MXPA97004296A - Cellular lines of condroc - Google Patents

Abstract

The invention relates to chondrocytes and chondrocyte clines for use, especially in the study of arthritic conditions and also for the development of skeletal tissue for use in repair and / or replacement surgery. The use of articular chondrocyte clines that mineralize calcium to determine potentially beneficial therapeutic agents is described. In addition, the means for producing such clines, as was the characteristics of such clines are also described. The use of hypertrophic chondrocyte clines to produce matrix tissue is also described. It also describes the means to produce hypertrophic chondrocyte clines, as was the characteristics of such clines

Description

CELLULAR LINES OF CONDROCITE DESCRIPTION OF THE INVENTION The invention relates to Chondrocytes and Lines Chondrocyte cells for use, particularly, but not exclusively for the study of arthritic conditions. In addition, the invention relates to chondrocytes that may be of particular advantage in the development of novel biomaterials for use in the replacement of skeletal tissues such as hips and joints. Arthritis is characterized by inflammation of the joints and therefore an inflammation of the tissue The cartilage is made from materials secreted by the cells, and specifically the chondrocytes, in the extracellular space.Two of the most prominent materials to be secreted by the cells are collagen and -copolysaccharides.The mucopolysaccharides are sugar molecules of high molecular weight that comprise carbohydrate chains that exist as repeating disaccharides.One of the two sugars is always an amino sugar, either N-acetyl-glucosamine or N-acetyl-galactosamine.In most cases , mucopolysaccharides exist in combination with proteins and thus form proteoglycans.

The collagen has a fibrous structure while the mucopolysaccharides are amorphous and extremely viscous and therefore can occupy large volumes for a given weight material. In many cases collagen and proteoglycans interact with each other to form a variety of extracellular structures. These structures seem to be self-organized; there is no evidence of enzymes or other materials that cause their polymerization, interaction or orientation. For example, a typical collagen proteoglycan found in the cartilage may consist of a central filament of hyaluronic acid several micrometers in length which has bound thereto, at regular intervals, binding proteins. The long-nucleated proteins Mucopolysaccharides such as chondroitin sulfate and keratan sulfate are linked at regular intervals. Thus, the extracellular matrix materials organize themselves to form cartilage. The formation of bones is characterized by calcification in the region of hypertrophic chondrocytes. While vascular invasion is important for in vivo ineralization, it is clear that the initiation of calcification may be independent of vascularization, since hypertrophic chondrocytes may spontaneously mineralize the matrix in vi tro in the absence of exogenous phosphate. However, articular cartilage in adults does not calcify normally, except in its articulation with the eubccr.iral bone where calcification forms a characteristic extreme limit. Therefore, it is interesting to note that chondrocalcinosis, the pathological calcification of "articular cartilage by calcium pyrophosphate, which occurs in different regions of articular cartilage and other types, increases in frequency with age and can be associated with osteoarthritis. It is therefore understood that understanding the mechanisms associated with osteoarthritis should involve an understanding of the mechanisms associated with the calcification of articular cartilage. - cellular models for this research. Instead, existing cell models employ the use of growth plate chondrocytes, that is, chondrocytes that mineralize calcium in order to produce bone tissue. With the use of this cellular model of chick has been possible to show that the "chondrocyte cell layers in chick growth plate are mineralized in the presence of ascorbate or beta-glycerophosphate (1) .The effects of ascorbate are associated with an increase in the synthesis of alkaline phosphatase and type X collagen, although it is not If both are essential for calcification to occur, beta-glycerophosphate acts independently of the regulation of these two molecules, presumably by providing a supply rich in exogenous phosphate for nucleation. -Other researchers have developed a pelleted culture system for rabbit growth platelets in which there is synthesis of alkaline phosphatase and type X collagen as well as calcification "in cultures with ascorbate (2). (3) suggests that the effects of ascorbate may be of a specific species, enhancing the need for a human chondrocyte model system if the processes of human diseases are to be studied. Matrix in vi tro through articular chondrocytes The researchers have compared chondrocytes -of growth plate and rabbit joints and have found that in high-density cultures the growth plate chondrocytes produce alkalinphosphatase and incorporate calcium in the cell layers, while the articular chondrocytes do not (4). However, two "Studies have shown that chick (5) or human (6) chondrocytes can express type X collagen and alkaline phosphatase in long-term cultures, but there is no evidence of calcification of the cell-layer matrix in any of the studies Other researchers have described in the International Patent Application WO9409118, the closest prior art, the way in which the human chondrocyte cell line can be produced by transfecting old primary chondrocytes using an SV Z shrink. Type II collagen, a specific material for articular chondrocytes, however, there is no reference to the matrix, matrix calcification using these cell lines, so it can be observed that existing models of calcification depend on the Long-term cultures of chondrocytes in animal growth plates There are currently no models of chondrocyte calcification. It has been surprisingly found that "mature articular chondrocytes and immortalized articular chondrocyte cell lines provide an extracellular matrix that rapidly calcifies. By the term "mature", it refers to the chondrocytes of an older individual such as an individual of propitious age to develop osteoarthritis such as, but not limited to, an individual over 50 years of age, and more preferably 65 years of age. Or, alternatively, chondrocytes cultured in functionally equivalent age. Therefore, the discovery represents a valuable opportunity for the study of the mechanisms associated with the calcification of articular cartilage and also for the regulation of such calcification. It is expected that the articular chondrocyte cell lines have both academic and commercial value in: to not only facilitate a superior understanding of calcification and that may be associated with the osteoarthritic condition, but also serve as valuable tools for the development of therapeutic agents to treat such a condition. The inventors of the present are not able to explain their discoveries, however they perform the following speculations. During development chondrocytes are typically consigned to one of two lineages. The first one refers to the production of bone and therefore to the differentiated chondrocytes that mineralize 'calcium. The second lineage refers to the production of cartilage tissue and therefore to differentiated cendrocytes that do not mineralize calcium. There may be some plasticity in the determination of lineage and, for example, this may be related to DNA de-ethylation with aging. Therefore, as articular chondrocytes age in the control mechanisms that prevent calcium mineralization, they operate less effectively and surprisingly the articular cartilage lineage chondrocytes actually mineralize calcium.

