DE102012201268B4 - Process for the production of hollow bodies from microbial cellulose - Google Patents

Abstract

A method for producing a preliminary product of a hollow body made of microbial cellulose, which comprises the following steps: a) bringing the surface of a template, which is a negative form of the cavity of the hollow body to be produced and the inner walls of the cavity, into contact with a mixture supply, which is a liquid Culture medium and a cellulose-forming microorganism comprises, b) interrupting the contact between the template and the mixture supply, a liquid film remaining on the surface of the template, which comprises the liquid culture medium and the microorganism, c) bringing the liquid film into contact with an oxygen-containing atmosphere and the formation of microbial cellulose in and / or on the liquid film, d) bringing the microbial cellulose obtained in step c) into contact with the mixture supply, e) interrupting the contact between the microbial cellulose and the mixture supply being on the surface of the microbial cellulose e a liquid film remains, which comprises the liquid culture medium and the microorganism, f) bringing the liquid film into contact with an oxygen-containing atmosphere and the formation of microbial cellulose in and / or on the liquid film, characterized in that the template at least during step c) and step f) is rotated around several spatial axes.

Description

The invention relates to a method for producing a hollow body made of microbial cellulose and a hollow body made of microbial cellulose which can be obtained by the method.

In order to replace blood vessels and other hollow organs in the human or animal body, prostheses made of synthetic polymers are used in clinical practice in addition to the body's own vessels. The preparation of the body's own vessels means an additional surgical procedure for the patient, which is also associated with considerable expenditure of time and money. In addition, many, especially older patients, do not have a sufficient amount of healthy vascular material suitable for revascularization. Commercial, synthetic prosthesis materials made of Dacron® and Teflon®, which are used in general vascular and thoracic surgery for the replacement of large-lumen vessels (vessel diameter> 6mm) such as the aorta or the iliac arteries (iliac arteries), are subject to insufficient mechanical due to their significant thrombogenicity Resilience, low elasticity / reduced compliance and insufficient resistance to infections with regard to their area of application and their duration of use, severe restrictions. For small-caliber vascular grafts (vascular diameter 3.0-6.0 mm) e.g. B. the coronary arteries as well as for the microvessel replacement (vessel diameter 1.0-3.0 mm) these materials can not be used so far.

• - Die Tunica intima besteht aus einer glatten, lumenseitigen Endothelschicht aus langgestreckten, parallel zur Blutstromrichtung angeordneten Zellen und einem daruntergelegenen Bindegewebe. Sie wird direkt über den Blutstrom mit Nährstoffen und Sauerstoff versorgt.
• - Die Tunica media ist die mittlere Wandschicht. Sie besteht aus dicht gefügten glatten Muskelzellen mit zirkulärem oder spiralförmigem Verlauf und elastischen und kollagenen Fasern.
• - Die Tunica externa (Adventicia) ist die äußere dünne Bindegewebsschicht, die das Gefäß in dem umgebenden Gewebe verankert. In dieser Schicht befinden sich kleinere Blutgefäße für die Ernährung der Gefäßwand (Ausnahme Intima) und Nervengeflechte.

All natural blood vessels are more or less elastic tubes with an endothelial lining. The structure of the vessel walls corresponds in their quantitative and qualitative nature to the different functions of the individual blood circulation sections. The walls of all natural arteries and veins are basically characterized by a 3-layer structure:

• - The tunica intima consists of a smooth, lumen-sided endothelial layer of elongated cells arranged parallel to the direction of the blood flow and a connective tissue underneath. It is supplied with nutrients and oxygen directly from the bloodstream.
• - The tunica media is the middle layer of the wall. It consists of tightly knit smooth muscle cells with a circular or spiral course and elastic and collagen fibers.
• - The tunica externa (adventicia) is the thin outer layer of connective tissue that anchors the vessel in the surrounding tissue. In this layer there are smaller blood vessels that nourish the vascular wall (with the exception of the intima) and nerve plexuses.

While the veins are characterized by only indistinctly differentiated layers, elastic layers exist between the intima and media (internal elastic membrane) and between media and adventitia (external elastic membrane) in the arteries. Since veins in the blood circulation are only exposed to low pressure loads, their vessel walls are thinner and muscular elements are reduced in favor of the connective tissue. The wall structure allows the veins to stretch strongly, venous valves prevent the blood from flowing back and ensure a blood flow directed towards the heart.

Arteries close to the heart have to withstand very high pressure loads. They are of the "elastic" type, i.e. their media consists of alternating layers of elastic membranes and smooth muscles. The arrangement of the elastic elements in crossing spiral systems enables the circular and longitudinal, rhythmic expansion of the vessel (elastic deformability). In the arteries distant from the heart, the structure of the vessel walls changes according to the different function. In the vessels of the “muscular” type, the elastic components decrease, the muscular ones increase until the elastic material is limited to the lamina elastica interna and externa.

The vessels are thus able to adapt to the prevailing blood pressure conditions (Roche Lexikon Medizin, Ed. Hoffmann-La Roche AG and Urban & Schwarzenberg, U & S Munich, Vienna, Baltimore, 1998, 4th edition; K. Sommer ( Ed.) Der Mensch-Anatomie, Physiologie, Ontogenie. Volk und Wissen, Berlin, 1983, 5th edition).

• - Es soll eine dem natürlichen Blutgefäß möglichst nahekommende Struktur (biomimetische Struktur) aufweisen.
• - Es soll biokompatibel sein, d.h. es darf keine Thrombogenität und Immunogenizität besitzen. Es sollte resistent gegenüber Infektionen sein und in den Körper integriert werden, so dass im Idealfall das Blutgefäßimplantat nicht mehr vom nativen Gefäß unterschieden werden kann.
• - Es soll über die biologische Funktionalität der nativen Blutgefäße verfügen und den Aufbau körpereigener Strukturen anregen (Bioaktivität). Die innere Oberfläche des Implantats soll so beschaffen sein, dass sie keine thrombogenen Eigenschaften aufweist, die Anlagerung von körpereigenen Endothelzellen und die Ausbildung einer glatten konfluierenden Endothelschicht im künstlichen Gefäß ermöglicht. Die Wandung des Implantats soll einen Stoffaustausch, vergleichbar mit den natürlichen Austauschprozessen, gestatten.
• - Es soll eine angemessene mechanische Stabilität aufweisen, um den statischen, dynamischen und punktuellen Belastungen (z.B. Blutdruck, Körperbewegungen, Kräfte an der Verbindungsstelle zu den natürlichen Gefäßen) standzuhalten. Darüber hinaus soll es in einem Hochdrucksystem dauerhaft funktionieren.
• - Es soll ausreichend elastisch, einfach sterilisierbar und chirurgisch sehr gut handhabbar sowie in den erforderlichen Abmessungen verfügbar und lagerfähig sein.

A blood vessel implant, also known as a "vascular graft", should therefore meet the following requirements:

• - It should have a structure (biomimetic structure) that comes as close as possible to the natural blood vessel.
• - It should be biocompatible, ie it must not have any thrombogenicity or immunogenicity. It should be resistant to infections and integrated into the body so that, ideally, the blood vessel implant can no longer be distinguished from the native vessel.
• - It should have the biological functionality of the native blood vessels and stimulate the development of the body's own structures (bioactivity). The inner surface of the implant should be designed in such a way that it does not have any thrombogenic properties, enables the body's own endothelial cells to accumulate and a smooth confluent endothelial layer to form in the artificial vessel. The wall of the implant should allow an exchange of substances, comparable to the natural exchange processes.
• - It should have adequate mechanical stability to withstand static, dynamic and punctual loads (e.g. blood pressure, body movements, forces at the connection point to the natural vessels). In addition, it should work permanently in a high pressure system.
• It should be sufficiently elastic, easy to sterilize and very easy to handle surgically, and it should be available and storable in the required dimensions.

Vascular implants of this type, which correspond to a biomimetic and bioactive implant in terms of structure, properties and functionality and whose dimensions (inner diameter of about 1–30 mm, length of 5–500 mm) can be varied with regard to the area of application, are not known to date.

Bacterial nanocellulose (BNC) - also called microbial cellulose and explained below in this description - offers ideal conditions for use as a vascular implant with the requirements described above due to its excellent material properties.

BNC differs significantly in its morphology from cellulose of vegetable origin. It consists of fibers with a diameter in the nanometer range (20-100nm), which are 100 times finer than conventional plant cellulose fibers (cellulose). The natural nanofiber network shows a framework structure comparable to human tissue (collagen). It contains up to 99% water and is able to enter into intensive interactions with the environment. BNC is mechanically stable even when it is wet. BNC is a highly pure polymer, free of accompanying vegetable components such as lignin, pectin and hemicelluloses. It is characterized by a high molecular weight (degree of polymerization of approx. 4,000-10,000) and high crystallinity (80-90%). BNC is not broken down in human or mammalian organisms, does not trigger any defense reactions in the body and is effectively colonized by the body's own cells (Klemm D, Kramer F, Moritz S, Lindström T, Ankerfors M, Gray D, Dorris A: Nanocelluloses: a new family of nature-based materials. Angew. Chem. Int. Ed. (2011) 50, 5438-5466) .

There are several known methods of forming bacterial nanocellulose (BNC), in particular as hollow bodies for surgical applications such as blood vessels or tissue implants. According to the shaping process described, different BNC hollow bodies with different dimensions, structure and surface quality as well as mechanical load capacity and strength are obtained.

• EP 396 344 A2 beschreibt die Herstellung eines mikrobiellen Cellulosehohlkörpers mittels zweier Glasröhren unterschiedlicher Durchmesser. Die Glasröhren werden ineinander gefügt, und im Raum zwischen den beiden Rohrwandungen wird die Kultivierung des Cellulose-bildenden Mikroorganismus statisch innerhalb von 30 Tagen durchgeführt. Dieses Verfahren erfolgt entsprechend der sogenannten horizontalen statischen Kultivierungsmethode. Es entsteht eine mikrobielle Cellulose mit hohlzylindrischer Gestalt. Das Beispiel zeigt allerdings, z.B. anhand von Thrombenbildung, dass die innere Oberfläche des nach diesem Verfahren mikrobiell hergestellten Hohlzylinders den Qualitätsansprüchen an ein Gefäßimplantat nicht umfassend genügt. Die übliche statische Kultivierung schließt das Abwärtsschieben des Celluloseproduktes entlang der Wandungen des verwendeten Kultivierungsgefäßes ein. Dieser Vorgang kann zur Bildung von Oberflächeninhomogenitäten wie z.B. Falten führen. Der Einsatz als Gefäßprothese ist daher eher bedenklich.

