AU759066B2

AU759066B2 - 3D matrix for producing cell transplants

Info

Publication number: AU759066B2
Application number: AU77861/00A
Authority: AU
Inventors: Birger Hahnemann; Thomas Lotzbeyer; Andrea Pahmeier
Original assignee: ALVITO BIOTECHNOLOGIE GmbH
Current assignee: Polypat Inc
Priority date: 1999-10-06
Filing date: 2000-10-06
Publication date: 2003-04-03
Anticipated expiration: 2020-10-06
Also published as: EP1232203B1; JP3471781B2; CA2386586A1; ATE271579T1; JP2003511120A; WO2001025321A1; DE19948120C2; EP1232203A1; AU7786100A; IL148853A0; DE50007153D1; DE19948120A1

Abstract

The invention relates to a method for producing a biologically compatible matrix, to a matrix that can be obtained using the method, and to the use thereof. The invention also relates to an implant. The inventive matrix can be produced by dissolving, in water, chitosan in acid, by freezing the solution and by subsequently lyophilizing the same. Excess acid can be removed by neutralizing with a base before or after the lyophilization. The inventive matrix is dimensionally stable and elastic. When implanted, it induces only a minor immunoreaction.

Description

3D MATRIX FOR PRODUCING CELL TRANSPLANTS The invention relates to a method for producing a biocompatible three-dimensional matrix, to a matrix obtainable with the method of the invention, and to the use thereof.

Considerable successes have been achieved in recent years in the area of medical transplants. However, problems arise through the small amounts of donor organs available, which has led to considerable waiting times for patients requiring transplants. A further problem is that pathogens may be transmitted with donor organs, as was the case at least occasionally with donors suffering from AIDS. In addition, transplanted organs are recognized as foreign by the recipient's immune system. After the transplantation the patient is therefore dependent on immunosuppressive medicaments to suppress rejection reactions. Attempts have recently been made to culture artificial organs from cell cultures, and considerable successes have been achieved in some areas. This entails cells being cultured on a three-dimensional matrix which can be shaped appropriate for the requirements, for example in the shape of an ear. This artificial organ or body part can then be transplanted, and if endogenous cells are used they are not recognized as foreign by the recipient's immune system, that is to say no rejection reaction can occur.

Examples of known materials for the matrix are collagen or alginate mixed with chitosan. However, collagen in particular is used only reluctantly for producing implants for human patients because it is produced from bovine cartilage and is thus associated with the risk of BSE.

Chitosan has attracted increasing interest as promising matrix material. Chitosan is a partially deacetylated 2 chitin and is obtained from the exoskeletons of arthropods. It is an aminopolysaccharide (poly-l-4glucosamine) which is used in the medical sector for example as suture material or for encapsulating drugs.

Its advantage is that it can be completely absorbed by the body. Chitosan can be dissolved in water under slightly acidic conditions (pH 6) through protonation of the free amino groups. It is reprecipitated from the aqueous solution under alkaline conditions (pH 7).

This pH-dependent mechanism means that chitosan can be purified and processed under mild conditions.

US 5,871,985 proposes a vehicle for transplantation into a patient which consists of a matrix into which cells have grown. For this purpose, first a chitosan solution containing living cells is produced. This solution is then enclosed in a semipermeable membrane to form the vehicle. The chitosan is precipitated and forms an uncrosslinked matrix in which the cells are distributed.

Madihally et al., (Biomaterials 1999: 20(12), pp. 1133- 42) describes a matrix for tissue regeneration.

