CARTILAGE GEL FOR CARTILAGE REPAIR, COMPRISING CHITOSAN AND CHONDROCYTES

Abstract

The present invention concerns a method for obtaining an implantable cartilage gel for tissue repair of hyaline cartilage, comprising particles of chitosan hydrogel and cells that are capable of forming hyaline cartilage, said method comprising a step for amplification of primary cells in a three-dimensional structure comprising particles of physical hydrogel of chitosan or a chitosan derivative, then a step for re-differentiation and induction of the synthesis of extracellular matrix by said amplified cells, in the same three-dimensional structure, wherein said cells are primary articular chondrocytes and/or mesenchymal stem cells differentiated into chondrocytes. The present invention also concerns the cartilage gel obtained thereby, and its various uses for cartilage repair following a traumatic lesion or an osteoarticular disease such as osteoarthritis. The invention also concerns a three-dimensional matrix comprising particles of physical hydrogel of chitosan or of chitosan derivative, optionally supplemented with an anionic molecule such as hyaluronic acid or a derivative of hyaluronic acid or a complex of hyaluronic acid.

Claims

1. An in vitro method for obtaining an implantable composition for cartilage tissue repair comprising particles of chitosan hydrogel and cells that are capable of forming hyaline cartilage, said method comprising the following successive steps: (i) amplification of primary cells in a three-dimensional structure comprising particles of physical hydrogel of chitosan or chitosan derivative, then (ii) induction of the synthesis of extracellular matrix by said amplified cells within the three-dimensional structure of step (i), in which said cells are primary articular chondrocytes and/or primary mesenchymal stem cells differentiated into chondrocytes and said cells are not human embryo stem cells, and in which said cells do not penetrate into the hydrogel particles.

2. The method according to claim 1, wherein the two steps (i) and (ii) are carried out within the same three-dimensional structure without any cell trypsinization step.

3. The method according to claim 1, wherein step (ii) for inducing the synthesis of extracellular matrix is carried out by changing the culture medium.

4. The method according to claim 1, wherein said composition is produced in less than 45 days, preferably less than 35 days, in particular less than 28 days.

5. The method according to claim 1, wherein the duration of the amplification step is between 1 and 3 weeks, preferably less than two weeks, and enables the number of cells to be multiplied by at least 4, preferably by 6.

6. The method according to claim 1, wherein the duration of the step for synthesizing extracellular matrix is 2 to 4 weeks, preferably less than three weeks.

7. The method according to claim 1, wherein the hydrogel particles have a mean size in the range 10 μm to 1.5 mm, preferably in the range 400 μm to 700 μm.

8. The method according to claim 1, wherein the chitosan has a weight average molecular weight higher than 50 kDa, preferably between 150 and 220 kDa.

9. The method according to claim 1, wherein the chitosan is extracted from fungi, preferably from Agaricus bisporus.

10. The method according to claim 1, wherein the three-dimensional structure also comprises an anionic polymer associated with particles of chitosan hydrogel at the surface, preferably hyaluronic acid, which is present during the two steps or is added at the end of step (i) or (ii).

11. The method according to claim 1, wherein the hyaluronic acid is extracted by bacterial fermentation, with a molecular mass by weight of more than 1 MDa, preferably more than 2 MDa.

12. The method according to claim 1, wherein the amplification step is carried out with a medium comprising FGF-2 (fibroblast growth factor) and insulin.

13. The method according to claim 1, wherein the step for inducing the synthesis of extracellular matrix is carried out with a medium comprising BMP-2 (bone morphogenic protein-2), insulin and triiodothyronine T3.

14. The method according to claim 1, wherein said cells are articular chondrocytes or are obtained from the nasal septum or atrial septum, and are of human, canine or equine origin.

15. The method according to claim 1, wherein the cell viability is more than 90%, preferably more than 93%.

16. The method according to claim 1, wherein the water content in the chitosan hydrogel is more than 70%, preferably more than 80%.

17. The method according to claim 1, wherein the physical hydrogel of chitosan is synthesized without a cross-linking agent.

18. The method according to claim 1, wherein the cells are added to the three-dimensional structure after gelling and formation of the physical hydrogel particles of chitosan or chitosan derivative.

