| Objective: As an endogenous bone engineering technology,Distraction Osteogenesis(DO)is widely used in congenital or acquired bone deficiencies and lager defects,which attributed to its easy operation and rapid osteogenesis.The osteogenesis of DO is closely related to angiogenesis.During DO,angiogenesis is prior to bone formation in both time and space.Tissue from the early stage of distraction is already rich in blood vessels and high flow.However,the specific mechanism of rapid angiogenesis in DO has not been fully elucidated.In our previous study,it was found that the angiogenesis in DO was related to the mobilization,recruitment and differentiation of EPCs.Results of miRNAs gene sequence analysis for DO new-formed tissue showed that the expression of miR-21 in consolidation 0,2 weeks groups was significantly increased with time and higher than that in adult mandible and newborn ones,while the angiogenesis decreased with the extension of consolidation time.Therefore,it is resonable to speculate that there is a negative correlation between miR-21 and angiogenesis in DO,but the regulatory media between them is not clear.Combined with evidence that EPCs have a positive impact on angiogenesis,it can be assumed that miR-21 may regulate DO angiogenesis through regulating some genes or signal pathways in EPCs.In this study,in vivo and vitro experiments will be conducted to investigate and verify the regulatory effect of miR-21 on EPCs,expound the angiogenesis mechanism of DO and supply the biological basis for the rapid formation of blood vessels during DO.Methods: 1.EPCs were isolated and cultured from adult dog bone marrow blood by density gradient centrifugation and differential attachment.Flow cytometry was used to detect the expression of CD34 and CD133 antigens on the surface of EPCs.The cultured EPCs were inoculated on Matrigel to observe tubules formation and verify their ability to form blood vessels.2.MiR-21 inhibition,miR-21 overexpression lentivirus and related plasmids for miR-21 target gene detection were constructed.The combination of miR-21 and TGFβ2was verified by double luciferase report detection system.The optimal infection complex number(MOI)of EPCs transfected by each virus was also detected.The experimental groups were divided into four groups: EPCs group(without treatment),Control group(EPCs transfected with control virus),miR-21 inhibition group(EPCs transfected with miR-21 inhibition virus),miR-21 overexpression group(EPCs transfected with miR-21 overexpression virus).After 48 hours of virus infection,the cells were cultured in puromycin medium for 24 hours.CCK-8 method was used to detect the effect of lentivirus infection on the proliferation of EPCs.Matrigel test was used to detect the effect of EPCs on angiogenesis.Immunofluorescence method was used to detect the expression of CD31,CD34,CD133 and VEGF.RT-q PCR was used to detect the gene expression of miR-21,TGF-β2,SMAD2,SMAD3,SMAD7,CD34,CD133,CD31,VEGF,S100A4.The expression of SMAD2,SMAD3,CD34,CD133,CD31,VEGF,S100A4 proteins were detected by WB.3.The dog mandible DO animal model was established.All the animals were divided into 6 groups: DO group(without no treatment in stretch space),Matrigel group(injection of Matrigel into stretch space),EPCs group(injection of EPCs-Matrigel mixture into stretch space),Control group(injection of control virus transfected EPCs-Matrigel mixture into stretch space),miR-21 inhibition group(injection of miR-21 inhibition virus transfected EPCs-Matrigel mixture into stretch space),miR-21 overexpression group(injection of miR-21 overexpression vvirus transfected EPCs-Matrigel mixture into stretch space).Mandible samples were collected at the end of the consolidation period of 1 week and 2 weeks and examined by X-ray.The new-formed tissues in the stretch space were taken out for HE staining,Masson staining and immunohis-tochemical staining to analyze the expression of CD31,VEGF,S100A4,CD133 and SMAD3.Results: 1.In the primary culture of EPCs,the cells grew as colonies,and the cell density on the culture dish was uneven.Because of the limitation of growth space,the cells exhibited different shapes such as short ellipse and long fusiform.But in subculture,after digestion and suspension,cell morphology was typical "paving stone" or "pebble" due to isotropic cell growth space.EPCs highly expressed CD34 and CD133 surface markers and formed lumina-like structure after cultured on Matrigel for 12 hours.All these results confirmed that the cells isolated and cultured in this experiment were EPCs,which could be used for subsequent experiments.2.MiR-21 can bind to 3’UTR of TGFβ2 and inhibit the expression of TGFβ2.The proportion of MOI = 20 could be used to infect EPCs by empty virus,miR-21 infection and miR-21 overexpression virus.CCK-8 results showed that the number of cells in EPCs group,Control group,miR-21 inhibition group and miR-21 overexpression group increased after 24 