DE102019124879A1 - Bioactive carrier - Google Patents

Abstract

Bioactive carrier for the settlement of living cells, with a porous three-dimensional bar framework (2) made of glass, comprising a porous reinforcing structure (3) made of a synthetic polymer which can be dissolved in the human or animal body.

Description

The invention relates to a bioactive carrier for the settlement of living cells, with a porous three-dimensional bar framework made of glass.

Osteoarthritis is a common joint disease that is very important for the economy as a result of treatment costs and incapacity for work, and its importance is increasing against the background of current demographic change. It is currently still incurable, characterized by a progressive course, whereby the cartilage in the joints, among other things, is attacked, loses its strength and is partially dissolved. Osteoarthritis often occurs as a result of articular cartilage injuries, because the articular cartilage and cartilage tissue in general only have an extremely low self-healing capacity. The treatment option for the reconstruction of the joint surfaces after cartilage injuries and advanced osteoarthritis is often only a corresponding surgical intervention, which is often accompanied by a partial or complete replacement of the joint with an implant. As an alternative to this, the aim is to cultivate the body's own cartilage tissue in vitro as a biological replacement. After cultivation, this replacement tissue is transferred into the patient's cartilage defect in order to replace the defective articular cartilage.

In order to ensure a three-dimensional arrangement of the cartilage cells, natural and synthetic biomaterials are used, which should dissolve as soon as the new tissue formation in the defect has progressed.

• Es muss ein geeignetes offenporiges Gerüst mit einer Porosität von wenigstens 60 % für das Wachstum der Zellen vorhanden sein, also ein bioaktiver Träger, der nicht nur die mechanischen Anforderungen, die an einen solchen Träger im zukünftigen Gewebe gestellt werden, erfüllt, sondern der auch hinreichend zyto- und biokompatibel ist, um das Zellüberleben zu gewährleisten und keine Entzündung auszulösen.

This process of obtaining tissue by culturing cultured cells in a suitable biomaterial is called "tissue engineering". In this way, initially in vitro, ie in a cell culture, cell proliferation of adherent cells and subsequent tissue formation on a three-dimensional bioactive carrier are achieved. In order to achieve this in vitro growth of cartilage cells, so-called chondrocytes, cells removed from the body must be stimulated in a suitable environment. A suitable geometric arrangement of the cells can support the formation of the new tissue. Several basic factors are of great importance for the in vitro growth of cells and tissue formation:

• There must be a suitable open-pore framework with a porosity of at least 60% for the growth of the cells, i.e. a bioactive carrier that not only meets the mechanical requirements that will be placed on such a carrier in future tissue, but also adequately is cyto- and biocompatible in order to ensure cell survival and not cause inflammation.

Living and reproducible cells must be able to settle on the bioactive carrier, the so-called scaffold. This means that sufficient cell adhesion to the scaffold must be possible. The cell surface receptors must therefore recognize structures in the scaffold as binding motifs and the necessary cofactors (including ions) must be sufficiently available.

Furthermore, it must be possible to stimulate and control a cell-type-specific signal transduction of the living cells, which can be achieved for example by growth stimulators (such as growth factors, ions, but also a suitable scaffold topology).

Finally, for an initial cell proliferation, that is to say cell multiplication and cell growth, as well as later differentiation of the cells, a beneficial extracellular milieu must be present in the scaffold and around the cells.

Of particular interest is the synthesis of a new extracellular cartilage matrix by the cells in the scaffold, which is controlled by the geometric scaffold structure (topology) and growth factors, since the articular cartilage consists of over 90% extracellular matrix (ECM = extracellular matrix). In terms of stimulation factors for cell growth and for matrix synthesis, one can essentially differentiate between electro- / biochemical, structure-associated and mechanical factors. If the colonized cells are to multiply well, these factors must be selected accordingly for the type of cell to be grown.

In the context of tissue engineering, the bioactive carrier represents a central element, because on the one hand it must be mechanically stable enough, on the other hand it must enable cell adhesion, cell growth and ECM synthesis. It must also be dissolvable (“degradable”), whereby the dissolving of the bioactive scaffold results in concentration gradients of ions with corresponding chemical and electrochemical potentials, cell proliferation, cell differentiation and ECM synthesis can support. In this context, glass is regarded as an excellent material for the production of such a bioactive carrier.

Such bioactive carriers, also called bio glasses, are known in various applications, for example from WO 2011/161422 A1 , WO 2016/089731 A1 or WO 2014/168631 A1 . These publications deal explicitly with the production of a scaffold. The bioactive carriers or bio glasses described there are based on alkaline earth and phosphate-containing glass systems which, in aqueous solutions, form a hydroxylapatite layer on the glass surface. This hydroxylapatite layer forms an interface as a so-called boundary layer to which the living cells to be multiplied must adhere and which is supposed to promote growth. The best known of these alkaline earth containing bio glasses is known under the designation 45 S5.

In DE 10 2018 114 946 B3 a bioactive carrier made of glass is described, which has excellent properties in terms of bioactivity and the required geometry for good cell growth. Bioactivity is achieved by leaching out the surface layer of the glass scaffold with associated gel formation on the surface. Due to the leaching and a leaching-related reduced content of alkali metal oxides in the leached surface and the inclusion of OH groups or water in the leaching-related "open" network in the leaching area, the corresponding assemblies within the glass matrix are only slightly linked.

This leads to a reduced chemical resistance and promotes the dissolution of the carrier during in vivo use, that is to say that the glass carrier also dissolves due to the hydrolytic acid-alkali attack of the surrounding medium during in vivo use. This ability to dissolve, i.e. biodegradation, is a central property of every bioactive glass carrier if it is to be used in vivo.

The specific glass structures of such a bioactive carrier, for example from DE 10 2018 114 946 B3 is known, are characterized by very thin webs in the porous three-dimensional bar framework, which are essential for the strength of the bar framework. To form an open-pored, quasi-sponge-like structure, web thicknesses of 10-400 μm are preferred, which means that it is a very finely structured framework. The strength of such glass structures is therefore limited, especially when the glass is dissolved in in vivo use, which is desired as described. However, a certain strength is required for the implant insert, so that short-term shear and pressure loads do not lead to a gap and thus to the destruction of the implant. In in vivo use, the strength of the implant is not only defined by the inorganic bar framework, but also by the ECM, which is supposed to take on part of the mechanical load, although the mechanical strength of the carrier or bar framework also plays a role.

The invention is thus based on the problem of specifying a bioactive carrier which is improved in comparison.

To solve this problem, a porous reinforcing structure made of a synthetic polymer that can be dissolved in the human or animal body is provided according to the invention in a bioactive carrier of the type mentioned at the beginning.

The bioactive carrier according to the invention is characterized by an additional reinforcement structure which serves to reinforce or stiffen the carrier itself, i.e. to give it greater mechanical strength as an overall component, so that it is able to withstand higher loads for a short time, without the bar framework opening up or breaking. In addition to the bar framework made of glass, which, with regard to in vivo use, can of course also basically be dissolved in the human or animal body over a corresponding period of time, a likewise porous reinforcement structure is provided on the bar framework, which consists of a synthetic, human or animal The body is also cell-compatible, i.e. non-toxic, dissolvable polymer. Like the bar framework, the reinforcement structure is also porous in order to retain the property of the carrier as a quasi sponge-like structure and to enable cells to settle within the entire volume of the three-dimensional carrier.

While a pure, unreinforced bar framework collapses under pressure because the bars break continuously, it has been shown, which will be discussed below, that applying such a polymer reinforcement structure increases the strength or resistance of the Bioactive carrier against such a mechanical load is significantly improved, since the carrier has quasi elastic properties and retains its shape under load and offers resistance to the load. This means that the glass framework or scaffold is essentially retained and does not break into its individual parts, so that the glass framework is stabilized and an increased resistance to pressure loading or compression is achieved.

This polymer reinforcement therefore enables the implementation of an implant that has an improved resistance to external loads and gives the bioactive carrier dimensional stability with elastic behavior without the advantageous properties of the bioglass being impaired.

