the second aspect of the invention

Surprisingly, the process according to the second aspect of the invention allows the production of diverse three-dimensional tissue geometries with high surface quality and homogeneous thickness distribution. By using granulates instead of a second rigid counterform, pressure peaks on the tissue surface can be completely avoided. As a result, only slight punctual fiber compaction may occur, which can have a locally negative effect on the mechanical properties. At the same time, a high crosslinking quality is achieved, since the interstices inherent in a granulate serve as quasi channels for a penetrating crosslinking solution, such as glutaraldehyde.

In contrast to the use of two rigid molded bodies as known from the prior art, the placement/arrangement of the tissue/tissue component to be crosslinked is considerably easier according to the second aspect of the invention, since both entrapment of air bubbles and slippage of the tissue are avoided when the second molded body is otherwise attached.

The process according to the second aspect of the invention can be applied, for example, to reduce the number of otherwise necessary surgical sutures, which are associated with a high expenditure of time and money in the manufacturing process, in order to achieve, among other things, a targeted 3D shaping of tissue components of a medical implant. In addition, the surgical nodes as mechanical weak points are eliminated. Consequently, the seamless 3D shaped tissue component according to the second aspect of the invention improves the fatigue strength of a medical implant.

The process of the second aspect of the invention further provides that by a suitable choice of the granulate per se, a specific microstructuring of the tissue surface of a tissue component can be realized. By means of such microstructuring, the mechanical properties of the tissue can also be specifically adapted to a wide variety of medical applications. Consequently, in the overall view, the second aspect of the invention provides permanently three-dimensionally shaped tissues/tissue components with a high surface and crosslinking quality, which are characterized, among other things, by a homogeneous thickness distribution and targeted microstructuring on the surface.

Description of the second aspect of the invention

In contrast to the use of molded bodies that are rigid on both sides, a penetrable granulate forms a flexible shell or covering from above around the tissue/tissue component to be molded during crosslinking, for example by means of glutaraldehyde, which optimally adapts to the molded geometry of the tissue/tissue component to be crosslinked and compensates for inhomogeneities in the tissue thickness. At the same time, the accessibility of the crosslinking solution to the tissue/tissue component is ensured by the cavities/interstices located in the granulate. In this way, various three-dimensional tissue geometries can be created for those skilled in the art.

The process described below is applicable to all tissues comprising crosslinkable groups, such as free amino groups in collagen, for chemical crosslinking:

Exemplary process for three-dimensional shaping of a tissue/tissue component, comprising at least the steps of: a) providing a tissue/tissue component, preferably pericardial tissue, comprising chemically and/or biochemically crosslinkable groups; b) Optionally stabilizing and/or drying the tissue/tissue component according to step a); c) Providing a rigid/solid molded body; d) Optional cutting of the tissue/tissue component to be crosslinked according to step a) or b) by means of a suitable cutting instrument and/or a suitable cutting device, preferably by means of laser cutting; e) placing/arranging the tissue/tissue component treated according to step a), b) or d) on the molded body according to step c) in such a way that the tissue/tissue component comes to rest on/in the molded body without wrinkles; f) providing a container or receptacle suitable for chemical crosslinking; g) placing/arranging the molded body occupied by the tissue/tissue component according to step e) in the container/receptacle according to step f); h) filling the container/ receptacle according to step g) with a granulate such that the molded body comprising the tissue/tissue component is substantially completely covered with the granulate; i) filling the container/receptacle according to step h) with a suitable crosslinking agent, such that the granulate is substantially completely covered and/or penetrated by the crosslinking agent; j) chemically crosslinking the granulate-covered tissue/tissue component according to step i); k) demolding/removal of the crosslinked tissue/tissue component according to step j), and substantial removal of the granulate from a tissue surface; l) optional purely chemical post-crosslinking of the tissue/tissue component according to step k) with a suitable crosslinking agent.

With respect to step c), said rigid/solid molded body may be produced, for example, via a suitable 3D printing process.

With respect to step d), the cutting of the tissue/tissue component can be performed, for example, via laser cutting or another suitable cutting instrument. An example is laser cutting of stabilized, dried pericardium for use in a TAVI / TAVR valve.

With respect to step f), the container/vessel may be, for example, a plastic container or, for example, a vessel made of a plastic.

With respect to step k), the substantial removal of the granulate from a tissue surface can be performed, for example, by means of a spray bottle filled with isotonic saline solution.

In the process according to the second aspect of the invention, the following influencing variables have a direct or indirect effect on the process results:

Shape and size of the selected granulates - using pericardium as an example.

For example, glass spheres with a diameter in the range of 100 pm - 500 pm have proven useful as granulates for porcine pericardium as tissue. Such glass spheres can be easily removed from the tissue surface during demolding of the tissue component by stripping with a suitable compress or by simple rinsing with NaCl. In contrast, glass beads of smaller diameter behave as granulates in such a way that they occupy fiber interstices of the tissue/tissue component and cannot be completely removed from the tissue surface after crosslinking. Glass beads of larger diameters than those described above are in principle suitable for crosslinking according to the second aspect of the invention, but they imprint themselves more visibly on the tissue surface and lead to a permanent surface structuring, which may be undesirable.

Sharp-edged granulates, such as quartz sand, on the other hand, are less suitable for the process according to the second aspect of the invention, since they become entangled in the collagen fibers of the tissue components, for example, and therefore also cannot be completely removed.

In general, it is obvious to the skilled person that depending on the tissue to be treated, the desired crosslinking results and the tissue properties to be achieved, a suitable granulate must be selected that meets the requirements for the tissue. Consequently, the above-described embodiments for pericardium are in no way to be interpreted as limiting a granulate within the scope of the second aspect of the invention.

Crosslinking time

Another influencing variable of the process according to the second aspect of the invention is the crosslinking time for shaping on the molded body, and it should be at least 1 minute, preferably at least 1 hour, preferably at least 4 hours, more preferably at least 12 hours, most preferably at least 24 hours. A crosslinking process of several days is also possible up to, for example, 12 days, but crosslinking can also be carried out beyond this time, but without expecting changes in the tissue state. After a certain saturation point, no substantial crosslinking reaction takes place. Nevertheless, crosslinked tissue is also stored in glutaraldehyde solution, for example, and thus also for several weeks and months, for example. Optionally, additional post-crosslinking lasting several days can take place following demolding.

For example, three-dimensional crosslinking of pericardial tissue according to the second aspect of the invention, e.g. for its use in a medical TAVI/TAVR valve, takes place using a rigid molded body manufactured in a 3D printing process. During crosslinking, the tissue is placed on the molded body without wrinkles. The aim is to precisely transfer the geometry of the molded body to the tissue. The basic prerequisite for this is that the tissue/tissue component is fixed/crosslinked on the molded body during crosslinking and also that accessibility of the crosslinking solution to the tissue is ensured; for example, of glutaraldehyde solution. In the above-described process for three-dimensional crosslinking, a rigid/solid counterform of a second molded body is thus completely dispensed with, and instead a granulate is used to fix the tissue on the molded body.

"Granulates", or also called "granular matter", in the context of the second aspect of the invention refers to a multi-particle system which is composed of individual, macroscopic, typically inelastic particles with expansions of more than one micrometer and between which there is no appreciable attractive interaction in the dry state. During crosslinking, the granulate in turn form a flexible shell/cover that optimally adapts to the molded part geometry and compensates for inhomogeneities in the tissue thickness. The voids between the particles in the granulate also ensure accessibility of the crosslinking agent, for example the glutaraldehyde solution, to the tissue.

Glass spheres with a diameter in the range of 500 - 600 pm, or in the range of 100 - 200 pm, are preferably suitable as granulates in the sense of the second aspect of the invention.

According to the second aspect of the invention, the initial state of the tissue/tissue component to be formed can vary. In one embodiment, the tissue for the process is in native form. In an additional or alternative embodiment, the tissue for the process is in stabilized form. In an additional or alternative embodiment, the tissue is present in a dried manner. In an additional or alternative embodiment, the tissue is present in a moist/wet form (so-called wet tissue). In a preferred embodiment, the tissue is in a stabilized, dried form.

According to the second aspect of the invention, the disclosed process results in increased stiffness of the tissue when the starting tissue is previously stabilized and dried. Without being bound by the following theory, it is believed that this effect is due to additional hydrogen bonding in the tissue that occurs during the drying process. Stabilization and drying of the starting tissue prior to processing according to the second aspect of the invention also leads to significantly weaker imprints of the granulate than in the native tissue.

According to the second aspect of the invention, the speed of crosslinking and shape stabilization qualitatively follows the same course. Particularly in the first seconds, minutes and hours, the tissue properties change drastically if the crosslinking solution is sufficiently accessible to the tissue. Basically, crosslinking starts ad hoc after addition of the crosslinking agent, and the crosslinking quality improves by the minute. The crosslinking reaction is largely completed after just one day, i.e. after approx. 24 hours. While the influence of the initial state of the tissue on the crosslinking rate appears negligible, significant differences were found with regard to the crosslinking variant itself. Compared to the crosslinking variants according to the second aspect of the invention, a significantly delayed crosslinking reaction is shown when using rigid molded bodies on both sides due to the liquid-impermeable plates. However, this very clearly illustrates the technical advantages of using a granulate instead of a rigid/solid counterform, as one of the cores of the second aspect of the invention.

In the context of the second aspect of the invention, the geometry of a molded body can be transferred to the tissue to be formed with a possible deviation of a few micrometers.

If the biological tissue components are stored in glutaraldehyde solution in the usual manner, the quality of the molded shape imprinted by means of granulates remains unchanged.

Transcatheter aortic valve implantation ("TAVI"), or transcatheter aortic valve replacement ("TAVR"), or percutaneous aortic valve replacement ("PAVR") is a minimally invasive procedure in which an artificial aortic valve prosthesis is placed and released within the native aortic valve in the collapsed (crimped; compressed) state.

The implant usually consists of individual, manually sutured, collagen-containing tissue components that are integrated into a suitable self-expanding or mechanically expandable stent (e.g. balloon-expandable) or a suitable support or retaining structure. Through the typically complex and error-prone suturing process, a complex, three-dimensional tissue geometry is thereby created, which is essential for the functionality of the prosthesis. At the same time, the expert is aware that the numerous surgical nodes/sutures represent mechanical weak points that can potentially lead to failure of the implant, and thus can also sometimes cause severe complications in the patient.

There are basically three different types of prosthetic heart valves, especially aortic valve prostheses: Prostheses with mechanical valves, which are manufactured artificially, mostly from graphite coated with pyrolytic carbon; prostheses with valves made from biological tissue (or partly biological tissue locally reinforced by artificial fibers, if necessary), mostly pericardial tissue typically derived from animal sources (e.g., porcine or bovine); and valves made from artificial materials such as polymers. The heart valve formed from the biological tissue is generally secured in a base body (e.g., a solid plastic scaffold or a self-expanding stent or a balloon-expanding stent) and this is implanted in the position of the natural valve. The second aspect of the invention describes, among other things, a method for sutureless and integral connect! on/jointing of such tissue for use in a prosthetic aortic valve to be implanted in place of a natural aortic valve.

Usually, the initial tissue must be thoroughly cleaned and prepared prior to implantation. As far as possible, the tissue is modified in such a way that it is not recognized by the body as foreign tissue, has as little calcification as possible, and has as long a service life as possible. Essentially, such a process for preparing tissue comprises several steps:

Preferably, according to the second aspect of the invention, the biological and/or artificial tissue is subjected to a pretreatment comprising an optional decellularization with a suitable detergent, preferably with a solution containing surfactin and deoxycholic acid. The decellularization can also be carried out in another way, for example by lysis of the cells or by osmotic digestion.

In the context of the second aspect of the invention, the expressions/terms "biological(s) and/or artificial(s) tissue" or similar terminology describe the tissue types suitable for the seamless joining/jointing processes according to the second aspect of the invention. That is, for example, purely biological tissue is tissue of purely natural origin, e.g., porcine pericardium taken from a porcine pericardium. Purely artificial tissue is tissue that has been artificially produced, for example, from one or more different polymer(s) - e.g., by means of suitable 3D printing processes or the like. Biological and artificial tissue refers to mixed forms of e.g. a biological basic substance such as porcine pericardium, but including artificial materials, e.g. for local reinforcement of certain tissue regions, which are exposed to e.g. enormous physiological pressure and/or tensile loads - e.g. leaflets of a TAVI/TAVR valve. However, in the context of the second aspect of the invention, common to all these tissue types, and essential, is that they comprise crosslinkable groups, e.g. free amino groups, in particular collagen fibers, which are chemically and/or biochemically crosslinkable.

It is also essential for the processes according to the second aspect of the invention that the starting tissue/tissue components are introduced into the processes according to the second aspect of the invention substantially non-crosslinked at least in the overlap region (i.e. the tissue region(s) to be joined/joined, but preferably in its entirety; i.e. that, if possible, no substantial pre-crosslinking has taken place, for example by means of glutaraldehyde solution. Substantially non-crosslinked tissue throughout the application means that the proportion of crosslinkable groups in the tissue to be treated (compared to non-crosslinkable groups) is greater than 50%, preferably greater than 60%, even more preferably greater than 80%, most preferably greater than 90%. However, this also means that lightly or only slightly pre-crosslinked or partially crosslinked tissue is suitable for the methods of the first aspect of the invention. However, this also means that lightly or only slightly pre-crosslinked or partially crosslinked tissue is suitable for the methods of the first aspect of the invention. However, this also means that lightly or only slightly pre-crosslinked or partially crosslinked tissue is suitable for the processes of the second aspect of the invention.

The processes according to the second aspect of the invention are thus suitable for seamless joining/joining of tissue, e.g. native tissue, non-crosslinked decellularized tissue or non- crosslinked non-decellularized tissue. Also suitable are natively dried tissues, which optionally have also been previously subjected to decellularization. The prerequisite is always that the tissue to be joined/joined must contain crosslinkable groups, e.g. free amino groups, in particular collagen, e.g. contained in collagen fibers.

After decellularization, as many cellular components as possible are removed from the tissue and the biological material consists exclusively of extracellular matrix. In pericardial tissue, the extracellular matrix is predominantly formed from said collagen fibers. In order to achieve a biological material with the best possible mechanical properties and to prevent defense reactions of the receiving body, in the prior art the collagen fibers are crosslinked by means of a suitable crosslinking agent through the incorporation of chemical bonds. The crosslinking agent specifically binds to free amino groups of the collagen fibers and forms chemically stable bonds between the collagen fibers. In this way, a long-term stable biological material is formed from the three-dimensionally arranged collagen fibers, which, moreover, is no longer recognized as foreign biological material. The three-dimensional crosslinking or linking of the individual collagen fibers via the crosslinking agent significantly increases the stability and stressability of the tissue. This is particularly crucial when used as the tissue of a heart valve, where the tissue must open and close as a valve every second.

According to the prior art, the tissue treated in this way is attached to a basic body (e.g., a hollow cylindrical nitinol stent), far predominantly by suturing using a plurality of surgical knots. The main body or scaffold is implantable by surgical techniques (mostly catheter-based). Frequently, the basic scaffold is self-expanding or mechanically expandable with the aid of a balloon, so that the prosthetic heart valve can be guided to the implantation site in a compressed state by means of a catheter and implanted within the natural valve.

A conventionally manufactured transcatheter aortic valve prosthesis typically consists of up to six individual tissue parts/components that are manually sutured together in a usually extremely time-consuming and cost-intensive process, and then integrated into a stent or other frame structure. This gives the implant a complex, three-dimensional geometry that is essential for the functionality of the prosthesis. The mostly three freely supported, inwardly directed leaflets form semilunar pockets that passively effect valve closure. The additional skirt components (inner and/or outer skirt) attached to the stent/frame structure serve to prevent or seal against paravalvular leakage (PVL).

Thus, the tissue portion of a TAVI/TAVR valve usually consists of a total of six individual tissue components cut from crosslinked tissue patches. The three leaflet parts, which functionally effect the opening and closing of the prosthesis, are called "leaflets". The three so-called inner skirt parts are immovably attached internally to the stent/frame structure in the final product and serve primarily to reduce paravalvular leakage. A shaping process, e.g. laser cutting or punching, is followed by a complex, multi-stage sewing process, which gives the valve implant its characteristic three-dimensional geometry. In some variants of the prior art, an outer skirt is additionally attached to the outside of the TAVI/TAVR valve, which is also mostly made of tissue and addresses PVL.

The entire suturing process of the valve is performed entirely manually under the microscope and is thus extremely time, cost and resource intensive. In total, several hundred individual surgical knots are tied, with about half of the knots being for suturing the above-mentioned tissue parts/components together and the other half for suturing the tissue components into the stent/frame structure. The difficulty here is that if a single knot is placed incorrectly, this immediately leads to rejection of the valve prosthesis and additional costs in the manufacturing process. Furthermore, sutures form mechanical weak points that can potentially lead to failure of the implant - as mentioned at the beginning.

Typically, the manufacturing of a TAVI/TAVR valve starts with the mechanical processing of the tissue (e.g. pericardium), where the required tissue component s) is/are prepared and cleaned (e.g. from the pericardium). In the subsequent crosslinking process, the tissue is usually placed and/or fixed (e.g., stretched at the edges) on a suitable planar mold (e.g., one or more plates or a plastic frame), and placed in a suitable crosslinking solution (e.g., glutaraldehyde solution comprising glutaraldehyde oligomers) for several days.

In this regard, it should be mentioned in general, and without attachment to this theory, that crosslinking in solutions comprising glutaraldehyde oligomers typically occurs via a plurality of glutaraldehyde macromolecules present in the solution. Due to the large number of molecular variants present, good crosslinking takes place. The spacing of the binding sites on the collagen fibers involved can therefore vary and yet chemically covalent binding can still occur due to the glutaraldehyde oligomers.

The background to the need for chemical crosslinking is that biological tissue, unless it is supplied by cells and endogenous processes in the body, is subject to natural decomposition and denaturation processes. Accordingly, it must be specifically processed for further processing into a functional long-term implant.

Glutaraldehyde, more correctly called glutardialdehyde, was first used for chemical fixation in the early 1960s and has since become the gold standard for crosslinking collagen-containing tissues. Chemical crosslinking of the collagen structure by glutaraldehyde reduces the immune response and prevents enzymatic degradation after implantation - without compromising the anatomical integrity of the tissue and the viscoelastic properties of the collagen. In addition to its crosslinking property, it can also be used as a sterilizing agent, as it has a killing effect against bacteria, viruses and spores. The great success of glutaraldehyde is due to its commercial availability at low cost, as well as its excellent solubility and high reactivity.

As exemplified above for TAVI/TAVR valves, artificial compounds of tissues/components (biological and/or artificial), especially tissues for medical use, are known. However, the connections of the prior art to that effect are far predominantly made of surgical materials; in particular, surgical sutures comprising one or more surgical knots.

As mentioned, such surgical sutures usually have to be placed manually. This process is very time-consuming, expensive and error-prone - to list just a few of the associated disadvantages. Surgical knots, for example, must be placed individually by personnel in a highly concentrated manner and must always be visually inspected. In addition, each individual knot represents a potential weak point of the medical tissue, since mechanical forces occurring under stress of a medical implant are focused on the knots. Surgical sutures also have a non-negligible space requirement (space requirement), which means that minimum structural sizes of a few millimeters cannot be undercut, especially in the case of medical implants. This noticeably restricts medical implants in their medical fields of application.

The connection of several tissue segments by sutures of surgical material to create a three- dimensional tissue geometry, e.g. of a TAVI/TAVR valve, are known. Furthermore, a process for three-dimensional shaping by means of rigid shaped bodies on both sides is known, for example, from US 8,136,218 B2. In this process, the tissue is placed between two rigid moldings and chemically crosslinked in this state so that the geometry of the moldings is permanently imprinted in the tissue.

However, the state of the art methods are also accompanied by disadvantages. For example, surgical sutures usually have to be placed manually. This process is very time-consuming, expensive and error-prone. The knots must be visually inspected individually. In addition, each individual knot represents a potential weak point, since mechanical forces that occur are focused on the knots. Surgical sutures also have a non-negligible space requirement, which means that minimum structural sizes of a few millimeters cannot be undercut for implants. Furthermore, the rigid molded bodies described above are not capable of compensating for inhomogeneities in tissue thickness that are naturally always present. In areas of higher tissue thickness, this results in pressure peaks which cause partial fiber compaction and the associated stiffening of the tissue. Visually, these pressure points can be identified as transparent areas on the tissue surface. Air bubbles trapped between the two moldings also have this effect. In addition, usually the rigid molded bodies hinder the access of the crosslinking solution to the tissue, resulting in poorer crosslinking quality of the tissue.

Furthermore, on the process side, the second aspect of the invention comprises a chemical crosslinking of tissue joining partners comprising crosslinkable groups, such as, for example, free amino groups, by means of a suitable crosslinking agent under static, quasi-static or periodic pulsatile pressure loading in a defined overlap region for seamless, dense and tight tissue closure disclosed - for example, for tissue closure for a one-piece valve component made of pericardial tissue for a TAVI/TAVR valve. Thereby, a seamless, homogeneous, and at the same time mechanically stable connect! on/jointing of tissue/tissue components is achieved.

That is, the second aspect of the invention exploits, among other things, for the first time in a targeted manner, in sufficient quantity and density, the effect that a crosslinking agent such as, for example, glutaraldehyde can also form interfibrillar connections/crosslinks between two joining partners such as, for example, tissue surfaces for a one-piece valve component, in order to realize a seamless, substance-locking and durable connect! on/joint.

The crosslinking agent is preferably an aldehyde-containing crosslinking agent, more preferably glutaraldehyde. In alternative embodiments of the second aspect of the invention, the crosslinking agent contains carbodiimide, formaldehyde, glutaraldehyde acetals, acyl azides, cyanimide, genepin, tannin, pentagalloyl glucose, phytate, proanthocyanidin, reuterin and/or epoxy compounds.

Glutaraldehyde, e.g. in aqueous solution, is a known crosslinking agent, in particular of free amino groups, proteins, enzymes, and e.g. collagen fibers (Isabelle Migneault, Catherine Dartiguenave, Michel J. Bertrand, and Karen C. Waldron: Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking; BioTechniques 37:790- 802 (November 2004).

A particular advantage of the processes disclosed herein is that, for example, a glutaraldehyde solution can be used as a crosslinking agent in principle independently of concentration.

In one embodiment, for example, the tissue/tissue components to be bonded is placed in a glutaraldehyde oligomer-containing solution at pH 7.4 for 48 hours at a temperature of 4°C during the chemical crosslinking step, and subjected to quasi-static or periodic pulsatile pressure loading/compression.

In general, the skilled person is aware that chemical crosslinking can also be regulated or controlled via the temperature, depending on the tissue to be treated and the desired properties of the crosslinked tissue. Crosslinking generally starts at a temperature above 0°C. Preferred temperature ranges for chemical crosslinking in the sense of the second aspect of the invention are 1 - 50°C, preferably 10 - 50°C, more preferably 20 - 50°C, even more preferably 25 - 40°C, most preferably 35 - 40°C, for example at 37°C.

In one embodiment of the second aspect of the invention, alpha-gal epitopes may additionally be removed from the tissue in a further treatment step, which may be performed after or before the optional decellularization step. Any suitable alpha-galactosidase can be used for such an additional treatment step, e.g., alpha-galactosidase from green coffee bean (GCB) or Cucumis melo.

As mentioned above, on the device side, the task posed is solved, inter alia, by a medical implant comprising the seamlessly and integrally bonded/joined tissue subjected to one of the processes according to the second aspect of the invention. With the context of the second aspect of the invention, the term "medical implant" or similar terms particularly includes stent-based implants and heart valve prostheses, particularly aortic valve prostheses, which are stent-based. According to the second aspect of the invention, the term "medical implant" also reads to any medical implant for which the suture-free joined/connected tissue is suitable as a process product, for example, to seal the implant against an anatomical structure.

Also included as a medical implant are pockets that can receive and be implanted with, for example, a pacemaker, an implantable leadless pacemaker, or a defibrillator.

Stents have become particularly popular for the treatment of vascular diseases. The use of stents can widen constricted areas in the vessels, resulting in a gain in lumen. Although the use of stents or other implants can achieve an optimal vessel crosssection, which is primarily necessary for the success of the therapy, the permanent presence of such a foreign body initiates a cascade of microbiological processes which, for example, promote inflammation of the treated vessel or necrotic vascular changes and which can lead to a gradual overgrowth of the stent through the formation of plaques.

Stent graft(s)" are stents that contain a fleece or other flat covering, such as a film or tissue, on or in their often grid-like basic structure. In this context, a "nonwoven" is understood to be a textile tissue formed by individual fibers.

In the context of the second aspect of the invention, the term "nonwoven" also includes the case in which the textile tissue consists of only a single "continuous" fiber. Such a stent graft is used, for example, to support weak points in arteries, esophagus, or bile ducts, for example in the area of an aneurysm or a rupture of the vessel wall (so-called bail-out device), especially as an emergency stent. Medical endoprostheses or implants for a wide variety of applications are known in great variety from the prior art and can be combined with the seamless and materially joined tissue of the second aspect of the invention for suitable purposes. Implants in the sense of the second aspect of the invention are in particular endovascular prostheses or other endoprostheses, e.g. stents (vascular stents, bile duct stents, vascular stents, peripheral stents or, e.g., mitral stents), endoprostheses, endoprostheses or endoprostheses, endoprostheses for closing persistent foramen ovale (PFO), pulmonary valve stents, endoprostheses for closing an ASD (atrial septal defect), as well as prostheses in the area of hard and soft tissue. Also possible as an implant is a left atrial appendage closure device (LAAC).

In all embodiments of the second aspect of the invention, the decellularization method, if performed, is applied to tissue that is not conventionally crosslinked after decellularization; rather, crosslinking occurs exclusively in the processes disclosed herein under quasi-static or periodic pulsatile pressure/compression in one or more selected overlap region(s) of the tissues involved.

After the optional decellularization and crosslinking processes disclosed herein, the tissue/tissue component can undergo a dimensional and structural stabilization step. It has also been shown that stabilization of the tissue can be significantly enhanced by exposure to certain stabilizing agents.

In a preferred stabilization step, the tissue is exposed to at least one solution containing glycerol and/or polyethylene glycol, wherein the tissue is exposed to either one of these solutions or to the two solutions sequentially in any order and composition as first and second solutions or to both solutions or even to multiple solutions with different molecular weights of PEG simultaneously as a mixture of solutions or sequentially in any order. When drying tissue, e.g., for storage or transportation of the tissue, the stabilization process is preferably carried out prior to drying.

As a non-limiting example, the stabilization process may be performed, for example, after decellularization and crosslinking by immersing the tissue in a series of one or more stabilizing solutions of glycerol and/or polyethylene glycol to sufficiently saturate the tissue with stabilizing agents, and ultimately to produce a stable, dry tissue with a seamless joint/joint. Saturation times can vary, but typically take about 5 minutes to 2 hours or 5 minutes to 15 minutes, depending on the properties of the tissue. The stabilized tissue can be dried by placing the tissue, for example, in a suitable environment with constant low relative humidity or, for example, controllable humidity and/or temperature, for example, in a climate chamber or desiccator and reducing the relative humidity. For example, from 95% to 10% over 12 hours at 37°C. It is obvious to the person skilled in the art that, depending on the circumstances, another suitable drying protocol may be applied.

The term "about" as used herein is intended to encompass a variation above and below the stated amount that would be expected in normal use, such as a variation of 5% or 10%. Glycerin may be added to any of the above stabilizing solutions to form a mixture, or it may be provided separately for stabilizing purposes, such as in aqueous solution.

In some embodiments, a subsequently applied polyethylene glycol-containing solution contains polyethylene glycol having a higher average molecular weight than a previously applied polyethylene glycol-containing solution. In some embodiments, the subsequently applied polyethylene glycol-containing solution contains polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol, or a mixture thereof. In some embodiments, the subsequently applied polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 300 g/mol and 1500 g/mol, or a mixture thereof.

In this regard, the skilled person is aware that the temperature during the stabilization step can affect the results. For example, too high a temperature (e.g., above about 85°C) will cause denaturation and irreversible damage to the tissue crosslinked, e.g., glutaraldehyde crosslinked, for the purpose of bonding/jointing. Again, however, too low a temperature can lead to a solution that is too viscous. Preferably, exposure to the stabilizing solutions is at 37°C, but temperatures from room temperature up to 60°C should be tolerable. As mentioned at the outset, the processes described in the second aspect of the invention are suitable for the preparation of substantially non-crosslinked tissue or, for example, decellularized, substantially non-crosslinked tissue - with the proviso that crosslinkable groups, e.g., free amino groups, must be present in the tissue. Optionally, all of the tissues addressed within the scope of the second aspect of the invention may be stabilized as described herein. Optionally, alpha-gal epitopes can be removed from all these tissues by a suitable alphagalactosidase treatment (preferably originating from GCB or Cucumis melo, see above).

With regard to the implant itself, the aforementioned problem is further solved by an implant containing biological tissue which has been subjected to one of the processes according to the second aspect of the invention and, if necessary, subsequently stabilized and/or dried.

A core of the process for seamless joining according to the second aspect of the invention lies in the surprising realization that various suitable crosslinking agents, such as for example and preferably glutaraldehyde, not only have the ability to form inter- and intramolecular crosslinks within a collagen fiber (see prior art above), but also interfibrillar crosslinks between individual fibers. Thus, it is possible for the first time to generate seamless and materially cohesive connect! ons/joints in an overlapping area of two tissue joining partners, which comprise crosslinkable groups, such as free amino groups, e.g. containing collagen, by simultaneously applying a static, quasi-static or periodic pulsatile pressure load/compression by means of a suitable device.

The basic requirement for this is that the distance between the collagen fibers is smaller than the length of the crosslinking molecules involved, such as the glutaraldehyde oligomers mentioned above, which form the actual crosslinks. Therefore, in the context of the second aspect of the invention, a pressure-generating device has been provided to generate a quasi-static or a periodic pulsatile vertical force application (pressure load/compression), with desired repetition cycles and over a desired time period, to a defined tissue region during the crosslinking process. According to the second aspect of the invention, the pressure generating device can be based on the physical principles of pneumatics, mechanics, and, for example, hydraulics, but is not limited in this respect. In the context of the second aspect of the invention, hydraulics is a particularly preferred embodiment for generating the pressure load/compression.

The basic requirement for said formation of interfibrillar crosslinks is that the distance between the collagen fibers and microfibrils involved is smaller than the length of the glutaraldehyde oligomers involved (see above), which essentially form the crosslink. Appropriate pressing parameters over suitable time periods to reduce the fiber spacing are thus essential to enable a stable, seamless and cohesive bond using glutaraldehyde oligomers. At the same time, however, a high pressing pressure potentially, and thus not necessarily, results in preventing accessibility of the crosslinking solution to the tissue during force application.

Therefore, according to the second aspect of the invention, in a preferred embodiment, not only a quasi-static pressure on the tissue over a suitable longer period of time during crosslinking is considered (quasi-static refers to a constant pressure over a longer period of time (e.g. 300 seconds), which may be less frequently interrupted by short and suitable pressure pauses (e.g. 1 or 2 second(s)), but a periodic-pulsatile pressure load/compression over suitable shorter periods, but with possibly more frequent repetition of the pressure phases, also interrupted by short pressure pauses (e.g. 30 seconds pressure, 1 or 2 second(s) pressure pause, followed by 30 seconds pressure, 1 or 2 second(s) pause, etc.).

