Regulatory Effects Of Demineralized Bone Scaffolds With Different Matrix Stiffness On The Repair Of Femoral Condylar Defects In Rabbit

Bone grafting ranks second in the number of various human organ transplantations.Besides tumor resection,pathological deformation,congenital deformation,sports injury,and infection all may cause bone defect,and even lead to osteoarthritis or other diseases,which can damage the normal musculoskeletal system of the human body.Although great progress has been made in bone repair in recent years,the real morphological and functional reconstruction of bone defects still faces enormous challenges.The current approaches still encounter obstacles such as limited sources,high cost,rejection response and difficulties in clinical transformation.Demineralized bone matrix(DBM)scaffold is considered as an appropriate bone grafting material for bone repair and regeneration for its good properties of osteoinductive,osteoconductive and osteogenesis.And it can be biodegradable without any toxic effects.Besides,the natural porous structure of the DBM scaffold provides sufficient surface area and internal space for the secretion of the extracellular matrix(ECM).A large number of studies have shown that matrix stiffness not only plays an important role in adhesion,proliferation,migration and differentiation of stem cells,but also regulates repair and regeneration of bone defect.In the previous study of our lab,three kinds of DBM scaffolds(?10 mm × 5 mm)with same three dimensional(3D)microstructure but different stiffness(low(0.67 ± 0.14 MPa),medium(26.90 ± 13.16 MPa)and high(66.06 ± 27.83 MPa))were successfully constructed by controlling the decalcification time.The results of the following in vitro experiments confirmed that the scaffolds could maintain the adhesion and growth of rat bone marrow mesenchymal stem cells(MSCs)and promote their osteogenic differentiation.The subcutaneous experiments indicated that endogenous cells were well-infiltrated on the scaffolds,and vessel-like structures appeared in the scaffolds with low stiffness.However,the in vivo repair effect of these scaffolds and its mechanisms are still unclear.To this end,this article further investigated the effect of 3D DBM scaffolds with different stiffness on bone repair and its possible mechanisms in vivo by establish rabbit femoral condyle bone defect models.The main contents and results are as follows:(1)DBM scaffolds with different stiffness but same microstructure were fabricated by controlling the decalcification time.The scaffolds were cylindrical with a diameter of 5 mm and a height of 5 mm(?5 mm × 5 mm).Compression modulus of the scaffolds with low stiffness was 0.68 ± 0.01 MPa,while medium and high stiffness contributed to 21.32 ± 1.74 MPa and 66.80 ± 21.60 MPa respectively.The compression modulus of each stiffness group of this size is consistent with the scaffolds with a size of ?10 mm × 5 mm that used in the cellular and subcutaneous experiments.(2)The defect model with the size of ? 5 mm × 5 mm was successfully established in the lateral condyle of the rabbit femur,and the corresponding scaffold was implanted.The fibrogenesis on the surface of the defect site,the exposed area of the implanted scaffold,the irregular hyperplastic bone in defect surface,the covered smooth and transparent soft tissue and apparent inflammation were analyzed by gross observation.X-ray and micro computed tomography(?-CT)were carried out to evaluate the fusion between the host bone and the scaffold.The results demonstrated that the bone repair and osseointegration effect of low stiffness group is slightly better than the medium stiffness group,and significantly better than the high stiffness group and control group.(3)H-E staining and Masson trichrome staining analysis of samples was performed after fully decalcification.The results indicated that the low stiffness group showed better osseointegration effect and lower inflammatory response than other groups in the early stage of repair,and obvious vascular structures could be observed in the low stiffness group.At 3 months,there was significantly enhanced angiogenesis and mature bone distribution in the defect repair area of the low stiffness group compared with the other groups.(4)The expression level and expression position of stromal cell-derived factor-1?/CXC chemokine receptor 4(SDF-1?/CXCR4)were detected by immunohistochemistry.The results showed that the expression of SDF-1?/CXCR4 was the lowest in the Low group(0.67 ± 0.14 MPa)with the best repair effect.And higher expression levels was found in the High group(66.80 ± 21.60 MPa)and the Control group(233.92 ± 34.02 MPa)with poor repair effect.In summary,the study proved that the 3D DBM scaffold in low stiffness(0.68 ± 0.01 MPa)could promote bone healing and integration in vivo.The SDF-1?/CXCR4 pathway can improve in situ osteogenic differentiation and participate in the bone repair process of rabbit femoral condyle defects.The findings contribute to further understand of the role of 3D matrix stiffness in bone repair and its mechanism.