Effects of hypoxia on the biological behavior of MSCs seeded in demineralized bone scaffolds with different stiffness

Matrix stiffness has been demonstrated in many studies to adjust the biological behaviors of mesenchymal stem cells (MSCs). However, in the initial phase of bone restoration, MSCs will encounter a hypoxic microenvironment. Studying the connection existing between the matrix stiffness and biological behavior of MSCs under hypoxic condition can better simulate the microenvironment at the prime period of bone repairment. In this work, three-dimensional (3D) decalcified bone scaffolds with diverse stiffness [high stiffness (66.06 ± 27.83) MPa, medium stiffness (26.90 ± 13.16) MPa, and low stiffness (0.67 ± 0.14) MPa] but same microstructure have been prepared by controlling decalcification time. In addition, the decellularized bone scaffold was regard as control group and its stiffness was (230.93 ± 72.65) MPa. The viability, proliferation, infiltration, and osteogenic differentiation of MSCs seeded into these 3D demineralized bone scaffolds were systematically investigated under 100 μM CoCl₂-simulated hypoxic and normoxic environments. The results showed that the viability, proliferation, and extracellular matrix (ECM) secretion of MSCs had no significant difference on scaffolds with diverse stiffness, but the degree of collagen deposition of MSCs gradually increased with the increase of scaffold stiffness both under normoxia and hypoxia. Compared to normoxia, the viability, proliferation, ECM secretion, vascular endothelial growth factor (VEGF) expression, and osteogenesis of MSCs on the scaffolds with the same stiffness were evidently inhibited by hypoxia. Additionally, under hypoxic condition, the expression of VEGF and hypoxia inducible factor 1α (HIF-1α) in MSCs on the low stiffness scaffold was markedly increased comparing to those on other groups. In summary, we found that the low-stiffness scaffold can improved the proliferation and osteogenic differentiation of MSCs under hypoxic environment, which may help to explore efficient methods for bone defect repairing.