As already mentioned, any bio-glass that can basically be dissolved in the human or animal body and that enables the settlement of living cells can be used as glass for forming the three-dimensional bar framework. In addition to glasses, as already described in the introduction, this is particularly suitable DE 10 2018 114 946 B3 described bioglass, which can be provided with the polymer reinforcement structure according to the invention and due to the superficial leaching of the bar framework, which can take place before or after application of the polymer reinforcement, has excellent support properties.

An essential property of the polymer reinforcement structure, in addition to giving it the appropriate strength or resistance to mechanical loads, is the ability of the polymer to dissolve in the human or animal body, i.e. in vivo use, i.e. that the polymer is correspondingly degradable, while during this degradation, no toxic substances that adversely affect cell growth or that again destroy cells that have grown should be emitted. A polymer that has been found to be particularly suitable for this is a polymer from the group of polylactides, ie polylactic acids (PLA, polylactic acid). These are synthetic polymers that count among the polyesters and that are made up of many chemically bonded lactic acid molecules. Due to their molecular structure, they are very readily biodegradable, which makes them ideal for in vivo use.

Among the polylactides are in particular poly (L-lactide), poly (L-lactide-co-glycolide), poly (L-lactide-co-D, L-lactide), poly (D, L-lactide) or poly (D , L-lactide-co-glycolide) suitable to produce the reinforcement structure. The table below shows the approximate degradation rates in the human body for the suitable biodegradable synthetic polymers and the inherent viscosity, which is a measure of the elasticity of the material. composition Rate of degradation in the human body "Inherent viscosity" [dl / g] Poly (L-lactide) longer than 2 years 0.8-4.3 Poly (L-lactide-co-glycolide) 1-2 years 1.7-7.0 Poly (L-lactide-co-D, L-lactide) 2-3 years 2.0-6.5 Poly (D, L-lactide) 5 months - 2 years 0.16-1.7 Poly (D, L-lactide-co-glycolide) 5 - 9 months 0.14-1.7

According to a first alternative of the invention, the reinforcement structure can be a fiber structure surrounding the bar framework on the outside. Accordingly, the reinforcement structure is attached quasi on the outside of the bar framework, quasi enclosing or surrounding it. This outer reinforcement structure is elastic and provides a corresponding resistance to an external load, so that the quasi-internal bar framework is protected.

According to the invention, the reinforcing structure can be designed in the form of a wrapping around the bar framework. This means that a thin polymer fiber is spun around the bar framework, which surrounds the bar framework in the manner of a very open-pored cocoon, with the fibers only contacting the bar framework at certain points. In this case, the wrapping is preferably attached in such a way that the bar framework is enclosed on all sides.

As described, the wrapping is to be attached in such a way that the open pores are still there. The wrapping or fiber structure is preferably applied in such a way that the surface it covers is a maximum of 10%. This means that 90% or more of the area enveloping the bar framework is not wrapped, that is to say over 90% of the surface of the outside defined by the bar framework is still open and an introduction of the nutrient solution and cell growth inside is possible without problems.

The fibers of the fiber structure themselves preferably have a diameter of 2-100 μm, in particular 5-75 μm and preferably 10-50 μm, that is to say that the fibers are extremely fine.

While, according to the first alternative of the invention, the reinforcement structure is applied on the outside of the bar structure, according to a second alternative of the invention, a fiber structure formed in the interior of the bar framework is applied as the reinforcement structure. Here the reinforcement structure is thus formed in the volume of the glass framework. The fibers forming the reinforcement structure here extend between the webs, and they can optionally also extend over several webs. The fiber structure preferably forms an at least partially connected fiber framework structure, which means that not only do individual fibers extend between the webs, stiffening them, but a real polymer framework is formed as an additional stiffening framework within the glass framework.

Here, too, the stiffening structure, for example the polymer framework, increases the strength so that the glass framework does not collapse in the event of a load, but rather a resistance is opposed to the load due to the elastic properties of the reinforcement structure, so that the carrier has a certain elasticity and is able to do so is to absorb any burdens.

In this second alternative of the invention, too, despite the integration of the reinforcement structure, a high degree of porosity is still maintained in order to enable cell growth in the interior of the carrier. The porosity of the carrier should be at least 80% in spite of the integrated reinforcement structure or integrated polymer framework.

Although of the two alternatives described above, either the outside fiber structure or the inside fiber structure can be applied as a reinforcement structure, it is also conceivable according to the invention to provide both an outside fiber structure and an inside fiber structure, i.e. to combine both reinforcement techniques. For example, the inner fiber structure, that is to say, for example, the cohesive polymer framework, is first formed in the glass framework, after which, in a second step, the external reinforcing structure is applied by spinning. The porosity is still sufficient to colonize the carrier volume with cells, the strength of the carrier in this case being even greater than when only one type of reinforcement structure is applied.

The bar framework itself should have a porosity of at least 80%, preferably more, wherein, as stated, the overall porosity of the carrier is only slightly reduced by applying the corresponding stiffening structures.

The pore size of the bar framework should be between 20-300 µm, in particular between 50-200 µm, while the bars of the bar framework can have a thickness of 10-400 µm, in particular 20-200 µm and preferably between 50-150 µm.

The bar framework itself can be produced in different ways. It is conceivable to produce the bar framework from glass fibers connected to one another by sintering. Here, individual glass fibers, either individually or, for example, in the form of prefabricated glass fiber mats or glass fiber layers or the like, are superimposed and connected to one another by sintering at the individual contact points, so that a corresponding glass framework is formed. Using such glass fibers, it is possible to produce various types of carrier geometries, for example in the form of plates, rectangular or square cubes, trapezoidal or spherical shapes, but also hollow bodies such as a tube or the like. This is because the glass fibers can be laid in different geometries and then sintered together, which makes it possible to design the basic shape of the bioactive carrier with regard to the desired geometry of the finished, populated implant.

As an alternative to the use of glass fibers, which are naturally very thin in accordance with the desired web thickness, a second alternative for producing the bar framework provides for it to be made from a sintered glass powder. In this alternative, a polymer carrier structure is used, for example, which is covered with a glass powder suspension. In a subsequent sintering process, the glass particles are connected to one another by sintering while the polymer support structure burns. This also results in a porous, three-dimensional bar framework, the geometry of which is different from the geometry of the polymer carrier structure depends. It is obvious that, depending on the shape of the polymer carrier structure, any desired carrier geometries can be produced here as well.

The bar framework particularly preferably has an alkaline earth metal oxide content of a maximum of 2.0 mass% and its alkali metal oxide content is reduced at least on the surface, preferably over the entire cross section of the bar, by leaching. Such a bar framework is particularly useful because it does not have a hydroxylapatite layer and on the one hand is very suitable for cell growth, but on the other hand it also dissolves well when used in vivo. Such a bar framework is in DE 10 2018 114 946 B3 described in detail. Reference is expressly made to this publication, the entire disclosure content with regard to the bioactive carrier or the bar framework, the production of a bioactive carrier with such a leached bar structure, the materials that can be used and the corresponding manufacturing processes, etc. being explicitly incorporated into the present disclosure .

1. a) Bereitstellen eines porösen dreidimensionalen Steggerüsts aus Glas,
2. b) Erzeugen einer das Steggerüst verstärkenden, porösen Verstärkungsstruktur aus einem synthetischen, im menschlichen oder tierischen Körper auslösbaren Polymer.

In addition to the bioactive carrier itself, the invention also relates to a method for producing a bioactive carrier, comprising the steps:

1. a) providing a porous three-dimensional bar framework made of glass,
2. b) Production of a porous reinforcement structure that reinforces the bar framework from a synthetic polymer which can be released in the human or animal body.

The method is thus characterized by two central steps, namely on the one hand the provision of the corresponding glass bar framework made of a biodegradable glass that can therefore dissolve in the human or animal body. Different glasses can be used as long as these requirements are met. Preference, but not limitation, is, as already described, a bar framework as in DE 10 2018 114 946 B3 disclosed used.

In the second central step, the porous reinforcement structure reinforcing the bar framework is applied, whereby this is also made from a material that is cell-compatible in the human or animal body, i.e. non-toxicly degradable. According to the invention, a synthetic polymer is used for this purpose.

