WO2006026981A1

WO2006026981A1 - Implant for treating osteochondral defects and method for producing the same

Info

Publication number: WO2006026981A1
Application number: PCT/DE2005/001585
Authority: WO
Inventors: Michael Gelinsky; Marlen Eckert
Original assignee: Technische Universität Dresden
Priority date: 2004-09-07
Filing date: 2005-09-07
Publication date: 2006-03-16
Also published as: DE102004044102B4; DE102004044102A1

Abstract

The invention relates to an implant for treating osteochondral defects and to a method for producing the same. The implant for treating osteochondral defects comprises at least two superimposed layers, a bottom-most porous bone matrix (2) and a porous cartilage matrix (1), the layer materials being locally mixed at the interfaces of the contacting layers. The inventive method for producing the implant comprises the following steps: a) producing the individual layers or the respective layer material, at least one layer or a layer material being converted to a pasty or liquid state using a solvent that is apt to partially dissolve the surfaces of the other layers, b) layering or putting the layers or layer materials one on top of the other, and c) lyophilizing the composite.

Description

Implant for the treatment of osteochondral defects, and process for its preparation

The invention relates to an implant for the treatment of osteochondral defects, and to a method for its production.

Osteochondral defects, ie defects that involve both the articular cartilage and the bone directly beneath it, can arise in two ways: on the one hand by advanced inflammatory processes such as arthrosis, which have already damaged the bone next to the cartilaginous tissue, and on the other hand by injuries or far advanced wear.

Osteochondral defects are also partly consciously produced by the doctor for therapeutic reasons: since a pure cartilage implant can hardly be anchored in the cartilage layer of the joints, which is only 2 mm to 5 mm thick, sometimes not only the damaged cartilage tissue is removed, but the defect as well intentionally enlarged in the direction of the underlying bone, in order then fix an implant then better.

To treat the defects, both implants derived from natural tissue and implants with tissue replacement materials, most commonly made by tissue engineering, are used. For the production of implants by tissue engineering, in the first step an extracellular structure-imitating matrix with many cavities of preferably biodegradable materials is created, the cavities of which are then colonized in the next step with predominantly autologous cells and filled by creating appropriate growth conditions. It is also known to immobilize cells in gelatinous carrier materials. Matrix material and cells are specifically selected according to whether a bony or a cartilaginous tissue is to be implanted.

Implants to be used at the interface of two or more different tissue types are optimally biphasic or polyphasic in order to meet the requirements of all the respective surrounding tissues with which the implant comes into contact.

Previously known biphasic implant materials for osteochondral defects each consist of two completely different material components, which are then assembled to form a workpiece. For the bone matrix z. B. porous Calcium phosphate ceramics (ß-TCP) and gels used for cartilage matrix. The problem with these composite implants is the connection of the different components. According to the prior art, this z. As by gluing with fibrin glue, which, however, is not equal to the occurring in the joint Scherbelasrungen in the long run.

DE 197 21 661, for example, describes a bone-cartilage implant which consists of a plurality of rods regularly arranged in a grid. The connection of the lattice structures happens on the one hand by gluing and on the other hand by fusing or welding of the mesh materials. The latter creates a firmer bond, but requires appropriate materials.

In WO 97/46665 of Fig. 2, a biphasic implant is described. A chamber is closed on one side by a porous bone matrix. The chamber is populated with cartilage cells. The cartilage cells grow into the bone matrix. As a result of this and the production of the cartilage matrix, a firm bond of both layers is produced without the need for an adhesive bond. The colonization with the cartilage cells must be done before the operation.

The object of the invention is to specify an implant for the treatment of osteochondral defects in which the bone and cartilage matrix are firmly connected to one another.

The object is achieved according to the invention by an implant having at least two superimposed layers, including a porous bone matrix and a porous cartilage matrix, wherein the layer materials are locally mixed at the interfaces of the contacting layers.

