La culture de chondrocytes : outil d'analyse de la différenciation et de l'organisation moléculaire du cartilage

Abstract

La recherche des gènes impliqués dans la régulation de la prolifération et de la maturation des chondrocytes se heurte aux difficultés d'extraction d'ARN à partir de cartilage, et à la perte du phénotype chondrocyte en culture traditionnelle sur plastique. Des méthodes de culture en susp ension e t des stratégies d'immortalisation ont été développées, qui ont servi à analyser la synthèse et l'assemblage de molécules de structure du cartilage. L'utilisation d'oncogènes a révélé que prolifération et maturation sont, pour les chondrocytes, deux phénomènes mutuellement exclusifs. En outre, l'immortalisation de chondrocytes humains permet de constituer un réservoir cellulaire précieux pour des études pharmacologiques et ouvre la voie à l'immortalisation de chondrocytes de cartilage pathologique. Ces lignées devraient permettre de cloner plus facilement les gènes contrôlant les différents étapes de différenciation du chondrocyte, ou impliqués dans le maintien ou le dérèglement du phénotype cartilagineux.

Cartilage differentiation is a multistep process starting from mesenchymal cells and eventually resulting in mature hypertrophic chondrocytes, a stage reached only in cartilage undergoing endochondral ossification. This differentiation is characterized by a switch of gene activation. Mesenchymal cells produce collagens I and III, whereas chondrocytes synthesize the large proteoglycan agrecan and collagens II, IX, and XI. Hypertrophic chondrocytes produce collagen X, a biochemical marker for this stage of cell maturation. This critical developmental pathway of the skeleton is under environmental control of growth factors, hormones, cytokines, extracellular matrix, which have been extensively studied these past years. However, the understanding of the molecular mechanisms of chondrocyte gene regulation or of the precise function of cartilage matrix components has been hindered because of the instability of primary chondrocytes in traditional culture. Suspension culture methods and immortalization strategies have been developed and have allowed a better insight into the mechanisms of synthesis and assembly of cartilage macromolecules. The use of immortalizing oncogenes have also shown that growth and maturation are mutually exclusive phenomena for chondrocytes. These chondrocyte cell lines could be used to clone genes controling the pathway of cartilage differentiation. Furthermore, immortalization of normal human chondrocytes represent a valuable tool to test pharmacological factors, and the potential exists for immortalizing chondrocytes from diseased cartilage. This approach should be useful to identify genes involved in maintenance or disruption of the cartilage phenotype. Taken together, in vitro chondrocyte studies supported by the recent developments of cell and molecular biology should help to better understand the mechanisms of skeletogenesis.