Effects Of Total Flavonoids Of Rhizoma Drynariae And Nano-bone Materials On Proliferation, Differentiation And Angiogenesis Of Osteoblasts

Objective:To study the osteopractic total flavone(OTF)with nano bone osteoblast proliferation differentiation and angiogenesis of MC3T3-E1.Methods:the study was divided into four parts.The first part focuses on the effects of total flavonoids and nHAC materials on morphological changes and apoptosis of MC3T3-E1 cells.Through the co culture of MC3T3-E1 osteoblasts and nHAC materials,the activity of osteoblasts was measured with the intervention of total flavonoids of osteoblast and CKK8 method.The pathological changes of osteoblasts were observed by histopathological staining,and the scanning electron microscopy was observed.Changes in the ultrastructure of osteoblasts,flow cytometry to detect the apoptosis rate of osteoblasts,observe the effects of the total flavonoids of different concentrations of nHAC on the morphological changes and apoptosis of MC3T3-E1 osteoblasts in vitro,to verify the good biocompatibility and non-toxic properties of nHAC materials,and to screen the best drug concentration after screening.Continue the experiment to do the foundation.The second part focused on the mechanism of OTF combined with nHAC to induce the osteogenic differentiation of MC3T3-E1 cells.The activity of alkaline phosphatase(ALP)was measured by PNPP,ELISA was used to determine Osteocalcin(OCN),alizarin red staining was identified,PCR was used to detect the expression of bone related genes,and the mechanism of osteoblast calcification was observed.The third part focuses on the mechanism of OTF combined with nHAC to regulate the Wnt/beta-catenin signaling pathway in MC3T3-E1 cells.In this part,MC3T3-E1 cells were co cultured with nHAC,and OTF was used as a drug intervention.Transforming growth factor(transforming growth factor-2,TGF-β)and Wnt pathway inhibitor Dickkopf-1(Dkk 1)were taken as positive and negative controls.Immunofluorescence markers were used to observe the osteoblasts In the combination of Wnt and LRP,the changes in gene and protein expression such as β-catenin,LRP 5,Gsk-3β Cyclin D1,Runx-2 and so on were detected and regulated by real-time PCR and Western-blot technology,and the target of promoting the proliferation and differentiation of osteoblasts and nHAC scaffolds was explored.The fourth part focuses on the effect of OTF combined with nHAC on the biological function of HUVEC in hFOB-HUVEC co culture system.In vitro,whether OTF can promote the reconstruction of vascular system during bone regeneration,whether it promotes the proliferation and migration of HUVEC,whether it changes the growth and morphology of HUVEC cells,explore the molecular mechanism of OTF’s function to HUVEC cells,and determine whether the OTF compound nHAC material promotes the secretion of vascular endothelial growth factor(vascu)by osteoblasts(vascu).Lar endothelial growth factor,VEGF)and fibroblast growth factor 2(Fibroblast growth factor-2,FGF-2),such as Fibroblast growth factor-2,FGF-2,and other factors that promote angiogenesis,provide ideas for the study of the interaction mechanism between bone cells and HUVEC cells after tissue engineering bone implantation.Results:in the first part:in the determination of CKK-8 cell activity,the proliferation of MC3T3-E1 osteoblasts was obviously promoted in the concentration group of 100 and 250μg/mL OTF,but there was no significant difference in the effect of two concentrations on cell proliferation,and the high concentration group inhibited the proliferation of cells.The results of cell culture under inverted microscope showed that the B group was DKK1 model group,the osteoblast growth was inhibited and the number of cell adherent decreased significantly.C(DKK 1+TGF-β group),E(DKK 1+OTF250 μg/mL group),F(DKK 1+nHAC+TGF-βgroup),and F(DKK 1+nHAC+TGF-β group)were superior to the normal group,the model group and the model group.In other experimental groups,48h was basically covered by the bottom.Under the microscope,the cells in the culture bottle grew well,the cell morphology became spindle or polygon,and the nucleus was round or oval.When SEM was cultured for 24 hours,the number of DKK1 models was significantly reduced than that in other groups.A large number of cell groups in the three groups had been adhered to and expanded and proliferating.The cells attached to the surface and pores of the material,and the cells had been extended into spindle or polygons,extending out of the pseudo foot and attachment materials,in which the F and H groups were more than the other groups.The cell growth was better in G group.The number of cell growth and surface filamentous extracellular matrix in the non material group C and E increased,and a large number of microvilli were visible on the surface of the cells.The cells in some pores and the secreted matrix were fused into