Chitosan which is 85-90% deacetylated is for this purpose dissolved in 0.2 M acetic acid to result in solutions with a chitosan content of from 1 to 3% by weight. The solution is frozen and the water and the excess acetic acid are removed by lyophilization. The shape of the pores which form can in this case be influenced by the lyophilization conditions. The average diameter of the pores in the resulting matrix is in the range 40-250 gm. The freshly lyophilized matrix is described as stiff and inelastic. On hydration in a neutral aqueous medium, the chitosan swells rapidly and finally dissolves. Renewed dissolving of the chitosan structure can be prevented by equilibration in dilute NaOH or by washing with an ethanol series, for example 100, 70, 50, The Slyophilized matrix is for this purpose equilibrated in 3 0.05 M NaOH for about 10 minutes and washed with water and PBS (phosphate-buffered physiological saline solution). On equilibration in dilute NaOH, however, the matrix shrinks and shows changes in its structure which are only partly reversible on reducing the pH to 7. In addition, the matrix shows numerous air inclusions. Better results are obtained on equilibration with an ethanol series. To remove air inclusions, initially a vacuum is briefly applied on washing with absolute alcohol. A further advantage mentioned is that the matrix is sterilized during the equilibration through the treatment with alcohol.

Nevertheless, the method is comparatively complicated.

Even if the membrane is stabilized by an ethanol series, a change in the pore structure occurs because the chitosan may be locally dissolved by the water and thus the pore structure is partly destroyed. The hydrated matrix is described as soft and flexible, but shows only low strength. This considerably impedes processing of the matrix. If the matrix is cut to the appropriate shape for example for culturing cartilage cells to form cartilage, it is very easily broken.

Likewise, culturing of the cells is made difficult because the matrix is easily broken into a plurality of fragments by manipulations, for example on transferring into fresh culture medium. This means that implants with a defined structure can be produced with known chitosan matrices only with great difficulty. In addition, Madihally et al. describe the production of microcarriers. For this purpose, either drops of the aqueous chitosan solution are frozen directly, for example in liquid nitrogen, or, before the freezing, are precipitated as gel under alkaline conditions with NaOH. Larger structures are not described.

The invention is based on the object of providing a method for producing a biocompatible, three-dimensional matrix which requires less effort and leads to stable soft matrices.

-4- The object is achieved according to a first embodiment of the invention with a method for producing a biocompatible three-dimensional matrix, where an aqueous solution is prepared from chitosan and an excess of an acid, the aqueous solution is neutralized, the neutralized aqueous solution is frozen, and the water is removed by sublimation under reduced pressure.

A three-dimensional matrix with comparable properties is obtained according to a second embodiment of the invention with a method for producing a biocompatible three-dimensional matrix, where an aqueous solution is prepared from chitosan and an excess of an acid which is selected from the group formed by alkyl and aryl hydroxy carboxylic acids, the aqueous solution is frozen, and the water is removed by sublimation under reduced pressure, with excess acid being removed before or after the removal by sublimation.

In the method of the invention, firstly an aqueous solution is prepared from a partially deacetylated chitosan and an acid which is present in excess. By excess is meant in this connection that the pH of the aqueous solution is in the acidic range, preferably below pH 4. By this means the free amino groups in the chitosan are at least partially protonated, which increases the solubility in water. The amount of acid is not critical. It must merely be chosen so that the chitosan dissolves. Excessive addition of acid is avoided where possible because excess acid must be removed again, and thus the working up is made difficult if the amounts of acid are large. Amounts of acid which result in a 0.05 to 1 N, preferably 0.1 to 0.5 N, in particular 0.1 to 0.3 N, solution are favorable.

SA particularly suitable acid is lactic acid. The finished matrix then contains lactate ions as 5 counterions of the chitosan. The matrix is particularly soft and elastic in this case.

The amount of chitosan is preferably chosen to result in a concentration of from 0.01 to 5% by weight, preferably 0.5 to 1% by weight. Influence can be exerted on the structure of the matrix, in particular its pore size, through the concentration of the chitosan solution. It is possible in this way to suit the pore size of the matrix to the particular, type of cell with which the matrix is intended to-be colonized.

