EP1372750A1

EP1372750A1 - Method for preparing a cartilaginous neo-tissue

Info

Publication number: EP1372750A1
Application number: EP02757749A
Authority: EP
Inventors: Alain Domard; Marie-Thérèse M. CORVOL; François Guillot; Xavier L. Chevalier; Isabelle Morfin; Dominique J. Vacher
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite Claude Bernard Lyon 1 UCBL; Institut National de la Sante et de la Recherche Medicale INSERM; Laboratoires Genevrier SAS
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite Claude Bernard Lyon 1 UCBL; Institut National de la Sante et de la Recherche Medicale INSERM; Laboratoires Genevrier SAS
Priority date: 2001-03-30
Filing date: 2002-03-29
Publication date: 2004-01-02
Also published as: FR2822842B1; FR2822842A1; CA2442732A1; US20040171151A1; WO2002078760A1

Abstract

The invention concerns the preparation of a cartilaginous neo-tissue capable of being grafted. The method consists in: a) culturing chondrogenic cells, which are either autologous chondrocytes or cells precursors of chondrocytes prepared in vitro from pluripotent stem cells; b) contacting said chondrogenic cells with a chitosan hydrogel, having preferably a degree of acetylation ranging between 40 and 60 %, whereof the amphiphilic properties and the degree of acetylation is such that said cells adhere naturally to the outer surface of said hydrogel; c) covering the hydrogel/cell assembly thus obtained with a culture medium; and d) allowing a cartilaginous neo-tissue to develop in contact with the chitosan hydrogel during a minimum period of two weeks, frequently renewing the culture medium.

Description

PROCESS FOR THE PREPARATION OF A CARTILAGINOUS NEO-TISSUE

The present invention relates to the field of repairing cartilage lesions by the implementation of a graft. It relates more particularly to a process for the preparation of a graftable cartilage tissue.

Cartilage is a tissue of mesenchymal origin consisting of a small percentage of chondrocytes distributed within an extracellular matrix which they ensure renewal. This matrix is composed of a network of collagen fibers, in particular type 11, and glycosaminoglycans associated with structural proteins to form proteoglycans. The whole by its amphiphilic nature and its ionic sites forms a physical gel ensuring the viscoelastic properties of the cartilaginous tissue.

The cartilage tissue disappears in adulthood except at the level of the joints, where its role is to ensure the movement of the articular surfaces and to support significant compressive loads. Joint cartilage is however not able to regenerate spontaneously. This is why in case of cartilage lesions recourse is had to transplant techniques, such as mosaic grafting or grafting of autologous cells.

Mosaic grafting involves taking samples of bone covered with cartilage in non-bearing regions and grafting them at the level of the lesion.

Autologous cell transplantation consists of taking a sample of healthy cartilage, subjecting it to an enzymatic digestion in order to free the chondrocytes from the extra cellular matrix, making the chondrocytes multiply ex-vivo until a sufficient number is obtained. chondrocytes, which are then reimplanted in the cartilage lesion. Chondrocytes in the form of a cell suspension in aqueous medium (dispersion in a liquid medium), first cover the previously unbridled lesion with a membrane made of periosteum sutured tightly at the edge of the cartilage and then inject the suspension (dispersion containing the culture) into the cavity thus created chondrocytes. After a certain time, these cells produce an extra-cellular matrix which does not however have the tissue organization of normal articular cartilage.

It should be noted that the mode of multiplication of the chondrocytes to be implanted must be determined so as to avoid a dedifferentiation of the cells. In particular if chondrocytes are made to proliferate on a support (synthetic polymer) like that of the bottom of the cell culture dishes, the chondrocytes become differentiated into fibroblastic cells. They are then spindle-shaped instead of being polygonal like chondrocytes and synthesize collagen I instead of collagen II.

It has already been proposed in document WO.00 / 56251 to make cells, including human chondrocytes, multiply on biodegradable polysaccharide beads crosslinked with polyamines. The polysaccharides are chosen in particular from the following compounds: dextran, cellulose, arabinogalactan, pullulan and amylose. The crosslinking agent is for example glutamic acid, lysine, albumin or gelatin.

According to this document, after being brought into contact with said polymer beads under mechanical agitation, the chondrocytes multiply while keeping their shape and their phenotype, and synthesize very particularly collagen II.

