Osteoblast-like Cell Activity On Porous Silicon-incorporated TiO₂ Coating Prepared By Micro-arc Oxidation

Objective: To prepare porous and silicon-incorporated TiO2 coating(Si-TiO2) by micro-arc oxidation (MAO) technique,and to detect the characterization of the novel coacting. Method: Porous and silicon-incorporated TiO2 coating was prepared on titanium by MAO technique in a Si, Ca and P containing electrolyte. The surface morphologies and microstructure of the coatings were observed by scanning electron microscopy (SEM). Phase compositions of the coatings were characterized using X-ray diffraction (XRD). The surface roughness of the coatings was measured by a surface profiler (HOMMEL TESTER T800 Wave, Germany). The elemental composition and distribution of the coatings were measured by energy-dispersive X-ray spectrometry (EDS) attached to electron probe X-ray microanalysis system (EPMA). The cross-sectional structure of the coatings was was observed by EPMA in back-scattering mode. Tris-HCl buffer solution immersion tests were conducted to evaluate the apatite forming ability and bioactivity of coatings. Result: Both the TiO2 and Si-TiO2 coatings mainly consist of anatase phases, their surfaces exhibit grains of about 30-50 nm in size and pores about 3μm in diameter. No significant difference of surface roughness (Ra) was found between TiO2 and Si-TiO2 coatings. Ti, O, Ca and P elements in both coatings as well as the Si element in the Si-TiO2 coatings distributed homogeneously in the whole coatings. Ca, P and Si begin to release from the coatings once the coatings were soaked in the buffer solution. Conclusion The Si-TiO2 coating is worth further consideration for orthopedic implant applications. Objective: To explore the influences of porous silicon-incorporated TiO2 coating on adhesion of MC3T3-E1 cells. Method:Osteoblast-like cells were cultured on Si-TiO2, TiO2 and Ti respcetively. After 1, 4, 12, 24 h of cultivation, samples were collected for detection. MTT and PI were used to mesure the number of cell attachment; Cell spreading was detected by scanning electron microscopy (SEM); The expression of actin on each sample was investigated by with a microscope Olympus BX 51equipped with epifluorescence. Result: The result of PI showed that at 4 and 12 h, cell attached on Si-TiO2 than that on TiO2 and Ti. The MTT showed that after 4 h of culture, cell cell attached on Si-TiO2 than that on TiO2 and Ti(p < 0.05). The SEM indicated that cell spreaded well on Si-TiO2; The fluorescence micrographs of actin stress fibers in the cells on various substrates after incubation of 4 and 12 h demonstrated that well-defined actin stress fibers distributed through the body of cells cultured on Si-TiO2 coatings. Conclusion: It was proved that the novel incorporated Si and porous surface were able to promote adhesion behavior of MC3T3-E1 cells, suggesting that the Si-TiO2 coating is worth further consideration for orthopedic implant applications.Objective: To explore the influences of porous silicon-incorporated TiO2 coating on proliferation of MC3T3-E1 cells. Method: Osteoblast-like cells were cultured on Si-TiO2, TiO2 and Ti respcetively. After 1, 3, 5, 7 days of cultivation respectively, samples were collected for detection. cell cycles were analyzed by flow cytometry, and MTT assay and PI were used to detect cell proliferation. Result: The result of PI showed that at 3 and 5 days, cell number on Si-TiO2 was much more than that on TiO2 and Ti. MTT value of Si-TiO2 group was significantly higher than that of TiO2 group and Ti group on day 3 and day 5. With the progression of culture time, no differences statistically significant were observed between the three groups on day 7. Flowcytometric analysis showed that the percentage of cells in S phase of the Si-TiO2 group was obviously higher than that of the TiO2 group and Ti group on day 3 and day 5.. At the same time, cells in G0/G1 phase of the Si-TiO2 group was significantly lower than that of TiO2 and Ti group. while cells in G2/M phase of the Si-TiO2 group was higher than that of the TiO2 group and Ti group. Conclusion: Si-TiO2 was able to promote the early but not the late proliferation of MT3T3-E1 cells, which may be intimately related to the high concentration of Si ion and the porous structur. And the ability of Si-TiO2 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.Objective: To explore the influences of porous silicon-incorporated TiO2 coating on differentiation of MC3T3-E1 cells. Method: Osteoblast-like cells were cultured on Si-TiO2, TiO2 and Ti respcetively. After 1, 3, 5, 7 days of cultivation respectively, samples were collected for detection. ALP activity and gene expression of 3 markers of osteoblastic differentiation, e.g type I collagen, ALP and osteocalcin were investigated. Result: From day 3 on, ALP activity in the Si-TiO2 group was higher than that of the TiO2 group and Ti group. With the progression of culture time, ALP activity increased in all groups but was higher in the Si-TiO2 group. Statistically significant differences were observed on timepoints of days 3 and 5. On day 7, although ALP activity was still higher in the Si-TiO2 group than those in TiO2 group and Ti group, the difference was already not significant. ALP mRNA expression in the TiO2 group and Ti group in the culture course showed statistically low than that of Si-TiO2 group on day 3 and 5 (p<0.05). The mRNA levels of type I collagen showed no significant difference in all groups on day 1 and 3, while was significantly higher than those of TiO2 group and Ti group on day 5 and 7. The mRNA levels of osteocalcin showed no significant difference in all groups on day 1,3 and 5, while was significantly higher than those of TiO2 group and Ti group on day and 7. Conclusion: Si-TiO2 coating was able to promote late differentiation of MC3T3-E1 osteoblast-like cells, which indicated that Si-TiO2 coating may be also a favorable bioactive biomaterial. Objective: To explore the possible signal transduction pathway which mediated enhanced osteoblast-like cell activity on porous silicon-incorporated TiO2. Method: Osteoblast-like cells were cultured on Si-TiO2, TiO2 and Ti respcetively. After 1, 3, 5, 7 days of cultivation respectively, samples were collected for detection. Gene expression of integrinβ1,α1,α3 andα5 , focal adhesion kinase (FAK), and extracellular regulated kinases (ERK, including ERK1 and ERK2) were measured by real-time PCR. Result: The obtained results showed that gene expression of integrinβ1 on Si-TiO2 was significantly higher than that of TiO2 and Ti at 4,12 and 24 h. Gene expression of integrinα5 on Si-TiO2 was significantly higher than that of TiO2 and Ti at 12 and 24 h. There was no significant difference of gene expression of integrinα1, andα3 between the three groups. The gene expression of FAK on Si-TiO2 was significantly higher than that of TiO2 and Ti at 4,12and 24 h. The gene expression of ERK1 on Si-TiO2 was significantly higher than that of TiO2 and Ti at 4,12and 24 h, while no significant difference of ERK2 gene expression was detected at each time-piont. Conclusion: The enhanced cell activity on Si-TiO2 coating may be mediated by integrin(subunitsβ1 andα5) binding and subsequent signal transduction pathway (involving FAK and ERK1).