Because chitosan is produced from natural sources, it has no uniform molecular weight. The molecular weight may be between 20 kDa to more than 1 000 kDa, depending on the source and preparation method. The chitosan for producing the three-dimensional matrix is not subject to any restrictions in relation to its molecular weight.

After the freezing, the water is removed by sublimation under reduced pressure. Suitable pressure ranges are from 0.001 to 3 hPa. The exact conditions are influenced by the composition of the aqueous solution.

Where a volatile acid has been used, it may be at least partly coevaporated during the lyophilization.

Before the freezing it is preferred to remove excess acid by adding a base to adjust the pH of the aqueous solution to from 5.0 to 7.5, preferably 5.5 to 7.0, in particular 6.0 to 7.0. A matrix with very good properties in relation to its dimensional stability is obtained. The matrix does not shrink on rehydration and does not show any structural changes either. This stable behavior is particularly marked when the aqueous solution has a pH of from 6.0 to 7.0 before the Sfreezing. The chitosan starts to precipitate in this range. It is assumed that under neutral conditions a gel or a fine suspension is formed, which has a 6 beneficial effect on the structure of the lyophilized matrix. The neutralized aqueous chitosan solution thus forms a suspension. The aqueous solution may therefore, depending on the chitosan concentration, become cloudy on neutralization.

In a method like that proposed by Madihally et al., the matrix experiences a large change in the structure on neutralization of excess acetic acid by equilibration with aqueous NaOH. Addition of the aqueous 'solution presumably leads to local dissolution of the chitosan, which is reprecipitated by the NaOH. The pore structure of the matrix is thus altered.

A possibility has now been found, with the method found by the inventors, for stabilizing the matrix so that no, or considerably smaller, structural changes occur on further processing of the matrix after removal of the water by lyophilization.

In the first embodiment of the method of the invention, the chitosan solution has a neutral pH even before the freezing and lyophilization. By "neutral pH" is meant in this connection a pH in the range indicated above.

It is therefore no longer necessary for the matrix to be equilibrated with base after the lyophilization. The essential point is that the chitosan solution is adequately neutralized and the chitosan precipitates as gel. If the neutralization is not carried out carefully, the resulting matrix redissolves on rehydration. In the first embodiment of the method of the invention, the acid is not in the first instance subject to any particular restrictions.

It is possible to use for preparing the aqueous chitosan solution an acid which is selected from the group formed by inorganic acids and organic acids, preferably alkyl- and arylcarboxylic acids, in particular hydroxy carboxylic acids. Lactic acid is 7particularly preferred.

Examples of inorganic acids are hydrochloric acid, phosphoric acid and acidic phosphate salts. Examples of organic acids are alkylcarboxylic acids having 1 to 12 carbon atoms, it being possible for the alkyl chain to be straight-chain or branched. Examples are formic acid, acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid and higher straight-chain carboxylic acids. Also suitable are dicarboxylic acids having 2 to 8 carbon atoms, such as oxalic acid, succinic acid or adipic acid. Also suitable are aromatic carboxylic acids such as benzoic acid or naphthoic acid. Furthermore, amino acids, such as glutamic acid or aspartic acid, are also suitable.

However, particularly good results are obtained with hydroxy carboxylic acids. Particularly suitable are hydroxy carboxylic acids having 2 to 12 carbon atoms.

There may be one or more hydroxyl groups and one or more carboxyl groups present in the molecule. Examples are glycolic acid, lactic acid, malic acid, tartaric acid and citric acid. It is also possible to use aromatic hydroxy carboxylic acids, for example mandelic acid.

Bases which can be used are conventional bases such as, for example, alkali metal hydroxides such as NaOH. The bases should be chosen so that a neutral pH can be adjusted without difficulty.

A further possibility for obtaining a matrix with improved structure is for the matrix to be stabilized even via the acids used for dissolving the chitosan in water.

