| Bone is a living organ with blood circulation and has a certain degree of self-healing ability.However,large bone defects caused by the trauma,tumor,and infection still have no effective solutions.Bone tissue engineering is considered to be the most promising approach for the treatment of large bone defects.Matrix mechanics have important effects on adhesion,migration,proliferation,and differentiation of stem cells.However,the existing study of matrix mechanics mainly focus on two-dimensional(2D)or quasi-3D(quasi-3D)environment,and most methods of preparing 3D scaffolds changed the 3D microstructure with changing stiffness of scaffolds.It is difficult to investigate the effects of single matrix mechanics factor on the biological behaviors of stem cells.In this paper,we constructed a new 3D demineralized bone scaffold with different stiffness but the same microstructure by controlling of the decalcification time of decellularized bone scaffold.Firstly,the effects of demineralized bone scaffolds with different stiffness on cell compatibility and osteogentic differentiation of mesenchymal stem cells(MSCs)were investigated in vitro.The effects of demineralized bone scaffolds with different stiffness on cell infiltration,deposition of collagen fibers,and osteogenic differentiation of endogenous cells were also studied in rat subcutaneous.The main contents and results of this paper are as follows:(1)Preparation and characterization of demineralized bone scaffolds with different stiffness but same microstructure.By controlling the degree of decalcification of decellularized bone scaffolds,we successfully prepared the 3D scaffolds with different stiffness but the same microstructures.The scaffolds had the best pore size and porosity,and retained the natural bone extracellular matrix components.The stiffness of the demineralized bone scaffods was 66.06 ± 27.83 MPa(high stiffness group,high),26.90 ± 13.16 MPa(medium stiffness group,medium),and 0.67 ± 0.14 MPa(low stiffness group,low),respectively.The results of micro-computed tomography(micro-CT)demonstrated that no significant changes in the microstructure of scaffolds before and after decalcification.(2)Effects of scaffolds with different stiffness on cell compatibility and osteogenic differentiation of MSCs in vitro.Live/dead staining showed that scaffolds with different stiffness were favorable for adhesion and growth of MSCs in vitro.Alkaline phosphatase(ALP)staining,immunohistochemical staining of osteopontin(OPN)and osteocalcin(OC)showed that low stiffness group scaffold was the most favourable for promoting the osteogenic differentiation of MSCs after cultured 21 days.(3)Effects of demineralized bone scaffolds with different stiffness on cell infiltration,collagen deposition,and osteogentic differentiation of endogenous cells in rat subcutaneous.Subcutaneous implantation experiments showed that these scacffolds have different cell infiltration abilities and the cell infiltration ability of demineralized bone scaffolds are better than the decellularized bone scaffolds(control group,control).Masson staining indicated that the low stiffness group can promote the collagen formation and depositon at 4 weeks,and the collagen formation and depositon was futher enhanced at 8 weeks.Immunohistochemical staining results showed that the low stiffness group had the strongest ability to promote the expression of OPN and OC at 4 weeks.These findings demonstrated that the low stiffness group was more favorable for promoting the osteogenic differentiation of endogenous cells.In summary,the demineralized bone scaffolds with different stiffness but same microstructure can promote adhesion,growth,and osteogenic differentiation of MSCs in vitro.These scaffolds were helpful to cell infiltration,deposition of collagen fibers and osteogenic differentiation of endogenous cells in rat subcutaneous.The results of this paper provide a new method for the study of matrix mechanics in 3D scaffold materials. |
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