WO2012038668A1 - Method for obtaining differentiated articular chondrocytes in vitro or ex vivo, and uses of same - Google Patents

Abstract

The present invention relates to a method for obtaining differentiated articular chondrocytes in vitro or ex vivo, and to the use of said chondrocytes for treating the loss of chondral substance possibly linked to a traumatic impact, osteoarticular diseases and, in particular, diseases that are characterised by a destruction or degeneration of cartilage, such as arthrosis.

Description

 PROCESS FOR IN VITRO OR EX VIVO PRODUCTION OF DIFFERENTIATED JOINT CHONDROCYTES AND USES THEREOF

 The present invention relates to the field of cell and tissue engineering of cartilage, and in particular the culture of chondrocytes or chondrocytes obtained from mesenchymal stem cells in vitro or ex vivo, in particular for the purpose of cell therapy.

 More specifically, the invention relates to a method for obtaining in vitro or ex vivo differentiated articular chondrocytes and the use of these chondrocytes for the treatment of chondral fat loss related or not to traumatic shock and osteoarticular diseases, especially diseases that are characterized by destruction or degeneration of cartilage, such as osteoarthritis.

 The significant incidence of pathologies characterized by cartilage destruction or degeneration is at least in part due to the fact that this tissue has a very limited spontaneous repair potential. Currently, medicinal treatments for cartilage degeneration are only palliative and do not prevent the evolution of the destruction. In the ultimate stages, prosthesis placement is unavoidable.

The autologous chondrocyte transplantation (TCA), initiated by Brittberg in 1994 for the treatment of articular cartilage lesions of the knee, consists of isolating by enzymatic extraction chondrocytes from a small sample of low morbidity. This therapeutic approach, used worldwide, involves cell amplification in culture on plastic. This technique is currently indicated for the treatment of limited cartilage lesions. To bridge larger lesions and to apply the technique to early osteoarthritic lesions, the technology is moving towards the combination of autologous chondrocytes with biomaterials for transplantation, such as collagen gels. However, the amplification of chondrocytes in monolayer culture for direct grafting or after combination with a biomaterial is accompanied by cell dedifferentiation, which is reflected, in vivo, by the formation of a non-functional fibrocartilage . Implantation of autologous chondrocytes is therefore a technique that remains perfectible in controlling the expression of the phenotype of chondrocytes, either during their amplification in culture, or within a biomaterial. She does not does not reproduce a functional hyaline cartilage, rich in collagen type II. The synthesized tissue is a fibrocartilage, rich in collagen type I, not having the same biomechanical properties that can confer a healthy cartilage rich in collagen type II, which requires renewal of the term intervention because this fibrocartilage substitution will be degraded in turn.

 Thus, the therapeutic needs for the treatment of these conditions are not met, particularly because there is currently no treatment to restore the chondrocyte phenotype. It is in this context that the present invention proposes to provide a method for obtaining in vitro or ex-vivo differentiated articular chondrocytes capable of synthesizing functional hyaline cartilage and thus imparting the improved biomechanical properties of this type of cartilage. compared to fibrocartilage.

 The present invention therefore relates to a process for obtaining in vitro or ex vivo differentiated articular chondrocytes characterized in that it comprises, or even consists of, the following steps:

 - to culture dedifferentiated or differentiated articular chondrocytes or mesenchymal stem cells,

 cultivating these cells under conditions of low oxygen tension, in the presence of BMP-2 ("Bone Mofphogenetic Protein-2") and specific inhibitor (s) of type I collagen.

 In the context of the invention, the inventors have demonstrated that the culture conditions used in the process as described above make it possible to promote a stable and most differentiated chondrocyte phenotype, and in particular characterized by a differentiation index. increased. In particular, the combination of low oxygen tension culture conditions in the presence of BMP-2 and type I collagen inhibitor promotes the increase of cartilage specific markers (type II collagen and aggrecan in particular). by decreasing non-specific markers of cartilage.

The conditions for carrying out the process according to the invention make it possible to limit the phenomenon of cell dedifferentiation inherent in the monolayer cell culture of chondrocytes in vitro or ex vivo, and to maintain and / or induce a stable chondrocyte phenotype. The method according to the invention thus makes it possible to obtain, in vitro, differentiated mature chondrocytes which exhibit the characteristics of cells derived from healthy and functional hyaline-type cartilage. As part of this method, it is possible to use articulated (i.e. cartilage derived from different anatomical locations) articular chondrocytes or differentiated or dedifferentiated mesenchymal stem cells which are by definition undifferentiated. Preferably, the cells which are used in the process according to the invention are differentiated or dedifferentiated articular chondrocytes, in particular adult articular chondrocytes, in particular human or equine chondrocytes. Joint chondrocytes or mesenchymal stem cells may be obtained by any method known to those skilled in the art to recover these cells from any biological sample that may contain them.

 The mesenchymal stem cells used may come from a variety of sources, such as bone marrow, adipose tissue, umbilical cord blood, muscle origin, germ line, embryonic, induced pluripotent stem (iPS) cells, and the like. According to a particularly advantageous embodiment, the cells used are chosen from bone marrow stem cells and non-hematopoietic progenitors of the cord blood.

 Cells that are cultured, i.e., differentiated or dedifferentiated articular chondrocytes or mesenchymal stem cells, may be of animal origin, and preferably human or equine.

 In the particular case of the use of human embryonic stem cells, stem cells that are not derived from the destruction of human embryos, such as induced pluripotent stem cells (iPS), will be used.

 Preferably, the mesenchymal stem cells used are non-human when they are embryonic or germ line mesenchymal stem cells.

The differentiated articular chondrocytes used in the context of the process of the invention are preferably derived from healthy subjects, but it is also possible to use dedifferentiated chondrocytes, and especially from patients, in particular patients suffering from osteoarticular disease. , in particular characterized by destruction or degeneration of the cartilage. According to a particular embodiment, the dedifferentiated articular chondrocytes used are derived from the patient intended to receive the chondrocytes that will be obtained by the implementation of the method of the invention (autologous transplant). he is also It is possible to perform an autologous transplant using mesenchymal stem cells to obtain chondrocytes according to the invention which will be implanted in a sick patient, and in particular suffering from chondral substance losses related or not to traumatic shock or suffering from osteoarthritis. articular, particularly characterized by destruction or degeneration of cartilage, such as osteoarthritis.

 In the context of the method according to the invention, it is also possible to use co-cultures of differentiated and / or dedifferentiated articular chondrocytes and / or mesenchymal stem cells.

According to the present invention, differentiated or dedifferentiated articular chondrocytes or mesenchymal stem cells are cultured under conditions of low oxygen tension, i.e. under hypoxic conditions. Preferably, culture conditions of less than or equal to 10% of O ₂ , preferably ranging from 1 to 10%, and in particular from 1 to 5% of O _{2, are used} .

 The cells used in the method of the invention are cultured in a conventional culture medium, for example DMEM, adapted to the selected cell type, namely differentiated or dedifferentiated chondrocytes or mesenchymal stem cells.

 The differentiated or dedifferentiated articular chondrocytes or the mesenchymal stem cells used in the method of the invention may be cultured in the form of a monolayer culture or preferably within a biomaterial. By "biomaterial" is meant any three-dimensional structure colonizable by cells, in particular by chondrocytes, and preferably capable of being implanted in vivo. Thus, the biomaterial used in the context of the invention is biologically inactive and preferably biodegradable. Examples of biomaterials that can be used include collagen gels, type I or type II collagen sponges, alginate gels, hyaluronic acid gels, or PLGA (poly) gels. (lactic-co-glycolic acid)), or other natural or synthetic matrices, or combinations of these different matrices. Preferably, the differentiated or dedifferentiated articular chondrocytes or the mesenchymal stem cells are cultured within type I collagen sponges.

According to the present invention, the culture of differentiated or dedifferentiated articular chondrocytes or mesenchymal stem cells is performed in the presence of BMP-2. To do this, it is possible to directly add the molecule BMP-2 in the culture medium. It is also possible to use means making it possible to maintain its presence in the culture medium, for example by stimulating its production and / or preventing its degradation and / or inactivation or that of its receptor. Therefore, recombinant BMP-2 (Inductos, Dibotermin-alpha, or other, for its drug form, or in its recombinant form), and preferably of the same species as the cells used, can be added directly to the culture medium. that is, human BMP-2 in human cells, equine BMP-2 in equine cells, etc. According to the invention, the reference human BMP-2 NP_001191.1 (GL4557369, September 11, 2011) (SEQ ID NO: 1) or the reference equine BMP-2 XP_001493945.1 will be used (GI: 149733087, June 27 2011) (SEQ ID NO: 2).

 According to a particularly preferred embodiment of the invention, any means is used to maintain the presence of BMP-2 in the culture medium, directly or indirectly, and in particular by stimulating its production and / or preventing the degradation of its receptor and / or its inactivation. For example, the presence of BMP-2 can be obtained by any means to prevent the presence or inactivation of the action of any molecule capable of degrading or inactivating BMP-2, such as proteases and in particular HTRA1 protease (Hig Temperature Requirement Protein) described by Zumbrunn J. and Trueb B., 1996, FEBLS Letter 398: 187-192, or members of the HTRA1 family. In particular, the action of BMP-2 can be maintained using an HTRA1 siRNA to prevent degradation of the BMP-2 receptor.

 Particularly advantageously, the presence of BMP-2 is ensured both by adding the BMP-2 molecule directly into the culture medium and by preventing its degradation and / or inactivation or that (s) of its receptor. In the context of this particular embodiment, the presence of BMP-2 is ensured both by adding the BMP-2 molecule directly into the culture medium and by preventing the HTRA1 protease from inactivating the action of the BMP. -2, preferably by the use of a siRNA HTRA1.