It is known that microbial cellulose can be shaped directly in the manufacturing process, in particular into a hollow body. The following methods of producing hollow cylindrical cellulose moldings are described:

• EP 396 344 A2 describes the production of a microbial cellulose hollow body using two glass tubes of different diameters. The glass tubes are joined together and the cellulose-forming microorganism is cultivated statically within 30 days in the space between the two tube walls. This method is carried out according to the so-called horizontal static cultivation method. A microbial cellulose with a hollow cylindrical shape is created. However, the example shows, for example on the basis of thrombus formation, that the inner surface of the hollow cylinder microbially produced by this process does not fully meet the quality requirements for a vascular implant. The usual static cultivation involves sliding the cellulose product down the walls of the cultivation vessel used. This process can lead to the formation of surface inhomogeneities such as wrinkles. Its use as a vascular prosthesis is therefore rather questionable.

WO 01/61026 A1 / US 2003/013163 A1 discloses a manufacturing method for shaped biomaterials, particularly for microsurgical applications as a replacement for blood vessels with a diameter of 1-3 mm and smaller. A cellulose hollow body is produced using two glass tubes of different diameters, which are immersed in the nutrient solution inoculated with the cellulose-forming bacteria, so that the nutrient solution is drawn into the space between the glass matrix by capillary force and the nutrient solution and air can circulate. A moist, aerobic environment for cellulose formation is guaranteed in the cultivation vessel during the entire cultivation process. After 7-14 On cultivation days, an anisotropic, layer-like BNC fleece forms in the cultivation vessel in accordance with the usual static cultivation. In the space between the two glass tubes, under the modified static cultivation conditions, a cellulose hollow cylinder from a microfibrillar / nanocellulose network is formed rotationally symmetrically around the glass core along the longitudinal axis of the hollow cylindrical interior of the glass matrix. The disadvantage of this method is that the hollow bodies produced in this way only reach a length of a few centimeters (up to 3 cm).

WO 2007/093445 A1 / US 2009/011161 A1 describes a process for producing a long hollow cellulose body by cultivating a cellulose-forming organism in the interior of a hollow molded body and growing the long hollow cellulose body in this interior. In the context of the invention, the rotational symmetry around the cylindrical axis of the hollow cylindrical interior is dispensed with, in that the cellulose hollow body is not created along the longitudinal axis of an interior but essentially perpendicular to it from one longitudinal side of the hollow molded body to the opposite longitudinal side. This means that the length of the cellulose hollow body is no longer limited by the layer thickness, which can be achieved by the static growth process and so any desired length is conceivable.

The disadvantage of this method is, on the one hand, the manufacturing expense. First, a usual static cultivation is used to build up a cellulose layer 7-10 Days carried out. Then the hollow molded body with the corresponding openings is placed on the growing cellulose layer supported by a carrier network, so that the bacteria penetrate the hollow body from the outside and the cellulose under modified static cultivation conditions is parallel but not rotationally symmetrical to the longitudinal axis of the molded body interior, i.e. from the bottom down above, in which the shaped body is formed within 2 to 3 weeks. During cultivation, care must be taken to ensure that used nutrient solution is replaced. The bacteria that migrate through a lower slit in the matrix have to adapt to the continuous change in the biosynthetic surfaces (change between enlargement and reduction) in the course of cultivation. In particular, the connection of the bacterial cellulose half-cylinders, which are built up separately on both sides of the matrix core, to form a homogeneous unit is problematic.

The structural features of the shaped body vary both along and perpendicular to the axis. Diffusion processes of the culture solution to the biosynthesis site through the primary cellulose layer and the secondary cellulose layer that builds up within the molded body lead to the formation of additional structural inhomogeneities. The isolated cellulose hollow bodies are therefore less dimensionally stable and insufficiently mechanically stable, which leads to major problems, e.g. pressure stability. The inner and outer boundaries of the cellulose hollow body are therefore also to be regarded as inhomogeneous and unstable, so that the inner and outer wall cannot counteract an applied pressure at all or cannot counteract it evenly. The lumen is widened, bumps and hollows appear, which should promote the formation of thrombi. In addition, the formation process described in the patent can only be made reproducible with great difficulty and can hardly be implemented for the production of large quantities.

There are also publications that deal with the production of BNC hollow bodies on gas-permeable surfaces.

After EP 186 495 A2 Formed microbial cellulose is built up on gas-permeable materials (e.g. PVC, cellulose, cellulose derivatives, polyethylene, silicone, PTFE) by having one side of the material in contact with an oxygen-containing gas and the other with the nutrient solution, so that the microbial cellulose is formed on this side and then isolated. If, for example, a suitable dialysis tube made of conventional cellulose is filled with nutrient medium and cellulose-forming microorganisms and the cultivation is carried out over a small amount of distilled water in closed glass vessels to ensure moist aerobic conditions, the dialysis tube becomes cylindrical within 2 days at 25 ° C shaped microbial cellulose. If, on the other hand, an air-filled dialysis tube is placed in a closed vessel filled with culture solution, the permeable material is encased with a thin cellulose layer after 3 days of cultivation.

The in EP 396 344 A2 The process described for the production of microbial cellulose hollow bodies also includes the cultivation of a cellulose-producing microorganism on the inner and / or outer surface of an oxygen-permeable hollow carrier made of cellophane, Teflon, silicone, ceramic or a non-woven or a woven material. A cellulose-producing microorganism and a culture medium are supplied to the inner and / or outer side of the hollow support. The cultivation takes place with the supply of an oxygen-containing gas (or liquid) likewise on said inner and / or outer side of the hollow carrier. A gelatinous cellulose with a layer thickness of 0.01 to 20 mm forms on the surface of the hollow support. Due to the interaction of the cellulose-producing microorganism, the cellulose produced and the hollow carrier, a composite of cellulose and hollow carrier is created within 1-2 months. If the cellulose is not bound to the carrier, it is removed after the cellulose has been synthesized. A hollow-shaped article composed entirely of cellulose can be obtained.

WO 2008/040729 A2 describes a method for the production of cellulose hollow bodies by cultivating cellulose-producing microorganisms on the outer, uniformly smooth surface of a highly oxygen-permeable, non-porous hollow carrier such as dimethyl silicone, vinylmethyl silicone, fluorosilicone, diphenyl silicone, nitrile silicone. The interior of the hollow support is supplied with an oxygen-containing gas, the oxygen content of which is higher than that of atmospheric oxygen (preferably 100% oxygen), so that the cellulose-producing microorganism is continuously supplied with oxygen to the extent necessary. The oxygen partial pressure is also varied. Cellulose hollow bodies of various dimensions and shapes as well as with branches can be built up, which are intended for use in human and animal surgery to replace or repair internal hollow organs such as the urethra, ureter, trachea, digestive tract, lymph vessels or blood vessels. The cellulose hollow bodies are built up from individual layers parallel to the vessel wall and have a dense inner and a porous outer surface. Burst pressures up to 880 mmHg are given in examples. It is stated that the strength of the built-up bacterial cellulose (BC) tubes can be controlled through the concentration of oxygen in the gas which is passed through the non-porous, oxygen-permeable hollow supports.

Bodin et al. (Bodin A, Bäckdahl H, Fink H, Gustafsson L, Risberg B, Gatenholm P: Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes. Biotech. Bioeng. (2007) 97, 425-434) concerns the same subject as WO 2008/040729 A2 which you can see when comparing the images of both publications. The potential vascular prostheses reached a burst value of 880 mmHg when produced in the presence of 100% oxygen. Within approx. 5-7 days of cultivation it is possible to produce BC tubes of variable lengths and diameters (eg 1.5-6.0 mm) as well as with branches. In addition to WO 2008/040729 A2 discloses Bodin et al. mechanical tests of the hollow bodies produced. A stress-strain test indicates that the hollow bodies are made up of layers that are not firmly connected to one another, recognizable by various peaks in the stress-strain diagram in 8th .

Backdahl et al. (Bäckdahl H, Risberg B, Gatenholm P: Observations on bacterial cellulose tube formation for application as vascular graft. Materials Science and Engineering (2011) 31, 14-21) concerns the same subject as WO 2008/040729 A2 what you can see in the Materials and Methods section. SEM examinations and examinations using confocal microscopy on uncleaned BC tubes show high bacterial concentrations close to the lumen.

The above-cited biosynthesis of bacterial cellulose on the surface of oxygen-permeable membranes is comparable to the static horizontal cultivation. In contrast to this, the biosynthesis does not take place directly at the nutrient medium-air interface but rather at the nutrient-oxygen-permeable hollow carrier interface. The BNC formation is no longer restricted to just one direction, but takes place in all spatial directions. The construction of BNC bodies with an almost unlimited hollow body length / dimension is thus possible, however, due to the diffusion-determined nutrient transport and the material-determined oxygen supply, limitations of the wall thickness are to be expected. In comparison to culture medium-air interface cultivation, Putra et al. a reduction of the cellulose layer thickness by two thirds (A Putra, A Kakugo, H Furukawa, JP Gong, Y Osada: Tubular bacterial cellulose gel with oriented fibrils on the curved surface. Polymer (2008) 49, 1885-1891).

Also known is the BNC production using reactors with rotating discs "rotating disc reactors" ( WO 9705271 A1 , Mormino R, Bungay H: Composites of bacterial cellulose and paper made with a rotating disk bioreactor. Appl. Microbiol. Biotechnol. (2003) 62, 503-506). The basic idea of this process is to attach growing cells to structural elements of a bioreactor to form a biofilm to build. By moving these structural elements through a stationary nutrient solution, cell growth continues to be promoted. One or more constantly rotating - for example perforated or roughened - disks are partially but permanently immersed in a culture solution containing cellulose-producing microorganisms. A strongly gelated and strongly hydrated cellulose forms on the surface of these discs, which is clearly different from microbial cellulose, which was produced under static conditions. This cellulose is characterized by a looser structure and a higher water absorption capacity as well as reduced mechanical parameters (modulus of elasticity, tensile strength). The external forces that occur due to the rotational movement during cultivation cause disturbances in the entire crystallization process and thus a looser and more disordered BC fiber structure. Replacing the disks with a horizontally arranged roller makes it possible to manufacture tubes of different diameters Krystynowicz A, Czaja W, Wiktorowska-Jezierska A, Goncalves-Miskiewicz M, Turkiewicz M, Bielecki S: Factors affecting the yield and properties of bacterial cellulose. Journal of Industrial Microbiology & Biotechnology (2002) 29, 189-195), however, it does not appear to be possible to use it as a vascular replacement due to the property profile. An improvement in the mechanical properties of the cellulose tubes manufactured under dynamic conditions could only be achieved by installing a reinforcement material (polyester hose) (Ciechanska D, Wietecha J, Kazmierczak D, Kazmierczak J: Biosynthesis of modified bacterial cellulose in a tubular form. Fibers and Textiles in Eastern Europe (2010) 18, 98-104) .