A polylactide is preferably used as the synthetic polymer, and within this group preferred and as the polymer poly (L-lactide), poly (L-lactide-co-glycolide), poly (L-lactide-co-D, L-lactide), Poly (D, L-lactide) or poly (D, L-lactide-co-glycolide) are used.

In terms of manufacturing technology with regard to the application or generation of the reinforcement structure, two fundamentally different manufacturing variants are conceivable. According to a first alternative of the invention, a fiber structure surrounding the web framework on the outside, preferably on all sides, is applied to the web framework in the form of a wrapping as a reinforcement structure. Accordingly, a very thin polymer fiber is placed around the bar framework, the fiber structure virtually envelops it, the degree of wrapping being of course very low with regard to the actually covered area, which is preferably a maximum of 10%, which means that it is ensured that, Seen from the outside, the braided carrier is still extremely open-pored, so that the corresponding solutions etc. can penetrate into the carrier volume during the cell colonization and the cells can settle there.

In a further specification of this variant of the invention, it is provided that a polymer thread is drawn or spun from a polymer solution, which is then spun around the bar framework, after which a heat treatment takes place, optionally after drying. A polymer solution is therefore first produced, which consists, for example, of double-distilled water, 1,4-dioxane and the desired polymer, for example the polylactide, which is added at 2-10% by weight. The ratio of water: dioxane should be, for example, 13:87, whereby the water content can also be higher or lower, up to a minimum of 0 mass%. 1,4-Dioxane is a suitable solvent, although it is also possible to use other organic solvents, optionally also as admixtures with 1,4-dioxane.

After the polymer solution has been produced, the corresponding polymer fiber is drawn and spun around the bar framework. This can be done manually, in particular on a laboratory scale, preferably by machine on a larger scale. If necessary, drying can then take place in order to drive off the water, which is followed by a heat treatment in order to finally remove the dioxane, that is to say the solvent, and crosslink the polymer. Annealing consequently takes place in this heat treatment.

The polymer solution can be heated to a temperature of 40 - 90 ° C in order to draw the polymer fiber. The heat treatment should be carried out at a temperature of 50 - 200 ° C for 0.2 - 4 hours.

The drawn polymer fiber should have a diameter of 2-100 μm, in particular 5-75 μm and preferably 10-50 μm, whereby when producing the polymer fiber it must be taken into account that during drying / sintering it results in a loss of water and dioxane Shrinkage can come by around 20%.

1. 1. Bereitstellen eines Steggerüsts aus einem biodegradierbaren Glas, insbesondere gemäß DE 10 2018 114 946 B3 ,
2. 2. Herstellen einer Polymerlösung aus bidestilliertem Wasser und 1,4-Dioxan im Verhältnis von ca. 13 : 87 sowie dem gewünschten Polymer (Polylactid) und Temperieren der Lösung auf eine Temperatur von 40 - 90° C, so dass eine viskose, zieh- oder spinnbare Polymerlösung entsteht,
3. 3. Ziehen oder Spinnen einer Polymerfaser aus der Lösung und Umspinnen der Polymerfaser um das poröse, dreidimensionale Steggerüst derart, dass die Polymerfaser mehrfach vollständig um das Steggerüst gewickelt wird, wobei die Umwicklung bevorzugt an allen Seiten des Steggerüsts erfolgt,
4. 4. Trocknung des umsponnenen Steggerüsts an Luft für mehrere Stunden bei Raumtemperatur,
5. 5. Wärmebehandlung des Steggerüsts samt Polymerfasern bei 50 - 200° C für 0,2 - 4 h zum Tempern.

A specific manufacturing variant is, for example, as follows:

1. 1. Providing a bar framework made of a biodegradable glass, in particular according to FIG DE 10 2018 114 946 B3 ,
2. 2. Preparation of a polymer solution from double-distilled water and 1,4-dioxane in a ratio of approx. 13:87 as well as the desired polymer (polylactide) and tempering the solution to a temperature of 40 - 90 ° C, so that a viscous, draw- or spinnable polymer solution is produced,
3. 3. Drawing or spinning a polymer fiber from the solution and spinning the polymer fiber around the porous, three-dimensional bar framework in such a way that the polymer fiber is completely wrapped around the bar framework several times, the wrapping preferably taking place on all sides of the bar framework,
4. 4. Drying of the covered bar framework in air for several hours at room temperature,
5. 5. Heat treatment of the bar framework including the polymer fibers at 50-200 ° C for 0.2-4 hours for tempering.

This process sequence is merely exemplary, but not restrictive.

According to a second fundamental alternative to the invention, the reinforcement structure can also be designed as a fiber structure, preferably forming a coherent fiber framework structure, in the interior of the bar framework. Accordingly, corresponding polymer fibers are formed in the interior of the three-dimensional bar framework, which preferably connect or crosslink to form a coherent fiber framework structure, i.e. an inner polymer framework, so that a double framework structure is created from the glass framework and the quasi-integrated, reinforcing polymer framework.

In a further specification of this variant of the method it can be provided that the bar framework is impregnated with a polymer solution, after which, if necessary after removing the polymer solution from the surface of the bar framework, the impregnated bar framework is placed in water so that the polymer precipitates like a foam, after which, if necessary, after a drying, a heat treatment takes place. By soaking the bar framework with the polymer solution, the polymer is brought into the interior of the bar framework. The polymer solution used is again preferably a mixture of double-distilled water and 1,4-dioxane in a ratio of approx. 13:87 and the desired polymer, i.e. preferably the polylactide, at 2-10% by weight, but here the water content also increases 0 can be reduced, since the water does not necessarily have to be contained within the polymer solution in order to precipitate the polymer in the later process.

After soaking, if necessary, for example using an absorbent cloth or the like, the polymer solution is drawn from the surface of the bar framework and, to a small extent, also from the immediately adjacent volume, which serves to prevent any skin formation that sometimes leads to extensive pore closure leads to avoid.

In the next step, the impregnated bar framework is placed in water in order to fall below the solubility limit, so that the polymer precipitates in the volume of the bar framework in a quasi-foam-like manner. These precipitations can optionally be followed by a drying step, but in any case there is a heat treatment in order to form the crosslinked polymer fibers from the scaffold-like structure of the foam-like precipitated polymer in a sintering process, which then extend from web to web or also along of the webs run and preferably form a connected fiber framework structure.

Here, too, a heated polymer solution can be used, which can have a temperature of 20 - 90 ° C. The water in which the impregnated carrier is added should have a temperature of 10 - 95 ° C, while the heat treatment should take place at a temperature of 50 - 200 ° C for 0.2 - 4 hours, the temperature during the heat treatment higher than the water temperature.

1. 1. Bereitstellen eines Steggerüsts aus einem biodegradierbaren Glas, insbesondere gemäß DE 10 2018 114 946 B3 ,
2. 2. Herstellen einer Polymerlösung aus bidestilliertem Wasser und 1,4-Dioxan im Verhältnis von ca. 13 : 87 sowie dem gewünschten Polymer (Polylactid) und Temperieren der Polymerlösung auf eine Temperatur von 20 - 90° C,
3. 3. Tränken des Steggerüsts mit der Polymerlösung,
4. 4. gegebenenfalls Entfernen der Polymerlösung von der Oberfläche respektive an der Außenseite des Steggerüsts,
5. 5. Einbringen des getränkten Steggerüsts in Wasser mit einer Wassertemperatur von 10 - 95° C zum Unterschreiten der Löslichkeitsgrenze der Polymerlösung zum Ausfällen eines eine Polymer-Scaffold-Struktur bildenden Polymerschaums,
6. 6. Trocknung des Steggerüsts an Luft für mehrere Stunden bei Raumtemperatur,
7. 7. Wärmebehandlung des Steggerüsts mit ausgefällter schaumartiger Polymer-Scaffold-Struktur bei 50 - 200° C und 0,2 - 4 h, wobei die Temperatur bei der Wärmebehandlung höher ist als die Wassertemperatur.