When the presence of BMP-2 is obtained by direct addition of this protein in the culture medium, it is added in a final concentration ranging from 10 to 200 ng / ml, and preferably of the order 50 ng / ml. The scope of the present invention is not limited to BMP-2 in particular. The effect of the presence of BMP-2 during the culture of chondrocytes or mesenchymal stem cells according to the invention can also be obtained by the presence of any other molecule, or cytokine in particular, likely to have the same effects or effects similar to those induced by BMP-2 on chondrocyte differentiation. In particular, the presence of BMP-2 in the context of the invention can be obtained by any other member of the BMP family capable of inducing the same effects or effects similar to those induced by BMP-2 on chondrocyte differentiation.

 Other cytokines may optionally be added to the culture medium, alone or as a mixture. For example, the culture medium may also contain TGF-βΙ for example at a final concentration ranging from 1 to 50 ng / ml, and preferably of the order of 10 ng / ml, of TGF-3. for example at a final concentration ranging from 1 to 50 ng / ml, and preferably of the order of 10 ng / ml, of 1 GF-1 for example to a final concentration in a range from 1 to 50 ng / ml, and preferably of the order of 10 ng / ml, estradiol for example at a final concentration in a range from 0.5 to 5 nM, and preferably of the order of 1 nM.

 Different combinations of these cytokines can thus be used when the method of the invention is implemented from differentiated and / or dedifferentiated chondrocytes and / or mesenchymal stem cells. For example, the presence of TGF-βΙ is preferred when the method according to the invention is implemented from mesenchymal stem cells, and in particular mesenchymal stem cells derived from bone marrow.

 According to the present invention, the culture of differentiated or dedifferentiated chondrocytes or mesenchymal stem cells is also carried out in the presence of specific inhibitor (s) of type I collagen.

"Type I specific collagen inhibitor (s)" means any molecule (s) capable of preventing the presence or inhibition of the activity, directly or indirectly, of collagen. of type I specifically. In particular, such a molecule will not significantly inhibit the presence and / or activity of other factors including factors essential for chondrocyte differentiation, such as type II collagen in particular. This specific inhibition of type I collagen can be achieved at all levels: gene, messenger, protein etc. In the case of inhibitors of type I collagen at the messenger level, RNA interference agents such as siRNAs may be used, and this is a particularly advantageous embodiment of the invention. It is also possible to use inhibitors that target transcription factors that modulate the expression of genes encoding type I collagen, and possibly modulate both expression of genes encoding type I and type II collagen. In particular, inhibitors that differentially modulate the expression of these genes may be used, for example the c-Krox "collagen-Krox" and Sp3 "Specify protein-3" factors which are activators of gene expression. encoding type I collagen and inhibitors of gene expression encoding type II collagen. It is also possible to target the p65 subunit of NF-kappaB factor "Nuclear Factor-kappaB" which inhibits the transcription of the IL-type collagen gene.

 The presence of BMP-2 and the inhibition of type I collagen can be obtained using any technique known to those skilled in the art. In particular, any method allowing the active or passive introduction of molecule (s) capable (s) to produce these effects. For intracellular action, any method of introducing the molecules necessary to achieve these effects can be used. Preferably, a method of active introduction of these molecules is used, in particular by transfection into the cells. For this purpose, any transfecting agent may be used, and in particular liposomes, polyethyleneimines, polymeric cations, and the like. It is also possible to use a method of passive introduction of these molecules, in particular by diffusion or by natural endocytosis of the molecules making it possible to carry out the desired effects mentioned above.

According to a particularly preferred embodiment of the invention, a strategy of interference with RNA, and in particular siRNA, is used to both maintain the action of BMP-2 and to inhibit the synthesis of collagen. type I in the culture medium to obtain articular chondrocytes according to the invention. In the context of this preferred embodiment, the presence of BMP-2 is itself ensured both by the addition of the BMP-2 molecule directly into the culture medium and also by the use of a medium. to prevent the protease HTRA1 inactivate the BMP-2 receptor, and in particular by the use of a siRNA HTRA1. Therefore, according to a particularly advantageous embodiment of the method of the invention, the differentiated articular chondrocytes or mesenchymate stem cells are cultured in conditions of low oxygen tension. In this method, the type I collagen inhibitors are siRNAs and the presence of BMP-2 is ensured by the addition of BMP-2 directly into the culture medium and by the use of a HTRA1 siRNA.

 Similarly, when mesenchymal stem cells are used, they are preferably cultured also in the presence of TGF-βΙ. More particularly, their culture is carried out under conditions of low oxygen tension, with siRNAs as inhibitors of type I collagen, with addition of BMP-2 directly into the culture medium and using a HTRA1 siRNA. , in combination with TGF-βΐ.

 Preferably, and particularly in the case of subsequent medical use of the chondrocytes obtained according to the method of the invention, the means used to maintain the action of BMP-2 in the culture medium and to inhibit collagen-type I are chosen so as to induce these effects transiently, in particular without involving the stable transformation of the chondrocyte genome, in order to be able to eliminate any trace of these means used during the in vitro or ex vivo culture before the implantation of the chondrocytes. within the recipient organism, including the pathological joint.

 Preferably, the different steps of the process according to the invention are carried out in the following order: oxygen level, presence of BMP-2 then inhibition of type I collagen. In the case where the cells are cultured within a biomaterial, the colonization of said biomaterial is preferably carried out first, before carrying out the three steps mentioned above. However, the order of realization of the steps of the process of the invention may be arbitrary and not limiting.

According to another of its aspects, the subject of the invention is also differentiated articular chondrocytes capable of being obtained by the implementation of the method according to the invention which is described above and which has a differentiation index, that is that is, a significantly increased type II collagen / type I collagen protein ratio, preferably at least a factor of 1.5 and / or a ratio of the average levels of collagen type II, IX, XI mRNAs. / COL1A1 or Agrecanne / COLIAI significantly increased, preferably at least 7.5 fold over non-re-differentiated cells. In particular, the invention relates to differentiated articular chondrocytes which are directly obtained by the method according to the invention which is described above and which have this differentiation index.

 These chondrocytes may be present within a biomaterial and the biomaterial / chondrocyte assembly is also another object of the present invention.

 Preferably, the differentiated articular chondrocytes capable of being obtained according to the invention are characterized by the fact that they exhibit no or little expression of type I collagen and / or do not produce it or little at the protein level but and preferentially express and synthesize collagen type II and aggrecan.

 According to another of its aspects, the subject of the invention is also the differentiated articular chondrocytes capable of being obtained according to the process of the invention described above for their use as a medicament.

 According to yet another of its aspects, the subject of the invention is also the differentiated articular chondrocytes that can be obtained according to the process of the invention described above for their use in the treatment of chondral loss of substance bound or not to traumatic shock, osteo-articular diseases, and in particular diseases that are characterized by destruction or degeneration of the cartilage, such as osteoarthritis.

 This aspect of the invention can also be defined as the use of the differentiated articular chondrocytes obtainable according to the process of the invention, for the preparation of a medicinal product intended to treat chondral losses, whether or not linked to traumatic shock, osteo-articular diseases, and in particular diseases that present a destruction or degeneration of the cartilage, such as osteoarthritis for example.

 In the context of the invention, the differentiated articular chondrocytes that can be obtained according to the method of the invention may be useful for or used for the preparation of a medicament for treating and / or preventing and / or slowing down loss of chondral substance related or not to traumatic shock, as well as destruction or degeneration of cartilage associated with osteoarticular diseases, such as osteoarthritis, for example.

In the context of these medical applications, the administration of the differentiated articular chondrocytes obtained according to the method of the invention can be administered directly within the lesions when they have been obtained. by monolayer culture. The differentiated articular chondrocytes which are obtained within a biomaterial by the method according to the invention can be implanted at the lesions to be treated. According to a preferred embodiment, the implanted chondrocytes are in the form of a biological substitute consisting of mature chondrocytes differentiated within a biomaterial according to the method of the invention. This may be positioned at the level of the lesion and possibly held in place in situ, for example with the aid of biological glue, especially fibrin-based glue.

 In general, all of the preferred embodiments which are mentioned for the method of obtaining chondrocytes are mutatis mutandis applicable for the differentiated articular chondrocytes obtainable by this method and for any composition or drug the containers and their uses.

 Other aspects and advantages of the invention will be better understood from the following examples which illustrate the invention, but are not limiting in nature. These examples refer to the appended figures:

 FIG. 1 represents the quantity of messengers, measured by quantitative RT-PCR, of collagen type I or COLIAI (A), collagen type II or COL2A1 (B) and on the messenger ratio of COL2A1 ZQLÎM genes (C). after the effect of siRNA COLIAI and / or BMP-2 in human articular chondrocytes (CAH).

 FIG. 2 represents the "Western Blot" detection of type I and type II collagen after the effect of RIMA COLIAI and / or BMP-2 in human articular chondrocytes (CAH).

 FIG. 3 represents the effect of COLIAI and / or BMP-2 siRNA on (A) quantification of protein levels of type I collagen by ELISA (n = 4), (B) quantification of protein levels of collagen of type II by ELISA (n = 4 to 24h and n = 3 to 48h) and (C) the protein ratio of collagen type II / type I by ELISA (n = 4 at 24h and n = 3 at 48h) after effect of COLIAI siRNA and / or BMP-2 in human articular chondrocytes (CAH).

FIG. 4 represents the amount of messengers, measured by quantitative RT-PCR, of collagen type I or COLIAI (A), of HTRA1 (B), and on the messenger ratio of genes COL2A1 / COL1A1 (C), Agrecan / COLIAI (D), COL9A1 / COL1A1 (E), COL11A1 / COL1A1 (F), COL2B / COL1A1 (G) after the effect of siRNA COLIAI and or Htral and / or BMP-2 in human articular chondrocytes (CAH).

 FIG. 5 represents the differentiation index of healthy cartilage chondrocytes or osteoarthritis (messenger ratio, measured by quantitative RT-PCR, of the COL2A1 / COL1A1, Agrecan (ACAN) / C0L1A1, COL9A1 / COL1A1 and COL11A1 / COL1A1 genes).