That is, in the pressureless phases (pressure pauses) during the crosslinking process, sufficient contact between the crosslinking agent, e.g. the glutaraldehyde oligomers, and the tissue to be joined/joined is ensured in this way. For this purpose, a suitable device is provided with which both a quasi-static, relatively constant pressure can be realized over longer period cycles, and a dynamic, periodic pulsatile pressure can be generated on the tissue, but over shorter and more frequent period cycles.

In an alternative embodiment, it is possible to achieve the crosslinking according to the second aspect of the invention for seamless and material-locking bonding/jointing via a static, i.e. permanent pressure, without breaks. The prerequisite for this is to provide a contact surface for the tissue to be joined/joined which is perforated, i.e. is open to the crosslinking solution, in order to ensure its access to the tissue to be crosslinked. In the context of the second aspect of the invention, the terms "amino group- containing(s)"/" comprising free amino groups" or similar terminology mean that the tissue to be joined/joined must comprise free amino groups that are chemically crosslinkable by means of a suitable crosslinking agent in order to be seamlessly and cohesively joined/joined via the processes described herein.

A preferred embodiment for amino group-containing tissue(s) are collagen-containing tissues such as connective tissue, skin, subcutaneous tissue, ligaments, cartilage, bone, tendons, teeth, and in particular pericardium (porcine and bovine for example), etc. Accordingly, the processes disclosed herein lend themselves particularly to the production of medical implants in the areas of: Skin, wound healing, therapies of bum patients, replacement of ligaments, cartilage, bone, or tendons, and in implantology. It is clear to the skilled person that due to the very broad medical application possibilities of compounds/joints of e.g. collagen-containing biological tissues, the aforementioned listing is by no means to be interpreted as exhaustive.

With this context, the term "collagen-containing(s)" or similar terms used in the context of the second aspect of the invention describes that the tissue(s) to be joined/joined must comprise free collagen fibers in order to be seamlessly and cohesively joined/joined via the processes described herein.

Suitable collagen-containing tissues within the scope of the second aspect of the invention are, for example, native collagen-containing tissues, moist collagen-containing tissues, already processed (but essentially non-crosslinked) collagen-containing tissues, such as, for example, already stabilized collagen-containing tissues, already preserved collagen-containing tissues, already dried (non-crosslinked) collagen-containing tissues, already decellularized tissues, as well as mixed forms of the aforementioned tissues. It is clear to the person skilled in the art that this list of suitable collagen-containing tissue forms is not exhaustive, but that further collagen- containing tissue types may be suitable for the disclosed process.

In accordance with the second aspect of the invention, bonding processes for stabilized, dried (non-crosslinked) tissue in particular have been tested.

In an alternative, even the seamless and material-locking bonding of already fully or partially crosslinked tissue is possible in principle, whereby, exceptionally, either no crosslinking solution such as, for example, glutaraldehyde solution or only a very low-concentration glutaraldehyde solution (0 - 1% glutaraldehyde) is additionally required.

In a further preferred variant, the medical implant is a vascular valve prosthesis, in particular a heart valve prosthesis. Suitable examples of a prosthetic heart valve include an aortic valve prosthesis, a tricuspid valve prosthesis, a mitral valve prosthesis and a pulmonary valve prosthesis. Typically, such prostheses or implants have a stent-like structure that carries a valve assembly inside it to replace a natural vascular or heart valve. In this regard, the seamless and materially bonded/joined tissue may be applied to a surface of the prosthetic heart valve (internal and/or external).

In a further variant, the heart valve prosthesis, in particular aortic valve prosthesis, comprising one or more of the seamlessly and integrally connected/jointed tissue/tissue components, is loaded in a dehydrated state into a so-called catheter delivery system and is delivered in this preloaded state to an operating room.

With the context of the second aspect of the invention, the terms/expressions "quasi-static compressive loading/compression" or similar terms/expressions denote a substantially vertical physical application of force to the tissue to be connected/joined, performed in such a way that it can be viewed solely as a sequence of equilibrium states. Thus, the time scale on which a quasi- static process occurs must be much slower than the time period in which equilibrium is reached (the so-called relaxation time). Although a respective state of equilibrium prevails to a large extent at each point in time of the process, it is nevertheless generally an objective of the process to obtain different states or a characteristic curve. This means that the equilibrium state at time tl (pressure load) may well differ considerably from the equilibrium state at time t2 (pressure relief or pressure pause). The above definition is merely intended to exclude the possibility that dynamic or more dynamic processes, e.g. a periodic pulsatile pressure load/compression, have any appreciable influence on the joining/joining behavior of the tissue components to be joined/joined. Specifically, in the context of the second aspect of the invention, this means that the relationship between "with pressure loading" and "pressure relief/pressure pause" during the chemical crosslinking process in the case of "quasi-static", is more protracted over time for the pressure loading, and with longer periods of time, possibly alternating The "periodic-pulsatile" relationship of "pressure load" and "pressure relief/pressure pause" is shorter for the pressure load, which means that the two states "with pressure" / "without pressure" are also shorter over time and, if necessary, are repeated alternately much more often.

Conversely, the terms/expressions "periodic-pulsatile pressure loading/compression" or similar terms/expressions denote that the relationship between "pressure loading" and "pressure relief/pressure pause" during the chemical crosslinking process is more short-lived over time, especially for the pressure loading, and thus the states "with pressure"/"without pressure" and with smaller time spans also alternate noticeably more often, in direct comparison to the "quasi- static" conditions described above.

Specifically, in the context of the second aspect of the invention, the terms/expressions "quasi- static pressure load compression" or similar terms/expressions can be used over a ratio of, for example, 300: 1 seconds with respect to "with pressure load" (300 seconds) vs. "pressure release/pressure pause" (for example. 1 or 2 second(s)), and thus differ from the terms/expressions "periodic-pulsatile pressure load/compression" or similar terms/expressions in such a way that in the latter case there is a ratio of e.g. 30:1 seconds with respect to "with pressure load" (e.g. 30 seconds) versus "pressure relief/pressure pause" (e.g. 1 or 2 second(s)).

This means that "quasi-static" includes, for example, a single, constant pressure load/compression on the tissue to be joined/joined for 5 minutes (= 300 seconds) in the presence of a suitable crosslinker solution with, for example, 1 or 2 second(s) pressure relief/pressure pause. Likewise, however, "quasi-static" also describes those cases in which two or more times of constant pressure load/compression with the pressure releases/pressure pauses as described above act on the tissue to be joined/joined. That is, even corresponding multiple cycles of this rather protracted "quasi-static" form of pressure loading and very short pressure pauses in between falls under these terms.

In contrast, this means, for example, that "periodic-pulsatile" includes at least two, but also several, short pressure loads/compressions on the tissue to be joined/joined of, for example, 30 seconds in the presence of a suitable crosslinker solution, but also always with 1 or 2 second(s) pressure relief/pressure pause. This means that multiple cycles of this rather short "periodic- pulsatile" form of pressure loading with short pressure pauses in between also fall under these latter terms.

It is obvious to the person skilled in the art that, within the scope of the second aspect of the invention, he should not slavishly adhere to the exact ratios of 300: 1 seconds for "pressure loading" (300 seconds) versus "pressure relief/pressure pause" (e.g. 1 second) in terms of "quasistatic", and 30: 1 for "pressure loading" (e.g. 30 seconds) versus "pressure relief (e.g. 1 second) in terms of "periodic-pulsatile", but rather the relations of the mentioned time spans to each other distinguish the variants "quasi -static" from "periodic-pulsatile" during chemical crosslinking, and the exact values may suitably deviate from the above examples. For example, for "quasi-static" the above-described ratios of 250: 1 seconds or, for example, 350: 1 seconds are also conceivable, and with regard to "periodic-pulsatile", for example, 15: 1 seconds or 30:2 seconds are conceivable.

At this point, the overlap length of the tissue components, the physical compression type (hydraulic, mechanical, etc.), cylinder force and the crosslinking time itself should be highlighted as other significant factors influencing the processes according to the second aspect of the invention. Thus, despite a lower breaking load, a reduction in the overlap length tends to result in a higher bond strength. In order to ensure the accessibility of the crosslinking agent, e.g. the glutaraldehyde solution, to the overlap area, a quasi-static or periodic pulsatile pressure load/compression is indispensable according to the second aspect of the invention.

The cylinder force has to be chosen appropriately, depending on the compression area, in order to cause significant (collagen) fiber densification.

With regard to the crosslinking time, a total period of static, quasi-static or periodic pulsatile pressure load/compression of three days is particularly preferred.

The crosslinking of overlapping tissue joining partners according to the second aspect of the invention is a valid concept for the seamless and material -locking connect! on/jointing of tissues, in particular tissues containing collagen. With regard to the application itself, however, the skilled person must always take into account the load limits of the bonded joint in different load cases, as well as the effects of the compression process on the properties of the tissue joining partners.

In light of the entire foregoing disclosure of the second aspect of the invention, the second aspect of the invention also encompasses the embodiments numbered in ascending order below:

1. Process for three-dimensional shaping of a tissue/tissue component, in particular for an artificial heart valve, the process comprising at least the following steps: a) providing a tissue/tissue component comprising chemically and/or biochemically crosslinkable groups; b) optionally stabilizing and/or drying the tissue/tissue component according to step a); c) providing a rigid/ solid molded body; d) optional cutting of the tissue/tissue component to be crosslinked according to step a) or b) by means of a suitable cutting instrument and/or a suitable cutting device; e) placing/arranging the tissue/tissue component treated according to step a), b) or d) onto the molded body according to step c); f) providing a container or receptacle; g) placing/arranging the molded body covered with the tissue/tissue component according to step e) in the container/receptacle according to step f); h) filling the container/receptacle according to step g) with a granulate so that the tissue/tissue component according to step g) is at least partially covered with the granulate; i) filling the container/receptacle according to step h) with a suitable crosslinking agent; j) chemically crosslinking the granulate-covered tissue/tissue component according to step h) for at least 1 minute; k) demolding/removal of the crosslinked tissue/tissue component according to step j); l) optional pure chemical post-crosslinking of the tissue/tissue component according to step k) with a suitable crosslinking agent.

2. The process according to embodiment 1, comprising at least the steps: a) providing a tissue/tissue component comprising chemically and/or biochemically crosslinkable groups; b) optionally stabilizing and/or drying the tissue/tissue component according to step a); c) providing a rigid/ solid molded body; d) optional cutting of the tissue/tissue component to be crosslinked according to step a) or b) by means of a suitable cutting instrument and/or a suitable cutting device; e) placing/arranging the tissue/tissue component treated according to step a), b) or d) on the molded body according to step c) in such a way that the tissue/tissue component comes to rest on/in the molded body without wrinkles; f) providing a container or receptacle suitable for chemical crosslinking; g) placing/arranging the molded body covered by the tissue/tissue component according to step e) in the container/receptacle according to step f); h) filling the container/ receptacle according to step g) with the granulate such that the molded body comprising the tissue/tissue component is at least partially covered with the granulate; i) filling the container/receptacle according to step h) with a suitable crosslinking agent, such that the granulate is at least partially covered and/or penetrated by the crosslinking agent; j) chemically crosslinking the granulate-covered tissue/tissue component according to step i); k) demolding/removal of the crosslinked tissue/tissue component according to step j), and removal of the granulate from a tissue surface; l) optional pure chemical post-crosslinking of the tissue/tissue component according to step k) with a suitable crosslinking agent.

3. The process according to embodiment 1 or 2, comprising at least the steps: a) providing a tissue/tissue component, preferably pericardial tissue comprising chemically and/or biochemically crosslinkable groups; b) optionally stabilizing and/or drying the tissue/tissue component according to step a); c) providing a rigid/ solid molded body; d) optional cutting of the tissue/tissue component to be crosslinked according to step a) or b) by means of a suitable cutting instrument and/or a suitable cutting device, preferably by means of laser cutting; e) placing/arranging the tissue/tissue component treated according to step a), b) or d) on the molded body according to step c) in such a way that the tissue/tissue component comes to rest on/in the molded body without wrinkles; f) providing a container or receptacle suitable for chemical crosslinking; g) placing/arranging the molded body covered by the tissue/tissue component according to step e) in the container/receptacle according to step f); h) filling the container/ receptacle according to step g) with the granulate such that the molded body comprising the tissue/tissue component is completely covered with the granulate; i) filling the container/receptacle according to step h) with a suitable crosslinking agent, such that the granulate is completely covered and/or penetrated by the crosslinking agent; j) chemically crosslinking the granulate-covered tissue/tissue component according to step i); k) demolding/removal of the crosslinked tissue/tissue component according to step j), and removal of the granulate from a tissue surface; l) optional pure chemical post-crosslinking of the tissue/tissue component according to step k) with a suitable crosslinking agent.

4. The process according to any of the preceding embodiments, wherein the crosslinking agent is an aldehyde-containing solution or is selected from the group consisting of glutaraldehyde, carbodiimide, formaldehyde, glutaraldehyde acetals, acyl azides, cyanimide, genipin, tannin, pentagalloyl glucose, phytate, proanthocyanidin, reuterin, and/or epoxy compounds.

5. The process according to any of the preceding embodiments, wherein the crosslinking agent is glutaraldehyde, preferably a 0.5-0.65% glutaraldehyde solution.

6. The process according to any one of the preceding embodiments, wherein the crosslinking agent is a 0.5% glutaraldehyde solution.

7. The process according to any of the preceding embodiments, wherein the tissue has been subjected to a pretreatment comprising an optional decellularization, preferably with a surfactin and deoxycholic acid containing solution, and optionally a pre-crosslinking, preferably with a glutaraldehyde containing solution.

8. The process according to any of the preceding embodiments, wherein the tissue is rinsed at least once with a suitable solution, in particular a salt solution and/or an alcohol solution, before and/or after the crosslinking, the optional pre-crosslinking and/or the optional post-crosslinking.

9. The process according to any of the preceding embodiments, wherein the process further comprises performing a structural stabilization step on the, optionally decellularized, tissue before or after the crosslinking, the optional pre-crosslinking and/or the optional postcrosslinking.

10. The process according to embodiment 9, wherein the structural stabilization step is performed on the, optionally decellularized, tissue after crosslinking, after the optional precrosslinking, or after the optional post-crosslinking.

11. The process according to any of the preceding embodiments 9 or 10, wherein the structural stabilization step comprises exposing the, optionally decellularized, tissue to at least one solution, but preferably at least two different solutions, wherein one solution comprises glycerol and another solution comprises polyethylene glycol.

12. The process according to embodiment 11, wherein exposure to one or more of the solutions lasts from 5 minutes to 2 hours.

13. The process according to any one of the preceding embodiments, wherein drying of the tissue/tissue component is carried out in a suitable environment with a constant low relative humidity or in a climate-controllable device/facility in a suitable manner, such as by reducing the relative humidity, for example from 95% to 10% over 12 hours at 37°C.

14. The process according to any of the preceding embodiments 11 to 13, wherein of the at least two different solutions, a first solution comprises polyethylene glycol having an average molecular weight between 150 g/mol and 300 g/mol; and a second solution comprises an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol. 15. The process according to any one of the preceding embodiments 11 to 14, wherein of the at least two different solutions, a first solution comprises polyethylene glycol having an average molecular weight between 200 g/mol and 600 g/mol; and a second solution comprises an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol.

16. The process according to any one of the preceding embodiments, wherein the process additionally comprises removal of alpha-gal epitopes by use of a suitable alpha-galactosidase.

17. The process according to embodiment 16, wherein the alpha-galactosidase is derived from green coffee bean (GCB).

18. The process according to embodiment 16, wherein the alpha-galactosidase is derived from Cucumis melo.

19. The process according to any of the preceding embodiments, wherein the chemical crosslinking or optional post-crosslinking takes place over a period of time in the range of at least 4 h to 12 days, preferably 12 h to 5 days, more preferably 3 days, even more preferably over 2 days.

20. The process according to any of the preceding embodiments, wherein the chemical crosslinking or optional post-crosslinking takes place at a temperature in the range of 1°C to 50°C, preferably 18°C to 40°C, more preferably 30°C to 38°C, even more preferably at or around 37°C.

21. The process according to any of the preceding embodiments, wherein the granulate is selected from the group comprising or consisting of glass spheres, metal spheres, ceramic spheres or plastic spheres or mixtures thereof.

22. The process according to any one of the preceding embodiments, wherein the granulate consists of glass spheres. 23. The process according to any one of the preceding embodiments, wherein the granulate consists of glass spheres having a diameter in the range of 50 - 800 pm, preferably 100 - 600 pm, more preferably 200 - 500 pm.

24. The process according to any one of the preceding embodiments, wherein the granulate consists of glass spheres having a diameter in the range of 500 - 600 pm or in the range of 100 - 200 pm.

25. The process according to any of the preceding embodiments, wherein the rigid/solid molded body is produced via a suitable 3D printing process, ablation process or milling process.

26. The process according to any of the preceding embodiments, wherein the tissue/tissue component according to step a) is in native form.

27. The process according to any of the preceding embodiments 1-25, wherein the tissue/tissue component according to step a) is in stabilized form.

28. The process according to any one of the preceding embodiments 1-25, wherein the tissue/tissue component according to step a) is in dried form.

29. The process according to any one of the preceding embodiments 1-25, wherein the tissue/tissue component according to step a) is in moist/wet form.

30. The process according to any one of the preceding embodiments 1-25, wherein the tissue/tissue component according to step a) is in stabilized, dried form.

31. Use of a granulate in combination with a suitable crosslinking agent and a rigid/solid molded body for three-dimensional shaping of a tissue/tissue component, in particular for medical applications.

32. The use according to embodiment 31, wherein the crosslinking agent is selected from the group consisting of glutaraldehyde, carbodiimide, formaldehyde, glutaraldehyde acetals, acylazides, cyanimide, genipin, tannin, pentagalloylglucose, phytate, proanthocyanidin, reuterin, and/or epoxy compounds. 33. The use according to embodiments 31 or 32, wherein the granulate is selected from the group comprising or consisting of glass spheres, metal spheres, ceramic spheres, or plastic spheres or mixtures thereof.

34. The use according to any of embodiments 31-33, wherein the crosslinking agent is glutaraldehyde, preferably glutaraldehyde in aqueous solution.

35. The use according to any of embodiments 31-34, wherein the granulate is glass spheres with a diameter of 50-800 pm.

36. Three-dimensionally shaped tissue obtained according to one of the processes according to embodiments 1-30 for medical use, in particular for use in an artificial heart valve, such as in a TAVI/TAVR valve.

37. Medical implant, characterized in that it comprises one or more three-dimensionally shaped tissue/tissue components, in their entirety or as a part thereof, obtained according to one of the processes according to embodiments 1-30.

38. The medical implant according to embodiment 37, further characterized in that the medical implant is selected from the group consisting of an artificial heart valve, in particular an artificial aortic valve, a vascular stent, in particular a covered stent or a stent graft, a venous valve, a LAAC device, or a pouch for a pacemaker, for an implantable leadless pacer, or for a defibrillator.

3D shaped tissue - three-dimensional shaping of a tissue or tissue component using hydrostatic column

The third aspect of the invention relates to a process that enables three-dimensional crosslinking and shaping of an substantially non-crosslinked tissue/tissue component, in particular an artificial heart valve, such as a TAVI/TAVR valve, adapted to physiological pressure conditions. For this purpose, for example, a prefabricated (possibly sutured or partially sutured) valve prosthesis with tissue component, e.g. with an already stabilized and dried collagen-containing tissue component (e.g. from bovine and/or porcine pericardium) is crosslinked under pressure load in a constant liquid column in the closed valve state - so-called hydrostatic crosslinking.

Transcatheter aortic valve implantation ("TAVI"), or transcatheter aortic valve replacement ("TAVR"), or percutaneous aortic valve replacement ("PAVR") is a minimally invasive procedure in which an artificial aortic valve prosthesis is placed and released in the collapsed (crimped; compressed) state within the native aortic valve.

The implant usually consists of individual, manually sutured, collagen-containing tissue components integrated into a suitable self-expanding or mechanically expandable stent (e.g., balloon-expandable) or support structure. Through the typically complex and error-prone suturing process, a complex, three-dimensional tissue geometry is thereby created, which is essential for the functionality of the prosthesis. At the same time, the expert is aware that the numerous surgical nodes/sutures represent mechanical weak points that can potentially lead to failure of the implant, and thus can also sometimes cause severe complications in the patient.

There are basically three different types of prosthetic heart valves, especially aortic valve prostheses: Prostheses with mechanical valves, which are manufactured artificially, mostly from graphite coated with pyrolytic carbon; prostheses with valves made from biological tissue (or partly biological tissue locally reinforced by artificial fibers, if necessary), mostly pericardial tissue typically derived from animal sources (e.g., porcine or bovine); and valves made from artificial materials such as polymers. The heart valve formed from the biological tissue is generally secured in a base body (e.g., a solid plastic scaffold or a self-expanding stent or a balloon-expanding stent) and this is implanted in the position of the natural valve. The third aspect of the invention describes, among other things, a method for sutureless and integral connect! on/jointing of such tissue for use in a prosthetic aortic valve to be implanted in place of a natural aortic valve.

Usually, the initial tissue must be thoroughly cleaned and prepared prior to implantation. As far as possible, the tissue is modified in such a way that it is not recognized by the body as foreign tissue, has as little calcification as possible, and has as long a service life as possible. Essentially, such a process for preparing tissue comprises several steps: One possible preparation step is the so-called decellularization of the tissue. In this step, cell membranes, intracellular proteins, cell nuclei and other cellular components are almost completely removed from the tissue to obtain an approximately pure extracellular matrix. Cells and cellular components remaining in the tissue represent in particular a possible cause of undesired calcification of the biological implant material. Decellularization should be carried out so gently that the structure of the extracellular matrix and in particular the collagen fibers in the extracellular matrix remain as unaffected as possible, while on the other hand all cells and cell components contained therein are removed from the tissue as completely as possible.

Preferably, the biological and/or artificial tissue is subjected to a pretreatment according to the third aspect of the invention, which comprises an optional decellularization with a suitable detergent, preferably with a solution containing surfactin and deoxycholic acid. The decellularization can also be performed otherwise, for example, via lysis of the cells or by an osmotic digestion.

In the context of the third aspect of the invention, the expressions/terms "biological and/or artificial tissue" or similar terminology describe the tissue genera suitable for the processes of the third aspect of the invention for seamless joining/joining. That is, for example, purely biological tissue is tissue of purely natural origin, e.g., porcine pericardium taken from a porcine pericardium. Purely artificial tissue is tissue that has been artificially produced, for example, from one or more different polymer(s) - e.g., by means of suitable 3D printing processes or the like. Biological and artificial tissue refers to mixed forms of e.g. a biological basic substance such as porcine pericardium, but including artificial materials, e.g. for local reinforcement of certain tissue regions, which are exposed to e.g. enormous physiological pressure and/or tensile loads - e.g. leaflets of a TAVI/TAVR valve. However, in the context of the third aspect of the invention, common to all these tissue types, and essential, is that they comprise crosslinkable groups, e.g. free amino groups, in particular collagen fibers, which are chemically and/or biochemically crosslinkable.

It is also essential for the processes according to the third aspect of the invention that the starting tissue/tissue components are introduced into the processes according to the third aspect of the invention substantially non-crosslinked at least in the overlap region (i.e. the tissue region(s) to be joined/joined, but preferably in its entirety; i.e. that, if possible, it has not been subjected to any substantial pre-crosslinking, for example by means of glutaraldehyde solution. In this context, non-substantial means that the proportion of reactive and thus crosslinkable groups in the tissue to be treated is greater than 50%, preferably greater than 60%, even more preferably greater than 80%, most preferably greater than 90%. However, this also means that lightly or only slightly pre-crosslinked or partially crosslinked tissue is suitable for the processes of the third aspect of the invention.

The processes according to the third aspect of the invention are thus suitable for seamless joining/joining of tissue, e.g. native tissue, non-crosslinked decellularized tissue or noncrosslinked non-decellularized tissue. Also suitable are natively dried tissues, which optionally have also been previously subjected to decellularization. The prerequisite is always that the tissue to be joined/joined must contain crosslinkable groups, e.g. free amino groups, in particular collagen, e.g. contained in collagen fibers.

The crosslinking agent specifically binds to free amino groups of the collagen fibers and forms chemically stable bonds between the collagen fibers. In this way, a long-term stable biological material is formed from the three-dimensionally arranged collagen fibers, which, moreover, is no longer recognized as foreign biological material. The three-dimensional crosslinking or linking of the individual collagen fibers via the crosslinking agent significantly increases the stability and stressability of the tissue. This is particularly crucial when used as the tissue of a heart valve, where the tissue must open and close as a valve every second.

According to the prior art, the tissue treated in this way is attached to a basic body (e.g., a hollow cylindrical nitinol stent), far predominantly by suturing using a plurality of surgical knots. The main body or scaffold is implantable by surgical techniques (mostly catheter-based). Frequently, the basic scaffold is self-expanding or mechanically expandable with the aid of a balloon, so that the prosthetic heart valve can be guided to the implantation site in a compressed state by means of a catheter and implanted within the natural valve. According to the state of the art, such catheter-implantable prosthetic heart valves are usually stored in a storage solution, correspondingly in a moist state. The storage solution serves to sterilely stabilize the biological tissue. One conceivable storage solution is, for example, glutaraldehyde.

A conventionally manufactured transcatheter aortic valve prosthesis typically consists of up to six individual tissue parts/tissue components that are manually sutured together in a usually extremely time-consuming and cost-intensive process, and then integrated into a stent or other frame structure. This gives the implant a complex, three-dimensional geometry that is essential for the functionality of the prosthesis. The mostly three freely supported, inwardly directed leaflets form semilunar pockets that passively effect valve closure. The additional skirt components (inner and/or outer skirt) attached to the stent/frame structure serve to prevent or seal against paravalvular leakage (PVL).

The entire suturing process of the valve is performed entirely manually under the microscope and is thus extremely time, cost and resource intensive. In total, several hundred individual surgical knots are tied, with about half of the knots being for suturing the above-mentioned tissue parts/tissue components together and the other half for suturing the tissue components into the stent/frame structure. The difficulty here is that if a single knot is placed incorrectly, this immediately leads to rejection of the valve prosthesis and additional costs in the manufacturing process. Furthermore, sutures form mechanical weak points that can potentially lead to failure of the implant - as mentioned at the beginning.

Typically, the manufacturing of a TAVI/TAVR valve starts with the mechanical processing of the tissue (e.g. pericardium), where the required tissue component(s) is/are prepared and cleaned (e.g. from the pericardium). In the subsequent crosslinking process, the tissue is usually placed and/or fixed (e.g., stretched at the edges) on a suitable planar mold (e.g., one or more plates or a plastic frame), and placed in a suitable crosslinking solution (e.g., glutaraldehyde solution comprising glutaraldehyde oligomers) for several days.

The background to the need for chemical crosslinking is that biological tissue, unless it is supplied by cells and endogenous processes in the body, is subject to natural decomposition and denaturation processes. Accordingly, it must be selectively processed for further processing into a functional long-term implant.

As exemplified above for TAVI/TAVR valves, artificial compounds of tissues/tissue components (biological and/or artificial), especially tissues for medical use, are known. However, the compounds of the prior art are predominantly made of surgical materials, in particular surgical sutures comprising one or more surgical knots.

The connection of several tissue segments by sutures of surgical material to create a three- dimensional tissue geometry, e.g. of a TAVI/TAVR valve, are known. Furthermore, a process for three-dimensional shaping by means of rigid shaped bodies on both sides is known, for example, from US 8,136,218 B2. In this process, the tissue is placed between two rigid molded bodies and chemically crosslinked in this state so that the geometry of the molded bodies is permanently imprinted in the tissue.

However, there are also disadvantages associated with the prior art methods. For example, surgical sutures usually have to be placed manually. This process is very time-consuming, expensive and error-prone. The knots must be visually inspected individually. In addition, each individual knot represents a potential weak point, since mechanical forces that occur are focused on the knots. Surgical sutures also have a non-negligible space requirement, which means that minimum structural sizes of a few millimeters cannot be undercut for implants.

Furthermore, the rigid molded bodies described above are not capable of compensating for inhomogeneities in tissue thickness that are naturally always present. In areas of higher tissue thickness, this results in pressure peaks that cause partial fiber compaction and the associated stiffening of the tissue. Visually, these pressure points can be identified as transparent areas on the tissue surface. Air bubbles trapped between the two moldings also have this effect. In addition, usually the rigid molded bodies hinder the access of the crosslinking solution to the tissue, which results in a poorer crosslinking quality of the tissue.

Technical problem of the third aspect of the invention

However, in the prior art solutions described above, especially the orientation of the collagen fibers of the tissue component is not adapted to the physiological pressure loads in the heart. Therefore, it can be assumed that pressure peaks occur in the tissue after implantation, which impair the fatigue strength of the prosthesis. Consequently, one task of the third aspect of the invention is to provide a process for a three- dimensional shaping and crosslinking of a tissue/tissue component, in particular for an artificial heart valve, such as a TAVI/TAVR valve, which is simultaneously also adapted to the physiological pressure conditions in the heart. Furthermore, this process can also be used in an adapted manner for all four valve types of a heart: Mitral, Tricuspid, Pulmonary, and the aforementioned Aortic valve.

Technical solution

For physiological shaping during chemical crosslinking of the tissue to be treated, an already sutured or otherwise assembled artificial heart valve, in particular a TAVI/TAVR valve, with tissue component, which optionally may already be stabilized and dried, is crosslinked under constant fluid pressure by the crosslinking agent in a suitable device, whereby a physiological valve closure is induced under crosslinking solely from the fluid pressure and gravity effect. The continuous crosslinking process under this pressure thereby has the effect of preserving the current elongation state of the collagen fibers (in the closed state of the valve), and following this process according to the third aspect of the invention, the leaflets are formed in their natural orientation, equivalent to the corresponding native valve.

Advantageous effects of the third aspect of the invention

The solution according to the third aspect of the invention allows the reproducible production of a three-dimensionally shaped tissue component, preferably for an artificial heart valve, such as a TAVI/TAVR valve, in which the orientation of the collagen fibers is adapted to the physiological pressure conditions in the heart. Consequently, pressure peaks in the tissue after implantation of the artificial heart valve are eliminated, which has a positive effect on the fatigue strength and thus service life of the heart valve implant.

By adapting the process, it is also possible to manufacture an artificial heart valve with a one- piece tissue component, as the surgical sutures to create the three-dimensional tissue geometry are unnecessary. This reduces manufacturing costs while eliminating the sutures as mechanical weak points in the implant.

Moreover, in contrast to the use of rigid molded bodies on both sides for three-dimensional shaping as in the prior art, no pressure points are observed on the tissue surface after the process disclosed herein, indicating local stiffening of the tissue. Furthermore, in the device according to the third aspect of the invention, the crosslinking liquid reaches the tissue unimpeded, which has a positive effect on the crosslinking quality.