A concrete manufacturing route for the formation of such an internal polymer framework structure can be as follows:

1. 1. Providing a bar framework made of a biodegradable glass, in particular according to FIG DE 10 2018 114 946 B3 ,
2. 2. Preparation of a polymer solution from double-distilled water and 1,4-dioxane in a ratio of approx. 13:87 and the desired polymer (polylactide) and tempering the polymer solution to a temperature of 20 - 90 ° C,
3. 3. Soaking the bar framework with the polymer solution,
4. 4. if necessary, removing the polymer solution from the surface or on the outside of the bar framework,
5. 5. Introduction of the soaked bar framework in water with a water temperature of 10 - 95 ° C to fall below the solubility limit of the polymer solution to precipitate a polymer foam forming a polymer scaffold structure,
6. 6. Drying of the bar framework in air for several hours at room temperature,
7. 7. Heat treatment of the bar framework with a precipitated foam-like polymer scaffold structure at 50-200 ° C and 0.2-4 hours, the temperature during the heat treatment being higher than the water temperature.

An alternative variant of the method for producing an internal polymer framework structure provides that the bar framework is impregnated with a polymer solution, after which it is cooled to differentiate the solubility limit, so that the polymer precipitates in the form of a foam, after which, if necessary after drying, a heat treatment takes place. Here, too, a polymer solution as described above is used (double-distilled water, 1,4-dioxane, polymer), with which the bar framework made of biodegradable glass is impregnated. In contrast to the above variant, in which the impregnated bar framework is added to water to differentiate the solubility limit, here the solubility limit is undershot by cooling, so that the polymer precipitates like a foam. After drying, if necessary, a heat treatment follows in order to form the crosslinked polymer fibers in the interior of the bar framework through final sintering.

The polymer solution here has a higher temperature of, for example, 50-90 ° C. The cooling takes place as quickly as possible to below the azeotropic point of the polymer solution, ideally 5 - 10 K below this azeotropic point, which is followed by a certain holding time. The heat treatment itself is again carried out at a temperature of 50-200 ° C for 0.2-4 hours.

1. 1. Bereitstellen eines Steggerüsts aus einem biodegradierbaren Glas, insbesondere gemäß DE 10 2018 114 946 B3 ,
2. 2. Herstellen einer Polymerlösung aus bidestilliertem Wasser und 1,4-Dioxan im Verhältnis von ca. 13 : 87 sowie dem Polymer (Polylactid) zu 2 - 10 Masse% und Temperieren der Polymerlösung auf 50 - 90° C,
3. 3. Tränken des Steggerüsts mit der Polymerlösung,
4. 4. gegebenenfalls Entfernen der Polymerlösung von der Oberfläche des Steggerüsts,
5. 5. schnelle Abkühlung des getränkten Steggerüsts mit Unterschreitung der Löslichkeitsgrenze, bevorzugt 5 - 10 K unterhalb des azeotropen Punkts, und Halten bei dieser Temperatur für 5 - 120 min zum Ausfällen eines eine Polymer-Scaffold-Struktur bildenden Polymerschaums,
6. 6. Trocknung des Steggerüsts an Luft für mehrere Stunden bei Raumtemperatur
7. 7. Wärmebehandlung des Steggerüsts mit Polymer-Scaffold-Struktur bei 50 - 200° C für 0,2 - 4 h.

A specific production route for this third variant can be as follows:

1. 1. Providing a bar framework made of a biodegradable glass, in particular according to FIG DE 10 2018 114 946 B3 ,
2. 2. Preparation of a polymer solution from double-distilled water and 1,4-dioxane in a ratio of approx. 13:87 and the polymer (polylactide) to 2 - 10% by mass and temperature control of the polymer solution to 50 - 90 ° C,
3. 3. Soaking the bar framework with the polymer solution,
4. 4. if necessary, removing the polymer solution from the surface of the bar framework,
5. 5. Rapid cooling of the impregnated bar framework, falling below the solubility limit, preferably 5 - 10 K below the azeotropic point, and holding at this temperature for 5 - 120 min to precipitate a polymer foam that forms a polymer scaffold structure,
6. 6. Drying of the bar framework in air for several hours at room temperature
7. 7. Heat treatment of the bar framework with polymer scaffold structure at 50 - 200 ° C for 0.2 - 4 h.

The inner polymer reinforcement structure or the polymer fiber framework should be designed in such a way that the porosity of the finished carrier, ie including the polymer framework, is at least 80%.

It is furthermore conceivable that both an outer fiber structure and an inner fiber structure are formed, that is to say both manufacturing techniques are used. The inner fiber structure is preferably produced first and then the outer one.

According to a particularly expedient development of the invention, a bar framework made of glass can be used which has an alkaline earth metal oxide content of at most 2% by mass, with the bar framework for at least superficial reduction of the strength before or after the production of the polymeric reinforcement structure Alkali metal oxides are acid-treated. A bar framework is therefore preferred, as in DE 10 2018 114 946 B3 described, used. The glass bar framework can be leached before the production of the polymeric reinforcement structure, either if the polymer does not withstand an acid attack for leaching, or if the bar framework is still easy to handle despite leaching in order to carry out the following steps for applying the polymer reinforcement structure, for example mechanical spinning or the like. Preferably, however, the leaching is carried out after the polymer reinforcement structure has been applied, since in particular the polylactides preferably used are sufficiently acid-stable and are not damaged by an acid attack used for the leaching. Since it has not yet been depleted, the bar framework for producing the polymeric reinforcement structure is easy to handle, which simplifies production.

The bar framework, with or without the reinforcement structure, is preferably leached with hydrochloric acid, preferably 0.1 molar hydrochloric acid. The temperature of the hydrochloric acid can be about 37 °, for example, and the acid attack can extend over several hours or days, depending on the degree of leaching.

In addition to the polymeric reinforcement framework, the glass bar framework naturally also plays a central role. This can be done in different ways. It is conceivable to use a bar frame made of glass fibers connected to one another by sintering, the fibers either being laid or spun individually or, for example, prefabricated fiber mats or fiber scrims or the like being used which are laid one on top of the other. The individual fibers are then firmly connected to one another by sintering at the corresponding contact points. Any desired carrier geometries are conceivable, since the fibers can be spun or cut to size and placed on top of one another in a corresponding, geometrically arbitrary manner.

Alternatively, a bar framework made of a sintered glass powder can be used, which is first applied in a suspension to a polymer support structure and is then sintered, the glass particles joining while the support structure burns so that a corresponding bar framework is also formed over this. However, the manufacturing variants are not limited to this; other manufacturing options are also conceivable.

• 1 eine Ansicht eines bioaktiven Trägers mit einem Steggerüst und einer durch Umspinnung erzeugten äußeren Verstärkungsstruktur vor der Wärmebehandlung,
• 2 eine REM-Aufnahme des Trägers aus 1,
• 3 eine Perspektivansicht eines bioaktiven Trägers einer zweiten Ausführungsform mit aus einzelnen Glasfasern gebildetem Steggerüst und Verstärkungsstruktur in Form einer Umspinnung,
• 4 eine REM-Aufnahme des Trägers aus 3,
• 5 eine Perspektivansicht eines bioaktiven Trägers einer dritten Ausführungsform mit im Inneren des Glasträgers ausgebildeter polymerer Verstärkungsstruktur vor der Wärmebehandlung,
• 6 eine REM-Aufnahme des Trägers aus 5,
• 7 eine REM-Aufnahme des Trägers aus 5 nach Durchführung der Wärmebehandlung,
• 8 eine REM-Aufnahme eines aus einzelnen Glasfasern gebildeten Steggerüsts mit darin ausgebildeter polymerer Verstärkungsstruktur nach der Wärmebehandlung,
• 9 ein Kraft-Weg-Diagramm zur Darstellung des Verhaltens unterschiedlicher bioaktiver Träger ohne und mit polymerer Verstärkungsstruktur.
• 10 eine Darstellung eines Trägers ohne polymerer Verstärkungsstruktur nach dem Belastungstest,
• 11 eine Darstellung eines Trägers mit Verstärkungsstruktur in Form einer Umspinnung nach dem Belastungstest,
• 12 eine Darstellung eines Trägers mit innerer polymerer Verstärkungsstruktur nach dem Belastungstest,
• 13 eine Laser-Scanning-Mikroskopaufnahme eines mit porcinen Gelenkchondrozyten besiedelten Trägers mit äußerer Verstärkungsstruktur in Form einer Umspinnung, nach 7 Tagen in Kultur,
• 14 den gleichen Trägers mit porcinen Gelenkchondrozyten nach 28 Tagen in Kultur,
• 15 eine Laser-Scanning-Mikroskopaufnahme eines Trägers mit polymerer Verstärkungsstruktur in Form einer Umspinnung und darauf angesiedelten humanen mesenchymalen Stromazellen nach 7 Tagen in Kultur,
• 16 den gleichen Träger mit Stromazellen nach 28 Tagen in Kultur,
• 17 ein Träger mit innerer polymerer Verstärkungsstruktur und darauf angesiedelten humanen mesenchymalen Stromazellen nach 7 Tagen in Kultur, und
• 18 den gleichen Träger mit Stromazellen nach 28 Tagen in Kultur.