The findings, therefore, suggest that, contrary to previous expectations, it is possible to provide a human cell model for arthritis. The cellular model can be provided using either mature or immortalized articular chondrocytes and ideally cell lines that belong thereto. According to a first aspect of the invention, therefore, an articular chondrocyte that mineralizes calcium is provided. Preferably a plurality of chondrocytes are provided which are arranged in pellet form. Preferably, such chondrocyte and chondrocytes are derived from an adult individual, and ideally from such an individual who appears or has a predisposition toward arthritis. Alternatively, such chondrocyte and chondrocytes are of fetal origin and preferably of human fetal origin. Even more preferably such chondrocyte and chondrocytes mature as defined herein. Even more preferred are cell lines of such chondrocytes. In preferred embodiments of the invention such cell lines are of human origin and ideally comprise an oncogene and ideally a temperature-sensitive oncogene such that at an initial permissive temperature an active oncogene product is expressed and at a second non-temperature expression. permissive of the active oncogene product is avoided. It has been found that the use of such oncogene product picpcrciona a cell line q ^ e at the non-permissive temperature shows an increase in the expression of type II collagen - a marker for the fully differentiated phenotype. Ideally, shrinkage is a temperature-sensitive mutant of the immortalizing SV40-T oncogene. The inventors of this are not sure, at this stage, whether calcium mineralization by articular chondrocytes is directly responsible for osteoarthritis, or alternatively, if calcium mineralization by articular chondrocytes is an indicator of "an as-yet-unidentified condition or marker that is responsible for osteoarthritis In any case, the cell lines of the present invention represent suitable cellular models for further investigation." As mentioned in the foregoing, the inventors of "the present are not sure." also, at this stage, about the nature of the mechanisms that lead to calcium mineralization through articular or hypertrophic chondrocytes. Other researchers have suggested that there is reactivation of genes (7) related to age. Therefore, as the present inventors speculated in the above, it may be that a differentiation process gives rise to osteoarthritis. It may be that the genes that are typically inactive during life change with aging and therefore are responsible for the aberrant phenotype of the mature articular chondrocyte. The previous speculation is provided with the "The sole purpose of construction is not that the application should be limited by any of the foregoing limitations, but that these speculations are provided for the purpose of understanding." There are obvious advantages derived from the use of in vi tro cellular models that simulate However, the inventors of the present invention have also produced chondrocyte cell lines of fetal tissue that express type X collagen, a marker of hypertrophic chondrocytes.Such chondrocytes are known to mineralize calcium and therefore once The phenotype of these cells was determined, the existence of the mineralization was less surprising, however, it is "important to note that no one has ever made a human hypertrophic chondrocyte cell line, although the advantages of understanding the Symptomatic process of mineralization of arthritis condition. In addition, no one has taught to make such a cell line for the study and / or production of joint matrix tissue for use in studies and / or replacement surgery and / or skeletal repair. Therefore, the present inventors also present therein the knowledge of, and methods relating to, the production of a human hypertrophic chondrocyte cell line. According to a second aspect of the invention, a hypertrophic chondrocyte-human cell line is provided. Even in a further preferred embodiment of the invention, such cell line is of human fetal origin and also ideally comprises an oncogene and ideally a temperature sensitive oncogene such that at an initial permissive temperature an active oncogene product is expressed and an Second non-permissive temperature expression of the active oncogene product is avoided. The inventors of the present have found that the use of such an oncogene product provides a cell line which at the non-permissive temperature shows an expression of type X