Interaction between synovium derived stem cells and chondrocytes in chondrogenesis and arthritis

Abstract

Abstract<br/> <br/> <br/> Purpose: The purpose of this study was to evaluate interaction between chondrocytes and synovium-derived stem cells (SDSCs) in chondrogenesis and arthritis. The author evaluated whether direct coculture of human chondrocytes and SDSCs can enhance chondrogenesis. Also, we analyzed the inflammation-related secretory proteins between chondrocytes and SDSCs by secretome antibody microarray and immunoblotting .<br/> <br/> Materials and Methods: To evaluate chondrogenic effect between human chondrocytes and SDSCs, cartilage and synovial tissues were harvested from patients undergoing total knee arthroplasty for osteoarthritis and expanded in monolayer. Passage 2 chondrocytes and SDSCs were directly cocultured with various mixed ratio (3:1, 1:1, or 1:3). GAG synthetic activity was accessed with GAG assay and Safranin–O staining. Expression of chondrogenesis-related genes (collagen types I, II, X, aggrecan, and SOX-9) were analyzed by RT-qPCR and immunohistochemistry staining. To investigate inflammation-related secretory mediators between human chondrocytes and SDSCs, passage 2 chondrocytes and SDSCs were obtained in the same way and cocultured indirectly. Secretomes obtained from monoculture or indirect coculture of each cells were analyzed by antibody microarray. Up or down regulated proteins more than 1.5 folds in coculture group compared with monoculture group were collected and extensive literature review was performed to select inflammation-related secretory proteins, as a candidate for inflammatory mediator between two cells. RT-PCR and immunoblotting (Western blot) was performed to confirm the expression of candidate mediators. <br/> <br/> Results : In direct coculture study, GAG/DNA ratio in 1:1 and 1:3 coculture groups were significantly increased compared to those in chondrocyte and SDSC monoculture groups. Type II collagen and SOX-9 were significantly up-regulated in the 1:1 coculture group compared to those in chondrocyte and SDSC monoculture groups. On the other hand, osteogenic marker (type I collagen) and hypertrophic marker (type X collagen) were significantly down-regulated in the coculture groups compared to those in the SDSC monoculture group. In secretome antibody microarray, 51 proteins were up-regulated and 36 proteins were down-regulated more the 1.5 folds in the coculture group compared with monoculture of each cell. Among these proteins, MMP-2, angiopoietin-1,Caveolin-1,ICAM-1,FABP4, Fibronectin, and VEGF-B were selected as up or down-regulated candidate for inflammatory mediators and subsequent RT-PCR and immublotting (Western blot) was performed. Finally, up-regulation of MMP-2 in cocultured chondrocyte and SDSCs, and down-regulation of FABP4 in cocultured SDSCs were consistently observed in all of microarray, RT-PCR, and immunoblotting. <br/> <br/> Conclusion: Direct coculture of human chondrocytes and SDSCs significantly enhanced chondrogenic<br/> potential, especially at 1:1 ratio, compared to chondrocyte or SDSC monocultures. In secretome microarray and immunoblotting under indirect coculture of chondrocytes and SDSCs, up-regulated MMP-2 and down-regulated FABP4 could be selected as possible inflammatory mediators. However, further study is necessary to clarify the role of MMP-2 and FABP4 in the interaction between two cells.<br/> <br/> Keywords: synovium derived stem cell, chondrocyte, coculture, chondrogenesis , secretome, microarray, inflammatory mediator <br/>