Further advantages and details of the invention emerge from the exemplary embodiments described below and with reference to the drawings. Show:

• 1 a view of a bioactive carrier with a bar framework and an outer reinforcement structure produced by wrapping before the heat treatment,
• 2 an SEM image of the carrier 1 ,
• 3 a perspective view of a bioactive carrier of a second embodiment with a bar framework formed from individual glass fibers and a reinforcement structure in the form of a wrapping,
• 4th an SEM image of the carrier 3 ,
• 5 a perspective view of a bioactive carrier of a third embodiment with a polymeric reinforcement structure formed in the interior of the glass carrier before the heat treatment,
• 6 an SEM image of the carrier 5 ,
• 7th an SEM image of the carrier 5 after carrying out the heat treatment,
• 8th an SEM image of a bar framework formed from individual glass fibers with a polymeric reinforcement structure formed therein after the heat treatment,
• 9 a force-displacement diagram to show the behavior of different bioactive carriers with and without a polymeric reinforcement structure.
• 10 a representation of a carrier without a polymeric reinforcement structure after the stress test,
• 11 a representation of a carrier with a reinforcement structure in the form of a wrapping after the load test,
• 12 a representation of a carrier with an internal polymeric reinforcement structure after the stress test,
• 13 a laser scanning microscope image of a carrier colonized with porcine joint chondrocytes with an external reinforcement structure in the form of a wrapping, after 7 days in culture,
• 14th the same carrier with porcine joint chondrocytes after 28 days in culture,
• 15th a laser scanning microscope image of a carrier with a polymeric reinforcement structure in the form of a wrapping and human mesenchymal stromal cells settled on it after 7 days in culture,
• 16 the same carrier with stromal cells after 28 days in culture,
• 17th a carrier with an internal polymeric reinforcement structure and human mesenchymal stromal cells settled thereon after 7 days in culture, and
• 18th the same carrier with stromal cells after 28 days in culture.

1 shows a bioactive carrier 1 a first embodiment for the settlement of living cells. The carrier consists of a porous three-dimensional bar framework 2 made of glass (see in particular 2 ), that accordingly DE 10 2018 114 946 B3 was produced. It is a quasi-random glass bar framework with a porosity> 80%, the bars having a thickness of 30-120 μm, for example. As will be discussed below, the webs are superficially leached to reduce the alkali metal oxide content, for which purpose a treatment with hydrochloric acid was carried out.

On the outside, the bar framework 2 surrounding it was a reinforcement structure 3 made of a synthetic polymer in the form of a fiber structure that can be dissolved in a cell-compatible manner in the human or animal body 4th applied by spinning, this fiber structure 4th respectively wrapping of one or more individual ones, completely around the bar framework 2 wound polymer fibers 5 consists.

A polymer solution with 10% by weight of the polymer and 1,4-dioxane was produced to produce this fiber structure or covering. A polylactide, specifically poly (D, L-lactide-co-glycolide), was used as the polymer. The polymer solution was heated to 70 °. Subsequently, fibers with a thickness of 5 to 80 µm were drawn from the solution with a syringe tip and around the bar framework 2 , so wrapped the bio-glass scaffold. The bar framework 2 was wound on all sides, with still a sufficiently large open pore volume, see 2 , remains to allow cells to settle inside.

After the spinning was complete, it was dried at room temperature for about 6 hours.

1 shows the carrier 1 after the drying step. The polymer fibers are a little thicker, they do not yet have the final geometry that they only obtain when a subsequent heat treatment has been carried out.

This heat treatment was carried out at 60 ° for 30 minutes. It drives off the dioxane and ultimately cross-links the polymer fiber, which means that it also shrinks and takes its final shape, as in 2 is shown in the enlarged micrograph. The polymer fibers can be seen to extend 5 in the form of turns around the bar framework 2 , so embrace this, but reduce the open pore volume on the outside of the bar framework 2 not worth mentioning. They form a kind of firm, but elastic cocoon around the bar framework, what the wearer 1 makes it much more stable against any shear or pressure loads.

3 Figure 3 shows a second embodiment of a bioactive carrier 1 , the same reference numerals being used for the same components. There is also a bar framework here 2 to use, according to DE 10 2018 114 946 B3 which was also superficially leached. However, it does not consist of a quasi spongy structure, but was made up of individual, randomly arranged glass fibers 6 (see. 6 ) that were sintered together. Independent of this, there is also a reinforcement structure here 3 in the form of a fiber structure 4th consisting of wrapped polymer fibers 5 provided, which in turn is on the outside around the bar framework 2 are spun.

While 3 the carrier 1 shows after spinning and drying, shows the enlarged microscope picture according to FIG 4th the carrier 1 excerpts after the heat treatment, which was also carried out at 60 ° for 30 minutes. As can be seen, a stable outer fiber structure, the glass bar structure, is also produced here 2 surrounds and stiffened on the outside.

The polymer solution used was the same as for the exemplary embodiment according to FIG 1 and 2 described.

The 5 - 7th show another embodiment of a bioactive carrier 1 , in which the reinforcement structure as inside the three-dimensional bar framework 2 formed polymer fiber structure or is designed as a polymer structure. This carrier too 1 has a glass bar framework 2 on, which is a one-piece three-dimensional bar framework and again according to DE 10 2018 114 946 B3 was produced.

Here, too, a polymer solution is produced which, in the example shown, contains 5% by mass of polymer (poly (D, L-lactide-co-glycolide) in a water / 1,4-dioxane mixture with a ratio of 13:87 is applied drop by drop onto the bar framework at approx. 50 - 60 ° with a syringe 2 , so given the bioglass scaffold so that the drops of solution sink in. As soon as the bar framework 2 is soaked with the polymer solution, the soaked bar framework 2 placed on an absorbent paper, whereby the polymer solution is removed from the framework surface and partly from the adjacent interior. This serves to avoid any skin formation in the area of the surface, which can lead to an at least partial pore closure on the outside.

The soaked bar framework is then quickly placed in a water bath heated to 50 ° C., which is placed on a temperature-controlled heating plate. After about 40 seconds, the bar framework is removed and placed on absorbent paper in order to pre-dry it there, after which the final drying takes place at room temperature.

By introducing the soaked bar framework into the water bath, the solubility limit is undershot, which leads to precipitation of the polymer in a foam-like consistency or form. 5 shows a photograph of the carrier after drying, in which the polymeric reinforcement structure 3 is visible in the form of the not yet fully crosslinked fiber structure. There are still relatively large zones with a foam-like polymer framework structure, as is also shown in the SEM image in 6 are shown. 6 also shows that this reinforcement structure or polymer foam structure is formed in the area of the surface as well as in the interior.

After drying, a heat treatment is carried out by removing the dried bar framework 2 heated to 95 ° C and held for about 30 minutes, after which it cools. 7th shows an enlarged SEM image of the carrier 1 with the now fully formed polymeric reinforcement structure 3 that have a fiber structure 4th forms their fibers 5 extend from jetty to jetty and virtually connect them. Nevertheless, the open pores are ensured, like 7th shows. The fibers 5 have, similar to the embodiment according to 1 - 4th , a diameter or a thickness of approx. 2 - 100 µm.

8th finally shows an embodiment of a carrier 1 in the form of an SEM image, again comprising a bar framework 2 , which here consists of a large number of individual glass fibers 6 is formed, which are connected to each other by sintering. Also this bar framework 2 is superficially leached to reduce the alkali metal oxide content.