After this multiplication, the chondrocytes are recovered by digesting the polysaccharide beads using specific enzymes, for example dextranase, which do not damage the chondrocytic cells. Said cells are then detached to be included in a chitosan matrix. To do this, chondrocytes are added to an acidic solution of chitosan, then the mixture is stirred until the formation of a three-dimensional structure which is placed in a 1N sodium hydroxide solution so as to obtain the precipitation of the chitosan. in a few minutes. The sodium hydroxide is then quickly eliminated and the conglomerate of chitosan and cells is cultured at 37 ° C., 5% CO 2 for a determined period.

Thus according to this document WO.OO / 56251, the chondrocytes mixed with the chitosan are incorporated, included in the three-dimensional structure of the precipitated chitosan, which structure would have a firm consistency resembling the texture of cartilage.

In document WO.OO / 56251, another possible variant is provided for at the level of the first step of multiplying chondrocytes, namely making the said cells multiply on a film of chitosan. The other two stages remain identical, the second consisting in extracting the chondrocytes thus multiplied by enzymatic digestion with collagenase or with trypsin and the third stage consisting in incorporating, including said chondrocytes in a three-dimensional matrix of chitosan, in the same conditions as above.

The applicant expresses doubts that the chondrocytes may still be viable after having been successively subjected to an acid medium and then to a strongly basic medium, as taught in document WO.00 / 56251 for the precipitation of chitosan around said cells.

Chitosan is obtained by deacetylation of chitin, the most widespread biopolymer in nature after cellulose. Chitin can be extracted from the exoskeleton of certain crustaceans such as lobster, crab or the squid endoskeleton, for example. The chitin and chitosan consist of the same two monomer units, N-acetylD glucosamine and D-glucosamine. When the polymer is strongly acetylated, that is to say when it comprises more than 60% of N-acetyl-D-glucosamine, it is called chitin. Both are biodegradable, bioresorbable and compatible with living tissue.

Chitosan is known to have a biostimulatory activity on the reconstitution of tissues. It is however generally used in combination with other elements. For example in document W0.96 / O2259, the chitosan is combined with another polysaccharide to form an agent for stimulating the regeneration of hard tissue at an integration site of an implant, for example made of titanium.

For example in document WO.99 / 47186, the chitosan is crosslinked with a glycosaminoglycan to constitute a biochemical environment close to the cartilaginous tissue, stimulating cell growth.

The process of the present invention is similar to document WO.OO / 56251 in that it uses chitosan which is not associated with any other constituent. However, it is distinguished by the fact that it does not require three successive operations.

The process of the present invention relates to the preparation of a graftable cartilage neo-tissue. It consists of: a) culturing chondrogenic cells, which are either autologous chondrocytes or chondrocyte precursor cells prepared in vitro from pluripotent stem cells, b) bringing said chondrogenic cells into contact with the outer surface of a hydrogel made exclusively of chitosan whose amphiphilic properties and degree acetylation are such that said cells adhere naturally to said outer surface, c) to cover the hydrogel / cells thus obtained with a culture medium and, d) to allow the development of a cartilage neo-tissue in contact with the surface of chitosan hydrogel, and without penetration of the cells for a minimum period of two weeks, by frequently renewing the culture medium. Thus, contrary to what is proposed in document WO.00 / 56251, the process of amplification of chondrogenic cells takes place either spontaneously in contact with the external surface of the chitosan hydrogel, or after prior amplification in conventional high-density growing conditions. In addition, the formation of the extracellular matrix takes place simultaneously, in the presence of the chitosan hydrogel.

The natural adhesion of the cells to the external surface of the chitosan hydrogel makes it possible to obtain a very good distribution of said cells and avoids the loss of cells during the operation, for example when the latter is carried out in wells. culture. The chitosan hydrogel plays an inducing role on the phenotype of chondrogenic cells, which proliferate without being indifferentiated, while retaining their chondrocyte phenotype and their cell maturation potential.

It should be noted that the chondrogenic cells do not penetrate directly inside the hydrogel, the latter having a pore size (at the surface and in the mass) insufficient with regard to the size of said cells. Chitosan hydrogel is gradually metabolized and / or replaced and / or invaded by matrix proteins of cartilage type, which are neo-synthesized by chondrocytes. After at least two weeks of culture, the whole - consisting of the chondrocytes and the extra-cellular matrix - produces a cartilaginous neo-tissue which can be grafted as it is, the chitosan hydrogel which temporarily serves as a support for this cartilaginous neo-tissue being partially or wholly biodegraded, and not participating in the graft, contrary to what is provided in documents WO.OO / 56251 and WO.99 / 47186 in which it is the three-dimensional structure in which the chondrocytes are included which acts as a graft.

