A Cancellous Bone Matrix System With Specific Mineralisation Degrees For Mesenchymal Stem Cell Differentiation And Bone Regeneration

Objective:This study aimed to develop a method to subject decellularised cancellous bone matrix(DCBM)to ultrasound to obtain specifc demineralisation to investigate the effect of DCBM with different degrees of mineralisation on proliferation and osteogenic differentiation in bone marrow-derived mesenchymal stem cells(BMSCs)and in repairing femoral bone defects in rabbits.Methods:Cylindrical cancellous bone matrix was drilled out from fresh porcine scapulae With a bone punch.After decellularised with 1%Triton-X100 and 1%SDS solutions,bone matrix was demineralised in EDTA-2Na solution iunder ultrasound condition for 0,4,8,12 h.Then content and distribution of calcium and phosphorus,mechanical properties,cellular toxicity of obtained DCBM of different mineralisation degrees were evaluated.By planting BMSCs on DCBM,cell adhesion,spread and proliferation was observed,and after 1,2,4 weeks,intracellular osteogenic gene expression was detected by qPCR and immunofluorescence,including Col-1α,ALP,BMP-2.20 healthy female New Zealand white rabbits were selected and about 4mm defects were drilled on epicondyles of both sides,and 4 groups of DCBM were implanted into defect.Athletic ability and surgery area condition were evaluated periodically and after implantation for 2 and 4 weeks,rabbits were euthanised and distal femurs were dissected out for micro-CT scan and histological evaluation.By analysing bone defect repair and vascularisation comprehensively,the effects that DCBM of different mineralisation degrees had on bone healing was evaluated.Results:1.Demineralising DCBM with EDTA-2Na solution under ultrasound condition was steady and effective,the concentration of calciun decreased gradually with demineralisation process going and by 12 h,cancellous bone matrix was demineralised completely(p<0.01).2.Degenerated mineralisation degree of DCBM would cause decreased content and scattered distribution of calcium and phosphorus.Atomic force microscopic analysis indicated that Young’s modulus,adhesive force were degraded,while porosity increased(p<0.01).Besides,BMP-2 reservation in materials was exposed more.3.Adhesion and spread ability of cells on partially demineralised DCBM(demineralised for 4 and 8 h)had no significant difference compared to undemineralised DCBM,while cell behavior was degraded on completely demineralised DCBM.4.On partially demineralised DCBM,BMSCs expressed higher level of osteogenic protein,including Col-la,ALP,BMP-2,than undemineralised or completely demineralised DCBM(p<0.05).5.Partially demineralised DCBM behaved better in promoting newly-formed trabeculae and vessels than undemineralised or completely demineralised DCBM.Conclusion:It is steady to obtain gradient demineralised DCBM system using condition of EDTA-2Na solution and ultrasound,and we found partially demineralised DCBM behaved better in promoting cell adhesion,spread,proliferation and differentiation,and bone repair than undemineralised or completely demineralised DCBM,and this mechaxnim may be connected to that concentration of calcium regulating the expression of BMP-2.