 FIG. 6 represents the "Western Blot" detection of type I, type II, HTRA1 and type X collagen collagen after the effect of COLIAI and / or Htral siRNA and / or BMP-2 in CAH.

 FIG. 7 shows the immunofluorescent detection of type I and type II collagen on articular chondrocytes (CAH) after the effect of COLIAI and / or Htral siRNA and BMP-2.

 FIG. 8 represents the immunohistochemical detection of collagen type I and type II on articular chondrocytes (CAH) in the presence or absence of BMP-2 (A), siRNA COLIAI in the presence or absence of the BMP-2 (B), Htral siRNAs in the presence or absence of BMP-2 (C), and COLIAI siRNAs and Htral siRNAs, in the presence or absence of BMP-2 (D).

 FIG. 9 represents the quantity of the messengers, measured by quantitative RT-PCR, of collagen type II or COL2A1 (A), collagen type I or COLIAI (B), HTRA1 (C), aggrecan (D). ), COL10A1 (E), COL3A1 (F) and the messenger ratio of the COL2A1 / COL1A1 (G) and Agrecan / COLIAI (H) genes, after the effect of COLIAI and / or Htral siRNA in the presence or absence BMP-2 and TGF-βΙ in mesenchymal stem cells.

 FIG. 10 represents the "Western Blot" detection of type I, type II, type III, type X and HTRA1 collagen, after the effect of COLIAI and / or Htral siRNAs in the presence or absence of the BMP-2 and TGF-βΙ in mesenchymal stem cells.

Figure 11 shows the established phenotypic profile for chondrocytes isolated from healthy cartilage and the phenotypic profiles of chondrocytes derived from mesenchymal stem cells following the effect of COLIAI and / or Htral siRNAs in the presence or absence of BMP-2 and TGF-βΙ. The results of this figure were obtained with a siRNA concentration of 100 nM. EXAMPLE 1 IMPLEMENTATION OF THE PROCESS ACCORDING TO THE INVENTION FROM HUMAN ARTICULAR CHONDROCYTES METHODOLOGIES

 SiRNAs:

 siRNA COL1A1 (collagen type I, alpha 1) (sense and antisense strands),

Hs_COLlAl_6 HP Validated SiRNA (SI02757363), Qiagen.

Direction: 5'-CAA CACC UGCG U ACAGAA-3 '(SEQ ID NO: 3)

Antisense: 5'- UUCUGUACGCAGGUGAUUG - 3 '(SEQ ID NO: 4)

Target sequence: ACC ACC CAC CTG CGT ACA GAA (SEQ ID NO: 5)

 siRNA control or "Negative Control" (double-stranded siRNA), Qiagen (No.

1022076)

 Direction: 5'-UUCUCCGAACGUGUCACGU-3 '(SEQ ID NO: 6)

Antisense: 5'-ACGUGACACGUUCGGAGAA-3 '(SEQ ID NO: 7)

Target sequence: AAT CCG TAC AAC GTG TCA CGT '(SEQ ID NO: 8)

 rhBMP-2 Recombinant human Bone Morphoqenetic Protein-2 "): The cDNA encoding human BMP-2 (R & D Systems) is expressed in CHO cells (" Chinese Hamster Ovary cells ") After proteolytic cleavage of the signal peptide and of the propeptide, the mature recombinant BMP-2 forms a homodimer or heterodimers with other members of its family (example with BMP-7), each monomer being each composed of 114 amino acids BMP-2 was added to the medium at a final concentration of 50 ng / ml.

 Type I collagen sponges (SYMATESE Biomaterials, Chaponost, France): They are characterized by a diameter of 5 mm and a thickness of 2 mm. They have a three-dimensional structure suitable for 3D culture. The collagen fiber networks I form pores with an average diameter of 150 μm. This biomaterial is of bovine origin, and with its crosslinked collagen I fibers, it is biologically inactive and biodegradable.

Cell culture: Human articular chondrocytes (CAH) were collected from femoral heads during prosthesis placement in patients with osteoarthritis. The cartilage was dissected into chips, then the latter were digested with two enzymes, pronase (Sigma-Aldrich, 2 mg / ml, 45 minutes) and collagenase (Sigma-Aldrich, 2 mg / ml, 15 hours). The next day, undigested cartilage chips were removed by passage of the 70 μm nylon cell suspension. After centrifugation, the cell pellet was taken up in DMEM (Dulbecco's Modified Eagle Medium, Fisher Bioblock Scientific "). The number of cells was determined by counting on a Malassez cell. The HPCs were then seeded in culture dishes at 4.10 4 cells / cm ² and cultured at 37 ° C in a 5% CO _{2 environment} . The chondrocytes were thus amplified in monolayer culture.

Nucleofection: RIMA si (siRNA control or siRNA COL1A1) were incorporated into chondrocytes using the nucleofection technique (Nucleofector ^® II, Lonza). Firstly, the cells were amplified in monolayer culture and only undergone one passage in order to limit their dedifferentiation. Once at confluence, they were peeled off with a trypsin-EDTA mixture (0.25% trypsin / 1 mM EDTA, Fisher Bioblock Scientific). Transfection was performed from 2 million cells using a nucleofection solution containing one microgram (50 nM) of siRNA. Once transfected, the chondrocytes remained for one hour at 37 ° C. in DMF medium (Fisher Bioblock Scientific) supplemented with 20% FCS (Fisher Bioblock Scientific). After centrifugation, they were seeded at 500,000 cells per collagen sponge and allowed to dry for one hour to allow adhesion to the collagen sponge. Finally, the chondrocytes were placed in hypoxia, in the presence or absence of 50 ng / ml rhBMP-2 for a period of 24 to 48 hours.

"Western-blot": The cells seeded in the type I collagen sponges were rinsed with PBS IX ("Phosphate Buffered Saline", Fisher Bioblock Scientific) and lysed at a rate of 100 μl of RIPA buffer ("Radioimmunoprecipitatton assay buffer" ) by sponge, then were shaken (vortexed) every 10 minutes for one hour. Protein extraction was continued by removing each sponge and pressing them one by one in order to recover the maximum amount of protein. The supernatant containing the cytoplasmic extracts was finally removed after centrifugation for 30 minutes at 14,000 g. The protein concentration of our samples was determined by the Bradford method. 20 μg of each sample was contacted with denaturation buffer (2.5 ml of 100% glycerol, 2 ml of 10% SDS, 1 ml of bromophenol blue 1%, 600 μl of 1M Tris-HCl pH 6 8, 0.5 ml of 13-mercaptoethanol) and heated at 95 ° C for 5 minutes. Polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) was performed. The proteins of the gel were then transferred onto a PVDF (Poly Vinylidene DiFluoride, Millipore) membrane by electro-transfer for 1 h 30. The membrane was previously activated by three successive baths: methanol 100% (1 min), distilled water (twice 1 min) and IX transfer buffer (15 min). At the end of the transfer, the membrane was washed with "Tris Buffered Saline-Tween" (TBS-T). Blocking of nonspecific sites of the membrane was achieved by incubation in TBS-T containing 10% skimmed milk for 1 h at room temperature and with stirring. The membrane was then washed with TBS-T and then incubated for 15 h at 4 ° C. in the presence of the primary antibody (Ac I ^domain ) directed against the protein of interest (Collagen type II, 1 / ^2000th , Novotec, Collagen type I, l / ^3000th, Novotec, GAPDH (Glyceraldehyde 3 Phosphate Dehydrogenase), l / 2000 ^th, Santa Cruz Biotechnology). The next day, a series of rinses of the membrane was carried out with TBS-T, then it was incubated in the presence of the secondary antibody (Ac II ^area , anti-rabbit, 1/6000 ^th , Tebu-Bio 1 hour at room temperature. The proteins were finally revealed using a detection kit ("Super Signal West Pico Chemiluminescent Substrate", PIERCE).

 ELISA ("Enzyme-linked immunosorbent assay", TECOmedical SARL): The ELISA experiments were carried out from the culture supernatants according to the instructions of the kit. The protein levels of the C-propeptide of Procollagene type II were evaluated according to the so-called "competition" method while those of the C-terminal part of type I collagen were evaluated via the so-called "sandwich" method.

 Real-time RT-PCR: The cells were lysed with TRIzol Reagent®,

Invitrogen. 1 μg of total RNA was treated with 1 μl of deoxyribonuclease I (DNase I, Invitrogen) for 15 min at 25 ° C. The reaction was stopped by the addition of 1 μl of 0.5 mM EDTA followed by a 10 min incubation at 65 ° C. The samples digested with DNase were then retro-transcribed into cDNA. The reaction was carried out in the presence of a mixture consisting of 5 μl of the 10X reverse transcriptase buffer, 1 μl of the "Moloney Murine Leukemia virus" reverse transcriptase at 200 units / μl (MMLV, Fisher Bioblock Scientific), 1 μl of oligodT, 2 μl of a mixture of the four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP, 20 μl final, Fisher Bioblock Scientific) and 1 μl of RNAse-out (40 U / μl, Invitrogen) . The volume was supplemented to 15 μΙ with water treated with DEPC (di-ethyl-pyrocarbonate). The reverse transcription reaction was carried out for a period of 1 h at 37 ° C, preceded by a 10 min heating cycle at 25 ° C. After reverse transcription, cDNA samples obtained were diluted to l / ^100th then used in PCR experiments in real time. To do this, the amplification of the gene studied was carried out in triplicate in 96-well plates, according to the instructions of the device "ABI Prism 7000 SDS" (Applied Biosystems). Samples were amplified using SYBR Green PCR Master Mix (Applied Biosystems) and primers as described in Table 1 below. The mRNA levels of the COLIAI and COL2A1 genes were estimated after normalization against the reference gene RPL13.

