The Influence Of Ionic Products Of Dicalcium Silicate Coating Dissolution On Bioactivity Of Osteoblasts In Vitro

Partâ… The effects of ionic products of dicalcium silicate coating dissolution on proliferation and cell cycle as well as cell apoptosis of osteoblastsObjective:To discuss the influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on proliferation,cell cycle and cell apoptosis of osteoblasts. Methods:The Ca₂SiO₄ conditioned DMEM was achieved by immersing plasma sprayed Ca₂SiO₄ coating in DMEM for 24h at 37℃.The ion concentration in DMEM was measured by ICP.MG63 cells cultured with the DMEM containing ionic products of dicalcium silicate coating dissolution was prepared to be experimental group,which cultured in normal DMEM was control groups.Cell proliferation was measure with MTT and cell counting at 6,12,24h,48h and 72h.Cell cycle distribution and cell apoptosis were investigated by flow cytometry at 12,24h,48h and 72h.Result: The results obtained from this work showed that Si ion concentration increased significantly about 17 folds in Ca₂SiO₄ conditioned DMEM(P<0.05),and Ca and P ion concentration decrease slightly and no significantly(P>0.05).MG63 cell proliferated more significantly in the group containing ionic products of Ca₂SiO₄ coatings than in the control group,and significantly at 24h and 72h.The cell cycle distribution indicated that the number of MG63 cell in each cycle phase showed no significantly difference at 12h and 24h.And the decreased osteoblast number in G₀/G₁ phase were detected in the cells cultured in the DMEM containing ionic products of Ca₂SiO₄ coatings compared with the control group at 48 and 72h.At 72h,the osteoblast number of S phase in experimental group increased about 2 folds when compared to control(P<0.05).G2/M phase osteoblast number increased significantly in experimental group at 48h(P<0.05).Cell apoptosis in experimental groups were significantly higher than control at 48 and 72h(P<0.05).Conclusion:The results suggest that the ionic products of Ca₂SiO₄ coating dissolution can promote MG63 cell proliferation which contributes to accelerating cell cycle. Partâ…¡The influence and possible mechanisms of ionic products of dicaicium silicate coating dissolution on differentiation and osteogenic gene expression of osteoblastsObjective:To discuss the influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on differentiation and osteogenic gene expression of osteoblasts. Methods:MG63 cells cultured with the DMEM containing ionic products of dicalcium silicate coating dissolution was prepared to be experimental group,which cultured in normal DMEM was control groups for 6,12,24h,48h and 72h.Cell differentiation was detected by measuring ALP activity in culture media.Osteogenic gene expression(ALP,COL-â… ,OC,Cbfal) was evaluated using Real time PCR technology.Result:The results obtained from this work showed that ALP activity increased in Ca₂SiO₄ conditioned DMEM when compared to control and significantly at 6h,48h and 72h(P＜0.05).Results from Real-time PCR showed that Cbfal transcriptional levels in experimental groups were up-regulated at all time points when compared with the control groups and significantly at 6h,12h and 24h(P＜0.05).When compared with control,ALP transcription in experimental groups were significantly up-regulated at 12h and 24h,and then decreased lower than control but no significantly at 48h and 72h(P＞0.05).COL-â… transcription were higher than control at all experimental periods and significantly at 24h(P＜0.05).OC transcription was not detected in both groups in early stage. Conclusion:The ionic products of Ca₂SiO₄ coating dissolution can up-regulate osteogenic-related gene expression and enhance differentiation of osteoblasts as well as new bone formation.Increased apoptosis of MG63 cells results from enhanced differentiation.Partâ…¢The influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on osteoclastogenie gene expression and their protein synthesis of osteoblasts Objective:To discuss the influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on osteoclastogenic gene expression and their protein synthesis of osteoblasts.Methods:MG63 cells cultured with the DMEM containing ionic products of dicalcium silicate coating dissolution was prepared to be experimental group,which cultured in normal DMEM was control groups for 6,12,24h,48h and 72h.Osteoclastogenic gene expression(OPG,RANKL, TNF-α) was evaluated using Real time PCR technology.OPG,RANKL and TNF-αprotein synthesis in culture media were measured by ELISA assay at 12,24h,48h and 72h.Result:Results from Real-time PCR showed that OPG transcriptional levels in Ca₂SiO₄ conditioned DMEM were promoted when compared to control and significantly at 6h,12h,24h and 48h(P<0.05).RANKL transcriptional levels in both groups were low at 6h,12h and 24h.and then up-regulated at 48h and 72h,but in experimental groups,RANKL transcription were significantly lower when compared with control at 48h and 72h(P<0.05).No TNF-αtranscription was detected in both groups.OPG protein synthesis in experimental