Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro

• Published in: Tissue & cell 2010-06, Vol.42 (3), p.151-157
• Main Authors: Steinhagen, J, Bruns, J, Niggemeyer, O, Fuerst, M, Rüther, W, Schünke, M, Kurz, B
• Format: Article
• Language: English
• Subjects: Advanced Basic Science; Animals; Cartilage, Articular - cytology; Cattle; Cell Culture Techniques - methods; Cell Proliferation; Cell Shape; Chondrocytes; Chondrocytes - cytology; Chondrocytes - enzymology; Chondrocytes - metabolism; Chondrocytes - secretion; Co-culture; Coculture Techniques; Collagen Type II - metabolism; Extracellular Matrix - metabolism; Extracellular Space - metabolism; Fibroblasts - cytology; Fibroblasts - metabolism; Immunohistochemistry; L-Lactate Dehydrogenase - secretion; Perfusion; Perfusion chamber; Protein Biosynthesis; Proteoglycans - secretion; Synovial fibroblasts; Synovial Fluid - cytology
• ISSN: 0040-8166; 1532-3072
• DOI: 10.1016/j.tice.2010.03.003

Abstract Objective To investigate the interactions of chondrocyte metabolism by synovial cells and synovial supernatants in a new perfusion co-culture system. Methods Chondrocytes and synovial fibroblasts were obtained from knee joints of slaughtered adult cattle. For experimental studies chondrocytes and synovial fibroblasts were placed together into a perfusion chamber (co-culture) or were placed into two different perfusion culture containers, which were connected by a silicone tube (culturing of chondrocytes with synovial supernatants). A control setup was used without synovial cells. Chondrocyte proliferation was shown by measurement of DNA content. The proteoglycan synthesis was quantified using35 SO42− -labelling and the dimethylmethylene blue assay.3 H-proline incorporation was used to estimate the protein biosynthesis. Type II collagen synthesis was measured by ELISA, furthermore extracellular matrix deposition was monitored immunohistochemically (collagen types I/II). Regarding to the role of reactive oxygen species LDH release before and after stimulation with hydrogen peroxide was measured. Results The proliferation of chondrocytes shows an increase in monoculture as well as in co-culture or in culture with synovial supernatants more than fivefold within 12 days.3 H-proline incorporation as a marker for chondrocytes biosynthetic activity decreases in co-culture system and in culture with synovial supernatants. A similar effect is seen measuring total proteoglycan content as well as the35 SO42− incorporation in chondrocytes. Co-culturing and culturing with synovial supernatants lead to a significant decrease of proteoglycan release and content. Quantification of collagen type II by ELISA shows significant lower amounts of native collagen type II in the extracellular matrix of co-cultured chondrocytes as well as in culture with synovial supernatants. The membrane damage of chondrocytes by hydrogen peroxide is reduced when chondrocytes are co-cultured with synovial fibroblasts. Conclusion The co-culture perfusion system is a new tool to investigate interactions of different cell types with less artificial interferences. Our results suggest that synovial supernatants and synovial fibroblasts modulate the biosynthetic activity and the matrix deposition of chondrocytes as well as the susceptibility to radical attack of reactive oxygen species.