 Table 1

 Statistical analyzes:

 For the RT-PCRs and the ELISAs, the results are represented in the form of "Box-plot" or box-to-mustache, representing n experiments carried out independently. This method makes it possible to represent on the graph the minimum and maximum values, the first quartile, the second quartile or the median, the third quartile, as well as the average of the experiments. The Mann-Whitney U test was used to determine significant differences between the control and treated groups. Values of P <0.05 are considered significant.

 RESULTS

 The inhibition of type I collagen synthesis and its effect were monitored at the messenger RNA level and at the protein level using real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) experiments. as well as "Western-blot" and ELISA ("Enzyme-linked immunosorbent assay"), as described above in the methodologies part (Example 1).

 Effect of COLIAI siRNA on messenger rates of type I collagen.

Human articular chondrocytes were cultured in type I collagen sponges at 500,000 cells / sponge after being transfected with siRNAs targeting type I collagen and cultured in hypoxic conditions (less than 5% 0 ₂ ). The mRNA levels of the COLIAl gene were estimated by RT-PCR in real time after normalization against the reference gene RPL13 (AU: Arbitrary Unit). The results are shown in FIG. 1A in the form of "box plots" corresponding to 4 independent experiments made in triplicate. For each incubation time, the significant differences between the control and the treated cases, and between the different cases were determined by the Mann-Whitney U test: * = p <0.05, ns = not significant.

 These results show that siRNA COLIAl strongly decreases the average levels of collagen type I mRNA when chondrocytes are cultured in collagen sponges, in hypoxia, in the presence of BMP-2 ("BMP-2") or without BMP. -2 ("Control") and for 24 and 48 hours. In addition, siRNA has a relative inhibition efficiency of 80 to 90%, and is able to block the slight increase in these average levels induced by BMP-2.

 Effect of COLIAL siRNA on collagen type II messenger rates COL2A1 gene mRNA levels were estimated by RT-PCR in real time as described above. The results are shown in Figure 1B. These results show that the mean levels of the type II collagen messengers are not affected by the presence of the siRNA control and the siRNA COLIAl at 24h culture, as at 48h.

Report of messenger rates of COL2A1 / COL1A1 genes

 The mRNA levels of the COL2A1 and COLIA1 genes were estimated as described above and the ratio of COL2A1 mRNA level to COLIA1 mRNA level was calculated under the different experimental conditions. This report makes it possible to define the index of differentiation of chondrocyte cultures.

 The results are shown in Figure 1C and show an increase in this ratio following transfection of chondrocytes with siRNA COLIAl and cultured for 24 and 48 h. In conclusion, the use of siRNA COLIAl combined with BMP-2 treatment induces and increases the stability of the chondrocyte phenotype.

Effect of siRNA COLIAl on protein levels of collagen type I and type II

Human articular chondrocytes were cultured in type I collagen sponges at 500,000 cells / sponge after being transfected with siRNAs targeting type I collagen or siRNA control and then grown under hypoxic conditions (less than 5% 0 ₂ ). Extracts Cytoplasmic cells were subjected to SDS-PAGE electrophoresis for "Western-Blot" analysis with anti-collagen II, I, and anti-GAPDH antibodies.

 The results corresponding to a representative Western blot are grouped together in FIG. 2. They show a significant decrease in the protein content of type I collagen in the presence of the COL1A1 siRNA whereas the collagen type II protein levels do not seem to be modified by significantly.

Quantification of protein levels of collagen type I by ELISA

 Human articular chondrocytes were cultured as described above. Quantification of the C-propeptide of type I collagen was performed by ELISA on the culture supernatants, and then reported to the total amount of protein in the sample. The results are represented in the form of "box plots" corresponding to 4 independent experiments for 24h and 3 experiments for 48h. For each incubation time, the significant differences between the control and the treated cases, and between the different cases were determined by the Mann-Whitney U test: * = p <0.05, ns = not significant.

 The results are grouped together in FIG. 3A and show a clear decrease in the protein levels of type I collagen, especially after 48 h of cell culture. Thus, the concentration of type I collagen passes on average from 0.8 mg of CICP / g of total protein in the presence of the negative control, to 0.4 mg of CICP / g of total protein in the presence of siRNA COL1A1.

 Quantification of protein levels of collagen type II by ELISA

 Human articular chondrocytes were cultured as described above. Quantification of the C-propeptide of type II collagen was performed by ELISA as described above.

 The results are grouped in Figure 3B. The ELISA quantification analyzes of the collagen type II protein levels are consistent with the results previously observed in "Western-blot". Indeed, COL1A1 siRNA does not seem to have a significant effect on collagen type II protein levels at 24 h or 48 h of collagen sponge culture in hypoxia with BMP-2 ("BMP-2 ") Or without B P-2 (" Control ").

 Quantification of the protein content ratio of collagen type II / type I by ELISA.

Quantification of the C-propeptide of type I and II collagen was performed by ELISA as described above and the protein ratio of collagen type II / type was calculated. The results are grouped together in FIG. 3C and show an increase in the collagen type II collagen protein ratio on type I collagen at 24 h of culture in the presence of siRNA COL1A1. This effect is also observed at 48 h of incubation and BMP-2 has an additive effect with siRNA COL1A1.

EXAMPLE 2: IMPLEMENTATION OF THE PROCESS ACCORDING TO THE INVENTION FROM HUMAN ARTICULAR CHONDROCYTES METHODOLOGIES

 SiRNAs:

 The siRNAs COL1A1 and control are described above in Example 1.

siRNA HTRA1 (siRNA double strand), eurogentec.

 Direction: 5'-CGGCCGAAGUUGCCUCUUUTT -3 '(SEQ ID NO: 15)

Antisense: 5 '- AAAGAGGC AACU U CGGCCGTT -3' (SEQ ID NO: 16)

Target sequence: CGG CCG AAG TTG CCT CTT T (SEQ ID NO: 17)

 rhBMP-2 "recombinant human Bone Morphoqenetic Protein-2": The cDNA encoding human BMP-2 (R & D Systems) is expressed in CHO ("Chinese Hamster Ovary cells") cells. After proteolytic cleavage of the signal peptide and the propeptide, the mature recombinant BMP-2 forms a homodimer or heterodimers with other members of its family (example with BMP-7), each monomer being each composed of 114 amino acids. BMP-2 was added to the culture medium at a final concentration of 50 ng / ml.

 Type I collagen sponges (SYMATESE Biomaterials, Chaponost, France): They are characterized by a diameter of 5 mm and a thickness of 2 mm. They have a three-dimensional structure suitable for 3D culture. The networks of collagen I fibers form pores with an average diameter of 150 m. This biomaterial is of bovine origin, and with its crosslinked collagen I fibers, it is biologically inactive and biodegradable.

Cell culture: Human articular chondrocytes (CAH) were collected from macroscopically healthy areas of cartilage from an osteoarthritic joint (non-bearing area of cartilage at a healthy joint) to obtain chondrocytes that were used throughout the experiment. The cartilage was dissected into chips, then the latter were digested with two enzymes, pronase (Sigma-Aldrich, 2 mg / ml, 45 minutes) and collagenase (Sigma-Aldrich, 2 mg / ml, 15 hours). The next day, undigested cartilage chips were removed by passage of the 70 μm nylon cell suspension. After centrifugation, the cell pellet was taken up in DMEM ("Dulbecco's modified Eagle Medium, Fisher Bioblock Scientific"). The number of cells was determined by counting on a Malassez cell. The HACs were then seeded in culture dishes at 4.10 ⁴ cells / cm ² and cultured at 37 ° C in an environment of 5% C0 ₂ . The chondrocytes were thus amplified in monolayer culture. The cells have only undergone one passage in order to limit their dedifferentiation. Once at confluence, they were peeled off with a trypsin-EDTA mixture (0.25% trypsin / 1 mM EDTA, Fisher Bioblock Scientific) and then centrifuged. After centrifugation, they are seeded at 400,000 cells per collagen sponge and allowed to dry for one hour at 37 ° C to allow adhesion to the collagen sponge. 16 h later (J0) the chondrocytes were placed in hypoxia, in the presence or absence of 50 ng / ml of rhBMP-2 in medium DMEM ("Dulbecco's Modified Eagle Medium", Fisher Bioblock Scientific) containing 2% of FCS (Serum of Fetal Calf) and then cultivated for a period of 7 days.

 At the same time, cartilage chips were prepared from samples taken within the osteoarthritic region on femoral heads when a prosthesis was placed in patients with osteoarthritis.

 The chondrocytes isolated after digestion were extracted with total or protein RNA and used in real-time RT-PCR and Western-blot experiments, with chondrocytes from osteoarthritis patients being used as controls. .

Transfection of siRIMAs by HNTE FE in ^® : si NA (siRNA control, siRNA COL1A1, siRNA HTRAÎ) were incorporated into chondrocytes using INTERFERin® as a transfection agent (Polyplus transfection ™). The siRNAs COL1A1 and HtrA1 were transfected into the chondrocytes at a concentration of 50 nM throughout the experiment, except for the studies shown in Figures 4 and 5 (5 nM) because the siRNAs at this low concentration turn out to be all also effective in blocking the expression of target mRNAs (results not shown).

