Epoxyeicosatrienoic Acids Promotes Bone Regeneration

Part 1:14,15-Epoxyeicosatrienoic Acid Promotes Bone Regeneration of Calvarial DefectObjective:To evaluate the effect of 14,15 epoxyeicosatrienoic acid(14,15-EET)on the healing of calvarial defect in C57BL/6 mice.Materials and Methods.Two circular bone defects(5 mm in diameter)were created on the calvarial bones of C57BL/6 mice.The one side of the bone defects were determined randomly as experimental sides,the other side as controls.14,15-EET(10 ?M,experimental side,n=10)or PBS(control sides,n =10)was administered by subcutaneous injection in calvarial defect area every other day from third post-operative day for two weeks.Mice were sacrificed at 8th post-operative week.Bone regeneration in the calvarial defect area was assessed with CBCT.HE and Masson's trichrome stain were performed to assess the newly formed bone and connective tissues.Immunostaining with anti-CD31 were performed to analysis the expression of CD31.Results:Compared with the control sides,the area of bone defect in the experimental sides treated with 14,15-EET was decreased at 8th post-operative week(p<0.05).14,15-EET treatment increased bone formation and blood vessels in the experimental sides compared with control sides at 8th post-operative week(p<0.05).In addition,The results of Masson's trichrome stain suggested that there were more new bone,osteoid and collagen fibers in the defect area treated with 14,15-EET.In addition,there were more CD31-positive microvessels in the defect area treated with 14,15-EET(p<0.05).Conclusions:EETs can increased bone formation and angiogenesis in C57BL/6 mice calvarial bone defect model.We speculate that 14,15-EET may promote bone regeneration via improvement of local microcirculation.Part 2:Soluble Epoxide Hydrolase Inhibitor Promotes Bone Regeneration of Calvarial DefectObjective:Inflammation,immunity,and nutrient supply are key issues for stem cell-based bone tissue engineering.Considering the short half-life of epoxyeicosatrienoic acid,we assessed the effects of soluble epoxide hydrolase inhibitor(sEHi)is on BMSC proliferation and osteogenesis,and evaluate the effect of sEHi combined with BMSC on the healing of calvarial defect in C57BL/6 mice to further confirm the study of EETs promoting bone regeneration.Materials and Methods:In vitro,BMSCs proliferation was examined with CCK8 assay.In addition,to evaluate the effect of sEHi on BMSCs osteogenesis,BMSCs were cultured in osteogenic-inducing medium.ALP activity was measured using ALP activity test kit.Alizarin Red staining was used to assess calcium content,and the expression of ALP,Runx2 and OPN were determinated by q-PCR technology.In vivo,two circular bone defects(5 mm in diameter)were created on the calvarial bones of the C57BL/6 mice.BMSCs treated with sEHi(1 ?M,experimental sides,n=10)or without sEHi(control sides,n =10)for 3 days,were mixed with ?-tricalcium phosphate(?-TCP)as a carrier and implanted into bone defect.sEHi(10 ?M,experimental side,n =10)or PBS(control sides,n =10)was administered by subcutaneous injection in calvarial defect area every other day from third post-operative day for two weeks.Mice were sacrificed at 8th post-operative week.Bone regeneration in the calvarial defect area was assessed with CBCT.HE and Masson's trichrome stain were performed to assess the newly formed bone and connective tissues.Immunostaining with anti-VEGF were performed to analysis the expression of VEGF.Results:In vitro,sEHi has no significant effect on BMSCs proliferation,and has no apparent influence on the osteogenesis of BMSCs shown by ALP activity test,Alizarin Red staining and the q-PCR results,respectively.In vivo,sEHi treatment increased bone formation of the experimental sides compared with control sides at 8th post-operative week(p<0.05).Masson's trichrome stain suggested that there were more new bone,osteoid and collagen fibers in the defect area treated with sEHi.In addition,there was more VEGF-positive expression in the defect area treated with sEHi(p<0.05).Conclusions:1.sEHi have no significant effect on proliferation and osteogenesis of BMSCs.2.sEHi can increase bone formation and VEGF production in C57BL/6 mice calvarial bone defect model.Our results further confirmed previous results,suggest that sEHi can increase the level of endogenous EETs to be used for bone tissue regeneration.