Another advantage is that in some embodiments of the process according to the third aspect of the invention, the entire suturing process of the heart valve implant takes place following stabilization and gentle drying of the tissue. In addition to an expected simplified handling, this reduces the risk of irreversible tissue damage due to desiccation during the suturing process.

Description of the third aspect of the invention

The three-dimensional crosslinking disclosed herein by means of a constant liquid column in a suitable device represents an advantageous possibility compared to the shaping of the tissue by rigid shaped bodies.

In particular, it is an objective of the third aspect of the invention to provide a functional artificial heart valve, preferably a TAVI/TAVR valve, more preferably with a one-piece or three- piece tissue component. However, according to the third aspect of the invention, a molded body is entirely omitted for three-dimensional crosslinking. Instead, a new process is provided that enables three-dimensional crosslinking in the closed and, for example, already sewn or otherwise prefabricated state of the valve component(s).

Briefly summarized, in the context of the third aspect of the invention, after mechanical preparation, the tissue to be formed is optionally stabilized and, for example, dried in a climatic chamber and, for example, processed/cut with a laser. In the cutting pattern of the laser, the leaflet sheets (e.g. three) as well as the skirt sheets (e.g. three or twelve) are already integrated, so that a further laser process is not necessary. In the subsequent sewing process, the tissue ends are first joined together to create a closed cylindrical valve shape. Then, the tissue component is sewn into the stent as the basic structure.

In other words, according to the third aspect of the invention, the entire suturing process of the artificial heart valve takes place before the tissue to be formed is crosslinked in the fluid column, and forms the basic prerequisite for the subsequent three-dimensional crosslinking of the valve in the closed state and thus quasi under simulated physiological pressure conditions following the native situation of the corresponding valve, for example a native aortic valve. For this purpose, a device is provided for forming a liquid column in which, for example, the sewn, partially sewn or even one-piece or three-piece and thus prefabricated/pre-assembled valve can be clamped essentially vertically, preferably vertically, so that the valve wings are oriented upwards. To generate a constant liquid column, a peristaltic pump connected to the device is used, for example, together with a supply and discharge hose system for a crosslinking solution, which continuously pumps the crosslinking liquid for shaping upwards into a hollow cylinder of the device (see Figure 3). The gravity pressure of the crosslinking liquid causes the artificial valve blades to be pressed against each other. The aim is to use a suitable crosslinking solution, such as a glutaraldehyde solution, to preserve this physiologically pressurized state of the collagen fibers, in accordance with the principle of three-dimensional crosslinking, and in this way to achieve a permanent shaping of the leaflets.

For positioning the valve prosthesis prefabricated as described above in the device according to the third aspect of the invention, a flexible hollow cylinder, e.g. a hollow cylinder produced by means of silicone casting, is inserted into the lower part of the sample chamber. This has an inner diameter of 26 mm, for example, and is thus adapted to the intended implantation diameter of the valve.

The pre-sewn prosthesis is inserted into the silicone ring in such a way that the stent struts in the lower valve area rest against an appropriately designed stop of the pressure part. This ensures reproducible alignment of the valve.

The remaining components are then assembled and the peristaltic pump connected. A crosslinking process lasting, for example, three days is started by filling, for example, 400 ml of glutaraldehyde solution into the device from above. The crosslinking liquid, which flows through or past the artificial valve into the collection basin, is immediately conveyed back into the hollow cylinder via the peristaltic pump - thus creating a continuous cycle of crosslinking solution. This generates a constant liquid column of about 10 cm, which induces valve closure under conditions similar to native physiology.

Optionally, fresh crosslinking solution could also always be supplied from above and the outflowed crosslinking solution then discarded. However, the technical solution involving the formation of a circuit is preferred within the scope of the third aspect of the invention. With the aforementioned context, a concrete and exemplary process for three-dimensional shaping of a tissue/tissue component under physiological pressure conditions, in particular for an artificial heart valve, comprises at least the following steps:

(a) providing a tissue/tissue component, preferably pericardial tissue, comprising chemically and/or biochemically crosslinkable groups; b) Optional stabilization and/or drying of the tissue/tissue component according to step a); c) Optional cutting of the tissue/tissue component to be crosslinked according to step b) by means of a suitable cutting instrument and/or a suitable cutting device, preferably by means of laser cutting; d) Optional joining/sewing of the tissue/tissue component according to step b) or c); e) arranging/placing and/or fixing/sewing the tissue/tissue component into a basic structure, in particular into a basic structure suitable for an artificial heart valve, such as a self-expanding or mechanically expandable stent; f) providing a device suitable and configured to form a constant liquid column of a crosslinking solution in a manner that loads and crosslinks the basic structure comprising the tissue/tissue component according to step e) in a liquid-tight manner and under physiological pressure conditions; g) arranging/placing the basic structure comprising the tissue/tissue component, in particular the prefabricated artificial heart valve, according to step e) in the device according to step f); h) assembling the device according to step f) and providing and connecting a pump unit, such as a peristaltic pump, in such a way as to be able to form the liquid column of crosslinking solution, preferably via a constant liquid circuit; i) filling the device according to steps f), g) and h) with a suitable crosslinking solution, preferably with a glutaraldehyde solution, more preferably with a 0.5 - 0.65% glutaraldehyde solution; j) Chemically crosslinking the tissue/tissue component according to step g) under physiological pressure conditions in the device according to steps f), g), h), and i) for at least 1 hour, preferably at least 4 hours, more preferably at least 12 hours, even more preferably at least 24 hours; k) Removal of the basic structure comprising the crosslinked tissue/tissue component, in particular the crosslinked artificial heart valve; l) optionally post-crosslinking the basic structure comprising the crosslinked tissue/crosslinked tissue component, in particular the crosslinked artificial heart valve, by means of a suitable crosslinking agent. Furthermore, on the process side, the third aspect of the invention comprises a chemical crosslinking of tissue joining partners comprising crosslinkable groups, such as, for example, free amino groups, by means of a suitable crosslinking agent under static, quasi-static or periodic pulsatile pressure loading in a defined overlap region for seamless, dense and tight tissue closure disclosed - for example, for tissue closure for a one-piece valve component made of pericardial tissue for a TAVI/TAVR valve. Thereby, a seamless, homogeneous, and at the same time mechanically stable connect! on/jointing of tissue/tissue components is achieved.

That means, the third aspect of the invention exploits, among other things, for the first time in a targeted manner, in sufficient quantity and density, the effect that a crosslinking agent such as, for example, glutaraldehyde can also form interfibrillar connections/crosslinks between two joining partners, such as, for example, tissue surfaces for a one-piece valve component, in order to realize a seamless, materially bonded and durable connect! on/joint.

The crosslinking agent is preferably an aldehyde-containing crosslinking agent, more preferably glutaraldehyde. In alternative embodiments of the third aspect of the invention, the crosslinking agent contains carbodiimide, formaldehyde, glutaraldehyde acetals, acyl azides, cyanimide, genepin, tannin, pentagalloyl glucose, phytate, proanthocyanidin, reuterin and/or epoxy compounds.

A particular advantage of the processes disclosed herein is that, for example, a glutaraldehyde solution can be used as a crosslinking agent in principle independently of concentration. In one embodiment, for example, the tissue/tissue components to be joined is placed in a glutaraldehyde oligomer-containing solution at pH 7.4 for 48 hours at a temperature of 4°C during the chemical crosslinking step, and subjected to quasi-static or periodic pulsatile pressure loading/compression.

In general, the skilled person is aware that chemical crosslinking, depending on the tissue to be treated and the desired properties of the crosslinked tissue, can also be regulated or controlled by temperature. Crosslinking generally starts at a temperature above 0°C. Preferred temperature ranges for chemical crosslinking in the sense of the third aspect of the invention are 1 - 50°C, preferably 10 - 50°C, more preferably 20 - 50°C, even more preferably 25 - 40°C, most preferably 35 - 40°C, for example at 37°C.

In one embodiment of the third aspect of the invention, alpha-gal epitopes may additionally be removed from the tissue in a further treatment step, which may be performed after or before the optional decellularization step. Any suitable alpha-galactosidase may be used for such an additional treatment step, e.g., alpha-galactosidase from green coffee bean (GCB) or Cucumis melo.

As mentioned above, on the device side, the task set is solved, inter alia, by a medical implant comprising the seamlessly and materially connected/joined tissue which has been subjected to one of the processes according to the third aspect of the invention.

With the context of the third aspect of the invention, the term "medical implant" or similar terms particularly includes stent-based implants and heart valve prostheses, particularly aortic valve prostheses, which are stent-based. According to the third aspect of the invention, the term "medical implant" also reads to any medical implant for which the suture-free joined/ connected tissue is suitable as a process product, for example, to seal the implant against an anatomical structure. Also included as a medical implant are pockets that can receive and be implanted with, for example, a pacemaker, an implantable leadless pacemaker, or a defibrillator.

Stent graft(s)" are stents that contain a fleece or other flat covering, such as a foil or tissue, on or in their often grid-like basic structure. In this context, the term "nonwoven" is understood to mean a textile tissue formed by individual fibers.

In the context of the third aspect of the invention, the term "nonwoven" also includes the case in which the textile sheet-like structure consists of only a single "continuous" fiber. Such a stent graft is used, for example, to support weak points in arteries, esophagus, or bile ducts, for example in the area of an aneurysm or a rupture of the vessel wall (so-called bail-out device), especially as an emergency stent.

Medical endoprostheses or implants for a wide variety of applications are known in great variety from the prior art and can be combined with the seamless and materially joined tissue of the third aspect of the invention for suitable purposes. Implants in the sense of the third aspect of the invention are in particular endovascular prostheses or other endoprostheses, e.g. stents (vascular stents, bile duct stents, vascular stents, peripheral stents or, e.g., mitral stents), endoprostheses, endoprostheses or endoprostheses, endoprostheses for closing persistent foramen ovale (PFO), pulmonary valve stents, endoprostheses for closing an ASD (atrial septal defect), as well as prostheses in the area of hard and soft tissue. Also possible as an implant is a left atrial appendage closure device (LAAC).

In an alternative, preferably the medical implant is a prosthetic heart valve, more preferably a TAVI/TAVR valve, which comprises an artificial heart valve made of sutureless and materially bonded/joined tissue and/or a seal made of said tissue, which is attached, preferably sutured, to an expandable or self-expanding and catheter implantable base frame, stent, or retaining device.

In all embodiments of the third aspect of the invention, the decellularization method, if performed, is applied to tissue that is not conventionally crosslinked after decellularization; rather, crosslinking occurs exclusively in the processes disclosed herein under quasi-static or periodic pulsatile pressure/compression in one or more selected overlap region(s) of the tissues involved.

In general, it is true for the entire present disclosure that the skilled person can suitably adjust the technical parameters such as times, amounts, concentrations, temperatures and, for example, pressures depending on the type of tissue to be treated and the desired crosslinking/bonding results.

In this respect, the skilled person is aware that the temperature during the stabilization step can influence the results. For example, too high a temperature (e.g., above about 85°C) leads to denaturation and irreversible damage to the tissue crosslinked for the purpose of bonding/jointing, e.g., glutaraldehyde crosslinked. Again, however, too low a temperature can lead to a solution that is too viscous. Preferably, exposure to the stabilizing solutions is at 37°C, but temperatures from room temperature up to 60°C should be tolerable.

As mentioned at the outset, the processes described in the third aspect of the invention are suitable for the preparation of substantially non-crosslinked tissue or, for example, decellularized, substantially non-crosslinked tissue - with the proviso that crosslinkable groups, e.g., free amino groups, must be present in the tissue. Optionally, all of the tissues addressed within the scope of the third aspect of the invention may be stabilized as described herein. Optionally, alpha-gal epitopes can be removed from all these tissues by a suitable alphagalactosidase treatment (preferably originating from GCB or Cucumis melo, see above).

As for the implant itself, the aforementioned problem is further solved by an implant containing biological tissue that has been subjected to one of the processes according to the third aspect of the invention and, if necessary, subsequently stabilized and/or dried.

A core of the process for seamless joining according to the third aspect of the invention lies in the surprising realization that various suitable crosslinking agents, such as for example and preferably glutaraldehyde, not only have the ability to form inter- and intramolecular crosslinks within a collagen fiber (see prior art above), but also interfibrillar crosslinks between individual fibers. Thus, it is possible for the first time to generate seamless and materially cohesive connect! ons/joints in an overlapping area of two tissue joining partners, which comprise crosslinkable groups, such as free amino groups, e.g. containing collagen, by simultaneously applying a static, quasi-static or periodic pulsatile pressure load/compression by means of a suitable device.

The basic requirement for this is that the distance between the collagen fibers is smaller than the length of the crosslinking molecules involved, such as the glutaraldehyde oligomers mentioned above, which form the actual crosslinks. Therefore, in the context of the third aspect of the invention, a pressure-generating device has been provided to generate a quasi-static or a periodic pulsatile vertical force application (pressure load/compression), with desired repetition cycles and over a desired time period, to a defined tissue region during the crosslinking process. According to the third aspect of the invention, the pressure generating device can be based on the physical principles of pneumatics, mechanics, and, for example, hydraulics, but is not limited in this respect. In the context of the third aspect of the invention, hydraulics is a particularly preferred embodiment for generating the pressure load/compression. The basic requirement for said formation of interfibrillar crosslinks is that the distance between the collagen fibers and microfibrils involved is smaller than the length of the glutaraldehyde oligomers involved (see above), which essentially form the crosslink. Appropriate pressing parameters over suitable time periods to reduce the fiber spacing are thus essential to enable a stable, seamless and cohesive bond using glutaraldehyde oligomers. At the same time, however, a high pressing pressure potentially, and thus not necessarily, leads to preventing accessibility of the crosslinking solution to the tissue during force application.

Therefore, according to the third aspect of the invention, in a preferred embodiment, not only a quasi-static pressure on the tissue over a suitable longer period of time during crosslinking is considered (quasi-static means a constant pressure over a longer period of time (e.g. 300 seconds), which may be interrupted less frequently by short and suitable pressure pauses (e.g. 1 or 2 second(s)), but a periodic-pulsatile pressure load/compression over suitable shorter periods, but with possibly more frequent repetition of the pressure phases, also interrupted by short pressure pauses (e.g. 30 seconds pressure, 1 or 2 second(s) pressure pause, followed by 30 seconds pressure, 1 or 2 second(s) pause, etc.).

In an alternative embodiment, it is possible to achieve the crosslinking according to the third aspect of the invention for seamless and material-locking connect! on/jointing via a static, i.e. permanent pressure, without pauses. The prerequisite for this is to provide a contact surface for the tissue to be joined/joined which is perforated, i.e. is open to the crosslinking solution, in order to ensure its access to the tissue to be crosslinked.

The terms "amino group-containing(s)"/" comprising free amino groups" or similar terminology mean, in the context of the third aspect of the invention, that the tissue to be joined/joined must comprise free amino groups that are chemically crosslinkable by means of a suitable crosslinking agent in order to be seamlessly and cohesively joined/joined via the processes described herein.

With this context, the term "collagen-containing(s)" or similar terms used in the context of the third aspect of the invention describes that the tissue(s) to be joined/joined must comprise free collagen fibers in order to be seamlessly and cohesively joined/joined via the processes described herein.

Suitable collagen-containing tissues within the scope of the third aspect of the invention are, for example, native collagen-containing tissues, moist collagen-containing tissues, already processed (but essentially non-crosslinked) collagen-containing tissues, such as, for example, already stabilized collagen-containing tissues, already preserved collagen-containing tissues, already dried (non-crosslinked) collagen-containing tissues, already decellularized tissues, as well as mixed forms of the aforementioned tissues. It is clear to the person skilled in the art that this list of suitable collagen-containing tissue forms is not exhaustive, but that further collagen- containing tissue types may be suitable for the disclosed process.

According to the third aspect of the invention, bonding processes for stabilized, dried (non- crosslinked) tissue were tested in particular.

In one alternative, even the seamless and material-locking connection of already fully or partially crosslinked tissue is possible in principle, whereby exceptionally either no crosslinking solution such as, for example, glutaraldehyde solution or only a very low-concentration glutaraldehyde solution (0 - 1% glutaraldehyde) is additionally required. In a further preferred variant, the medical implant is a vascular valve prosthesis, in particular a heart valve prosthesis. For example, an aortic valve prosthesis, a tricuspid valve prosthesis, a mitral valve prosthesis and a pulmonary valve prosthesis are suitable examples of a heart valve prosthesis. Typically, such prostheses or implants have a stent-like structure that carries a valve assembly inside it to replace a natural vascular or heart valve. In this regard, the seamless and materially bonded/joined tissue may be applied to a surface of the prosthetic heart valve (internal and/or external).

With the context of the third aspect of the invention, the terms/expressions "quasi-static compressive loading/compression" or similar terms/expressions denote a substantially vertical physical application of force to the tissue to be connected/joined, performed in such a way that it can be viewed solely as a sequence of equilibrium states. Thus, the time scale on which a quasi- static process occurs must be much slower than the time period in which equilibrium is reached (the so-called relaxation time). Although a respective state of equilibrium prevails to a large extent at each point in time of the process, it is nevertheless generally an objective of the process to obtain different states or a characteristic curve. This means that the equilibrium state at time tl (pressure load) may well differ considerably from the equilibrium state at time t2 (pressure relief or pressure pause). The above definition is merely intended to exclude the possibility that dynamic or more dynamic processes, e.g. a periodic pulsatile pressure load/compression, have any appreciable influence on the joining/joining behavior of the tissue components to be joined/joined.

Specifically, in the context of the third aspect of the invention, this means that the relationship between "with pressure loading" and "pressure relief/pressure pause" during the chemical crosslinking process in the case of "quasi-static", is more protracted over time for the pressure loading, and with longer periods of time, possibly alternating The "quasi-static" case is more protracted over time for the pressure load, and takes place over longer periods of time, possibly alternating several times, than in direct comparison with a "periodic pulsatile" relationship between "pressure load" and "pressure relief/pressure pause", which in contrast is more shortlived for the pressure load; i.e. the two states "with pressure" / "without pressure" are also shorter over time and are repeated alternately noticeably more often, if necessary.

Conversely, the terms/expressions "periodic-pulsatile pressure loading/compression" or similar terms/expressions denote that the ratio between "pressure loading" and "pressure relief/pressure pause" during the chemical crosslinking process is more short-lived over time, especially for the pressure loading, and thus the states "with pressure "/"without pressure" and with smaller time spans also alternate significantly more times, in direct comparison to the "quasi-static" ratio described above.

Specifically, in the context of the third aspect of the invention, the terms/expressions "quasi- static pressure load compression" or similar terms/expressions can be used over a ratio of, for example, 300: 1 seconds with respect to "with pressure load" (300 seconds) vs. "pressure release/pressure pause" (for example. 1 or 2 second(s)), and thus differ from the terms/expressions "periodic-pulsatile pressure load/compression" or similar terms/expressions in such a way that in the latter case a ratio of e.g. 30: 1 seconds exists with respect to "with pressure load" (e.g. 30 seconds) versus "pressure relief/pressure pause" (e.g. 1 or 2 second(s)).

It is to be understood by the person skilled in the art that, within the scope of the third aspect of the invention, he should not slavishly adhere to the exact ratios of 300: 1 seconds for "pressure loading" (300 seconds) versus "pressure relief/pressure pause" (e.g. 1 second) in terms of "quasistatic", and 30: 1 for "pressure loading" (e.g. 30 seconds) versus "pressure relief (e.g. 1 second) in terms of "periodic-pulsatile", but rather the relations of the mentioned time spans to each other distinguish the variants "quasi-static" from "periodic-pulsatile" during chemical crosslinking, and the exact values may suitably deviate from the above examples. For example, for "quasi-static" the above-described ratios of 250: 1 seconds or, for example, 350: 1 seconds are also conceivable, and with regard to "periodic-pulsatile", for example, 15: 1 seconds or 30:2 seconds are conceivable.

The above-mentioned alternative case of static crosslinking - without a pressure pause - but with a perforated/hole-shaped counterform for accessibility of the crosslinking solution is appropriately delimited with the above definition of the quasi-static case.

The skilled person is generally aware that the above times can vary significantly depending on the tissue to be treated and the crosslinking agent to be used. Too short times are likely to lead to insufficient stability, too long times are likely to end in a waste of time, and the skilled person would optimize the parameters (time, temperature, concentrations, etc.) depending on the material.

At this point, the overlap length of the tissue components, the physical compression type (hydraulic, mechanical, etc.), cylinder force and the crosslinking time itself should be highlighted as other significant factors influencing the processes according to the third aspect of the invention. Thus, despite a lower breaking load, a reduction in the overlap length tends to result in a higher bond strength. In order to ensure the accessibility of the crosslinking agent, e.g. the glutaraldehyde solution, to the overlap area, a quasi-static or periodic pulsatile pressure load/compression is indispensable according to the third aspect of the invention.

With regard to the crosslinking duration, a total period of static, quasi-static or periodic pulsatile compressive loading/compression of three days is particularly preferred.

The crosslinking of overlapping tissue joining partners according to the third aspect of the invention is a valid concept for the seamless and material -locking joining/joining of tissue, in particular tissue containing collagen. With regard to the application itself, however, the skilled person must always take into account the load limits of the bonded joint in different load cases, as well as the effects of the compression process on the properties of the tissue joining partners.

Example - Hydrostatic crosslinking of a TAVI/TAVR valve together with a device for forming a constant fluid column

In the human body, the pressure gradient between the ventricle and the aorta leads to valve leaflet closure. According to the third aspect of the invention, a suitable method to cause valve closure in vitro is to apply pressure to an artificial heart valve by means of the formation of a constant liquid column of crosslinking solution, which fixes/crosslinks the tissue/tissue component(s) of the artificial heart valve following physiological conditions. This concept of the third aspect of the invention is described below on the basis of an exemplary process for a TAVI/TAVR valve with porcine pericardium as biological tissue, but is not to be understood as limited in this respect, but is rather also suitable for other types of heart valves but also, for example, venous valves:

The device used in the following for the constant formation of a liquid column of crosslinking solution, as shown in Figures 2-4, was manufactured, for example, using a suitable 3D printing process.

The collection basin (8) acts as a reservoir and collection container for the crosslinking solution. A built-in viewing window (12) is used to check the liquid level and to guide the lower liquidcollecting hoses (13). Via the connected peristaltic pump (e.g. Cartridge Pump 4.7519-06; Masterflex; 15, 16, 17), the crosslinking solution is continuously conveyed from the collection reservoir (8, 12) via the liquid-feeding hoses (14) at the top through bores into the hollow cylinder for constant generation of the liquid column. The hollow cylinder itself is connected via a thread to the two-part sample chamber and this in turn is connected via a further thread to the collecting basin.

The connected peristaltic pump continuously conveys the crosslinking solution into the hollow cylinder so that a constant liquid column is generated.

After mechanical preparation and optional stabilization and drying of the tissue/tissue component, a cut is made, e.g. a laser cut. The corresponding cutting pattern already contains the twelve skirt and three leaflet sheets provided. The tissue component is then sutured at the open ends and integrated into a suitable self-expanding stent. Laser and suturing processes are performed, for example, in the dry, non-crosslinked state of the tissue/tissue component.

To position the prefabricated valve prosthesis in the test set-up, a (flexible) hollow cylinder made, for example, by silicone casting is inserted into the lower part of the sample chamber. This has an inner diameter of, for example, 26 mm and is thus adapted to the intended implantation diameter of the valve. The sutured prosthesis is inserted into a silicone ring in such a way that the stent struts in the lower valve area rest against an appropriately designed stop of the pressure part. This ensures reproducible alignment of the valve. The remaining components are then assembled and the peristaltic pump connected.

The exemplary three-day crosslinking process is started by filling 400 ml of glutaraldehyde solution into the set-up from above. The liquid flowing through or past the valve into the collecting basin (12) is immediately conveyed back into the hollow cylinder (6) via the pump (15, 16, 17). This generates a constant liquid column of about 10 cm, which induces the valve closure.

Three-dimensional crosslinking by means of a liquid column differs significantly from the prior art crosslinking variant with rigid molded bodies. In liquid column crosslinking, the crosslinking solution reaches the tissue unimpeded. Due to the pumping process and the associated liquid circulation, there is also a dynamic crosslinking process. It is therefore of interest to know how these effects affect the denaturation temperature and the number of free amino groups, since both properties are decisive in characterizing the degree of crosslinking of the biological tissue. The results of corresponding tests of tissue crosslinked according to the third aspect of the invention in direct comparison with conventionally freely crosslinked porcine pericardium are summarized in Table 1.

Table 1: Denaturation temperature and number of free amino groups of porcine pericardium crosslinked according to the third aspect of the invention under a liquid column with glutaraldehyde in direct comparison with equivalent freely crosslinked porcine pericardium (reference/standard); (n = 4).

Sample type Denaturation temperature Free Amino groups

Crosslinking free

88,7 ± 0,6 [°C] 4,9 ± 1,4 [pg/mg]

(Standard)

Crosslinking under

89,0 ± 0,7 [°C] 6,1 ± 1,6 [ug/mg] liquid column

It is shown that the tissue crosslinked according to the third aspect of the invention is completely and uniformly crosslinked. There are no indications of a significant difference compared with conventionally freely crosslinked reference tissue. With regard to the crosslinking quality, the three-dimensional crosslinking by means of a liquid column is qualitatively identical to the conventional tissue used so far.

Not only in water, but also in air, the tissue component exhibits remarkable dimensional stability and self-aligns according to its imprinted geometry. This proves that three-dimensional shaping of the tissue can also be realized by crosslinking by means of a liquid column.

That is, in summary, according to the third aspect of the invention, a pre-assembled/prefabricated artificial TAVI/TAVR valve is oriented in the device disclosed herein such that the freely movable leaflets (e.g., three) point upward toward the hollow cylinder. The pressure gradient/gravity pressure (resulting from the action of gravity on the liquid column) induces the valve closure, and is thereby essentially adjustable by the height of the formed liquid column, the flow velocity, and the liquid amount of crosslinking solution.

It should be noted that the device described here is merely a proven example, and many other embodiments of the setup for generating a constant gravity pressure on the valve blades, in particular via a liquid column of crosslinking solution, are conceivable. In view of the above disclosure, the third aspect of the invention further comprises the following embodiments numbered in ascending order:

1. Process for three-dimensional shaping of a tissue/ tissue component under physiological pressure conditions, in particular for an artificial heart valve, wherein the process comprises at least the following steps: a) providing a tissue/ tissue component, preferably pericardial tissue, comprising chemically and/or biochemically crosslinkable groups; b) optional stabilization and/or drying of the tissue/tissue component according to step a); c) optional joining/sewing of the tissue/tissue component according to step a); d) arranging/placing and/or fixing/sewing the tissue/tissue component into a basic structure, in particular into a basic structure suitable for an artificial heart valve, such as a self-expanding or mechanically expandable stent; e) providing a device (6, 7, 8, 13, 14, 15, 16, 17) suitable and configured to form a constant liquid column of a crosslinking solution in a manner that fluid-tightly loads and crosslinks the basic structure comprising the tissue/tissue component according to step d) under physiological pressure conditions; f) arranging/placing the basic structure comprising the tissue/tissue component, in particular the prefabricated artificial heart valve, according to step d) in the device according to step e) (18, 19, 20, 21, 22); g) filling the device according to steps e) and f) with a suitable crosslinking solution; h) chemically crosslinking the tissue/tissue component according to step f) under physiological pressure conditions in the device according to steps e), f) and g) for a suitable period of time; i) removal of the basic structure comprising the crosslinked tissue/tissue component, in particular the crosslinked artificial heart valve (23, 24, 25); j) optional post-crosslinking of the basic structure comprising the crosslinked tissue/crosslinked tissue component (23, 24, 25), in particular the crosslinked artificial heart valve, by means of a suitable crosslinking agent.

2. The process according to embodiment 1, wherein the process comprises at least the following steps: a) providing a tissue/tissue component, preferably pericardial tissue, comprising chemically and/or biochemically crosslinkable groups; b) stabilization and/or drying of the tissue/tissue component according to step a); c) optional cutting of the tissue/tissue component to be crosslinked according to step b) using a suitable cutting instrument and/or a suitable cutting device; d) optional joining/sewing of the tissue/tissue component according to step b) or c); e) arranging/placing and/or fixing/sewing the tissue/tissue component into a basic structure, in particular into a basic structure suitable for an artificial heart valve, such as a self-expanding or mechanically expandable stent; f) providing a device (6, 7, 8, 13, 14, 15, 16, 17) suitable and configured to form a constant liquid column of a crosslinking solution in a manner that fluid-tightly loads and crosslinks the basic structure comprising the tissue/tissue component according to step e) under physiological pressure conditions; g) arranging/placing the basic structure comprising the tissue/tissue component, in particular the prefabricated artificial heart valve, according to step e) in the device according to step f) (18, 19, 20, 21, 22); h) filling the device according to steps (f) and (g) with a suitable crosslinking solution; i) chemically crosslinking the tissue/tissue component according to step g) under physiological pressure conditions in the device according to steps f), g), and h) for a suitable period of time, preferably at least 1 hour, more preferably at least 4 hours; j) removal of the basic structure comprising the crosslinked tissue/tissue component, in particular the crosslinked artificial heart valve (23, 24, 25); k) optional post-crosslinking of the basic structure comprising the crosslinked tissue/crosslinked tissue component, in particular the crosslinked artificial heart valve by means of a suitable crosslinking agent.

3. The process according to embodiment 1 or 2, wherein the process comprises at least the following steps: a) providing a tissue/tissue component, preferably pericardial tissue, comprising chemically and/or biochemically crosslinkable groups; b) stabilization and/or drying of the tissue/tissue component according to step a); c) optional cutting of the tissue/tissue component to be crosslinked according to step b) by means of a suitable cutting instrument and/or a suitable cutting device, preferably by means of laser cutting; d) optional joining/sewing of the tissue/tissue component according to step b) or c); e) arranging/placing and/or fixing/sewing the tissue/tissue component into a basic structure, in particular into a basic structure suitable for an artificial heart valve, such as a self-expanding or mechanically expandable stent; f) providing a device (6, 7, 8, 13, 14, 15, 16, 17) suitable and configured to form a constant liquid column of a crosslinking solution in a manner that fluid-tightly loads and crosslinks the basic structure comprising the tissue/tissue component according to step e) under physiological pressure conditions; g) arranging/placing the basic structure comprising the tissue/tissue component, in particular the prefabricated artificial heart valve, according to step e) in the device according to step f) (18, 19, 20, 21, 22); h) assembling/putting together the device according to step f) and providing and connecting a pump unit (13, 14, 15, 16, 17), such as a peristaltic pump, in such a way as to be able to form the liquid column of crosslinking solution via a constant liquid circuit; i) filling the device according to steps f), g) and h) with a suitable crosslinking solution; j) chemically crosslinking the tissue/tissue component according to step g) under physiological pressure conditions in the device according to steps f), g), h), and i) for a suitable period of time, preferably at least 1 hour, more preferably for at least 4 hours; k) removal of the basic structure comprising the crosslinked tissue/ tissue component, in particular the crosslinked artificial heart valve (23, 24, 25); l) optional post-crosslinking of the basic structure comprising the crosslinked tissue/crosslinked tissue component, in particular the crosslinked artificial heart valve, by means of a suitable crosslinking agent.