"Western-blot": The cells seeded in type I collagen sponges were rinsed with PBS IX ("Phosphate Buffered Saline" Fisher Bioblock Scientific) and lysed at 100 μΙ of RIPA buffer ("radioimmunoprecipitation assay buffer" ) by sponge, then were shaken (vortexed) every 10 minutes for one hour. Protein extraction continued by removing each sponge and squeezing them one by one to get the most protein. The supernatant containing the cytoplasmic extracts was finally removed after centrifugation for 30 minutes at 14,000 g. The protein concentration of our samples was determined by the Bradford method. 20 μg of each sample was contacted with denaturation buffer (2.5 ml of 100% glycerol, 2 ml of 10% SDS, 1 ml of bromophenol blue 1%, 600 μl of 1M Tris-HCl pH 6 0.8 ml of β-mercaptoethanol) and heated at 95 ° C for 5 minutes. Polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) was performed. The proteins of the gel were then transferred to a PVDF (Poly Vinylidene DiFluoride, illipore) membrane by electro-transfer for 1 h 30. The membrane was first activated by three successive baths: 100% methanol (1 min), water distilled (twice 1 min) and IX transfer buffer (15 min). At the end of the transfer, the membrane was washed with "Tris Buffered Saline-Tween" (TBS-T). Blocking of nonspecific sites of the membrane was achieved by incubation in TBS-T containing 10% skimmed milk for 1 h at room temperature and with stirring. The membrane was then washed with TBS-T and then incubated for 15 h at 4 ° C. in the presence of the primary antibody (Ac I ^domain ) directed against the protein of interest (Collagen type II, 1 / ^2000th , Novotec, collagen type I, l / ^3000th, Novotec, HtrAl, l / ^2000th, Millipore, ref. AB15852, Collagen type X, l / ^1000th, Sigma, C7974-ZML and GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase) , l / 2000 ^th , Santa Cruz Biotechnology). The next day, a series of rinses of the membrane was performed with TBS-T and then it was incubated with secondary antibody (Ab IP ^ire anti-rabbit, l / ^6,000th, Tebu- Bio or Ac II anti-mouse ^area , 1/6000 ^th , Tebu-Bio) 1 h at room temperature. The proteins were finally revealed using a detection kit ("Super Signal West Pico Chemiluminescent Substrate", PIERCE)

Real-time RT-PCR: Cells were lysed with TRIzol Reagent ^®, Invitrogen. 1 μg of total RNA was treated with 1 μl of deoxyribonuclease I (DNase I, Invitrogen) for 15 min at 25 ° C. The reaction was stopped by the addition of 1 μl of 0.5 mM EDTA followed by a 10 min incubation at 65 ° C. The samples digested with DNase were then retro-transcribed into cDNA. The reaction was carried out in the presence of a mixture consisting of 5 μl of the 10X reverse transcriptase buffer, 1 μl of the "Moloney Murine Leukemia virus" reverse transcriptase at 200 units / μl (MMLV, Fisher Bioblock Scientific), 1 μl of oligodT, 2 μl of a mixture of the four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP, 20 μl final, Fisher Bioblock Scientific) and 1 μΙ RNAse-out (40 U / μΙ, Invitrogen). The volume was supplemented to 15 μΙ with water treated with DEPC (di-ethyl-pyrocarbonate). The reverse transcription reaction was carried out for a period of 1 h at 37 ° C, preceded by a 10 min heating cycle at 25 ° C. After reverse transcription, cDNA samples obtained were diluted to l / ^100th then used in PCR experiments in real time. To do this, the amplification of the gene studied was carried out in triplicate in 96-well plates, according to the instructions of the device "ABI Prism 7000 SDS" (Applied Biosystems). Samples were amplified using SYBR Green PCR Master Mix (Applied Biosystems) and primers as described in Table 1 above (COLIAI, COL2A1 and RPL13) and Table 2 below. The mRNA levels of the COLIAI, HTRA1, COL2A1, Agrecan, COL9A1, COL111A and COL2B genes were estimated after normalization against the reference gene RPL13.

 Table 2

Statistical analyzes: For RT-PCR, the results are represented as histograms of a representative experiment and represent the mean of three measurements ± the standard deviation. The significance of the values was evaluated using a Student's t-test (* p <0.05; ** p <0.01; *** p <0.001).

Immunofluorescence: At the end of the incubations, the sponges are rinsed in 0.1M phosphate buffer, pH 7.4, then fixed in 4% paraformaldehyde for 16 h at 4 ° C., and finally rinsed with phosphate buffer. 1M. The sponges are then included in 5% agar (Super LM, Roth, Karlsure, France). Sections of 50 μm are made with a vibratome (MICROM HM 650V, Francheville, France) and mounted on a blade. They are incubated for 5 minutes in 0.5% triton, rinsed with PBS, incubated for 30 minutes at 37 ° C. with 0.2% hyaluronidase in PBS, rinsed and treated with 10% BSA. in PBS. The markings are made using polyclonal antibodies specific for collagen type I and II (dilution l / ^100th, Novotec, Lyon, France). These primary antibodies were revealed with anti secondary antibody rabbit IgG coupled with a fluorochrome Alexa Fluor 546 (l / ^2000th, InVitrogen). Primary antibodies are not applied to the control slides. The sections are then mounted between slides and coverslips with a mounting medium containing DAPI (Ultracruz ™ Mounting Medium, Santa Cruz Biotechnology Inc.). The observations are made with a confocal microscope (Olympus FV1000, Rungis, France). The same acquisition parameters are used for all images that are representative of several experiments.

 In vivo study: The chondrocytes transferred into the collagen sponges are cultured in the presence of DMEM 2% of SVF +/- BMP-2 (50 ng / ml) for 4 days at low oxygen tension (<5%). A change of medium is made with the addition of siRNA COL1A1 +/- siRNA HtrAl from j4 to j6 (50nM). After 2, 4 and 6 days of culture, the medium is changed. After 7 days of culture, the collagen sponges colonized by chondrocytes were implanted subcutaneously (dorsal part, at the rate of 4 sponges per mouse) in the nude mouse for a period of 28 days. The sponges are then recovered after euthanasia of the mice.

- Fixation, dehydration and inclusion of samples: The collagen sponges are first immersed in a solution of 4% paraformaldehyde in PBS for at least 18 hours at room temperature, in order to fix the antigens. The samples are then dehydrated by successive baths of 100% ethanol (1 bath of 30 min followed by 5 baths of 1 h) and toluene (1 bath of 30 min, followed by 23 FR2011 / 052198

2 baths of 60 min) and finally immersed in paraffin (1 bath of 15 min, 1 bath of 30 min, 1 bath of 60 min, 1 bath of 90 min). These steps were performed by an automaton (Laboratory of Pathological Anatomy (CHU, Caen)). The samples are finally placed in cassette and included in paraffin blocks. Sections of 5 μιη are then made by microtome and deposited on silanized slides for immunostaining. Before any marking of the slides, the sections are deparaffinized by successive baths of toluene (2 baths of 5 min), alcohol 100% (5 min), alcohol 90% (5 min), alcohol 70% ( 5 min) and finally distilled water.

Immunolabeling: After dewaxing, an enzymatic pretreatment with hyaluronidase (0.5% in 1X PBS + 3% BSA, 30 minutes at room temperature) makes it possible to unmask the antigenic sites. The polyclonal antibodies (type I collagen, 1/1000 ^th , Novotec Lyon France, type II collagen, 1/250 ^th , Novotec, Lyon France) diluted with 3% PBS-BSA are deposited on the sections for 3 hours at room temperature. After rinsing in PBS and 0.2% PBS Tween, the endogenous peroxidases are inhibited by incubation with 1.5% hydrogen peroxide in PBS-3% BSA. Then the secondary antibody (anti-rabbit, undiluted, Envision DAKO) is applied on each cut. The antigen-antibody complexes are then revealed by enzymatic reaction through the action of the secondary antibody-coupled peroxidase, which, once the diaminobenzidine substrate is applied to the sections, will give a brown precipitate, the slides are then counter-attacked. stained with Mayer's hematoxylin and then mounted in an aqueous medium. For negative controls, the primary antibody has been omitted.

RESULTS

 The in ibition of type I collagen synthesis and its effect were monitored at the messenger RNA level and at the protein level using RT-PCR (Reverse Transcription-Polymerase Chain Reaction) experiments in time. actual as well as "Western-blot" as described above in the methodologies part (Example 2). The detection of type I collagen and type II collagen has also been visualized in immunofluorescence and in vivo in immunohistology.

Effect of COL1A1 and / or HTRA1 siRNAs and / or recombinant BMP-2 on messenger rates of collagen type I and HtrAl.

Human articular chondrocytes were cultured in type I collagen sponges at 400,000 cells / sponge after being transfected with siRNAs (5 n between days 0-2) targeting type I collagen. and / or HtrAl. Cells were cultured under hypoxic conditions (<5% 0 ₂ ) in the presence or absence of BMP-2.

 The results obtained shown in FIGS. 4A to 4G in which the average mRNA levels of the COLIAI (A), HtrAl (B), COL2A1, Agrecan (ACAN), COL2B, COL9A1 and COL11A1 gene were estimated by RT-PCR. in real time after normalization against the reference gene RPL13 {k: Arbitrary unit). The ratio of COL2A1 / COL1A1 (C), Agrecan / COLIAI (D), COL9A1 / COL1A1 (E), COL11A1 / COLIAI (F), and COL2B / COLIAI (G) mRNA levels under the different experimental conditions are represented and allow to define the index of differentiation of chondrocyte cultures. The results of a representative experiment are presented.

 The results obtained on the mRNA levels of the COLIAI and HtrAl genes, evaluated by RT-PCR in real time, show that the two siRNAs are effective in blocking the expression of these two target genes (FIGS. 4A and B respectively). In addition, HtrAl siRNA decreases COLIAI mRNA levels and acts synergistically with COLIAI siRNA and BMP-2 to reduce the expression of COLIAI mRNA levels (FIG. 4A). Moreover, FIG. 4A and B shows that articular chondrocytes obtained from an injured arthritic cartilage region, but not cultured, overexpress COLIAI and HtrA1 mRNAs, with regard to the redifferentiated chondrocytes according to the method of the invention.

 Effect of siRNA COLIAI and / or siRNA HTRA1 and / or recombinant BMP-2 on the ratio of messenger levels of different markers of chondrocyte differentiation.

