The Osteogenic Effect Of Different Silicon Content Of Silicon-doped Titanium Dioxide Nanotubes

Part I Preparation of graded silicon loaded nanotubular structuresObjective:To prepare different silicon content of silicon-doped titanium dioxide nanotubes?Si-TiO₂-NTs?by anodic oxidation and electron beam evaporation?EBE?technique,and to detect the characterization of the novel film.Method:Firstly,the titanium dioxide nanotube layers?TiO₂-NTs?were prepared on titanium by anodic oxidation.And then,the different content of silicon was doped into TiO₂-NTs by EBE.The surface morphologies were observed by scanning electron microscopy?SEM?and atomic force microscopy?AFM?.The elemental compositions of the samples were measured by X-ray photoelectron spectroscopy?XPS?.The wettability of the samples was illustrated by water contact angle measurement using a camera system.Result:The Si-TiO₂-NTs films were prepared successfully and the concentration of Si was about0.81wt%,3.86 wt%?6.48 wt%,respectively.The surface topography was not altered by the addition of Si.The contact angle was 36.9±2.03°on TiO₂-NTs,and 32.2±1.64°,26.2±5.4°and 20.7±4.6°on EBE-2 min,EBE-5 min and EBE-8 min,respectively,which indicated that the Si-doped show the better wettability with a lower contact angle.Conclusion:The surface topography was not altered apparently after the doping of Si,but chemical composition of the Si-TiO₂-NTs was changed.Water contact angle of the surface decreases and shows better hydrophilicity with the doping of silicon.Part II The influence of different silicon content of silicon-doped titanium dioxide nanotubes on osteoblastic adhesionObjective: To study the influences of different silicon content of silicon-doped titanium dioxide nanotubes on adhesion of osteoblast-like cells.Method:EBE-2 min,EBE-5 min and EBE-8 min served as experimental group;TiO₂-NTs and Ti served as control group.The release curve of silicon was determined by ICP-AES.The protein adsorption and LDH activity were measured by the kit.After incubation for 30 min,60 min and 120 min,the cells were stained with DAPI and then counted for cell adhesion.At 2 and 24 h,actin stress fibers and nucleus were double labeled with fluorescent dye to observe the actin cytoskeleton.At 24 h,cell spreading was detected by SEM.At 24 h,the cell migration was observed by transwell plate.Result: The cumulative release of EBE-2 min,EBE-5 min and EBE-8 min was 1.32 ppm,2.49 ppm and 3.14 ppm,respectively after culturing 14 days.There was no significant in protein adsorption and LDH activity among all samples.Compared with the control group,the early cell adhesion of the experimental groups was not significantly affected.However,the number of cells adhered to the EBE-8 min group was higher than that of the other groups after 120 min incubation.After 2 h of culture,no significant difference of actin stress fibers was observed.When the incubation time was increased to 24 h,actin stress fibers and vinculin immunofluorescence on Si-TiO₂-NTs films were significantly stronger than those on TiO₂-NTs,and the EBE-8 min was the best.The SEM observed that cellular filopodia spreaded better and the coverage area covered larger on Si-TiO₂-NTs surfaces after 24 h of culture,and EBE-8 min was the best.Cell migration experiments showed that the number of cells in the Si-TiO₂-NTs was more than that in the TiO₂-NTs and Ti groups,and the number of EBE-8 min migrated was the highest.Conclusion: Si-TiO₂-NTs had no obvious cytotoxicity but had not significant promote the early adhesion of osteoblasts.Si-TiO₂-NTs can also promote the spread of osteoblasts and the expression of cytoskeleton,indicating that Si-TiO₂-NTs have good biocompatibility.Si-TiO₂-NTs can significantly promote the cell migration with the increase of silicon content.Among them,EBE-8 min group showed the best performance in promoting cell spreading,skeleton expression and cell migration.Part III The influence of different silicon content of silicon-doped titanium dioxide nanotubes on osteoblastic proliferation and apoptosisObjective: To study the influences of different silicon content of silicon-doped titanium dioxide nanotubes on proliferation and apoptosis of osteoblast-like cells.Method: EBE-2 min,EBE-5 min and EBE-8 min served as experimental group;TiO₂-NTs and Ti served as control group.MC3T3-E1 cells were seeded on TiO₂-NTs and Si-TiO₂-NTs surfaces at the same density.After 1,4 and 7 days of cultivation,MTS assay was used to detect the number of cell proliferation.PI single staining was used to detect the cell cycle.The percentages of viable and apoptotic cells were determined from the plotting pattern of the cellular population by a flow cytometry analysis.Result: At 4 days,The MTS results showed that the number