collagen, a marker for hypertrophic chondrocytes. Therefore, alternative cell lines derived from human fetal tissue that mineralize calcium can be provided. According to yet a further aspect of the invention there is provided a method for producing articular chondrocyte cell lines, such method comprising: a) immortalizing at least one articular chondrocyte cell of mature human using an immortalized agent !; and b, culturing the immortalized cell in order to produce a population of human articular chondrocyte cells. Preferably, the immortalizing agent is an oncogene and even more preferably a temperature-sensitive oncogene which, at a non-permissive temperature, results in an improved expression of the phenotypic characteristics of the cnndrocyte. In a preferred embodiment of the invention, immortalization is achieved using transfection techniques Conventionally and preferably the immortalizing agent is an immortalizing gene such as an oncogene and even more preferably a temperature-sensitive mutant of the SV40-T oncogene. Ideally such a method further comprises culturing such an immortalized cell at the non-permissive temperature of the oncogene. According to a further aspect of the invention there is provided a method for producing hypertrophic chondrocyte cell lines, such method comprises: a) immortalizing at least one human fetal chondrocyte cell, using an immortalizing agent; and b) culturing such an immortalized cell in order to produce a population of chondrocyte cells derived from human fetus. Preferably, the immortalizing agent is an oncogene and even more preferably a temperature-sensitive oncogene, which at a non-permissive temperature results in an improved expression of the phenotypic characteristics of the chondrocyte. In a preferred embodiment of the invention, immortalization is achieved using transfection techniques "conventional and preferably the immortalizing agent is an inorterizing gene such as an oncogene and even more preferably a temperature-sensitive mutant of the oncogene SV40-T immortalizer. Even more preferred transfection is carried out using transduction of oncogene sensitive to retroviral temperature. Ideally such a method further comprises culturing such an immortalized cell at the non-permissive temperature of the oncogene. According to yet a further aspect of the invention chondrocytes, or at least one chondrocyte cell line, according to the invention are provided for use in the development of therapeutic agents designed to treat arthritis. In accordance with a further aspect of the invention there is provided a method for developing therapeutic agents designed to treat arthritis comprising: a) exposing chondrocytes, or at least one chondrocyte cell line, according to the invention to at least one test agent; and b) observe the nature of the response to such test agent. In a preferred method of the invention such an observation comprises an assessment of the amount of calcium mineralization associated with such chondrocytes or with such chondrocyte cell lines. According to an alternative aspect of the invention, hypertrophic chondrocytes, or at least one hypertrophic chondrocyte cell line, which is used to provide articulation matrix tissue, are provided. According to an alternative aspect of the invention there is provided a method for producing articulation matrix tissue for use in repair and / or replacement of injured tissue comprising: a) providing a hypertrophic chondrocyte cell line; b) cultivating such a cell line under conditions that promote matrix growth; and c harvesting such cells and / or matrix tissue and / or selected portions thereof. The invention will now be described only by way of example with reference to the following examples, in which; Figure 1 shows up-regulation of type II collagen expression in human articular chondrocytes. Figure 2 shows the extracellular calcification achieved by these cells using a normal histological procedure (Von Kassa staining, 11) for the identification of calcification in tissue matrix. Figure 3 illustrates collagen (12) of type X located immunocytochemically in these cells suggesting in-vitro expression of the expected characteristics of hypertrophic chondrocytes. Figure 4 shows a femur derived from a human fetus that has been cultured for 14 days. Figure 5 shows 3 cloned chondrocyte cell lines from fetal femur when cultured either in the presence of 1, 25 (OH) 2D3 (top line) or in the absence of 1, 25 (OK) 2D3.