1 It has emerged, surprisingly, that the properties of

-V

Sthe chitosan matrix can be influenced by the choice of Sthe counterion. This is utilized in the second 8 embodiment of the method of the invention. The acids used in this case are hydroxy carboxylic acids. Very good properties are obtained in particular on use of lactic acid, with which the lactate anion forms the counterion to the protonated amino group of chitosan.

The matrix shows a high temperature resistance and can, for example, be autoclaved without difficulty. No discolorations or other signs of decomposition of the matrix appear during this. The matrix also shows a high elasticity and mechanical strength. Thus, a sheet-like matrix obtained by pouring the aqueous chitosan/lactic acid solution into a Petri dish and then freezing and lyophilizing, can be rolled up without difficulty and without fracturing the matrix.

In the second embodiment of the method of the invention, the stabilizing effect of the hydroxyalkylor -arylcarboxylic acids appears even if the chitosan solution is not neutralized before the freezing. After the lyophilization, residues of excess acid which are still present are removed by equilibration with base, for example 0.05 N NaOH. The matrix is then washed with water, which may contain a phosphate buffer (0.1 N, pH about The structural changes in the matrix are in this case considerably less than with the matrix described by Madihally. It is particularly advantageous in the second embodiment of the method of the invention for the chitosan solution to be neutralized even before the freezing. Equilibration under alkaline conditions is then no longer necessary. The matrix can be further processed directly and, for example, colonized by cells.

Both embodiments of the method of the invention result in a matrix with a uniform pore structure, which is soft and pliable. If, for example, the matrix is compressed by pressure from the fingers, it resumes its original shape again elastically after the pressure is removed. The matrix can be molded without breaking or 9 crumbling and can, for example, be rolled up.

To form coherent assemblages of cells it is necessary for the matrix to have sufficient strength during the colonization by cells and to be broken down only over a very prolonged period. The rate of breakdown can be controlled via the degree of deacetylation of chitosan.

On use of a chitosan which still has a very high degree of acetylation it is possible for breakdown times of more than one month to be achieved.

The matrix is observed to be particularly highly absorbable when the chitosan has a degree of deacetylation of 80%. The degree of deacetylation relates to the N-acetyl groups of chitosan. By degree of deacetylation is meant the ratio of unacetylated amino groups to the total of amino groups present (total of free amino groups and acetylated amino groups). It is assumed that at a degree of deacetylation of 80% amorphous structures are formed in wide areas of the matrix. These amorphous areas can presumably be attacked better by enzymes. It is thus possible to influence the rate at which the matrix is absorbed in the body and adjusted to the particular needs.

The pore size of the matrix can further be influenced by the rate at which the aqueous chitosan solution is frozen. In this connection, rapid freezing, for example in liquid nitrogen, results in small pore diameters, whereas slow freezing, for example in the range from to -15 0 C, results in larger pores. The temperature for freezing the chitosan solution is preferably chosen in the range from -2000C to -OOC, preferably -90 0 C to -100C, particularly preferably -40 0 C to The matrix obtainable using the method described above has excellent properties by comparison with matrices Sknown in the prior art. The invention therefore also 10 relates to a matrix as obtained using the method described above. The matrix is dimensionally stable, soft and flexible, and can thus easily be processed and, for example, be cut to the desired shape simply.

It can be autoclaved and thus sterilized simply without decomposition reactions occurring. No further substances such as collagen or alginate are necessary for stabilizing the matrix. The degree of acetylation and thus, for example, the absorbability of the matrix can be chosen without restrictions and can, thus be adjusted to the particular requirements. The structure of the matrix, in particular the pore size, can be influenced through the concentration of the aqueous chitosan solution and the freezing conditions.

A very dimensionally stable and flexible matrix is obtained when the matrix has a lactate anion as counterion.

The matrix can be modified as desired through the large number of amino groups. In a preferred embodiment of the three-dimensional matrix, ligands are covalently or noncovalently bound to the chitosan matrix, preferably to the free amino groups of chitosan. Ligands which can be used are, for example, growth promoters, proteins, hormones, heparin, heparan sulfates, chondroitin sulfates, dextran sulfates or a mixture of these substances. The ligands preferably serve to control and improve cell proliferation.