The local mixing causes the layers to intermesh at their points of contact down to the molecular level. In a narrow area, the layer materials penetrate. In the interface region, comparatively similar linkages of the matrix molecules are present as within the layers. As a result, the layers are strongly bonded even without glue. The compound is also resilient to shear forces.

Such an implant with at least two layers, including a porous bone and a porous cartilage matrix, is produced according to the invention with the steps of a) producing the individual layers or the respective layer material, wherein at least one layer or a layer material with a solvent which at least the Can dissolve surfaces of the other layers, is converted into a pasty or liquid state, b) superimposing or layers of the layers or layer materials and c) freeze-drying of the composite.

Because the layers are at least partially in flowable or pasty form and contain a solvent that can also dissolve the surface of the adjacent layer, the layers mix locally at the contacting interfaces.

The solvent of the layers is selected so that during freeze-drying, i. H. by applying a vacuum to the frozen material, passes directly from the frozen state to the gaseous state. Besides water, acetic acid and cyclohexane are further preferred solvents.

The pore size of the matrix layers is preferably adjusted by freezing. By freeze-drying, pores remain at the points where solvent crystals were contained in the frozen state. A sponge-like, foam-like material is formed which contains a plurality of randomly distributed pores arranged in an outwardly open pore system. (See M. Gelinsky, U. König, A. Sewing, W. Pompe: Porous scaffolds of mineralized collagen - a biomimetic bone substitute material., Materials Science., 2004, 35, 229-233).

The implant can advantageously be shaped as desired and thus adapted in its outer geometry to the size and structure of the bone and cartilage tissue to be replaced.

The implant according to the invention is elastic in the wet state and thus adapts to the defect geometry, which guarantees good contact between the tissues and the implant material. In addition, the elasticity allows precultivation with cells under cyclic mechanical stimulation. Its compressive strength is higher than that of conventionally produced, uncrosslinked collagen sponges of comparable porosity, the pores of which usually collapse when moistened; It is therefore preferably used for the therapy of minor to moderate defects. In cyclic pressure experiments, an implant with cylinder geometry with a 30% uniaxial compression in the direction of the cylinder axis preferably has an E-mode of 10-40 kPa, more preferably 15 to 25 kPa.

The implant is porous, with outwardly open and interconnecting pores, preferably completely resorbable, and may be either cell-free or in vitro pre-populated with one or more cell types. The average pore size is preferably at least 80 .mu.m, more preferably 100 to 200 .mu.m, a diameter which is optimal for colonization with cells. Bone content and cartilage proportion preferably have a comparable porosity (same average pore size, distribution of pores). By resorbable material is meant here and in the following text that the material is slowly degraded after implantation in the body by biological or chemical processes or slowly dissolves. Partial absorption of the material may also be used during cell culture in vitro.

The implant is fully resorbed in the body within 3 to 24 weeks, preferably between 4 and 8 weeks, and converted into living tissue.

For treating an osteochondral defect, the non-mineralized portion of cartilage replacement is preferably populated with autologous cartilage cells (chondrocytes). The bone content is in a particular embodiment pre-colonized with autologous bone cells (osteoblasts) or autologous mesenchymal stem cells of the patient.

The bone matrix consists essentially of fibrillar collagen type I, which is preferably mineralized with calcium phosphate, more preferably with hydroxyapatite (HAP) - and thus mimics the composition of the extracellular matrix of the bone tissue.

The mineralized collagen is preferably prepared by the process already known from EP0945146 or EP0945147.

The cartilage matrix preferably consists of fibrillar, non-mineralized collagen type I and / or collagen type II or hyaluronic acid or hyaluronic acid derivatives or of a composite of collagen and hyaluronic acid or hyaluronic acid derivatives and optionally other constituents of the extracellular cartilage matrix, such as chondroitin sulfate, dermatan sulfate , Keratan sulfate and / or heparan sulfate.

Preferably, the composite of fibrillar collagen and hyaluronic acid is formed in that the hyaluronic acid is present during fibrillogenesis of the collagen and thus at least partially incorporated into the collagen fibrils forming during fibrillogenesis. This creates a composite material in which both components are homogeneously distributed and interconnected.

