Effects Of Hypoxia On The Proliferation,Infiltration And Osteogenic Differentiation Of BMSCs On Demineralized Bone Scaffolds With Different Stiffness

Trauma,infection and cancer et al.may cause large bone defects.Bone defects are difficult to repair and treat,and bring patients great pain.As a new bone defect treatment strategy,bone tissue engineering methods bring new hope for patients.Based on the bone tissue engineering,researchers applied the three dimension?3D?decellulaized bone scaffolds into bone repair and showed some positive effects.Previous studies in our lab demonstrated that biomechanical modification of the 3D scaffolds could further accelerate the osteogenic differentiation of the mesenchymal stem cells?MSCs?.Since the seed cells in the early stage of bone repair will face a hypoxic microenvironment,studying matrix mechanics to regulate the biological behavior of seed cells in a hypoxic environment can simulate the matrix microenvironment in the early stage of bone repair.In order to investigate the effects of hypoxia on the infiltration,proliferation and osteogenic differentiation of MSCs on decalcified bone scaffolds with different stiffness,by controlling the decalcification time,we first prepared three 3D decalcified bone scaffolds with different stiffness but consistent microstructure?pore size and porosity?,among them,high stiffness was 66.06±27.83 MPa,medium stiffness was 26.90±13.16 MPa and low stiffness was 0.67±0.14 MPa.Then the viability,proliferation,infiltration and osteogenic differentiation of MSCs seeded into the 3D demineralized bone scaffold with changing stiffness were systematically detected in the hypoxic condition simulated by 100?M CoCl₂.The following main results were gotten:?1?Effects of hypoxia on the cell viability and proliferation of MSCs seeded on demineralized bone scaffolds with different stiffness.Theresultsoflive/deadstainingand3-?4,5-dimethylthiazol-2-yl?-5-?3-carboxymethoxyphenyl?-2-?4-sulfophenyl?-2H-tetrazo lium?MTS?analysis showed that cell survival and proliferation of MSCs on demineralized scaffolds with same stiffness was inhibited by the hypoxia condition simulated by 100?M CoCl₂.But it had no significant effect on demineralized scaffolds with different stiffness under hypoxic and normoxic conditions.?2?Effects of hypoxia on the adhesion,infiltration and extracellular matrix?ECM?deposition of MSCs seeded in demineralized bone scaffolds with different stiffnessHematoxylin eosin?HE?staining showed that the infiltration capacity of MSCs in low-stiffness demineralized bone scaffolds was significantly increased compared with the other groups under normoxic and hypoxic conditions,but there was no significant difference in ECM deposition in all demineralized bone scaffolds.Masson staining showed that the collagen expression and deposition capacity of MSCs increased significantly with the increase of scaffolds stiffness after cultured by normoxia and hypoxia.?3?Effects of hypoxia on the osteogenic differentiation of MSCs seeded in demineralized bone scaffolds with different stiffness.Alkaline phosphatase?ALP?staining and immunohistochemical staining of osteopontin?OPN?and osteocalcin?OC?showed that osteogenic differentiation of MSCs was also inhibited by hypoxia.However,the osteogenic differentiation ability of MSCs on low-stiffness demineralized bone scaffolds was higher than other groups in both normoxic and hypoxic conditions.?4?Effects of hypoxia on the expression of hypoxia inducible factor 1??HIF-1??and vascular endothelial growth factor?VEGF?of MSCs seeded in demineralized bone scaffolds with different stiffness.This article also confirmed that the expression of HIF-1?in MSCs was significantly raised in hypoxic microenvironment mimicked by 100?M cobalt chloride,and the expression of HIF-1?and VEGF in MSCs on low-rigid demineralized bone scaffolds was significantly enhanced.Hypoxic culture inhibited the expression of VEGF in MSCs on demineralized bone scaffold with same matrix stiffness.In summary,the biological property of MSCs implanted on the demineralized bone scaffolds with different stiffness but same microstructure?pore size and porosity?was systematically analyzed under 100?M CoCl₂ simulated hypoxic condition.It was found the low stiffness matrix was more conducive for MSCs viability,proliferation,infiltration and osteogenic differentiation under hypoxic condition.