Cell growth on the matrix is further improved if the matrix is coated with autologous fibrin.

The three-dimensional matrix of the invention can be used as solid phase in a culture reactor (cell factory). The matrix shows a very high strength in culture medium. It has also emerged that the matrix promotes cell growth.

11 The matrix stimulates cell growth. It can therefore be implanted directly, i.e. without previous colonization by cells. After the implantation, endogenous cells grow in, with the matrix gradually dissolving.

The matrix is also suitable for use as cell implant, in particular for cartilage-forming cells. In this connection it is preferred not to use any genetically modified cells. The cells are preferably taken from the patient by biopsy and cultured on the cell matrix, and the cell implant is then implanted into the patient.

Transplant rejection reactions are substantially precluded owing to the colonization of the threedimensional matrix with endogenous stem cells (bone substitute) which stimulated by the respective growth factors of the surrounding tissue differentiate only at the site of the transplant, and owing to colonization with cartilage cells for renewed formation of hyaline cartilage. This is a great advantage compared with previous matrices made of various plastics or ceramics. These materials remain in the body and may even after some years be recognized as foreign and be rejected. The three-dimensional matrix can be colonized both by human and by animal cells (for example from horse, dog or shark). Shark cells are particularly suitable because they induce a negligible immunological response in the recipient. Shark cells are already used as organ replacement, for example for the lenses of eyes.

An implant of the invention which stimulates only a slight or no immune response is advantageously composed of the three-dimensional matrix described above, which is preferably colonized by endogenous cells. Thus, for example, a thin sheet-like matrix which has been colonized with keratocytes under sterile conditions in a suitable culture medium can serve as skin transplant.

The colonized matrix can be removed from the culture medium under sterile conditions in the operating I 12 theater and be placed on the cleaned wound surface. It is then possible for endogenous cells to migrate into the matrix to form tissue.

An embodiment as artificial liver is also possible. The matrix in this case is colonized with liver cells in a suitable culture medium. The assemblage of cells, which may be also be stabilized by the matrix, is then implanted in the patient's body and there can, for example, assist the function of a damaged liver.

Good results have been achieved when the threedimensional matrix is colonized by autologous chondrocytes. The matrix can then be transplanted to the appropriate places on the cartilage where new cartilage substance can then develop. This provides great advantages compared with the method currently in use, of transplantation of cartilage cells without support matrix. The high probability existing in this method that the periosteum will be damaged and bone cells will then grow into the new cartilage layer, with subsequent degeneration of the new cartilage, is avoided by the implant of the invention.

Examples of other cells which are suitable for colonizing the matrix are osteocytes, keratinocytes, hepatocytes, bone marrow stem cells or neuronal cells.

The invention is explained in more detail below with reference to several figures and by means of examples.

The figures show specifically: Fig. 1 an image of a matrix with lactate as counterion, the aqueous chitosan solution having been neutralized before the freezing in the production of the matrix; Fig. 2 an image of a matrix, the matrix having been equilibrated after lyophilization with NaOH by 13 weight) in the production of the matrix; the counterion is formed in by lactate and in by phosphate; Fig. 3 a micrograph of the matrix colonized by chondrocytes.

Example 1: General method for producing a threedimensional matrix (1st embodiment) Chitosan is dissolved by stirring in dilute' aqueous acid at a pH between 3 and 6. The desired pH is then adjusted with diluted NaOH. The solution is transferred into a vessel of a suitable shape and frozen at -30 to 0 C. The vessel with the frozen solution is transferred into a lyophilizer and the water is removed by sublimation under reduced pressure (0.01 to hPa). The spongy residue can be employed without further purification for colonization by cells.