Again, there is a reinforcement structure 3 formed in the form of an inner polymer framework structure using the same polymer solution and the same procedure as for the embodiment according to FIG 5 - 7th was produced. 8th shows the carrier 1 after the heat treatment, during which the formerly foam-like polymer scaffold structure becomes the finished fiber structure 4th with the individual who is about the fibers 6 formed webs connecting polymer fibers 5 has changed.

As already described in the introduction, the bar framework is leached out by acid attack in order to reduce the alkali metal oxide concentration at least on the surface. This acid treatment can either be done before applying the reinforcement structure 3 , ie before the spinning around or before the formation of the inner polymer framework structure, if the bar framework is still sufficiently stable and thus manageable after this acid treatment, and the application of the reinforcement structure 3 is possible without any problems. Should be due to the filigree structure of the bar 2 If the acid attack leads to an unstable bar framework, this leaching can also occur after the polymeric reinforcement structure has been applied 3 take place, i.e. after the last heat treatment has been completed. This is because the polylactides which are preferably used and which are used to form the polymeric reinforcement structure are stable against such acid attack, in which, for example, 0.1 molar hydrochloric acid is used. So they are not damaged by the acid attack and still have their mechanical properties.

As described, the production of the polymeric reinforcement structure is used 3 to do this, the carrier 1 as such, to make it more stable, so that it withstands any mechanical loads better than a pure carrier that does not have a polymeric reinforcement structure and that therefore only consists of the glass bar framework. The aim of polymer reinforcement is that when an implant is used in the human body, the shape of the implant is basically retained under load and that it offers resistance to the load.

To demonstrate this, three different carriers were made. All three examined carriers have the same bioglass bar framework. For this purpose, a bio glass as in DE 10 2018 114 946 B3 and processed accordingly in order to produce a leached bioglass bar framework, as described in this publication.

Embodiment I:

In this exemplary embodiment, the pure glass bar framework was used as the carrier. That according to DE 10 2018 114 946 B3 The bar framework produced was treated with 0.1 molar hydrochloric acid at 37 ° C. for 5 days at a constant pH of 1 without applying a polymeric reinforcement structure.

Embodiment II:

In this exemplary embodiment, the glass bar framework according to FIG DE 10 2018 114 946 B3 provided with a reinforcement structure in the form of a fiber structure produced on the outside by wrapping prior to leaching.

For this purpose, a polymer solution with 10% by mass of polymer (poly (D, L-lactide-coglycolide), double-distilled water and 1,4-dioxane (ratio 13 : 87), which was heated to 70 °. Fibers with a diameter of 5-80 µm were drawn from the solution with a syringe tip and wrapped around the bioglass bar framework.

After the spinning was complete, drying was carried out at room temperature for 6 hours and a subsequent temperature treatment at 60 ° for 30 minutes. After this temperature treatment, the bar framework including the polymer reinforcement was treated with 0.1 molar hydrochloric acid at 37 ° C. for 5 days at a constant pH of 1.

Embodiment III:

Here, too, a bioglass bar framework was used DE 10 2018 114 946 B3 used, which was provided with an internally formed polymer reinforcement structure prior to leaching. For this purpose, a polymer solution with 5% by weight of poly (D, L-lactide-co-glycolide) and a mixture of double-distilled water and 1,4-dioxane in the ratio 13 : 87 made.

The solution was then added drop by drop to the bioglass bar framework at approx. 60 ° with a syringe so that the drops of solution sink in. After the bar framework was completely soaked, the bar framework was placed on absorbent paper so that the polymer solution was removed from the framework surface and adjacent volume areas in order to avoid skin formation. The soaked bar framework was then placed in a water bath which was heated to about 50 ° and stood on a temperature-controlled heating plate. After about 40 seconds, the bar framework was removed and placed on absorbent paper for predrying, after which it was finally dried at room temperature. This was followed by a temperature treatment at 60 ° C. for 30 minutes in order to form the final polymer bar structure integrated in the interior of the glass bar structure. After this treatment, the carrier including the polymer backbone reinforcement was treated with a 0.1 molar hydrochloric acid at 37 ° C. for 5 days at a constant pH of 1.

These three different carriers were then subjected to a load test in which a stamp was pressed with a defined force on the respective carrier. The result is in 9 shown, where in 9 along the abscissa the compression in µm, that is to say the path of the pressure stamp with which the carrier is pressed, is plotted, while the force in mN is plotted along the ordinate.

Three curves I, II, III are shown corresponding to the exemplary embodiments I, II, III.

Curve I shows the force-displacement curve for exemplary embodiment I, that is to say for the unreinforced, leached carrier consisting only of the glass bar framework. It can be seen that a pressure load on the leached bar framework leads to the bars breaking continuously, as the jagged course of curve I over the compression path clearly shows. The webs collapse under relatively low loads elastic behavior cannot be recognized. The bar breaks apart and is pressed apart, so that at the end of the experiment many individual parts are left over.

10 shows a picture of the carrier 1 according to embodiment I after carrying out the experiment. The glass carrier can be seen 1 broken into a multitude of small pieces.

Curve II shows the force-displacement curve of a leached bar framework with a polymeric reinforcement structure obtained by wrapping. The course of the curve shows that the bar framework can be easily compressed at the beginning of the compaction, but then the effect of the wrapping as a framework stabilization comes into play. A driving apart or pressing apart of the bar framework is avoided, and increased resistance to compression is achieved. The lack of serrations compared to curve I shows that there is no fracturing of the bar framework. This is also evident in the 11 who have favourited the carrier 1 shows according to embodiment II after carrying out the experiment. The carrier 1 The glass bar framework shows no impairment or damage 2 is intact, the carrier 1 consequently retains its shape. A fracture is avoided, the dimensional stability is due to the elastic behavior and the stiffening due to the polymeric reinforcement structure 3 ensured.

A similar, even slightly better result is shown by curve III for embodiment III. This carrier has a polymeric reinforcement structure integrated in the glass bar structure, that is to say that a polymeric reinforcing bar structure is integrated in the glass bar structure. The course of curve III shows that the bar structure shows a comparatively elastic behavior after the start of the load, consequently a structure stabilization is given and, as the steep curve towards the end shows, an increased resistance against further compaction is achieved. Here, too, the lack of serrations compared to curve I shows that there is no fracturing of the glass bar framework. This also shows here 12 who have favourited the carrier 1 with its bar framework 2 and the integrated polymer framework 3 shows in undamaged form after carrying out the test.

This is also achieved here through the integration of the polymeric framework-like reinforcement structure 3 a clear stiffening and thus stabilization of the leached glass bar framework 2 reached and the carrier 1 given an elastic behavior that brings about dimensional stability.

As described, a bioactive carrier according to the invention with polymer reinforcement serves to colonize and multiply living cells on it. To demonstrate that this is successful, colonization attempts were made on various carriers 1 , which were produced according to embodiments II and III either with fiber braiding or with an integrated polymer fiber framework structure, carried out using different cells.

The cell types used were either porcine joint chondrocytes (pGC) or mesenchymal stromal cells from human bone marrow (hMSC).

To carry out the experiments, appropriately prepared cell suspensions were pipetted onto appropriately prepared bioactive carriers according to the invention and then incubated in an incubator.