Figure 6 shows a single-layer, actively mineralizing culture of a chondrocyte cell line derived from human fetus cells.

Inpiortalization of Articular Chondrocytes Reference 13 is provided as a general method for preparing human articular chondrocytes for the primary culture, but any published method will suffice. In short chondrocytes taken from sections of mature tibial and femoral condyles, from human patient biopsies, they were digested enzymatically (14) and placed in tissue culture flasks (Costar Ltd. UK) in Eagle's minimal essential medium (Gibco Ltd UK) containing antibiotics and L-glutamine as well as 10% hot inactivated fetal bovine serum. The cultures were maintained in a humidified atmosphere of 5% C02 at 37 ° C and the medium was changed at regular intervals. The adherent cell population was then transfected with a temperature-sensitive mutant of the large tumor (T) antigen derived from the simian virus using retroviral transduction. Any normal method of transfection of this sequence (together with the binding to a suitable promoter to drive its expression, for example, the LTR promoter) will suffice, such as precipitation of calcium phosphate DNA, electroporation or micro-injection, but chose the ictroviral transduction because of its simplicity of use.

Immunization of Huinano Fetal Tissue Fetal rib or femur tissue was taken from 7 to 9 weeks' gestation and placed in tissue culture flasks (Costar Limited, UK) in Eagle's minimal essential medium (Gibco Co Limited, UK) containing antibiotics and L-glutamine. , as well as 10% hot activated fetal bovine serum. Cultured cells were maintained in a humidified atmosphere at 5% C02 and 37 ° C and the medium was changed at regular intervals (2 to 3 days). As usual -for the primary cultures of resected tissue, the cells within the sample move out of the body of the tissue, adhere to the surface of the flask and potentially replicate, to cover the entire surface of the tissue culture slide. After an initial 2-week period in the primary culture, the cell population was transfected with a temperature-sensitive mutant of the large tumor (T) antigen derived from the simian virus using retroviral transduction.

Cultivation of Immortalized Cells in order to produce a Homogeneous Cell Population In summary, amphotropically packed retroviral particles that comprise this construct and a geneticin resistance marker, G418 (kindly donated by Dr. M O'Hare, Institute of Cancer Research, Royal Marsden Hospital, Lincoln's Inn, Sutton , Surrey, alternatively, essentially the same construct expressing a temperature-sensitive mutant of the SV40-T oncogene was kindly donated by Professor Phil Gaillimore, CRC Laboratories, University of Birmingham, UK) was added to the medium along with polybrene (Sigma Chemicals) at a final concentration of 0.8 mg / ml. The viral titer was adjusted to give a low transduction efficiency of 0.0002% yielding an average of 20 colonies of cells immortalized per flask, each colony derived from an individual cell. Two hours after the addition of the virus, the culture medium was replaced with fresh medium. The cultures were maintained at 33 ° C, the permissive temperature for the active form of the SV40-T oncogene product. Five days after transduction, geneticin was added to the medium (0.4 mg / ml) for an additional 10 days to eradicate the cells that had not been incorporated into the retroviral vector.

Differentiation of such cells Between 14 and 20 days after transduction, the individual colonies of replication cells were identifiable. Clones were selected on the basis of being "well separated from other replication colonies, the cells in each colony being from 100 to 1000 cells in number.These were collected by ring cloning and expanded to near confluence in 75cm2 flasks ( Costar, UK Ltd) that were matched to approximately 22 divisions from a single cell, before freezing the lots in aliquots, samples were also used for cell characterization and to determine that they had the ability to express specific markers of articular chondrocyte The expression was made by exposing the cells to the non-permissive temperature of the oncogene (39 ° C.) The clones were derived from the routine method of individual cell expansion.

