Extraculuar material promoted C3H10T1/2 chondrogenic differentiation than Pellet cultured in vitro

Abstract

Objective In order to understand the chondrogenesis differentiation of mesenchymal stem cells in either hydrogel or pellet culture, we applied the two methods and reveal the possible mechanism and for further investigation. Methods In C3H10T1/2 chondrogenic differentiation, we apply extracellular matrix hydrogel mixed the cell suspensions of freshly prepared (including scaling chondroitin sulfate, sodium hyaluronate synthesis and cross-linking agent) co-culture system and high cell density pellet formed by centrifugation. Chondrogenic differentiation of C3H10T1/2 was induced by treatment with TGF-β3 (10 ng/ml), dexamethasone (100 nmol/L), ascorbic acid (50 ug/ml), 1∶100 dilution ITS+Premix and high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum. And high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum is for control group. Histochemistry staining was utilized to identify extracellular proteoglycan and real-time PCR was performed to assess gene expression of SOX9, collagen IIa1/Xa1 and aggrecan for the 1st, 2nd and 3rd week respectively. Results In the hydrogel model for 3 weeks chondrogenic differentiation, the expression of master transcription factor SOX9 was upregulated in both culture models. While the marker genes of collagen IIa1 and collagen Xa1 were all promoted in hydrogel culture, the aggrecan gene expression was peaked in pellet culture. In addition, immunocytochemistry analysis of the hydrogel and pellet for 3 week illustrated the expression of extracellular matrix and more obviously in the hydrogel model. Conclusion In compared with pellet culture, the MSCs in the hydrogel were more likely promoted chondrogenesis leading to the eventual expression of marker genes. And the hydrogel would be applied in regeneration of cartilage injury. Key words: Mesenchymal stem cells; Cellular microenvironment; Chondrocytes