Construction Of An Artificial Bone Graft Recruiting Host Endothelial Progenitor Cells And Its Effect On Vascularization And Bone Regeneration

The artificial bone graft is a promising alternative to traditional autografts and allografts for repairing bone defects(especially the large ones)in clinic.Unfortunately,issues of poor vascularization at the implantation sites of artificial graft impede its clinical application severely.Currently,using grafts to activate endogenous signals for vascularization has been considered as an emerging strategy,receiving wide attention in the field of regenerative medicine.Increasing evidence suggests that as a response to certain stimuli(e.g.,injuries,hypoxia,ischemia,and chemotactic cytokines),endothelial progenitor cells(EPCs)locating in patients'body(host/endogenous EPCs)are able to specifically migrate(recruit)to sites releasing these signaling,and finally promote vascularization and tissue regeneration(e.g.,self-healing of fractures).The aim of this study was to immobilize both WKYMVm and YIGSR peptides onto electrospun poly-?-caprolactone(PCL)/poliglecaprone(PGC)nanofiber surface to construct an artificial bone graft which is able to recruit host EPCs,and evaluate the effect of as-prepared graft on in situ vascularization and bone regeneration.The three main contents and results of this study are listed as follows:(1)The eletrospun PCL/PGC nanofibers with both WKYMVm and YIGSR immobilized on their surface(PCL/PGC-W+Y)was constructed.PCL/PGC nanofibers was generated by electrospinning technology,followed by being immersing into solution of WKYMVm and YIGSR peptides,which were designed with the PGA-binding motif sequence to bring about immobilization of PCL/PGC with two peptides.Results from X-ray photoelectron spectroscopy(XPS)showed that after peptide immobilization,chemical element contents of nitrogen(N)significantly increased.Observation via laser scanning confocal microscopy(LSCM)illustrated that WKYMVm and YIGSR peptides were deposited on the surface of fibers uniformly,indicating the feasibility of using PGA-binding motif to combine peptides with PCL/PGC.Interestingly,such an immobilization method didn't alter the original structure of electrospun nanofibers.Further more,water contact angle measurements suggested the improved hydrophilicity after peptide combination.Results above all indicated that PCL/PGC nanofibers with WKYMVm and YIGSR immobilized on their surface was constructed successfully,laying favorable foundation for stimulating EPCs-participating vascularization.(2)The regulation effect of as-prepared PCL/PGC-W+Y on EPCs behaviors was investigated both in vitro and in vivo.PCL/PGC-W+Y was found to encourage EPCs proliferation.Besides,results from the in vitro cell adhesion assay suggested that peptide immobilization benefited adhesion of substantial EPCs in a short time.Results from the Transwell migration assay demonstrated that PCL/PGC nanofibers significantly induced EPCs migration because of peptide combination.Meanwhile,in vivo studies confirmed that PCL/PGC-W+Y grafts were able to recruit a large number of host EPCs after their implantation into the critical-sized cranial bone defects.Furthermore,enhanced tube formation by EPCs was observed on Matrigel in the presence of PCL/PGC-W+Y.These results demonstrated that PCL/PGC-W+Y possessed biological functionality of inducing EPCs adhesion,recruitment and tube formation,strongly indicating their potential of promoting vascularization and bone regeneration during bone defect repair.(3)The abilities to promote in situ vascularization and bone regeneration of PCL/PGC-W+Y were tested in vivo.Results from immunofluorescence staining showed substantial newly formed CD31~+Endomucin~+blood vessels at 4 weeks'post-implantation.Micro-CT measurements suggested that PCL/PGC-W+Y promoted bone regeneration in the critical-sized cranial bone defects.H&E staining assays confirmed the accelerated vascularization and bone formation after 8and 12 weeks of implantation and revealed contribution of the fast vascularization to bone regeneration.In summary,based on the sober fact that host EPCs can be specifically recruited to the injured,hypoxia,as well as ischemic sites to participate in vessel formation,this thesis takes advantages of stimulation effect of peptides on EPCs as well as biofunctionalization of artificial materials and yields fast in situ vascularization at implantation sites and promoted bone regeneration,offering a new forward-looking strategies of theoretical and practical value to design and develop next generation artificial bone graft.