The collagen for the bone matrix is preferably Type I which has been recombinantly produced as human collagen or isolated from bovine tendons or other animal sources and is approved for clinical use. Preferably, an acid-soluble collagen is selected, such as. B acid-soluble bovine collagen I from the manufacturer Syntacoll (Saal / Danube, Germany).

The collagen for the cartilage matrix is preferably type I or II or a mixture of both types. It is also preferably recombinantly produced as human collagen or isolated from calf skin or other animal sources and should be approved for clinical use, such as the product Collaplex 1.0 from the manufacturer GfN (Waldmichelbach, Germany).

The hyaluronic acid for the cartilage matrix is preferably one produced by microorganisms (eg Streptococcus equi). The term hyaluronic acid also includes its salts, such as. As the product offered by Fa. Fluka product hyaluronic acid sodium salt having a solubility in water of 5 mg / ml. Preferred hyaluronic acid derivatives are hyaluronic acid esters (eg benzyl esters).

In a particular embodiment of the implant lies between the layers for the bone substitute and for the cartilage replacement a film-like, resorbable biopolymers existing pore-less or -arme intermediate layer.

The intermediate layer preferably consists of a biopolymer which is dissolvable at least on the surface by the solvent of the layers for the bone substitute and the cartilage replacement. As a result, the materials of the contacting layers are also locally mixed at the respective interfaces.

Preferably, the intermediate layer consists of a film of hyaluronic acid or hyaluronic acid Drivaten or a film of another glycosaminoglycan, such as. B. chondroitin sulfate, dermatan sulfate, keratan sulfate and heparan sulfate, the intermediate layer is preferably dried in air. Compared to the bone or cartilage matrix, it is largely pore-free and thus impermeable to chondrocytes and / or osteoblasts or mesenchymal stem cells. It thus fulfills a dual function. In terms of strength of the implant connects them and in terms of ingrowth of the cells acts as a release layer.

In a preferred embodiment, the implant includes a support structure with openings. The support structure stiffens the implant. Through the openings, the layer materials can touch each other directly and also mix locally Preferably, a two- or three-dimensional embroidery of resorbable and biocompatible polymer fibers (preferably of poly-L-lactide, poly-DL-lactide, polyglycolide, polyhydroxybutyrate or copolymers thereof) is used as the support structure.

In a further embodiment, the support structure is ordered from a fiber-flocked support material according to DE 103 12 144.7, wherein the distance between the flock fibers must be dimensioned so that the liquid matrix materials can completely fill the interstices. The preferred spacing of the fibers is approximately 200-1000 μm.

The parallel arrangement of the flock fibers achieves mechanical stabilization in the direction of the fibers.

As materials of the base material, of the fibers and of the adhesive, it is possible to use in principle all absorbable materials mentioned in DE 103 12 144.7.

Preferred materials for the fibers are polyhydroxybutyrates or polylactides, and gelatin is preferably used as the adhesive.

The carrier material according to DE 103 12 144.7 preferably consists of a layer of a base material which is flocked on one side with fibers. Preferably, the base material itself consists of a tape of mineralized collagen and forms the lowest layer of the bone replacement portion. The porous bone substitute material portion is preferably on the mineralized collagen tape and surrounds the fibers, but preferably only the lower part or ends (i.e., the ends of the fibers bonded to the base material). The cartilage replacement share layer preferably adjoins the bone substitute material portion layer in such a way that some of the fibers or the tips of the fibers still protrude into the cartilage matrix.

In a particular variant of the implant according to the invention, the bone replacement component and / or the cartilage replacement component contain active substances, such as, for example, As pharmaceutical substances, amino acids or amino acid derivatives, peptides, proteins, growth factors, cytokines, antibodies, oligonucleotides, cDNA, DNA fragments, nucleic acids or nucleic acid derivatives.

