Effect Of The Combination Of WKYMVm And YIGSR Peptides On Bone Defect Repair And Its Mechanism

Background:Bone defect is a common problem in orthopedics,which is characterized by high incidence,low cure rate and many complications in clinic.In the process of bone defect healing,there are four crucial stages,including inflammation,revascularization,recruitment of cells and bone regeneration.Regulating these stages precisely plays a key role in the treatment of this disease.Among these processes,revascularization is prerequisite for bone repair.The sufficient blood flow not only ensures enough nutrition and oxygen,but also creates an optimal microenvironment for osteogenesis.Conversely,cumulative studies have implied that slow and incomplete vascularization is disadvantageous for bone defect healing.Therefore,a primary concren is to address the issue of the limited revascularization in defect region.Some recent researches show that EPCs are responsible for vessel development and(re)vascularization after bone defect,exhibiting their angiogenic ability in two populations of differentiation,known as early and late EPCs.Circulating angiogenic cells,identified as early EPCs,could not become endothelial cells but promote re(vascularization)by secreting angiogenic factors,such as vascular endothelial growth factor(VEGF)-A,platelet-derived growth factor,and insulin-like growth factor.Late EPCs,also known as endothelial outgrowth cells,have a more important function in re(vascularization).Compared with early EPCs,they represent a more typical endothelial cell type and possess highly proliferative characteristics,performing vascular repair by incorporating into newly forming vasculature.During bone regeneration,EPCs are recruited to the intervention site and prepared for neovascularization.However EPCs are such critical for neovascularization in bone regeneration,their therapeutic potential is limited because of their low migration capability.Given the importance of revascularization in tissue-engineered bone,an increasing number of studies have focused on the modulation of migration and tube formation of EPCs.In addition,enough and mature bone is of great importance to treatment of bone defect.The regeneration of bone relies on the balance between osteoclastogenensis and osteoblastognesis,which can be ascribed to osteogenic-related cells and osteoclasts.There are two different types of bone regeneration: intramembranous ossification and entochondrostosis.In the process of intramembranous ossification,osteoclast precursors and MSCs differentiate into osteoblasts directly,resulting in the formation of hard bone callus.In the latter,MSCs differentiate into chondrocytes which could secrete cartilage matrix before the formation of bones.Either way,osteoclasts will be recruited to defect sites,absorbing the redundant or necrotic bone,which creates an optimal micro-environment for bone regeneration.But in view of the inflammatory environment in bone defect sites,over-activated osteoclasts do more harm to bone regeneration than good,even leading to the failure of bone healing.Therefore,the modulation of balance between osteoclastogenesis and osteoblastogenesis is essentially far-reaching.Modulating osteoblastogenesis is relatively difficult due to its complicated mechanism.Compared with the osteoblastogenesis process,osteoclastogenesis is easier to be regulated.The regulation of osteoclastogenesis can also contribute to maintain the balance between bone regeneration and resorption.Biomimetic peptides,the peptides which are designed containing certain sequences,always have their special function by binding to activate a certain membrane protein of cells.They have many biological functions,such as promoting cell proliferation and differentiation.Now,the biomimetic peptides are researched in a majority of domains because of their advantages of low immune response,high effectiveness and inexpensiveness.WKYMVm was previously demonstrated to stimulate neovasculogenesis in animal ischemic limb by promoting EPC migration.We thus hypothesized that WKYMVm could also perform this function in bone regeneration via positively modulating EPC migration and tube formation.Additionally,WKYMVm is the agonist of FPR2.A research shows that some other agonists of FPR2 could inhibit the maturation of osteoclasts.Therefore,we supposed that WKYMVm also could negatively regulate osteoclast maturation.The pentapeptide YIGSR(Tyr-Ile-Gly-Ser-Arg)is a functional motif that binds to the laminin receptor and can promote cellular attachment.This peptide is widely used to facilitate the migration and adhesion of target cells in cell-free scaffolds.Several studies have demonstrated that YIGSR may regulate cell migration by activating laminin receptors.On the other hand,the combination of YIGSR and RGD peptides was found to enhance the migration of microvascular endothelial cells compared to that of RGD alone.Based on these facts,we hypothesized that the combination of WKYMVm and YIGSR may promote the migration and tube formation ability of EPCs more effectively for bone healing.In this study,we researched effects of WKYMVm and YIGSR on EPCs in vitro firstly.Next,we explored the combined effect and mechanism of WKYMVm and YIGSR on EPCs in vitro.And then we also observed that if WKYMVm could inhibit osteoclastogenesis or not.Lastly,femur defect module was used to research the WKYMVm combined with YIGSR in vivo.Method:1.Isolation,culture and identification of EPCs.2.Cytotoxicity of WKYMVm and YIGSR on EPCs in vitro is detected by CCK8-kits.3.The effect of WKYMVm and YIGSR on the migration ability of EPCs in vitro is detected by transwell migration assay and Scratching migration assay.4.The effect of WKYMVm and YIGSR on tube formation of EPCs in vitro is detected by Matrigel.5.The change of EPCs RNA and protein is detected by Western Blot and q RT-PCR.6.The effect of WKYMVm on osteoclastogenesis in vitro is detected by Trap-staining,Western Blot,qRT-PCR and bone resorption assay.7.Mouse femur defect model is used to detect the function of WKYMVm and YIGSR in vivo.Results:1.We isolated,cultured and identified EPCs successfully.2.WKYMVm and YIGSR have no effect on EPCs proliferation when the concentrations are under 10?M.3.WKYMVm could positively regulate the tube formation of EPCs dose-dependently,when the concentration is under 1?M.But when the concentration is at 10?M,the effect is worse than that of at 1?M.YIGSR has no effect on the tube formation ability of EPCs,when the concentration is under 0.1?M.But when the concentration is higher than 1?M,YIGSR could suppress the tube formation ability of EPCs.The effect of WKYMVm combined with YIGSR on EPCs is better than that of one peptide alone.4.Both of WKYMVm and YIGSR could promote the migration of EPCs.And the effect of combination is better than that of one peptide only.5.The combination of WKYMVm and YIGSR could promote the angiogenesis-related gene expression of ECPs.6.The combination of WKYMVm and YIGSR works throughout FPR2/PI3K/AKT/VEGF-A pathway to promote angiogenesis of EPCs.7.WKYMVm can inhibit the maturation and bone resorption ability of osteoclasts.8.WKYMVm and YIGSR could accelerate bone defect healing through recruiting EPCs in vivo.Conclusion:1.The combination of WKYMVm and YIGSR could modulate EPCs behavior positively through FPR2/PI3K/AKT/VEGF-A pathway in vitro.2.WKYMVm could suppress osteoclastogenesis and the resorption ability of osteoclast in vitro.3.The combination of WKYMVm and YIGSR could accelerate bone healing in mouse femur defect module in vivo.