The Influence Of Silicon Incorporated Porous Tio₂Coating On Bioactivity Of Osteoblast-like Cell In Vitro

Objective: To detect the characterization of the porous and silicon incorporated TiO₂coating (Si-TiO₂),and to explore the effects of the novel coactings on the adhesion and proliferation of MC3T3-E1cells.Method: Si-TiO₂coating was prepared on titanium by micro-arc oxidation (MAO) technique in a Si, Ca,and P containing electrolyte. The microstructure, composition and phase of all the samples werecharacterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and Energy dispersivespectroscope (EDS), respectively. Osteoblast-like cells were cultured on Si-TiO₂, TiO₂and Ti,respectively. After one-day culture, cell morphology and cytoskeletal arrangement were observed by aconfocal laser scanning microscopy; the cell spreading was detected by scanning electron microscopy(SEM) after12and24h of culture; and the proliferation rate of cells after1,4,7, and14days ofculture was determined using MTT assay.Result: Homogeneous layers appeared on the surface of TiO₂and Si-TiO₂coatings without crack, andall the pores on the oxidized layers were spherical with inner diameters of about3μm. No obviousdifferences in topography were observed between the TiO₂and Si-TiO₂coatings. XRD results indicatedthat the incorporation of Si did not alter the phase composition of the TiO₂and Si-TiO₂coatingsapparently. Results from confocal laser scanning microscopy showed that the well-developed actinfilaments were apparent within MC3T3-E1cells on Si-TiO₂coatings; The SEM indicated that cellsspreaded well on Si-TiO₂coatings; The MTT results illustrated that Si-TiO₂coating could stimulate thecells to proliferate faster than Ti plate and TiO₂coating.Conclusion: Porous and silicon-incorporated TiO₂coatings can be prepared on titanium by MAOtechnique without changed its characterization, and we proved that the Si-TiO₂coating was able topromote the adhesion and the early but not the late proliferation of MT3T3-E1cells. Objective: To evaluate the effects and possible mechanisms of porous silicon incorporated TiO₂coating(Si-TiO₂) on the early differentiation and osteogenic gene expression of osteoblasts.Methods: Osteoblast-like cells were cultured on Si-TiO₂, TiO₂and Ti for1,4,7and14days,respectively. Then, cell differentiation was detected by measuring ALP activity in cultured cells.Osteogenic gene expression (ALP, Runx2, COL-I) was evaluated using real time PCR technology.Result: There was a low level of ALP activity on day1under three culture conditions followed by anincrease on day4and day7. Of note, there was a significant increase of ALP activity of cells onSi-TiO₂coating on day7and a weak decrease on day14. Additionally, there was no difference for ALPactivity of cells on day14among these samples. The gene expression of ALP on the TiO₂group and Tigroup in the culture course showed statistically lower than that of Si-TiO₂group on day4and7(p<0.05). On day14, although ALP activity was still higher in the Si-TiO₂group than those in TiO₂group and Ti group, the difference was already not significant. The gene expression of Runx-2on Tiplate, TiO₂coating, and Si-TiO₂coating had low and similar expression levels on day1and day4.However, there was a significant increase on day7and day14(p<0.05). The expression levels of type Icollagen on Ti plate, TiO₂coating, and Si-TiO₂coating showed no significant difference on day1and4,and were significantly higher than those of TiO₂group and Ti group on day7and day14(p<0.05).Conclusion: The Si-TiO₂coatings were able to promote early differentiation and up-regulateosteogenic-related gene expression of MC3T3-E1osteoblast-like cells as well as new bone formation,indicating that Si-TiO₂coating may be a favorable bioactive biomaterial. Objective: To evaluate the effects and possible mechanisms of porous silicon incorporated TiO₂coating(Si-TiO₂) on mineralization and specific protein expression of