ECM coating modification generated by optimized decellularization process improves functional behavior of BMSCs.

Bone mesenchymal stem cells (BMSCs) have been widely applied in tissue engineering and regenerative medicine. However, small number of BMSCs and loss of stem cell characteristics after expansion in vitro limited clinical use of BMSCs. In the present study, osteoblasts were cultured to lay down extracellular matrix (ECM) and then the cells were removed (decellularization) to generate ECM coating substrates. The decellularization process was optimized to maximally remove cells and cellular components, along with integrated ECM retained which was demonstrated to be beneficial for BMSCs expansion in vitro. After decellularization, only less than 2% of residual DNA and cellular proteins were detected in TFFF-ECM (decellularized by triton X-100 (T) and three freeze/thaw cycles (FFF)), which was much less than that in TN-ECM generated by traditional decellularization method (triton X-100 (T) and NHOH (N)). Meanwhile, ECM components and structure were preserved best after decellularization by TFFF method. More ECM proteins were detected, and structure proteins (fibronectin and collagen) exhibited as classic network fibers in TFFF-ECM. Functionally, all kinds of decellularized ECM (dECM) were demonstrated to promote BMSCs proliferation and osteogenic differentiation capacity, thus maintain the stemness of BMSCs. Importantly, cells cultured on TFFF-ECM grew faster than the cells on other kinds of dECM at early stage and TFFF-ECM was beneficial to preserve stemness of BMSCs with high expression of OCT4 and NANOG when cultured in vitro. Proteomic analysis showed the proteins in ECM functioned in multiple biological activities and signaling pathways, which contributed to stemness maintenance of BMSC. Thus, the mild decellularization process optimized in this study enhanced the effectiveness of dECM for BMSCs culture in vitro and maybe further applied to BMSCs based tissue repair.