In WO 2000/023516 A1 describes a process for making an extruded cellulosic product (film, tube / tube, fiber) which includes making a cellulosic solution from non-bacterial and bacterial cellulose in an amine oxide solvent. The proportion of bacterial cellulose influences the mechanical properties of the end products. The above-mentioned products are formed using a purely technical process. The dissolution process required for this destroys the unique structure of the bacterial cellulose.

The CN 201 809 342 U relates to an apparatus for the dynamic production of a bacterial cellulosic material with a profiled cavity. The device comprises a thermometer opening, an acid-base liquor supply opening, a handle, a vent opening, a pH meter opening, a nutrient supply opening, a rotating shaft, a fermentation tank, a motor, a water bath device and a nozzle, one end of which is rotating Shaft is fixedly connected to an output shaft of the motor and the other end of the rotating shaft protrudes into the fermentation tank through the central position of an end face of the fermentation tank. The nozzle is attached to the rotating shaft in the fermentation tank. In the utility model, the die or die rotates on a turntable around the center of the rotating shaft so that the die coils bacterial cellulose in a fermentation liquid to form the hollow bacterial cellulosic material in a certain shape on the surface.

The invention is based on the object of specifying an improved method with which hollow bodies can be produced from microbial cellulose. The hollow bodies should, if possible, be producible in any shape. It was also an object to provide an improved hollow body made of microbial cellulose, in particular for medical applications.

One or more of these objects are achieved with a method and a hollow body as specified in the independent claims, or with advantageous embodiments of the invention as specified in the subclaims.

1. a) das in Kontakt bringen der Oberfläche eines Templates, das eine Negativform des Hohlraums des herzustellenden Hohlkörpers und der inneren Wände des Hohlraums ist, mit einem Gemischvorrat, der ein flüssiges Kulturmedium und einen Cellulose bildenden Mikroorganismus umfasst,
2. b) die Unterbrechung des Kontakts zwischen dem Template und dem Gemischvorrat, wobei auf der Oberfläche des Templates ein Flüssigkeitsfilm zurückbleibt, der das flüssige Kulturmedium und den Mikroorganismus umfasst,
3. c) das in Kontakt bringen des Flüssigkeitsfilms mit einer sauerstoffhaltigen Atmosphäre und die Bildung mikrobieller Cellulose in und/oder auf dem Flüssigkeitsfilm,
4. d) das in Kontakt bringen der in Schritt c) erhaltenen mikrobiellen Cellulose mit dem Gem ischvorrat,
5. e) die Unterbrechung des Kontakts zwischen der mikrobiellen Cellulose und dem Gemischvorrat wobei auf der Oberfläche der mikrobiellen Cellulose ein Flüssigkeitsfilm zurückbleibt, der das flüssige Kulturmedium und den Mikroorganismus umfasst,
6. f) das in Kontakt bringen des Flüssigkeitsfilms mit einer sauerstoffhaltigen Atmosphäre und die Bildung mikrobieller Cellulose in und/oder auf dem Flüssigkeitsfilm,

wobei das Template zumindest während Schritt c) und Schritt f) um mehrere Raumachsen rotiert wird.A method is specified for the production of a preliminary product of a hollow body made of microbial cellulose, which comprises the following steps:

1. a) bringing the surface of a template into contact, which is a negative form of the cavity of the hollow body to be produced and the inner walls of the cavity, with a mixture supply comprising a liquid culture medium and a cellulose-forming microorganism,
2. b) the interruption of the contact between the template and the mixture supply, a liquid film remaining on the surface of the template, which comprises the liquid culture medium and the microorganism,
3. c) bringing the liquid film into contact with an oxygen-containing atmosphere and the formation of microbial cellulose in and / or on the liquid film,
4. d) bringing the microbial cellulose obtained in step c) into contact with the mixture stock,
5. e) the interruption of the contact between the microbial cellulose and the mixture supply, whereby a liquid film remains on the surface of the microbial cellulose which comprises the liquid culture medium and the microorganism,
6. f) bringing the liquid film into contact with an oxygen-containing atmosphere and the formation of microbial cellulose in and / or on the liquid film,

wherein the template is rotated around several spatial axes at least during step c) and step f).

The sequence of steps d), e) and f) can optionally be repeated one or more times.

• g) die Trennung der mikrobiellen Cellulose von dem Template.

As a further optional step, the method has:

• g) the separation of the microbial cellulose from the template.

The term "microbial cellulose" describes a cellulose that is produced by a microorganism. Exemplary microorganisms are fungi, bacteria and algae. A number of microorganisms are able to produce cellulose. These include, but are not limited to, algae such as Valonia and Boergesenia, fungi such as Dictyostelium discoideum and bacteria such as Gluconacetobacter, Enterobacter, Agrobacterium, Sarcina, Pseudomonas, Rhizobium and Zoogloea. Examples of Gluconacetobacter species are Acetobacter xylinum, Acetobacter pasturianus, Acetobacter aceti, Acetobacter ransens. A particularly suitable microorganism is Gluconacetobacter, in particular Gluconacetobacter xylinus.

Microbial cellulose is conventionally produced by microorganisms at the interface between air and a nutrient medium containing D-glucose, for example, in the form of a biofilm (fleece). The bacteria expel the cellulose as fibrils. These accumulate to form fibers. The interweaving of the fibers creates a three-dimensional, highly water-containing nanofiber network, which consists of approx. 99% water and 1% cellulose (Jonas R, Farah LF: Production and application of microbial cellulose. Polym. Degrad. Stab (1998), 59 (1-3), 101-106; Hirai A, Horii F: Cellulose assemblies produced by Acetobacter xylinum. ICR Annual Report (1999) 6, 28-29; Klemm D, Heublein B, Fink HP, Bohn A: Cellulose: fascinating biopolymer as sustainable raw material. Angew. Chem. Int. Ed. (2005) 44, 3358-3393) .

The culture medium, also referred to as "nutrient solution" or "nutrient medium", contains the usual components for cultivating a cellulose-producing microorganism, such as glucose, peptone, yeast extract, sodium hydrogen phosphate and citric acid in aqueous solution (Hestrin-Schramm medium). An alternative acidic medium consists of an aqueous solution of glucose, peptone, yeast, acetic acid and ethanol.

The process is preferably carried out at a temperature of 20 to 40 ° C.

If it is produced by bacteria, microbial cellulose is also referred to using the terms “bacterial cellulose” and “bacterial nanocellulose” (BNC). The term “bacterial nanocellulose” (BNC) is derived from the fact that bacterially produced cellulose, as mentioned above, forms a nanofiber network.

1. I. Wird die Cellulose an der inneren Seite eines permeablen Hohlträgers synthetisiert, wird die innere Formkörperwandung ohne Begrenzung aber in ständigem Kontakt zur Kulturlösung „frei“ im Raum entwickelt und stellt die ursprünglich erste (älteste) und lockerere Cellulosegelschicht dar. Im Verlauf der Kultivierung steht der an der inneren Hohlträgeroberfläche gebildeten Celluloseschicht, die sich in die Nährlösung hineinbewegt, immer weniger Platz zur Verfügung. Die Ausbildung von Störstellen (Faltungen, Verdichtungen) innerhalb des Schichtsystems und am Lumen ist die Folge. Weitere wesentliche Nachteile dieser Methoden bestehen demnach darin, dass die auf diese Weise hergestellten Hohlzylinder keine hinreichend glatte, homogene innere Oberfläche aufweisen. Da neben der Ausbildung von gravierenden Oberflächeninhomogenitäten durch Falten- oder Muldenbildung auch die Gefahr der Ablösung von Teilen der Cellulose gegeben ist, bergen diese Hohlzylinder ein sehr großes Thromboserisiko. Die Qualität des inneren Lumens kann den Anforderungen an einen Blutgefäßersatz nicht entsprechen.
2. II. Wird die Cellulose an der äußeren Seite eines permeablen Hohlträgers synthetisiert, wird die jüngste Celluloseschicht immer zwischen bereits gebildeten Celluloseschichten und der äußeren Oberfläche des Hohlträgers gebildet. Daher entspricht das Lumen des BNC-Hohlkörpers, wenn der Hohlträgers entfernt wurde, der aktuell synthetisierten jüngsten, festeren Celluloseschicht, die eine hohe Bakterienzahl enthält (Bäckdahl H, Risberg B, Gatenholm P: Observations on bacterial cellulose tube formation for application as vascular graft. Materials Science and Engineering (2011) 31, 14-21 ). Im Verlauf der Kultivierung muss die an der äußeren Hohlträgeroberfläche gebildete Celluloseschicht, die sich in die Nährlösung hineinbewegt und damit nach außen ortsverlagert wird, eine immer größere Fläche abdecken. Morphologische Veränderungen können die Folge sein. Bodin et al. (Bodin A, Bäckdahl H, Fink H, Gustafsson L, Risberg B, Gatenholm P: Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes. Biotech Bioeng (2007) 97, 425-434) berichten von einem, die Gefäßwand aufbauendem Schichtensystem aus sehr vielen, nicht fest miteinander verbundenen Einzelschichten ähnlicher Morphologie, so dass die Gefahr des Ablösens bzw. Verschiebens einzelner Schichten gegeben ist.
3. III. Außerdem kann davon ausgegangen werden, dass die Oberflächentopgraphie (Rauhigkeit) des inneren Lumens sehr stark von der Oberflächenstruktur des eingesetzten gas-permeablen Materials (Poren, Spalten, Kanäle und andere Unregelmäßigkeiten) bestimmt wird. Poröses Material erlaubt nur die Passage von Mikrosauerstoffblasen, die die Sauerstoffversorgung der Mikroorganismen nicht gleichmäßig über die gesamte Wachstumsebene gewährleisten und somit die Cellulosebildung stören und zu Defekten/Inhomogenitäten in der gesamten aufgebauten hohlen Cellulose, insbesondere aber auch im Lumen der hohlen Cellulose führen können.
4. IV. Nicht-poröses Material soll die gleichmäßige Sauerstoffversorgung der Mikroorganismen gewährleisten und die Gasblasenbildung unterbinden. Als mögliche Folge kann eine zu starke Haftung der BNC am Trägermaterial angenommen werden. Die Isolierung des Cellulosetubes führt zu Verletzungen der inneren Oberfläche.
5. V. Durch die Erhöhung des angelegten partiellen Sauerstoffdrucks an der äußeren Wandung eines gaspermeablen, nicht-porösen Hohlträgers wie in WO 2008/040729 A2 beschrieben, wird ein Gasüberdruck an der inneren Wandung des gebildeten Cellulosehohlkörpers initiiert. Dieser Gasüberdruck wirkt zum einen der Diffusion der Kulturlösung durch die gebildete Cellulose entgegen, zum anderen können auch Ablöseprozesse der sich bildenden Cellulose vom Träger während der Kultivierung nicht ausgeschlossen werden. Das Einschließen von Gasblasen führt zu Fehlstellen/Inhomogenitäten innerhalb der Cellulose sowie an der inneren Oberfläche.
6. VI. Die hohe Konzentration an Cellulose-synthetisierenden Bakterien über die gesamte innere Oberfläche der Cellulosehohlkörper stellt hohe Anforderungen an eine sich an Produktion und Isolierung anschließende Reinigung, da bei Verwendung der so hergestellten Cellulosehohlkörper als Blutgefäßersatz diese innere Oberfläche der Kontaktfläche mit dem Blut entspricht. Trotz intensiver Reinigung können Reaktionen des Immunsystems auf Verunreinigungen nicht ausgeschlossen werden.
7. VII. Wird BNC an der Oberfläche von sauerstoffdurchlässigen Membranen kultiviert, ist von verminderter Biokompatibilität, -stabilität sowie fehlender Bioaktivität des Materials auszugehen. Außerdem ist das Kultivierungsverfahren an sich nur sehr eingeschränkt steuerbar. Lediglich die Sauerstoffzufuhr und der Durchmesser der Hohlkörper-Membranen bieten die Möglichkeit, auf die Morphologie des BNC-Hohlkörpers und damit auf mechanische Eigenschaften Einfluss zu nehmen.

