Study On Multilayer Film Modified 3D Printing Apatite Composites Scaffolds Loaded With Dual Growth Factors For Repairing Rabbit Skull Defect

Bone tissue damage caused by infection,aging,bone tumor resection and trauma often needs implantation of bone grafts for defect repair.Due to many drawingbacks of autologous and xenogenic bone,such as the shortage of source and immune rejection,degradable scaffolds with inter-conntected porosity play important roles in the applications of bone tissue engineering.In this study,by using gelatin(Gel)and hydroxyapatite(HAP)as raw materials,HAP composite scaffolds with inter-connected pores were successfully fabricated by using 3D printing technology.Moreover,sodium hyaluronate/chitosan(SH/CHI)polyelectrolyte multilayer film was coated on the surface of HAP composite scaffolds via layer-by-layer self-assembly.The results of fluorescence microscopy,scanning electron microscopy(SEM)and thermal analysis(TG)demonstrated that the SH/CHI multilayer film has successfully coated on the surface of HAP scaffolds.In addition,the structure of SH/CHI multilayer film enhanced the mechanical properties and mineralization ability of HAP composite scaffolds.The degradation experiment showed SH/CHI multilayer film had excellent stability and could reduce the degradation rate of HAP composite scaffolds in PBS and SBF.HAP composite scaffolds can be loaded with drugs and growth factors by the dipping method.The controlled drug release behavior of HAP composite scaffolds was investigated by ultalizing small molecule rhodamin b(RHB)and macromolecule bovine serum albumin(BSA)as model drugs.The releasing behavior of HAP composite scaffolds is pH-sensitive.Subsequently,the bone morphologenetic protein-2(BMP-2)and vascular endothelial growth factor(VEGF)were loaded on the scaffolds,respectively.Dual-loading of BMP-2 and VEGF was carried out at the same time.The result suggested that the SH/CHI multilayer film can slow down the release of BMP-2 and VEGF.According to the results of cell behavior on HAP scaffolds,SH/CHI multilayer film modified HAP composite scaffolds loaded with BMP-2 and VEGF could promote the cytocompatibility of scaffolds,including increasing the proliferation rate of MC3T3-E1,enhancing the expression of ALP activity and osteogenic related genes(Runx-2,OPN,Osterix and Col Ⅰ),which shows great bone tissue engineering potential.The rabbit skull defect with a diameter of 15 mm was used to evaluate the osteogenic effect of HAP composite scaffolds.Tissue samples of rabbit skull defect areas were collected at 4,8,12 weeks and characterized.The results of micro-CT analysis,HE,Masson and immunohistochmical staining showed SH/CHI multilayer film modified HAP composite scaffolds loaded with BMP-2 and VEGF could significantly improve the quality of bone regeneration,which are potential promise in bone tissue engineering.