The degree of acetylation of chitosan, used for the preparation of the hydrogel, is between 30 and 70%; preferably this is between 40 and 60%.

According to a first alternative embodiment, the chondrogenic cells are brought into contact with the outer surface of the chitosan hydrogel which is in the form of small particles of a few millimeters. According to a second alternative embodiment, the chondrogenic cells are spread in the form of at least one layer between at least two layers of chitosan hydrogel, each layer being of the order of a few millimeters thick. This particular arrangement makes it very easy to obtain a large cartilage neo-tissue, after complete disappearance of the chitosan hydrogel.

The neo-cartilage tissue formed by the process of the invention is characterized in that it consists of more or less parallel rows of cells, having a cell maturation gradient oriented from a determined zone towards its periphery, the determined zone. corresponding to the junction area of the cells with the chitosan hydrogel. When this neo-tissue is analyzed in histology, its morphological appearance is close to a normal cartilage tissue. The present invention will be better understood on reading the description which will be given of several examples of preparation. of a cartilaginous neo-tissue using as an amplification support a chitosan hydrogel whose degree of acetylation is between 40 and 60%.

PURIFICATION ni,) CHITOSANF The reference chitosan used is obtained from endoskeletons of squid. It has an acetylation degree of 5.2%. It is first of all purified in order to remove the insoluble particles, by implementing the following stages: dissolution, filtration, precipitation, washing and lyophilization. For its dissolution, a solution of low viscosity is prepared, with a concentration of the order of 0.5% by weight of chitosan in an acid solution. More specifically, acetic acid is put in a stoichiometric amount relative to the amino groups of chitosan. The polymer solution is filtered by successive passages on membranes of decreasing porosity (1.2; 0.8 and 0.45 μm) under a maximum pressure of 3 bars.

The polymer is precipitated by raising the pH of the solution by adding a concentrated ammonia solution, with stirring. Several washing operations are then necessary to lower the pH of the suspension by eliminating the excess ammonia. After each washing, the suspension is centrifuged and the pellet recovered. Washing takes place until the pH of the washing water stabilizes at a value which is a function of the degree of acetylation. Lyophilization makes it possible to obtain the chitosan in solid form.

ACFTYI ATION ni l CHITOSANF The chitosan is then re-acetylated to obtain the desired degree of acetylation. This reacetylation is carried out by reaction of the amino function with acetic anhydride in a hydro-alcoholic medium. The ratio between the number of amino functions and the number of anhydride molecules present in solution determines the degree of acetylation of the chitosan produced. For a chitosan having a given degree of acetylation, a hydroalcoholic solution is used which comprises, in addition to chitosan, water, 1,2-propanadiol and the necessary quantity of acetic acid to have a quantity stoichiometric with respect to the amino functions of chitosan. In a specific embodiment, the hydro-alcoholic solution included 3 g of chitosan, 323 g of water and 272 g of 1,2-propanadiol. The acetylating mixture comprises acetic anhydride and propanediol. For example, for 62.38 g of propanediol, the mixture included 1.26 ml of acetic anhydride to obtain a degree of acetylation of chitosan of 50% and 1.62 ml of acetic anhydride to obtain a degree of acetylation of 60% .

FORMATION DF the HYπROGFI PHYSIQUF RF CHITOSANF This formation requires the passage from a liquid state to a state of gel. This passage corresponds to an anterior situation (liquid state) where the hydrophilic interactions dominate to a posterior situation (hydrogel state) where the hydrophobic interactions become strong enough so that there is no more dissolution without however being strong enough to cause complete precipitation of the polymer. The preferred preparation method, according to the invention, starts with an initial solution of chitosan. If necessary, depending on the degree of acetylation, it will be an acid solution, the c hitosane being dissolved in hydrochloric acid in stoichiometric amount with the amino groups of chitosan. After complete dissolution of the chitosan, a certain volume of 1.2 propanediol is added dropwise to the solution which is then degassed under vacuum for a period of approximately one hour. The solution is then poured into a container allowing a large free surface to volume ratio and is placed in an oven at 45 ° C. for the time necessary for it to gel. Thus the formation of the chitosan hydrogel is obtained by a physicochemical process. It is not a precipitate but a real physical hydrogel, which is a viscoelastic material containing only chitosan and more than 90% water. Unlike a precipitate, such a hydrogel does not break up by stirring or dilution.

To obtain a hydrogel which is not soluble in water at pHs of the order of 6 or 7, neutralization of the hydrogel thus obtained is carried out for approximately one hour in basic medium, for example sodium hydroxide. O, 1 molar.