The advantages of the method according to the invention over the biosynthesis of bacterial cellulose on the surface of an oxygen-permeable membrane according to the prior art include the following:

1. I. If the cellulose is synthesized on the inner side of a permeable hollow carrier, the inner wall of the molded body is “freely” developed in space without limitation but in constant contact with the culture solution and represents the originally first (oldest) and looser cellulose gel layer The cellulose layer formed on the inner surface of the hollow carrier and moving into the nutrient solution has less and less space available. The result is the formation of imperfections (folds, densities) within the layer system and on the lumen. Further significant disadvantages of these methods consist in the fact that the hollow cylinders produced in this way do not have a sufficiently smooth, homogeneous inner surface. Since, in addition to the formation of serious surface inhomogeneities due to the formation of folds or hollows, there is also the risk of parts of the cellulose becoming detached, these hollow cylinders involve a very high risk of thrombosis. The quality of the inner lumen cannot meet the requirements for a blood vessel replacement.
2. II. If the cellulose is synthesized on the outer side of a permeable hollow carrier, the youngest cellulose layer is always formed between already formed cellulose layers and the outer surface of the hollow carrier. Therefore, the lumen corresponds to the BNC hollow body when the hollow beam is removed the newest, firmer cellulose layer currently being synthesized, which contains a high number of bacteria (Bäckdahl H, Risberg B, Gatenholm P: Observations on bacterial cellulose tube formation for application as vascular graft. Materials Science and Engineering (2011) 31, 14-21 ). In the course of cultivation, the cellulose layer formed on the outer surface of the hollow support, which moves into the nutrient solution and is thus relocated to the outside, must cover an ever larger area. Morphological changes can be the result. Bodin et al. (Bodin A, Bäckdahl H, Fink H, Gustafsson L, Risberg B, Gatenholm P: Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes. Biotech Bioeng (2007) 97, 425-434) report of a layer system that builds up the vessel wall and consists of many individual layers of similar morphology that are not firmly connected to one another, so that there is a risk of individual layers becoming detached or displaced.
3. III. It can also be assumed that the surface topography (roughness) of the inner lumen is very much determined by the surface structure of the gas-permeable material used (pores, crevices, channels and other irregularities). Porous material only allows the passage of micro-oxygen bubbles, which do not ensure the oxygen supply of the microorganisms evenly over the entire growth plane and thus disrupt cellulose formation and can lead to defects / inhomogeneities in the entire hollow cellulose, but especially in the lumen of the hollow cellulose.
4. IV. Non-porous material should ensure the uniform supply of oxygen to the microorganisms and prevent the formation of gas bubbles. As a possible consequence, too strong an adhesion of the BNC to the carrier material can be assumed. The isolation of the cellulose tube leads to damage to the inner surface.
5. V. By increasing the applied partial oxygen pressure on the outer wall of a gas-permeable, non-porous hollow support as in WO 2008/040729 A2 described, a gas overpressure is initiated on the inner wall of the cellulose hollow body formed. On the one hand, this excess gas pressure counteracts the diffusion of the culture solution through the cellulose formed; on the other hand, processes of detachment of the cellulose formed from the carrier during cultivation cannot be excluded. The inclusion of gas bubbles leads to defects / inhomogeneities within the cellulose and on the inner surface.
6. VI. The high concentration of cellulose-synthesizing bacteria over the entire inner surface of the cellulose hollow body places high demands on subsequent cleaning in terms of production and isolation, since when the cellulose hollow body produced in this way is used as a blood vessel substitute, this inner surface corresponds to the contact surface with the blood. Despite intensive cleaning, reactions of the immune system to contamination cannot be ruled out.
7. VII. If BNC is cultivated on the surface of oxygen-permeable membranes, reduced biocompatibility and stability as well as a lack of bioactivity of the material can be assumed. In addition, the cultivation process itself can only be controlled to a very limited extent. Only the oxygen supply and the diameter of the hollow body membranes offer the possibility of influencing the morphology of the BNC hollow body and thus the mechanical properties.

In the method according to the invention, the cultivation does not take place purely statically, but in what is known as “moving-static” form, with the template or the mixture containing culture solution and microorganism, or both, being moved in a controlled manner so that the surface of the template is wetted. A permanent contact of the template with the mixture supply is excluded. The template and the mixture supply, which comprises the culture medium and the microorganism, are moved relative to one another and, in the process, are brought into contact at times, but not constantly.

Characteristics of the process are a template that is periodically but not permanently brought into contact with culture solution and microorganism, the formation of a film containing culture medium and microorganism on the template and the biosynthesis of cellulose on the template exclusively inside and / or on the film - outside of the mixture supply. The term “interruption of contact” means that the contact between template and mixture supply is interrupted in such a way that no part of the surface of the template has contact with the mixture supply during the interruption.

In the process, the inner contour of the hollow body is given by a suitably shaped template, on the surface of which there is a liquid film in which the biosynthesis of the cellulose takes place when the process is carried out. Thus, the cellulose produced directly on the template surface later forms the inner surface of the hollow body. The process can produce further cellulose on this cellulose produced on the template surface.

A supply of the microorganisms with nutrients etc. is ensured through periodic, but preferably short-term contact with the culture medium. According to the invention, the outer shaping of the hollow body takes place without contact, exclusively through the influence of gravity. After the wetting process, the wetted template is free in the surrounding oxygen-containing atmosphere and the cellulose formation process takes place in and / or on the film.

The process does not require an external molded body, as is the case, for example, with processes of static cultivation, where microbial cellulose is formed by cultivating a cellulose-forming microorganism in a space between two walls. The external shape of the hollow body is defined exclusively by the choice of cultivation conditions. The cultivation conditions include, for example, the direction of the action of gravity, the frequency and the distance between individual rotations, the time interval between wetting, the wetting time, the residence time, as will be explained below, the temperature and the cultivation time. During the process, there is an exchange of culture solution and oxygen between the layer of the hollow body that is forming and the cultivation medium, so that an optimal supply of nutrient medium, air and microorganisms is guaranteed.

The method avoids processes in which substances have to pass through an already formed cellulose layer, which can lead to disturbances in the structure of these layers.

According to the invention, there is brief wetting and thus the necessary supply of the culture solution to the site of biosynthesis. Thus, in contrast to the publications mentioned and discussed, the supply of the culture solution is no longer determined by diffusion. When wetting, all nutrients and bacteria necessary for biosynthesis are transferred.

In the case of BNC production using reactors with rotating disks, the diffusion barrier is indeed overcome, which is achieved by a constant rotating movement of the template in the culture solution. The permanent contact of the microorganisms with the culture solution leads to BNC products with structural disadvantages, as mentioned in the appraisal of the prior art. By the controlled supply of the culture solution, the cellulose formation rate is regulated according to the invention so that optimal compression and solidification processes are made possible.

Surprisingly, the method according to the invention offers optimal conditions for the accumulation of bacteria on a template and their uniform supply with nutrients and oxygen. At the same time, the BNC is loaded with freely moving bacterial cells in the culture solution.

The location of the biosynthesis and thus the location of the highest bacterial concentration are located in the outer boundary layer of the hollow body and thus not on the side of the cavity which, in an artificial blood vessel, forms the lumen in contact with the blood.

The method according to the invention can be carried out in the device described in the exemplary embodiments. It is preferably carried out in a previously sterilized device and with sterile culture medium. In a variant, the culture medium and the device can be sterilized separately from one another. In another variant, the culture medium and the device are sterilized together and then the microorganism is added.

The process enables the production of hollow cellulose bodies with various micro and nanostructures. It is possible to build up micro and nano structures in a targeted manner. Biomimetic hollow bodies can be produced. The term “biomimetic” means that a human or animal hollow organ, for example a blood vessel, is modeled structurally and / or functionally by the hollow body produced according to the invention, the structural / functional properties of the natural template not necessarily having to be met exactly. The term “biomimetic structure” particularly refers to a structure that comes as close as possible to the natural blood vessel.

BNC implants manufactured using this process are characterized by improved mechanical properties and bioactive surfaces.

A hollow body is understood to mean a body that has a wall that surrounds a cavity. The wall can have one or more openings through which the cavity is accessible.

In principle, the hollow body can be shaped as desired. The shape is adapted to the later application. For medical uses as an implant, the hollow body can, for example, have the shape of a blood vessel, an esophagus, part of the digestive tract, a windpipe, a urethra, a bile duct, a ureter, a lymphatic vessel or a cuff. These natural forms can be achieved exactly or approximately.

In one embodiment, the hollow body has the shape of a tube or a hollow cylinder. Furthermore, the hollow body can have one or more bends, for example a tube with one or more bends. Furthermore, the hollow body can have one or more branches or ramifications, for example the hollow body can have a Y-shape.

The length and inner diameter of the hollow body can be variably and variably combined with one another. Exemplary inner diameters are 1-30 mm, preferably 2 to 8 mm, and exemplary lengths are 5-500 mm, preferably 100-200 mm. The length / diameter ratio is preferably greater than 1. The inside diameter can also vary within a hollow body.

The hollow body, in particular its hollow space, can also have a differently shaped cross section, for example a square, rectangular, triangular or star-shaped cross section, instead of a round one.