19. An implantable composition comprising a three-dimensional structure formed from particles of physical hydrogel of chitosan or chitosan derivative, associated with an anionic polymer, preferably hyaluronic acid, and differentiated chondrocytes or any cells differentiated into chondrocytes.

20. The implantable composition according to claim 19, wherein the chondrocytes preferentially synthesize type II collagen proteins and messenger RNA of type II collagen, expressing a COLII/COLI ratio higher than 1.

21. The implantable composition according to claim 19, obtainable by an in vitro method for obtaining an implantable composition for cartilage tissue repair comprising particles of chitosan hydrogel and cells that are capable of forming hyaline cartilage, said method comprising the following successive steps: (i) amplification of primary cells in a three-dimensional structure comprising particles of physical hydrogel of chitosan or chitosan derivative, then (ii) induction of the synthesis of extracellular matrix by said amplified cells within the three-dimensional structure of step (i), in which said cells are primary articular chondrocytes and/or primary mesenchymal stem cells differentiated into chondrocytes and said cells are not human embryo stem cells, and in which said cells do not penetrate into the hydrogel particles.

22. The composition implantable by arthroscopy according to claim 19, for use in cartilage repair.

23. The implantable composition according to claim 19, wherein the water content in the chitosan hydrogel is more than 70%, preferably more than 80%.

24. The implantable composition according to claim 19, wherein the physical hydrogel of chitosan is synthesized without a cross-linking agent.

25. A three-dimensional structure formed by particles of physical hydrogel of chitosan or of chitosan derivative, associated with a hyaluronic acid polymer, wherein said chitosan is extracted from fungi, and wherein said hydrogel particles have a mean size in the range 400 μm to 700 μm.

26. Use of a three-dimensional structure formed by particles of physical hydrogel of chitosan or of chitosan derivative, associated with an anionic polymer, preferably hyaluronic acid, to seed chondrocytes for the purposes of proliferation and synthesis of extracellular matrix by said chondrocytes.

Description

FIGURES

[0140] FIG. 1: scanning electron microscopy micrograph of a physical hydrogel of pure chitosan.

[0141] FIG. 2: scanning electron microscopy micrograph of a physical hydrogel of pure dehydrated chitosan

[0142] FIG. 3: optical microscopy image of a physical hydrogel of pure chitosan after treatment with eosin.

[0143] FIG. 4: shows the viability rates for cells seeded into a M1-type 3D structure as a function of the initial density of chondrocytes, measured with the Live and Dead kit on 7-day culture fractions (during amplification, in FI medium). The count of the dead cells (in black) and live cells (grey) was carried out with ImageJ software from fluorescence microscopic images with a magnification of ×20. The percentage of dead cells was calculated for each condition by calculating the dead cell/total cell ratio.

[0144] FIG. 5: shows the evolution as a function of time of the population of chondrocytes in 3D structures based on particles of physical hydrogel of chitosan extracted from fungi supplemented or not with hyaluronic acid, with different particle sizes or in 3D structures based on particles of physical hydrogel of chitosan extracted from squid, or also of chondrocytes cultured in monolayers under “FI” culture conditions. The number of cells is along the ordinate; the culture time in days is along the abscissa. p FIG. 6: shows the evolution in the population of chondrocytes as a function of time for different initial densities of chondrocytes cultured as monolayers, under “FI” culture conditions, confirming that the cell population obtained is identical beyond 7 days irrespective of the initial density.

[0145] FIG. 7: optical microscopic images as a function of time of chondrocytes cultured inside the three-dimensional structures of pure hydrogel particles (M1).

[0146] FIG. 7A: represents cells obtained from the amplification step in FI medium 14 days after seeding.

[0147] FIG. 7B: represents cells during the ECM synthesis step in BIT medium 24 days after seeding, i.e. 10 days after inducing re-differentiation and ECM synthesis. The magnification ratio is ×20.

[0148] FIG. 8: shows the quantity of messenger RNA of type I collagen and type II collagen relative to the GAPDH gene, measured by quantitative RT-PCR for chondrocytes cultured in a 3D structure (M1) with several initial cell densities compared with the monolayer technique, after 35 days of culture.

[0149] FIG. 9: shows the ratio of messenger RNA for the COLII/COLI genes obtained by quantitative RT-PCR, for chondrocytes seeded in a 3D structure (MD with several initial cell densities compared with the monolayer technique, after 35 days of culture.