Example 2: General method for producing a threedimensional matrix (2nd embodiment) Chitosan is dissolved by stirring in dilute aqueous hydroxy carboxylic acid at a pH between 3 and 6. The clear solution is frozen in a vessel of suitable shape at a temperature of from -30 to -150C. The vessel with the frozen solution is transferred into a lyophilizer, and the water is removed by sublimation under reduced pressure (0.01 to 0.5 hPa). The spongy residue remaining after removal of the water by sublimation is basified in sodium hydroxide solution by weight) for 24 hours. The spongy matrix is then washed with deionized water until the pH is tolerable for cells.

Example 3: Production of a porous chitosan matrix (counterion: lactate) 0.1 g of chitosan and 0.1113 g of lactic acid (90% in water, purchased from Merck KGaA, Darmstadt) were 14 dissolved by stirring in 9.79 g of deionized water, and the pH was adjusted to 7 with 1 M sodium hydroxide solution. The clear solution was initially frozen at -24 0 C and then the solvent was removed by sublimation under a pressure of 0.05 hPa.

A white, fine-pore foam with a spongy elasticity was obtained. The foam was used without further purification for colonization by cells. The matrix is depicted in Fig. i. It has a uniform, finely sitructured surface. No distortions are detectable, and the matrix shows a uniform fine-pore structure throughout.

Example 4: Production of a porous chitosan matrix (counterion: acetate) (Ist embodiment) 0.1 g of chitosan and 0.1 g of acetic acid (100%) were dissolved by stirring in 9.79 g of deionized water, and the pH was adjusted to 7 with 1 M sodium hydroxide solution. The clear solution was firstly frozen at -24 0 C and then the solvent was removed by sublimation under a pressure of 0.05 hPa.

A white, fine-pore foam was obtained.

Example 5: Production of a porous chitosan matrix (counterion: phosphate) 0.1 g of chitosan and 0.037 ml of ortho-phosphoric acid (85% Sigma P 6560) were dissolved by stirring in 7.00 g of deionized water, and the pH was adjusted to 7 with 1 M sodium hydroxide solution. The clear solution was firstly frozen at -24 0 C and then the solvent was removed by sublimation under a pressure of 0.05 hPa.

A white, fine-pore foam with a spongy elasticity was obtained. The foam was used without further purification for colonization by cells.

15 Example 6: Production of a porous chitosan matrix (counterion: glutamate) 0.1 g of chitosan and 0.1 g of glutamic acid (Sigma G 6904) were dissolved by stirring in 10.00 g of deionized water, and the pH was adjusted to 7 with 1 M sodium hydroxide solution. The clear solution was firstly frozen at -24 0 C and then the solvent was removed by sublimation under a pressure of 0.05 hPa.

A white, fine-pore foam having a spongy elasticity was obtained.

Example 7: 0.1 g of chitosan and 0.1113 g of lactic acid were dissolved by stirring in 9.79 g of deionized water, and the clear solution was firstly frozen at -24 0 C. The solvent was then removed by sublimation under a pressure of 0.05 hPa. The spongy residue was basified in sodium hydroxide solution by weight) for 24 hours and then washed with deionized water until neutral. Drying resulted in an elastic white foam. The matrix is depicted in Fig. 2a.

Comparative example 1: 0.1 g of chitosan and 0.037 ml of ortho-phosphoric acid were dissolved by stirring in 7.00 g of deionized water, and the clear solution was firstly frozen at -240C. The solvent was then removed by sublimation under a pressure of 0.05 hPa. The spongy residue was basified in sodium hydroxide solution for 24 hours and then washed with deionized water until neutral. Drying resulted in a brittle white foam. The matrix is depicted in Fig. 2b.