6. The process according to any one of the preceding embodiments, wherein the crosslinking agent is a 0.5% glutaraldehyde solution. 7. The process according to any one of the preceding embodiments, wherein the tissue has been subjected to a pretreatment comprising an optional decellularization, e.g. with a suitable detergent, preferably with a solution containing surfactin and deoxycholic acid, and optionally a pre-crosslinking, preferably with a solution containing glutaraldehyde.

9. The process according to any of the preceding embodiments, wherein the stabilization step comprises exposing the, optionally decellularized, tissue/tissue component to at least one solution, but preferably at least two different solutions, wherein one solution comprises glycerol and another solution comprises polyethylene glycol.

10. The process according to embodiment 9, wherein exposure to one or more of the solutions lasts from 5 minutes to 2 hours.

11. The process according to any of the preceding embodiments, wherein drying of the tissue/tissue component is carried out in a suitable environment with a constant low relative humidity or in a climate-controllable device/facility in a suitable manner, such as by reducing the relative humidity, for example from 95% to 10% over 12 hours at 37°C.

12. The process according to any one of embodiments 9 to 11, wherein of the at least two different solutions, a first solution comprises polyethylene glycol having an average molecular weight between 150 g/mol and 300 g/mol; and a second solution comprises an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol.

13. The process according to any one of embodiments 9 to 12, wherein of the at least two different solutions, a first solution comprises polyethylene glycol having an average molecular weight between 200 g/mol and 600 g/mol; and a second solution comprises an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol.

14. The process according to any of the preceding embodiments, wherein the process additionally comprises removal of alpha-gal epitopes by use of a suitable alpha-galactosidase. 15. The process according to embodiment 14, wherein the alpha-galactosidase is obtained from green coffee bean (GCB).

16. The process according to embodiment 14, wherein the alpha-galactosidase is obtained from Cucumis melo.

17. The process according to any one of the aforementioned embodiments, wherein the chemical crosslinking or the optional post-crosslinking takes place over a period of time in the range of at least 4 h to 12 days, preferably 12 h to 5 days, more preferably 3 days, even more preferably over 2 days.

18. The process according to any of the above embodiments, wherein the chemical crosslinking or optional post-crosslinking takes place at a temperature in the range of 1°C to 50°C, preferably 18°C to 40°C, more preferably 30°C to 38°C, even more preferably at or around 37°C.

19. Basic structure comprising a tissue/ tissue component obtained according to one of the aforementioned processes for medical application, in particular for application as a vascular implant, preferably as an artificial heart valve, more preferably as an artificial aortic valve.

20. Device for forming a constant liquid column from a crosslinking solution, comprising the components:

- a hollow cylinder (6), preferably flexible hollow cylinder (6), with an inlet for a crosslinking agent (9);

- a sample chamber (7) suitable for receiving and holding a base structure comprising a tissue/tissue component to be crosslinked;

- a collecting basin (8) having an opening/outlet (12) for a crosslinking agent;

- a supply and removal hose system for the crosslinking agent (13, 14);

- a controllable pump unit (15, 16, 17) with suitable connections for the hose system, wherein the pump unit in connection with the hose system is configured to provide continuous fluid circulation from the hollow cylinder (6) through the sample chamber (7) to the collection basin (8) and back to the hollow cylinder. Seamless connecting of tissue

The fourth aspect of the invention relates to a process for seamlessly joining biological and/or artificial tissue comprising crosslinkable groups, e.g. free amino groups, for use as a component of a medical implant, in particular for use as a component of a covered stent or an artificial heart valve, as well as a medical implant, which contains such seamlessly joined biological and/or artificial tissue.

More specifically, the fourth aspect of the invention relates to a process for seamlessly and materially bonded joining/connecting tissues suitable for chemical crosslinking; for example, elastin-containing tissues and tissues containing free amino groups - in particular, collagen- containing tissues. Preferred in the context of the fourth aspect of the invention are amino group- containing, more preferably collagen-containing, biological and/or artificial tissue components, such as e.g. biological pericardial tissue components (skirt/leaflets etc.) of a TAVI/TAVR valve or preferred are amino group-containing, more preferably collagen-containing, biological and/or artificial tissue components of a so-called covered stent.

The fourth aspect of the invention is described herein essentially using the example of a method for the sutureless and material bonded connect! on/joining of tissue for use for an artificial aortic valve (TAVI/TAVR). While the fourth aspect of the invention is particularly well suited for joining such tissue, it is not limited to such application(s). For example, the fourth aspect of the invention is also applicable to the sutureless and material bonded ex vivo connection/ joining of (artificial) blood vessels, (artificial) bone cartilage, (artificial) ligaments, (artificial) skin or the like.

Transcatheter aortic valve implantation ("TAVI"), or transcatheter aortic valve replacement ("TAVR"), or percutaneous aortic valve replacement ("PAVR") is a minimally invasive procedure in which an artificial aortic valve prosthesis is placed and released in a collapsed (crimped; compressed) state within the native aortic valve.

The implant usually consists of individual, manually sutured, collagen-containing tissue components integrated into a suitable self-expanding or mechanically expandable stent (e.g., balloon-expandable) or support structure. Through the typically complex and error-prone suturing process, a complex, three-dimensional tissue geometry is thereby created, which is essential for the functionality of the prosthesis. At the same time, a skilled person is aware that the numerous surgical nodes/sutures represent mechanical weak points that can potentially lead to failure of the implant, and thus can also sometimes cause severe complications in the patient.

There are basically three different types of prosthetic heart valves, especially aortic valve prostheses: Prostheses with mechanical valves, which are manufactured artificially, mostly from graphite coated with pyrolytic carbon; prostheses with valves made from biological tissue (or partly biological tissue locally reinforced by artificial fibers, if necessary), mostly pericardial tissue typically derived from animal sources (e.g., porcine or bovine); and valves made from artificial materials such as polymers. The heart valve formed from the biological tissue is generally secured in a base body (e.g., a solid plastic scaffold or a self-expanding stent or a balloon-expanding stent) and this is implanted in the position of the natural valve. The fourth aspect of the invention describes, among other things, a method for sutureless and integral connection/ joining of such a tissue for use in a prosthetic aortic valve to be implanted in place of a natural aortic valve.

Usually, the initial tissue must be thoroughly cleaned and prepared before implantation. As far as possible, the tissue is modified in such a way that it is not recognized by the body as foreign tissue, calcifies as little as possible and has the longest possible service life. Essentially, such a process for preparing tissue comprises several steps:

Preferably, according to the fourth aspect of the invention, the biological and/or artificial tissue is subjected to a pretreatment comprising an optional decellularization with a suitable detergent, preferably with a solution containing surfactin and deoxycholic acid. The decellularization can also be performed otherwise, for example, via lysis of the cells or by an osmotic digestion. In the context of the fourth aspect of the invention, the expressions/terms "biological and/or artificial tissue" or similar terminology describe the tissue genera suitable for the processes of the fourth aspect of the invention for seamless joining/connecting. That is, for example, purely biological tissue is tissue of purely natural origin, e.g., porcine pericardium taken from a porcine pericardium. Purely artificial tissue is tissue that has been artificially produced, for example, from one or more different polymer(s) - e.g., by means of suitable 3D printing processes or the like. Biological and artificial tissue refers to mixed forms of e.g. a biological basic substance such as porcine pericardium, but including artificial materials, e.g. for local reinforcement of certain tissue regions, which are exposed to e.g. enormous physiological pressure and/or tensile loads - e.g. leaflets of a TAVI/TAVR valve. However, in the context of the fourth aspect of the invention, common to all these tissue types, and essential, is that they comprise crosslinkable groups, e.g. free amino groups, in particular collagen fibers, which are chemically and/or biochemically crosslinkable.

It is also essential for the processes according to the fourth aspect of the invention that the starting tissue/components are introduced into the processes according to the fourth aspect of the invention substantially non-crosslinked at least in the overlap region (i.e. the tissue region(s) to be joined/connected, but preferably in its entirety; i.e. that, if possible, no substantial precrosslinking has taken place, for example by means of glutaraldehyde solution. Substantially non-crosslinked tissue throughout the application means that the proportion of crosslinkable groups in the tissue to be treated (compared to non-crosslinkable groups) is greater than 50%, preferably greater than 60%, even more preferably greater than 80%, most preferably greater than 90%. However, this also means that lightly or only slightly pre-crosslinked or partially crosslinked tissue is suitable for the methods of the first aspect of the invention.

However, this also means that lightly or only slightly pre-crosslinked or partially crosslinked tissue is suitable for the processes of the fourth aspect of the invention.

The processes according to the fourth aspect of the invention are thus suitable for seamless joining/connecting of substantially non-crosslinked tissue, native tissue, non-crosslinked decellularized tissue or non-crosslinked non-decellularized tissue. Also suitable are natively dried tissues, which optionally have also been previously subjected to decellularization. The prerequisite is always that the tissue to be joined/connected must comprise crosslinkable groups, e.g. free amino groups, in particular collagen, e.g. contained in collagen fibers. After decellularization, as many cellular components as possible are removed from the tissue and the biological material consists exclusively of extracellular matrix. In pericardial tissue, the extracellular matrix is predominantly formed from the said collagen fibers. In order to achieve a biological material with the best possible mechanical properties and to prevent defense reactions of the receiving body, in the prior art the collagen fibers are crosslinked by means of a suitable crosslinking agent through the incorporation of chemical bonds.

A conventionally manufactured transcatheter aortic valve prosthesis typically consists of up to six individual tissue parts/components, which are manually sutured together in a usually extremely time-consuming and cost-intensive process, and then integrated into a stent or other frame structure. This gives the implant a complex, three-dimensional geometry that is essential for the functionality of the prosthesis. The mostly three freely supported, inwardly directed leaflets form semilunar pockets that passively effect valve closure. The additional skirt components (inner and/or outer skirt) attached to the stent/frame structure serve to prevent or seal against paravalvular leakage (PVL).

Thus, the tissue portion of a TAVI/TAVR valve usually consists of a total of six individual tissue components cut from crosslinked tissue patches. The three leaflet parts, which functionally effect the opening and closing of the prosthesis, are called "leaflets". The three so-called inner skirt parts are immovably attached internally to the stent/frame structure in the final product and serve primarily to reduce paravalvular leakage. A shaping process, e.g. laser cutting or punching, is followed by a complex, multi-stage sewing process, which gives the valve implant its characteristic three-dimensional geometry. In some prior art variants, an outer skirt is additionally attached to the outside of the TAVI/TAVR valve, which is also mostly made of tissue and addresses PVL.

The entire valve suturing process is performed entirely manually under a microscope, making it extremely time-, cost-, and resource-intensive. In total, several hundred individual surgical knots are tied, with approximately half of the knots involved in suturing together the aforementioned tissue parts/components and the other half involved in suturing the tissue components into the stent/frame structure. The difficulty here is that if a single knot is placed incorrectly, this immediately leads to rejection of the valve prosthesis and additional costs in the manufacturing process. Furthermore, sutures form mechanical weak points that can potentially lead to failure of the implant - as mentioned at the beginning.

As mentioned, such surgical sutures usually have to be placed manually. This process is very time-consuming, expensive and error-prone - to list just a few of the associated disadvantages. Surgical knots, for example, must be placed individually by personnel in a highly concentrated manner and must always be visually inspected. In addition, each individual knot represents a potential weak point of the medical tissue, since mechanical forces occurring under stress of a medical implant are focused on the knots. Surgical sutures also have a non-negligible space requirement (space requirement), which means that minimum structural sizes of a few millimeters cannot be undercut, especially in the case of medical implants. This noticeably limits medical implants in their medical application areas.

Technical problem

Against the background described above, a technical problem of the fourth aspect of the invention is to provide processes that enable, in particular, a seamless and material bonded joining/connecting of tissue/tissue components in a defined area (e.g. one or more overlap area(s)) for its application in medical implants, in particular covered stents and TAVI/TAVR valves. Technical solution

To solve the technical problem, a chemical crosslinking of tissue joining partners comprising crosslinkable groups, such as free amino groups, is disclosed on the process side by means of a suitable crosslinking agent under static, quasi-static and periodic pulsatile pressure loading, respectively, in a defined overlap region for seamless, dense and firm material closure. As a result, a seamless, homogeneous, and at the same time mechanically stable connection/ joining of tissue/tissue components is achieved.

In other words, the fourth aspect of the invention for the first time specifically exploits, in sufficient quantity and density, the effect that a crosslinking agent such as, for example, glutaraldehyde can also form interfibrillar bonds/crosslinks between two joining partners such as, for example, tissue patches, in order to realize a seamless, tight and stable bond/joint.

The crosslinking agent is preferably an aldehyde-containing crosslinking agent, more preferably glutaraldehyde. In alternative embodiments of the fourth aspect of the invention, the crosslinking agent includes carbodiimide, formaldehyde, glutaraldehyde acetals, acyl azides, cyanimide, genepin, tannin, pentagalloyl glucose, phytate, proanthocyanidin, reuterin, and/or epoxy compounds.

In one embodiment, for example, the tissue/components to be joined is placed in a glutaraldehyde oligomer-containing solution at pH 7.4 for 48 hours at a temperature of 4°C during the chemical crosslinking step, and subjected to quasi-static or periodic pulsatile pressure loading/compression.

In general, the skilled person is aware that chemical crosslinking, depending on the tissue to be treated and the desired properties of the crosslinked tissue, can also be regulated or controlled by temperature. Crosslinking generally starts at a temperature above 0°C. Preferred temperature ranges for chemical crosslinking in the sense of the fourth aspect of the invention are l-50°C, preferably 10-50°C, more preferably 20-50°C, even more preferably 25-40°C, most preferably 35-40°C, for example at 37°C.

Advantageously, the tissue is rinsed at least once, preferably several times, with a suitable solvent, in particular a buffered salt solution and/or an alcohol solution, before and particularly preferably after the decellularization (provided that it is decellularized tissue). Buffered sodium chloride solutions and/or an ethanol solution are particularly advantageous.

In one embodiment of the fourth aspect of the invention, alpha-gal epitopes may additionally be removed from the tissue in a further treatment step, which may be performed after or before the optional decellularization step. Any suitable alpha-galactosidase can be used for such an additional treatment step, e.g., alpha-galactosidase from green coffee bean (GCB) or Cucumis melo.

As mentioned above, on the device side, the problem posed is solved, inter alia, by a medical implant comprising the seamlessly and material bonded connected/joined tissue subjected to one of the processes according to the fourth aspect of the invention.

With the context of the fourth aspect of the invention, the term "medical implant" or similar terms particularly includes stent-based implants and heart valve prostheses, particularly aortic valve prostheses, which are stent-based. According to the fourth aspect of the invention, the term "medical implant" also reads to any medical implant for which the suture-free joined/ connected tissue is suitable as a process product, for example, to seal the implant against an anatomical structure.

Also included as a medical implant are pockets that can receive and be implanted with, for example, a cardiac pacemaker, an implantable leadless pacemaker, or a defibrillator. Nowadays, stents are used particularly frequently as implants for the treatment of stenoses (narrowing of blood vessels). They have a body in the form of a possibly perforated tubular or hollow cylindrical basic structure, which is open at both longitudinal ends. The basic structure of the stent may be composed of individual meshes formed by zigzag or meander-shaped webs. The tubular basic structure of such an endoprosthesis is inserted into the vessel to be treated and serves to support the vessel.

In the context of the fourth aspect of the invention, the term "nonwoven" also includes the case in which the textile sheet-like structure consists of only a single "continuous" fiber. Such a stent graft is used, for example, to support weak points in arteries, esophagus, or bile ducts, for example in the area of an aneurysm or a rupture of the vessel wall (so-called bail-out device), especially as an emergency stent.

Medical endoprostheses or implants for a wide variety of applications are known in great variety from the prior art and can be combined with the seamless and materially joined tissue of the fourth aspect of the invention for suitable purposes. Implants in the sense of the fourth aspect of the invention are in particular endovascular prostheses or other endoprostheses, e.g. stents (vascular stents, bile duct stents, vascular stents, peripheral stents or, e.g., mitral stents), endoprostheses, endoprostheses or endoprostheses, endoprostheses for closing persistent foramen ovale (PFO), pulmonary valve stents, endoprostheses for closing an ASD (atrial septal defect), as well as prostheses in the area of hard and soft tissue. Also possible as an implant is a left atrial appendage closure device (LAAC). In an alternative, preferably the medical implant is a prosthetic heart valve, more preferably a TAVI/TAVR valve, comprising an artificial heart valve made of sutureless and material bonded connected/joined tissue and/or a seal made of said tissue attached, preferably sutured, to an expandable or self-expanding and catheter implantable base frame, stent, or retaining device.

In an alternative, preferably the medical implant is a covered stent or a so-called stent graft, which has one or more tissue components of seamless and material bonded connected/joined tissue and/or a seal of said tissue, which is attached, preferably sutured, to the corresponding basic framework, stent, or holding device, and wherein said covered stent or stent graft is implantable by catheter.

With the context of the fourth aspect of the invention, the term "covered stent(s)" or similar terms describes an intraluminal endoprosthesis, with a preferably hollow cylindrical basic structure (e.g. made of nitinol), which is covered/sheathed by a further structure and/or one or more material layer(s) on a surface (inside and/or outside), preferably with a seamless and material bonded connected/joined tissue according to the fourth aspect of the invention.

Conceptually, a distinction is to be made in the context of the fourth aspect of the invention between "covered" in the sense of "covered/jacketed" and "coated" in the sense of "covered with a substance or an alloy". According to the fourth aspect of the invention, covered stents refer to stent implants or implants with a retaining structure, wherein the stent or the retaining structure itself is covered or sheathed by the tissue bonded/joined according to the fourth aspect of the invention, quasi as one or more "layers". That is, the stent or the retaining structure can, for example, be covered/sheathed from the outside and/or from the inside with the tissue connected/joined according to the fourth aspect of the invention. This may be realized in the form of one or more layers of the tissue joined/jointed according to the fourth aspect of the invention; or an inner and an outer layer of this tissue may also be joined/jointed with the joining/ joining methods according to the fourth aspect of the invention, and may also include, for example, an envelope of the tissue according to the fourth aspect of the invention at one end of the stent/holding structure. For example, an inner layer of the tissue of the fourth aspect of the invention may be folded over outwardly at both ends of the stent/holding structure, thus becoming an outer layer. The foregoing examples are not limiting, and the person skilled in the art may anticipate several different configurations and possible applications of the tissues joined/jointed according to the fourth aspect of the invention in light of the present disclosure. "Coated," on the other hand, rather focuses on a direct chemical, physical, or pharmaceutical coating of the stent structure. For example, prior art stents can be coated with silicon carbide or a so-called drug eluting stent is coated with a physiologically compatible pharmaceutical agent.

The person skilled in the art is aware here that numerous other conceivable possible applications of the seamlessly and material bonded connected/joined tissue for medical implants according to the fourth aspect of the invention are to be considered.

In all embodiments of the fourth aspect of the invention, the decellularization method, if performed, is applied to tissue that is not conventionally crosslinked after decellularization; rather, crosslinking occurs exclusively in the processes disclosed herein under quasi-static or periodic pulsatile pressure/compression in one or more selected overlap region(s) of the tissues involved.

In a preferred stabilization step, the tissue is exposed to at least one solution containing glycerol and/or polyethylene glycol, wherein the tissue is exposed to either one of these solutions or to the two solutions sequentially in any order and composition as first and second solutions or to both solutions or even to multiple solutions with different molecular weights of PEG simultaneously as a mixture of solutions or sequentially in any order. When drying tissue, e.g. for storage or transport of the tissue, the stabilization process is preferably carried out before drying.

As a non-limiting example, the stabilization process can be performed, for example, after decellularization and crosslinking by immersing the tissue in a series of one or more stabilizing solutions of glycerol and/or polyethylene glycol to sufficiently saturate the tissue with stabilizing agents and ultimately produce a stable, dry tissue with a seamless joint/joint. Saturation times can vary, but typically take about 5 minutes to 2 hours or 5 minutes to 15 minutes, depending on the properties of the tissue. The stabilized tissue can be dried by placing the tissue, for example, in a suitable environment with constant low relative humidity or, for example, controllable humidity and/or temperature, for example, in a climate chamber or desiccator and reducing the relative humidity. For example, from 95% to 10% over 12 hours at 37°C. The person skilled in the art is aware of the fact that, depending on the circumstances, another suitable drying protocol may be applied.

In general, throughout the present disclosure, the skilled person can suitably adjust the technical parameters such as times, amounts, concentrations, temperatures and, for example, pressures depending on the type of tissue to be treated and the desired crosslinking/bonding results.

The polyethylene glycol-containing solutions typically contain polyethylene glycol with an average molecular weight between 150 g/mol and 6000 g/mol, or a mixture thereof. As used herein, the term "between" includes the upper and lower specified values. Thus, an average molecular weight between 150 g/mol and 6000 g/mol is intended to include 150 g/mol and 6000 g/mol.

In this regard, the skilled person is aware that the temperature during the stabilization step can affect the results. For example, too high a temperature (e.g., above about 85°C) will cause denaturation and irreversible damage to the tissue crosslinked, e.g., glutaraldehyde crosslinked, for the purpose of bonding/ joining. Again, however, too low a temperature can lead to a solution that is too viscous. Preferably, exposure to the stabilizing solutions is at 37°C, but temperatures from room temperature up to 60°C should be tolerable.

As mentioned at the outset, the processes described in the fourth aspect of the invention are suitable for the preparation of substantially non-crosslinked tissue or, for example, decellularized, substantially non-crosslinked tissue - with the proviso that crosslinkable groups, e.g., free amino groups, must be present in the tissue. Optionally, all of the tissues addressed within the scope of the fourth aspect of the invention may be stabilized as described herein. Optionally, alpha-gal epitopes can be removed from all these tissues by a suitable alphagalactosidase treatment (preferably originating from GCB or Cucumis melo, see above).

As for the implant itself, the aforementioned problem is further solved by an implant containing biological tissue that has been subjected to one of the processes according to the fourth aspect of the invention and, if necessary, subsequently stabilized and/or dried.

Detailed description of the fourth aspect of the invention

The core of the process according to the fourth aspect of the invention lies in the surprising realization that various suitable crosslinking agents, such as and preferably glutaraldehyde, not only have the ability to form inter- and intramolecular crosslinks within a collagen fiber (see prior art above), but also interfibrillar crosslinks between individual fibers. Thus, it is possible for the first time to generate seamless and materially material bonded connect! ons/joints in an overlapping area of two tissue joining partners, which comprise crosslinkable groups, such as free amino groups; e.g. containing collagen, by simultaneously applying a quasi-static or periodic pulsatile pressure load/compression by means of a suitable device.

The basic requirement for this is that the distance between the collagen fibers is smaller than the length of the crosslinking molecules involved, such as the glutaraldehyde oligomers mentioned above, which form the actual crosslinks. Therefore, in the context of the fourth aspect of the invention, a pressure-generating device has been provided to generate a quasi-static or a periodic pulsatile vertical force application (pressure load/compression), with desired repetition cycles and over a desired time period, to a defined tissue region during the crosslinking process. According to the fourth aspect of the invention, the pressure generating device can be based on the physical principles of pneumatics, mechanics, and, for example, hydraulics, but is not limited in this respect. In the context of the fourth aspect of the invention, hydraulics is a particularly preferred embodiment for generating the pressure load/compression. The basic requirement for said formation of interfibrillar crosslinks is that the distance between the collagen fibers and microfibrils involved is smaller than the length of the glutaraldehyde oligomers involved (see above), which essentially form the crosslink. Appropriate pressing parameters over suitable time periods to reduce the fiber spacing are thus essential to enable a stable, seamless and material bonded bond using glutaraldehyde oligomers. At the same time, however, a high pressing pressure potentially, and thus not necessarily, results in preventing accessibility of the crosslinking solution to the tissue during force application.

Therefore, according to the fourth aspect of the invention, in a preferred embodiment, not only a quasi-static pressure on the tissue over a suitable longer period of time during crosslinking is considered (quasi-static refers to a constant pressure over a longer period of time (e.g. 300 seconds), which may be less frequently interrupted by short and suitable pressure pauses (e.g. 1 or 2 second(s)), but a periodic-pulsatile pressure load/compression over suitable shorter periods, but with possibly more frequent repetition of the pressure phases, also interrupted by short pressure pauses (e.g. 30 seconds pressure, 1 or 2 second(s) pressure pause, followed by 30 seconds pressure, 1 or 2 second(s) pause, etc.).

That is, in the pressureless phases (pressure pauses) during the crosslinking process, sufficient contact between the crosslinking agent, e.g. the glutaraldehyde oligomers, and the tissue to be joined/connected is ensured in this way. For this purpose, a suitable device is provided with which both a quasi-static, relatively constant pressure can be realized over longer period cycles, and a dynamic, periodic pulsatile pressure can be generated on the tissue, but over shorter and more frequent period cycles.

In an alternative embodiment, it is possible to achieve the crosslinking according to the fourth aspect of the invention for seamless and material bonded connecting/ joining via a static, i.e. permanent pressure, without pauses. The prerequisite for this is to provide a support surface for the tissue to be joined/connected which is perforated, i.e. is continuous for the crosslinking solution, in order to ensure its access to the tissue to be crosslinked.

In some embodiments, the disclosed processes are used to prepare a coronary artery bypass graft. In other embodiments, the processes of the fourth aspect of the invention are used to prepare biological and/or artificial tissue/tissue components for a heart valve replacement. In other embodiments, said processes are used for seamless and material bonded joining/connecting of tissue, grafts or even substrates for use in a wound treatment process, e.g., for treating lacerations or bums - e.g., wound patches joined/connected according to the fourth aspect of the invention.

In some embodiments, the processes are used to provide a sutureless and material bonded connect! on/joint to treat an inguinal hernia. In some embodiments, the processes disclosed herein are used for endogenous tissue regeneration using the patient's body to naturally restore tissue via a biodegradable scaffold. As stated, basic requirements for the foregoing and herein disclosed uses of the processes of the fourth aspect of the invention are: i) substantially non-crosslinked starting material/starting tissue, and ii) that the substantially non-crosslinked starting material/ starting tissue comprises crosslinkable groups, e.g., comprises free amino groups, and is thus suitable for chemical crosslinking, preferably using glutaraldehyde.

In the context of the fourth aspect of the invention, the terms "comprising amino group(s)" / "comprising free amino groups" or similar terminology mean that the tissue(s) to be joined/connected must comprise free amino groups that are chemically crosslinkable by means of a suitable crosslinking agent in order to be seamlessly and materially bonded joined/connected via the processes described herein.

With this context, the term "collagen-containing(s)" or similar terms used in the context of the fourth aspect of the invention describes that the tissue(s) to be joined/connected must comprise free collagen fibers in order to be seamlessly and materially bonded joined/connected via the processes described herein.

Suitable collagen-containing tissues within the scope of the fourth aspect of the invention are, for example, native collagen-containing tissues, moist collagen-containing tissues, already processed (but essentially non-crosslinked) collagen-containing tissues, such as, for example, already stabilized collagen-containing tissues, already preserved collagen-containing tissues, already dried (non-crosslinked) collagen-containing tissues, already decellularized tissues, as well as mixed forms of the aforementioned tissues. It is clear to the person skilled in the art that this list of suitable collagen-containing tissue forms is not exhaustive, but that further collagen- containing tissue types may be suitable for the disclosed process.

In accordance with the fourth aspect of the invention, bonding processes for stabilized, dried (non-crosslinked) tissue in particular have been tested.

In an alternative, even the seamless and material bonded connection of already fully or partially crosslinked tissue is possible in principle, whereby, exceptionally, either no crosslinking solution such as, for example, glutaraldehyde solution or only a very low-concentration glutaraldehyde solution (0-1% glutaraldehyde) is additionally required.

This means that a seamless compound in the sense of the fourth aspect of the invention can not only be formed by the direct, bilateral bonding of free glutaraldehyde oligomers (as described above), but in principle also by polymerization of oligomers already bonded on one side in the overlap region.

This means that fully or partially pre-crosslinked tissue can also be bonded/joined in pure Dulbecco's phosphate-buffered saline (DPBS) in the sense of the fourth aspect of the invention. Therefore, it can be assumed that the joining mechanism is indeed not exclusively due to the direct, bilateral bonding of free amino groups between the joining partners (tissue/tissue components), but likewise to the polymerization of unilaterally bonded crosslinking molecules in the overlap region.

In a further preferred embodiment, the processes according to the fourth aspect of the invention provide medical implants having a base structure, wherein a tissue or tissue component obtained according to one of the processes according to the fourth aspect of the invention is attached/fixed in and/or on the base structure. In an additional or alternative embodiment, a tissue or tissue component obtained according to one of the processes according to the fourth aspect of the invention is attached/fixed in at least one section of the stent implant, preferably at the proximal and/or distal end of the implant.

Thereby, the tissue or tissue component can be connected/joined, for example, over the entire length of the implant or, for example, only at the proximal and/or distal ends of the implant by means of the process according to the fourth aspect of the invention, in such a way that there is a seamless and material bonded connect! on/joint, for example, between an inner and an outer side of the implant through the meshes/cells of the implant. This reduces/prevents entirely the suturing of the tissue/tissue component to the implant; e.g. a stent-graft or a so-called covered stent (see above).

Such stent-based implants described above can be used, for example, as a so-called bail-out stent, neurostent, drug eluting stent, graft on balloon (PEB), PTA (percutaneous transluminal angioplasty), artery replacement or vein replacement.

The basic structure of such an implant can preferably be a metal or a metal alloy, preferably stainless steel, CoCr, a magnesium alloy (implant designed as a stent consisting of a magnesium alloy is also called AMS = absorbable metal stent) and/or nitinol, and/or a polymer from the class of biodegradable polymers, preferably polylactic acids, polycaprolactones and/or mixtures or copolymers thereof, and/or a polymer from the class of biocompatible polymers, preferably UHMWPE and/or PEEK.

In a further embodiment, a metallic base structure/stent implant may additionally be provided with a coating of amorphous silicon carbide (aSiC coating).

In a further preferred variant, the medical implant is a vascular valve prosthesis, in particular a heart valve prosthesis. For example, an aortic valve prosthesis, a tricuspid valve prosthesis, a mitral valve prosthesis and a pulmonary valve prosthesis are suitable examples of a heart valve prosthesis. Typically, such prostheses or implants have a stent-like structure that carries a valve assembly inside it to replace a natural vascular or heart valve. In this regard, the seamless and material bonded connected/joined tissue may be applied to a surface of the prosthetic heart valve (internal and/or external). In a further variant, the medical implant is a dry-stored and/or dry-delivered complete system, in particular a dry-stored/dry-delivered heart valve prosthesis, in particular an aortic valve prosthesis.

In a further variant, the heart valve prosthesis, in particular aortic valve prosthesis, comprising one or more of the sutureless and tissue-joined/tissue components, is loaded in a dehydrated state into a so-called catheter delivery system and is delivered in this preloaded state to an operating room.

All variations of the sutureless and tissue bonded/joined tissue/tissue component(s) may be combined in any manner and may be transferred in any combination to the medical implant described herein, and vice versa.