[0150] FIG. 10: shows Western blot analysis of the protein counts for type I and type II collagen, for chondrocytes cultured in a three-dimensional structure M1 compared with chondrocytes cultured in monolayers, after 35 days. The level of expression of actin acts as a control.

[0151] FIG. 11: shows the analysis, by immunohistochemistry, for chondrocytes cultured in 3D structure M1 and M2 compared with those cultured in monolayers (×20) (MC) after 35 days, for the same initial cell density (6×10.sup.5 cells) by HES and SO staining, as well as immunolabelling of type I collagen and of type II collagen.

EXAMPLES

Example 1

Synthesis of Physical Chitosan-Based Hydrogel.

[0152] The chitosan used was from non-animal origin, extracted from the cell wall of the common mushroom, Agaricus bisporus. Its weight average molecular weight (Mw) was 170 g/mol; and its degree of acetylation (DA) was 32%. It was used in the form of a powder.

[0153] The pure chitosan was dissolved in an acidic solution of acetic acid (1% in water), in stoichiometric amounts with the amine groups of the chitosan. The solution was stirred at 10 room temperature until the chitosan had completely dissolved, i.e. for at least 3 h, preferably at least 6 h.

[0154] Next, 1,2 propanediol was added in a quantity identical to that of the acetic acid and stirring was continued for at least 30 min, preferably 1 h at room temperature. The mixture could then be degassed at room temperature, or under vacuum if necessary if the solution shows a lot of air bubbles.

[0155] The solution was then poured into containers like multi-well plates or 3 cm petri dishes, then it was left to stand, preferably overnight. The solution was then placed in a vacuum oven, preferably at 50° C., for the time necessary to allow a gel to form, preferably at least 20 hours.

[0156] The gelling step could also be carried out at room temperature, but then would have required longer times (5-8 days depending on the intrinsic characteristics of the chitosan).

[0157] The thickness of the solution before gelling could be in the range 2 to 7 mm, preferably in the range 3 to 6 mm, in order to favour evaporation and hydrophobic-like interactions for good gel setting.

[0158] The physical hydrogel obtained was then neutralized in a basic medium with a 0.1 N sodium hydroxide solution, preferably for 1 h. Next, several washes with water were carried out, preferably with sterile water. Each wash preferably lasted approximately 1 hour in order to 15 remove excess alcohol and bring the hydrogel to a neutral pH. In general, at least 6 washes were carried out.

[0159] The gel obtained thereby had a water content of approximately 80% by weight. The final concentration by weight of chitosan in the hydrogel was in the range 1% to 4.5% before neutralization, preferably between 3.4% and 4.2% before neutralization.

[0160] It is important to control the temperature and humidity conditions during the synthesis of the chitosan-based hydrogels, more particularly when it is extracted from fungi, preferably under room temperature conditions which are below 25° C.

[0161] The hydrogel obtained at the end of these various steps had a thickness of 3 to 6 mm, preferably between 4 and 5 mm thick, and was a translucent white colour and its surfaces were smooth and regular. However, its appearance could vary as a function of the intrinsic properties of the basic chitosan, in particular the degree of acetylation, the molar mass and the concentration. It was in the form of a viscoelastic block with mechanical properties which depended on the intrinsic characteristics of the starting chitosan, in particular and once again the degree of acetylation, the molar mass and the concentration.

[0162] The hydrogel obtained was easy to manipulate, detached without difficulty and without tearing the flat surface on which it had been produced.

[0163] Conventional scanning electron microscopic observation of the dehydrated hydrogel showed a fibrillar 3D structure, porous, similar to that of a living tissue, as can be seen in FIG. 2. Scanning electron cryomicroscopic observation of the hydrated hydrogel, as can be seen in FIG. 1, showed a pore size between 1 -3μm, which did not allow cells to penetrate inside the hydrogel but allowed free diffusion of nutrients and cellular waste.

Example 2

Synthesis of Particles/Fragments of Chitosan Hydrogel in Order to Produce the 3D Structure (Structure M1 and Structure M2).