osteoblasts.Methods: Osteoblast-like cells were cultured on Si-TiO₂, TiO₂and Ti for1,4,7and14days,respectively. Then, RT-PCR and Western-blot were applied to detect the mRNA and protein levelsof BSP and OCN, respectively.Result: Results of Real-time PCR showed that the mRNA levels of BSP and OCN from cells on Si-TiO₂coating are similar on day1and day4as those of on Ti plate and TiO₂coating. However, there was asignificant increase on day7and day14. Compared with Ti plate and TiO₂coating, Si-TiO₂coating tended to stimulate their expression, especially BSP mRNA expression. Results of Western-blot showedthat the protein levels of OCN and BSP from cells on Si-TiO₂coating increased more significantly thanthose on TiO₂coating and Ti plate on day7and remained high at day14. These results further indicatedthat the Si-TiO₂coating was superior to the TiO₂coating and Ti plate in supporting the mineralization ofMC3T3-E1cells.Conclusion: The Si-TiO₂coatings were able to promote mineralization and up-regulate specific proteinexpression of MC3T3-E1osteoblast-like cells, which may be related to the introduction of silicon in theSi-TiO₂coatings. Objective: To investigate the effects and possible mechanisms of porous silicon incorporated TiO₂coating on cell apoptosis of osteoblastsMethods: Osteoblast-like cells were cultured on Si-TiO₂, TiO₂and Ti for1,4,7and14days,respectively. Cell apoptosis was detected by measuring caspase-3activity in cultured cells. The numberof apoptotic cells was evaluated using Hoechst staining, and the apoptosis rate was detected using flowcytometry.Result: Results from Hoechst staining showed that the number of apoptotic cells on Ti plate, TiO₂coating, and Si-TiO₂coating had an increase trend from day1to day7, and had a decrease trend fromday7to day14. Compared with Ti plate and TiO₂coating, Si-TiO₂coating tended to stimulate theirapoptosis. There was no difference for caspase-3activity of cells on day1among these samples.However, the protein levels of caspase-3from cells on Si-TiO₂coating were significantly higher in theSi-TiO₂group than in the two other groups on day4,7, and14(p<0.05). The apoptosis rate of cells onTi plate, TiO₂coating, and Si-TiO₂coating was low and similar on day1. However, there was asignificant increase on day4and day7with the Si-TiO₂group increasing most obviously. These resultsindicated that the Si-TiO₂coating was superior to the TiO₂coating and Ti plate in inducing theapoptosis of MC3T3-E1cells.Conclusion: The Si-TiO₂coatings were able to induce apoptosis of MC3T3-E1osteoblast-like cells in the early and mid-term. Objective: To detect the possible signaling pathway which enhanced osteogenic activity on poroussilicon incorporated TiO₂.Method: Osteoblast-like cells were cultured on Si-TiO₂, TiO₂and Ti for1,4,7and14days,respectively; and the mRNA expression of Lrp5,Dkk1,ERK1,ERK2,c-fos were measured by real-timePCR.Result: The obtained results showed that the Lrp5mRNA level had a weak increase on day4and asignificant increase on day7and day14. Compared with TiO₂and Ti coating, Si-TiO₂coating tended tostimulate the expression of Lrp5to a higher extent. In contrast, the increased Dkk1mRNA levels wereonly observed for cells on Ti plates, while decreased Dkk1expression of cells on the TiO₂and Si-TiO₂coatings was observed on days4,7and14. Also, the Dkk1expression levels in Si-TiO₂group weresignificantly lower when compared with those in TiO₂and Ti coating (p<0.05). The mRNA levels ofERK1were significantly higher in the Si-TiO₂group than those of TiO₂group and Ti group on day7and14but not on day1. No significant difference of ERK2mRNA levels was detected at each timepoint. The c-fos mRNA levels on Si-TiO₂were significantly higher than those of TiO₂and Ti on day1,4,7,14, and the highest c-fos mRNA level was detected on Si-TiO₂coating on day4.Conclusion: The enhanced osteogenic activity on the Si-TiO₂coating may be mediated by the Wnt andMAPK signaling pathway.