As already mentioned, the template, or “template”, is the negative form of the cavity of the hollow body and the inner walls of the cavity. The term “negative form” describes the aid, the positive form is the desired result, in this case the hollow body / cavity / cavity wall. The template is shaped complementary to the shape of the desired cavity to be produced and is specified accordingly. Correspondingly, the shape of the template is also defined via the shape of the hollow bodies specified above. The template specifies the internal geometry of the hollow body. For example, the template is cylindrical, with a diameter of 1-30 mm, preferably 2-8 mm, and a length of 5-500 mm, preferably 100-200 mm. Like the hollow body or cavity, however, the template can have any cross-section, such as round, angular, in particular square, rectangular, triangular, or star-shaped or snowflake-shaped. Just like the hollow body, the template can have branches. In a further embodiment, the template has a 2-dimensional or 3-dimensional grid-like structure.

In one embodiment, the template has a surface which has structures in the millimeter, micrometer and / or nanometer range. The structures are, for example, elevations or depressions or both. The structures can have different geometries. Similar or different structures can be distributed regularly or irregularly over a template surface.

The template surface can be designed in such a way that the internal surface structure generated in the hollow body, i.e. the structure of the surface that adjoins the cavity, is adapted to the later function of the hollow body. If, for example, hollow bodies are to be synthesized as blood vessel replacements, the surface of the template can be structured in such a way that the inner surface of the synthesized hollow body later enables good endothelialization.

The material from which the surface of the template is made is in principle not restricted. In one embodiment, the template has a surface made of wood, metal such as aluminum, stainless steel or titanium, plastic, ceramic, synthetic polymers such as polypropylene, polyesters, polyamides or Teflon, paper, textile fabric or glass. The entire template can also consist of one of the substances mentioned. The template itself can be a hollow body or solid (solid body).

The surface of the template can be untreated or pretreated. For example, the pretreatment can be a change in the surface morphology, e.g. by etching, polishing or roughening. The surface can be coated or pretreated or coated with chemical compounds

The template is designed in such a way that, on the one hand, optimal conditions for binding and supplying the microorganism to its surface and adhesion of the cellulose are achieved. On the other hand, the template is chosen so that the product can be easily detached from the template in order to obtain the hollow body. The surface of the template is such that the surface quality of the surface of the hollow body which comes into contact with body fluid, in particular blood, during implantation is reproducible.

In a special embodiment, an arrangement of several templates is used in the method, also referred to as a template matrix. Templates with the same or different geometry, in particular different cross-sections, and / or made of the same or different material can be used. As a result, several identical or different hollow bodies can be obtained in the method according to the invention. An example of an arrangement made up of several templates is an arrangement made up of several cylindrical templates for producing several hollow cylindrical or tubular hollow bodies. Several templates of the same or different geometry can be fixed in a clamping device (template matrix) which is connected to a wetting device and / or moving device, as explained in the exemplary embodiments.

In the method, the template is periodically, preferably briefly, wetted with the mixture comprising the culture solution and the microorganism. A liquid film forms on the surface of the template. The shape of this liquid film is determined by the position of the template in space, since gravity acts on this film.

A liquid film is formed on the surface of the template, which includes the liquid culture medium and the microorganism. Microbial cellulose is formed in and / or on the liquid film. The liquid film is distributed on the template by rotating the template around several spatial axes, e.g. by means of the movement device as explained in the examples. A predetermined movement of the template achieves an even better distribution of the liquid on the surface of the template. The distribution of the liquid can be influenced by the type of predetermined rotational movement of the template, with the rotational movement also being able to be interrupted. The outer geometry of the hollow body is thus determined by a defined distribution of a liquid film and by a defined movement under the influence of gravity.

The template preferably has a geometry with a length-to-diameter ratio of greater than 1. For example, the template has a rod-shaped, conical or cylindrical geometry with a length-to-diameter ratio of greater than 1, for the production of a tubular or hollow-cylindrical hollow body, branches can be provided. With this geometry, the template has a longitudinal axis. The method is then carried out in such a way that the template is rotated around a plurality of spatial axes which are preferably transverse, more preferably perpendicular, to the longitudinal axis of the template.

As mentioned, a liquid film is formed on the surface of the template. The liquid film is formed in that the template and the mixture, which comprises the culture medium and the microorganism, are moved relative to one another and thereby brought into contact. In principle, the relative movement can be such that the template, or the mixture comprising the culture medium and microorganism, or the template and the mixture are moved.

In one variant, the surface of a template is brought into contact with the mixture in such a way that the template is immersed in and out of the mixture which comprises the culture medium and the microorganism. A movement of the template around several spatial axes can be superimposed on an inward and outward movement of the template.

In one variant, the surface of a template is brought into contact with the mixture in such a way that the mixture from the first mixture supply is poured over the template. After the mixture has been poured over the template, the mixture that does not remain on the surface of the template is preferably collected and reused for further pouring.

In one variant, the surface of a template is brought into contact with the mixture in such a way that the mixture of culture medium and microorganism is sprayed onto the template.

The oxygen-containing atmosphere is preferably air or pure oxygen or an oxygen-containing gas mixture. Microbial cellulose is formed in and / or on the liquid film when it comes into contact with oxygen.

The contact between the template and the mixture supply is interrupted in the process and the synthesis of the cellulose takes place only in or on the liquid film which was separated from the mixture supply. As a result, any diffusion processes of culture solution and oxygen play a subordinate or no role at all. Surprisingly, it has been found that no cellulose formation takes place within or on the liquid surface of the mixture reservoir.

At the end of the process, the template and cellulose can be separated from one another in order to obtain the hollow body without a core. Therefore, as an optional step, the template is separated from the cellulose formed so that a hollow body made of microbial cellulose without a template is obtained. In this case, the method according to the invention is referred to as a “method for producing a hollow body from microbial cellulose”. After separation from the template, the BNC hollow body can be cleaned and sterilized.

Alternatively, the microbial cellulose can remain on the template without the described optional separation step being carried out. In this case, the method according to the invention is referred to as a “method for producing a preliminary product of a hollow body from microbial cellulose”. The preliminary product describes the template with microbial cellulose on it. The microbial cellulose can be cleaned and sterilized on the template without first being removed from the template. Furthermore, the microbial cellulose can be stored together with the template without it being removed from the template. The cleaning is preferably carried out with water, an aqueous acidic or alkaline solution, or an organic solvent, or a combination thereof. For the final use, the microbial cellulose can then be separated from the template in order to obtain a hollow body. The separation takes place, for example, in that the cellulose formed is removed from the template or the template is removed in another way. For example, the cellulose is stripped from a cylindrical template and a hollow cylinder without a template core is obtained. When the cellulose is cleaned on the template, the detachment of the cellulose from the template can be promoted by a cleaning liquid. Depending on the material used, the template can be reused after carefully cleaning its surface. But it is also possible to destroy the template for the purpose of separating it from the cellulose.

The template is rotated about several spatial axes at least during step c) and / or step f), or one or more of steps f) if step f) is carried out several times. With this measure, the shape and distribution of the liquid film and the shape of the product being formed can be influenced. In other words, the template is covered with a defined liquid film, which in turn leads to a defined shape of the product being formed. The rotation can also take place during steps a) and b) and / or during steps d) and e). If, for example, the template is moved relative to the mixture supply for wetting and is brought into contact with it, then a relative movement about several spatial axes superimposed on the movement of the template can also take place.

This sequence of steps d) -f) can be repeated one or more times until a desired amount of cellulose has been produced on the surface of the template and the cellulose has reached a desired total layer thickness. The so-called total layer can be composed of several individual layers or phases. In this process variant, further microbial cellulose is synthesized on cellulose that has already formed.

The rotation described above can take place in each of the steps f) (first step f and repetitions thereof if the sequence of steps d) -f) is repeated). In other words, the rotation about several spatial axes does not necessarily take place after each wetting process. In one variant of the method, the sequence of steps d) -f) is run through 1-40 times, preferably 1-30 times, without a rotational movement of the template for distributing the liquid film taking place and in at least one subsequent step f) when the sequence of steps d ) -f) is run through again, there is a rotation around several spatial axes.

The times of bringing the surface of a template into contact (step a) with a mixture supply and of bringing the microbial cellulose produced in step c) into contact with the mixture supply (step d) are referred to as “wetting times”. The times when the liquid film is brought into contact with an oxygen-containing atmosphere (steps c and f) are referred to as “residence times”. Wetting times and dwell times can be controlled independently of each other. In one embodiment, the residence time is 1-60 minutes, preferably 5-40 minutes.

The total cultivation time is preferably 1-7 days. The total cultivation time corresponds to the total process time, within which all steps of the process, such as turning, wetting and other steps, take place. The duration of the process determines the thickness of the total cellulose layer formed on the template, which corresponds to the wall thickness of the insulated hollow body and which, as mentioned above, can be composed of several individual layers.

The hollow bodies produced with the method can be cleaned in order to remove residues and components of the culture medium and microorganisms. The cleaning is preferably carried out with water, an aqueous acidic or alkaline solution, or an organic solvent, or a combination thereof.

The hollow bodies obtained with the method can be used as moist implants after a cleaning process and sterilization without drying.

On the other hand, the hollow bodies can be gently dried for storage, for example by freeze drying or critical point drying, the structure and the resellability of nanocellulose being retained. Before a surgical application, the implant can be swollen again, for example in physiological saline solution or the patient's own or allogeneic (pharma-grade) serum.

In a further aspect, the invention relates to a hollow body made of microbial cellulose, which can be obtained by the method described above.

As already described above, the method differs fundamentally from the methods known from the prior art. Cellulose is built up according to a completely different mechanism. During the biosynthesis of bacterial cellulose on the surface of an oxygen-permeable membrane WO 2008/040729 A2 the youngest layer is formed at the interface between the nutrient medium and the oxygen-permeable hollow carrier during the biosynthesis process. The youngest layer is thus formed on the side of the later cavity of the hollow body and older layers migrate outwards. Oxygen is introduced from the side of the lumen and nutrients must be supplied from the outside and penetrate cellulose layers that have already formed. With this method, no external supply of microorganisms is possible, as they do not fit through the pores. Cellulose is always formed by the same microorganisms that are already localized at the site of cellulose formation. There could only be an increase through cell division.

In contrast to this, the biosynthesis in the present process takes place on the outside of the hollow body, at the interface between the mixture film and the surrounding oxygen-containing atmosphere. As a result, the recently formed cellulose is localized on the outside and cellulose already formed remains stationary during the synthesis. The oxygen entry takes place in contrast to WO 2008/040729 A2 from the outside, and the microorganisms and nutrients are also supplied to the outside, so cellulose layers that have already formed do not have to penetrate. In the present method, a supply of microorganisms from the outside is in contrast to WO 2008/040729 A2 possible.