The decrease in the number of positive charges due to the increase in pH promotes hydrophobic interactions and the formation of hydrogen bonds, and therefore the stability of the gel. The hydrogel is then washed to remove the alcohol and obtain a pH of approximately 7. It is this chitosan hydrogel, washed, which will be used for the cultivation of the chondrogenic cells. || it should be noted that the gel setting corresponds to a determined aqueous solution / 1-2 propanediol ratio, a ratio which depends on the degree of acetylation of the chitosan. In addition, the gelation being accompanied by a loss of water, it is important that the operating conditions favor this evaporation of the water. Several types of containers can be used during gelation, whether they are petri dishes, multi-well dishes or inserts specially designed to be housed in the wells of multi-well dishes. For example, a 24-well plate equipped with inserts is used, each insert consisting of a plastic cone, the bottom of which is formed by a membrane permeable to nutrition liquid and which is arranged to be deposited in each well without touching the bottom thereof.

MISF FN ΓUI TNRF

The chondrogenic cells can be autologous chondrocytes or chondrocyte precursor cells prepared, in vitro, from pluripotent stem cells.

Whatever the container used for the formation of the hydrogel, the hydrogel obtained is in the form of a visco-elastic and translucent block, the capacity and resistance of which depend in particular on the concentration of chitosan in the initial solution. . Preferably, this concentration is 0.5 to 4%. For the cultivation of chondrogenic cells, it is first of all necessary to increase the surface of contact between the chitosan hydrogel and said cells. For this, according to a first variant, the block of chitosan hydrogel is cut into small fragments whose external dimensions are of the order of a few millimeters. These fragments are placed in the wells of a multi-well plate or possibly in the inserts fitted to such a plate. The chondrogenic cells are introduced in the form of a suspension and mixed delicately with the hydrogel fragments. The whole is covered with an appropriate culture medium. It is found that the chondrogenic cells spontaneously adhere to the outer surface of the hydrogel fragments and do not fall into the bottom of the wells. The cultivation is carried out by placing the plates thus garnished in an atmosphere of 10% CO 2 at 37 ° C. The nutrition medium is renewed twice a week. The cultivation continues for a variable time which can range from 2 to 6 weeks depending on the size desired for the cartilage neo-tissue which forms on contact with the chitosan hydrogel. You have to choose a proportion or ratio: "number of cells / chitosan hydrogel fragment "to minimize the risk of some cells falling to the bottom of the well. In a specific embodiment, 5.10 ⁵ chondrogenic cells were placed for about thirty chitosan hydrogel fragments per insert or 1 to 3.10 ⁶ chondrogenic cells for a hundred chitosan hydrogel fragments per well, not equipped with insert.

The degree of acetylation, between 30 and 70%, but preferably between 40 and 60% induces optimal amphiphilic conditions favorable to the establishment of an environment conducive to the synthesis of the cartilage neo-tissue. Indeed, by increasing the degree of acetylation, one contributes to reinforcing the hydrophobic interactions due to the N-acetamide functions. Simultaneously, the cationicity of the residual ane sites is also increased, thereby strengthening their hydrophilic nature and their ability to create electrostatic interactions. All these conditions are favorable for the establishment of interactions with the proteoglycans of the extracellular matrix neo-formed by the chondrogenic cells.

In addition, the pH conditions, of the order of 7, are favorable to the action of enzymes, for example the lysosyme, secreted by the chondrocytes and allowing the degradation by hydrolysis of glycosuric bonds constituting the chitosan chain.

Under the conditions indicated above, the chondrocytes multiply and simultaneously synthesize an important matrix which accumulates around the cells and gradually replaces or covers the chitosan hydrogel. It is also possible to follow the formation of this cartilage neo-tissue depending on the culture time. At the early stage of culture, the chondrogenic cells adhere to the hydrogel without ever penetrating it, they secrete matrix proteins of the collagen type and proteo- glycans which accumulate around the cells and form a denser layer along the hydrogel, between the cells and the hydrogel which retains its original appearance. When the culture continues, especially for four to six weeks, the chondrogenic cells multiply from the cells bordering the hydrogel and the matrix proteins continue to accumulate. The structure of the hydrogel changes gradually taking on colors which are specific for collagen proteins and proteoglycans. A block of neoformed tissue is obtained at the end of the culture, consisting of several colonies of cells organized in more or less parallel rows and having a cell maturation gradient which is oriented from the junction zone of the cells with the hydrogel towards its periphery. . By analyzing this block of neo-tissue in histology, we note that its morphological aspect is close to a normal cartilaginous tissue. A molecular analysis by RT-PCR was carried out after five weeks of culture, for the expression of collagens I and II, agrecan, biglycan and decorin. The messenger RNAs of collagen II, agrecane, biglycan and decorin are expressed when those of collagen I are not detectable. At the protein level, the synthesis of proteoglycan was studied after incorporation of sulfur 35. The proteoglycans were extracted from the neo-tissue with guanidine chloride 4M, purified and analyzed by chromatography on a column of sepharose 2B. The elution profiles obtained show that the cells have synthesized and secreted proteoglycans which have gathered in the matrix in the form of high molecular weight aggregates, with a profile similar to those synthesized and secreted in vivo.