Advantageously, the method can easily implants in any shape and size and thus - for the production of implants for osteochondral defects - also produce an individual thickness ratio of bone replacement and cartilage replacement. The stacking is preferably done by pouring over in a three-dimensional container. At the time of stacking, the matrix materials are preferably present in pasty or liquid form.

The geometry and size of the implant are determined by the geometry and size of the container used for stacking the layers, which can advantageously be shaped as desired. In addition, both the frozen body (before lyophilization) and the freeze-dried implant can be machined by cutting, milling, drilling, or the like, and thus advantageously further adjusting the size and shape of the implant.

The microstructure of the pores, in particular the pore size, of the implant is dependent on the size of the solvent crystals formed during the freezing. This can be advantageously adjusted by the speed and temperature of the freezing. Pores having an average diameter of 100 to 250 μm, which is optimal for ingrowth of cells, are advantageously formed by slow freezing at -5 ° C to -50 ° C, preferably -15 ° C to -35 ° C. The slow freezing is achieved, for example, by placing the stacked bone substitute portion and the cartilage replacement portion, or the container containing it, in a freezer preheated to the corresponding temperature.

The microstructure of the pores is also influenced by the solvent content of the layers for the bone substitute fraction and the cartilage replacement fraction. In order to obtain the desired, high porosity, both layers preferably contain 75 to 98%, particularly preferably 80 to 90%, of solvent before freezing

In a particular variant of the method, first the one layer is poured into a container and frozen. Before or during the superposition with the second layer, the first (frozen) layer on the surface is then thawed. The second layer then mixes locally with the thawed surface. For this, the second layer is either poured onto the thawed surface, or even slightly heated, so that the surface of the first layer is thawed by the superposition itself. Preferably, the frozen layer is adapted to the desired shape by cutting, milling, drilling or the like before the superimposing. This variant thus has the advantage that the composite surface between bone replacement portion and cartilage replacement portion designed arbitrarily three-dimensional and adapted to the natural shape of the joint can be. In addition, the strength of the implant over a two-dimensional composite surface can be increased by a three-dimensional composite surface.

In a preferred embodiment of the invention, the implant obtained after lyophilization is chemically cross-linked to stabilize the layers in themselves by the introduction of chemical bonds and to firmly bond them together. The introduction of chemical bonding is preferably done with a chemical crosslinker, such. As glutaraldehyde or a carbodiimide derivative, particularly preferably with EDC (N-dimethylaminopropyl-N'-ethyl-carbodiimide hydrochloride). For example, by using EDC as a chemical crosslinker, amino groups of collagen are covalently linked to aryl bonds with carboxyl groups and carboxyl groups of collagen or hyaluronic acid are covalently linked to hydroxyl groups of hyaluronic acid to form ester bonds.

For mechanical reinforcement, a support structure, preferably of resorbable and biocompatible polymer fibers (preferably of poly-L-lactide, poly-DL-lactide, polyglycolide, polyhydroxybutyrate or copolymers of these), are introduced into the implant. For this purpose, a two- or three-dimensional fiber structure, preferably produced by embroidering, is placed in the mold before filling in the first phase. The pourable solution or suspension of the first phase is then over poured so that the spaces between the fibers are filled. For this purpose, a slight negative pressure can be applied or briefly centrifuged. Then, as described, the second phase is overcoated, the construct is frozen and freeze-dried. The fiber structure can either be in the entire implant or only in one of the two phases.