of cells was the highest on the TiO₂-NTs,but the number of cells on EBE-5 min was less than that of the EBE-8 min and EBE-2 min.When cultured to 7 days,the number of cells TiO₂-NTs and EBE-8 min was the highest,and EBE-8 min was slightly higher than TiO₂-NTs.The percentage of S+G2M was slightly higher than EBE-2 min and EBE-5 min,but there were no significant difference?p>0.05?.Flowcytometric analysis showed that no the apoptotic rate of EBE-8 min was lowest?p<0.05?,and there was no significant difference in the early apoptosis rate among other groups?p>0.05?.Conclusion: MC3T3-E1 cells on the EBE-8 min displayed better proliferation than those on the other samples.Moreover,EBE-8 min promote osteoblast proliferation ability by up-regulating the proportion of S + G2 / M phase cells and inhibiting the early apoptosis rate.Part IV The influence of different silicon content of silicon-doped titanium dioxide nanotubes on osteoblastic differentiationObjective: To study the influences of different silicon content of silicon-doped titanium dioxide nanotubes on differentiation of osteoblast-like cells.Method: EBE-2 min,EBE-5 min and EBE-8 min served as experimental group;TiO₂-NTs and Ti served as control group.The ALP activity of samples was detected after culturing 7 and 14 days.The expression levels of the five differentiation-specific genes including ALP,Col-I,Runx2,OPN and OCN were measured by real-time quantitative PCR.The expression of OPN protein was determined by El ISA protein assay kit.Alizarin red staining was used to observe the mineralization of extracellular matrix on different samples.Result: After 7 day of culture,the ALP activity of EBE-8 min was the highest,and that of EBE-2 min was higher than that of EBE-5 min and TiO₂-NTs.The ALP activity of EBE-5 min was lower than that of TiO₂-NTs,but there was no significant difference between the EBE-5 min and TiO₂-NTs?P > 0.05?.The ALP activity of all samples was slightly decreased after culturing 14 days.The ALP activity of EBE-8 min was highest,and the difference was statistically significant?p <0.01?.Runx2,Col-1,OCN and OPN genes expression levels were similar in EBE-2 min and EBE-5 min by RT-PCR at 7 days.Those were lower than EBE-8 min and TiO₂-NTs,and the difference was statistically significant?p <0.05?.When culture to 14 days,the expression of related osteogenic genes was slightly decreased.However,the Runx2,Col-1,OPN and OCN expression levels of EBE-8 min were still higher than those of other groups.Samples EBE-2 min,EBE-5 min and EBE-8 min induce mineralization and the effects on EBE-8 min are most obvious due to the nanostructure and proper amount of incorporated Si.Conclusion: EBE-8 min which shows excellent osteogenic properties is very attractive and has larger clinical potential.Part V The differential expression of micro RNA with silicon-doped titanium dioxide nanotubes on osteoblastic differentiationObjective: To study the differential expression of micro RNA with silicon-doped titanium dioxide nanotubes on osteoblastic differentiation and further analyze the biological function and possible mechanism of silicon in osteoblast differentiation.Method: EBE-8 min served as experimental group,TiO₂-NTs served as control group.After 7 days of culturing,the cell were collected,and the expression of miRNA was analyzed by miRNA chip.The difference of miRNA was screened.miR-125b-5p,miR-199a-5p,miR-331-3p,miR-125a-5p,miR-345-5p;miR-760-3p,miR-324-3p,miR-139-3p,miR-345 and miRNA-709 was measured by RT-PCR.Through the target gene prediction,and further analysis of GO,Pathway and other Bioinformatics.Result: The results of miRNA microarray showed that there were 92 differentially expressed between EBE-8 min and TiO₂-NTs,of which the expression of the 38 genes was up-regulated and the expression of was down regulated.The results of RT-PCR that 8 micro RNAs,including miR-125b-5p,miR-199a-5p,miR-331-3p,miR-125a-5p,miR-345-5p;miR-760-3p,miR-324-3p and miR-139-3p,was consistent with the results of the chip detection.The coincidence rate is 80%.Target Scan,PITA,micro RNAorg databases were used to predict target miRNAs.There were 1962 pairs of miRNAs.Mi R-324-5p,miR-199a-5p,miR-331-3p,miR-125a-5p;miR-760-3p,miR-324-3p,miR-139-3p,miR-125b-5p and miRNA-542-5p can predict the target genes.GO and pathway bioinformatics analysis of the target gene indicates that most target genes are involved in gene transcription and PI3K-Akt signaling pathway.Conclusion: The mechanism of Si regulation of osteoblast differentiation may be up-regulated expression miRNAs,including miR-125a-5p,miR-125b-5p,miR-199 a and miR-331-3p,and down-regulation in miR-760-3p,miR-324-3p and miR-139-3p.Most target genes were involved in gene transcription and PI3K-Akt signaling pathways.