Experiments to show the functional characteristics of the differentiated articular chondrocyte cell line A specific marker of articular chondrocytes is the ability to express collagen (8) of Type II. The cells of the present invention were therefore investigated in order to show the expression of Type II collagen. Immortalized human chondrocytes were exposed at 39 ° C, the non-shrinking temperature of the shrinkage, after pellet culture (9,10) for six weeks. Figure 1 shows the media of 6 pellets incubated either at the permissive temperature of 33 ° C or at the non-permissive temperature of 39 ° C. The error bars represent the normal deviation. Figure 1 clearly shows ascending regulation of type II collagen expression at the non-permissive temperature of 39 ° C. These data indicate that the cell lines of the present invention comprise differentiated functional joint chondrocytes.

Extracellular Calcification Figures 2 and 3 show the extracellular calcification achieved using cells according to the invention. Specifically, Figure 2 shows a normal histological procedure (Von Kossa stain 11) for the identification of calcification in tissue matrix. The dark areas (Spotted Von Kossa Positive) represent the calcified matrix produced by these articular chondrocytes, these being separated by chondrocyte cells stained or not purposely stained in the culture. Figure 3 is included to show X-type collagen located immunocytochemically (12) in these cells suggesting in-vitro expression of the expected characteristics of hypertrophic chondrocytes. These data tend to suggest that the cells of the invention can be differentiated in order to show the characteristics associated with hypertrophic chondrocytes.

Longevity of the Human Articular Chondrocyte Cell Line Using the method of the invention, the inventors of the present surprisingly have found that their human articular chondrocyte cell lines "successfully differentiate to produce functional cells with articular chondrocyte characteristics." The cells survive and have passed through more than 60 divisions over a period of 2 years yielding 1018 cells of a variety of clones.The cell lines continue to survive and furthermore the cells of the line continue to exhibit functional characteristics typical of the tissue type of the differentiated cell.In addition, the inventors of the present invention have were able to produce immortalized human articular chondrocyte cell lines comprising differentiated cells that surprisingly mineralize calcium.

Experiments to Show the Functional Characteristics of the Cell Line of Chondrocyte "Derived from Differentiated Hypertrophic Fetus.

A specific marker of hypertrophic chondrocytes is the ability to express type X collagen. The cells of the present invention were therefore investigated in order to show the expression of type X collagen. All chondrocytes derived from fetus of the present invention expressed this cellular marker and further tests were performed and it was concluded that collagen type I was not expressed in these cells clearly showing that they were not of osteoprogenitor origin. Figure 4 shows a femur derived from a human fetus from 7 to 9 weeks of gestation and approximately 600 microns in length. The femur has been cultured in the culture medium described above for a period of 14 days to allow the growth and expansion of replication cell populations as would be the case for any primary cell culture. In this case, no previous treatment with enzymes or enzymes is required, however, to help cellular recovery. Cell expansion for 14 days is carried out before transduction with retroviral temperature sensitive oncogene.

Extracellular Calcification Hypertrophic chondrocytes derived from the immortalized fetus also responded to l, 25 (OH) 2D3 and expressed high levels of alkaline phosphatase activity, two additional markers of the hypertrophic chondrocyte-like phenotype. When the cells were left in the single-layer cell for 10 to 14 days at 39 ° C, the non-permissive temperature of the oncogene and in the absence of added β-glycerophosphate, the cultures were mineralized. This can be clearly seen in Figure 5 as it shows three cloned chondrocyte cell lines from fetal femur after retroviral transduction with an oncogene sensitive to the "temperature Each cell line has a basal level of alkaline phosphatase activity and some expression of type X collagen. If the cells are plated and incubated at the non-permissive temperature of the oncogene, and in the presence of 1, 25 ( OH) 2D3, upper line, each clone actively mineralizes calcium as illustrated by the normal histological staining procedure of von Kossa as described above.The cells plated and incubated at the non-permissive temperature of the oncogene in the absence of l, 25 (OH) 2D3, lower row, do not actively mineralize calcium.

