Effects Of Astragalus Polysaccharide On Seed Cells Of Tissue Engineering Bone And Its Mechanism

Osteoblasts and vascular endothelial cells are important seed cells for tissue-engineered bone.In the implemention of bone tissue engineering,the seed cells need to be cultured in vitro,and compounded on the bone scaffold material of tissue engineer together with bioactive stimulation factors to form a graft body compound.Then the graft body compound will be transplanted to the bone defect part for further proliferation and activation.After gradual vascularization and ossification process,the bone tissue trauma repair is completed.The bioactive factors are used mainly to promote the survival and proliferation of seed cells and accelerate the process of bone regeneration.Therefore,the selection of bioactive factors has always been a heated topic.Astragalus polysaccharide(APS)is an active ingredient extracted from the Chinese medicinal Radix Astragali seu Hedysari and has various biological activities.It has been reported that APS can promote the proliferation and migration of vascular endothelial cells to initiate rapid vascularization,and significantly promotes the proliferation of osteoblasts.In the previous studies of our research group,we observed that APS had an activation effect on both human peripheral bloods separated overgrowth endothelial cells(OECs)and human primary osteoblasts(hPOBs).In this study,based on the comparative observation of the effects of APS on OECs,hPOBs single culture and the coculture system of OECs and hPOBs,we further observed and verified the angiogenesis and ossification effects of APS on tissue-engineered bone in vivo,and explored its mechanism based on TLR4 signaling pathway,to provide a theoretical and experimental basis for the successful application of APS on construction of tissue-engineered bone as a bioactive stimulator.The research mainly includes the following four parts:Part One The observation of effect of Astragalus Polysaccharide on endothelial cells and its dose-effect relationship in vitroObjective:The aim of this part was to observe the effects of APS on promoting OECs proliferation and vascularization,and its dose-effect relationship.Moreover,we’d like to determine the optimal concentration of APS intervention and its correlation with angiogenesist at cellular level.Methods:1.Cytomorphology,immunofluorescence and flow cytometry were employed to identify the surface antigens of OECs from human peripheral blood derived from different donors;CCK8 was used to test the effects of APS at different concentrations on the viability of OECs to determine the optimal concentration.2.OECs at passages P8 to P13 were stimulated with pure cell culture medium(without APS)and 0.4 mg/mL APS,respectively,serving as the control group and experimental group.The effect of APS on the proliferation of OECs was detected by Ki67 immunofluorescence staining;The effect of APS on the migration of OECs was detected by cell scratch test and the effect of APS on the apoptosis of OECs was determined by flow cytometry.3.OECs at passages P8 to P13 were stimulated with pure medium and 0.4 mg/mL APS for 24 h,respectively,serving as the control group and the experimental group;The effect of APS on tubular formation of OECs was detected by tubular formation experiment.The immunofluorescence staining was used to detect the expression changes of angiogenic factors such as angiopoietin-1 and-2(Ang-1 and Ang-2)and vascular endothelial growth factor(VEGF)in OECs to determine the effect of APS on the angiogenesis of OECs.Results:1.The results of morphology,immunofluorescence and flow cytometry surface antigen labeling indicated that OECs were successfully isolated;The results of CCK8 cell viability test showed that APS had a proliferation promoting effects on OECs,and the effect was bidirectional with the appropriate concentration was 0.4 mg/mL.2.The results of OECs scratch test,flow cytometry analysis of apoptosis and Ki67 immunofluorescence assay indicated that compared with control group,the mobility of OECs significantly increased after 6 h and 12 h stimulation by APS(6 h,P<0.01;12 h,P<0.001).Furthermore,compared with control group,the apoptosis of OECs induced by APS for 24 h significantly decreased(P<0.05)and the expression of Ki67 significantly increased(P<0.05)3.The results of OECs tubular formation experiment and immunefluorescence staining of Ang-1,Ang-2 and VEGF demonstrated that compared with control group,the total length of tubular formation and the total number of nodes in APS experimental group significantly increased(P<0.05),and the expressions of Ang-1(P<0.01),Ang-2(P<0.01)and VEGF(P<0.05)in the APS experimental group significantly increased.Conclusions:1.The cells we isolated were OECs derived from human peripheral blood.2.APS can accelerate the proliferation and migration of OECs,reduce apoptosis,and promote angiogenesis.3.APS angiogenesis had a concentration-dependent effect,and the appropriate concentration is 0.4 mg/mL.Part Two The observation of consistent effect of Astragalus Polysaccharide on osteoblast