Roderick H.M., J Stassen, Guus G.H. Van Den Akker, Don A.M Surtel, Andy Cremers, Marjolein M.C. Caron, Lodewijk W. Van Rhijn, Tim J.M. Welting
Objectives: Establish a phosphate-driven in vitro chondrocyte mineralization model and determine the influence of TGF-β on phosphate-driven mineralization of articular chondrocytes.
Status: Mineralization is an intrinsic property of normal physiology, but also occurs in pathologies such as osteoarthritis (OA). Several mechanisms have been shown to play a potential role in the development of OA, such as an excess of inorganic phosphate. We aimed to utilize this disbalance in inorganic phosphate to develop an in vitro mineralization model for human OA articular chondrocytes (HACs). Moreover, considering the ambivalent role of TGF-β in cartilage homeostasis and disease, we investigated the effect of TGF-β in our in vitro model.
Methods: OA HACs were isolated from surgical waste material originating from total knee arthroplasty. A pool of HACs (N=6 donors) was cultured in DMEM/F12 supplemented with 10% FCS, 1% P/S and 1% NEAA. Inorganic phosphate disbalance was induced by addition of ATP (1 mM) and β-glycerophosphate (BGP, 10 mM) in the absence or presence of 10 ng/ml rhTGF-β. Following successful mineralization, cell cultures were analysed by von Kossa staining and SEM-EDX. Quantification of total calcium and phosphate was performed after crystal hydrolysis with the Randox calcium and phosphate colorimetric assays (Sigma). Gene expression analysis was performed by RT-qPCR and normalized to PPIA as a reference gene.
Findings: HACS were stimulated with ATP and BGP to induce phosphate-driven mineralization. The presence of calcium-containing crystals was evidenced by von Kossa staining and increased amounts of calcium and phosphate deposition. Further confirmation of the presence of calcium-containing crystals was obtained with SEM-EDX (Figure 1A). Since formation of mineral nodules is largely facilitated by the composition of the cartilage extracellular matrix (ECM), we investigated chondrocyte gene expression changes induced by the mineralization medium. A significant decrease of COL2A1 and ACAN accompanied by a decreased expression of fibrotic collagens was observed (Figure 1B). Additionally, a significant increase in SOX9 and MMPs was found. Addition of rhTGF-β3 enhanced mineralization as evidenced by increased total calcium and phosphate. This could be inhibited by the ALK5 inhibitor SB505124 in a dose-dependent manner. To explore the potential underlying molecular mechanisms, we determined gene expression of ECM markers and MMPs. Here we observed an increased expression of COL2A1 and fibrotic collagens, accompanied by decreased expression of ACAN and MMP’s (Figure 1C).
Significance: Due to the prevalence of calcium-containing crystals in articular cartilage at the time of total knee arthroplasty, there is a need for better understanding of the mechanisms involved in cartilage mineralization. In this study, we developed a phosphate-driven in vitro mineralization model for OA HACs. Our results show substantial chondrocyte-driven mineralization over a time course of 7 days. We found a pro-mineralizing role for rhTGF-β3 in an imbalanced phosphate environment. This was potentially caused by increased fibrotic collagen gene expression. Future studies will focus on validating these findings on the protein level and determining the influence of TGF-β on phosphate homeostasis by analyzing phosphate transporter levels in chondrocytes.