Finally, Figure 6 shows, once again, using Von Kossa staining, a single layer culture that actively mineralizes a chondrocyte cell line derived from human fetus cells. The dark patches represent the mineralized matrix that develops and "spreads over the cultured cells that adhere to the surface of the culture slide." The inventors hereby have clearly demonstrated that it is possible to produce articular chondrocyte cell lines, - either derived from adult tissue, or alternatively, immortalized younger human tissue - which mineralizes calcium.The cell lines of the present invention therefore have the use of "identifying agents that affect calcium mineralization by these tissues. The inventors of the present invention have also demonstrated that it is possible to provide human hypertrophic chondrocyte cell lines for use as mentioned above and also for use in the development of biomaterials for use in any number of applications, but particularly for use in the development of materials for use in the replacement of joints.

REFERENCES 1. Leboy P. S., L. Vaiae, B. Usch ann, E. Golub, S. L. Adams and M. Pacifici 1989. Ascorbic acid induces alkalinphosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes. J. Biol. chem. 264: 17281-17286. 2 . Kato Y., M. Iwamoto, T. Koike. F. Suzuki, and Y. Takano. 1988. Terminal differentiation and growth calcification of rabbit chondrocyte cultures in centrifuge tubes: Regulation by transforming growth factor ß and serum factors. Proc. Na ti, Acad, Sci, -USA 85: 9552-9556. 3. Sandell L. J., and J. C. Daniel. 1988. Effects of ascorbic acid on the levels of collagen mRNA in short-term chondrocyte cultures. Tiss. Res. 17: 11-22. 4. Jikko A., T. Aoba. H. Murakami, Y. Takano, M.

Iwamoto and Y. Kato. 1993. Characterization of the mineralization procedure in cultures of rabbit growth plate chondrocytes. Dev. Biol. 156. - 372-380.

. Pacifici M., E. B. Golden S. L. Adams and I. M. Shapiro. 1991. Cellular hypertrophy and synthesis of type X collagen in cultured articular chondrocytes. Exp. Cell Res. 912: 266-270. 6. Stephens M., A. P. L. Kwan, M. T. Bayliss and C.

W. Archer. 1992. Articular surface chondrocytes from humans initiate the synthesis of alkali phosphatase and type X collagen in suspension culture. "Cell. Sci. 103.- 1111-1116. 7. areham K.A. , M. F. Lyon. H. Glenister and E. D.

Williams. 1987. Reactivation related to the age of a gene linked with X. Nature 327: 725-727. 8. Hollander A.P., Heathfield T.F., Webber C, Iwata Y, Bourne R, Rorabeck C and Poole A.R., 1994. Increased damage to type II collagen in osteoarthritic articular cartilage detected by a novel immunoprotection. "Clin. Invest. 93: 1722-1732. 9. Kato Y, Iwamoto M, Koike T, Suzuki F and Takano Y 1988. Terminal differentiation and growing calcification of rabbit chondrocyte cultures in centrifugation tubes: Regulation by transforming growth factor-b and serum factors. Proc. Nat. Acad. Sci (USA) 85: 9552-9556.

. Jikko A, Aoba T, Murakami H, Takano Y, I amoto M and Kato Y, 1993. Characterization of the mineralization procedure in cultures of rabbit growth plate chondrocytes. Dev. Biol. 156.- 372 -380. 11. Clark G, 1961. Miscellaneous Staine in Staining Procedures Eds. Williams and Wilkins. publ Balti pray. New York 12. Stephens M, Kwan A.P.L., Bayliss M.T., and Archer C.W., 1992. Articular surface chondrocytes from humans initiate the synthesis of alkaline phosphatase and type X collagen in suspension culture. J. Cell. Sci. 103: 1111-1116. 13. Frazer A, Bunning R. Thavarajah M, Seid J and Russell R.G.G., 1994. Studies on type II collagen and aggrecan production in human chondrocytes in vitro and effects of transforming growth factor-b and interleukin-lb. Osteoarthri tis and Cartilage 2: 235-245. 14. Go on M, Wood D.D., Ihrie E.J., Meats E.J., and Russell R.G.G., 1984. Stimulation by human interleukin-1 of cartilage decomposition and production of collagenase and proteoglycanase by human chondrocytes, but of non-human in vitro osteoblasts. Biochim. Biophye. Acta 797: 186-193.