and endothelial cellsObjective:The single culture system of hPOBs,and the co-culture system of OECs and hPOBs were employed to observe whether APS had a consistent effect on OECs and hPOBs.At the same time,we investigated the dose-effect relationship between APS and cells.Methods:1.hPOBs surface markers were identified by cell morphology and immunofluorescence staining.The CCK8 method was used to test the effect of APS on the activity of hPOBs to determine its optimal concentration.2.hPOBs at passages P2 to P5 were stimulated with pure medium and 0.4 mg/mL APS,respectively,serving as the control group and the experimental group.The immunofluorescence staining experiment was conducted to compare the expression levels of osteopontin,osteocalcin,Ki67 and alkaline phosphatase(ALP)in the control group and the experimental group to determine the effects of APS on the proliferation and ossification of hPOBs.3.The co-culture system constructed with OECs from different donors and hPOBs from the same donor was the research object.After the co-culture system was stimulated with pure medium and 0.4 mg/mL APS for 24 h,the microvascular formation in the co-culture system was observed by laser scanning confocal microscope to determine the effect of APS on the microvascular formation in the co-culture system and whether individual difference was existed.4.The coculture system constructed by OECs and hPOBs from one source was the research object.After the coculture system was stimulated with different concentrations of APS(0.2,0.4 and 0.8 mg/mL)and pure medium for 24 h,the microvascular formation in the coculture system was observed by laser confocal microscope to determine the effect of APS on the microvascular formation in the coculture system and the optimal concentration.5.The coculture system constructed by OECs from single source and hPOBs from single source was the research object.The coculture system was stimulated with 0.4 mg/mL APS and pure medium for 96 h,respectively.The microvascular formation in the coculture system was observed by laser confocal microscope to determine the optimal action time of APS on microvascular formation in the coculture system.Results:1.Morphological and immunofluorescence staining results of hPOBs showed that hPOBs was successfully isolated;The results of CCK8 experiments showed that APS had a pro-proliferation effect on hPOBs,and the effect was bidirectional.The optimal concentration was 0.4 mg/mL,which was consistent with the effect of APS on OECs.2.The immunofluorescence staining results of osteopontin,osteocalcin,Ki67 and ALP of hPOBs showed that compared with control group,the expression levels of osteopontin(P<0.05),osteocalcin(P<0.01),Ki67(P<0.05)and ALP(P<0.05)in APS group were increased.3.Compared with control group,the microvessel formations in the coculture systems of OECs and hPOBs from three different donor sources were all increased under the intervention of 0.4 mg/mL APS,and two of which were statistically significant as compared with control group(P<0.05),while the another one showed no individual difference as a whole.4.Various concentrations of APS(0.2,0.4,and 0.8 mg/mL)promoting OECs and hPOBs microvessel formation was observed in the coculture system,and the effect was most significant at 0.4 mg/mL(P<0.001).5.There was a time-dependent relationship on APS promoting microvascular formation in OECs and hPOBs co-culture systems.After the coculture system was stimulated with 0.4 mg/mL APS for 96 h,angiogenesis was found to be increased(P<0.05),but the effect was not as significant as that at 24 h.Conclusions:1.The cells we isolated were human hPOBs.2.APS(0.4 mg/mL)significantly promoted the proliferation,differentiation and osteogenic capability of hPOBs,which was consistent with that of OECs.3.APS promoted the microvessels formation in the co-culture system of OECs and hPOBs,and the effect of 0.4 mg/mL APS stimulation for 24 h was the most significant.Part Three The mechanism of Astragalus Polysaccharide intervention on the biological activity of endothelial cells and osteoblastsObjective:The molecular mechanism of APS in promoting the biological activities of OECs and hPOBs was preliminarily explored based on TLR4 signaling pathway.Methods:1.After co-culture of OECs and hPOBs for 7-9 days,the control group and the experimental group were stimulated with pure medium and 0.4 mg/mL APS for 24 h,respectively.ELISA was performed to detect the expression levels of IL-6,TNF-α,IFN-γ and E-Selectin in the supernatant of co-culture system of OECs and hPOBs to determine the effect of APS on inflammatory factors in the co-culture system.2.After co-culture of OECs and hPOBs for 7-9 days,the cells were stimulated with pure medium and 0.4 mg/mL APS for 24h,respectively,acting as the control group and experimental group.The mRNA expression levels of VEGF and PDGF-BB in the two groups were detected by RT-qPCR technique,to determine the effect of APS on the expression of pro-angiogenic factors in the co-culture system.3.The OECs at passages P8 to P13 or hPOBs at passages P2 to P5 was stimulated by pure