In particular, the fourth aspect of the invention discloses processes based on which crosslinking by means of a suitable crosslinking agent, such as, for example, glutaraldehyde solution comprising glutaraldehyde oligomers, in combination with a quasi-static or preferably periodic pulsatile pressure load/compression, enables a seamless, material bonded and durable connect! on/joint between the tissue/components (biological and/or artificial) defined above. The joining techniques disclosed herein can achieve, among other things, sutureless, material bonded and durable medical implants, such as, for example, sutureless covered stents or a sutureless TAVI/TAVR valve (each with respect to the tissue components, such as, for example, skirt and/or leaflet elements). In particular, suture-free skirt tissue components of a TAVI/TAVR valve (inner and/or outer skirt) can lead to an improved seal against paravalvular leakage (PVL).

With the context of the fourth aspect of the invention, the terms/expressions "quasi-static compressive loading/compression" or similar terms/expressions denote an essentially vertical physical application of force to the tissue to be joined/connected, carried out in such a way that it can be considered exclusively as a sequence of equilibrium states. Thus, the time scale on which a quasi-static process occurs must be much slower than the time period in which equilibrium is reached (the so-called relaxation time). Although a respective state of equilibrium prevails to a large extent at each point in time of the process, it is nevertheless generally an objective of the process to obtain different states or a characteristic curve. This means that the equilibrium state at time tl (pressure load) may well differ considerably from the equilibrium state at time t2 (pressure relief or pressure pause). The above definition is merely intended to exclude the possibility that dynamic or more dynamic processes, e.g. a periodic pulsatile pressure load/compression, have any appreciable influence on the joining/connecting behavior of the tissue components to be joined/connected.

Specifically, in the context of the fourth aspect of the invention, this means that the relationship between "with pressure loading" and "pressure relief/pressure pause" during the chemical crosslinking process in the case of "quasi-static", is more protracted over time for the pressure loading, and with longer periods of time, possibly several times alternating, than in the direct The "periodic-pulsatile" relationship of "pressure load" and "pressure relief/pressure pause" is shorter for the pressure load, which means that the two states "with pressure" / "without pressure" are also shorter over time and, if necessary, are repeated alternately much more often.

Specifically, in the context of the fourth aspect of the invention, the terms/expressions "quasi- static pressure load compression" or similar terms/expressions can be used over a ratio of, for example, 300: 1 seconds with respect to "with pressure load" (300 seconds) vs. "pressure release/pressure pause" (for example. 1 or 2 second(s)), and thus differ from the terms/expressions "periodic-pulsatile pressure load/compression" or similar terms/expressions in such a way that in the latter case a ratio of e.g. 30: 1 seconds exists with respect to "with pressure load" (e.g. 30 seconds) versus "pressure relief/pressure pause" (e.g. 1 or 2 second(s)).

That is, "quasi-static" includes, for example, a single, constant pressure load/compression on the tissue to be joined/connected of 5 minutes (= 300 seconds) in the presence of a suitable crosslinker solution with, for example, 1 or 2 second(s) pressure relief/pressure pause. Likewise, however, "quasi-static" also describes those cases in which two or more times of constant pressure load/compression with the pressure releases/pressure pauses as described above act on the tissue to be joined/connected. That is, even corresponding multiple cycles of this rather protracted "quasi-static" form of pressure loading and very short pressure pauses in between falls under these terms.

In contrast, this means, for example, that "periodic-pulsatile" includes at least two, but also several, short pressure loads/compressions on the tissue to be joined/connected of, for example, 30 seconds in the presence of a suitable crosslinker solution, but also always with 1 or 2 second(s) pressure relief/pressure pause. This means that even correspondingly multiple cycles of this rather short "periodic-pulsatile" form of pressure loading with short pressure pauses in between fall under these latter terms.

The skilled person is aware of the fact that within the scope of the fourth aspect of the invention he does not have to slavishly adhere to the exact ratios of 300: 1 seconds for "pressure load" (300 seconds) versus "pressure relief/pressure pause" (e.g. 1 second) in terms of "quasi-static", and 30: 1 for "pressure load" (e.g. 30 seconds) versus "pressure relief (e.g. 1 second) in terms of "periodic-pulsatile". 1 second) in terms of "periodic-pulsatile", but rather the relations of the mentioned time spans to each other distinguish the variants "quasi-static" from "periodic- pulsatile" during chemical crosslinking, and the exact values may suitably deviate from the above examples. For example, for "quasi-static" the above-described ratios of 250: 1 seconds or, for example, 350: 1 seconds are also conceivable, and with regard to "periodic-pulsatile", for example, 15: 1 seconds or 30:2 seconds are conceivable.

The above-mentioned alternative case of static crosslinking - without pressure pause - but with perforated/hole counterform for accessibility of the crosslinking solution is appropriately delimited with the above definition of the quasi-static case.

In the context of the fourth aspect of the invention, quasi-static pressure loading/compression is preferred over static pressure loading/compression, and periodic-pulsatile pressure loading/compression is the most preferred embodiment for the processes disclosed herein. Another factor of the disclosed joining/connecting processes is the total time period over which the static, quasi-static, or periodic-pulsatile pressure loading/compression acts on the tissue being joined/connected during chemical crosslinking.

A static, quasi-static or periodic pulsatile pressure load/compression over a total time duration of 1 to 3 days is preferred. A total time duration that falls below 4 hours may indeed result in a bond/join of the tissue partners involved; however, this appears too unstable to bring about a permanence of the bond/join. According to the fourth aspect of the invention, sufficient durability of the seamless and integral joints/junctions of the tissue partners is only given from at least 12 hours, preferably at least 24 hours, more preferably at least 36 hours, more preferably at least 48 hours, even more preferably from 72 hours of the static, quasi-static or periodic pulsatile pressure load/compression under the chemical crosslinking by means of a suitable crosslinking agent.

The skilled person is generally aware that the above-mentioned times can vary considerably depending on the tissue to be treated and the crosslinking agent to be used. Too short times are likely to lead to insufficient stability, too long times are likely to end in a waste of time, and the skilled person would optimize the parameters (time, temperature, concentrations, etc.) depending on the material.

At this point, the overlap length of the tissue components, the physical compression type (hydraulic, mechanical, etc.), cylinder force and the crosslinking time itself should be highlighted as other significant factors influencing the processes according to the fourth aspect of the invention. Thus, despite a lower breaking load, a reduction in the overlap length tends to result in a higher bond strength. In order to ensure the accessibility of the crosslinking agent, e.g. the glutaraldehyde solution, to the overlap area, a quasi-static or periodic pulsatile pressure load/compression is indispensable according to the fourth aspect of the invention.

The cylinder force must be selected appropriately, depending on the compression area, in order to bring about significant (collagen) fiber densification.

With regard to the crosslinking duration, a total period of static, quasi-static or periodic pulsatile compressive loading/compression of three days is particularly preferred. The crosslinking of overlapping tissue joining partners according to the fourth aspect of the invention is a valid concept for the seamless and material bonded joining/connecting of tissue, in particular tissue containing collagen. With regard to the application itself, however, the skilled person must always take into account the load limits of the bonded joint in different load cases as well as the effects of the compression process on the properties of the tissue joining partners.

The exemplary process described below, represents an embodiment of the fourth aspect of the invention, and is particularly, but not exclusively, suitable for native (biological) as well as for stabilized (e.g. dried) and/or decellularized tissue. In general, the disclosed processes are suitable for tissues containing collagen.

Thus, the fourth aspect of the invention provides a process for seamless, material bonded, and durable joining/connecting of tissue or a tissue component, preferably substantially noncrosslinked tissue/tissue compenents, for medical applications, in particular for use as a component of a medical implant, preferably a vascular implant, more preferably an artificial heart valve or a covered stent, wherein the process comprises at least the following steps:

(a) providing one or more tissue(s) to be joined, preferably substantially non-crosslinked tissue(s) comprising crosslinkable groups, in particular free amino groups, and having an overlap region;

(b) providing a suitable container, mold and/or support surface for the tissue/tissue component(s);

(c) providing a device capable of receiving the container, mold and/or support surface of step (b) in a form-fit manner, and further capable of providing controllable static, quasi-static or periodic pulsatile and substantially vertical compressive loading/compression of the overlap region(s) of the tissue/tissue component(s) to be joined of step (a), wherein the pressure load/compression is applied in a range of 0.01 - 10 N/mm², preferably 0.1 - 1 Nmm², over a time in the range of 1 second to 15 minutes, preferably with pressure relief/pressure pauses of 1 to 60 seconds, and this over a total period of at least 4 hours to a maximum of 12 days; d) Optional cutting of the tissue/tissue component(s) to be joined/connected after step a) by means of a suitable cutting instrument and/or a suitable cutting device; e) Placement/arrangement of the tissue/tissue component(s) after step a) or d) in the container, in the mold and/or on the support surface after steps b) and c) for joining/connecting the overlap area; f) chemical crosslinking of the tissue/component(s) after step e) in the device after step c) with addition of a suitable crosslinking agent into the container, mold and/or support surface and subsequent application of a quasi-static or periodic pulsatile compressive load/compression to the overlap area(s);

(g) demol ding/removal of the tissue/tissue component(s) bonded/joined after step (f); h) Optional purely chemical post-crosslinking using a suitable crosslinking agent.

According to the fourth aspect of the invention, said container, mold, support surface may be a two- and/or three-dimensional mold suitable for chemical crosslinking and static, quasi-static, or periodic pulsatile compressive loading/compression, for example, produced by a known 3D printing process (e.g., tooth-lifting process such as CNC milling). The material of the mold must be suitable to enable the process steps disclosed herein without negatively affecting the integrity of the tissue/component(s) to be joined.

A suitable device for the processes disclosed herein is, for example, a pneumatic cylinder and/or inflation sleeve in combination with at least one control element comprising electronics configured to control a static, quasi-static and/or periodic pulsatile, time-dependent and substantially vertical pressure/compression movement in the overlap region(s) of the tissue/component(s). Substantially vertical or orthogonal means with a deviation of ± 5°.

A preferred device for the processes disclosed herein is, for example, a hydraulic cylinder and/or a hydraulic inflation sleeve in combination with at least one control element comprising electronics configured to control a static, quasi-static and/or periodic pulsatile, time-dependent and substantially vertical pressure/compression movement in the overlap region(s) of the tissue component(s).

As a suitable cutting method of tissue in the sense of the fourth aspect of the invention, for example, laser cutting by means of a suitable laser cutting device such as a CO2 laser or a femtolaser is suitable; however, this is always in combination with a suitable positioning unit for the tissue/tissue component(s). Wateijet cutting is also conceivable.

A suitable cutting instrument in the sense of the fourth aspect of the invention is, for example, a pair of scissors, a scalpel, a knife, etc. With the above context of the fourth aspect of the invention, the disclosed processes comprise the following essential influencing parameters on the quality of the seamless, material bonded and durable connect! on/joint of the tissue/tissue component(s):

Static compressive load/compression

Appropriate compression loading is essential for the seamless, integral and durable joining/connecting of the tissue/tissue component(s) in the static regime. For the static regime, a time interval in the pressure phase of at least 3 minutes up to at least 15 minutes has proven to be suitable. A static pressure load of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 up to at least 15 minutes is therefore suitable; for example, also comprising 20, 25 or 30 minutes of constant pressure; depending on the dependence of the starting tissue to be joined/connected.

Pressure loading/release times of quasi-static pressure loading/compression

Suitable pressure-change times are essential for seamless, material bonded and consistent joining/connecting of the tissue/tissue component(s). For the quasi-static regime, a time interval in the pressure phase of at least 60 seconds up to 15 minutes has proven to be suitable. A quasi- static pressure load of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 up to 15 minutes is therefore suitable; e.g. 60 seconds of pressure as the lower limit and a maximum of 15 minutes of pressure per cycle as the upper limit; e.g. and particularly preferably 5 minutes.

For the pressure-relieving phase/pressure pause, a time interval of at least 1 second but not more than 10 seconds per cycle has proven suitable in the quasi-static regime; e.g. and preferably 1 to 2 seconds.

Pressure load/pressure release cycling times of the periodic pulsatile pressure load/compression. Appropriate pressure-change times are essential for seamless, integral and consistent joining/connecting of the tissue/tissue component(s). For the periodic pulsatile regime, a time interval in the pressure phase of at least one second up to 1 or 4 minutes has proven to be suitable. Suitable is therefore a pressure load of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 seconds or 1, 2, 3, 4 minutes; e.g. 1 second of pressure as lower limit and a maximum of 4 minutes as upper limit; e.g. and preferably 30 seconds. For the pressure-relieving phase/pressure pause, a time interval of at least 1 second but not more than 10 seconds per cycle has proven to be suitable in the periodic pulsatile regime; e.g. and preferably 1 to 2 seconds.

Compression pressure

For both the static, the quasi-static and the periodic pulsatile regime, values in the range of 0.01 - 10 N/mm², in particular 0.1 - 1 N/mm², have been found to be advantageous as suitable compression pressures for the seamless, integral and durable joining/connecting of the tissue/tissue component in the overlap region according to the fourth aspect of the invention. At lower compression pressures, the mechanical stability of the joint decreases. Higher compression pressures are rather ineffective or even ineffective with regard to the bond strength of the joint. It should be noted that compression of the tissue/tissue component to be bonded results in fiber compaction and thus also a reduction in thickness, which leads to increased mechanical stiffness and optical transparency of the tissue.

Crosslinking time - total time for the process according to the fourth aspect of the invention.

For both the static, quasi-static and the periodic-pulsatile regimes, a total crosslinking time of in particular at least 4 hours, preferably at least 12 hours to 3 days crosslinking (without optional post-crosslinking) with a suitable crosslinking agent, in particular glutaraldehyde, has been proven according to the fourth aspect of the invention. In principle, a further increase in the crosslinking time is rather ineffective or even ineffective with regard to the adhesive strength of the seamless joint/joint. However, this does not exclude a post-crosslinking in e.g. glutaraldehyde in the "free-floating" state, which typically lasts at least 5 days; but is no longer relevant for the tissue connection/ joining according to the fourth aspect of the invention, only for the final state of the tissue/component(s) as completely reacted biological material.

Furthermore, in embodiments of pericardial tissue/pericardial tissue components, it has been found that the choice of overlapping pericardial sides (rough or smooth; pericardium fibrosum or lamina parietalis, respectively) has no significant influence on the adhesive strength of the sutureless, material bonded and durable joint according to the fourth aspect of the invention.

Advantageous effects of the fourth aspect of the invention

The processes of the fourth aspect of the invention generate a seamless, materially bonded, homogeneous and at the same time mechanically stable, i.e. durable, connection between individual joining partners of tissue or one or more (free amino group-containing; collagen- containing) tissue component(s); for example, in the case of pericardium. The process enables a connection of two ends of one piece of tissue with each other or a connection of more two or more pieces of tissue with each other. A piece of tissue to be connected or joined preferably has an area of more than 0.5 mm²

The joining processes described above are all based on chemical crosslinking by means of a suitable crosslinking agent, such as glutaraldehyde. Since in the case of pericardial components, for example, this is in any case a mandatory process step for medical implants based thereon, the actual tissue connection/ joining is thus realized without any additional material component in the end product, which is clearly a technical advantage of the disclosed processes. Beside the crosslinking agent, e.g. Glutaraldehyde, no other chemicals are required for the process using static and/or quasi-static and/or periodic pulsatile and vertical/orthogonal compressive loading/compression.

The use of these processes for medical applications significantly reduces the need for and number of surgical sutures/nodes in the case of implants based on, for example, collagen- containing tissue, as a result of which the sutures/nodes as the weak points described at the beginning are greatly reduced or at least eliminated at certain points, and at the same time the manufacturing costs of such implants are noticeably reduced.

The seamless, material bonded and mechanically resilient and durable connect! on/joint also makes it possible to achieve medical implants of smaller diameter, since, for example, the surgical sutures/nodes that would otherwise be necessary are no longer required.

Furthermore, it is possible to enclose alloplastic support structures/stents by means of the connect! ons/joints of tissue/tissue components according to the fourth aspect of the invention in a seamless, material bonded and durable manner. In this regard, reference is made to the embodiments cited below; in particular, to an inflatable inner balloon or an inflatable cuff with an outer shape.

Advantageously, an average tensile shear strength (adhesive strength) of 14.82 cN (breaking load 7.4 N) could be achieved for stabilized tissue, for example (see embodiment examples below). Decisive influencing factors here, in addition to the initial condition of the tissue, are the compression type, cylinder force, overlap length and crosslinking time. In contrast, no significant influence can be determined for the choice of overlapping pericardial sides (see above).

In contrast to tensile stress, peel stress leads to failure of the material bond/joint already at a force of a few centi-newtons. In this case, the load-bearing crosslinks in the overlap area are unable to dissipate stress peaks, so that a brittle adhesive bond must be assumed. This lesson is given to the skilled person.

It should also be mentioned that the joining process according to the fourth aspect of the invention can possibly lead to a reduction in the water content in the tissue/component(s), which can possibly also affect the optical, structural and mechanical tissue properties. This may possibly lead to optically transparent overlap areas after the process.

Examples of TAVI/TAVR heart valve replacements and covered stents

For the following application examples, exemplary tissue components from porcine pericardium have been seamlessly, materially bonded, and durably bonded/joined in accordance with the processes of the fourth aspect of the invention. However, it is clearly evident to the skilled person that this process can be generally transferred to biological tissues and/or artificial tissues comprising free amino groups, which are correspondingly suitable for chemical crosslinking by means of, for example, glutaraldehyde.

Examples A) - Tissue processing

The starting material for the following experiments is porcine pericardium from approximately six-month-old pigs, which are obtained fresh from the slaughterhouse as required. During transport, the tissue is stored in isotonic saline (NaCl rinsing solution, sterile) at a mass concentration of 0.9% and initially cooled at 4 C for 1-2 hours prior to mechanical preparation.

Mechanical preparation

In the first step of mechanical preparation, the pericardium is dissected along the pericardial cavity. Subsequently, the stable fibrous composite of pericardium fibrosum and lamina parietalis required for the heart valve replacement is freed from coarse fat and muscle remnants using surgical scissors or a scalpel. Adherent fatty tissue on the rough pericardial side can be wiped off with a compress soaked in saline solution. During the entire preparation process, the tissue must always be prevented from drying out in order to avoid irreversible damage to the tissue. After mechanical preparation, rinse the tissue three times in saline for 5 minutes to completely clean it.

Glutaraldehyde as an exemplary chemical crosslinking agent

For crosslinking the tissue, a phosphate-buffered saline solution, DPBS for short (Dulbecco's Phosphate Buffered Saline w/o Ca and Mg), with a mass fraction of glutaraldehyde of 0.5% is always used in this work. For this purpose, 9 ml of a 50% glutaraldehyde solution is pipetted into pure DPBS per liter and dissolved in it.

Stabilization and laser cutting

A pulsed CO2 laser (Epilog Zing 24; Epilog) with a maximum power of 30 W is used to shape the tissue. Cutting in the non-crosslinked tissue state requires, in addition to adjusting the laser power, additional stabilization and drying of the tissue, as the process can otherwise lead to significant internal stresses in the tissue and resulting high distortion of the samples. Without a preceding stabilization process, reproducible tissue cutting may not be possible.

A process for the preservation of biological tissues by controlled dehydration is used for stabilization. The three stabilization solutions used here are each composed of the stabilizer glycerol, PEG200 or PEG400 and ultrapure water with different mass fractions.

For the laser process in the non-crosslinked state, the tissue is rinsed for 15 minutes each in glycerol 30%, PEG200 40% and PEG400 40% after mechanical preparation and then dried in a suitable climatic chamber at 40°C for a period of 12 hours, with the humidity being reduced linearly from 95% to 10%. The tissue is spread out on a ceramic plate, which can also serve as a base for the laser cut. Due to the low water content of the dried tissue, the laser power is reduced from 12% to 6% with otherwise unchanged conditions.

Uniaxial tensile tests and thickness measurement

In general, pericardial tissue is characterized by its viscoelastic material behavior. Despite the low thickness, this tissue shows a high mechanical load capacity and is elastic at the same time. Depending on the application of the tissue (e.g. as heart valve replacement material), it is sometimes exposed to high mechanical loads. Tensile tests to determine the mechanical properties are therefore a fundamental tool to assess the stability and stiffness of the tissue. The aim is always to process the tissue in such a way that mechanical integrity is maintained. Uniaxial tensile tests to characterize the mechanical behavior of the biological tissue were performed on a test rig that allows both uniaxial and biaxial tensile tests. This measurement apparatus consists of four identical and independently controllable drive units. The clamping of the tissue to be tested (e.g. the seamlessly joined/connected tissue according to the fourth aspect of the invention) is realized by clamping jaws which are fixed to the carriage in a roller guide. Platform load cells with a measuring range of 0.01 - 85 N serve as force sensors.

Unless otherwise specified, a specimen geometry of 21 mm x 3 mm is used for the tensile tests. The zero length of the tissue in the tensile test is determined automatically at a preload of 2 g. The travel speed of the jaws is, for example, 12 mm/min. In order to prevent the tissue from drying out, a Plexiglas tub is attached in the area of the clamping jaws, which is filled with isotonic saline solution during the measurement. In addition to the breaking force Fmax and the breaking strain Emax, the breaking stress cm ax and the modulus of elasticity (Young's modulus) E can also be determined from the stress-strain diagram if the thickness of the specimen is known.

The thickness is measured tactilely with a circular measuring plunger (0 5 mm), which presses with a weight of 30 g for 2 s on the tissue to be tested. The arithmetic mean of three thickness measurements at different points on the specimen is always used.

Concept description and test setup

The basic requirement for the formation of interfibrillar crosslinks is that the distance between the collagen fibers and microfibrils involved is smaller than the length of the molecules of the crosslinking agent, e.g. the glutaraldehyde molecules, which form the crosslink. The identification of suitable pressing parameters to reduce the fiber spacing is thus essential to enable a stable, seamless and material bonded bond by e.g. glutaraldehyde. At the same time, too high a pressing pressure (an excessive pressure load) during the quasi-static or periodic pulsatile pressure load/compression according to the fourth aspect of the invention potentially results in preventing accessibility of the crosslinking solution to the tissue/components to be bonded.

Therefore, not only static or quasi-static pressure on the tissue during crosslinking is possible, but also periodic pulsatile compression. In the pressureless phases during the crosslinking process, this ensures sufficient contact between the crosslinking agent (e.g. glutaraldehyde) and the tissue. For this purpose, a corresponding device has been developed according to the fourth aspect of the invention, with which both a quasi-static, relatively constant pressure and a more dynamic, periodic pulsatile pressure on the tissue/components can be generated.

The device consists, for example, of two double-acting pneumatic cylinders, connected by solenoid valves, which are suspended vertically in a framework of aluminum profiles.

The distance between the cylinders and the base plate can be adjusted by means of telescopic locking sets. Each solenoid valve is connected to a double-acting cylinder via two connections.

The control thereby causes each valve to be either statically, quasi-statically or periodically- pulsatilically alternately open, resulting in a likewise static, quasi-static or periodically-pulsatilic retraction or extension of the cylinder piston rods in order to exert the essentially vertical pressure load/compression on the tissue(s) to be joined/connected. Both cylinders can be controlled independently, allowing two series of tests with different parameters to be performed simultaneously.

By controlling the solenoid valves and the applied air pressure, either a static, quasi-static or a periodic-pulsatile, defined force application essentially vertical/orthogonal to the base plate (42) of the assembly is thus generated for each cylinder. To generate a static or quasi-static, rather constant force input, the compressed air hose of the cylinder is directly connected to the compressed air source without an intermediate valve. The air pressure is continuously adjustable up to a maximum pressure. The force of an idealized piston is calculated according to:

F = p x A

Where p is the applied air pressure and A is the piston area of the cylinder. It should be noted that due to frictional effects, the effective piston force is about 10 % less than the theoretical piston force. The support surface for the tissue samples is formed by exemplary laser-cut, crossshaped acrylic parts of 3 mm thickness. The two lateral holes are used to clamp the acrylic parts in an appropriately designed and 3D-printed holder made of polylactide. This allows the acrylic parts to be stacked exactly vertically, preventing horizontal movement. A stamp, also 3D- printed, is connected to the cylinder piston rod via a thread. This punch is used to transmit force between the cylinder and the specimen stack. At the same time, it prevents the acrylic parts from tilting sideways during the piston movement. By extending the cylinder piston rod, pressure/compression is exerted on the specimen stack (pressure phase/compression). When the cylinder is in the retracted state (pressure release/pressure pause), the chemical crosslinking solution used, e.g. 0.5 % glutaraldehyde solution, easily reaches the tissue.

Seamless, material bonded and durable tissue connection

According to the fourth aspect of the invention, a seamless, interlocking and durable connection/ joining of tissue/tissue components is most preferable when a suitable crosslinking agent, such as glutaraldehyde, is involved in the process and forms crosslinks between the joining partners.

In the present embodiment example, porcine pericardial tissues are first cut into rectangles (30 mm x 10 mm) by laser and placed on the acrylic parts as a support surface (4) in such a way that an overlapping tissue area of 10 mm x 10 mm is formed between every two specimens. A rough pericardial side (pericardium fibrosum) is always brought to overlap with a smooth pericardial side (lamina parietalis). The overlapping tissue samples are additionally enclosed in laser-cut rectangular filter paper strips (50 mm x 10 mm). The absorbency of the filter paper strips ensures accessibility of the crosslinking solution, in this case glutaraldehyde, to the tissue samples during the pressureless phases of periodic pulsatile pressure loading/compression.

After the tissue is placed, ten acrylic parts covered with tissue and a final acrylic part are placed on top of each other on the 3D-printed holder. The holder with the tissue samples is then placed in a plastic vessel and aligned vertically under the punch of the cylinder piston rod. The height of the pneumatic cylinder is adjusted via the telescopic locking sets so that the plunger rests on the uppermost acrylic part without pressure in the retracted cylinder state. The process according to the fourth aspect of the invention is started by connecting the system to the compressed air source and filling the plastic vessel with the crosslinking solution, in this case glutaraldehyde. For periodic pulsatile pressure loading/compression, the control system in this embodiment example is programmed such that the duration of pressure loading or pressure relief/pressure pause is 30 seconds each. The applied air pressure is controlled to 4.8 bar. This corresponds to a theoretical piston force of the cylinder of about 150 N. The curing time is set at 24 hours (1 day).

At the end of this total period of periodic pulsatile pressure/compression, the tissues are removed from the holder and transferred to saline as quickly as possible. To remove any residual unbound glutaraldehyde, the specimens are rinsed three times for five minutes in isotonic saline. Uniaxial tensile tests are used to check whether a seamless, materially bonded and durable connection/joint has been established between the tissue pieces. For this purpose, it is determined that tissue pieces are to be regarded as successfully joined if a short-term tensile shear load with a force of 1 N does not completely break the bond.

Results

The results of the tensile tests described above, among others, are plotted in Tab. 1 below. It can be seen that the joining/connecting of stabilized, non-crosslinked tissue by mere crosslinking with glutaraldehyde is possible in principle, but dynamic compressive loading/compression is required (quasi-static or periodic pulsatile) to generate a reproducible, stable joining/connecting. In contrast, both a mere static and dynamic pulsatile compression of the tissue samples with pure DPBS, i.e. without crosslinking agent, do not cause tissue bonding, as expected. Here, already the rinsing process of the tissues in saline leads to the failure of the bond in most cases.

In contrast, a significant influence of the compression type on the connection/ joining of crosslinked tissue cannot be determined. Moreover, in this case, tissue bonding is in principle feasible both in a glutaraldehyde solution and in pure DPBS, but about half of the specimens fail at a force of 1 N already. This suggests that the glutaraldehyde molecules of the frame- crosslinked tissue, which are unilaterally bound in the tissue, generally have the ability to form interfibrillar crosslinks. It can be assumed that this is not only explained by direct binding of the unilaterally bonded glutaraldehyde molecules to free amino groups of the joining partner, but is largely due to the polymerization of the glutaraldehyde between the joining partners.

Overall, these results confirm the theory that the formation of interfibrillar crosslinks by glutaraldehyde between two pieces of tissue is possible in principle and that only in this way can a sutureless, material bonded and durable joining of pericardial tissue be achieved.

Table 1: Results of sutureless, material bonded and durable tissue bonding/joining according to the embodiment example explained above.

Tissue- Compression Number of successful condition type Tissue connections

DPBS Glutaraldehyde solution stabilized statisch 0/10 4/10 pulsatil 0/10 10/10 crosslinked statisch 6/10 5/10 pulsatil 5/10 6/10 Influence of the crosslinking time

The measurement results with variation of the curing time are listed in Table 2 below. Tripling the curing time from one day to three days increases the breaking load and shear strength by about 65%.

Tab. 2: Influence of the crosslinking time on the adhesive strength, thickness and crosslinking quality in the overlap and edge areas (indicated in brackets) in the joining of pericardial tissues according to the fourth aspect of the invention (n = 10).

Crosslinking Adhesive- Denaturation- Amino- Thicknesstime strength temperature groups coefficient in - ^cN i ¹n¹¹ °C i inn — mm² mg

, 96,0 ± 0,7 5,7 ± 1,4

1 day 9,0 ± 1,7 ’ ’ ’ ’ 1,66 ± 0,23 y (93,4 ± 0,3) (5,9 ± 1,2) 96 5 ± 0 7 4 7 ± 0 5

(93,2 ± 0,9) (5,1 ± 0,8)

, 96,9 ± 0,7 4,6 ± 0,7

8 days 14,7 ± 2,1 ’ ’ ’ ’ 1,72 ± 0,25 y ’ ’ (94,3 ± 1,0) (5,1 ± 1,7)

Together with the results of the amino group detection, the following theory can be put forward: In the first 24 hours, the majority of free amino groups are occupied by glutaraldehyde oligomers. The probability for the formation of interfibrillar crosslinks is maximal during this period. Subsequently, the number of free amino groups decreases only insignificantly, and the polymerization of glutaraldehyde dominates during this period. Unilaterally bonded as well as free glutaraldehyde oligomers combine and form additional load-bearing interfibrillar bonds between the tissue joining partners, which are reflected in a significant increase in the breaking strength. A further increase in the crosslinking time is ineffective in that no more load-bearing bonds are generated by polymerization either.

Within the scope of the fourth aspect of the invention, parameters could be determined as optimal with which overlapping tissues/tissue components can be joined in a reproducible, seamless, material bonded and stable manner. With the process parameters listed in Tab. 3, an average shear strength of 14.8 cN/mm² can be achieved. This corresponds to a breaking load of the overlap of 7.4 N. Frame-crosslinked reference tissue (stabilized and dried before the start of crosslinking) has an average breaking load of 19.7 N (n=30) for the same crosslinking time and a tissue width of also 10 mm. This means that chemical crosslinking of overlapping tissue samples can generate a joint/joint whose breaking force (for an overlap area of 50 mm²) under tensile shear stress corresponds to about 38 % of the breaking force of conventional tissue.

Tab. 3: Process parameters for maximizing the shear strength in the seamless, materially bonded and durable joining of overlapping tissue areas/tissue components

Process parameters Determined optimum

Pressure change times 30 s / 2 s

Cylinder force 225 N

Tissue condition stabilized

Overlapping pericardial sides smooth-smooth

Overlapping length 5 mm

Crosslinking time 3 days

Optical properties and surface topography

The effects of the bonding process of overlapping tissue samples on the collagen fiber structure were analyzed, for example, in this embodiment. First, a visual inspection was performed, focusing on the influence of pressure on the optical properties of the tissue. This is followed by a detailed examination of the surface topography under a scanning electron microscope. Of particular interest here is the extent to which the bonding process of the tissue influences the arrangement of the collagen fibers in the overlap and edge regions. For the preparation of the tissue samples, the optimal process parameters for tissue bonding are used according to Table 3 (see above).