To produce an implant which contains a carrier material produced by means of flock technology according to DE 103 12 144.7, the procedure is analogous: the carrier material, with the base material downwards, is placed in the mold in which the implant is to be produced. Then, as described, the two solutions or suspensions are successively übereinandergegossen. For a better penetration of the solutions or suspensions in the fiber interstices, a slight negative pressure can be applied or briefly centrifuged. The flock fibers can either penetrate the implant in its entire height, or only a part of the same. In direct contact with the base material, either the bone replacement portion or the cartilage replacement portion may be present. For the preparation of implants which have been functionalized with active ingredients, various methods can be used. The active ingredients can be added either one or both phases before stacking, or after the first freeze-drying (and thus before the chemical cross-linking). The active compounds can be suspended in solid form in the solutions or suspensions or else applied as a solution to the freeze-dried implant. Sensitive active substances, in particular growth factors, which could be impaired by the process steps in their biological activity, are preferably applied only after the chemical crosslinking and the second freeze-drying. Another variant represents the functionalization of the implant with active ingredients directly before the operation: in this case, the active ingredient or the active ingredients are applied in sterile form to the likewise sterile implant, preferably as a solution dripped. The bone replacement portion and the cartilage replacement portion can be functionalized in all cases either with the same active ingredients, or with different - or else only the bone or cartilage replacement portion is functionalized with one or more active ingredients.

The invention will be explained in more detail with reference to the figures and exemplary embodiments described below. In detail these are:

Fig. 1 shows a schematic representation of an osteochondral defect A, in which both the articular cartilage B and the underlying (subchondral) bone C is affected.

2 shows schematically on the left the construction of a biphasic implant for osteochondral defects with cylinder geometry, which is constructed from a cartilage matrix 1 and a bone matrix 2. The cartilage matrix 1 consists of a collagen-hyaluronic acid composite, the bone matrix 2 of mineralized collagen. On the right, in the form of a longitudinal section, the structure of an individually shaped, biphasic implant is shown schematically, in which the bone matrix 2 is connected only to partial regions of the cartilage matrix 1.

For the production of these implants according to FIG. 2, a suspension of mineralized collagen fibrils and for the cartilage matrix 1 a suspension of collagen-hyaluronic acid is first prepared for the construction of the bone matrix 2. a.) Preparation of the Hydroxylapatite Mineralized Collagen Fibril Suspension:

In a 2 liter Erlenmeyer flask, 1 g of acid-soluble collagen (collagen type I isolated from bovine tendons from Innocoll in Saal / Donau, Germany) in 1 l of 10 mmol / l HCl (prepared from 100 ml of 0.1 mol / 1 HCl and 900 ml of deionized water) with vigorous stirring (large stirring / magnetic stirrer). With vigorous stirring, it is then added in order:

1) 180 ml of 0.1 mol / 1 CaCl ₂ solution

2) 120 ml 2 mol / 1 NaCl solution

3) 168 ml of 0.5 mol / l TRIS buffer solution (pH = 7.5)

4) 500 ml of deionized water

5) 22.6 ml Sorbensen phosphate buffer (0.5 mol / l, pH = 7.4)

Upon addition of the phosphate buffer, a milky, colorless turbidity of the solution occurs (precipitated calcium phosphates). Finally, it is made up to 2.0 liters with deionized water. After still for about 1 min. vigorously stirred, the flask is sealed with paraffin or aluminum foil and placed him for 12-24 h in a heated to 35 ⁰ C heat bath.

The mineralized collagen is separated by centrifugation. For this purpose, the gelatinous precipitate is stirred vigorously and then filled into suitable centrifuge tubes (eg metal cup for 200 ml or glass tube for 50 ml). It is centrifuged for 15 min at 2500 rpm in a refrigerated centrifuge (to prevent heating of the product) and then poured off the supernatant; 120 ml of the supernatant are stored for later use. Now refill the collagen suspension and repeat the centrifugation until all 2 liters have been treated appropriately. After the last time the supernatant is poured off, the pellets are removed from the tubes with a spatula and collected in a 125 ml beaker. While stirring with a heavy stirring fish just enough mother liquor is added dropwise until a pourable suspension is formed.

This process for the production of mineralized collagen substantially corresponds to EP0945146 (J.-H. Bradt, K. Weis, W. Pompe). Alternatively, the mineralized collagen is prepared from recombinant collagen according to the method described in EP0945147. b.) Preparation of the suspension of collagen and hyaluronic acid:

To build up the cartilage replacement portion 1, a suspension of collagen and hyaluronic acid with 20% by weight hyaluronic acid is first prepared. For this purpose, 200 mg of hyaluronic acid (hyaluronic acid sodium salt from Streptococcus equi, Fa. Fluka (Sigma / Aldrich, Taufkirchen) dissolved in 40 ml of deionized water and stirred for 1 h until a colorless, transparent suspension of high viscosity is formed.