medium and 0.4 mg/mL APS for 24h,resprctively,serving as control group and experimental group.The mRNA expression levels of TLR4,MyD88 and NF-κB in two groups were measured by RT-qPCR.Western Blot(WB)was used to detect the protein expression levels of TLR4,MyD88 and NF-κB,which were the main node molecules in the TLR4 signaling pathway,to determine the effect of APS on the TLR4-MyD88-NF-κB signaling pathway in OECs/hPOBs.Results:1.The results of ELISA indicated that compared with control group,the cytokine IL-6(P<0.01),TNF-α(P<0.05),IFN-γ(P<0.05)and E-Selectin(P<0.05)were all significantly increased.2.The results of RT-qPCR experiment showed that compared with the control group,the mRNA expression levels of VEGF(P<0.05)and PDGF-BB(P<0.05)significantly increased in the APS group.3.RT-qPCR results showed that compared with control group,the mRNA expression levels of TLR4(P<0.05),MyD88(P<0.01)and NF-κB(P<0.05)in OECs of APS group significantly increased,as well as the hPOBs.4.The results of WB experiment showed that the protein expressions of TLR4(P<0.001),MyD88(P<0.05),and NF-κB(P<0.001)increased significantly,as well as the hPOBs.Conclusions:APS might up-regulated the expressions of inflammatory factors and angiogenesis-related factors by activating the TLR4-MyD88-NFκB signaling pathway,thus promoting the biological activities of OECs and hPOBs.Part Four In vivo validation of Astragalus Polysaccharidepromoting vascularization and ossification of tissue-engineered Bone Objective:The aim of this part was to observe the effects of APSintervention on vascularization and ossification of tissue-engineered bone in animal experiments.Methods:1.In vivo vessel formation:PBS,FGF-2 and 0.4 mg/mL APS wererespectively added into the matrix of vascular reactor as negative group,positive group and experimental grou.Then we implanted the vascular reactors subcutaneously into 4-week-old nude mice,and removed them 22 days later to evaluate the angiogenesis.Moreover,FITC-Lectin immunofluorescence staining was employed to detect the relative invasion value of vascular endothelial cells that migrated into vascular reactor in nude mice,to determine the in vivo effect of APS on vascular endothelial cells.2.The chitosan scaffold was prepared by freeze-drying.3.Cell morphology and immunofluorescence were applied for identifying the surface markers of rPOBs.4.rPOBs with chitosan scaffolds were subcutaneously applied to SD rats.First,rPOBs were inoculated into chitosan scaffoalds as a tissue-engineered bone graft.The experiments were divided into three groups:the group that the chitosan scaffold was not inoculated with rPOBs as a simple scaffold group;the group that the chitosan scaffold was inoculated with rPOBs as a cell scaffold group;the group that the chitosan scaffold was inoculated with rPOBs and rats were given intraperitoneal injection of 50 mg/kg/d APS as a cell scaffold+APS group.They were respectively implanted into the subcutaneous layer of SD rats,and after 40 days,the implants were taken out.HE staining,immunofluorescence and immunohistochemical staining were performed to detect the differences in osteogenesis and angiogenesis of the constituent osteoblasts,to determine the effect of APS on angiogenesis and bone formation in tissue-engineered bone.Results:1.The results of direct angiogenesis experiment in vivo showed that although the experimental group was not as good as the positive group when observed with the naked eye,the angiogenesis was also significantly increased in the experimental group when compared with that in the negative control group.The results of FITC-Lectin immunofluorescence staining were consistent with those observed by the naked eye.The OD value of the contents of the vascular reactor was detected by ELISA reader,and the result was consistent with the macroscopic observation.Compared with the negative control group,the angiogenesis in the experimental group was significantly increased(P<0.05).2.The cells we isolated were rPOBs.3.The chitosan scaffold we prepared were highly porous,and the diameter of pore was about 20-100 μm.The rPOBs were successfully inoculated into the stent.4.rPOBs were inoculated into chitosan scaffolds and subsequently implanted subcutaneously in SD rats.The in vivo results indicated that compared with stent group,rPOBs in the cell scaffold group significantly increased(P<0.05),and increased more significantly in the cell scaffold+APS group(P<0.01).Compared with the cell scaffold group,the rPOBs in the cell scaffold+APS group increased significantly(P<0.05).For angiogenesis,compared with stent group,the vascular density and percentage in cell scaffold group and cell scaffold+APS group significantly increased(P<0.05),especially in the cell scaffold+APS group.Meanwhile,compared with the cell scaffold group,the vascular density and percentage of blood vessels in the cell scaffold+APS group also significantly increased(P<0.05).Conclusions:1.APS promoted the migration of vascular endothelial cells and angiogenesis in vivo.2.APS promoted the ossification and angiogenesis of tissue-engineered bone in vivo.