The Osteogensis And Vascularization Of Bioactive Calcined Bone Scaffold

OBJECIVEThe treatment of large segment or large bone defect caused by trauma,tuberculosis and tumor is a common problem in orthopedics clinic,since improper treatment often results in delayed or nonunion of the defect site and local dysfunction.In this study,we used the hydrothermal calcination method in the Mg2+?PO43-and SO42-multiphase system to transform the bovine cancellous bone into macroporous scaffold.The water hydrothermal reaction could generate nano-crystal micro structure on materials surface with retaining the 3D interconnected porous structure,as well as the ability of releasing bioactive ions.A series of in vitro and in vivo tests were conducted to comprehensively evaluate the material science and biological characteristics in order to provide valuable bone substitution materials or tissue engineering scaffolds for clinical application.METHODS1.Hydrothermal calcination method was used to transform bovine cancellous bone into bioactive macroporous scaffold.The chemical composition,elemental energy spectrum distribution,porosity,pore size and pore connectivity of the scaffold were characterized.The mechanical properties and surface micro structure were observed and evaluated.The degradation and release of bioactive ions were analyzed.2.The scaffold was co-cultured with cells in vitro,then the survival,proliferation and focal adhesion formation of cells were evaluated by the CCK-8,cytoskeleton fluorescence staining,focal adhesion staining,scanning electron microscopy and other techniques on the scaffold surface.The angiogenesis on Matrigel and ELISA test were used to evaluate the effect of the released ions on HUVECs.The differentiation ability of MC3T3-E1 pre-osteoblasts was observed to indicate the osteoblastic activity of the scaffold in vitro.3.A mouse subcutaneous implantation model was established to evaluate the compatibility of bioactive scaffold in vivo by histological analysis.An animal model of radial segmental bone defect in rabbits was established,and the vascularization and bone repair in the defect were evaluated by radiology,micro-CT,histology(decalcified and undecalcified bone tissue sections),immunohistochemistry of related proteins,fluorescence labeling and other techniques.RESULTS1.The mechanical properties of scaffold materials have been improved after hydrothermal calcining,and new components of Ca(3-n)Mgn(PO4)2 and CaSO4 were generated by hydrothermal reaction,which giving artificial bone scaffolds the characteristics of degradability and releasing active Mg2+,Ca2+and PO43-.The porous biological scaffolds still maintained the natural pore structure of cancellous bone.The porosity is 82.4±4.1%,the porosity connectivity is 100%and the pore diameter is 370.3±37.6?m.After the hydrothermal reaction,the unique nano-crystal micro structure was generated on the surface of the artificial bone scaffold material.2.It was found that the surface nano-crystal micro structure effectively promoted the focal adhesion formation of MC3T3-E1 pre-osteoblasts in vitro,thus promoting their adhesion,survival and proliferation on the scaffold surface.At the same time,bioactive ions released by the scaffold could effectively enhance the osteogenesis and vascularization of cells in vitro.3.The results of the mouse subcutaneous implantation model indicated that the calcined scaffold material had excellent histological compatibility in vivo.The results of rabbit radial segmental bone defect model indicated that the new bone formation,the level of vascularization and bone incorporation were significantly enhanced after the treatment of hydrothermal calcining.CONCLUSIONSThe hydrothermal calcination method was used to transform the bovine cancellous bone into a novel bioactive macroporous scaffold in the multiphase system containing Mg2+,PO43-and and SO42-,which retained its natural 3D interconnected porous structure and endowed it with new characteristics(biodegradable and bioactive ions release).The in vitro and in vivo tests showed that calcined artificial bone scaffolds can significantly promote the adhesion,survival and proliferation of cells on the scaffold surface.The scaffold also showed great ability to promote cells osteogenesis and vascularized activity.The scaffold had excellent biocompatibility and can effectively promote the repair of bone defects in vivo.These results indicated that the bioactive scaffolds may be new bone substitution materials or tissue engineering scaffold that have a good prospect in clinical application.