16 Example 8: Production of a porous chitosan matrix with anionic characteristics (immobilization of L-glutamic acid) 0.1 g of chitosan were put in 10 ml of water and the pH was adjusted to 4 with 1 M hydrochloric acid, and the mixture was stirred for 24 hours. The pH was adjusted to 5.8 by adding 1 N NaOH and then, while stirring, 0.05 g of glutamic acid and 0.05 g of l-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDAC) were added and dissolved. After having been stirred at room temperature for a further 3 hours, the pH was adjusted to 7 with 1 M NaOH, and the precipitate was filtered off and washed with 2 1 of deionized water. The washed precipitate was dissolved with 0.1113 g of lactic acid in water, purchased from Merck KGaA, Darmstadt) by stirring in 9.79 g of water. The pH was adjusted to 7 with 1 N sodium hydroxide solution, and the solution was frozen at -24 0 C. The water was then removed by sublimation under a pressure of 0.05 hPa.

A white spongy residue was obtained. The sponge can be used without further purification.

Example 9: Production of a three-dimensional matrix with immobilized cell adhesion factors L-Arg-L-Gly-L- Asp 0.1 g of chitosan were put in 10 ml of water and the pH was adjusted to 4 with 1 M hydrochloric acid. After stirring at room temperature for 24 hours, the clear solution was adjusted to pH 5.8 with 1 M NaOH and then 0.01 g of L-Arg-L-Gly-L-Asp and 0.05 g of l-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDAC) were successively added and dissolved by stirring. After having been stirred at room temperature for a further 3 hours, the pH was adjusted to 7 with 1 N NaOH. The precipitate' was filtered off and washed with 2 1 of water. The precipitate was then dissolved with 0.1113 g 17 of lactic acid (90% in water, purchased from Merck KGaA, Darmstadt) by stirring in 9.79 g of water, the pH was adjusted to 7 with 1 N sodium hydroxide solution, and the solution was frozen at -24 0 C. Finally, the water was removed by sublimation under a pressure of 0.05 hPa.

Example 10: General procedure for seeding and incubating cells on the three-dimensional matrix A three-dimensional matrix obtained as in Examples 1 to 6 is autoclaved in PBS at 1210C for 20 minutes. The matrix is then sterile and completely wetted with liquid. Cells which have been cultured in a suitable culture medium are then put directly onto a matrix which has been cut appropriate for requirements and incubated at the suitable temperature. The matrix with growth can be removed under sterile conditions and, for example, implanted.

Example 11: Culturing of chondrocytes on the threedimensional matrix Autologous chondrocytes obtained by biopsy were cultured on chondrocyte growth medium (Cell Applications Inc., USA).

A three-dimensional matrix produced as in Example 3 was initially autoclaved at 121 0 C for 20 minutes. The completely wetted matrix was cut appropriately under sterile conditions and put in a culture vessel. The cultured chondrocytes with the culture medium were then put on the three-dimensional matrix and incubated at 37 0 C for 14 to 21 days. During this period, the cells Zrew into the three-dimensional matrix. The matrix with rowth was removed under sterile conditions and can 18 then be implanted.

Fig. 3 shows the colonized chitosan matrix as microscopically magnified image. The darker regions indicated by an arrow and composed of spherical aggregates are aggregates of cells which have developed on the matrix during culturing. The cells grow into the pores of the matrix and form three-dimensional aggregates of cells there.

The properties of the chitosan matrix are strongly influenced by the production method and by the counterion used. The effect of the counterion is clear from comparison of Fig. 2a (Example 7) and 2b (Comparative example Both matrices were produced by dissolving chitosan and the appropriate acid in water, freezing the solution and removing the solvent by sublimation. The foam-like residues were then equilibrated in aqueous NaOH. On use of lactate as counterion (Fig. 2a), the structural change in the matrix on equilibration with NaOH is distinctly less than on use of phosphate (Fig. 2b). The surface of the matrix is smoother and shows less pronounced structuring. The matrix obtained in Example 7 is distinctly more elastic and stable than the matrix from Comparative example 1. It can be compressed and, after the pressure is removed, elastically resumes the original shape again. It can likewise be bent elastically without breaking.

