Investigating the role of the extracellular matrix on differentiation of human mesenchymal stem cells and MC3T3 cells

Human mesenchymal stem cells (hMSCs) are a promising cell source for bone tissue engineering, but due to their limited number and donor variation, other cell types are used to answer relevant questions in bone tissue engineering. Since the extracellular matrix (ECM) is a complex entity with instructive properties, it is of key importance to analyze its role in osteogenic differentiation in different cell types used for bone tissue engineering. To investigate the role of the ECM on osteogenic differentiation, we interfered with collagen assembly using an irreversible inhibitor of lysyl oxidase, the enzyme responsible for collagen crosslinking, and analyzed its effect on differentiation of MC3T3 cells and hMSCs. MC3T3 cells and hMSCs were differentiated into the osteogenic lineage using BMP-2 and dexamethasone, respectively. β-aminopropionitrile (BAPN) was added to the medium at different concentrations and alkaline phosphatase expression and calcium deposition were quantified. To analyze whether collagen crosslinking is essential for osteogenic differentiation, we exposed MC3T3 cells and hMSCs to BAPN. In the case of MC3T3 cells, we observed a decrease in ALP expression with increasing concentrations of BAPN. In contrast, different concentrations of BAPN did not affect ALP expression of hMSCs when compared to the control. Next, we analyzed whether differences in the level of collagen crosslinking, due to the presence of BAPN, would have an effect on matrix mineralization. To analyze this, cells were grown in the presence or absence of dex and with or without BAPN. Surprisingly, when BAPN was added to the medium in combination with dex, hMSCs showed a 5-fold increase in calcium deposition. Nevertheless, BAPN alone is not able to induce mineralization. These results demonstrated that BAPN does not affect ALP expression in hMScs, in contrast to MC3T3 cells. Interestingly, BAPN treatment greatly enhances mineralization of hMSCs. These data are in line with our previous results that show that collagen does not affect ALP expression by hMSCs besides the one provided by dex (data not shown).