Structure M1:

[0164] The chitosan hydrogel obtained at the end of Example 1 was cut into small squares with 1 mm sides then placed in 10 mL of water, preferably sterile water. The hydrogel was then ground with the aid of an Ultra Turrax, at 6000 to 17000 rpm for 10 seconds, carrying this out 2 -4 times. In order to obtain particles with a homogeneous size and the expected diameter, grinding was preferably carried out at 6000 rpm for 10 seconds repeated 3 times, in order to obtain particle sizes of: 400-700 μm (50%), or in fact 250-900 μm (>80%), with a mean of the order of 650 microns.

[0165] The solution obtained was centrifuged, preferably at 1375 g for 7 minutes, in order to recover the pellet constituted by particles of chitosan hydrogel. FIG. 3 illustrates an example of the chitosan particles obtained. A mini-spoon was used to measure the quantity of particles of chitosan which would be brought into contact with the chondrogenic cells. Preliminary tests 25 validated the reproducibility of the measurement.

Structure M2:

[0166] In order to reinforce the viscoelastic properties of the 3D structure in which the chondrocytes 30 were seeded, the inventors also produced a second structure (M2) by adding an anionic constituent, interacting with the cationic functions of the chitosan. The selected polymer was hyaluronic acid, preferably from bacterial origin, since such a constituent is known for its better biocompatibility properties, in order to avoid allergies or any rejections. The molecular mass by weight of the hyaluronic acid used in producing the structure M2 was approximately 2 MDa. The hyaluronic acid was added after preparing the chitosan hydrogel particles.

Example 3

Culture of Cells in 3D Structure

[0167] The cells used in the context of this example were human chondrocytes obtained from human samples and treated in accordance with the protocol described in the document FR 2 965 278 (University of Caen Basse-Normandie, et al).

[0168] The hydrogel particles obtained at the end of Example 2, with hyaluronic acid (3D structure M2) or without hyaluronic acid (3D structure MD were sterilized, for example at 121° C. for 15 minutes, prior being brought into contact with the cells. Several mini-spoons of hydrogel particles were removed, preferably 2 mini-spoons corresponding to 80 to 84 particles, which were introduced into the wells of a 24-well culture plate which had been covered with an insert (pore size 8 μm). The cells were added thereto, between 10.sup.5 and 10.sup.7, preferably of the order of 6×10.sup.5 cells/wells, per 80-84 particles of hydrogel, which were mixed carefully with the chitosan hydrogel particles. This proportion of cells with respect to the 3D structure corresponded to approximately 6.7 x 10.sup.6 cells per gram of 3D structure at the moment of seeding.

[0169] Culture was carried out in a controlled atmosphere in an oven at 37° C., with a CO.sub.2 percentage of 5% under normoxic conditions.

[0170] The cells adhered spontaneously to the chitosan hydrogel particles. The quantity of cells falling to the bottom of the well was considered to be negligible.

[0171] As a control, 6×10′ cells obtained as described above were cultured in monolayers on plastic in 24-well plates under culture conditions which were identical to those described above for the 3D structures (controlled atmosphere in an oven at 37° C., with a CO.sub.2 percentage of 5%, normoxia). The cells also adhered there spontaneously.

Example 4

Cell Proliferation Step

[0172] In this step, the selected culture medium was favourable to the multiplication of cells.

[0173] The selected medium was a 50/50 solution of DMEM-HAM F12 +1% AB (streptomycin/penicillin)+10 FCS supplemented with “Fl” solution comprising FGF-2 in a concentration of 5 ng/mL +insulin in a concentration of 5 μg/mL. (Claus et al; 2012).

[0174] After a short period, the mixture described in the preceding step was recovered from this culture medium which is known to favour the proliferation of cells.

[0175] During this amplification phase, the culture medium was renewed 3 times per week for the 10 cultures in the 3D structures, as was the case for the monolayer cultures. The proliferation period lasted between one and two weeks in order to obtain a sufficient number of cells.

[0176] The inventors observed that the amplification phase lasted approximately two weeks when the cells were seeded into a structure constituted by particles of pure hydrogel (3D structure M1) and could be shortened to 1 week in the presence of hydrogel particles supplemented by linear chains of an anionic molecule such as hyaluronic acid (3D structure M2).

