Controlled Release Of BMP-2 From Collagen-mimetic Peptide Modified Silk Fibroin-nanohydroxyapatite Scaffold For Bone Regeneration

[Objective]To establish a new type ofcomposite scaffoldof silk fibroin / nano hydroxyapatite combined with bone morphogenetic protein 2 loaded on microspheres andGFOGER,a collagen-mimetic peptide.And to study the scaffold about the effect of promoting celladhesion,proliferation and osteogenic differentiation of bone marrow mesenchymal stem cell and whether promotingthe repairof bone defect.[Methods]The silk fibroin(SF)microspheres were prepared by the micro-fluid method,and the bone morphogenetic protein-2(BMP-2)was loaded onto the microspheres by physical adsorption method.Nano hydroxyapatite(nHAP)was dispersed homogeneouslyinto SF solution by using carboxylated cellulose nanofibrils(cCNF),and then the microspheres were added to the solution and then mixed to obtain the initial scaffold.Polypeptide GFOGER was grafted onto the scaffolds by a bifunctional crosslinking agent Sulfo-Lc-SPDP.Composite scaffolds loading two biomolecules was characterizedby ELISA,X-ray photoelectron spectroscopy,scanning electron microscopy and Fourier tranSForm infrared spectroscopy.The ability of promoting cell adhesion,proliferation,migration and osteogenic differentiation was examined by CCK-8 assay,cell migration assay,ALP and alizarin red staining,and RT-qPCR assay.Through the establishment of the rat model of skull defect and implanting scaffolds into defect areas,the skull specimens were collected after 8 weeks and 12 weeks.And Micro-CT reconstruction and analysis and histological section staining was taken to assess the ability of composite scaffolds to promote bone regeneration in vivo.[Results]The size of the prepared microspheres was uniform with a diameter around 4.2?m,and the pore size of the composite scaffold was uniformtoo and was about 51.19?m.The sustained release of BMP-2was more than 28 days.The results of experiments in vitro showed that the scaffolds loaded with two factors had the best ability to promote cell adhesion,proliferation and osteogenesis,but no obvious ability to promote migration.Experiments in vivo showed that the scaffolds loaded with two factors could brige the defect areas at 12 weeks,while the other groups couldn't.[Conclusion] The microspheres prepared by the micro fluidic method have better property for sustained release,and can improve the biocompatibility of the composite scaffold and provide a better osteogenic environment for the cell.GFOGER can be succesSFully grafted to scaffolds by Sulfo-Lc-SPDP and its original efficacy retained.The composite scaffold loading two biomolecules has the best ability to promote cell adhesion,proliferation and osteogenic differentiationand can complete the repair of skull defects in rats at 12 weeks.This newcomposite scaffold provides a prospect for the bone tissue engineering and repair of clinical bone defects.