• - Einlegen der bioaktiven Träger unter der Sterilbank in ein steriles 50 ml Bioreaktorröhrchen mit Filtercap
• - Behandlung der für die Scaffoldbesiedlung vorgesehenen Zellen (pGC und hMSC) nach einem Spülschritt mit PBS für 5 min in einem Inkubator mit Trypsin/EDTA-Lösung (0,05/0,02 %) zum Ablösen der Zellen vom Kulturboden der für die Zellvermehrung verwendeten Zellkulturflaschen
• - pGC: Zugabe von 5 ml 10 % fetales Kälberserum (fetal calf serum: FCS) enthaltendes Kulturmedium (Ham's F-12/Dulbecco's Modified Eagle's) [DMEM] Medium 1: 1), 10.000 IU/mL Penicillin/10.000 µg/mL Streptomycin, 2,5 µg/ml Amphotericin B, nicht essentielle Aminosäuren und 25 µg/ml Ascorbinsäure
• - hMSC: Zugabe von 5 ml Stammzell-spezifischem Expansionmedium (FG0445/Dulbecco's Modified Eagle's [DMEM]/HG) mit 5 % Plättchenlysat (PL), 50 IU/ml Streptomycin, 50 IU/ml Penicillin,
• - 2,5 µg/ml Amphotericin B, 100 mM Natriumpyruvat und 5000 U/ml Heparin
• - Stoppen des Ablösevorgangs durch die Zugabe von 5 ml 10 % FCShaltigem Medium, wobei das FCS bzw. das PL die enzymatische Aktivität des Trypsins beendet
• - Zentrifugation der Zellsuspension bei 300 U/min oder 484 g für 5 min
• - Resuspension des sich aus der Zentrifugation ergebenden Zellpellets in 1 ml Kulturmedium
• - Mischen von 10 µl Trypanblau-Lösung (1 %) mit 10 µl Zellsuspension, so dass sich tote Zellen anfärben
• - Pipettieren von 10 µl aus dieser Mischung in eine Neubauer-Zählkammer und Zählen der lebenden Zellen
• - Pipettieren der Zellen in einer Konzentration von 500.000 bis 1.000.000

Zellen pro Scaffold in einem Volumen von 1 mL pro Scaffold

• - Nach 20 min Auffüllen des Kulturmediums auf 10 ml Volumen zur Sicherstellung einer Versorgung in der nachfolgenden dynamischen Kultur
• - Kultivierung der besiedelten bioaktiven Träger im Inkubator bei 37 °C und 5 % CO₂ auf einem Orbitalschüttler (dynamisch)
• - bei längerer Inkubationsdauer Wechsel des Kulturmediums alle zwei Tage.

The implementation was as follows:

• - Place the bioactive carriers under the sterile bench in a sterile 50 ml bioreactor tube with a filter cap
• Treatment of the cells intended for scaffold colonization (pGC and hMSC) after a rinsing step with PBS for 5 min in an incubator with trypsin / EDTA solution (0.05 / 0.02%) to detach the cells from the culture soil for cell reproduction used cell culture flasks
• - pGC: addition of 5 ml of 10% fetal calf serum (fetal calf serum: FCS) containing culture medium (Ham's F-12 / Dulbecco's Modified Eagle's) [DMEM] medium 1: 1), 10,000 IU / mL penicillin / 10,000 µg / mL streptomycin , 2.5 µg / ml amphotericin B, non-essential amino acids and 25 µg / ml ascorbic acid
• - hMSC: addition of 5 ml stem cell-specific expansion medium (FG0445 / Dulbecco's Modified Eagle's [DMEM] / HG) with 5% platelet lysate (PL), 50 IU / ml streptomycin, 50 IU / ml penicillin,
• - 2.5 µg / ml amphotericin B, 100 mM sodium pyruvate and 5000 U / ml heparin
• - Stopping the detachment process by adding 5 ml of 10% FCS-containing medium, the FCS or the PL ending the enzymatic activity of the trypsin
• - Centrifugation of the cell suspension at 300 rpm or 484 g for 5 min
• - Resuspension of the cell pellet resulting from centrifugation in 1 ml of culture medium
• Mixing 10 µl trypan blue solution (1%) with 10 µl cell suspension so that dead cells are stained
• - Pipetting 10 µl of this mixture into a Neubauer counting chamber and counting the living cells
• - Pipetting the cells in a concentration of 500,000 to 1,000,000

Cells per scaffold in a volume of 1 mL per scaffold

• - After 20 min, the culture medium is made up to a volume of 10 ml to ensure a supply in the subsequent dynamic culture
• - Cultivation of the colonized bioactive carriers in the incubator at 37 ° C and 5% CO ₂ on an orbital shaker (dynamic)
• - Change the culture medium every two days for longer incubation periods.

• - Herstellung einer Vitalitätsfärbe-Lösung aus 15 µg/ml Fluoreszeindiazetat (FDA) und 10 µg/ml Propidiumiodid (PI) gelöst in PBS
• - Entnahme des jeweiligen bioaktiven Trägers aus der dynamischen Kultivierung im Inkubator
• - Pipettieren von 50 µl Vitalitäts-Lösung auf den besiedelten bioaktiven Träger
• - Begutachtung der Zellen: auf dem bioaktiven Träger sind lebende Zellen aufgrund der Einfärbung mit der Vitalitäts-Lösung grün und tote Zellen rot mit Hilfe eines konfokalen Laser-Scanning-Mikroskops differenzierbar.

To check that the polymer reinforcement had no negative effects, pictures were taken of the in vitro culture after various times / days using a laser scanning microscope, for which a vitality staining was necessary. This was done as follows:

• - Preparation of a vitality staining solution from 15 µg / ml fluorescein diacetate (FDA) and 10 µg / ml propidium iodide (PI) dissolved in PBS
• - Removal of the respective bioactive carrier from the dynamic cultivation in the incubator
• - Pipetting 50 µl vitality solution onto the colonized bioactive carrier
• - Assessment of the cells: on the bioactive carrier, living cells can be differentiated due to the coloring with the vitality solution green and dead cells red with the help of a confocal laser scanning microscope.

The PBS serves as a buffer, and FDA is used as the dye. This dye penetrates the living cells and is enzymatically converted into a fluorescent compound, which is excited with a wavelength of 525 nm. PI intercalates into the DNA of dead cells because their cell and nuclear membranes become permeable. PI is excited with a wavelength of 600 nm.

The corresponding results are in the 13 - 18th shown.

Basically show in the 13-18 the light areas the areas in or on the wearer 1 those with living, multiplying cells 8th are populated. The dark areas are primarily unpopulated parts of the wearer 1 .

13 shows a representative vitality record for porcine joint chondrocytes after 7 days in culture, which are settled on a bioglass bar framework with a polymer reinforcement formed by spinning. A web can be seen, for example 7th of the bar framework 2 on which there are cells 8th have settled. A pore can also be seen 9 within the bar framework 2 as well as an imprint of a polymer fiber 5 in the cell layer. One can also see one with cells 8th colonized polymer fiber 5 .

14th shows the carrier 13 with the porcine joint chondrocytes settling on it after 28 days in culture. Cell growth has continued continuously, the bioactive carrier is more densely populated.

15th shows a representative vitality record for human mesenchymal stromal cells after 7 days in culture, which have also grown on a bioglass bar framework with polymer reinforcement formed by spinning, ie on the same carrier as in the 13 and 14th .

15th shows that this other cell type after the same culture time ( 7th Days) has spread much more widely than the joint chondrocytes according to the 13 . Almost complete closure of the pores of the bar framework, which as such can no longer be identified exactly, can be seen after just 7 days. A larger pore can also be seen here 9 as well as several smaller pores 9 of the bar framework, as well as impressions of the polymer fibers 5 within the cell layer.

16 shows the carrier 15th with mesenchymal stromal cells after 28 days in culture. Obviously one can hardly see individual cells here. The entire carrier 1 is as good as filled with grown cells, the cell lawn now covers the surface of the carrier 1 .

Both experiments show that a carrier reinforced by wrapping is ideally suited for colonization with cells and their reproduction, with different cell types growing at different speeds.

17th shows a representative vitality record for human mesenchymal stromal cells after 7 days in culture on a bioglass bar framework with integrated polymer reinforcement produced from a foam precipitation.

18th shows the carrier 17th after 28 days in culture. A comparison of 17th and 18th clearly the increase in cell area over time. Already after 7 days, larger cell clusters form on both the glass bars and the polymer fibers, such as the light spots in 17th demonstrate. These cell clusters enlarge significantly after 14 days in culture, which means that a carrier with integrated polymer reinforcement is also very well suited for the colonization and multiplication of cells.

