Three-dimensional Spheroid Culture Combined with Hypoxic Preconditioning Can Promote the Proliferation and Osteogenic Differetiation of BMSCs in Low-oxygen Environment in Vitro

Abstract

Background: Presently, it is difficult to use BMSCs in bone regeneration because the cells are severely weakened or even die after transplantation into the body after in vitro culture. This is due to the difference between the living environment of cells cultured in vitro and in vivo. This study explored a fast and efficient method for developing BMSCs with stronger survival and osteogenesis ability in a harsh environment through hypoxic preconditioning and three-dimensional (3D) culture to transform BMSCs into cell therapy. Methods: BMSCs were treated with various hypoxic preconditioning processes in vitro, including sustained hypoxia, intermittent hypoxia, and sustained normoxia, in planar culture and 3D spheroid culture. Cell Counting Kit-8 assay, apoptosis determination assay, and live-dead staining were used to evaluate the proliferation and survival abilities of BMSCs. Alkaline phosphatase, alizarin red staining, and osteogenic-related proteins were used to assess the osteogenic differentiation abilities of BMSCs. Meanwhile, the proliferation, survival, and osteogenesis abilities of BMSCs between planar culture and 3D spheroid culture were compared. Results: BMSCs showed stronger proliferation and survival abilities after intermittent hypoxic preconditioning and 3D spheroid culture. Moreover, cells cultured based on intermittent hypoxic preconditioning and 3D spheroid culture showed less apoptosis and higher osteogenic differentiation abilities than those cultured via conventional treatment in the hypoxic microenvironment simulated by matrix gel placed in a three-gas incubator, including alkaline phosphatase activity, cell calcium content, and expression of osteogenic-related molecules. Conclusions: Intermittent hypoxic preconditioning and 3D spheroids culture at specific stages of BMSCs significantly increase the proliferation, survival, and osteogenic differentiation abilities of BMSCs, thus improving the application potential of seed cells in bone regeneration.