Screening Of A Specific Affinity Cyclic Peptide Of Rat Bone Mesenchymal Stem Cells And Its Modification On ?-tricalcium Phosphate Scaffolds

Background:Osteonecrosis of the Femeral Head(ONFH)is a disabling disease that occurs in young and middle-aged people,leading to structural changes in the femoral head,eventually resulting in femoral head collapse and osteoarthritis.At present,due to the unclear pathogenesis of ONFH and the lack of effective prevention and treatment measures,its incidence has increased year by year,which has brought a heavy burden to society.Therefore,how to treat ONFH early and effectively,prevent the collapse of the femoral head,and delay the time of total hip arthroplasty is a hotspot and a difficult point in the field of joint surgery.In recent years,several studies have shown that tissue engineering therapy using Bone Marrow Mesenchymal Stem Cells(BMSCs)combined with biological scaffolds has a good application prospect.However,the current difficulty of this method lies in the low utilization efficiency of bone marrow mesenchymal stem cells,which is mainly due to the low adhesion efficiency of bone marrow mesenchymal stem cells on biological scaffolds and poor extension morphology.Therefore,this study screened a specific affinity cyclic peptide for rat bone marrow mesenchymal stem cells by phage display technology and explored whether it promoted the adhesion,extension and proliferation of BMSCs on the cyclic peptide-modified p-tricalcium phosphate(?-TCP)scaffolds.Objective:Screening of a specific affinity cyclic peptide for rat bone marrow mesenchymal stem cells by phage display technology,verifying its affinity for BMSCs in vitro,and exploring its effect on adhesion,stretching and proliferation of BMSCs to the cyclic peptide-modified ?-tricalcium phosphate scaffolds.Methods:A SD rat bone marrow mesenchymal stem cell-specific affinity cyclic peptide was screened by phage display technology using a phage random loop heptapeptide display library(Ph.D.-C7CTM).The affinity cyclic peptide and the control polypeptide were synthesized by gene sequencing.The affinity of the affinity cyclic peptide and the control groups for BMSCs were detected by fluorescence staining and flow cytometry.The fluorescently labeled affinity cyclic peptide was further modified onto the ?-tricalcium phosphate scaffolds by natural adsorption and low temperature freeze-drying technique,and BMSCs were observed about the adhere and extension to the cyclic peptide-modified ?-tricalcium phosphate scaffold by fluorescence staining and confocal microscopy.The proliferation of BMSCs on the cyclic peptide-modified ?-tricalcium phosphate scaffolds was detected by CCK-8 technique.Results:A bone marrow mesenchymal stem cell-specific affinity loop heptapeptide(C7)was successfully screened by phage display technology.The gene was sequenced and the cyclic peptide was synthesized.Fluorescence staining and flow cytometry showed that the cyclic peptide had better affinity for BMSCs than the control groups(p<0.05).The fluorescently labeled cyclic peptide was successfully modified to the(3-tricalcium phosphate scaffold by natural adsorption and low temperature lyophilization.Fluorescence staining and confocal microscopy showed that BMSCs adhered better on the cyclic peptide-modified ?-tricalcium phosphate scaffolds than the control group,and the extension morphology was better.The CCK-8 results showed that the affinity cyclic peptide significantly promoted the proliferation of BMSCs on the ?-tricalcium phosphate scaffolds compared with the control groups(p<0.05).Conclusion:Using phage display technology,a bone marrow mesenchymal stem cells affinity cyclic peptide can be successfully screened,which has a good affinity for BMSCs.By modifying the affinity cyclic peptide to the ?-tricalcium phosphate scaffolds,it was found that the affinity cyclic peptide can promote the adhesion,extension and proliferation of BMSCs on the ?-tricalcium phosphate scaffolds,and has no obvious promoting effect on the proliferation of BMSCs.This study may provide a new idea for biofunctionalized scaffolds in bone tissue engineering to improve the utilization efficiency of BMSCs,and provide theoretical support for bone tissue engineering methods for the treatment of ONFH.