[0177] Furthermore, the initial quantity of 6×10.sup.5 cells/insert could of course be increased provided that the condition regarding number of cells/mass of hydrogel or number of cells/volume of hydrogel or number of cells/ number of hydrogel particles is adhered to. By way of example, it is entirely possible to seed 1 to 1.5×10.sup.6 cells/insert, provided that the necessary quantity of 3D structure is added in order to obtain more than 4×10.sup.6 cells/insert at the end of the method, or even 10.5×10.sup.6 cells/insert, or more.

Analysis by Optical Microscopy:

[0178] Analysis by phase contrast microscopy was carried out. It confirmed that the cells adhered well to the particles of chitosan hydrogel and that this environment was favourable to their culture. The culture conditions (three-dimensional structure M1 or M2, and FI culture medium) favoured the proliferation and division of the chondrocytes.

[0179] The cells observed could proliferate either in an isolated manner or in clusters/bunches. The cultures carried out in 3D structures of hydrogel particles exhibited mainly round cells. The elongated form, characteristic of fibroblasts, was not observed in the 3D structures M1 and M2, except occasionally at the periphery, i.e. at the interface between the structure and the external medium.

[0180] As a control, the inventors carried out monolayer cultures at the same time. After 24 hours of culture, in the same FI medium as the cells seeded into the structures M1 and M2, the chondrocytes adopted an elongated morphology characteristic of fibroblastic cells.

Viability of cells:

[0181] The viability of the chondrocytes seeded inside the three-dimensional structures was measured with the Live and Dead kit on fractions of cultures at 7 days (during amplification, in FI medium). The dead cells (red) and live cells (green) were counted using ImageJ software from fluorescence microscope images, magnification ×20. The percentage of dead cells was estimated for each condition by calculating the dead cell/total cell ratio. The viability was more 15 than 93%, or in fact more than 97%, which demonstrated good compatibility with the 3D structure.

[0182] FIG. 4 illustrates the obtained results.

Proliferation Tests:

[0183] Proliferation tests were carried out by counting the total cells using the Cellometer T4 after detaching the cells with trypsin and staining the dead cells with trypan blue.

[0184] The measurements were carried out after 1 day (DI), 14 days (D14) and 21 days (D21) of culture after seeding the primary chondrocytes at D0.

[0185] FIG. 5 illustrates the evolution in the cellular population.

[0186] After 7 days, the increase in the number of cells was clearly observed. The cells survived and proliferated very well in the 3D structure as well as in monolayers (MC).

[0187] In the three-dimensional structures, M1 or M2, the cells remained round during the multiplication step, while they adopted an elongated shape like fibroblasts in monolayers.

[0188] Further, FIG. 6 illustrates that in monolayers, the cell population was identical from 7 days, irrespective of the initial density of the seeded cells.

[0189] In conclusion, at the end of this proliferation step, amplification of the cells in a three-dimensional structure composed of particles of pure chitosan hydrogel (structure M1) were observed to be almost as productive as in monolayers (MC), which is the reference protocol for the multiplication of cells such as chondrocytes, but it does involve a trypsinization step which can be avoided by using the structure M1.

[0190] Adding hyaluronic acid to the three-dimensional structure of chitosan hydrogel particles (structure M2) induced a very strong acceleration of cell proliferation, much greater than the M1 structure or the monolayer culture, in particular by a ratio of two.

Example 5

Differentiation and Production of Extracellular Matrix

[0191] In the context of the present invention, the steps for multiplication and differentiation were preferably distinct: firstly, the cells are multiplied and secondly, they are differentiated and produce extracellular matrix. The culture medium used for the preceding multiplication step was modified after 15 days. The culture medium “FI” was replaced by a medium “BIT” with the aim to favouring the step for differentiation of cells and the production of extracellular matrix.

[0192] Thus, the culture medium was preferably composed of: 50/50 DMEM-HAM F12+1% AB (streptomycin/penicillin)+10 FCS, to which a BIT solution composed of the following was added:

[0193] BMP-2, 200 ng/mL+insulin, 5 μg/mL+triiodothyronine, T3, 100 mM (Claus et al, 2012).

[0194] This fresh culture medium was renewed every two or three days. The period for re-differentiation and chondrogenesis preferably lasted 3 weeks. As a control, the same medium change was employed as for the cells cultured in monolayers.