Even without optical aids, the influence of the joining process on the optical properties of the tissue can be clearly seen. Compared to frame-crosslinked tissue, the tissue samples bonded under pressure (quasi-static or periodic pulsatile) are almost completely transparent in the overlap area as well as in the single-layer tissue area. Individual fibers are not discernible. The rough tissue side (pericardium fibrosum) is visually indistinguishable from the smooth tissue side (lamina parietalis), and the overlap region is also barely visually distinguishable from the single-layer tissue region.

The increased transparency of the tissue can basically be explained by the removal of water. In frame-crosslinked tissue, there is free or bound water between the individual collagen fibers. At each interface between collagen (refractive index 1.4 - 1.55) and water (refractive index 1.3), light is refracted as it passes through the tissue. Due to the inhomogeneous distribution of collagen in pericardium, a chaotic refraction pattern results, and the tissue appears opaque. Pressure loading/compression according to the fourth aspect of the invention forces the interfibrillar water out of the tissue, so that the number of interfacial junctions decreases and the transparency of the tissue increases.

Mechanical properties and water content

The transparency of the tissue already indicates that the quasi-static or periodic pulsatile compression significantly decreases the amount of interfibrillar water. In the following embodiment example, it will be shown to what extent this affects the mechanical properties of the tissue. In addition to the breaking stress, the elongation at break and the modulus of elasticity, the water content of the tissue is determined. To produce the tissue samples bonded according to the fourth aspect of the invention, the optimum process parameters from Table 3 are used and applied as described above. Sampling for uniaxial tensile tests is performed in the single-layer tissue section. The water content is measured differentially for the overlap area as well as the single-layer tissue area. Frame-crosslinked tissue, which was also subjected to a stabilization and drying process before the start of the three-day crosslinking process, serves as a reference.

Uniaxial tensile tests

The results of the uniaxial tensile test are shown in Table 4 below. Obviously, the periodic pulsatile compressive loading/compression in particular causes a significant thickness reduction of the tissue. The nearly identical breaking forces of the compressed tissue and the reference tissue suggest that the collagen fibers are not damaged by the compaction process. Due to the increased density of the collagen fibers, the breaking stress and Young's modulus increase considerably. At the same time, a much lower elongation at break is observed compared to the frame crosslinked reference tissue. It can be assumed that the individual fibers do not lose their load-bearing capacity as a result of the pressing process, but that the sliding of the collagen fibers against each other is impeded by dehydration.

Tab. 4: Mechanical properties and thickness of the tissue crosslinked under periodic pulsatile compression in the single-layer tissue section (composite single-layer) : frame-crosslinked tissue serves as reference, (n = 30) Sample type Thickness Breaking Breaking Elongation Young's force stress at break modulus in N in MPa in % in MPa

Reference 0,07 ± 0,01 4,4 ± 1,7 22,1 ± 8,4 22,7 ± 3,2 150,3 ± 62,4

Composite 0,05 ± 0,01 4,5 ± 1,5 30,5 ± 8,5 16,3 ± 3,6 255,3 ± 82,3 single layer

Water content

The results of water content determination in Table 5 confirm the structural changes of the tissue due to crosslinking under periodic pulsatile compression. Compared to frame-crosslinked tissue, the water content is much lower in both the single-layer tissue and the overlap region.

In principle, there are two explanations for this: First, it can be assumed that the water removal is not limited to unbound water molecules. If bound water molecules are also removed from the tissue, it can be assumed that hydrogen bonds cause embrittlement of the tissue and preclude complete rehydration. Second, it is conceivable that the higher collagen fiber density increases the likelihood of interfibrillar crosslinks due to reaction with glutaraldehyde. That is, the mechanism used to join two tissue samples also leads to adhesion of the collagen fiber layers within the individual tissue samples and in this way impedes the reincorporation of water molecules.

Tab. 5: Water content of tissue crosslinked under periodic pulsatile compressive loading/compression in the single-layer tissue region (composite single-layer) and overlap region (composite overlap) : frame crosslinked tissue serves as reference. (n=10)

Sample type Water content in %

Reference 74,3 ± 0,9

Composite single layer 55,2 ± 2,6

Composite overlap 53,8 ± 2,8

Summary of embodiments A)

In the above-mentioned embodiments, a process (essentially the periodic-pulsatile variant) for seamless, material bonded and durable joining was illustrated using pericardial tissue. According to the fourth aspect of the invention, a pneumatic assembly was used as an exemplary device to achieve both static, quasi-static and periodic-pulsatile pressure loading/compression of overlapping tissues/ tissue regions/tissue components can be achieved. In the above-mentioned embodiments relating to the tissue per se, it was shown that the joining process according to the fourth aspect of the invention is fundamentally based on the formation of interfibrillar crosslinks between the joining partners. Accordingly, a suitable chemical crosslinking solution, preferably glutaraldehyde solution, is always required far preferentially for joining non-crosslinked, stabilized tissues. In contrast, pre-crosslinked tissues can in principle be joined even in pure DPBS. It can therefore be assumed that the bonding mechanism is not exclusively due to the direct, bilateral bonding of free glutaraldehyde oligomers between the joining partners, but also to the polymerization of unilaterally bonded glutaraldehyde molecules in the overlap region.

With optimum parameters, an average tensile shear strength (adhesive strength) of 14.82 cN/mm² (breaking load: 7.4 N) could be achieved for stabilized tissue according to the fourth aspect of the invention. In addition to the initial condition of the tissue itself, the compression type (quasi-static or periodic-pulsatile), cylinder force, overlap length and crosslinking time should be mentioned as decisive influencing factors.

Furthermore, it was found that the processes according to the fourth aspect of the invention lead to a noticeable reduction of the water content in the tissue, which has a massive effect on the optical, structural and mechanical tissue properties. For example, the joints produced in the above-mentioned embodiment examples are almost completely transparent in the overlap area as well as in the single-layer edge area. Although the collagen fibers are not destroyed by the pressure load/compression of the tissue and the associated reduction in thickness, their freedom of movement is considerably restricted. As a result, the breaking stress and modulus of elasticity increase, and the elongation at break is reduced.

Overall, the processes according to the fourth aspect of the invention offer an applicable technical solution for seamless, material bonded and durable bonding of tissues comprising free amino groups, in particular tissues containing collagen. However, the application of these processes must always take into account the load limits of the joint in different load cases, as well as the altered mechanical and structural tissue properties due to the compression/compression process. Examples of embodiments B) - Suture reduction in a TAVI/TAVR valve.

Using an exemplary self-expanding TAVI/TAVR valve, the following demonstrates how the processes according to the fourth aspect of the invention can be integrated into the manufacturing process of a cardiovascular implant. The aim is to achieve a reduction in the number of surgical knots/sutures by bringing about one or more sutureless connect! ons/joints of one or more tissue component(s) of a TAVI-TAVR valve - without compromising the functionality of the valve prosthesis.

After mechanical preparation (see above), the pericardial tissue is first stabilized and dried in a climate chamber. The tissue is then cut with a suitable laser in such a way that a defined overlap of the tissue ends of what is in this case a one-piece tissue component is created by placing it on a suitable mold. This is located exclusively in the skirt area, so that the function of the leaflets is unaffected.

After the tissue has been clamped in a forming structure, the chemical crosslinking process begins; in this case with glutaraldehyde solution. During the crosslinking with glutaraldehyde, the overlap area is periodically pulsatilized with pressure load by a punch (78) adapted to the recess in combination with the device according to the fourth aspect of the invention in order to realize the seamless connection of the tissue ends. After the crosslinking process, the side parts are removed and the excess tissue is removed by the second laser process on the molded body. In the last step, the valve is connected to the stent, equivalent to the conventional manufacturing process. That is, the goal of this embodiment is to avoid any suturing of the tissue component per se; however, the sutures for placement/fixation to the stent remain.

To realize the integral connection of the tissue ends during the three-dimensional crosslinking process, a molding construction is provided as described below. The aim of the design is to enable vertical force/pressure to be applied to the overlap area(s), while at the same time fixing the tissue to the molded body during the crosslinking process. For this purpose, both the side parts and the molded body are modified, and a holder and a punch are also provided, which is used to transfer force from the pneumatic cylinder to the overlap area of the tissue.

In the case of two of the three side parts, a recess is created in the web area instead of the extension; this recess is precisely matched to the dimensions of the punch and serves as a guide for it. The punch is connected via a thread to the pneumatic cylinder of the device for periodic pulsatile pressure loading/compression and is adapted to the curvature of the molded part. A corresponding support prevents tilting of the molded body during periodic pulsatile force application.

Tissue processing

For the fabrication of the exemplary TAVI/TAVR valve, the pericardial tissue is first stabilized after mechanical preparation and dried in a climate chamber. A suitable cutting pattern is used for the first laser cutting process.

A suitable tissue geometry makes it possible to place the tissue on the molded body without wrinkles and at the same time generate a defined, overlapping tissue area. This has a width of 10 mm in the skirt area and tapers to a minimum width of 1.2 mm between the leaflets. A reproducible lay-up of the tissue in its native state is not recommended, as it tends to wrinkle in the edge area. In this case, too, the molded part is wrapped with self-adhesive aluminum foil to prevent it from being damaged by the subsequent laser process.

After tissue placement, the side parts are attached so that the recesses created enclose the overlap area. To fix the side parts, rubber rings are attached to the grooves provided. The assembled structure is then inserted into the holder with the overlapping tissue area facing upwards. An appropriately cut filter paper strip is placed on the overlap area to promote accessibility of the glutaraldehyde solution to the tissue. An additional thin silicone pad has proven effective to ensure homogeneous pressure distribution over the entire overlap area. The edges of the recess serve as a guide for the plunger, which is positioned via the telescopic locking sets so that it rests on the silicone layer without pressure when the cylinder plunger is retracted. Three-day crosslinking using glutaraldehyde is performed with a periodic pulsatile pressure load/pressure pause of the cylinder at a ratio of 30:2 seconds; i.e., 30 seconds of pressure load/compression per cycle and 2 seconds of pressure pause per cycle. The theoretical cylinder force is 150 N, which corresponds to a pressure of 0.71 N/mm² in the overlap area. The subsequent laser process in combination with the turning device gives the tissue its final shape.

Due to the seamless, material bonded and durable connection of the tissue ends of the TAVI- TAVR valve tissue component obtained, only the cutting of the e.g. three leaflet and the e.g. twelve skirt sheets is required according to the fourth aspect of the invention. The tissue can then be carefully pulled off over the three-sided prism of the molded body. Thus, only a simplified suturing process is used for suturing the TAVI/TAVR tissue component(s) into the stent, since the suturing of individual tissue components to each other, such as individual leaflet and skirt components, is omitted. In another embodiment, an approach is disclosed for achieving a sutureless connection of the tissue component(s) of a TAVI/TAVR valve to the stent.

In addition to the classic internal skirt component, aortic valve implants optionally contain another pericardial strip attached to the outside of the stent (external skirt). This additional border serves to reduce paravalvular leakage (PVL) and is crucial for the approach described below.

The basic idea is to generate a sutureless connection between inner and outer skirt part with stent in between.

Mechanical preparation and stabilization and drying of the pericardial tissue is followed by a two-part laser process. First, the inner tissue component is cut. In addition, a second tissue component is cut out that corresponds to the skirt area of the first tissue component. The tissue components are then placed in an inflation sleeve device so that the stent is enclosed from both sides in the skirt area and an overlap area is formed between the stent struts.

Radial compression is thus required to generate a circumferential connection between the inner and outer skirt components. According to the fourth aspect of the invention, an annular, doublewalled silicone sleeve (inflation sleeve device) is provided specifically for this purpose, which inflates radially inward in a time-dependent manner. Thus, a homogeneous, quasi-static or periodic pulsatile pressure load/compression of the tissue in the overlap area is achieved and in this way a seamless connection of the tissue component to both sides of the stent (inner and outer side) is realized.

For reproducible production of an inflatable cuff in the sense of the fourth aspect of the invention with a defined wall thickness.

Four symmetrically arranged holes are provided in the otherwise closed bottom of the lower casting for subsequent demolding. These are closed with screws before the casting process begins in order to produce a flush bottom surface. For the casting process, a 3D-printed hollow cylinder made of water-soluble polyvinyl alcohol (PVA) is first suspended symmetrically in the mold via additionally designed and 3D-printed PLA rods so that a defined gap dimension is created between the hollow cylinder and the mold on each side. Corresponding holes in the mold as well as recesses in the PVA hollow cylinder are provided for correct positioning of the rods. At the beginning of the two-stage casting process, the mold is half filled with silicone. After the silicone has cured, the PLA rods are moved outward until they also flush the wall of the mold. At this point, the PVA hollow cylinder is self-supportingly embedded in the silicone compound. The entire mold is then filled with silicone up to a designated edge on the lid so that the PVA core is completely enclosed. During the entire casting and curing process, an outwardly directed compressed air hose is attached to the side of the PVA hollow cylinder in a further recess. This is also embedded in the silicone compound through a bulge in the two-part mold.

After curing of the silicone compound and demolding of the sleeve, the water-soluble PVA core is finally washed out. This is designed to give a wall thickness of 3 mm for the outer wall of the inflatable sleeve and for the base and lid. The thickness of the inner wall is set at 4 mm, since the sleeve is exposed to the highest stresses in this area during the crosslinking process.

A suitable control system in combination with a solenoid valve is used to control the inflation process.

The starting material for this embodiment is two mechanically prepared, stabilized and dried tissue patches, which are first processed with the laser to provide a suitable cutting geometry.

The sequence of subsequent tissue placement includes the steps of rolling the first tissue component onto the lower, thin-walled support structure so that the leaflets are freely supported for movement. Subsequently, the shape memory effect of the nitinol is exploited to achieve the correct placement of the stent. For this purpose, the support structure including the tissue is centered on the guide plate. Meanwhile, the stent is radially expanded in ice water with an auxiliary body and then swiftly slipped over the tissue-covered component.

While the stent regains its original geometry upon heating to room temperature, the gaps on the guide plate ensure proper alignment of the stent relative to the support structure. The outer skirt is then rolled flush on the outside of the stent. The tissue components are then sutured to the eyelets of the stent. The individual surgical knots do not serve to connect to the stent, but are essential for proper alignment of the tissue components to each other. Subsequently, the remaining support components are assembled into a hollow cylinder with the leaflets facing inward through appropriately provided gaps. Essentially, tissue placement to join the inner and outer skirts includes the following steps:

- Placement of the inner tissue component onto the lower support structure.

- Center placement of the lower support structure on the guide plate

- Placement of the nitinol stent

- Placement of the outer tissue component

- Removal of the prefabricated, sutured valve prosthesis

- Assembly of the final construction.

After the tissue placement described above, the experimental construct is assembled as follows: First, the inflatable sleeve is inserted into the lower specimen chamber so that the compressed air port occupies the designated lateral hole. The prefabricated valve together with the support cylinder is enclosed with a suitably cut rectangular filter paper strip and then sunk centrally into the sample chamber. In order to prevent vertical displacement of the valve during the compression process, the thin-walled lower support structure is firmly screwed to the sample chamber via holes provided.

Now the inflation collar is connected to the compressed air hose of the solenoid valve and the control system is connected. The periodic-pulsatile pressure-change time for this embodiment is set at 30:2 seconds; i.e., 30 seconds of pressure load/compression per cycle and 2 seconds of pressure pause per cycle. To avoid failure of the inflation cuff, the pressure is controlled at 2 bar. Due to the liquid-like behavior of the silicone, the inflation of the sleeve generates a pressure greater than 0.1 N/mm² inside the specimen chamber.

After three days of crosslinking in this case (total duration of crosslinking) under periodic pulsatile radial compression, the valve prosthesis is demolded. Radial expansion of the stent in the skirt region to remove the support structures is not appropriate here, as this potentially damages the adhesive bond. While the bottom plate as well as the upper support structure and the thick-walled inner core can be easily removed, an additional process step is therefore necessary to remove the thin-walled inner core. For this purpose, the structure is heated to 70°C in a water bath. This temperature is above the softening temperature of the thin-walled PLA component, so that it can be plastically deformed without high force. At the same time, the denaturation temperature of the crosslinked tissue is not exceeded. This makes it possible to detach the support structure from the construction without changing the conformation of the collagen fibers and damaging the adhesive bond.

Thus, in the latter embodiment, a concept according to the fourth aspect of the invention was illustrated, which for the first time enables a seamless connection of the tissue component to the stent by generating a tissue connection between the inner and outer skirt with the stent in between.

Overall, it can be seen that the seamless, interfacing and durable tissue connection of the fourth aspect of the invention can be profitably integrated into the manufacturing process of e.g. TAVI/TAVR valves without compromising the functionality of the implant.

Example C) - Biological stent graft

In the context of the fourth aspect of the invention, a biological stent graft may be prepared as follows:

(1) A layer of tissue is wrapped around an outer surface of a stent graft and folded over inwardly;

(2) A balloon that can be dilated inside the stent is inserted;

(3) The stent is wrapped on its outer side with a technical fabric as an interlayer;

(4) A cylindrical perforated outer shape, i.e., with holes, is mechanically fixed on the outside;

(5) Dilatation of the inner balloon with a static pressure;

(6) Fixation of the tissue of the stent framework in 0.5% glutaraldehyde for three days;

(7) Demolding/removal of a stent seamlessly encased with tissue.

The embodiments described herein are primarily for exemplary illustration of the fourth aspect of the invention. The number and/or design of compressive loads/compressions of the tissues to be joined/connected in the presence of a suitable crosslinking agent (particularly with respect to the concentrations and compositions thereof of the crosslinking agent solution) may be identified as suitable and varied by the skilled person within the scope of his knowledge.

With reference to the above disclosure, the fourth aspect of the invention further comprises the embodiments numbered in ascending order below:

1. Process for the seamless, material bonded and durable joining or connecting of tissue or one or more tissue component(s) (1, 2, 7), preferably (substantially non-crosslinked) tissue or tissue component(s), for medical applications, in particular for use as a component of a medical implant, preferably a vascular implant, more preferably an artificial heart valve (27, 28) or a covered stent, the process comprising at least the following steps: a) providing one or more tissue(s) to be connected or joined (1, 2, 7), which may have or form one or more overlap region(s) (3); b) providing a suitable container, mold and/or support surface for the tissue component(s) (4, 11, 12, 13, 53, 54); c) providing a device (37, 38) capable of receiving the container, mold and/or support surface (4, 11, 12, 13, 53, 54) in a form-fit manner, and further capable of providing controllable static and/or quasi-static and/or periodic-pulsatile and (substantially) vertical or orthogonal compressive loading or compression of said overlap area(s) (41, 42); d) optional cutting of the tissue or tissue component(s) (1, 2, 7) to be joined or connected by means of a suitable cutting instrument and/or a suitable cutting device; e) placing or arranging the tissue or tissue component(s) according to step a) or d) in the container, in the mold and/or on the support surface according to step b) (4, 11, 12, 13, 53, 54) in such a way that the overlapping areas (3) intended for joining or connecting come to lie on top of one another in the device according to step c); f) chemical crosslinking of the tissue(s) or tissue component(s) according to step e), in particular of said overlap areas, in the device according to step c) with addition of a suitable crosslinking agent into the container, into the mold and/or onto the support surface and under the action of said static, quasi-static or periodic pulsatile pressure load or compression on the overlap area(s) (41, 42); g) demolding or removal of the tissue or tissue component(s) bonded or joined after step f) (6); h) optional purely chemical post-crosslinking using a suitable crosslinking agent.

2. The process according to embodiment 1, wherein the process comprises at least the following steps:

(a) providing one or more tissue(s) or tissue component(s) (1, 2, 7) to be joined or connected, preferably (substantially non-crosslinked) tissue(s) or tissue component(s), which may have or form one or more overlap area(s) (3); b) providing a suitable container, mold and/or support surface for the tissue/tissue component(s) (4, 11, 12, 13, 53, 54); c) providing a device (37, 38) capable of receiving said container, mold and/or support surface (4, 11, 12, 13, 53, 54) in a form-fit manner and further capable of providing controllable static and/or quasi-static and/or periodic-pulsatile and (substantially)vertical/orthogonal compressive loading or compression of said overlap area(s), wherein the force input of said compressive loading or compression is exerted in a range of 0.01 N/mm² to 10 N/mm² (41, 42); d) optional cutting of the tissue or tissue component s) (1, 2, 7) to be joined/connected by means of a suitable cutting instrument and/or a suitable cutting device; e) placing or arranging the tissue or tissue component(s) according to step a) or d) in the container, in the mold and/or on the support surface according to step b) (4, 11, 12, 13, 53, 54) in such a way that the overlapping areas (3) intended for joining/connecting come to lie on top of one another in the device according to step c); f) chemical crosslinking of the tissue(s) or tissue component(s) according to step e), in particular of said overlap areas, in the device according to step c) with addition of a suitable crosslinking agent into the container, into the mold and/or onto the support surface according to step b) and under the action of said static or quasi-static or periodic pulsatile pressure load or compression on the overlap area(s) (41, 42); g) demolding or removal of the tissue or tissue component s) bonded/joined after step f) (6); h) optional purely chemical post-crosslinking using a suitable crosslinking agent.

3. The process according to embodiment 1 or 2, wherein the process comprises at least the following steps: a) providing one or more tissue(s) or tissue component(s) (1, 2, 7) to be joined or connected, preferably (substantially non-crosslinked) tissue(s) or tissue component(s) comprising crosslinkable groups, and which may have or form one or more overlap areas (3); b) providing a suitable container, mold and/or support surface for the tissue/tissue component(s) (4, 11, 12, 13, 53, 54); c) providing a device (37, 38) capable of receiving said container, mold and/or support surface (4, 11, 12, 13, 53, 54) in a form-fit manner and further capable of providing controllable static and/or quasi-static and/or periodic-pulsatile and (substantially) vertical or orthogonal compressive loading or compression of said overlap area(s), wherein the force application of the pressure load or compression is applied in a range of 0.01 N/mm² to 10 N/mm², preferably 0.1 N/mm² to 1 N/mm², over a time in the range of 1 second to 15 minutes in combination with a corresponding pressure release or pressure pause of 1 to 60 seconds, and this over a total period of at least 4 hours up to a maximum of 12 days (41, 42); d) optional cutting of the tissue or tissue component(s) (1, 2, 7) to be joined or connected by means of a suitable cutting instrument and/or a suitable cutting device; e) placing or arranging the tissue or tissue component(s) according to step a) or d) in the container, in the mold and/or on the support surface (4, 11, 12, 13, 53, 54) in such a way that the overlapping areas intended for joining or connecting come to lie on top of one another in the device according to step c); f) chemical crosslinking of the tissue(s) or tissue component(s) according to step e), in particular of said overlap areas, in the device according to step c) with addition of a suitable crosslinking agent into the container, into the mold and/or onto the support surface and under the action of said static or quasi-static or periodic pulsatile pressure load/compression on the overlap area(s) (41, 42); g) demolding or removal of the tissue or tissue component(s) bonded or joined after step f) (6); h) optional purely chemical post-crosslinking using a suitable crosslinking agent.

4. The process according to any of embodiments 1 to 3, wherein the static compressive load/compression is applied via a force application in the range of 0.01 N/mm² to 10 N/mm², preferably 0.1 N/mm² to 1 N/mm², over a total period of at least 4 hours to 5 days, preferably 2 days.

5. The process according to any one of embodiments 1 to 3, wherein the quasi-static pressure load/compression is applied via a force application in the range of 0.01 N/mm² to 10 N/mm², preferably 0.1 N/mm² to 1 N/mm², over a time in the range of 200 seconds to 400 seconds, preferably 300 seconds, in combination with a corresponding pressure release/pressure pause of 1 to 10 seconds, preferably 1 or 2 seconds, and this over a total period of at least 4 hours to 5 days, preferably 2 days.

6. The process according to any of embodiments 1 to 3, wherein the periodic pulsatile pressure load/compression is applied over a force input in the range of 0.01 N/mm² to 10 N/mm², preferably 0.1 N/mm² to 1 N/mm² over a time in the range of 10 seconds to 60 seconds, preferably 30 seconds, in combination with a corresponding pressure release/pressure pause of 1 to 10 seconds, preferably 1 or 2 seconds, and this over a total period of at least 4 hours to 5 days, preferably 2 days. 7. process according to any of embodiments 1 to 6, wherein the device according to step c) comprises an electronically controllable pneumatic cylinder (41), hydraulic cylinder or inflation sleeve (21), which can be controlled via a suitable control element comprising suitable electronics in such a way that said quasi-static or periodic pulsatile pressure/compression movement can act on the overlap area(s) of the tissue/component(s) (3) (substantially) vertically/orthogonally.

8. The process according to any of embodiments 1 to 7, wherein the crosslinking agent is an aldehyde-containing solution or is selected from the group consisting of glutaraldehyde, carbodiimide, formaldehyde, glutaraldehyde acetals, acyl azides, cyanimide, genipin, tannin, pentagalloyl glucose, phytate, proanthocyanidin, reuterin, and/or contains epoxy compounds.

9. The process according to any of embodiments 1 to 8, wherein the crosslinking agent is glutaraldehyde, preferably a 0.5% to 0.65% glutaraldehyde solution.

10. The process according to any of embodiments 1 to 9, wherein the tissue/component(s) has been subjected to a pretreatment comprising optional decellularization with a suitable detergent, preferably with a solution containing surfactin and deoxycholic acid, and optionally precrosslinking, preferably with a solution containing glutaraldehyde.

11. The process according to any one of embodiments 1 to 10, wherein the tissue/tissue component(s) is rinsed at least once with a suitable solution, in particular a salt solution and/or an alcohol solution, before and/or after the crosslinking, the optional pre-crosslinking and/or the optional post-crosslinking.

12. The process according to any of embodiments 1 to 11, wherein the process further comprises performing a structural stabilization step on the, optionally decellularized, tissue/component(s) before or after the crosslinking, the optional pre-crosslinking and/or the optional postcrosslinking.

13. The process according to embodiment 12, wherein the structural stabilization step is performed on the, optionally decellularized, tissue/tissue component(s) after crosslinking, after optional pre-crosslinking, or after optional post-crosslinking.

14. The process according to any of embodiments 12 or 13, wherein the structure stabilization step comprises exposing the, optionally decellularized, tissue/component(s) to at least one solution, but preferably at least two different solutions, wherein one solution comprises glycerol and another solution comprises polyethylene glycol. 15. The process according to embodiment 14, wherein exposure to one or more of the solutions lasts from 5 minutes to 2 hours.

16. The process according to any of embodiments 1 to 15, further comprising drying the tissue/tissue component(s) in a suitable controllable environment, such as a climatic chamber or desiccator, for example at constant low relative humidity or by reducing the relative humidity, optionally from 95% to 10% over 12 hours at 37°C.

17. The process according to any one of embodiments 12 to 16, wherein of the at least two different solutions, a first aqueous solution comprises polyethylene glycol having an average molecular weight between 150 g/mol and 300 g/mol; and a second solution is an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol.

18. The process according to any one of embodiments 12 to 17, wherein of the at least two different solutions, a first solution comprises aqueous polyethylene glycol having an average molecular weight between 200 g/mol and 600 g/mol; and a second solution is an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol.

19. The process according to any one of embodiments 1 to 18, wherein the process additionally comprises removing alpha-gal epitopes by using a suitable alpha-galactosidase.

20. The process according to embodiment 19, wherein the alpha-galactosidase is obtained from green coffee bean (GCB).

21. The process according to embodiment 19, wherein the alpha-galactosidase is obtained from Cucumis melo.

22. Use of an aldehyde-containing crosslinking agent or a crosslinking agent selected from the group consisting of glutaraldehyde, carbodiimide, formaldehyde, glutaraldehyde acetals, acyl azides, cyanimide, genipin, tannin, pentagalloyl glucose, phytate, proanthocyanidin, reuterin, and/or epoxy compound-containing crosslinking agents in combination with a static or quasi- static or periodic pulsatile compressive load/compression to form interfibrillar crosslinks for seamless, material bonded and durable joining/connecting of tissues and/or one or more tissue component s) for medical applications.

23. The use according to embodiment 22, wherein the crosslinking agent is glutaraldehyde.

24. Seamlessly joined tissue/tissue component s) obtained according to any of the processes according to embodiments 1 to 21 for medical application, in particular for application in vascular implants, preferably in an artificial heart valve or in a covered stent.

25. Medical implant, preferably with a hollow cylindrical base structure, wherein in and/or on a surface of the base structure the seamlessly, materially and permanently connected/joined tissue or tissue component according to embodiment 24 is arranged/fixed, and which in the implanted state of the medical implant is intended and arranged to contact an anatomical structure of a patient, in particular a vessel wall, in particular a vessel, to which the medical implant has been implanted.

26. The medical implant according to embodiment 25, wherein the implant is a prosthetic heart valve comprising an artificial heart valve made of said tissue/component and/or a seal made of said tissue/component, which is attached, preferably sutured, to an expandable or self-expanding and catheter implantable base body.

27. The medical implant according to any of embodiments 25 or 26, wherein the medical implant is selected from the group consisting of an artificial heart valve, in particular an artificial aortic valve, a coronary or peripheral vascular stent, in particular a covered stent and/or a stent graft.

28. The medical implant according to embodiment 25, wherein the tissue/tissue component is selected from the group consisting of pericardium, ligaments, tendon, cartilage, bone, skin.

Stabilized dry Tissue - preservation and stabilization of tissue

The fifth aspect of the invention relates to a process for (direct) preservation and stabilization of (substantially non-crosslinked) tissue, preferably native biological tissue, or pretreated but (substantially non-crosslinked) tissue (e.g. decellularized), which is characterized by an at least partial substitution of the (native) tissue water with a hygroscopic exchange material and a subsequent controlled drying. The process according to the fifth aspect of the invention enables direct stabilization of substantially non-crosslinked tissue, preferably native biological tissue - without prior substantial chemical fixation/crosslinking, such as by means of a glutaraldehyde solution, thereby opening up new fields of application, in particular medical fields of application.

The fifth aspect of the invention is described herein essentially using as an example a process for the preservation and stabilization of substantially non-crosslinked tissue for use in an artificial aortic valve (TAVI/TAVR). While the fifth aspect of the invention is particularly well suited for preservation/stabilization of such tissue, it is not limited to such application(s). For example, the fifth aspect of the invention is also applicable to the preservation/stabilization of, for example, blood vessels, bone, cartilage, ligaments, skin or the like.

There are basically three different types of prosthetic heart valves, especially aortic valve prostheses: Prostheses with mechanical valves, which are manufactured artificially, mostly from graphite coated with pyrolytic carbon; prostheses with valves made from biological tissue (or partly biological tissue locally reinforced by artificial fibers, if necessary), mostly pericardial tissue typically derived from animal sources (e.g., porcine or bovine); and valves made from artificial materials such as polymers. The heart valve formed from the biological tissue is generally secured in a base body (e.g., a solid plastic scaffold or a self-expanding stent or a balloon-expanding stent) and this is implanted in the position of the natural valve. The fifth aspect of the invention describes, among other things, a method for sutureless and integral connection/jointing of such tissue for use in a prosthetic aortic valve to be implanted in place of a natural aortic valve.