In the meantime, 1 g of acid-soluble collagen (eg Collaplex 1.0, calfskin type I collagen isolated from GfN, Waldmichelbach) is added to 1 l of 10 mM HCl in an Erlenmeyer flask and dissolved with stirring (magnetic stirrer). Thereafter, 80 ml of Sörensen's phosphate buffer (0.5 M, pH = 7.4) and 130 ml of 2 M NaCl are added, the solution is made up with 750 ml of deionized water and stirred again. With vigorous stirring, the hyaluronic acid solution is added. The flask is then placed in a heated to 37 ⁰ C heat bath for about 14 h. The resulting composite of fibrillar collagen and hyaluronic acid precipitates as an insoluble precipitate. The suspension is stirred up, filled into suitable centrifuge glasses and centrifuged for 15 min at 2500 rpm. From the resulting supernatant about 100 ml are stored. The pellets of the collagen-hyaluronic acid composite produced by the centrifugation are thoroughly stirred in a beaker with just enough supernatant (magnetic stirrer) until a homogeneous suspension which can be pipetted is formed. c.) Production of the biphasic, porous implant:

To prepare the biphasic, porous implant, approximately 1 ml of the suspension of mineralized collagen fibrils prepared according to a) is now introduced with the aid of a pipette into the well of a 24-well plate. Then, carefully and in such a way that the two phases do not mix, pipette 1 ml of the collagen / hyaluronic acid suspension described under b). The corrugated sheet is then frozen at about -25 ° C and then freeze-dried in an oil pump vacuum (about 0.1 mbar). The resulting porous, sponge-like scaffold is removed from the corrugated sheet and chemically cross-linked for one hour in a 1% solution of N-dimethylaminopropyl-N'-ethyl-carbodiimide hydrochloride (EDC) in 80% by volume of ethanol. Following the cross-linking, the scaffold is first thoroughly distilled. Water, then with a 1% aqueous glycine solution and finally again with dist. Washed water. Finally, the implant is frozen again and freeze-dried.

Fig. 3 shows a light micrograph of a thin section in the longitudinal direction by - as described for Fig. 2 produced - implant (magnification 10 *). The mineralized bone matrix 2 (top) is dark colored - the non-mineralized cartilage matrix 1 (bottom) is much brighter. It can be clearly seen that the matrix materials are directly connected to each other and penetrate each other at the interface 3. Both phases have a pore diameter of about 50-200 microns to a comparable porosity.

4 shows a scanning electron micrograph (SEM) of a longitudinal section through an implant produced as described for FIG. 2 (magnification 15x). The mineralized (appearing brighter) Knochematrix 2 part is on the left in the picture, the cartilage matrix 1 on the right. It is again clearly recognizable that the matrix materials are directly connected to one another and penetrate one another at the interface 3, wherein both layers have a comparable porosity.

FIG. 5 shows SEM element mapping of the interface between the mineralized bone matrix 2 (left) and the non-mineralized cartilage matrix 1 (right) of the implant fabricated as described with reference to FIG. 2. The figures are each a detail of FIG (Magnification: 5Ox each). The mineralized (appearing brighter) bone matrix 2 is located on the left side of the image, cartilage matrix 1 on the right.

At the top left of Fig. 5, a normal SEM image is shown (Fig. 5A). At the top right is a BSE image (Figure 5B - detection of the backscattered electrons = backscattered electrons). The light coloration to be seen in the left-hand area of FIG. 5B detects the presence of atoms of higher order number. This light coloration is due to the calcium and phosphorus atoms of the calcium phosphate contained in the bone matrix.