The following is a list of the materials and equipment used in the course of the colonization tests carried out and the vitality recordings: Material / device Order number company HERAcell 150i incubator Thermo Electron LED GmbH, DE Centrifuge 5810 R Eppendorf AG, DE Diavert microscope Leitz GmbH, Wetzlar, DE Dulbecco's Modified Eagle's Medium FG4815 Biochrom AG, DE Essential amino acids, 50x MEM-AS K0363 Biochrom AG, DE Ethidium bromide solution, 1% 2218.1 Carl Roth GmbH & Co. KG, DE Fetal calf serum (FBS superior) S0615 1000F Biochrom AG, DE Fluorescein acetate (FDA) F7378-5G Sigma-Aldrich, USA Heraeus Multifuge X1R Centrifuge Thermo Elektron LED GmbH, DE Confocal laser scanning microscope DMi8 Leica, Wetzlar, DE Neubauer counting chamber, improved Brand, DE Paraformaldehyde, 4% (PFA) USBM199431LT VWR, DE Penicilin-streptomycin (10,000 U / ml / 10,000 µg / ml) A2212 Biochrom AG, DE Phosphate Buffered Saline (PBS) L1825 Biochrom AG, DE Orbital shaker, SU1400 for dynamic scaffold cultivation Sustainable Lab Instruments, DE HERAsafe KS sterile bench Thermo Elektron LED GmbH, DE TBS (Tris Buffered Saline) 09-7500-10 Medicago, SWE In tablet form: - Trypan blue, 1% L6323 Biochrom AG, DE - Trypsin / EDTA L2153 Biochrom AG, DE Tubespine®Bioreactor 50 (50 ml volume) cell culture vessels for dynamic culture TPPO, CH Vitamin C (ascorbic acid) A5960 Sigma-Aldrich, USA Tissue culture flask Cell plus T25 / T75 / T175 Sarstedt AG, DE

QUOTES INCLUDED IN THE DESCRIPTION

This list of the documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent literature cited

• WO 2011/161422 A1 [0010]
• WO 2016/089731 A1 [0010]
• WO 2014/168631 A1 [0010]
• DE 102018114946 B3 [0011, 0013, 0019, 0034, 0036, 0044, 0051, 0054, 0057, 0062, 0068, 0071, 0080, 0081, 0082, 0085]

Claims (35)

Bioactive carrier for the settlement of living cells, with a porous three-dimensional bar framework (2) made of glass, characterized by a porous reinforcing structure (3) made of a synthetic polymer that can be dissolved in the human or animal body. Bioactive carrier after Claim 1 , characterized in that the polymer is a polylactide. Bioactive carrier after Claim 2 , characterized in that the polymer is poly (L-lactide), poly (L-lactide-co-glycolide), poly (L-lactide-co-D, L-lactide), poly (D, L-lactide) or poly (D, L-lactide-co-glycolide). Bioactive carrier according to one of the preceding claims, characterized in that the reinforcement structure (3) is a fiber structure (4) surrounding the bar framework (2) on the outside. Bioactive carrier after Claim 4 , characterized in that the reinforcement structure (3) is designed in the form of a wrapping around the web frame (2). Bioactive carrier after Claim 5 , characterized in that the wrapping surrounds the web framework (2) on all sides. Bioactive carrier according to one of the Claims 4 to 6 , characterized in that the surface covered by the fiber structure (4) is a maximum of 10%. Bioactive carrier according to one of the Claims 4 to 7th , characterized in that the fibers (5) of the fiber structure (4) have a diameter of 2-100 µm, in particular 5-75 µm and preferably 10-50 µm. Bioactive carrier according to one of the Claims 1 to 3 , characterized in that the reinforcement structure (3) is a fiber structure (4) formed in the interior of the bar framework (2). Bioactive carrier after Claim 9 , characterized in that the fiber structure (4) is an at least partially connected fiber framework structure. Bioactive carrier after Claim 10 , characterized in that the porosity is at least 80%. Bioactive carrier according to one of the Claims 4 to 8th and one of the Claims 9 to 11 , characterized in that both an outer fiber structure (4) and an inner fiber structure (4) are provided. Bioactive carrier according to one of the preceding claims, characterized in that the bar framework (2) has a porosity of at least 80%. Bioactive carrier according to one of the preceding claims, characterized in that the pore size of the bar framework (2) is between 20-300 µm, in particular 50-200 µm. Bioactive carrier according to one of the preceding claims, characterized in that the webs of the bar framework (2) have a thickness of 10-400 µm, in particular 20-200 µm, preferably between 50-150 µm. Bioactive carrier according to one of the preceding claims, characterized in that the bar framework (2) consists of glass fibers (6) connected to one another by sintering or of sintered glass powder. Bioactive carrier according to one of the preceding claims, characterized in that the bar framework (2) has an alkaline earth metal oxide content of a maximum of 2.0 mass% and is reduced in its alkali metal oxide content at least on the surface, preferably over the cross section of the bar, by leaching. A method of making a bioactive carrier comprising the steps of: a) providing a porous three-dimensional bar framework (2) made of glass, b) producing a porous reinforcing structure (3) that reinforces the bar framework (2) from a synthetic polymer which can be dissolved in the human or animal body. Procedure according to Claim 18 , characterized in that a polylactide is used as the polymer. Procedure according to Claim 19 , characterized in that poly (L-lactide), poly (L-lactide-co-glycolide), poly (L-lactide-co-D, L-lactide), poly (D, L-lactide) or poly (D, L-lactide-co-glycolide) is used. Method according to one of the Claims 18 to 20th , characterized in that a fiber structure (4) surrounding the web framework on the outside, preferably on all sides, in the form of a wrapping around the web framework (2) is applied as the reinforcement structure (3). Procedure according to Claim 21 , characterized in that the wrapping is applied in such a way that the covered surface is a maximum of 10%. Method according to one of the Claims 21 or 22nd , characterized in that a polymer fiber (5) is drawn or spun from a polymer solution, which is then spun around the bar framework (2), after which, optionally after drying, a heat treatment takes place. Procedure according to Claim 23 , characterized in that a polymer solution heated to a temperature of 40-90 ° C is used and / or the heat treatment is carried out at a temperature of 50-200 ° C for 0.2-4 h. Method according to one of the Claims 18 to 21st , characterized in that a fiber structure (4), preferably forming a coherent fiber framework structure, is formed in the interior of the bar framework as the reinforcement structure (3). Procedure according to Claim 25 , characterized in that the bar framework (2) is impregnated with a polymer solution, after which, if necessary after removing the polymer solution from the surface of the bar framework (2), the impregnated bar framework (2) is placed in water so that the polymer precipitates like a foam, after which, if necessary after drying, a heat treatment takes place. Procedure according to Claim 26 , characterized in that the polymer solution has a temperature of 20-90 ° C, and / or that the water has a temperature of 10-95 ° C and the heat treatment at a temperature of 50-200 ° C for 0.2-4 h takes place, the temperature during the heat treatment being higher than the water temperature. Procedure according to Claim 25 , characterized in that the bar framework (2) is impregnated with a polymer solution, after which cooling takes place, so that the polymer precipitates like a foam, after which a heat treatment takes place, optionally after drying. Procedure according to Claim 28 , characterized in that the polymer solution has a temperature of 50-90 ° C and the cooling takes place below the azeotropic point of the polymer solution, and / or that the heat treatment takes place at a temperature of 50-200 ° C for 0.2-4 h . Method according to one of the Claims 25 to 29 , characterized in that the reinforcement structure (3) is formed in such a way that the porosity of the carrier is at least 80%. Method according to one of the Claims 21 to 24 and one of the Claims 25 to 30th , characterized in that both an outer fiber structure (4) and an inner fiber structure (4) are formed. Method according to one of the Claims 18 to 31 , characterized in that a bar framework (2) made of glass is used, which has an alkaline earth metal oxide content of a maximum of 2 mass%, the bar framework (2) being acid-treated before or after generating the polymeric reinforcement structure to reduce the alkali metal oxides at least on the surface. Procedure according to Claim 32 , characterized in that the bar framework (2), with or without the reinforcement structure, is leached with hydrochloric acid, preferably 0.1 molar hydrochloric acid. Method according to one of the Claims 18 to 33 , characterized in that a bar framework (2) with a pore size of 300 µm, in particular 50-200 µm and / or a web thickness of 10-400 µm, in particular 20-200 µm, preferably between 50-150 µm, is used. Method according to one of the Claims 18 to 34 , characterized in that a bar framework (2) made of glass fibers (5) connected to one another by sintering or of sintered glass powder is used.

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