The effect of the production method is clear by comparing Fig. 1 and 2a. In the case of Fig. 1 (Example the lactic acid is neutralized before the lyophilization, whereas with the matrix from Fig. 2a the lactic acid was removed after the lyophilization by basification with aqueous NaOH. The uniform structure of the surface of the matrix is clearly evident in Fig. 1, being virtually free of major pits. The matrix depicted in Fig. 1 shows far better softness and 19 elasticity than the matrix from Fig. 2a.

The properties of the matrices are summarized in a qualitative comparison in Table 1.

20 Table 1 of the properties Comparison of porous threedimensional chitosan matrices Counterion Production Color of the method* matrix Example 3 Lactate A white Example 4 Acetate A white Example 5 Phosphate A white Example 6 Glutamic acid A white Example 7 Lactate B white Comparative Phosphate B white example 1 Flexibility of Behavior on Sterilization the matrix addition of (20 min at water 1200C) Example 3 soft, stable, white, flexibly scarcely no change rollable any particles dissolve Example 4 soft, fibers and slight flexible, particles brown breaks with dissolve coloration relatively at the severe edges stress Example 5 soft, particles white, no flexible dissolve change Example 6 soft, particles white, no flexible dissolve change Example 7 elastic, scarcely slight flexible any brown particles coloration dissolve Comparative brittle, many distinct example 1 breaks particles brown easily dissolve coloration *A Dissolving of chitosan at acidic pH Neutralization of the aqueous solution Freezing Removal of water by sublimation B: Dissolving of chitosan at acidic pH Freezing 21 Removal of water by sublimation Equilibration with NaOH

Claims

1. A method for producing a biocompatible three- dimensional matrix, where an aqueous solution is prepared from chitosan and an excess of an acid, the aqueous solution is neutralized, the neutralized aqueous solution is frozen, and the water is removed by sublimation under reduced pressure.

2. A method for producing a biocompatible three- dimensional matrix, where an aqueous solution is prepared from chitosan and an excess of an acid which is selected from the group formed by alkyl and aryl hydroxy carboxylic acids, the aqueous solution is frozen and the water is removed by sublimation under reduced pressure, with excess acid being removed before or after the removal by sublimation.

3. A method as claimed in claim 1 or 2, where the acid is lactic acid.

4. A method as claimed in any of the preceding claims, where the acid is removed or neutralized by adjusting the pH of the aqueous solution by adding a base to from 5.0 to 7.5, preferably 5.5 to 7.0, in particular 6.0 to A three-dimensional matrix obtainable by a method as claimed in any of claims 1 to 4.

6. A three-dimensional matrix as claimed in claim where adhesion factors, hormones and/or growth factors are bound to the chitosan framework.

7. A three-dimensional matrix as claimed in claim where the matrix is coated with autologous fibrin.

8. The use of the three-dimensional matrix as claimed 23 in any of claims 5 to 7 as solid phase in a culture reactor.

9. The use of the three-dimensional matrix as claimed in any of claims 5 to 7 as matrix for implants. An implant comprising a three-dimensional matrix as claimed in any of claims 5 to 7. 3310A -24

11. A method for producing a biocompatible three-dimensional matrix, said method being substantially as herein described with reference to any one of the Figures and/or to any one of the Examples.

12. A biocompatible three-dimensional matrix obtained by use of the method as claimed in claim 11.

13. A biocompatible three-dimensional matrix substantially as herein described with reference to any one of the Figures and/or to any one of the Examples.

14. An implant comprising the matrix as claimed in claim 12 or 13. A method of surgery including the step of implanting a three-dimensional matrix fabricated by the method of any one of claims 1-4 and 11 or as claimed in any one of claims 5-7 and 12 and 13. Dated this 11 day of April 2002 ALVITO BIOTECHNOLOGIE GMBH S** By: 0o Patent Attorneys for the Applicant