Surface Modification Of Silk Fibrin Electrospun Scaffold Based On Functional Peptide Modification And Its Application In Bone Tissue Repair

ObjectiveOral and maxillofacial bone defects caused by tumors,trauma and other factors will seriously affect the language,eating and other physiological functions.Traditional repair of bone defect with autogenous bone graft will bring secondary damage,insufficient bone mass,etc.With the rapid development of bone tissue engineering,the applications of the artificial bone substitute materials for the repair and regeneration of bone tissue defects have been extensively and deeply studied.Good bone substitute materials should not only have good biocompatibility,but also have a three-dimensional fiber network structure that can mimic the natural extracellular matrix.Therefore,this study took full advantage of the advantages of silk fibroin(SF)natural polymer materials and electrospinning technology to prepare silk fibroin electrospun scaffolds.The surface of the scaffold was functionally modified by electrostatic self-assembly technique and polydopamine(PDA)coating technology.It is hoped to provide a new composite scaffold material for the repair of maxillofacial bone defect and provide some theoretical basis for surface modification of scaffold material.Materials and MethodsIn this experiment,the SF electrospun scaffolds were prepared and functionalized with different treatment methods.1)Firstly,graphene oxide(GO)was prepared and BMP-2 peptide(P24)was grafted onto the surface of graphene oxide(GO-P24)by chemical grafting,and the properties of GO-P24 was characterized by using transmission electron microscopy(TEM)and atomic force microscopy(AFM).Then the negatively charged GO-P24 were loaded onto the chitosan(CS)coated SF electrospun scaffolds by electrostatic self-assembly to constitute composite scaffolds(SF-CS-GO-P24).The effects of SFCS-GO-P24 scaffolds on adhesion,proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)were investigated at the cellular and molecular levels by Live/Dead staining,CCK-8 detection,cytoskeleton staining,q RTPCR and immunofluorescence staining.2)Firstly,polydopamine(PDA)coating was formed on the surface of SF scaffolds by oxidative self-polymerization of dopamine,and then E7 peptide was grafted onto the surface of scaffolds(SF-PDA-E7)to form a composite scaffold through the chemical activity of PDA.The surface characteristics of the prepared SF-PDA-E7 scaffolds were characterized by SEM,XPS,hydrophilicity test,etc.And then,the effect of SF-PDA-E7 scaffolds on adhesion,proliferation and osteogenic differentiation of BMSCs were explored by using Live/Dead staining,MTT assay,q RT-PCR and alkaline phosphatase(ALP)semi-quantitative detection experiments.In addition,the recruitment of BMSCs by SF-PDA-E7 scaffolds was verified both in vitro and in vivo animal experiments,we also demonstrated the main mechanism of the biological function of E7 peptide through molecular biology testing.Finally,we implanted the SFPDA-E7 scaffolds into the rat skull defect model and evaluated the ability of the composite scaffolds for promoting new bone formation in vivo by Micro-CT analysis,H&E staining and Masson staining histological analysis.Results1)The preparation of GO-P24 and the loading on the surface of SF electrospun scaffolds by electrostatic self-assembly were confirmed by TEM and SEM measurements.In vitro Live/Dead staining,CCK-8 assay,q RT-PCR and immunofluorescence results showed that the good biocompatibility of SF-CS-GO-P24 scaffolds and their promoting effect on osteogenic differentiation of BMSCs.2)Firstly,the SEM,XPS,MTT assay and q RT-PCR showed that SF-PDA-E7 scaffolds could not only promote adhesion and proliferation of BMSCs and also can promote the up-regu Lation of osteogenic related genes in the osteogenic induction environment.In addition,SF-PDA-E7 scaffolds were able to recruit BMSCs in situ to migrate and grow into the scaffolds under both in vitro and in vivo.Western Blot,immunofluorescence and other molecular biological tests confirmed that E7 peptide could increase the protein expression of SDF-1? and CXCR4,and the phosphorylation levels of p38,ERK and Akt signaling pathway-related proteins.Finally,the Micro-CT and histological analysis demonstrated that the SF-PDA-E7 scaffolds could significantly promote new bone formation and repair bone defects in vivo.In addition,ConclusionsIn this study,SF scaffolds were prepared by electrospinning technique,and functionalized modification of the surface of SF electrospun scaffolds by electrostatic self-assembly technique and PDA coating technology.The prepared composite scaffolds had good biocompatibility and osteogenic induction activity,which could provide a biomimetic microenvironment suitable for survival and play an active role in promoting the repair and regeneration of bone tissue defects.It is expected to become a biomaterial with broad application prospects in the field of bone tissue engineering.