The results presented in Figure 4C show that the COL2A1 / COLIAI differentiation index in chondrocytes is increased when COLIAI siRNA alone, or HtrAl siRNA alone, or both types of siRNAs are transfected into cells under basal conditions. BMP-2 significantly increases this differentiation index, whether added alone or simultaneously with each siRNA. Interestingly, the treatment of chondrocytes in the concomitant presence of BMP-2 and the two siRNAs makes it possible to obtain a synergistic effect on the increase of the differentiation index which is of the order of 60 (FIG. this index is only in the range of about 5 in the osteoarthritic chondrocytes and of the order of 200 in healthy articular cartilage chondrocytes (FIG. 5). In order to better characterize the functionality of the extracellular matrix of neo-cartilage formed under these experimental conditions, the expression of the mRNAs encoding the mature form of collagen II (COL2B) and other phenotypic markers of differentiation of articular chondrocytes, namely aggrecan and collagenic isotypes IX and XI have been studied. The corresponding differentiation indices at the level of mRNA levels, AGR / COL1A1, COL9A1 / COL1A1, COL11A1 / COL1A1 and C0L2B / C0L1A1 (respectively Figures 4D, 4E, 4F and 4G) were calculated. The results presented in these figures make it possible to conclude quite similarly with respect to the results presented in FIG. 4A where the COL2A1 / COL1A1 index had been evaluated. Thus, both siRIMAs used alone or in combination favor these various differentiation indices; BMP-2, alone or in combination with siRIMAs, increases these indices. Nevertheless, the BMP-2 combinatorial and the COL1A1 and HtrA1 siRNAs exert a synergistic effect, of at least 20 on all the indexes, which is very much higher than the index obtained in osteoarthritic chondrocytes, and close to the differentiation indices. obtained with healthy cartilage chondrocytes (Figure 5).

 Effects of COL1A1 and HtrA1 siRNAs and / or recombinant BMP-2 on the protein levels of collagens I, II, X and HtrAl

Human articular chondrocytes were cultured in type I coliagene sponges at 400,000 cells / sponge after being transfected with siRNAs (5 nM, between days 0 to 2) targeting type I collagen and / or HtrAl. The cells were cultured under hypoxic conditions (3% 0 ₂ ) in the presence or absence of BMP-2. Total protein extracts prepared from the sponges colonized by the cells were subjected to SDS-PAGE electrophoresis for Western blot analysis with anti-collagen II, I, X, HtrAl and anti-collagen antibodies. GAPDH. The figure corresponds to a representative "Western-Blot".

 In order to partially validate the mRNA level results, "Western-blot" experiments were undertaken to confirm the effects at the protein level. The experimental conditions are identical to those described in FIG. 4. At the end of the incubation, total protein extracts prepared from the sponges colonized by the cells were prepared and subjected to "Western-blot" analysis with antibodies. anti-collagen II, I, X and anti-HtrAl.

The results corresponding to a representative Western-blot are presented in FIG. 6. They demonstrate the functionality of siRNAs, since transfection a COL1A1 siRNA in articular chondrocytes virtually eliminates the expression of type I collagen. It is the same for the HrAl siRNA which blocks the protein expression of HtrAl. The combination of the two siRNAs is just as effective in preventing the corresponding protein expression. The effect of the two siRNAs appears to be specific, since they do not affect the protein expression of GAPDH and type II collagen at all.

 BMP-2, in the absence of siRNA, increases the expression of type II collagen and also collagen type I. Combinatorial BMP-2 + siRNA alone or BMP-2 + both siRNAs increases collagen production II without induction of type I collagen, thus confirming the synergistic effect of the two siRNAs + BMP-2 on the differentiation index evaluated in FIG. 4C.

 The results also show that articular chondrocytes cultured under these experimental conditions do not undergo hypertrophy (absence of X-type collagen expression, regardless of the samples presented in FIG. 6), which is frequent in osteoarthritic chondrocytes. , suggesting that the redifferentiated chondrocyte phenotype is optimal.

Expression of type I and II collagens after effect of COL1A1 and HtrA1 siRNA and / or recombinant BMP-2

Human articular chondrocytes were cultured in type I collagen sponges at 400,000 cells / sponge for 16 h in DMEM + 2% FCS (J0), and then treated or not with 50 ng / ml BMP-2 and transfected with siRNA (50 nM, between days 4 to 6) targeting type I and / or HtrAl collagen. Cells were grown under hypoxic conditions (<5% 0 ₂ ). An immuno-cytological analysis in confocal microscopy with anti-collagen II and I antibodies was performed. Day 0: D0; day 7: J7.

In order to partially validate the "Western-blot" studies, immuno-cytology experiments were performed. The results are presented in FIG. 7 and show that the chondrocytes at time D0 express the collagenic isotypes I and II. Chondrocyte culture under hypoxic conditions in collagen sponges for 7 days in the presence of siRNA COL1A1 leads to a decrease in the expression of type I collagen and BMP-2 significantly increases collagen type II levels ( Figure 7), confirming the results obtained in the "Western-blot" experiments (Figure 6). The same conclusions can be drawn when HtrAl siRNA is transfected into chondrocytes and the combination of this siRNA with BMP-2 increases 27 T / FR2011 / 052198

type II collagen while reducing those of type I collagen, reflecting an increased differentiation index (Figure 7). Finally, the joint transfection of the COLIAI and HtrA1 siRNAs decreases the expression of type I collagen and increases the expression of isotype II and the simultaneous addition of BMP-2 with these siRNAs synergizes the effects observed in the presence of siRNAs alone. (Figure 7).

 Effects of COLIAI and HtrA1 siRNAs and / or recombinant BMP-2 on the neo-cartilage phenotype reconstructed in vivo in nude mice

Human articular chondrocytes were cultured in type I collagen sponges at 400,000 cells / sponge after being transfected with siRNAs (50 nM, between days 4-6) targeting type I collagen and / or HtrAl. Cells were cultured under hypoxic conditions (<5% 0 ₂ ) in the presence or absence of BMP-2. At day 7, the sponges colonized by the cells and having undergone the various treatments are implanted subcutaneously in the dorsal position in the nude mouse (4 sponges per mouse). After 28 days, the mice are euthanized, the sponges collected and analyzed by immunohistochemistry with anti-collagen II and I antibodies.

Following the results obtained in the in vitro studies demonstrating a significant and synergistic effect of the COLIAI and HtrAl siRNAs in the presence of BMP-2 on the chondrocyte phenotype of the neo-cartilage reconstructed under hypoxic conditions, preclinical studies were performed. They consisted of grafting collagen sponges colonized by articular chondrocytes and subjected to the same incubation protocol for 7 days as that described in FIGS. 4 to 7. At the end of the in vitro culture period, the sponges were grafted subcutaneously on the back of nude mice (4 sponges per mouse). After 28 days, the mice were euthanized, the sponges collected and subjected to an immunohistochemical study. The results of these experiments, presented in FIG. 8, show that there remains a weak expression of type I collagen (image from the top left) within the neo-cartilage and a consequent expression of collagen II (image from the bottom to left) in the absence of BMP-2 and presence of the siRNA control (negative control) (Figure 8A). BMP-2 very significantly increases the synthesis of isotypes I and II (FIG. 8A, the two images on the right). In the presence of BMP-2, treatment with siRNA COLIAI alone, or HtrAl siRNA alone, or with the combination of the two siRNAs significantly increases the expression of type II collagen at the expense of type I collagen, materializing a synergistic effect on the differentiation index of neo-cartilage (FIG. 8B, 8C, 8D). 28 11 052198

These results show that transfection of articular chondrocytes with COLIAI and HtrA1 siRNA in the presence of BMP-2 under hypoxic conditions is capable of causing the increase of the expression of cartilage specific markers (collagens II, IX and XI, aggrecan ) and the decrease in nonspecific markers (collagens I and X), which leads to a sharp increase in the differentiation index of chondrocytes cultured in vitro and the neo-cartilage reconstructed in vitro retains similar characteristics in vivo.

 EXAMPLE 3: IMPLEMENTATION OF THE PROCESS ACCORDING TO THE INVENTION FROM MESENCHYMAL STEM CELLS fCSM

 METHODOLOGIES

 Mesenchymal stem cells derived from bone marrow: induction of chondrocyte lineage and phenotype study.

Isolation and amplification: The bone marrow sample is centrifuged at a speed of 200 g, then the cell pellet is directly inoculated into a culture dish of 25 cm ² . The amplification medium [α-MEM ("Modification of Eagle's Medium", Fisher Bioblock Scientific), containing 10% FCS, 2 mM L-Glutamine and FGF-2 ("Fibroblast Growth Factor-2", Sigma chemical Co) 1 ng / ml] is changed every 2 to 3 days. When the cells reach 80% confluency, they are detached by trypsin-EDTA (0.25% trypsin / 1 mM EDTA, Fisher Bioblock). Thus, the CSMs are amplified in monolayer culture up to a minimum of five passages (P5) in the presence of α-MEM medium + 10% FCS.

 - Induction of the chondrocyte lineage in vitro: After 4 or 5 passages, the CSM are seeded in type I collagen sponge at a rate of 500,000 cells per sponge. The latter are cultured in an ICM medium ("Incomplete Chondrogenic Medium") [medium for inducing chondrogenic differentiation containing DMEM, dexamethasone 500 nM, ascorbic acid-2-phosphate 50 g / ml, proline 40 μg / ml, 1 mM sodium pyruvate] and under hypoxic conditions (<5% oxygen). The culture extends over a period of 14 days with a change of medium every 3 or 4 days.

In order to reduce the amount of type I collagen, siRNAs targeting type I and / or HtrA1 collagen (Qiagen) are added by transfection during the MSC differentiation step (100 nM each) on day 0 and at day 7. the biomaterial with cells is then recovered at ^day 14 of differentiation. The total proteins or RNA are then extracted and studied by "Western-blot" and RT-QPCR respectively, according to the methods described for chondrocytes. The stem cells obtained by monolayer culture are characterized by immunophenotyping (CD44, CD73, CD90, CD105) and their multipotency is validated by differentiation, in parallel, of the stem cells into adipocytes, osteoblasts and chondrocytes (results not shown). For the definition of the chondrocyte lineage, are associated for the cultivation of chondrogenic factors (TGF-βΙ and BMP-2), a biomaterial (collagen type I sponge), an oxygen tension and interfering RNAs targeting collagen type I and / or HtrAl.

