Wnt10b Overexpressing Umbilical Cord Mesenchymal Stem Cells Promote Bone Regeneration Partially By Ameliorating Osteogenesis And Angiogenesis Coupling

Of more than 5 million fractures that occur each year in China,resulting in 3-7%of delayed or nonunion healing [1].These failures are a substantial physical,medical,and financial burden for affected individuals and our society.Therefore,strategies for promoting bone regeneration is a crucial issue for both surgeons and basic scientists.A large number of studies have found that Wnt signaling pathway plays an important role in the process of bone formation and bone regeneration.The Wnt10 b can promote the osteogenic differentiation and inhibit adipogenic differentiation of mesenchymal stem cells.In Wnt10 b overexpressed transgenic mice(FABP-Wnt10 b mouse),bone mass in long bone was tripled [2].In addition,the change of serum Wnt protein content is highly related to the progress of fracture healing [3],while the enhancement of Wnt signaling pathway can enhance the proliferation ability of periosteal stem cells [4],and promote the fracture healing of osteoporosis rats [5].Meanwhile,Wnt signaling pathway is important in angiogenesis and homeostasis [6].Activation of Wnt / β-Catenin pathway can promote the expression of VE-Cadherin to enhance the formation of new blood vessels [7].Therefore,activation of local Wnt signal may enhance the function of periosteal stem cells and local angiogenesis at the same time,thus inducing bone regeneration.Tissue-specific mesenchymal stem cells share self-renewal and multidifferentiation capacity,so they are a good source of cells for transplantation to regenerate tissue [8].In recently,human umbilical cord mesenchymal stem cells(HUCMSCs)have become the first choice of treatment cells in regenerative medicine research because of its advantages of non-invasive material collection,convenient transformation and application [9].HUCMSCs coming from the umbilical cord are reported to be highly proliferative,they represent a noncontroversial source of tissuespecific stem cells that are noninvasively collected [10].Therefore,HUCMSCs are probably more suitable for clinical application,such as heart disease [11],Alzheimer’s disease [12] and osteoarthritis [13].Exosomes from HUCMSCs rat long bone fracture healing [14] possibly via wnt signaling pathway [15].However,in a vitro study HUCMSCs showed relatively weak osteogenic differentiation ability compared to bone marrow MSCs(BMSCs)[16].A clinical study demonstrated that HUCMSCs combined with BMPs could effectively promote femoral shaft fracture healing [17].Thus,it is of great interest to improve the osteogenic differentiation ability of HUCMSCs to obtain robust bone healing effects.In this study,we generated HUCMSCsWnt10 b,which were transduced with a lentivirus that enabled overexpression of the Wnt10 b ligand,and we hypothesized that these cells possess an improved bone regeneration ability.Even though HUCMSCsWnt10 b possess a robust CFU-F and osteogenic differentiation ability,they have a limited adipogenic differentiation capacity in vitro.In a critical size rat calvarial defect model,treatment with HUCMSCsWnt10 b can significantly accelerate the bone healing process s and induce increased VEGF-A expression and formation of CD31+blood vessels;yet these effects may be greatly diminished by treatment with IWR-1,which is a Wnt signaling pathway inhibitor.A tube formation assay was performed and showed that human umbilical vein endothelial cells(HUVECs)cultured in conditioned media from HUCMSCsWnt10 b exhibited more angiogenesis than HUVECs cultured in empty vector transduced HUCMSC(HUCMSCsEmp)conditioned media that is abolished by the angiogenesis inhibitor : SU5416.These findings suggest that HUCMSCsWnt10 b can promote bone regeneration partially through enhanced osteogenesis and VEGF-mediated angiogenesis.In reality,however,long bone fracture is more common than the calvarial defect,and it has different mechanism to be healed.Therefore,we made a preliminary research about it.A rat femoral fracture model was made and treated with HUCMSCsWnt10 b on the fracture point.The result shows that HUCMSCsWnt10 b can also promote the long bone fracture healing,but whether it has same mechanism with calvarial defect repair is still being studied.