The lower images in Fig. 5 show the detection of calcium (lower left, Fig. 5C) and the lower right detection of phosphorus (Fig. 5D) individually. The light color indicates calcium (FIG. 5C) or phosphorus (FIG. 5D).

Fig. 5 illustrates the only partial mineralization of the implant: the components of calcium phosphate (calcium and phosphorus) can be detected only in the mineralized bone matrix 2 (in the pictures on the left). The interface 3 is clearly visible only in FIGS. 5B-D.

Fig. 6 shows a detail of the interface between the non-mineralized cartilage matrix 1 (top) and the mineralized bone matrix 2 (bottom). The figure shows a detail of Fig. 4 (magnification: 500x). Clearly visible are the bright appearing calcium phosphate crystallites in the lower part of the image, which characterize the mineralized phase and make their surface look rougher. The above-mentioned cartilage matrix 1, consisting of a composite of collagen I and hyaluronic acid, on the other hand, has a smooth surface. Fig. 7 shows an enlarged section of the direct contact area between the cartilage matrix 1 (top) and the mineralized bone matrix 2 (bottom) (magnification: 100Ox). The intimate connection of both phases in the area of the composite surface 3 can be clearly seen. d.) colonization of the implant with chondrocytes

For vzϊrσ colonization of the cartilage replacement portion with primary human chondrocytes, a sterile cylindrical biphasic implant having a diameter of 10 mm and a height of 6 mm (3 mm cartilage replacement and 3 mm bone replacement phase) for 24 hours in 5 ml of modified DMEM cell culture medium ( Gibco Life Technologies GmbH, Karlsruhe) and swelled, the medium is changed 4 times. For cell colonization, the implant is removed from the medium and placed on a sterile filter paper, so that the medium is largely sucked out of the porous structure. Now 5 × 10 ⁵ primary human chondrocytes, suspended in 200 μl of cell culture medium, are pipetted onto the cartilage replacement phase located above. After 2 hours storage in the incubator, the colonized implants are transferred to a 24 well plate with fresh cell culture medium and cultured according to standard conditions until implantation. e.) Producing an implant with an intermediate layer of a hyaluronic acid film

To produce an implant with an intermediate layer of a hyaluronic acid film, suspensions of mineralized collagen or a collagen-hyaluronic acid composite are prepared as described under a.) And b.). A hyaluronic acid film is produced by drying a saturated aqueous solution of hyaluronic acid sodium salt from Streptococcus equi (Fluka, Sigma / Aldrich, Taufkirchen) in air. For this purpose, a hyaluronic acid solution is conveniently poured into a Petri dish. As described under c.), 1 ml of the mineralized collagen suspension is pipetted into the well of a 24 well plate. A suitably cut piece of the hyaluronic acid film is now placed on the suspension, whereupon in turn the suspension of the collagen-hyaluronic acid composite is pipetted. As described under c.), The resulting biphasic construct is frozen, freeze-dried, chemically cross-linked, washed and finally freeze-dried again.

Fig. 8 shows schematically the cross-section through such an implant, with 1 the cartilage matrix (top), with 2 the mineralized bone matrix (bottom) and 4 the Hyaluronsäurefolie referred to as a release layer.

'f.) Producing an implant with a three-dimensional embroidery as a support structure

/ As described under a.) ~ Α), a biphasic implant is produced by stacking two suspensions, with the difference that a bead of polyhydroxybutyrate threads is inserted into the latter before the first phase is filled into the mold provided for this purpose. For better penetration of the suspension in the interstices of the embroidery is centrifuged after pipetting up the two suspensions each for 5 min. For 1000 rev / min.

