Research Of Application And Mechanism Of CBD-IKVAV-cRGD Peptide In Selective Cell Retention Technology

Background and purpose:The therapy of massive bone defects is a difficult problem in the field of orthopedic research and medical service.The key problem is that it is difficult to obtain sufficient high-activity bone repair materials.Selective cell retention(SCR)is a useful strategy for bone tissue engineering.Improving the adhesion efficiency of mesenchyma stem cells(MSCs)to matrix is the key to enhance the osteogenic activity of bone grafts constructed by SCR.Demineralized bone matrix(DBM)is a commonly used SCR enrichment material in clinic,which could improve the adhesion and osteoinductivity to cells through adding additional adhesion sites,positive charge or surface modification.Therefore,we designed a peptide(CBD-IKVAV-cRGD)composing of collagen-binding domain(CBD)and IKVAV-cRGD domain to modify collagen-based DBM,which contains multiple integrin ligands and binds to the subunit of integrin expressed in MSCs.It could enhance the specific adhesion of cells to scaffolds and osteogenic induction ability.Methods:(1)We designed CBD-IKVAV-cRGD peptides in the case group,CBD-IKVAV and CBD-cRGD peptides in the control group,respectively.Among the three peptides,the IKVAV and cRGD peptides could specifically adhere to MSCs and have osteogenic induction ability,and the CBD could bind to DBM.The three groups of peptides were prepared,purified and validated by solid-state synthesis and HPLC,and then modified into DBM.The validity and stability were verified by laser confocal and environmental scanning electron microscopy(ESEM).(2)The adhesion and osteogenic induction ability of CBD-IKVAV-cRGD peptides to MSCs were tested by shock and centrifugal adhesion test,Western Blot,CCK-8,qPCR and alkaline phosphatase activity detection.(3)The adhesion and osteogenesis ability of CBD-IKVAV-cRGD scaffolds to nucleated cells of bone marrow were detected by flow cytometry,osteogenesis test of parafemoral defect in nude mice,and micro-CT.Results:(1)High purity CBD-IKVAV,CBD-cRGD,CBD-IKVAV-cRGD peptides were successfully prepared,and the morphological characteristics of the scaffolds were detected by ESEM.The results show that the level of surface microroughness of DBM/CBD-IKVAV-cRGD,DBM/CBD-IKVAV,and DBM/CBD-cRGD has been more compared with that of DBM alone.The effect of modified DBM of three groups of peptides with FITC fluorescence was verified by laser confocal microscopy,which remained stable on the surface of DBM after high-intensity washing using PBS buffer.(2)Cell centrifugal and concussion adhesion test showed that CBD-IKVAV-cRGD peptide had stronger adhesion ability to MSCs compared with CBD-IKVAV and CBD-cRGD modified groups.CCK-8 experiment confirmed that DBM/CBD-IKVAV-cRGD scaffolds retained more MSCs after SCR compared with DBM/CBD-IKVAV and DBM/CBD-cRGD,and cell density reached its peak at the earlier time.The expression levels of some biomarkers of osteoblastic in MSCs,containing Runx 2,ALP,OPN and OCN,in DBM/CBD-IKVAV-cRGD group were higher than those in control group by Western Blot,qPCR and alkaline phosphatase activity detection kit after induction with MSCs cell suspension to simulate SCR.Additional,higher levels of phosphorylation of FAK and ERK1/2 were detected.These results indicated that CBD-IKVAV-cRGD peptide has better ability to induce osteogenic differentiation of MSCs.(3)Flow cytometry showed that the enrichment efficiency of nuclear cells,CD90+/CD105+,CD271+ cells in DBM/CBD-IKVAV-cRGD group was significantly higher than that in control group using healthy adult bone marrow.DBM/CBD-IKVAV-cRGD scaffolds had stronger osteogenesis ability compared with DBM scaffolds in nude mice with parafemoral defect.Conclusion:We have successfully designed and synthesized the CBD-IKVAV-cRGD peptides,which contained the ligands of integrin subunit expressed in MSCs,and could be used for surface modification of DBM scaffold materials.In vitro experiments,the peptides could enhance the adhesion ability of MSCs to scaffold materials and osteogenic differentiation induction activity,and promote the selective retention of MSCs in scaffold materials and promote their proliferation and osteogenesis.The osteogenesis was also confirmed in vivo experiments.The study indicated that the CBD-IKVAV-cRGD artificial peptides provide a novel idea for enhancing the specific adhesion of cells to scaffolds,and show the application prospects of developing new bioactive bone repair materials.