Analysis by Optical Microscopy:

[0195] Cells with a fibrillar appearance in monolayers in Fl medium are known to become round after 1 week of culture in BIT medium. The cells cultured in monolayers (corresponding to the control) changed appearance after changing to BIT medium, indicating that the change in culture medium indeed induced a change in the behaviour of the cells.

[0196] In the hydrogels (structures M1 and M2), the cells were primarily round at the end of the amplification phase and continued to be round during the entire phase for the production of extracellular matrix. This point is illustrated in particular in FIG. 7.

[0197] At the end of culture, D35, the cells were all round in the hydrogels (structures M1 and M2), less so in monolayers.

[0198] In the three-dimensional structures, the cells could agglutinate the particles of hydrogel and form a kind of “bead” with a compact form to a greater or lesser extent. This observation was made under the majority of conditions containing the hydrogels, but not in the monolayers, however, which acted as the control. This observation constitutes a proof of the strong production of extracellular matrix which had accumulated around the cells. The cells produced more extracellular matrix in a three-dimensional environment than in monolayers.

[0199] It should be noted that under certain conditions, however, the composition constituted by said beads remained injectable despite the synthesis of a large quantity of extracellular matrix. Whatever the case, the composition was implantable.

PCR Tests:

[0200] PCR was used to quantify the degree of transcription of the following proteins: COLI, COLII and GAPDH. The degree of transcription acts as a reference for comparing the levels of transcription of COLI and COLII. In fact, it is well known that the chondrocyte phenotype and the production of extracellular matrix are accompanied by a strong synthesis of COLII transcripts, while the COLI transcripts generally accompany the process of dedifferentiation, in particular into a fibroblast phenotype. The results are presented in FIG. 8.

[0201] The COLII/GAPDH results produced at the end of the ECM synthesis step, show that there are more COLII transcripts for the cultures in hydrogel than for the monolayer cultures. The COLI/GAPDH results show that, in contrast, there are more COLE transcripts in the monolayer cultures than in the cultures within the three-dimensional structures.

[0202] The result of the calculation of the COLII/COLI ratio is illustrated in FIG. 9. It shows that the ratio is indeed better, and in fact that re-differentiation is much better after dedifferentiation within the 3D structures constituted by particles of chitosan hydrogel compared with the monolayers.

[0203] The three-dimensional environment tested thus substantially favours the re-differentiation of dedifferentiated chondrocytes following prior intense multiplication, by a ratio of at least 6. This 3D structure favours the expression of chondrocyte phenotype.

[0204] The culture conditions (three-dimensional structure based on chitosan hydrogel, and BIT culture medium) thus favour the re-differentiation of chondrocytes and the production of cartilaginous matrix, compared with monolayer culture.

Western Blot Tests:

[0205] After having verified the degrees of transcription of the Coll and ColII genes as a function of the culture conditions (3D or monolayers), the degree of synthesis of the corresponding proteins was verified using the Western blot technique. The results of the various Western blots are illustrated in FIG. 10.

[0206] The results for the anti-COLII Western blot revealed the presence of COLII in all conditions. The results for the anti-COLI WB revealed more intense spots in monolayers. This observation corroborates the fact revealed in Q-PCR: the COLII/COLI ratio is higher in 3D hydrogel structures than in monolayers.

[0207] The Western blot analysis showed the expression of characteristic proteins of articular cartilage in the 3D structure.

Immunohistochemistry Results:

[0208] The compositions obtained were then observed using immunohistochemistry in order to compare the implementation of the novel method using the three-dimensional structure, and the traditional culture using monolayers, at the level of the synthesis of ECM, proteoglycans, and collagen types I and II. The results are illustrated in the photos of FIG. 11.

[0209] The presence of more proteoglycans in the three-dimensional structures (M1 and M2 in FIG. 11) than in the monolayers (MC in FIG. 11) are clearly observed because of the Safranin O staining (SO), which demonstrates the presence of GAG. Furthermore, the production of a lot of extracellular matrix and type II collagen was observed on the immunohistochemistry images for cells in the three-dimensional environment respectively evidenced with HES staining, which demonstrates the presence of nuclei and ECM, and by collagen II immunolabelling, which demonstrates the presence of COLII. Highlighting of the cells in the matrix by collagen I immunolabelling also confirms the quasi-absence of collagen I when the chondrocytes are cultured in the three-dimensional structures.