A product with a new structure is obtained through these different educational processes.

In particular, the hollow body has a wall with an inner surface and an outer surface, the inner surface and the outer surface having an identical or similar coverage by fibers of microbial cellulose per unit area. As mentioned, BNC fibers form more or less dense or more or less porous networks. Using image evaluation methods, it is possible to demarcate surface areas in which there are fibers (e.g. lighter in an SEM image) from surface areas in which there are no fibers (gaps, in an SEM image, for example, darker) and the areas in relation to the observed Total area to set. The coverage per unit area is given as $$\begin{array}{l}\text{cover}=\\ \text{Part of the area under consideration},\text{covered by fibers / total area of}\\ \text{observed area section}\end{array}$$

The coverage can be given as a percentage. The term “similar” in this context means that the coverage on the inner surface and the outer surface differ relative to one another by a maximum of 20%, preferably a maximum of 10%, most preferably a maximum of 5%. The percentage of the difference between the values is determined as follows: Difference (%) = (larger number - smaller number) / smaller number * 100.

A similar coverage as defined above means a similar porosity of the inner and outer surfaces, the porosity with respect to a surface being defined two-dimensionally as $$\begin{array}{l}\text{porosity}\left(\text{two-dimensional}\right)=\\ \text{Part of the area under consideration},\text{that is not covered by fibers /}\\ \text{Total area of the area under consideration}\end{array}$$

The area not covered by fibers can be referred to as "open-pore area" or "pore area".

oder ausgedrückt in Prozentzahlen: Porosität (%) + Bedeckung (%) = 100 %,The following applies to the relationship between coverage and two-dimensional porosity: $$\text{porosity}+\text{cover}=\mathrm{1,}$$

or expressed in percentages: porosity (%) + coverage (%) = 100%,

In terms of coverage and porosity, BNC fibers can be taken into account that lie somewhat outside of an inner / outer surface assumed to be two-dimensional. An electron microscope image gives a two-dimensional representation of the outer or inner surface of the wall of the hollow body. However, the BNC fibers shown do not always lie in one plane, since the surfaces can have a certain roughness and / or BNC fibers may also be shown that are behind the surface when viewed from the observer. It has been shown that, in the case of the hollow body according to the invention, the outer surface of the wall can be rougher than the inner surface. When determining the coverage and porosity, all BNC fibers that are visible on an SEM image (10,000x magnification) are preferably taken into account and they are treated as if they were in one plane, since the coverage / porosity is based on a unit area.

Thus, the hollow body has a wall with an inner surface and an outer surface, wherein the inner surface and the outer surface have a similar porosity as defined above. With reference to the porosity, the term “similar” means that the two-dimensional porosity of the inner surface and the outer surface differ relative to one another by a maximum of 20%, preferably a maximum of 10%, most preferably a maximum of 5%. The percentage of the difference between the values is determined as follows: Difference (%) = (larger number - smaller number) / smaller number * 100.

A similar porosity of the inner and outer surface of its wall makes the hollow body of the present invention particularly suitable for use as tunica media, in particular of arteries near the heart, due to the uniform fiber structure. The invention provides a hollow body that is limited by layers of high and functionally relevant surface quality. The surfaces are preferably free from imperfections and inhomogeneities.

In the procedure after WO 2008/040729 A2 one obtains exclusively hollow bodies with a smooth inner surface and a relatively very porous outer surface, as in the 4A and 4B shown. The porosity of the inner and outer surfaces, or their coverings with fibers, are assessed as not being similar. This may be due to the fact that the procedure after WO 2008/040729 A2 the youngest layer is formed on the side of the later cavity of the hollow body and older layers migrate outwards and are stretched due to the increasing radius. The wall structure generally consists of many layers that are clearly distinguishable / delimited from one another using a scanning electron microscope ( 5 the WO 2008/040729 A2 ).

In particular, the hollow body according to the invention has a wall with an inner surface and an outer surface, the wall comprising several layers of microbial cellulose which run parallel to the inner and outer surface of the wall. These layers are also referred to below as “phases”. The layers correspond to the above-mentioned “individual layers or phases” of the wall of the hollow body. The phases of the wall of the hollow body according to the invention are in contrast to those in WO 2008/040729 A2 The layers disclosed represent uniform structures over larger areas than in laser electron microscopy, which is why it is differentiated from WO 2008/040729 A2 the term "phase" is used here

These phases do not have to be obtained by a process cycle of wetting / film formation and subsequent cellulose formation in or on the film. A phase can be formed by several such process cycles. The phases are by means of scanning electron microscopy, for example at 24 times Magnification, visually distinguishable from each other. The phases are made up of a network of fibers from bacterial nanonocellulose, whereby the fiber structures of the phases can be the same or different when the phases are compared.

The phases are preferably homogeneous in their density over the entire phase thickness. In other words, they do not have a density gradient. Furthermore, the phases are preferably also free from defects (see also 4th to 6th , 2 .000x magnification).

The invention also relates to a hollow body, the wall of which is made up of layers, also referred to as phases, as described above, one of the layers having the inner surface, ie the surface on the cavity side, of the wall and a further layer having the outer surface of the wall and where these two layers have an identical or similar porosity. The layer / phase comprising the inner surface of the wall is also referred to as the “lumen-side layer / phase” or “cavity-side layer / phase”. The layer / phase that has the outer surface of the wall is also referred to as the "outside layer / phase".

With reference to the three-dimensional layers / phases, the porosity is spatially defined as: $$\text{porosity}\left(\text{spatial},\text{three dimensional}\right)=\text{Void volume / total volume}$$

For the total volume, the volume of the entire layer / phase can be used. However, it is also possible to consider only part of the volume of the layer / phase and to consider this part of the volume as the “total volume” for the purpose of measuring the porosity. With reference to the spatial porosity, the term “similar” means that the porosity of the lumen-side phase and the porosity of the outer-side phase differ relative to one another by a maximum of 20%, preferably a maximum of 10%, most preferably a maximum of 5% Percentage of the difference between the values is determined as follows: Difference (%) = (larger number - smaller number) / smaller number * 100.

An identical or similar three-dimensional / spatial porosity as defined above means that the lumen-side layer / phase and the outer layer / phase have an identical or similar density. Furthermore, an identical or similar three-dimensional / spatial porosity as defined above means that the lumen-side layer / phase and the outer layer / phase have an identical or similar fiber density. The density is preferably given as mass / volume and the fiber density is given as the number of fibers / volume. The volume of the entire layer / phase can be used for the volume. However, it is also possible and preferred to consider only part of the volume of the layer / phase in order to determine the density or the fiber density. With reference to the density and fiber density, the term "similar" means that the density / fiber density of the lumen-side phase and the density / fiber density of the outer phase "differ relative to one another by a maximum of 20%, preferably a maximum of 10%, most preferably maximum 5%. The percentage of the difference between the values is determined as follows: $$\begin{array}{l}\text{difference}\left(\text{\%}\right)=\\ \left(\text{number greater value}-\text{lower number value}\right)\text{/ lower number}*100.\end{array}$$

One or more further phases are preferably arranged between the phase on the lumen side and the phase on the outside. These one or more further phases particularly preferably have an identical or similar porosity, density and fiber density to the lumen-side phase and the outside phase.

The phases are preferably connected to one another so firmly that they do not delaminate from one another in a tensile test when subjected to mechanical stress. This means in particular that no delamination in the form of various peaks is visible in the tensile strain diagram when a tensile test is carried out using the method as described in Bodin A et al., Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes. Biotech. Bioeng. (2007) 97, 425-434 on page 427 under the heading "Tensile Measurement". A ring made of BNC with an inside diameter of 4 mm and a width of 5 mm is cut and stretched radially at a speed of 0.25 mm / s.

In particular, the hollow body is a hollow cylinder with a central axis which runs through the cavity in the center and along the extent of the cylinder. In particular, the hollow body is characterized by at least two BNC phases which are arranged rotationally symmetrically about the main axis, run parallel to one another and are connected to one another. In this way, a biomimetic structure of the wall of the hollow body can be produced. In contrast to WO 2008/040729 A2 the phases are firmly connected to each other. Furthermore, the phases preferably have a homogeneous structure, without defects and structural gradients.

Surprisingly, it has been found that this structure gives the hollow body dimensional stability and uniformly high mechanical tear and compressive strength as well as sufficient elasticity regardless of the inner diameter of the hollow body over the entire length. The phases are preferably characterized by a uniform (isotropic), well-branched fiber network. The number and strength of the phases can be adjusted in a controlled manner. They are arranged in such a way that they correspond to the structure that comes close to a natural vessel, especially the media (biomimetic structure) and stimulate the development of the body's own structures (adventitia and intima) as well as the exchange of substances comparable to natural exchange processes (bioactive material). The structure as a layered phase structure presumably results in mechanical stability, biocompatibility through endothelizability of the inner surface, and an integration into the body's own tissue structure, without being bound by this explanation.

The hollow body preferably has one or more openings through which the cavity of the hollow body is accessible. The openings can be, for example, the inflow and outflow opening of a part of the blood vessel.

Geometries of the hollow body have already been explained above using the manufacturing method. In a special embodiment, the hollow body is selected from a tube or a hollow cylinder, which can have one or more branches.

The hollow bodies according to the invention are characterized by improved mechanical properties, specifically adjustable microstructures (biomimetic structure) and bioactive surfaces. Further preferred mechanical properties, which can be combined as desired with the mechanical, structural and geometric properties already described above, are described below.

The invention also relates to a hollow body with a seam tear strength in the range of 5-15 N, preferably 8-10 N, in particular a hollow cylinder or a tube with such a seam tear strength. The seam pull-out strength is determined according to the method given in the examples.

A hollow body with a bursting pressure of at least 400 mm Hg, preferably at least 600 mm Hg and most preferably at least 800 mm Hg, is also specified. The bursting pressure is determined by the method which is given in the examples.

In one aspect, the invention also relates to an artificial vessel, in particular for use as an implant in the human or animal body, having a hollow body as described above. The hollow body can be present as a composite with other substances, for example with polymers other than BNC such as cellulose derivatives, alginate or proteins, for example for the purpose of improving the mechanical properties. The hollow body can additionally contain growth and / or recruitment factors and / or other biologically active substances, for example for the purpose of improving their bioactivity through the accumulation and immigration of the body's own cells.

If the hollow body is to be used as an artificial blood vessel, then it can be inserted directly into the body. The hollow body can also be subjected to a pretreatment, for example endothelial cells can be adhered to the surface of the hollow body.