pieces,indicating that the cells were in good condition.At the time of 48h,the number of cells increased.There was granular calcium crystal deposition on the cell surface,and some cells were covered by more minerals.Annexin V/PI double staining flow cytometry was used to detect apoptosis rate in each group.We evaluated the apoptotic rate of MC3T3-E1 cells in 24h and 48h groups.From 24h to 48h,with the increase of OTF concentration,the apoptosis rate of MC3T3-E1 cells showed a steady trend.The apoptosis rate of C,E,F and H groups was significantly lower than that in the B model group and the normal group(P<0.01).The apoptosis rate in D and G groups was significantly lower than that in the model group(P<0.05),and there was no significant difference in the apoptosis rate between the experimental groups(P>0.05).The second part:OTF can induce MC3T3-E1 cells to enter osteoblasts,improve the activity of ALP and the concentration of OCN,and increase the expression of Col-I,OPN and OCN mRNA in the MC3T3-E1 cell culture and the expression of protein.There was no significant difference in osteogenic potential of OTF between 100 μg/mL and 250 μg/mL concentration,but it was significantly higher than that of other groups.Therefore,we believe that OTF with a concentration of 100 μg/mL and 250 μg/mL can effectively promote the differentiation and mineralization of MC3T3-E1 cells.The third part:This study showed that the changes in the expression of gene and protein levels of beta-catenin,LRP5,GSK-3 beta,RUNX2 and Cyclin D1 were detected after 24 hours and 48 hours.As shown in the picture,we found that the gene and protein expression of beta-catenin,LRP5,Runx2 and Cyclin D1 in C,D and E three groups increased significantly after 24 hours and 48 hours,and the expression of GSK3 beta gene and protein was significantly decreased.There was a significant difference in the expression of GSK3 beta gene and protein(P<0.05).C,E two groups The expression level of D1 gene and protein was significantly higher than that in group D,and the difference was statistically significant(P<0.05).The fourth part:CKK8 results showed that the supernatant of osteoblast culture was more likely to promote the proliferation of HUVEC cells,and there was no statistical difference in promoting the proliferation of HUVEC cells by OTF.NHAC material had no effect on the attachment,growth and proliferation of the two cells,indicating that the material had good biocompatibility.ELISA results showed that osteoblasts secreted FGF-2,and combined with the results of CCK8,the content of FGF-2 and the proliferation of HUVEC cells showed the same trend,and had no obvious effect on VEGF secretions.Conclusion:(1)a preliminary study on the combined culture of rat osteoblasts in vitro by OTF combined with nHAC material,the results of cell proliferation activity test,cell apoptosis detection and morphological observation are consistent and verified,indicating the good functional state of the cells in the experimental group,and the observation of the concentration group of OTF 100 and 250μg/mL.To improve the adhesion and proliferation of osteoblasts and to maintain their normal cell morphology,the growth trend is better with the prolongation of culture time,which has obvious dose dependence and time dependence;(2)nHAC has good biocompatibility and non-toxic to cells.It is an ideal bone substitute material,and the OTF compound nHAC material can promote osteoblast proliferation,inhibit apoptosis and maintain normal cell morphology,but its osteogenesis effect remains to be further studied;(3)OTF can induce osteogenic differentiation of MC3T3-E1 cells,improve ALP activity and OCN concentration,and increase the expression of Col-I,OPN and OCN mRNA in MC3T3-E1 cell culture and the expression of protein.OTF with a concentration of 100μg/mL and 250 μg/mL can effectively promote the differentiation and mineralization of MC3T3-E1 cells;(4)through the culture of MC3T3-E1 cells and nHAC complex in vitro,OTF has the activity and biological characteristics of osteoblast after induction and culture.It is suitable to be used as the seed cell for constructing tissue engineering bone and the research object of cell experiment under three dimensional conditions;(5)OTF can obviously promote the differentiation and proliferation of osteoblasts,promote bone formation and bone mineralization,and increase the expression of β-catenin,LRP5 and RUNX2,and down regulate the expression of GSK3β.It is possible to speculate that OTF can promote osteoblasts by activating the classical Wnt/β-catenin signaling pathway,which is one of the possible mechanisms of action;(6)nHAC material has good cell binding ability.HFOB-HUVEC can be co cultured on scaffold materials,and can be well attached,grown and proliferated;(7)osteoblasts may promote the proliferation of HUVEC by secreting FGF-2,while OTF does not promote FGF-2 secretion by osteoblasts.