groups were higher than the control groups at all time points and significantly at 48h and 72h(P<0.05).RANKL protein synthesis in both groups showed no significantly difference but lower in experimental groups(P>0.05).TNF-αsynthesis of MG63 cells in experimental groups was lower than control and significantly at 72h(P<0.05).Conclusion:The ionic products of Ca₂SiO₄ coating dissolution can up-regulate OPG gene expression and down-regulate RANKL gene expression,more importantly increase OPG protein synthesis and inhibit TNF-αsynthesis,which influence the ratio of OPG/RANKL that inhibiting osteoclast differentiation and activity,finally lead to diminishing bone resorption and increasing bone formation.Partâ…£The influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on proliferation and differentiation as well as TGF-β1 synthesis of osteoblasts derived from old patientsObjective:To discuss the influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on proliferation and differentiation as well as TGF-β1 synthesis of osteoblasts derived from old patients.Methods:The primary cultured osteoblasts were obtained from bone slice which were discarded from old patients suffered from total hip arthroplasty(older than 60 years).The Ca₂SiO₄ conditioned DMEM was achieved by immersing plasma sprayed Ca₂SiO₄ coating in DMEM for 72h at 37℃.The ion concentration in DMEM was measured by ICP.Osteoblasts cultured with the DMEM containing ionic products of dicalcium silicate coating dissolution was prepared to be experimental group,which cultured in normal DMEM was control groups for 1,3,7 and 14 days.Cell proliferation was measure with MTT assay.Osteoblast differentiation was detected by ALP activity.ELISA was used to measure TGF-β1 synthesis in culture media.Result:The results obtained from this work showed that Si ion concentration increased significantly about 19 folds in Ca₂SiO₄ conditioned DMEM(P＜0.05),and Ca and P ion concentration decrease slightly and no significantly(P＞0.05).Primary cultured osteoblast proliferated more significantly in the group containing ionic products of Ca₂SiO₄ coatings than in the control group,and significantly at 3 days. ALP activity in experimental groups were enhanced when compared to that in control groups at all time points,and significantly at 7 days(P＜0.05).ELISA results showed that TGF-β1 synthesis in experimental groups was promoted significantly when compared to control(P＜0.05) at 1 and 3 days, and decreased which lower than control but no significantly at 7 days,and no difference was found in 14 days.Conclusion:The results suggest that the ionic products of Ca₂SiO₄ coating dissolution can promote primary culture human osteoblast derived from old patient proliferation and differentiation which contributes to increasing TGF-β1 synthesis.Partâ…¤The influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on differentiation and TGF-β1 synthesis as well as its receptors(TGF-βRâ… and TGF-βRâ…¡) gene expression of MG63 cellsObjective:To discuss the influence and possible mechanisms of ionic products of dicalcium silicate coating dissolution on differentiation and TGF-β1 synthesis as well as its receptors(TGF-βRâ…  and TGF-βRâ…¡) gene expression of MG63 cells.Methods:The medium containing ionic products of dicaicium silicate(Ca₂SiO₄) for culturing MG63 cells was prepared by immersing the plasma sprayed Ca₂SiO₄ coatings in DMEM solution.The effect of the ionic products on cellular differentiation and local growth factor(TGF-β1) of osteoblast-like MG63 cell were investigated.The normal DMEM was also used to culture MG63 cells as the control group.Differentiation of cell was evaluated by detecting alkaline phosphatase(ALP) activity and osteocalcin(OC) synthesis as well as their gene expression. The levels of TGF-β1 in culture medium were measured using Enzyme-linked immunosorbent assay (ELISA).The gene expressions of TGF-β1 receptors(TGF-βRâ… and TGF-βRâ…¡) were also measured by Real time PCR technology.Result:MG63 cells cultured in DMEM containing ionic products of Ca₂SiO₄ coating showed enhanced differentiation.The results obtained from ELISA showed that the levels of TGF-β1 in experimental group were higher than that in control.Compared with cultured in normal DMEM medium,ALP activity of MG63 cells in Ca₂SiO₄-DMEM was promoted from day 3 and significantly in day 7 and 14(P＜0.05);OC synthesis was significantly enhanced at day 14.Their gene expression was shown as identical pattern.ELISA results showed that TGF-β1 synthesis in experimental groups was promoted significantly when compared to control(P＜0.05) at 1 and 3 days, and decreased significantly which lower than control at 7 days,and no difference was found in 14 days. TGF-βRâ… gene expression in experimental groups was enhanced at day 3 and reached its tip at day 7 which significantly higher than control(P＜0.05).TGF-βRâ…¡gene expression was higher than control but no significantly(P＞0.05).Conclusion:It is concluded that ionic products of Ca₂SiO₄ coating may enhance cellular differentiation and collagen production by influencing TGF-β1 pathway.