In another aspect, the invention relates to the use of a hollow body or an artificial vessel, as described above, as a medical implant. The hollow bodies and artificial vessels can be used in medical applications as inner hollow structures and vessels, such as blood vessels, esophagus, digestive tract, trachea, urethra, bile duct, ureter, lymphatic vessels or as a cuff to enclose the body's own structures such as hollow organs or nerve fibers, or as Interponate can be used, whereby the hollow bodies can be used directly or after adaptation to the organ specifics. Further uses are the use as medical exercise material, in particular for the realistic training of surgical techniques, in cardiovascular medicine and visceral surgery, for which purpose the hollow body can also be processed mechanically.

• 1 eine erste Vorrichtung zur Durchführung des erfindungsgemäßen Verfahrens mit dynamischer Anordnung des Templates und eine statische Anordnung des Gemisches, in einer Schnittansicht
• 2 eine alternative Vorrichtung zur Durchführung des erfindungsgemäßen Verfahrens mit dynamischer Anordnung des Templates und dynamischer Anordnung des Gemisches, in einer Schnittansicht
• 3 rasterelektronenmikroskopische (REM) Aufnahme eines erfindungsgemäßen BNC-Hohlzylinder-Querschnitts mit 3-Phasenaufbau
• 4 REM-Aufnahme des BNC-Netzwerks der Phase a
• 5 REM-Aufnahme des BNC-Netzwerks der Phase b
• 6 REM-Aufnahme des BNC-Netzwerks der Phase c

The invention is explained in more detail below using exemplary embodiments. Show it

• 1 a first device for performing the method according to the invention with a dynamic arrangement of the template and a static arrangement of the mixture, in a sectional view
• 2 an alternative device for carrying out the method according to the invention with dynamic arrangement of the template and dynamic arrangement of the mixture, in a sectional view
• 3 Scanning electron microscope (SEM) image of a BNC hollow cylinder cross-section according to the invention with a 3-phase structure
• 4th SEM image of the BNC network of phase a
• 5 SEM image of the BNC network of phase b
• 6th SEM image of the BNC network of phase c

Example 1: Devices for carrying out the process

The device 1 the 1 is designed as a bioreactor with a housing 15th , which from a vessel 9 and a cover 8th consists and the interior 14th encloses. The container 9 is with the cover 8th lockable and shown here in the closed state. The container 9 and the cover 8th can be made of stainless steel. There is a separate reservoir in the bioreactor 2 for the mixture 10 , consisting of a culture solution and a microorganism. The reservoir 2 and the mixture 10 can through the opening 17th of the vessel 9 be introduced when the cover 8th from the vessel 9 is removed.

Another lockable opening 19th is in the cover 8th provided and with a closure 20th locked. Alternatively, it is therefore also possible to use the reservoir 2 into the vessel 9 insert the cover 8th to close and the culture solution or the mixture through the opening 19th admit.

Multiple templates 3 , here cylinders made of metal or wood, are between two clamping devices 4th clamped and form an arrangement of several templates (template matrix).

The templates 3 are about a clutch 6th , here rod-shaped, with a motor 7th connected. The motor 7th can the rod 6th and the templates 3 lower and into the mixture 10 in the reservoir 2 immerse. In a countermovement, the templates can be lifted again and replaced. When immersed, a film remains from the mixture 10 on the templates 3 . The motor 7th and the clutch 6th form a wetting device. In the schematic representation shown here, which is not to scale, the coupling is 6th drawn greatly simplified. It is designed so long that the immersion and replacement movement is possible. This embodiment of the device according to the invention is characterized by a dynamic arrangement of the templates 3 and a static arrangement of the mixture 10 characterized from culture solution and microorganism: The mixture 10 is not moved while for wetting the templates 3 by moving it up and down into the mixture 10 be immersed and removed. The movement in and out takes place in a translatory manner along the Z-axis shown.

The coupling 6th is through the opening in the cover 8th guided. The gap between the edge of the opening and the rod is through the seal 16 locked so the interior 14th against the environment 18th is tightly closed.

By a movement device shown schematically 5 becomes a movement of the templates 3 inside the interior 14th realized. The movement device is driven by the motor 7th about the clutch 6th . In the embodiment shown, the clutch 6th rotates around its longitudinal axis and thus has the function of a shaft. The shaft is connected to a gear (not shown) which is part of the movement device 5 is and the assembly of jigs 4th and templates 3 rotates clockwise, represented by an arrow. The assembly from jigs 4th and templates 3 is rotated around a spatial axis that is perpendicular to the plane of the drawing (X axis). The X-axis is perpendicular to the longitudinal axes of the templates 3 which are aligned lengthways in the direction of the drawn Z-axis. The movement device can be designed in such a way that the assembly can be rotated instead of about the X-axis, or in addition to this, about the drawn Y-axis or about further axes. For example, a cardanic suspension of the assembly is conceivable.

In the 2 instructs the device 1 a housing 15th on which from a vessel 9 and a cover 8th consists and the interior 14th encloses. In the interior 14th the assembly is off Jigs 4th and templates 3 , a first reservoir 2 and a second reservoir 2 'for the mixture 10 . The first reservoir 2 and the second reservoir 2 'are opposite, on both sides of the jig assembly 4th and templates 3 arranged. The reservoirs 2 '2' can be opened in the direction of the templates. With locking devices 13th , 13 'can be the openings of the reservoirs 2 , 2 'can be opened or closed as required. The locking devices 13th '13' are not a mandatory feature of the device according to the invention, but are advantageous to prevent the mixture from escaping inadvertently 10 , 10 'from the reservoirs 2 To prevent '2'. The locking devices shown schematically 13th , 13 'can for example be mechanically or electromagnetically controlled valves.

The assembly from jigs 4th and templates 3 , the first reservoir 2 , the second reservoir 2 'and the closure devices 13th '13' are of a wrapper 12 surrounded and fixed relative to this and relative to each other. The movement device 5 grabs the wrapping 12 at.

The drive of the movement device 5 takes place with the engine 7th about the clutch 6th . In the embodiment shown, the clutch 6th rotates around its longitudinal axis and thus has the function of a shaft. The shaft is connected to a gear (not shown) which is part of the movement device 5 and the assembly from the shell 12 , Jigs 4th , Templates 3 , Reservoirs 10 , 10 'rotates clockwise, represented by an arrow. The jig 4th with the templates 3 are rotated around a spatial axis that is perpendicular to the plane of the drawing (X axis). The X-axis is perpendicular to the longitudinal axes of the templates 3 which are aligned lengthways in the direction of the drawn Z-axis.

In the illustrated state of the device is the first reservoir 2 with mixture 10 filled. When rotated clockwise by 180 ° around the X-axis, the first reservoir becomes 2 including mixture 10 moved into the upper position, where the reservoir 2 'is still in this illustration. The locking device 13th opened and the mixture 10 pours over the templates 3 . Not on the surface of the templates 3 The remaining mixture pours into the second reservoir 2 ', which has been moved into the lower position, the closure device 13' of which has also been opened, and is collected therein as a mixture 10 '(in the illustration in FIG 2 10 'still denotes the space into which the mixture 10' flows, not yet the mixture itself). On the surface of the templates 3 any remaining mixture forms a film there. The described process can be repeated any number of times and takes place in the opposite direction with the next rotation around the X-axis by 180 °: the mixture 10 'is made from the second reservoir 2' via the templates 3 poured and not on the surface of the templates 3 remaining mixture is in the first reservoir 2 caught.

The wetting device ( 6th , 7th ) and the movement device 5 which the templates 3 rotates are in the embodiment of 2 in summary: The movement device ( 5 ) rotates the reservoirs 2 , 2 'and the templates 3 as indicated above, whereby wetting takes place as explained. In addition to the mentioned rotation around the X-axis, the templates 3 and the reservoirs 2 , 2 'also rotated about other spatial axes, for example about the Y-axis. In the periods between the wetting processes, the reservoirs 2 , 2 'by means of the closure devices 13th , 13 'closed and the templates 3 can with the movement device 5 be rotated without causing mixture 10 pours over the templates. During this time it is in and / or on the film on the surface of the templates 3 the cellulose formed.

The device of the 2 is characterized by a dynamic arrangement of the templates and a dynamic arrangement of the culture solution. The device enables a method for the construction of BNC implants with the templates at the same time 3 and the mixture 10 moved periodically.

Example 2: Process - production of a hollow body from microbial cellulose:

General procedure:

Templates are used to manufacture the products 3 between jigs 4th arranged and in the moving device 5 used. The reactor will then come with the cover 8th sealed and sterilized. After the entire reactor has been sterilized, the reservoir is / will be 2 or the reservoirs 2 , 2 'under sterile conditions with a mixture 10 Cellulose-forming microorganisms and separately sterilized culture solution 10 filled.

It is also possible to use the reservoir 2 / the reservoirs 2 '2' to be filled with culture solution before the sterilization process and to be sterilized together with all other internals in the bioreactor and then Admit microorganisms, for example through a closable opening in the housing under sterile conditions.

The engine will then 7th started the movement device 5 and the wetting device 6th , 7th set in motion and the template matrix 3 , 4th moved according to the specified cultivation conditions. The types of movement of the various embodiments of a device were explained in Example 1. The movement is preferably carried out discontinuously with frequencies, preferably less than 0.01 Hz, in such a way that the templates in a defined manner with culture medium 10 are wetted. The movement device takes care of this or at the same time 5 through targeted superimposed movements for a defined distribution of the liquid film. During this movement, the bacterial nanocellulose is cultivated on the template 3 at the interface between the liquid film and the oxygen-containing atmosphere in the interior 14th . This process is repeated until the product has taken on the desired shape.

Then the reactor can be opened, the templates 3 with the hollow bodies formed thereon made of bacterial nanocellulose and fed to the processing. The hollow bodies are stripped from the templates, cleaned and stored moist or dried, as described in the general part of the description.

With a modified device, it is also possible to sterilize and work up the synthesized hollow bodies in the reactor in such a way that the finished product can be removed when the reactor is opened. The culture medium could through a further opening by a device not shown 10 be replaced by appropriate cleaning or rinsing liquids.

Methods:

Seam tear strength:

Surgical suture material (e.g. PROLENE 5/0 (1 metric)) is used to attach loops through the wall of a tubular cellulose hollow body, as described in the example, in such a way that it pierces from the outside in the direction of the lumen through the wall of the hollow body, the suture material is pulled through and then knotted .
This procedure corresponds to the production of a so-called single button seam. This process is repeated a few times (eg six times) so that six loops are attached to the circumference of the hollow body.
The end of the hollow body opposite the loops is fixed in a tensile testing machine and one of the loops is grasped with a hook that is connected to the load cell of the testing machine. The testing machine is activated so that the force transducer and the fixing point move away from each other. This results in a pull on the suspended loop, which is torn out of the hollow body when a certain tensile force is exceeded. This force is read off on the testing machine and the test for statistical validation of the measured value is repeated with all other loops in the manner described. The mean value of the forces determined is the seam pull-out strength of the hollow body.

