Role of insulin in Sox9 mediated COL2A1 expression in passaged articular chondrocytes

Abstract

Purpose: One of the factors preventing clinical application of regenerative medicine to degenerative cartilage diseases is a suitable source of cells. Cartilage tissue is sparsely cellular and chondrocytes are the only cell type but under in vitro culture conditions they lose their phenotype along with the ability to generate hyaline cartilaginous tissue. Here we describe a reliable serum and growth factor free culture system to reverse this change in phenotype. Methods: Bovine articular chondrocytes (BAC) were passaged twice to allow for cell number expansion (P2) and cultured at high density on 3D collagen type II-coated membranes in media supplemented with ITS+ with 4.5g/l glucose, proline and 10-8 M dexamethasone (SF3D). Cultures were grown for up to 4 weeks. Inhibition studies were initiated on day 2 with 100uM HNMPA-(AM3). For FACS cells were harvested with Trypsin, stained with antibodies reactive with CD105-PE or CD44-PE (eBioscience, US) and analyzed by EPICS XL FACS and Kaluza analysis software (Beckman Coulter, US). ChIP-qPCR was carried out after in-situ cross-linking with 0.75% formaldehyde. Cells were sonicated in 1% Triton-100 and 1% SDS lysis buffer (Vibracell 30sec ON and 60sec OFF, 20x) and 250-900 bp DNA fragmentation was confirmed by agarose gel electrophoresis. Purified DNA was analyzed by qPCR using primers within the enhancer region of COL2A1. For immunoprecipitation cells were extracted in Ripa buffer and incubated with Sox9 antibody, the immune complex was harvested with Protein A/G beads. Extracts were immunoblotted with antibodies reactive with Sox6 and Med12. Results: 99% of P2 cells in monolayer culture were CD44+ and 40% were CD105+. They showed differential gene expression with high type 1 collagen and low type II collagen. Gene expression of other cartilage associated genes was lower in P2 cells when compared with the primary P0 chondrocytes. When placed in SF3D the P2 cells expressed chondrogenic genes and accumulated extracellular matrix (ECM) rich in proteoglycans (PGs) and type II collagen. Decreasing insulin receptor (IR) with HNMPA-(AM3) inhibited collagen and PGs synthesis and reduced expression of col II, col XI, IR and IGF1R genes as well as Sox6 and 9 proteins. Co-IP and ChIP analysis on inhibited cells showed binding of co-activators Sox6 and Med12 with Sox9 but reduced Sox9-Col2A1 binding. Figure 1. Photomicrographs of the tissue formed by P2 when cultured in SC3D culture show no matrix accumulation (far left panel) SF3D show accumulation of matrix rich in PGs evident by Toluidine Blue staining (second panel). SF3D tissue stained positively for collagen type II (red) and were predominantly negative for collagen type I (green) (third panel) similar to native cartilage tissue stained with the same antibodies (fourth panel). Conclusions: Here we describe a novel serum/ xenogeneic compound/ growth factor- free culture system which promotes hyaline-like cartilage tissue formation by insulin mediated regulation of Sox9-Col2A1 binding. This is a critical step towards the ultimate goal of developing autologous patient specific tissue engineered hyaline-like cartilage tissue suitable to use for repair/replacement of damaged articular cartilage. The suitability of this system for use in regenerative medicine approaches needs to be examined in vivo. Acknowledgements: Supported by DoD.