 SiRNAs:

 The siRNAs COL1A1 and control are described above in Example 1.

SiRNA HTRA1 is described above in Example 2.

 rhBMP-2 Recombinant Human Bone Morphogenetic Protein-2 '): The cDNA encoding human BMP-2 (R & D Systems) is expressed in CHO ("Chinese Hamster Ovary cells") cells. After proteolytic cleavage of the signal peptide and the propeptide, the mature recombinant BMP-2 forms a homodimeric or heterodimers with other members of its family (example with BMP-7), each monomer being each composed of 114 amino acids. BMP-2 was added to the culture medium at a final concentration of 50 ng / ml.

 TGF-βΙ "Transformina arowth factor-B"): The cDNA encoding TGF-βΙ is expressed in CHO ("Chinese Hamster Ovary") cells. TGF-βΙ was added to the culture medium at a final concentration of 10 ng / ml.

 Type I collagen sponges (SYMATESE Biomaterials, Chaponost, France): they are characterized by a diameter of 5 mm and a thickness of 2 mm. They have a three-dimensional structure suitable for 3D culture. The collagen fiber networks I form pores with an average diameter of 150 μm. This biomaterial is of bovine origin, and with its crosslinked collagen I fibers, it is biologically inactive and biodegradable.

Cell culture: After 5 passages and once at confluence, the MSCs were peeled off with a trypsin-EDTA mixture (0.25% trypsin / 1 mM EDTA, Fisher Bioblock Scientific) and then centrifuged. After centrifugation, they are seeded at 500,000 cells per collagen sponge and allowed to dry for one hour at 37 ° C to allow adhesion to the collagen sponge. Finally, the stem cells were placed in hypoxia (<5%), in the presence or absence of 50 ng / ml of rhBMP-2 and 10 ng / ml of TGF-βΙ in ICM medium and then cultured for a period 14 days. At the same time, cartilage chips were prepared from human articular chondrocytes (CAH) that were collected from areas of healthy cartilage (a non-bearing area of cartilage at a healthy joint).

 The chondrocytes isolated after digestion were extracted with total or protein RNA and used in real-time RT-PCR and Western-blot experiments, with chondrocytes derived from healthy cartilage being used as controls. .

 Transfection of siRNAs by INTERFERin®:

The siRNA (control siRNA, siRNA COL1A1, siRNA HTRAJ were incorporated into stem cells using lINTERFERin ^® as transfection agent. (Polyplus transfection ™).

"Western-blot": The cells seeded in type I collagen sponges were rinsed with PBS IX ("Phosphate Buffered Saline" Fisher Bioblock Scientific) and lysed at 100 μΙ of RIPA buffer ("radioimmunoprecipitation assay buffer" ) by sponge, then were shaken (vortexed) every 10 minutes for one hour. Protein extraction was continued by removing each sponge and pressing them one by one in order to recover the maximum amount of protein. The supernatant containing the cytoplasmic extracts was finally removed after centrifugation for 30 minutes at 14,000 g. The protein concentration of our samples was determined by the Bradford method. 20 μg of each sample was contacted with denaturation buffer (2.5 ml of 100% glycerol, 2 ml of 10% SDS, 1 ml of bromophenol blue 1%, 600 μl of 1M Tris-HCl pH 6 0.8 ml of β-mercaptoethanol) and heated at 95 ° C for 5 minutes. Polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) was performed. The proteins of the gel were then transferred to a PVDF (Poly Vinylidene DiFluoride, illipore) membrane by electro-transfer for 1 h 30. The membrane was first activated by three successive baths: 100% methanol (1 min), water distilled (twice 1 min) and IX transfer buffer (15 min). At the end of the transfer, the membrane was washed with "Tris Buffered Saline-Tween" (TBS-T). Blocking of nonspecific sites of the membrane was achieved by incubation in TBS-T containing 10% skimmed milk for 1 h at room temperature and with stirring. The membrane was then washed with TBS-T and then incubated for 15 h at 4 ° C. in the presence of the primary antibody (Ac I ^aira ) directed against the protein of interest (Collagen type II, l / 2000 ^th , Novotec, Collagen of Type I, l / ^3000th, Novotec, HtrAl, l / ^2000th, Millipore, ref. AB15852), Collagen Type X (l / ^1000th, Sigma, C7974-ZML) Collagen type III (l / ^1000th + 5% skimmed milk, Santa Cruz Biotechnology sc-28888) and GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase ), l / 2000 ^th , Santa Cruz Biotechnology). The next day, a series of rinses of the membrane was performed with TBS-T, and then it was incubated in the presence of the secondary antibody (Ac IF ^Iire , anti-rabbit, 1/6000 ^th , Tebu-Bio or Ac II ^area , anti-mouse, 1/6000 ^th ) 1 h at room temperature. The proteins were finally revealed using a detection kit ("Super Signal West Pico Chemiluminescent Substrate", PIERCE).

Real-time RT-PCR: The cells were lysed with TRIzol Reagent ^® ,

Invitrogen. 1 μg of total RNA was treated with 1 μl of deoxyribonuclease I (DNase I, Invitrogen) for 15 min at 25 ° C. The reaction was stopped by the addition of 1 μ! 0.5 mM EDTA followed by a 10 min incubation at 65 ° C. The samples digested with DNase were then retro-transcribed into cDNA. The reaction was carried out in the presence of a mixture consisting of 5 μl of the 10X reverse transcriptase buffer, 1 μl of the "Moloney Murine Leukemia virus" reverse transcriptase at 200 units / μl (MMLV, Fisher Bioblock Scientific), 1 μl of oligodT, 2 μl of a mixture of the four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP, 20 μl final, Fisher Bioblock Scientific) and 1 μl of RNAse-out (40 U / μl, Invitrogen) . The volume was supplemented to 15 μl with water treated with DEPC (ethyl-pycarbonate). The reverse transcription reaction was carried out for a period of 1 h at 37 ° C, preceded by a 10 min heating cycle at 25 ° C. After reverse transcription, cDNA samples obtained were diluted to l / ^100th then used in PCR experiments in real time. To do this, the amplification of the gene studied was carried out in triplicate in 96-well plates, according to the instructions of the device "ABI Prism 7000 SDS" (Applied Biosystems). The samples were amplified using a SYBR Green PCR Master Mix (Applied Biosystems) and using the primers as described in Tables 1 (COLIAI, COL2A1 and RPL13) and 2 (COL9A1, COL11A1, Agrecan, HTRA1 and COL2B) above and in Table 3 below. The mRNA levels of the COLIAI, COL2A1, C0L3A1, COL9A1, COL11A1, COL10A1, HTRA1, Agrecan, Sox9, HIF-α, HIF-2a, COL2A, COL2B, Integrin-β, Osteocalcin and Cbfa-1 genes were estimated. after normalization against the reference gene RPL13. Table 3

Statistical analyzes: For RT-PCs, the results are represented as histograms of a representative experiment and represent the average of three ± SD measures. Significance of values was assessed using Student's t-test (* p <0.05; ** p <0.01; *** p <0.001) RESULTS

 Effects of COL1A1 and / or HtrAl siRNA on induction of chondrocyte lineage of mesenchymal stem cells derived from bone marrow

Bone marrow derived human mesenchymal stem cells were cultured in type I collagen sponges at 500,000 cells / sponge in the presence of siRNA COL1A1 and / or HtrA1 (100 nM, between days 0 and 3 days and then 100 nM between 7 and 10 days). Cells were cultured under hypoxic conditions (<5% O ₂ ) in the presence of BMP-2 (50 ng / ml) and TGF-βΙ (10 ng / ml) for 14 days. The average mRNA levels of the COL1A1, C0L2A1, HtrA1, Agrecan, COL10A1, COL3A1 genes were estimated by RT-PCR in real time after normalization against the reference gene RPL13 (AU: Arbitrary Unit). The results are collated in Figures 9A-9F. The ratio of the COL2A1 / COL1A1 and Agrecan / COLIAI mRNA levels making it possible to define the chondrocyte culture differentiation index were also measured under the various experimental conditions and are represented in FIGS. 9G and 9H. The results of a representative experiment are presented.

Effects of COL1A1 and / or HtrAl siRNA on protein levels of type I, II, III, X and HtrAl collagens.

Bone marrow-derived human mesenchymal stem cells were cultured in type I collagen sponges at 500,000 cells / sponge in the presence of siRNAs targeting type I and / or HtrA1 collagen (100 nM, between 0 days). and 3 days then 100 nM between 7 and 10 days). Cells were cultured under hypoxic conditions (<5% O ₂ ) in the presence of BMP-2 (50 ng / ml) and TGF-βΙ (10 ng / ml) for 14 days. Total protein extracts isolated from the sponges colonized by the cells were subjected to SDS-PAGE electrophoresis for Western blot analysis with anti-collagen I, II, III, X, anti-collagen antibodies. HtrAl and anti-GAPDH. The results are grouped in Figure 10 which corresponds to a representative "Western-Blot". Phenotypic profile of differentiated cells after induction of chondrocyte lineage in hypoxia under the effect of siRNAs COL1A1 and HtrAl.