Burst pressure

A tubular hollow cellulose body as described in the example is fixed horizontally on two hose nozzles with cable ties in such a way that a non-slip and largely liquid-tight connection is provided. The distance between the olives is 100 mm. One of the hose nozzles is connected to a valve, the other to a reservoir that contains water and can be pressurized by compressed air. The pressure in this reservoir is increased to 200 mbar (g) until the cellulose hollow body is completely filled with water. The valve is then closed and the pressure in the reservoir is increased at a rate of 0.1-0.2 bar / s until the cellulose hollow body fails, which can be seen from the strong leakage of water at a narrowly limited point. The pressure in the system at the time of failure is read on a manometer.

SEM

SEM examinations were carried out to prove the biomimetic structure of the BNC hollow cylinder. For this purpose, the material was subjected to freeze drying for 24 hours and sputtered with gold.

Example 2.1

4950 ml nutrient solution, which per liter of deionized water contains 20.00 g glucose anhydrous, 5.00 g bactopeptone, 5.00 g yeast extract, 3.40 g di-sodium hydrogen phosphate dihydrate and 1.15 g citric acid monohydrate as well as a pH value between 6.0 and 6.3 (Hestrin-Schramm medium) and was steam-sterilized at 121 ° C. for 20 min in the autoclave, 247.5 ml of a preculture of a bacterium of the genus Gluconacetobacter were inoculated. The sterilized device 1 was filled with the mixture thus prepared.

As a template 3 a bacteriophilic round material (wood) with a diameter of 5 mm was used. The periodic wetting of the templates combined in a matrix arrangement with the culture solution containing the microorganism was carried out with the aid of the motor 7th realized. During the cultivation process, the templates combined in a matrix arrangement were wetted in a time interval of 10 minutes. The superimposed rotary movement around the rotary axes 11 took place after each wetting process.

After the biosynthesis period of 6 days had elapsed, the bioreactor 1 including the template matrix, dismantled the BNC hollow cylinders from their templates 3 isolated, sterilized and cleaned.

The synthesis yielded BNC hollow cylinders with an inner diameter of 5 mm, a wall thickness of 5 mm and a length of 15 cm.

The structure of the BNC hollow cylinder obtained is shown in 3 , 5-9 shown, which represent scanning electron microscope images.

3 shows the SEM image of the cross section of a BNC hollow cylinder in the overview. The hollow cylinder is made up of 3 parallel layers (a, b, c) that are rotationally symmetrical to the axis and are firmly connected to one another, here referred to as phases (a, b, c). The phase transitions are clearly visible.

Phase a has the inner surface (surface on the cavity side) of the wall and is also referred to as the “lumen-side phase”. Phase c has the outer surface of the wall and is also referred to as the "outside phase".

In the case of phase a on the lumen side, both the cross-sectional area and the side area angled to it, which adjoins phase b, are visible. The lettering "Phase a" is drawn on the cross-sectional area. The side surface of phase a is adjacent to it on the right. Only cross-sectional areas of phases b and c are visible which, in this preparation, are offset towards the rear, into the image plane, compared to the cross-sectional area of phase a. The strip to the right of phase c is a preparation artifact and not part of the wall or any surface thereof. The BNC hollow cylinder is limited by 2 layers / interfaces of high surface quality.

4-6 show SEM images of the fiber network structures of the phases ac making up the BNC hollow cylinder. All phases are characterized by a very uniform fiber structure of similar fiber density. All three phases a, b, c have a similar spatial porosity, defined as void volume / total volume. The structure of the BNC hollow cylinder is comparable to that of the tunica media of arteries near the heart, made up of alternating layers of elastic (the phase transitions we have described) and muscular components (the phases we have described).

The lumen-side phase a has the inner lumen-side surface of the hollow body, and the outer phase c has the outer surface of the hollow body. Due to the very uniform fiber structure and the similar fiber density of phases a and c, the inner lumen-side surface of the hollow cylinder and the outer surface very likely have a similar porosity and a similar coverage with fibers, the porosity being defined two-dimensionally with regard to the surfaces as in FIG Description given.

Application tests:

The intended use is as a vascular prosthesis. Investigations into the bursting strength of the vascular prosthesis showed bursting pressures in the range of 800mmHg and higher. The seam pull-out strength was in the range of 8-10N.

The blood compatibility of the vascular prosthesis from Example 2 was assessed by means of an experimental animal test in which parts of the common carotid artery of sheep were replaced with the BNC implant produced. The BNC implants were convincing in terms of surgical handling. They were characterized by complete stability in the high pressure animal (no cracks or ruptures). Immediately after the operation, unhindered blood flow could be observed. After 3 months, the artificial blood vessel was removed, which was very well integrated into the animal body because it was embedded in connective tissue and the formation of small blood vessels within the connective tissue. A “biologization” of the implant material could be proven histologically.

Light microscopic examinations after animal experiments show a 3-layer wall structure of the removed BNC-implant complex, whereby the BNC was retained as media, an inner endothelial layer and an outer connective tissue layer formed.

Example 2.2

4950 ml nutrient solution, which per liter of deionized water contains 20.00 g glucose anhydrous, 5.00 g bactopeptone, 5.00 g yeast extract, 3.40 g di-sodium hydrogen phosphate dihydrate and 1.15 g citric acid monohydrate and a pH value between 6.0 and 6.3 (Hestrin-Schramm medium) and was steam-sterilized in the autoclave at 121 ° C. for 20 minutes, 247.5 ml of a preculture of a bacterium of the genus Gluconacetobacter were inoculated. The sterilized device 1 was filled with the mixture thus prepared.

As templates ( 3 ) a bacteriophilic round material (wood) with a diameter of 5mm was used. The periodic wetting of the templates combined in a matrix arrangement 3 with the culture solution containing the microorganism was with the help of the motor 7th realized.

During the cultivation process, the templates combined in a matrix arrangement 3 wetted in a time interval of 30 minutes. The superimposed rotary movement around the rotary axes 11 took place after every 30th wetting process.

After the 10 day biosynthesis period, the bioreactor 1 including the template matrix, dismantled the BNC hollow cylinders from their templates 3 isolated, sterilized and cleaned.

The synthesis yielded vascular prostheses with an inner diameter of 5 mm, a wall thickness of 3 mm and a length of 15 cm. Investigations into the bursting strength of the vascular prosthesis showed bursting pressures greater than 800mmHg.

List of reference symbols

1

contraption

2

Reservoir for culture solution / mixture

3

Template

4th

Jigs

5

Movement device

6th

coupling

7th

engine

8th

cover

9

vessel

10

Mixture containing culture solution and microorganism

11

Axes of rotation

12

Wrapping

13

Locking device

14th

Interior (with an oxygen-containing atmosphere)

15th

casing

16

poetry

17th

opening

18th

Surroundings

19th

opening

20th

Clasp

Claims (16)

A method for producing a preliminary product of a hollow body made of microbial cellulose, which comprises the following steps: a) bringing the surface of a template, which is a negative form of the cavity of the hollow body to be produced and the inner walls of the cavity, into contact with a mixture supply, which is a liquid Culture medium and a cellulose-forming microorganism comprises, b) interrupting the contact between the template and the mixture supply, a liquid film remaining on the surface of the template, which comprises the liquid culture medium and the microorganism, c) bringing the liquid film into contact with an oxygen-containing atmosphere and the formation of microbial cellulose in and / or on the liquid film, d) bringing the microbial cellulose obtained in step c) into contact with the mixture supply, e) interrupting the contact between the microbial cellulose and the mixture supply, whereby on the surface of the microbial cellulose lulose, a liquid film remains which comprises the liquid culture medium and the microorganism, f) bringing the liquid film into contact with an oxygen-containing atmosphere and the formation of microbial cellulose in and / or on the liquid film, characterized in that the template at least during step c) and Step f) is rotated around several spatial axes. Method according to one of the preceding claims, wherein the template has a surface which has structures in the millimeter, micrometer and / or nanometer range. Method according to one of the preceding claims, wherein the template has a surface made of wood, metal, plastic, ceramic, synthetic polymer, paper, textile fabric or glass. Method according to one of the preceding claims, wherein in steps a) and d) an arrangement of several templates is immersed in the mixture supply, and in steps b) and e) the arrangement of several templates is removed from the mixture supply. Method according to one of the preceding claims, comprising repeating the sequence of steps d), e) and f) once or several times. A method for producing a hollow body from microbial cellulose, comprising the method steps according to one of the Claims 1 - 5 , and further comprising separating the microbial cellulose from the template. Hollow body made of microbial cellulose, obtainable by a process according to Claim 6 . Hollow body after Claim 7 , comprising a wall having an inner surface and an outer surface, the inner surface and the outer surface having an identical or similar degree of coverage by microbial cellulose fibers per unit area, the coverage being indicated as part of a considered surface section, that of fibers is covered / total area of the area under consideration, and a similar degree of coverage means that the coverage on the inner surface and the outer surface differ relative to one another by a maximum of 20%, the percentage of the difference being determined as follows: $$\begin{array}{l}\text{difference}\left(\text{\%}\right)=\\ \left(\text{number greater value}-\text{lower number value}\right)\text{/ lower number}*100.\end{array}$$

Hollow body after Claim 7 or 8th , comprising a wall having an inner surface and an outer surface, the wall comprising multiple layers of microbial cellulose, the layers being parallel to the inner and outer surfaces of the wall. Hollow body after Claim 9 , the layers being homogeneous in their density over the entire layer thickness. Hollow body after Claim 9 or 10 , wherein one layer has the inner surface of the wall and another layer has the outer surface of the wall, and these two layers have an identical or similar porosity, the porosity being given as void volume / total volume and a similar porosity means that the Differentiate the porosities of the layers by a maximum of 20%, the percentage of the difference being determined as follows: $$\begin{array}{l}\text{difference}\left(\text{\%}\right)=\\ \left(\text{number greater value}-\text{lower number value}\right)\text{/ lower number}*100.\end{array}$$

Hollow body according to one of the Claims 7 - 11 , which has one or more openings through which the cavity of the hollow body is accessible. Hollow body according to one of the Claims 7 - 12 selected from a tube or a hollow cylinder. Hollow body after Claim 13 that has one or more branches. Artificial biological vessel, in particular an implant, having a hollow body according to one of the Claims 7 - 14th . Use of a hollow body according to one of the Claims 7 - 14th or a vessel Claim 15 as a medical implant, in particular as a hollow vessel, blood vessel, esophagus, digestive tract, trachea, urethra, bile duct, ureter, lymph vessel or cuff.

Images