According to a second alternative embodiment, the chondrogenic cells have been spread, in the form of a sheet, between layers of hydrogel, each layer having a thickness of the order of a few millimeters. For example, four layers of cells have been spread in combination with three layers of hydrogel, namely two layers respectively on the outer faces of the first and third layers of hydrogel and two layers sandwiched between the first and the second respectively. second layer and the second and third hydrogel layer.

The cells were cultured under the same conditions as above and the same observations were made as regards the formation of a cartilage neo-tissue. The cell colonies which have formed either from the cells in contact with the outer face of the first and third layers of hydrogel or from the cells spread between two layers of hydrogel all have a morphological gradient similar to that which has been described above. The hydrogel layers, inserted between the layers of cells, have disappeared and are replaced by a very alkyanophilic fibrillar structure whose thickness corresponds approximately to the superposition of two to three layers of cells. It is understood that this last variant using a superposition of layers of chitosan hydrogel and chondrogenic cells makes it possible very easily to obtain a larger cartilage neo-tissue.

Whatever the variant used, the cartilaginous neo-tissue which is obtained according to the method of the invention is capable of being grafted as it is for the repair of cartilaginous, meniscal or intervertebral disc lesions, notably lesions of size important.

Claims

1. Method for preparing a graftable cartilage tissue characterized in that it consists of: a) culturing chondrogenic cells, which are either autologous chondrocytes or chondrocyte precursor cells prepared in vitro from cells pluripotent strains, b) bringing said chondrogenic cells into contact with the outer surface of a hydrogel made exclusively of chitosan, the amphiphilic properties and the degree of acetylation are such that said cells adhere naturally to the outer surface of said hydrogel, c) covering the whole hydrogel / cells thus obtained with a culture medium and, d) allowing a cartilage neo-tissue to develop in contact with the surface of the chitosan hydrogel and without penetration of the cells inside said hydrogel for a minimum period of two weeks, frequently renewing the culture medium.

2. Method according to claim 1 characterized in that the chitosan hydrogel has a degree of acetylation of between 30 and 70%, preferably between 40 and 60%.

3. Method according to one of claims 1 and 2 characterized in that the chitosan hydrogel is prepared by implementing the following steps:

- adding to a chitosan solution a determined amount of

1.2 propanediol, an amount which depends on the degree of acetylation of chitosan, - degassing under vacuum,

- the degassed solution is poured into a container making it possible to have a large free surface to volume ratio which is placed in an oven at 45 ° C. until setting in gel, - it is neutralized and the chitosan hydrogel is washed got.

4. Method according to one of claims 1 to 3 characterized in that the contacting of the chondrogenic cells with the chitosan hydrogel is done by mixing said suspended cells with chitosan hydrogel fragments whose dimensions make the order of a few millimeters.

5. Method according to claim 4 characterized in that the contacting of the chondrogenic cells with the chitosan hydrogel fragments taking place in a well, the proportion of the number of cells relative to the hydrogel fragments is determined so as to Avoid as much as possible that some cells fall to the bottom of the well.

6. Method according to one of claims 1 to 3 characterized in that the contacting of the chondrogenic cells with the chitosan hydrogel is done by spreading the cells in the form of a sheet on the chitosan hydrogel in the form layer a few millimeters thick.

7. Method according to claim 6 characterized in that the chondrogenic cells are spread in the form of several layers in contact with several layers of chitosan hydrogel.

8. A graftable cartilage tissue, obtained by the method of one of the preceding claims, characterized in that it consists of more or less parallel rows of cells, having a cell maturation gradient oriented from a determined zone towards its periphery, the determined area corresponding in particular to the junction area of the cells with the chitosan hydrogel.