Study Of Ionic Products Of Plasma Sprayed Dicalcium Silicate Coating On Osteogenic Capacity In Vitro

Partâ… Study of ionic dissolution products of dicalcium silicate coating on osteoblastic proliferatoinObjective To explore the dissolving properties of plasma sprayed dicalcilum silicate coating on Ti plates and accordingly the concentrations of the main ions in different dissolutions,as well as the influences of ionic dissolution products of dicalcium on proliferation of MG63 cells.Method 10 pieces of Ti plates with plasma sprayed dicalcium silicate coating were soaked in 200ml DMEM and double dilluted H₂O, respectively,at 37℃for 72h in static conditions,then ICP-OES was employed to detect the concentrations of Si,Ca and P.MG63 cells were cultured in DMEM and conditioned DMEM respectively.Cell mophorlogy was observed under microscopes,cell cycles were analyzed by flow cytometry,and MTT assay was used to detect cell proliferation on days 3, 6,9 and 12.Results Si concentrations in DMEM and double dilluted H₂O after soaking with dicalcium silicate coating were increased,markedly when compared with normal DMEM;no differences of Ca and P ionic concentrations were observed between normal DMEM and dicalcium silicate conditioned DMEM.No differences in cell morphorlogy were existed between the cells of the two groups.MTT value of experimental group was significantly higher than that of control group on day 3.With the progression of culture time,no differences statistically significant were observed between the two groups,and MTT values on days 9 and 12 in the experimental group were even a little lower than those of the control group.Flowcytometric analysis showed that the percentage of MG63 cells in S phase of the experimental group was obviously higher than that of the control group.At the same time,cells in G0/G1 phase of the experimental group was significantly lower than that of the control group.While cells in G2/M phase of the experimental group was a little higher than that of the control group with no statistically significant difference.No differences in different phases between the two groups could be observed thereafter. Conclusions Plasma sprayed dicalcium silicate coating on Ti plates lead to increase in Si ion concentration after soaked in DMEM for 72h.Dicalcium silicate conditioned DMEM was able to promote the early but not the late proliferation of MG63 cells,which may be intimately related to the high concentration of Si ion.And the ability of C2S conditioned culture medium to promote osteoblastic proliferation was intimately related to the upregulated percentage of cells in S and G2/M phases as well as the downregulated percentage in G0/G1 phase.Partâ…¡Study of ionic dissolution products of dicalcium silicate coating on osteoblastic differentiationObjective To explore the influences of ionic dissolution products of dicalcium silicate coating on differentiation of MG63 cells.Method MG63 osteoblast-like cells were cultured in normal DMEM and conditioned DMEM,respcetively. After 3,6,9,12 days' cultivation,respectively,samples were collected for detection of the ALP activity and gene expression of core binding factorα1(Runx2) and 3 markers of osteoblastic differentiation,e.g typeâ… collagen,ALP and osteocalcin.Results From day 3 on,ALP activity in the experimental group was higher than that of the control group. With the progression of culture time,ALP activity increased in both of the two groups but was higher in the experimental group.Statistically significant differences were observed on timepoints of days 3,6 and 9.On day 12,although ALP activity was still higher in the experimental group than those in control group,the difference was already not significant. Although there were no significant differences at days 3 and 6,mRNA levels of Runx2 were higher in the experimental group on days 9 and 12.ALP mRNA expression in the control group in the culture course showed no signifant differences,but its expression markedly upregulated(p＜0.05) in the experimental group from day 3 on and increased persistently till the end of the study.When considering the mRNA levels of typeâ… collagen and osteocalcin,the changes were similar in these two markers,which showed no significant changes in the control group but increased from 3 day on and was markedly higher on day 9 in the experimental group,then decreased slightly on day 12 but was still markedly higher than that of the control group.Conclusions Ionic dissoluiton products of dicalcium silicate coating was able to promote late differentiation as well as early proliferation of MG63 osteoblast-like cells,which indicated that dicalcium silicate may be also a favorable bioactive biomaterial.Partâ…¢Study of the ionic dissolution products of dicalcium silicate coating on osteoblastic mineralisationObjective To explore the influences of ionic dissolution products of dicalcium silicate coating on osteoblastic mineralisation.Method MG63 cells were cultured in 24 wells plates accordingly and samples were collected and detected on days 3, 6,9 and 12,respectively.Alizarin Red S was used for staining of mineralisation for morphorlogic observation,and calcium mineral deposition was detected by a quantifying AR-S assay.Results AR-S staining showed that on day 3,cells in the experimental group already exhibited obvious staining,while those in the control group was very faint. On day 6,faint staining in the control group could be observed,while cells of the experimental group showed of much obvious staining compared to that in the control group,and also was stronger than that on day 3 in the same group.On day 9,cells of the control group increased a little when compared to that on day 6,while increased markedly in the experimental group during the same period.Although AR-S staining increased a lot when comparing cells in the control group on day 12 to day 9,the staining was still a little faint.While there was much stronger AR-S staining in the experimental group,and some portions with dense staining were also could be observed on day 12.Importantly,some very dense accumulation of AR-S staining which have formed focuses could be randomly observed in the experimental group that mostly like to represented the centre of nodule on day 12.What is more,calcium mineral content quantification showed that little mineral depostion was formed in the control group,while more mineral formation could be found in the experimental group on day 3.Then the mineral contents increased in both groups, but much more mineral deposition have formed in the experimental group when compared to those of the control group from day 6 to 12.Differences between the two groups on the 4 timepoints were all statistically significant.Conclusion Except for the ability of promoting proliferation and differentiation,ionic dissolution products of dicalcium silicate coating was also able to promote mineralisation of MG63 osteoblast-like cells,which may be mainly because of the high Si concentration in the conditioned DMEM.Partâ…£Study of the mechanisms of osteogenic capacity of ionic dissolution products of dicalcium silicate coatingObjective To explore the possible mechanisms of ionic dissolution products of dicalcium silicate coating in promoting osteogenic capacity in vitro.Method MG63 cells was cultured accordingly and samples were collected on the predetermined timepoints respectively.ELISA assay was used to detect BMP2 and PGE2 expression,biochemistry method was used for the detection of NO production,and quantitative RT-PCR was used to detect the gene expression of BMP2 and its' signal transducers Smadl,6,7,respectively. Results BMP2 protein and mRNA expression were all increased in the two groups, which were higher on all of the time points in the experimental group although were not significant between the two groups.Gene expression of Smadl,which is a Co-Smad for BMPs signal tranduction increased markedly on day 9 and day 12 in the experimental group when compared with that of the control group(p＜0.05).And no obvious changes of the two inhibitary Smads,Smad6 and 7 existed in the two groups on all of the 4 time points.Accordingly,NO production in the control group exhibited no obvious change with the progression of culture time,but was increased with the progression of culture time in the experimental group and begin to decrease on day 12,with significant differences exist on days 6,9 and 12 between the two groups.PGE2 contents in the two groups were all decreased with the progression of culture time,but which were higher in the experimental group,especially on the late 3 timepoints.Conclusion Ionic dissolution products of dicalcium silicate coating may promote the osteogenic capacity of MG63 osteoblast-like cells via upregulate expression of BMP2 and Smadl,while have no influences on the gene expression of Smad6 and Smad7.What is more,NO and PGE2 are also likely contributed to the positive effects of ionic dissolution products of dicalcium silicate coating in promoting osteoblastic metabolism.