Bone marrow derived human mesenchymal stem cells were cultured in type I collagen sponges at 500,000 cells / sponge in the presence of siRNAs targeting type I and / or HtrA1 collagen (5, 25, 50). 4

and 100 nM between days 0 and 3 days). The cells were cultured under hypoxic conditions (3% O ₂ ) in the presence of BMP-2 (50 ng / ml) and TGF-βΙ (10 ng / ml for 14 days) The average levels of mRNA of 16 genes COL1A1, COL2A1, COL2A1-2A, COL2A1-2B, HtrAl, Agrecan, COL10A1, COL3A1, COL9A1, COL11A1, SOX9, HIF-lalpha, HIF-2alp, Integrin-β, Cbfal and osteocalcin were estimated by RT-PCR in real time after normalization against the reference gene RPL13 (AU: Arbitrary Unit) and allow to establish the phenotypic profile of the cells shown in Figure 11. The phenotypic profile is also established for chondrocytes isolated from cartilage healthy.

 Preliminary studies have shown that BMP-2 alone is not able to induce the synthesis, by hypoxic-sponge collagen-derived stem cells, of phenotypic markers of chondrocytes. The chondrocyte lineage is therefore not engaged under these conditions whereas these same conditions stabilize the phenotype of the differentiated cells that are the chondrocytes.

 In contrast, the addition of another cytokine, TGF-βΙ, to the combination [BMP-2 + sponge + hypoxia] induces the differentiation of MSCs into chondrocytes, as evidenced by the results in Figure 9. However, cells after 14 days of culture always synthesize type I collagen, hence the use of RNA interference targeting type I collagen and / or HtrA1, to stabilize the chondrocyte phenotype.

More specifically, the combination [BMP-2 + TGF-β + sponge + hypoxia] induces a very strong expression of the type II collagen gene and to a lesser extent that of the aggrecan gene (FIGS. 9A and 9D). Nevertheless, the high level of messenger type I collagen associated with these experimental conditions is very significantly reduced in the presence of siRNA COL1A1 or siRNA COL1A1 + HtrAl (Figure 9B). However, the use of siRNA COL1A1 alone reduces the messenger rates of agrecan (Figure 9D) while that of HtrAl siRNA, beyond the inhibition of HtrAl messenger rates (Figure 9C), is associated with increased messenger aggrecan (Figure 9D) and has no impact on the expression of collagen type I. The combined use of siRNA COL1A1 and HtrAl thus allows a strong expression of messengers type II collagen , aggrecan while reducing the rates of messengers of type I collagen, whose value is lower than that found in healthy cartilage. The index of 35 FR2011 / 052198

Differentiation with both siRNAs, evaluated by the collagen type II / collagen type I ratio (Figure 9G), is therefore improved to reach that of chondrocytes derived from healthy cartilage. This differentiation index is also supported by the aggrecan / collagen type I ratio which is most significantly increased by the combination [B P-2 + TGF-1 + sponge + hypoxia] + siRNA COLIAI and HtrAl. Under these same conditions, the expression of type III collagen is contained in the messenger (Figure 9F) and is only slightly noticeable at the protein level (Figure 10). In addition, the expression of X-type collagen, although increased at the level of messengers (Figure 9E), is only slightly noticeable at the protein level (Figure 10). For type II collagen, higher than normal healthy cartilage levels in the form of 220 kDa were observed in Western blot, significantly increased but lower than in healthy cartilage, for the mature form. of 135 KDa. Finally, RNA interference using both COLIAI and HtrAl siRNA virtually prevents protein synthesis of type I collagen (Figure 10).

 These results show that the stem cells are well engaged in the chondrocyte lineage, acquiring with the experimental conditions a differentiation index comparable to that of chondrocytes derived from healthy cartilage.

 Gene analysis has been expanded to 16 genes defining the chondrocyte phenotypic profile to be reached by stem cells in their chondrocyte lineage.

 This phenotypic chondrocyte profile is established on the basis of 3 major gene families: i: the characteristic genes of articular chondrocytes; ii: the characteristic genes of hypertrophic chondrocytes; iii: the characteristic genes of osteoarthritic chondrocytes.

Chondrocytes Chondrocytes Hypertrophic and even osteoarthritic articular chondrocytes

 osteoblasts

 COL2A1, COL2A1-2A * COL10A1, Osteoca / cine, COLIAI, COL3A1, HtrA1 COL2A1-2B **, COL9A1, Cbfal, HIF-2a

COL11A1, Agrecanne,

Integrin-βΐ, SOX9, HIF-la

* Immature and immature chondrocytes The phenotypic profile obtained from chondrocytes from healthy cartilage (Figure 11) is characterized by / ^'- a strong expression of the messenger of the major constituents of the cartilage matrix: aggrecan, COL2A1, COL11A1, COL9A1 and signaling activators the synthesis of specific matrix constituents: integrin-βΐ, HIF-Ια, SOX9. low expression / basal expression of the messengers of the characteristic molecules of osteoarthritic chondrocytes: COL1A1, COL3A1 and HtrA1 and iih low expression / basal expression of the messengers of the molecules characteristic of hypertrophic chondrocytes (with the exception of COL10A1).

 Specifically, the major constituents of hyaline cartilage, aggrecan and collagen type II, are distributed on a crown / scale ranging from 100 to 100,000 whereas the constituents of fibrocartilaginous arthritic cartilage COL1A1 and COL3A1 are on the crown / scale 1 at 10 as well as markers of hypertrophic chondrocytes with the exception of X-type collagen which is between 10 and 100. For cells obtained with the combination [B P-2 + TGF-βΐ + sponge + hypoxia] + siRNA COL1A1 and HtrAl, the profile is superimposable with a distribution of the different constituents very close to that observed with healthy cartilage. If we compare this latter profile with that of the cells cultured in the absence of B P-2, TGF-βΙ and siRNA COLlAl / HtrAl, a real difference is observed corresponding to the acquisition of the chondrocyte phenotype sought by the stem cells. with the combination [BMP-2 + TGF-pi + sponge + hypoxia] + siRNA COL1A1 and HtrAl, also with regard to the healthy cartilage profile.

 These results, in which the index of differentiation by the ratios COL2A1 / COL1A1 and Agrecanne / COLIAI supported by the study of protein levels, and in parallel, the phenotypic chondrocyte profile, show that the bone marrow stem cells acquire a phenotype. chondrocyte and that the cells at the end of the culture (14 days) with the combination [BMP-2 + TGF-βΐ + sponge + hypoxia] + siRNA COL1A1 and HtrAl, synthesize an extremely qualitative matrix, hyaline cartilage type.

All the results obtained show a significant decrease in type I collagen, both in terms of its average levels of mRNA and protein level. In addition, they demonstrate that interference with mRNA is an effective approach to decrease the production of this atypical collagenic isotype of healthy joint cartilage that is induced when articular cartilage becomes osteoarthritis. Our results also show an increase in collagen type II / type I ratio, and ratios of mRNA levels C0L2A1 / C0L1A1, COL9A1 / COL1A1, COL11A1 / COL1A1, Agrecan / COLIAI, etc. reflecting a marked improvement in the state of differentiation of chondrocytes.

 The method according to the invention therefore makes it possible to induce the increase of cartilage-specific markers and the decrease of non-specific markers, which leads to a sharp increase in the differentiation index of chondrocytes cultured in vitro.

Claims

1. Process for obtaining in vitro or ex vivo differentiated articular chondrocytes characterized in that it comprises the following steps:

 - to culture dedifferentiated or differentiated articular chondrocytes or mesenchymal stem cells,

 culturing these cells under conditions of low oxygen tension, in the presence of BMP-2 ("Bone Morphogenetic Protein-2") and specific inhibitor (s) of type I collagen.

 2. Method of obtaining according to claim 1, characterized in that the articular chondrocytes which are cultured are adult articular chondrocytes.

 3. Process for obtaining according to any one of the preceding claims, characterized in that the differentiated or dedifferentiated chondrocytes or the mesenchymal stem cells which are cultured are of human or equine origin.

4. Process according to any one of the preceding claims, characterized in that the differentiated or dedifferentiated chondrocytes or the mesenchymal stem cells are cultured under culture conditions of less than or equal to 10% of O ₂ .

 5. Production method according to any one of the preceding claims, characterized in that the differentiated or dedifferentiated chondrocytes or mesenchymal stem cells are cultured within a biomaterial, preferably within type I collagen sponges. .

 6. Method of obtaining according to the preceding claim, characterized in that the mesenchymal stem cells are non-human when they are embryonic mesenchymal stem cells or germ line.

 7. Production process according to any one of the preceding claims, characterized in that the presence of BMP-2 is ensured both by adding the molecule BMP-2 directly into the culture medium and preventing its degradation and / or its inactivation or that (s) of its receiver.

8. Production process according to claim 7, characterized in that the presence of BMP-2 is ensured both by adding the molecule BMP-2 directly into the culture medium and using a siRNA HTRA1.

9. Production method according to any one of the preceding claims, characterized in that the type I collagen inhibitors are RIMA si.

 10. Production method according to any one of the preceding claims, characterized in that the inhibitors of type I collagen are siRNA and the presence of BMP-2 is ensured by the addition of BMP-2 directly into the medium. culture and use of a siRNA HTRA1.

 11. differentiated articular chondrocytes characterized in that they are obtainable, preferably directly obtained, by the implementation of the method according to any one of claims 1 to 10 and which have a ratio of collagen type II protein / type I collagen significantly increased by at least a factor of 1.5 and / or a ratio of the average collagen, II, IX, XI / COL1A1 or Agrecan / COLIAI collagen mRNA levels significantly increased by at least a factor of 7 Compared to non-differentiated cells.

 12. Chondrocytes according to claim 11, characterized in that they exhibit little or no expression of type I collagen and / or do not produce or little protein level but express and synthesize significantly preferentially the collagen of type II and aggrecan.

 13. Chondrocytes according to one of claims 11 or 12 for their use as a medicament.

 14. Chondrocytes according to any one of claims 11 to 13 for their use in the treatment of chondral loss of substance related or not traumatic shock, osteoarticular diseases, and in particular diseases that are characterized by destruction or degeneration of the cartilage.

 Chondrocytes according to claim 14 for use in the treatment of osteoarthritis.