Usually, the initial tissue must be thoroughly cleaned and prepared prior to implantation. As far as possible, the tissue is modified in such a way that it is not recognized by the body as foreign tissue, has as little calcification as possible, and has as long a service life as possible. Essentially, such a tissue preparation process comprises several steps:

One possible processing step is the so-called decellularization of the tissue. In this step, cell membranes, intracellular proteins, cell nuclei and other cell components are almost completely removed from the tissue to obtain an almost pure extracellular matrix. Cells and cellular components remaining in the tissue represent in particular a possible cause of undesired calcification of the biological implant material. Decellularization should be carried out so gently that the structure of the extracellular matrix and in particular the collagen fibers in the extracellular matrix remain as unaffected as possible, while on the other hand all cells and cellular components contained therein are removed from the tissue as completely as possible.

Preferably, according to the fifth aspect of the invention, the biological and/or artificial tissue is subjected to a pretreatment comprising an optional decellularization with a suitable detergent, preferably with a solution containing surfactin and deoxycholic acid. The decellularization can also be performed otherwise, for example, via lysis of the cells or by an osmotic digestion.

In the context of the fifth aspect of the invention, the expressions/terms "biological and/or artificial tissue" or similar terminology describe the tissue species suitable for the processes according to the fifth aspect of the invention. That is, for example, purely biological tissue is tissue of purely natural origin, e.g., porcine pericardium taken from a porcine pericardium. Purely artificial tissue is tissue that has been artificially produced, for example, from one or more different polymer(s) - e.g., by means of suitable 3D printing processes or the like. Biological and artificial tissue refers to mixed forms of e.g. a biological basic substance such as porcine pericardium, but including artificial materials, e.g. for local reinforcement of certain tissue regions, which are exposed to e.g. enormous physiological pressure and/or tensile loads - e.g. leaflets of a TAWTAVR valve. In the context of the fifth aspect of the invention, however, all these tissue types have in common, insofar as they are to be subjected to chemical crosslinking according to the processes of the fifth aspect of the invention, that they also comprise crosslinkable groups, e.g. free amino groups, in particular collagen fibers, which are chemically and/or biochemically crosslinkable.

According to the prior art, the tissue treated in this way is attached to a basic body (e.g., a hollow cylindrical nitinol stent), far predominantly by suturing using a plurality of surgical knots. The main body or scaffold is implantable by surgical techniques (mostly catheter-based). Frequently, the basic scaffold is self-expanding or mechanically expandable with the aid of a balloon so that the prosthetic heart valve can be guided to the implantation site in a compressed state by means of a catheter and implanted within the natural valve.

According to the state of the art, such catheter-implantable prosthetic heart valves are usually stored in a storage solution, correspondingly in a moist state. The storage solution serves to sterilely stabilize the biological tissue. One conceivable storage solution is, for example, glutaraldehyde.

As mentioned, such surgical sutures usually have to be placed manually. This process is very time-consuming, expensive and error-prone - to list just a few of the associated disadvantages. Surgical knots, for example, must be placed individually by personnel in a highly concentrated manner and must always be visually inspected. In addition, each individual knot represents a potential weak point of the medical tissue, since mechanical forces occurring under stress of a medical implant are focused on the knots. Surgical sutures also have a non-negligible space requirement (space requirement), which means that minimum structural sizes of a few millimeters cannot be undercut, especially in the case of medical implants. This noticeably restricts medical implants in their medical fields of application. In order to achieve a treated biological material with the best possible mechanical properties and to prevent defense reactions of the receiving body, in the prior art the collagen fibers are crosslinked by means of a suitable crosslinking agent through the incorporation of chemical bonds. The crosslinking agent typically binds to free amino groups of the collagen fibers and forms chemically stable bonds between the collagen fibers. In this way, the three-dimensionally arranged collagen fibers form a biological material that is stable over the long term and, moreover, is no longer recognized as foreign biological material. The three-dimensional crosslinking or linking of the individual collagen fibers via the crosslinking agent significantly increases the stability and stressability of the tissue. This is particularly crucial when used as the tissue of a heart valve, where the tissue must open and close as a valve every second.

Consequently, native biological tissues, with or without previous decellularization, are crosslinked with a suitable crosslinking agent, such as glutaraldehyde, as standard for their use as implant material, for example, shortly after the tissue is removed. The actual shaping for e.g. medical purposes takes place only after crosslinking, since according to the state of the art no dimensionally stable cutting is possible in the native state.

With regard to an application as a biocompatible and biodegradable tissue patch, for example, the use of bacterial cellulose and decellularized collagen matrices is known.

However, in terms of process technology, biological tissues in their native, completely noncrosslinked state are always subject to natural decomposition processes, e.g. as a result of the action of proteinases contained in the tissue. The time window between tissue removal and chemical fixation/crosslinking is therefore very limited. Cutting of the native tissue is not possible in a dimensionally stable manner due to the locally varying internal mechanical stresses.

However, in terms of material technology, the use of exclusively decellularized tissue as collagen matrices has the disadvantage of a very limited shelf life due to the lack of chemical fixation/crosslinking. The use of alternative biological materials, such as bacterial cellulose, again has the disadvantage of a novel material with clinically still largely unknown long-term behavior.

In the case of preservation and stabilization of essentially non-crosslinked, preferably native biological tissue, water plays a crucial role in the specific conformation of collagen molecules. A distinction is made between structural water, bound water and free water. Structural water, also called tightly bound water, is associated with stabilization of the triple helix structure. Intermolecular water molecules between triple helices and microfibrils are called bound water or contact water. Between the microfibrils and fibrils is free, unbound water. As water is removed, the collagen fibers lose their flexibility. If the process is limited to free, unbound water only, this process is reversible. However, if the drying process results in the removal of bound water molecules, the fiber network is irreversibly damaged. The absence of water causes a change in tissue conformation, which is accompanied by an increase in intra- and possibly also intermolecular hydrogen bonding. Complete rehydration of the fiber composite is thus precluded. The elastic properties of the collagen fibers are lost, and the tissue becomes brittle and fragile.

Consequently, the present fifth aspect of the invention is based on a task to provide a direct preservation and stabilization process of essentially non-crosslinked, in particular native or decellularized biological tissue in order to enable i) any further processing and ii) at the same time also shaping directly in an substantially non-crosslinked or native state.

With the context of the fifth aspect of the invention, the term "substantially non-crosslinked" means that the corresponding tissue may be, for example, slightly pre- or partially crosslinked, but at least 50%, preferably at least 60%, more preferably at least 70%, still more preferably at least 80%, most preferably at least 90% of crosslinkable groups are still present in the tissue to be subjected to the processes according to the fifth aspect of the invention.

In order to enable structure-preserving drying of the tissue, i.e. preservation/stabilization, according to the fifth aspect of the invention a hygroscopic exchange material (e.g. glycerol and/or polyethylene glycol, if necessary as mixture(s)) is/are used, which stabilizes the collagen structure when water is removed. In this way, rehydration makes it possible to restore the tissue to its initial state.

According to the fifth aspect of the invention, the substantially non-crosslinked starting tissue, such as native biological tissue selected from the group consisting of: porcine, bovine or equine pericardium, ligaments, tendons, cartilage, bone, and skin (this is not to be understood as limiting) is first mechanically processed to remove unwanted tissue residues. Then, optionally, decellularization known in the prior art, e.g., using surfactin and deoxy cholic acid, DNAse, alpha-galactosidase treatment, etc., can be performed to generate a collagen-containing tissue matrix with reduced immune response in vivo. Preferably, according to the fifth aspect of the invention, the biological and/or artificial tissue is subjected to a pretreatment comprising an optional decellularization with a suitable detergent, preferably with a solution containing surfactin and deoxycholic acid.

Advantageously, the tissue is rinsed at least once, preferably several times, with a suitable solvent, in particular a buffered saline solution and/or an alcohol solution, before and particularly preferably after the decellularization (if the tissue is decellularized). Buffered sodium chloride solutions and/or an ethanol solution are particularly advantageous.

The substantially non-crosslinked or native tissue, if necessary decellularized or otherwise pretreated, is then preserved with a hygroscopic substitute such as glycerol, polyethylene glycol (PEG) or a mixture of both, also comprising various mixtures of PEG, preferably with a group of substitutes consisting of glycerol, polyethylene glycol 200 and polyethylene glycol 400, and then subjected to gentle drying in a climatic chamber (stabilized). However, according to the fifth aspect of the invention, a less controlled drying, for example at constant low relative humidity in a controllable environment, is also possible.

In one embodiment of the present fifth aspect of the invention, alpha-gal epitopes may additionally be removed from the tissue in a further treatment step, which may be carried out after or before the optional decellularization step. Any suitable alpha-galactosidase can be used for such an additional treatment step, e.g., alpha-galactosidase from green coffee bean (GCB) or Cucumis melo.

This process according to the fifth aspect of the invention results in a flexible, soft (biological) tissue whose water content and activity are reduced to such an extent that storage and, if necessary, dimensionally stable cutting are possible immediately, i.e. directly.

On the device side, the task posed is solved by a medical implant comprising the directly preserved and stabilized tissue - which may have been further processed as cut and installed; for example, a biocompatible and biodegradable tissue patch.

With the context of the present fifth aspect of the invention, the term "medical implant" or similar terms particularly includes stent-based implants and heart valve prostheses, particularly aortic valve prostheses, which are stent-based. Also conceivable implants within the scope of the fifth aspect of the invention are biodegradable patches/pockets/envelopes for e.g. pacemakers or defibrillators.

According to the fifth aspect of the invention, the term "medical implant" also refers to any medical implant for which the directly preserved and stabilized tissue is suitable as a process product, e.g. the above-mentioned biocompatible and biodegradable tissue patch.

Nowadays, stents are used particularly frequently as implants for the treatment of stenoses (narrowing of blood vessels). They have a body in the form of a possibly perforated tubular or hollow cylindrical basic structure, which is open at both longitudinal ends. The basic structure of the stent may be composed of individual meshes formed by zigzag or meander-shaped webs. The tubular basic structure of such an endoprosthesis is inserted into the vessel to be treated and serves to support the vessel. Stents have become particularly popular for the treatment of vascular diseases. The use of stents can widen constricted areas in the vessels, resulting in a gain in lumen.

Although the use of stents or other implants can achieve an optimal vessel cross-section, which is primarily necessary for the success of the therapy, the permanent presence of such a foreign body initiates a cascade of processes which, for example, promote inflammation of the treated vessel or necrotic vascular changes and which can lead to a gradual overgrowth of the stent through the formation of plaques.

Stent graft(s)" are stents that contain a fleece or other flat covering, such as a foil or tissue matrix, on or in their often grid-like basic structure. In this context, a "nonwoven" is understood to be a textile fabric, for example, which is formed by individual fibers. Such a stent graft is used, for example, to support weak points in arteries, e.g. in the area of an aneurysm or a rupture of the vessel wall (so-called bail-out device), especially as an emergency stent.

Medical endoprostheses or implants for the most diverse applications are known in great variety from the prior art and can be combined with the directly preserved and stabilized tissue of the fifth aspect of the invention for suitable purposes - if necessary after further processing of the tissue such as, for example, cutting. Implants in the sense of the present fifth aspect of the invention are in particular endovascular prostheses or other endoprostheses, e.g. stents (vascular stents, bile duct stents, vascular stents, peripheral stents, coronary stents or e.g. mitral stents), endoprostheses for closing persistent foramen ovale (PFO), stents for all four heart valves such as e.g. pulmonary valve stents (PFO), stents for all four heart valves such as e.g. mitral stents). Endoprostheses for closing an ASD (atrial septal defect), as well as prostheses in the area of hard and soft tissue.

In one alternative, the medical implant is preferably an artificial heart valve prosthesis, e.g. a TAVI/TAVR valve, which comprises an artificial heart valve made of the directly preserved and stabilized tissue of the fifth aspect of the invention - possibly after suitable further processing such as, for example, chemical crosslinking - and/or a seal made of said tissue, which is attached, preferably sutured, to an expandable or self-expanding and catheter-implantable basic scaffold, stent, or holding device.

In an alternative, preferably the medical implant is a covered stent or a so-called stent graft, which has one or more tissue components from the directly preserved and stabilized tissue of the fifth aspect of the invention - possibly after suitable further processing - and/or a seal from said tissue, which is attached, preferably sutured or also seamlessly joined, to the corresponding basic framework, stent, or the holding device, and wherein said covered stent or stent graft is implantable by catheter.

With the context of the fifth aspect of the invention, the term "covered stent(s)" or similar terms describes an intraluminal endoprosthesis, with a preferably hollow cylindrical basic structure (e.g. made of nitinol), which is covered/sheathed by a further structure and/or one or more material layer(s) on a surface (inside and/or outside), preferably with a seamless and materially joined/joined tissue according to the fifth aspect of the invention.

In the context of the fifth aspect of the invention, a distinction must be made between "covered" in the sense of "covered" and "coated" in the sense of "covered with a substance or an alloy". According to the fifth aspect of the invention, covered stents refer to stent implants or implants with a retaining structure, wherein the stent or the retaining structure itself is covered or sheathed by the tissue bonded/joined according to the fifth aspect of the invention, quasi as one or more "layers". That is, the stent or the retaining structure can, for example, be covered/sheathed from the outside and/or from the inside with the tissue connected/joined according to the fifth aspect of the invention. This may be realized in the form of one or more layers of the tissue joined/jointed according to the fifth aspect of the invention; or an inner and an outer layer of this tissue may also be joined/jointed with the joining/jointing methods according to the fifth aspect of the invention, and may also include, for example, an envelope of the tissue according to the fifth aspect of the invention at one end of the stent/holding structure. For example, an inner layer of the tissue of the fifth aspect of the invention may be folded over outwardly at both ends of the stent/holding structure, thus becoming an outer layer. The foregoing examples are not limiting, and the person skilled in the art may anticipate several different configurations and possible applications of the tissues joined/jointed according to the fifth aspect of the invention in light of the present disclosure. "Coated," on the other hand, rather focuses on a direct chemical, physical, or pharmaceutical coating of the stent structure. For example, prior art stents may be coated with silicon carbide (aSiC) or a so-called drug eluting stent is coated with a physiologically acceptable pharmaceutical agent.

The tissue directly preserved and stabilized according to the fifth aspect of the invention - possibly with suitable further processing - could be used, for example, in cases where cellular ingrowth is preferred, such as in the treatment of a wound or burn with a porous matrix or in use as a means of sealing an implant or graft.

The person skilled in the art is aware that numerous other conceivable applications of the tissue for medical implants, which is seamlessly bonded/joined according to the fifth aspect of the invention, are to be considered.

Detailed description of the fifth aspect of the invention

In the context of the fifth aspect of the invention, a hygroscopic exchange material is used for the direct preservation and stabilization of non-crosslinked, in particular native biological tissue, in the sense that it can be a type of hygroscopic exchange material, such as e.g. glycerol, or two or more types of hygroscopic exchange materials, such as glycerol and polyethylene glycol (possibly with different molecular weights) can be added either individually and, for example, one after the other, or as a mixture of the two substances, for example glycerol dissolved in polyethylene glycol. Preferred in the context of the fifth aspect of the invention is a sequence of glycerol in a first stabilization step, followed by a light PEG stabilization (e.g. with PEG 200), followed by a heavier PEG stabilization (e.g. with PEG 400). Under the second mentioned variant of two or more hygroscopic substitutes also fall embodiments in which the same base material is used, such as for example polyethylene glycol, but in different embodiments, such as for example with a different molecular weight. With this context of the fifth aspect of the invention, a group of hygroscopic substitutes consisting of: Glycerin, Polyethylene glycol 200 (PEG200) and Polyethylene glycol 400 (PEG400) is preferred.

All three aforementioned substances are characterized by their hygroscopic properties, low toxicity values and excellent water solubility. The ability to absorb and bind moisture from the environment opens up a wide range of applications for glycerol and polyethylene glycol (PEG) in a wide variety of suitable mixing ratios to bring about the preservation of non-crosslinked, in particular native, biological tissue according to the fifth aspect of the invention.

In a preferred preservation/stabilization step, the tissue is exposed to at least one solution containing glycerol and/or polyethylene glycol, the tissue being exposed either to one of these solutions or to the two solutions successively in any order and composition as first and second solution or to both solutions simultaneously as a mixture of solutions - or also to a solution of glycerol and PEG in water. When drying tissue, e.g., for storage or transport of the tissue, the stabilization process is preferably carried out prior to drying.

As a non-limiting example, the preservation/stabilization process may be performed, for example, after decellularization by immersing the tissue in a series of one or more stabilizing solutions of glycerol and/or polyethylene glycol to sufficiently saturate the tissue with stabilizing agents, and ultimately to introduce a stable tissue into the ensuing drying. Saturation times may vary, depending on the properties of the tissue; but typically last from about 5 minutes to 2 hours, or 5 minutes to 15 minutes for, e.g., porcine pericardium as a non-limiting embodiment. The stabilized tissue may be dried, for example, by placing the tissue in a suitable climate chamber and suitably reducing the relative humidity, for example, from about 95% to 10% over, for example, about 12 hours at about 37° - 40°C. It is also possible to dry under constant low relative humidity in a controlled environment.

It is obvious to the skilled person that the parameters for the reduction of humidity, time and temperature must always be selected appropriately depending on the tissue to be treated. The polyethylene glycol-containing solutions generally contain polyethylene glycol with an average molecular weight between 150 g/mol and 6000 g/mol. As used herein, the term "between" also includes the upper and lower specified values. Thus, an average molecular weight between 150 g/mol and 6000 g/mol is intended to include 150 g/mol and 6000 g/mol.

In some embodiments, at least one polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 150 g/mol and 200 g/mol, between 150 g/mol and 300 g/mol, between 200 g/mol and 300 g/mol, between 200 g/mol and 600 g/mol, between 200 g/mol and 400 g/mol, between 150 g/mol and 400 g/mol, or between 400 g/mol and 600 g/mol. According to a particularly preferred embodiment, the polyethylene glycol-containing solution provided alone or before or after a glycerol solution contains polyethylene glycol at or about 150 g/mol to 300 g/mol or at or about 200 g/mol (e.g., PEG200), and in an even more preferred embodiment, the polyethylene glycol-containing solution contains 40% PEG200 or about 40% PEG200; for example, in aqueous solution.

The term "about" as used herein is intended to encompass a variation above and below the specified amount that would be expected in normal use, such as a variation of 5% or 10%.

Glycerin may be added to any of the above stabilizing solutions to form a mixture, or it may be provided separately for stabilizing purposes.

In some embodiments, a subsequently applied polyethylene glycol-containing solution includes polyethylene glycol having a higher average molecular weight than a previously applied polyethylene glycol-containing solution. In some embodiments, the subsequently applied polyethylene glycol-containing solution contains polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol. In some embodiments, the subsequently applied polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 300 g/mol and 1500 g/mol. In some embodiments, the subsequently applied polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 300 g/mol and 1200 g/mol. In some embodiments, the subsequently applied polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 400 g/mol and 800 g/mol. In some embodiments, the subsequently applied polyethylene glycol-containing solution comprises polyethylene glycol having an average molecular weight between 400 g/mol and 600 g/mol. In some embodiments, the subsequently applied polyethylene glycol-containing solution contains polyethylene glycol having an average molecular weight of 400 g/mol (PEG400) or about 400 g/mol.

Again, glycerol can be added to any of the above stabilizing solutions to form a mixture, or it can be provided separately as a stabilizing solution.

In the context of the fifth aspect of the invention, glycerol is preferably provided in aqueous solution. An exemplary and preferred glycerol solution contains 30% glycerol in water. In this regard, it is also apparent to the skilled person that the glycerol concentration must be selected depending on the tissue to be treated, and for example depending on whether glycerol alone is used as a hygroscopic exchange material according to the fifth aspect of the invention, or in combination with one or more hygroscopic exchange materials, either in mixture(s) or used sequentially.

In this regard, the skilled person is aware that the temperature during the stabilization step may influence the results. For example, too high a temperature (e.g., above about 85°C) will cause denaturation and irreversible damage to the tissue. Again, however, too low a temperature may result in a solution that is too viscous. Preferably, exposure to the stabilizing solutions is at 20- 50°C, preferably at 37-40°C, but temperatures from room temperature up to 60°C should be tolerable.

In this case, the drying of the tissue is always controlled and managed in such a way as to ensure a slow and gentle removal of the water in the liquid state from the tissue. This is advantageously achieved by controlled reduction of the ambient humidity of the biological tissue in, for example, a desiccator or a climatic chamber with controlled adjustment of the parameters of the ambient atmosphere of the biological tissue.

Thus, the fifth aspect of the invention provides a process for direct preservation and stabilization of non-crosslinked, preferably native biological tissue, which in a specific embodiment comprises the following steps: a) providing a, preferably native biological tissue, comprising chemically and/or biochemically crosslinkable groups, optionally present in a suitable storage solution; b) Optionally rinsing the tissue with a suitable rinsing solution; c) Moist preparation of the tissue, in particular mechanical removal of unwanted tissue, optionally followed by further cutting of said tissue; d) Optional decellularization of the tissue with a suitable decellularizing agent, preferably surfactin and deoxycholic acid; e) Optional irrigation of the tissue with a suitable irrigation solution (e.g. 0.9% isotonic saline) to remove detergents; f) Optional irrigation of the tissue in a suitable glycerol solution, with appropriate mechanical agitation of said tissue; g) Optional rinsing of the tissue in a first suitable polyethylene glycol (PEG) solution, preferably in a suitable PEG200 solution, optionally with suitable mechanical agitation of said tissue; h) optionally rinsing the tissue in a second suitable polyethylene glycol (PEG) solution, preferably in a suitable PEG solution having a higher molecular weight than said first PEG solution, further preferably in a suitable PEG400 solution, optionally with suitable mechanical agitation of the tissue; followed by i) drying the tissue in a suitable environment at constant low humidity or a suitable desiccator or climatic cabinet with reduction of an initial relative humidity, preferably 95%, to a lower target relative humidity, preferably 10%, for a suitable period of time at a suitable ambient temperature in said climatic cabinet; j) Optional cutting of the tissue; optionally followed by a k) storage of the tissue; and/or optionally followed by a l) Sterilization of the tissue; m) Optional further processing of the tissue, preferably a three-dimensional crosslinking of said tissue.

All variants of the directly preserved and stabilized tissue can be combined in any way and can be transferred in any combination to the medical implant described herein, and vice versa.

Advantageous effects of the fifth aspect of the invention

According to the fifth aspect of the invention a (stabilized) dried tissue comprising (chemically and/or biochemically) crosslinkable groups, preferably having a proportion of crosslinkable groups in the tissue to be treated compared to non-crosslinkable groups of more than 50%, can be obtained. This (stabilized) dried tissue is still biodegradable and can be colonized by cells. In contrast crosslinked tissue, e.g. crosslinked by the use of glutaraldehyde, (comprising much less than 50% chemically and/or biochemically crosslinkable groups) is not biodegradable and cannot be colonized by cells.

Substantially non-crosslinked tissue throughout the application means that the proportion of crosslinkable groups in the tissue to be treated (compared to non-crosslinkable groups) is greater than 50%, preferably greater than 60%, even more preferably greater than 80%, most preferably greater than 90%. However, this also means that lightly or only slightly pre-crosslinked or partially crosslinked tissue is suitable for the methods of the first aspect of the invention.

The disclosed preservation process of the fifth aspect of the invention enables storage of tissue comprising (chemically and/or biochemically) crosslinkable groups, preferably having a proportion of crosslinkable groups in the tissue to be treated compared to non-crosslinkable groups of more than 50%, in particular native, biological tissues - without loss of quality and mechanical stability.

The disclosed preservation process of the fifth aspect of the invention enables storage of substantially non-crosslinked, in particular native, biological tissues - without loss of quality - for further processing as desired.

Furthermore, the above-described process can be integrated into existing tissue processes, for example, to extend the critical time period between removal and crosslinking of the tissue or to enable shaping in the non-crosslinked, e.g. native, state, or also for the development of new products such as a non-crosslinked and storable biological matrix structure, e.g. for use as a biocompatible and possibly biodegradable tissue patch.

Furthermore, the present process provides tissue that is preserved and stabilized in such a way that it can be stored in its native state and cut to a stable shape. For the person skilled in the art, this opens up new options for tissue processing, which can be integrated, for example, in the existing processes of tissue processing, such as in the case of heart valve production for a TAVI/TAVR valve, or for the production of a covered stent.

Furthermore, the tissue treated according to the disclosed process of the fifth aspect of the invention can optionally be used for a new product in the sense of a biocompatible and possibly biodegradable carrier structure in temporary implants, since it is converted into a stable storage state by drying. Relatively, a biodegradable cover on a biodegradable carrier structure can thus be provided.

A major advantage of the solution according to the fifth aspect of the invention is the use of essentially non-crosslinked, e.g. native, biological tissue, which has distinct advantages with respect to thromboembolic complications and potential biocompatibility problems compared to artificial materials. The resulting properties of the tissue processed as described above are not significantly different in the rehydrated state from those of a non-stabilized and non-dried native tissue.

In view of the foregoing disclosure, the present fifth aspect of the invention further comprises the following embodiments, numbered in ascending order:

1. Process for preservation and stabilization of tissue, in particular native biological tissue, for medical applications, in particular for use as a component of a medical implant, preferably a vascular implant, more preferably an artificial heart valve or a covered stent, comprising at least the steps of:

- at least a partial substitution of the tissue water of the tissue, in particular native biological tissue, by means of a hygroscopic substitute in solution, preferably in aqueous solution; optionally under a suitable mechanical agitation acting on the tissue, such as shaking, panning and/or stirring; followed by

- suitable drying in a suitable controlled environment, such as a suitable climatic chamber or desiccator.

2. The process according to embodiment 1, wherein the hygroscopic exchange material comprises glycerol and/or polyethylene glycol.

3. The process according to embodiment 1, wherein the hygroscopic exchange material comprises at least two different solutions, preferably comprises at least three different solutions, and wherein a first solution comprises glycerol and a second and a third solution comprises polyethylene glycol.

4. The process according to any of the preceding embodiments, wherein the exposure to one or more of the solutions lasts from 5 minutes to 2 hours. 5. The process according to embodiment 4, wherein exposure to one or more of the solutions lasts from 5 minutes to 45 minutes.

6. The process according to any of the preceding embodiments, wherein the drying of the tissue is carried out in a suitably controlled environment, such as in the air, with a constant low relative humidity, or is carried out in the suitable climatic chamber or desiccator stepwise by reducing the relative humidity at a constant temperature.

7. The process according to embodiment 6, wherein the drying of the tissue in the suitable climatic chamber or the suitable desiccator is carried out stepwise by reducing the relative humidity optionally from 95% to 10%, preferably over 12 hours, at 35 - 60°C, preferably at 35 - 40°C.

8. The process according to any of the preceding embodiments, wherein the tissue, in particular the native biological tissue, has been subjected to a pretreatment comprising an optional decellularization, preferably with a surfactin and deoxycholic acid containing solution.

9. The process according to any of the preceding embodiments, wherein the tissue, in particular the native biological tissue, is rinsed at least once with a suitable solution, in particular a salt solution and/or an alcohol solution, before and/or after the preservation and stabilization and/or the optional decellularization.

10. The process according to any of the above embodiments, wherein glycerol is present as an aqueous solution, and is preferably an aqueous solution with 1 - 70% glycerol in water.

11. The process according to any of the preceding embodiments, wherein glycerol is present as an aqueous solution, and preferably is an aqueous solution with 10 - 50% glycerol in water.

12. The process according to any of the above embodiments, wherein glycerol is present as an aqueous solution, and is preferably an aqueous solution containing 30% glycerol in water.

13. The process according to any one of the preceding embodiments, wherein polyethylene glycol is present as two different solutions, and a first solution comprises an aqueous solution of polyethylene glycol having an average molecular weight between 150 g/mol and 300 g/mol; and a second solution is an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol.

14. The process according to any one of embodiments 1 to 12, wherein polyethylene glycol is present as two different solutions, and a first solution comprises an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 600 g/mol; and a second solution is an aqueous solution of polyethylene glycol having an average molecular weight between 300 g/mol and 6000 g/mol.

15. The process according to any of the preceding embodiments, wherein polyethylene glycol is present as two different solutions, and a first solution is an aqueous solution of polyethylene glycol having an average molecular weight around 200 g/mol, preferably 200 g/mol; and a second solution is an aqueous solution of polyethylene glycol having an average molecular weight around 400 g/mol, preferably 400 g/mol.

16. The process according to any of the preceding embodiments, wherein the process additionally comprises removal of alpha-gal epitopes by use of a suitable alpha-galactosidase.

17. The process according to embodiment 16, wherein the alpha-galactosidase is obtained from green coffee bean (GCB).

18. The process according to embodiment 16, wherein the alpha-galactosidase is obtained from Cucumis melo.

19. Use of a hygroscopic exchange material for direct preservation and stabilization of a tissue, in particular a native biological tissue, in combination with subsequent drying in a suitable environment with constant low relative humidity or under a regulable and reducible relative humidity.

20. The use according to embodiment 19, wherein the hygroscopic exchange material is selected from the group comprising or consisting of glycerol, preferably glycerol in aqueous solution; polyethylene glycol, preferably polyethylene glycol in aqueous solution; PEG 200, preferably PEG 200 in aqueous solution; and/or PEG 400, preferably PEG 400 in aqueous solution. 21. Preserved and stabilized tissue obtained according to any of the processes according to embodiments 1 to 18 for medical applications, in particular for use as a component of a medical implant, preferably a vascular implant, more preferably an artificial heart valve, a covered stent, a vascular patch, a pacemaker pocket, an implantable leadless pacer sheath, a LAAC cover or a biocompatible and optionally biodegradable tissue patch.

22. The medical implant, in particular comprising a basic structure, preferably a biocompatible and biodegradable basic structure, wherein the medical implant and/or the basic structure comprises the directly preserved and stabilized tissue according to embodiment 21, preferably at an inner and/or outer surface thereof, and which, in the implanted state of the medical implant, is intended and arranged to contact an anatomical structure of a patient, in particular a vessel wall, in particular a vessel, to which the medical implant has been implanted.

23. The medical implant, in particular comprising a biocompatible and biodegradable tissue matrix, wherein the medical implant and/or the tissue matrix comprises the directly preserved and stabilized tissue according to embodiment 21, preferably at an inner and/or outer surface thereof, and which in the implanted state of the medical implant is intended and adapted to contact an anatomical structure of a patient, in particular a vessel wall, in particular a vessel, to which the medical implant has been implanted.

24. The medical implant according to any one of embodiments 22 or 23, wherein the medical implant is selected from the group consisting of an artificial heart valve, in particular an artificial aortic valve, a coronary or peripheral vascular stent, in particular a covered stent and/or a stent graft, a vascular patch, a pacemaker pocket, an implantable leadless pacer sheath, a LAAC cover or a biocompatible and optionally biodegradable tissue patch.