Fig. 9 demonstrates the schematic structure of such an implant in the form of a cross section through a cylindrical sample. 1 is the cartilage matrix (top), 2 is the mineralized bone matrix (below), and 5 is the polyhydroxybutyrate threads that penetrate both phases. g.) Producing an implant with a support structure made by flocking technique

The procedure is analogous to that described under f.), If one intends to integrate a carrier material produced in accordance with DE 103 12 144.7 by means of flocking technology into the implant as a support structure. The support material - consisting of a base material of a mineralized collagen membrane, an adhesive layer of gelatin and flock fibers of polyhydroxybutyrate - is placed, with the base material down, in the recess of a 24-well plate in which the implant is to be produced. Then, as described under a.) - c.), In each case 1 ml of the two suspensions poured successively übereinandergegossen. For better penetration of the suspensions in the fiber interstices, as described in f.) Briefly centrifuged. Continue as described under c.) And f.).

10 shows the result as a schematic longitudinal section, with 1 the cartilage matrix (top), with 2 the mineralized bone matrix (bottom), with 6 the base material (membrane of mineralized collagen), with 7 the adhesive layer (gelatin) and with the 8 Flock fibers (polyhydroxybutyrate) of the carrier material are designated.

Claims

claims

1) implant for the treatment of osteochondral defects with at least two superimposed layers, including a porous bone matrix (2) and a porous cartilage matrix (1), wherein the layer materials are locally mixed at the interfaces (3) of the contacting layers.

2) Implant according to claim 1, characterized in that between the bone and the cartilage matrix (2 or 1) is a film-like, resorbable biopolymers existing, pore-free or low-intermediate layer (4).

3) Implant according to claim 1 or 2, characterized in that the implant additionally contains a support structure with openings and the layer materials are mixed together at the interface (3) of the layers through the openings of the support structure.

4) Implant according to one of claims 1 to 3, characterized in that the support structure consists of a two- or three-dimensional knit (5) of resorbable polymer fibers.

5) Implant according to one of claims 1 to 4, characterized in that the support structure consists of a fiber-flocked carrier material (6-8).

6) Implant according to one of claims 1 to 5, characterized in that the materials of the layers are crosslinked.

7) Implant according to one of claims 1 to 6, characterized in that the cartilage matrix (1) with chondrocytes and / or the bone matrix (2) is populated with osteoblasts or mesenchymal stem cells.

8) Implant according to one of claims 1 to 7, characterized in that at least one of the layers contains active ingredients.

9) A process for producing an implant for the treatment of osteochondral defects having at least two layers, including a porous bone and a porous cartilage matrix (2 or 1), comprising the steps of a) producing the individual layers or the respective layer material, wherein at least a layer or a layer material with a solvent which can dissolve at least the surfaces of the other layers, is converted into a pasty or liquid state, b) superimposing or laying the layers or layer materials and c) freeze-drying of the composite. 10) A method according to claim 9, characterized in that the bone and the Korpelmatrix (2 or 1) are stacked directly above one another.

11) A method according to claim 9, characterized in that between the bone and the cartilage matrix (2 or 1) an intermediate layer (4) is inserted.

12) Process according to claims 9 - 11, characterized in that the bone matrix (2) is essentially made of mineralized collagen.

13) Process according to claims 9 - 11, characterized in that the cartilage matrix (1) is essentially made of non-mineralized collagen and / or hyaluronic acid or derivatives.

14) Method according to one of claims 9 to 13, characterized in that the pore size of the matrix materials is set by freezing.

15) Process according to claims 9 and 11, characterized in that the intermediate layer (4) is prepared essentially from glycosaminoglycan and pore-less or -arm dried.

16) A method according to claim 15, characterized in that the intermediate layer (4) is made of Hyaluronsäurefolie.

17) Method according to one of claims 9 to 16, characterized in that the materials are cross-linked.

18) A method according to claim 9, characterized in that before or when stacking or laying the layers, a support structure is inserted with openings.

19) Method according to claim 18, characterized in that the support structure consists of a two- or three-dimensional knitted fabric (5) of resorbable polymer fibers.

20) Method according to claim 18, characterized in that the support structure consists of a fiber-flocked carrier material (6-8).

21) Method according to one of claims 9 or 10, characterized in that the cartilage matrix (1) is colonized with chondrocytes and / or the bone matrix (2) with osteoblasts or mesenchymal stem cells.