hs cultured,and there was no significant difference among all the groups,indicating that the lentivirus transfection and the change of miR-21 expression did not affect the proliferation and viability of EPCs.Matrigel tube forming experiment showed that nodes,junctions,meshs and mesh area of miR-21 inhibition group were significantly higher than those of the other three groups,while miR-21 overexpression group was significantly lower than the other three groups,and there was no significant difference between the EPCs group and the Control group,indicating that inhibition of miR-21 enhanced the ability of EPCs tube forming,which was weakened after miR-21 overexpression.The results of immunofluorescence staining showed that CD34 was expressed in most of the cells in each group,and CD133 was expressed in less cells,but there was no significant difference among the four groups.The expression of CD31 and VEGF in miR-21 inhibition group was stronger than that in other three groups,while that in miR-21 overexpression group was weaker than that in other groups.There was no difference between EPCs group and Control group,and the expression level was between the former two groups.The results of RT-q PCR showed that the expression of miR-21 decreased significantly after the transfection of miR-21 inhibition virus,while it increased after the transfection of miR-21 overex-pression virus.There was no significant difference between the expression of miR-21 between Control and EPCs group,indicating that the transfection system was stable and could effectively control the expression of miR-21.miR-21 was negatively correlated with TGFβ2,SMAD2 and SMAD3,but positively correlated with SMAD7.There was no difference in the expression of CD34 and CD133 in all groups.However,CD31,VEGF and S100A4 were significantly increased in miR-21 inhibition group but decreased in miR-21 overexpression group.There was no difference between the EPCs group and the Control group.WB detection showed that the expression of SMAD2,SMAD3,CD34,CD133,CD31,VEGF,S100A4 was consistent with the expression of the corresponding genes of RT-q PCR.3.It was found that the new-formed tissues from the distraction gaps in all six group were red-and-white with slight elasticity and no significant difference was observed among the groups.X-ray examination showed that the distraction gap had sign of low-density area with no definite image of bone formation and there was no significant difference among the groups.Results of HE showed that abundant vessels could be observed in all groups after 7 days consolidation.However,there were fewer vessles in miR-21 inhibition than that in other groups,and cartilage islands appeared in the new-formed tissues.After consolidation for 14 days,a lot of bone trabecula could be seen in the pink matrix composed of capillaries,fibers and cells in DO group,Matrigel group,EPCs group,Control group and miR-21 inhibition group,while abundant vascular components were still found in miR-21 overexpression group,and a few soft cartilage like structures were occasionally seen with no bone trabecula being formed in miR-21 overexpression group.HE results suggested that the process of vascularization in miR-21 inhibition group was shortened and advanced to the stage of ossification.However the vascularization process was prolonged and the ossification time was delayed in miR-21 overexpression.According to the results of Masson,the collagen fiber from highest to lowest was: miR-21 inhibition group,EPCs and Control group,DO and Matrigel group,miR-21 overexpression group.According to the IHE results of CD31,VEGF and S100A4,the sequence of vessels from most to least was,miR-21 overexpression group,DO and Matrigel group,EPCs and Control,miR-21 inhibition group.There was no significant difference in CD133 expression among all the groups at 7 days and14 days consolidation.The results of SMAD3 staining showed that SMAD3 was mainly distributed in the blood vessel and its surrounding cells.At 7 days,its expression trend was similar to that of CD31,VEGF and S100A4.But at 14 days,the expression of SMAD3 was slightly higher in miR-21 overexpression group than that in other groups and it was nearly negative in other groups,especially except weaker,especially miR-21 inhibition group.Conclusions: 1.In vitro,EPCs can rapidly differentiate into endothelial cells and exhibited strong angiogenic ability.Furthermore,it can promote angiogenesis in DO animal model.2.MiR-21 is negatively correlated with angiogenesis.Overexpression of miR-21 weakens angiogenic ability of EPCs,leading to postponed and poor neovascularization.However,inhibition of miR-21 results in the opposite results,which evidenced by accelerated angiogenesis.3.MiR-21 can directly bind to TGFβ in EPCs and inhibit the activation of TGF-β/Smad signaling pathway,suggesting that miR-21 may control the angio-genesis function of EPCs by regulating TGF-β/Smad.. |
PDF Full Text Request