Claims (29)

1. The use of an articular chondrocyte as a cellular model for matrix calcification.

2. The use of an articular chondrocyte according to claim 1, characterized in that a plurality of such chondrocytes is arranged in the form of pellets.

3. The use of an articular chondrocyte according to claims 1 or 2, characterized in that the chondrocyte or chondrocytes are mature or derived from an adult individual and preferably an individual suffering from or predisposed to arthritis.

4. The use of an articular chondrocyte according to claims 1 or 2, characterized in that the chondrocyte or chondrocytes are of fetal origin.

5. The use of an articular chondrocyte according to claims 1 to 4, characterized in that the chondrocyte or chondrocytes are provided as a cell line.

6. The use of an articular chondrocyte according to claim 5, characterized in that the cell line is of human origin.

7. The use of an articular chondrocyte in compliance "with any of the preceding claims, characterized in that the chondrocyte comprises an oncogene.

8. The use of an articular chondrocyte according to claim 7, characterized in that the oncogene is sensitive to temperature.

9. The use of an articular chondrocyte according to claim 8, characterized in that the oncogene is an SV40T antigen.

10. A method for producing an articular chondrocyte cell line that mineralizes calcium, characterized in that it comprises: a) immortalizing at least one articular chondrocyte cell of human precursor using an immortalizing agent; and b) culturing such an immortalized cell in order to produce a population of human articular chondrocyte cells.

11. A method in accordance with the claim 10, characterized in that the inortising agent is an oncogene.

12. A method in accordance with the claim 11, characterized in that the oncogene is sensitive to temperature.

13. A method according to claim 12, characterized in that the oncogene is an SV40T antigen.

14. A method according to claim 12 or 13, characterized in that the method further comprises culturing the immortalized cell at the non-permissive temperature of the oncogene.

15. The use of at least one articular chondrocyte, characterized in that it mineralizes calcium to identify therapeutic agents designed to treat arthritis.

16. A method for developing therapeutic agents designed to treat arthritis, characterized in that it comprises: a) exposing chondrocytes, or at least one chondrocyte cell line, comprising articular chondrocytes that mineralize calcium to at least one test agent; and b) observe the nature of the response to the test agent.

17. A method according to claim 16, characterized in that the observation comprises an assessment of the amount of calcium mineralization associated with the chondrocytes or with the chondrocyte cell line.

18. A hypertrophic chondrocyte cell line from 'human, characterized because the cell line is of fetal origin.

19. A cell line according to claim 18, characterized in that the cell line comprises an oncogene.

20. A cell line according to claim 19, characterized in that the oncogene is sensitive to temperature.

21. A cell line according to claim 19 or 20, characterized in that the oncogene is an SV40T antigen.

22. A method for producing human hypertrophic chondrocyte cell lines, characterized in that the method comprises: a) immortalizing at least one human fetal chondrocyte cell using an immortalizing agent; and b) culturing such an immortalized cell in order to produce a population of chondrocyte cells derived from human fetus.

23. A method according to claim 22, characterized in that the immortalizing agent is an oncogene.

24. A method according to claim 23, characterized in that the oncogene is sensitive to temperature.

25. A method according to claim 23 or 24, characterized in that the oncogene is an SV40T antigen.

26. A method according to claim 24 or 25, characterized in that the method further comprises culturing the immortalized cell at the non-permissive temperature of the oncogene.

27. The use of hypertrophic chondrocytes, or at least one hypertrophic chondrocyte cell line, characterized in that it is used in the provision of articulation matrix tissue.

28. A method for producing articulation matrix tissue for use in the replacement and / or placement of damaged tissue, characterized in that it comprises: a) providing a hypertrophic chondrocyte cell line; b) culturing the cell line under conditions that promote matrix growth; and c) harvesting the cells and / or matrix tissue and / or selected portions thereof.

29. Hypertrophic chondrocytes, or at least one hypertrophic chondrocyte cell line, characterized in that they are used in the identification of therapeutic agents designed to treat arthritis.