25. The medical implant according to any of embodiments 22 to 24, wherein the directly preserved and stabilized tissue is selected from the group consisting of pericardium, ligaments, tendon, cartilage, bone, skin. Example A) - Preservation/stabilization of a native pericardium.

The following exemplary process describes step-by-step the preservation/stabilization of a native heart sac (pericardium) in the sense of the fifth aspect of the invention, in order to subsequently enable a dimensionally stable cutting in the native state:

Example of embodiment B) - Preparation of a non -fixed. decellularized biological tissue matrix from pericardium.

The following exemplary process describes the preparation of a noncrosslinked, decellularized biological tissue matrix from pericardium, e.g. for application as a biocompatible and biodegradable tissue patch for cardiovascular applications:

Combination of ultrathin, 3D-shaped, (stabilized) dried and/or seamless connected tissue technologies

Another aspect of the invention is to provide a combination of the aforementioned aspects of the invention. Thus, (decellularized) ultrathin tissue and/or 3D-shaped and/or (stabilized) dried tissue and/or seamless connected tissue is disclosed herein as well as methods for making the same.

In one embodiment, a process for treating tissue, preferably pericardial tissue, comprising (chemically and/or biochemically) crosslinkable groups comprises the following process steps: optionally decellularizing the tissue, preferably by using a surfactin and deoxycholic acid containing solution and/or by using a galactosidase and/or DNAse treatment, crosslinking at least a part of the crosslinkable groups of the tissue by using at least one crosslinking agent, e.g. glutaraldehyde or a glutaraldehyde solution, while optionally 3D- shaping the tissue using a molded body and/or applying a pressure load on the tissue, preferably by using at least one pressure compensation layer and/ or at least one permeable material layer and/or perforated or counterforms, optionally at least partially substituting tissue water of the tissue by means of a hygroscopic substitute, preferably using one or more solutions containing glycerol and/or polyethylene glycol, and optionally drying the tissue, optionally sterilizing the obtained tissue, preferably by using ethylene oxide.

Crosslinked tissue, e.g. crosslinked by the use of glutaraldehyde, (comprising a proportion of crosslinkable groups in the tissue to be treated compared to non-crosslinkable groups being much less than 50%, preferably less than 20% or less than 10% chemically and/or biochemically crosslinkable groups) is not biodegradable and cannot be colonized by cells.

Furthermore, the use of the aforementioned (decellularized) crosslinked tissue, which is optionally a 3D-shaped tissue and/or an ultrathin tissue (having a thickness of less than 80 pm, preferably between 80 and 20 pm, more preferably between 60 and 20 pm or even between 25 pm and 20 pm) for a medical implant, is disclosed herein as well. The medical implant may be a (cardio)vascular implant, a endovascular prostheses, an endoprostheses, an esophageal implant, a bile duct implant, a dental implant, an orthopedic implant, a sensory implant or a neurological implant a microchip containing implant.

The medical implant may be a stent, a vascular stent, a drug eluting stent, a pulmonary valve stent, a bile duct stent, a peripheral stent, a mitral stent, a stent graft, a drug eluting stent, a covered stent, a vascular patch, a tissue patch, a venous valve, a tooth implant, a bone implant, a glucose sensor implant, a neurostimulator, a cochlear implant, an endoprostheses for closing persistent foramen ovale, an endoprostheses for closing an atrial septal defect, a left atrial appendage closure device, a pacemaker, a leadless pacemaker, a defibrillator, a prosthetic heart valve, preferably a TAVI/TAVR valve.

The tissue obtained by the aforementioned process is a (decellularized) crosslinked tissue, which is optionally a 3D-shaped tissue and/or an ultrathin tissue (having a thickness of less than 80 pm, preferably between 80 and 20 pm or between 25 pm and 20 pm).

The aforementioned medical implant can comprise a biodegradable or non-biodegradable support structure and/or fixation means, e.g. a plate, a bone screw, a mesh or an open or closed framework comprising struts (e.g. a stent of LAAC device). In case the support structure is an open or closed framework comprising struts (e.g. a stent of LAAC device) the support structure may be a self-expanding or self-expandable support structure. A non-biodegradable support structure may be made of a metal (e.g. titanium) or metal alloy (e.g. stainless steel, titanium comprising alloys like Nitinol) or a polymer or reinforced polymer.

In another embodiment, a process for treating tissue, preferably pericardial tissue, comprising (chemically and/or biochemically) crosslinkable groups, preferably comprising a proportion of crosslinkable groups compared to non-crosslinkable of more than 50%, comprises the following process steps:

- optionally decellularizing the tissue, preferably by using a surfactin and deoxycholic acid containing solution and/or by using a galactosidase and/or DNAse treatment,

- optionally crosslinking at least a part of the crosslinkable groups of the tissue by using at least one crosslinking agent, e.g. glutaraldehyde or a glutaraldehyde solution, while optionally 3D- shaping the tissue using a molded body and/or applying a pressure load on the tissue, preferably by using at least one pressure compensation layer and/ or at least one permeable material layer and/or perforated or counterforms,

- at least partially substituting tissue water of the tissue by means of a hygroscopic substitute, preferably using one or more solutions containing glycerol and/or polyethylene glycol, and optionally drying the tissue,

- optionally sterilizing the obtained tissue, preferably by using ethylene oxide.

Polyethylene glycol preferably has a molecular weight being between 150 g/mol and 600 g/mol.

The tissue obtained by the aforementioned process is a (decellularized) tissue, comprising chemically and/or biochemically crosslinkable groups, preferably more than 50% or more than 80% chemically and/or biochemically crosslinkable groups, which is optionally a 3D-shaped tissue and/or an ultrathin tissue (having a thickness of less than 80 pm, preferably between 80 and 20 pm or between 25 pm and 20 pm). Tissue comprising more than 50% chemically and/or biochemically crosslinkable groups, is biodegradable and can be colonized by cells.

Furthermore, the use of the aforementioned (decellularized) tissue, comprising chemically and/or biochemically crosslinkable groups, preferably more than 50% or more than 80% chemically and/or biochemically crosslinkable groups, which is optionally a 3D-shaped tissue and/or an ultrathin tissue (having a thickness of less than 80 pm, preferably between 80 and 20 pm or between 25 pm and 20 pm) for a medical implant, preferably a vascular implant, more preferably an artificial heart valve, a venous valve, a covered stent, a vascular patch, a pacemaker pocket, an implantable leadless pacer sheath, a LAAC cover or a tissue patch is disclosed herein as well. Also a medical implant preferably a vascular implant, more preferably an artificial heart valve, a venous valve, a covered stent, a vascular patch, a pacemaker pocket, an implantable leadless pacer sheath, a LAAC cover or a tissue patch comprising the aforementioned (decellularized) tissue, comprising chemically and/or biochemically crosslinkable groups, preferably more than 50% or more than 80% chemically and/or biochemically crosslinkable groups, which is optionally a 3D-shaped tissue and/or an ultrathin tissue (having a thickness of less than 80 pm, preferably between 80 and 20 pm or between 25 pm and 20 pm) is disclosed herein.

The aforementioned medical implant can comprise a biodegradable or non-biodegradable support structure, e.g. a plate, a mesh or an open or closed framework comprising struts, preferably a stent. A non-biodegradable support structure may be made of a metal or metal alloy (e.g. a metal alloy of nickel and titanium like Nitinol) or a polymer or reinforced polymer.

Unless otherwise explicitly stated the tissue to be treated throughout the application is to be understood as biological tissue. The biological tissue may be an autologous, xenogeneic or allogeneic tissue. In principle, all types of tissue e.g. from non-mammalian or mammalian tissue including human tissue can be used. The tissue may be derived from pig (porcine tissue), sheep, goat, horse, crocodile, kangaroo, ostrich, monkey, preferably primate, octopus, rabbit or cattle (bovine tissue). Tissue that can be used may be collagen containing tissue, pericardial tissue, skin, ligament, connective tissue, tendons, peritoneal tissue, dura mater, tela submucosa, in particular of the gastrointestinal tract, or pleura. The tissue can be in its native form or in a processed form or can comprise combinations thereof.

Autologous tissue (in medicine) refers to tissue that was isolated from the human or animal body and is to be re-transplanted elsewhere in the same human or animal body (i.e. originating from the same human or animal body or in other words donor and recipient are the same). The autologous tissue can be in its native form or in a processed form or can comprise combinations thereof. The autologous tissue to be used comprises chemically and/or biochemically crosslinkable groups.

Allogeneic tissue (in medicine) refers either to material that was isolated from a(nother) human or animal body that is genetically distinct from the human or animal body, but of the same species. Thus, allogeneic (also denoted as allogenic or allogenous) tissue is tissue that was isolated from a human or animal body which is different from the human or animal body where the implant is to be implanted. Allogeneic tissue can be not from the patient itself (but from a genetic different donor of the same species). Allogeneic here also includes hemiallogeneic (genetically different because of being derived from one parent of the same species and one parent from another species). The allogeneic tissue can be in its native form or in a processed form or can comprise combinations thereof. The allogenic tissue to be used comprises chemically and/or biochemically crosslinkable groups.

Xenogeneic tissue (in medicine) refers to tissue that was isolated from a human or animal body of a different (heterologous) species. Thus, xenogeneic (also known as xenogenous or xenogenic) tissue is material that was isolated form a human or animal body which is different from the human or animal body where the implant is to be implanted. Xenogeneic tissue may also refer to tissue based on human or animal donor cells (cells obtained from a or the human or animal donor) being cultivated in a bioreactor or being obtained via 3D printing. The xenogeneic material, e.g. tissue, can be in its native form, in a fixed form, in a processed form or can comprise combinations thereof.

Biological tissue preferably has an organizational level intermediate between cells and a complete organ.

Description of the figures

Embodiments as well as further features and advantages of the invention will be explained hereinafter with reference to the drawings, in which:

Fig. 1 shows a tissue placement of planar tissue patches/components porcine pericardium for a subsequent seamless joining/connecti on of two tissue patches porcine pericardium according to a process according to the invention. By means of tweezers 5, for example, a first rectangular joining partner 1 - tissue patch of porcine pericardium - is placed on a suitable support surface 4 with a part forming the desired overlap area of the first joining partner 3, whereupon, by means of tweezers 5, for example, a second rectangular joining partner 4 is placed on the support surface 4. A second rectangular joining partner 2 - tissue patch from porcine pericardium - is placed on the suitable support surface 4 in such a way that a part of the second joining partner 2 comes to lie overlapping with the first joining partner 1 in the desired overlap area 3. Fig. 1 thus represents an exemplary initial shape for the subsequent static, quasi-static or periodic pulsatile pressure loading/compression of porcine pericardial tissue components in the presence of a suitable crosslinking agent. The support surface 4 has two holes 4a, 4b for fixing and stacking the support surface(s).

Fig. 2 shows a planar seamlessly connected/joined porcine pericardium 1 + 2 with a crosslinked overlap region 6 after passing through a periodic pulsatile pressure loading/compression of porcine pericardial tissue components according to the invention in the presence of a suitable crosslinking agent; in this case glutaraldehyde solution. The joined pericardium rests on a support 4. Fig. 3 shows tissue placement of a one-piece, complete tissue component of porcine pericardium 7 on a three-dimensional device 13 for the valve component of an artificial aortic valve (TAVI/TAVR) with negatives for three leaflets 8 and an inner skirt 9, which is used for subsequent three-dimensional crosslinking by means of static, quasi-static or periodic pulsatile pressure loading/compression, for example, in order to join the open tissue ends of the tissue component in the area 10 in a subsequent seamless joining/connecting according to one of the processes according to the invention. Fig. 3 thus represents, inter alia, an exemplary three- dimensional initial shape for the subsequent quasi-static or periodic pulsatile pressure loading/compression of porcine pericardial tissue components in the presence of a suitable crosslinking agent. The 12 is a holder of the device for clamping/fixing into a suitable crosslinking device.

Fig. 4 shows a one-piece, seamlessly connected/joined tissue component (here a one-piece valve component) with a leaflet portion 14 and an inner skirt 15, which is suitable for realizing a valve function in an artificial aortic valve arranged/fixed to a suitable support structure/stent. The valve component includes individually imprinted leaflets 16 as well as a continuous inner skirt 17, as well as recesses in the lower region for a precisely fitting insertion in an inlet region of a stent; 18 and 19.

Fig. 5 shows an exemplary construction of an inflatable sleeve 21 for the sutureless joining/connection of a TAVI/TAVR valve 20, which is suitable for radial static, quasi-static or periodic pulsatile pressure loading/compression. Shown is a stent framework 22 comprising a valve component 23 fixed into a pressure cylinder 24 of the inflatable cuff, wherein compressed air can be injected via a nozzle 25 with a channel 26. For example, to expand a balloon located in the center, which in turn exerts the pressure load, for example from the inside, on the valve component to be connected.

Fig. 6 shows an exemplary TAVI/TAVR valve with a self-expanding nitinol stent 27 having struts 35, a seamlessly joined valve component 28 according to the invention, in such a way that the stent component 27 is completely enclosed in the tissue of the valve component in the valve region 32 and has been completely enclosed by the static, quasi-static or periodic pulsatile pressure load according to the invention during chemical crosslinking with glutaraldehyde. Furthermore, in the lower region of the stent component (in the direction of influence), an inner skirt 33, 34 is shown as a dashed line in its contours. Thus, in this exemplary embodiment of a self-expanding TAVI/TAVR valve, as a technical effect, the surgical sutures typically required for the arrangement/fixation of the valve and skirt components could be significantly reduced, since both the one-piece valve component 28 as well as the one-piece inner skirt component 33, 34 have been connected/joined by means of the crosslinking technique according to the invention, and in particular the stent in the valve area has been inserted into the tissue used 36 and firmly enclosed via the seamless connections of the inner and outer sides of the double-layer tissue used here. If necessary, surgical sutures may still be required at the commissures of a stent 31 to suspend the valve component, or individual surgical sutures in the lower area of the stent inlet to additionally fix the respective one-piece valve or inner skirt component here. Furthermore, the exemplary TAVI/TAVR valve shown in Fig. 6 can additionally comprise an outer skirt component, which is also seamlessly connected/joined to the outer side of the tissue valve component via a process according to the invention. The outer skirt may have one or more three-dimensional protrusions, protrusions, or protrusions around its circumference, all of which are suitable for sealing against paravalvular leakage.

Figure 7 shows an embodiment of an implant 51, here in form of tubular implant, (e.g. a covered stent or stent graft), wherein the implant 51 has a non-biodegradable tubular support structure 52 and a biological covering material 53 being a biodegradable or non-biodegradable tissue, preferably pericardial tissue, covering the inner and/or outer side of the tubular support structure 52. The covering material 53 may be a (decellularized) dried pericardial tissue and the support structure 52 may be a nitinol scaffold.

Fig. 8 shows another embodiment of an implant 51 having at least one non-biodegradable support structure 52 and a biological covering material 53 being a biodegradable or non- biodegradable tissue, preferably pericardial tissue, covering the biodegradable support structure 53 only on one side. The covering material 52 may be a (decellularized) dried tissue and the support structure 53 may be a nitinol scaffold. The support structure 53 may optionally be coated with a biodegradable polymer or co-polymer, for example PLLA or PLLA-PCL. For example, the support structure 53 has a planar form and/or can be porous support structure or a mesh. The implant 51 may be in the form of a vascular patch or tissue patch.

Fig. 9 shows a drug loaded implant 51 having at least one non-biodegradable support structure 53 and a biological covering material 52 being a biodegradable or non-biodegradable tissue, preferably pericardial tissue, covering the non-biodegradable support structure only on one side. The covering material may be a (decellularized) dried tissue and the support structure may be a nitinol scaffold. The covering material 52 comprises at least one drug 54. The drug may be a proliferative or cell growth-promoting drug or an anti-inflammatory drug. The support structure 53 may optionally be coated with a biodegradable polymer or co-polymer 55, for example PLLA or PLLA-PCL. The implant 51 may be in the form of a vascular patch or tissue patch.

Fig. 10 shows another embodiment of an implant 51 having at least one non-biodegradable support structure 53 and at least one biological covering material 52 being a biodegradable or non-biodegradable tissue, preferably pericardial tissue, covering the non-biodegradable support structure 53. The implant 51 may have one covering material 52 fully covering the non- biodegradable support structure 53 or several covering materials covering different parts of the support structure. The implant 51 may have more than one non-biodegradable support structure 53. The covering material 52 may be a (decellularized) dried tissue and the support structure 53 may be made from nitinol. The support structure 53 may optionally be coated with a biodegradable polymer or co-polymer, for example PLLA or PLLA-PCL. The support structure 53 may have a planar form and/or may be a porous support structure or a mesh.

Fig. 11 shows another embodiment of a method for making an implant 51 having a non- biodegradable support structure 53 and a biological covering material 52 being a biodegradable or non-biodegradable tissue, preferably pericardial tissue, covering at least one side of the support structure, preferably fully covering the support structure. A piece of the biological covering material 52, preferably a dried (decellularized) pericardial tissue, is folded and the support structure 53 is placed between the folded covering material 52. The support structure 53 is at least partially or fully covered with the covering material 52. The covering material 52 has regions not being in contact with the supports structure 53 but with itself. These regions can be affixed to each other, for example by suturing, gluing or by chemical cross-linking.

Fig. 12 shows another embodiment of a method for making an implant 51 having a biodegradable support structure 53 being fully covered by a biological covering material being a biodegradable or non-biodegradable tissue, preferably pericardial tissue. The covering material 52 is in form of a pocket 56 and the support structure 53 is inserted in the pocket formed by the covering material. The support structure 53 is at least partially or fully covered with the covering material 52. The covering material 52 has regions not being in contact with the supports structure 63 but with itself. These regions can be affixed to each other, for example by suturing, gluing or by chemical cross-linking.

Fig. 13 shows a device for an alternative manufacturing variant of a 3D outer skirt using two rigid mold bodies. The device for imprinting a 3D shape to the biological tissue has a top-plate 43 with holes 45 enabling a solvent to pass through, a shaping mold holder 41 having a 3D shaping mold 33. The shaping mold holder 41 and the 3D shaping mold 33 can be one or more pieces. The top-plate 43 can be affixed on top of the 3D-shaping mold 33 and/or the shaping mold holder 41 by fixation means 40, 44. Between the top plate 43 and the 3D-shaping mold 33 and/or the shaping-mold-holder 41 a sponge or solid foam 42 (e.g. made from polyurethane) can be arranged. The sponge may have a compression hardness of 60 kPa.

Fig. 14 shows in schematic cross-section the structure/arrangement of various material layers in a device/crosslinking unit suitable for a basic embodiment of the methods of the invention with pressure loading (the arrows represent the pressure loading schematically). Essential to these methods is a further modification of the crosslinking step. As can be seen in Fig. 14, the crosslinking of the exemplary pericardial tissue 380 takes place while it is arranged/placed in a device/crosslinking unit consisting of two rigid but optionally perforated counterforms 310, 311, a polyurethane foam (e.g. 10 - 30 mm in height) as an additional pressure compensation layer 312, and two permeable material layers of e.g. technical fabric 77a, 77b (with drainage function; e.g. 50 pm polyester with 40 pm meshes). The device/crosslinking unit assembled in this way is placed in a suitable container with, for example, 0.5% glutaraldehyde solution for chemical crosslinking so that the mold is completely covered by the crosslinking solution.

According to the first aspect of the invention, the technical fabric as a permeable material layer has essentially two functions: (i) Draining the water from the tissue when the external pressure is applied; (ii) Improving the accessibility of a crosslinking solution to the tissue during the pressed crosslinking. So not only a drainage function, because of the fiber structure, the technical fabric, such as mesh, additionally "conducts" liquid from one position to another.

The use of the permeable material layers 77a, 77b, e.g. made of technical fabric, enables through its permeable properties on the one hand that water present in the pericardial tissue can be removed with comparatively low pressure (in the sense of drainage), and on the other hand ensures sufficient accessibility of the crosslinking solution to the pericardial tissue 380. This can optionally be promoted by the fact that the rigid counterforms 310, 311 are either both or only one of them, preferably the upper one, additionally perforated and also facilitate access to the pericardial tissue 380.

By compressing the e.g. polyurethane foam as a pressure compensation layer 312 under an applied pressure load between the rigid counterforms 310, 311, a desired and continuously adjustable pressure is exerted on the pericardial tissue 380. The water present in the pericardial tissue escapes via the layers of the technical fabric as permeable material layers 77a, 77b already during the assembly of the previously described device/crosslinking unit. Since the entire device/crosslinking unit is placed in a container with, for example, 0.5% glutaraldehyde solution directly after assembly, by means of which, among other things, interfibrillar crosslinks are formed, the compacted state of the pericardial tissue (8) is permanently maintained. For example, according to the first aspect of the invention, ultra-compact and thus very thin tissue can be provided by applying an appropriately high pressure to the tissue to be treated via the aforementioned device/crosslinking unit; for example, pericardium with an initial thickness of 200 pm, which can be compacted to a final thickness of up to 20 pm.

The use of the pressure compensation layer 312, e.g. polyurethane foam or a silicone mat, serves to transfer the force of the applied pressure of the rigid counterforms 310, 311 and ensures compensation of the natural inhomogeneity of the tissue 38, thus avoiding local stress peaks. The resulting tissue thus exhibits improved thickness homogeneity. Furthermore, in addition, such a pressure compensation layer promotes the wetting of the pericardial tissue 380 with the crosslinker solution, thus ensuring a high crosslinking quality. By continuously reducing the plate spacing of the counterforms 310, 311 and the associated compression of the foam 312, the final thickness of the pericardial tissue 380 can be specifically adjusted to the requirements of a subsequent application, in particular for a medical application as an implant

Fig. 15A-H schematically show the individual method steps for setting up the device/crosslinking unit described in Fig. 14 for producing a planar, e.g. ultra-compact pericardial tissue. Fig. 15A shows a lower rigid counterform 411with several holes 413 as an initial stage. Fig. 15B shows the lower counterform 411, whereby all holes 413 are equipped with a continuously adjustable connecting means, e.g. a screw 414, in such a way that the screws themselves embody quasi joining rails, by means of which the above counterform 410 can be connected to the lower counterform 410, e.g. with an accurate fit and a positive fit. In Fig. 15C, a first layer of permeable material 87b, e.g. of technical fabric, is placed in the center of the device without folds. In Fig. 15D, the pericardial tissue 480 is placed on top of the first layer of permeable material 87b, e.g. of technical tissue, without folds in the center. In Fig. 15E, a second layer of permeable material 87a, e.g. of technical fabric, is placed without folds centrally in the device over the pericardial tissue 480 and thus also over the first permeable material layer 87b. In Fig. 15F, a pressure compensation layer of polyurethane foam 412 is placed centrally in the device, and thus comes to rest above the second permeable material layer 87a, above the pericardial tissue 480, and above the first permeable material layer 87b. In Fig. 15G, the upper rigid counterform 410 with perforations 410a is precisely and positively joined over the holes also present in 410 and the screws 414. In Fig. 15H, the screws 414 are each connected with a nut 414a as a connecting means in such a way that the screw connection via the nuts 414a allows a pressure load to be set on all material layers and the tissue in the device and also to be released again. In other words, the distance between the rigid counterforms 410, 411 can be selectively and continuously adjusted via the connecting means 414, 414a to suit the situation, depending on the tissue to be treated and the crosslinking solution. With the structure of Fig. 15H, an exemplary device is completed in such a way that it can be positioned either substantially vertically or alternatively substantially horizontally in a container with suitable crosslinking agent, and can be completely covered by crosslinking agent.

Fig. 16 shows an example of a rigid molded body 66 for fabricating a TAVI/TAVR valve with a one-piece tissue component made from porcine pericardium in preparation for a granular crosslinking process disclosed herein. Surgical sutures, which usually represent a high cost factor in the manufacturing process and at the same time form mechanical weak points, can be efficiently reduced in the following manner without affecting the functionality of the prosthesis. For this purpose, a one-piece tissue component 69 with three imprinted leaflets 68 is placed centrally on the rigid molded body 66, which contains a negative of a TAVI/TAVR tissue component that serves as a support surface. The negative is characterized by three upper indentations in the form of three leaflets and a cylindrical lower part as inner skirt elements. According to the invention, when this molded body is covered with a suitable granulate, such as the glass beads described herein, and (substantially) completely covered and crosslinked by a suitable crosslinking solution, such as a 0.5-0.65% glutaraldehyde solution, a one-piece tissue component with an imprinted three-dimensional shape for a TAVI/TAVR valve is obtained. The molded body can have molded body holders 67a, 67b at its ends. Fig. 17 schematically shows the basic structure of a suitable device in the sense of the invention for the hydrostatic crosslinking disclosed herein with formation of a constant liquid column of crosslinking solution, e.g. glutaraldehyde solution. The exemplary device essentially comprises a hollow cylinder for generating the liquid column 60, 100 with holes 90 for a hose passage for crosslinking agent supply, a two-part sample chamber 70 with a tapered neck 110, and a collecting basin 80 comprising a type of window/opening for a hose passage for crosslinking agent discharge 120, as well as a stand 130 for safe vertical standing of the device.

Fig. 18 shows an exemplary finally assembled device 120 in the sense of the invention for the hydrostatic crosslinking disclosed herein, which is suitable for forming a constant liquid column of crosslinking solution, for example glutaraldehyde solution. Fig. 18 thus shows the device of Fig. 17 with connected feeding hoses of crosslinking agent 140 and connected outgoing hoses of crosslinking agent 130, which are all in turn connected to a pump unit 150, 160, 170, which comprises controllable electronics, in order, by means of an adjustable pumping capacity (e.g., pumping speed and liquid quantity), to achieve a constant liquid column of crosslinking solution, e.g., glutaraldehyde solution.

Fig. 19 shows the lower part of the two-part sample chamber 190, 220 with placement/arrangement of a prefabricated TAVI/TAVR valve 180 with a self-expanding stent scaffold 200 and sewn-in valve component comprising three tissue leaflets 210. This TAVI/TAVR valve is arranged to undergo a three-dimensional forming process under hydrostatic meshing in accordance with the invention.

Claims

1. Process for preservation and stabilization of tissue comprising crosslinkable groups, in particular a proportion of crosslinkable groups compared to non-crosslinkable groups of greater than 50%, in particular native biological tissue, for medical applications, in particular for use as a component of a medical implant, preferably a vascular implant, more preferably an artificial heart valve, a left atrial appendage closure device, a pacemaker, a leadless pacemaker or a covered stent, comprising at least the steps: decellularizing the tissue by using a surfactin or surfactin salt and deoxycholic acid or deoxycholic acid salt containing solution followed by a DNAse treatment and optionally an alpha-galactosidase treatment, at least a partial substitution of the tissue water of the tissue comprising crosslinkable groups, in particular a proportion of crosslinkable groups compared to non- crosslinkable groups of greater than 50%, in particular native biological tissue, by means of a hygroscopic exchange material, wherein the hygroscopic exchange material comprises glycerol and polyethylene glycol preferably in aqueous solution; optionally under a suitable mechanical agitation acting on the tissue, such as shaking, panning and/or stirring; followed by drying of the tissue by stepwise reduction of the relative humidity, to obtain a dried tissue comprising crosslinkable groups, in particular a proportion of crosslinkable groups compared to non-crosslinkable groups of greater than 50%.

2. The process according to claim 1, wherein the decellularized tissue is not subjected to a crosslinking using a crosslinking agent, preferably glutaraldehyde or glutaraldehyde solution, before the partial substitution of the tissue water.

3. The process according to claim 1, wherein the hygroscopic exchange material consists of at least two different solutions, wherein a first solution comprises glycerol and a second solution comprises polyethylene glycol, preferably polyethylene glycol with a molecular weight being between 150 g/mol and 600 g/mol, or the hygroscopic exchange material preferably consists of at least three different solutions, and wherein a first solution comprises glycerol and a second and a third solution comprises polyethylene glycol, preferably the second and third solution comprise polyethylene glycol with different molecular weights being between 150 g/mol and 600 g/mol. The process according to any one of the preceding claims, wherein the exposure to one or more of the solutions lasts up to 2 hours, preferably from 5 minutes to 2 hours. The process according to claim 4, wherein the exposure of one or more of the solutions is up to 45 minutes, preferably lasts from 5 minutes to 45 minutes. The process according to any one of the preceding claims, wherein the drying of the tissue takes place in a suitably controlled environment, such as in the air, with a constant low relative humidity, or takes place in the suitable climatic chamber or desiccator stepwise by reducing the relative humidity at a constant temperature. The process according to claim 6, wherein the drying of the tissue is carried out in a climatic chamber or a desiccator by stepwise reduction of the relative humidity from 95% to 10% or less over 12 hours, at 35°C to 60°C, preferably at 35°C to 40°C. The process according to any of the preceding claims, wherein the dried tissue is further sterilized, preferably using ethylene oxide. The process according to any one of the preceding claims, wherein the tissue comprising crosslinkable groups, in particular a proportion of crosslinkable groups compared to non- crosslinkable groups of greater than 50%, in particular the native biological tissue, is rinsed at least once with a suitable solution, in particular a salt solution and/or an alcohol solution, before and/or after the preservation and stabilization and/or the decellularization. The process according to any of the preceding claims, wherein glycerol is present as an aqueous solution, and is preferably an aqueous solution with 1% to 70% glycerol in water. The process according to any one of the preceding claims, wherein polyethylene glycol is present as two different solutions, and a first solution comprises an aqueous solution of polyethylene glycol having an average molecular weight between 150 g/mol and 300 g/mol; and a second solution is an aqueous solution of polyethylene glycol having an average molecular weight between 200 g/mol and 6000 g/mol. 195 Dried tissue comprising crosslinkable groups, in particular a proportion of crosslinkable groups compared to non-crosslinkable groups of greater than 50%, obtained by a process according to one of the preceding claims. Use of a hygroscopic exchange material for direct preservation and stabilization of a tissue comprising chemically and/or biochemically crosslinkable groups, in particular a native biological tissue, in combination with subsequent drying in a suitable environment with constant low relative humidity or under a controllable and reducible relative humidity, wherein the hygroscopic exchange material is PEG 200, preferably PEG 200 in aqueous solution; and PEG 400, preferably PEG 400 in aqueous solution. Use of the dried tissue according to claim 12 or obtained according to any one of the process according to claims 1 to 11 for medical applications, in particular as a component of a medical implant, preferably a vascular implant, more preferably an artificial heart valve, a venous valve, a covered stent, a vascular patch, a pacemaker pocket, an implantable leadless pacer sheath, a left atrial appendage closure device cover or a biocompatible and optionally biodegradable tissue patch, an endoprostheses for closing persistent foramen ovale, an endoprostheses for closing an atrial septal defect. Medical implant comprising the dried tissue according to claim 12 or obtained according to any one of the process according to claims 1 to 11, wherein the medical implant is a cardiovascular implant, a endovascular prostheses, an endoprostheses, a prosthetic heart valve, a TAVI/TAVR valve, an esophageal implant, a bile duct implant, a stent, a vascular stent, a drug eluting stent, a pulmonary valve stent, a bile duct stent, a peripheral stent, a mitral stent, a stent graft, a venous valve, a dental implant, a bone implant, a cochlear implant, an endoprostheses for closing persistent foramen ovale, an endoprostheses for closing an atrial septal defect, a left atrial appendage closure device, a pacemaker, a leadless pacemaker or a defibrillator.