Example 6

Chondrocytes Implantation

[0210] The assembly of the cells and the 3D structure (i.e. either the structure M1 constituted by particles of chitosan hydrogel or the structure M2, constituted by particles of chitosan hydrogel supplemented with an anionic molecule like hyaluronic acid) constitutes, at the end of culture, i.e. between 3 and 6 weeks, a cartilaginous neo-tissue which may be injected or implanted by arthroscopy.

[0211] It is clearly possible to increase the number of cells in an insert by increasing the quantity of hydrogel, keeping however the same conditions for the number of cells with respect to the mass of hydrogel or the number of hydrogel particles constituting the 3D structure.

[0212] By way of example, for a sample of 0.3 g−0.5 g of human cartilage, 1−1.5×10.sup.6 cells (chondrocytes) may be extracted. Since in the preceding examples the inventors have demonstrated that, starting from 6×10.sup.5 initial cells per insert, it is possible to obtain from them 3.6×10.sup.6 cells/insert in 0.09 g of hydrogel structure, corresponding to 80 -84 particles of hydrogel, the following concentration data were obtained: [0213] initial concentration of 6.7×10.sup.6 cells/g of biomaterial, [0214] final concentration of more than 40×10.sup.6 cells/g of biomaterial (3D structure).

[0215] For a sample from 1 to 1.5×10.sup.6 cells, then, more than 3×10.sup.6 cells can be obtained, or even more than 10.5×10.sup.6 cells at the end of the method, after 2-5 weeks, which is amply sufficient for a construct where the recommended quantities are 3.2−6.5×10.sup.6 cells.

[0216] In conclusion for the preceding examples, the following points are observed:

[0217] During the amplification/multiplication phase: [0218] an equivalent yield or in fact a greater yield in the three-dimensional structure comprising particles of pure chitosan hydrogel compared with monolayer culture, [0219] a much higher yield in three-dimensional structure comprising hydrogel particles supplemented with hyaluronic acid than in monolayers.

[0220] During the phase for differentiation and ECM production: [0221] a re-differentiation of cells inside 3D structures and in monolayers, as proved by the PCR, WB and immunohistochemistry analyses; [0222] a ratio of COLII/COLI messenger RNA which is significantly higher in the three-dimensional structure compared with monolayer culture, [0223] a COLII/COLI protein ratio which is significantly higher in three-dimensional structure than in monolayers, [0224] a stable chondrocyte phenotype in the 3D structure, [0225] abundant production of cartilaginous matrix in the 3D structure.

[0226] The succession of steps in the same three-dimensional medium comprising particles of chitosan hydrogel with/without structuring molecule, amplification then differentiation/chondrogenesis is highly favourable to the production of an injectable or implantable cartilaginous neo-tissue with excellent mechanical and biological properties.

[0227] The addition of hyaluronic acid improves the system by accelerating the process for the amplification of cells and meaning that the number of cells to be implanted can be increased, or a week can be saved over the overall protocol.

[0228] The configuration of the structure can be used to optimize the contact surface with the cells.

Example 7

Comparison Between Various 3D Structures

[0229] The inventors reproduced the 3D structures described in the preceding examples, in particular in Example 2, by varying the type of chitosan constituting the hydrogel particles, the size of the hydrogel particles and the presence and the concentration of hyaluronic acid. The proliferation ratios were compared and the results obtained are illustrated in Table 1. The value 1 was attributed to the structure M1 corresponding to particles of several hundred microns obtained from chitosan of fungi.

[0230] The proliferation ratio obtained with the structure M2 (extracted from fungi and supplemented with 2M hyaluronic acid) was twice as high as the structure M1 (extracted from fungi, not supplemented with hyaluronic acid), which itself makes it possible to obtain a proliferation rate 1.5 times higher than chitosan extracted from squid, or in fact with particles with a size of the order of tens of μm.

TABLE-US-00001 TABLE 1 Proliferation ratios Fragments, hundreds Fragments, of μm tens of μm Chitosan extracted from fungi supplemented 2** with 2M HA Chitosan extracted from fungi supplemented 1.2 with 1M HA Chitosan extracted from fungi 1* 0.67 Chitosan extracted from squid 0.67 *structure M1; **structure M2

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