Osteoblast-like Cell Activity On Silicon-doped TiO₂Film Prepared By Cathodic Arc Deposition

Part â…  Preparation of silicon-doped TiO₂film prepared bycathodic arc depositionObjective: To prepare silicon-doped TiO₂（Si-TiO₂） and pure TiO₂film on titaniumsubstrates with strong bond by filtered cathodic arc deposition （FCAD） technique, and to detect thecharacterization of the novel film. Method: Si-TiO₂films were prepared on titanium by filtered Ti andSi cathodic arc plasma sources co-deposition while these deposited with double Ti cathodes werelabeled as TiO₂for comparison. The surface morphologies both films were observed by scanningelectron microscopy （SEM）. Phase compositions of both films were characterized using X-raydiffraction （XRD）. The elemental compositions of both films were measured by X-ray photoelectronspectroscopy （XPS）. The wettability of the samples was illustrated by water contact angle measurementusing a camera system. The surface energy was determined with one non-polar liquid and two polarliquids. Result: The Si-TiO₂films were prepared successfully and the concentration of Si was about4.6%. The XRD patterns showed that both films were amorphous and the peaks of the titanium substratewere clear. There was no difference in topography between the films, which showed that, the surfacetopography was not altered by the addition of Si. The contact angle was94±0.78°on TiO₂films and83±0.74°on Si-TiO₂films, which indicated that the Si-TiO₂films show the better wettability with a lowercontact angle. The corresponding surface energy of Si-TiO₂films was increased than that of TiO₂films.Conclusion: The surface topography and phase composition were not altered apparently after thedoping of Si, but chemical composition of the Si-TiO₂film surface was changed. Water contact angle ofthe surface decreases and shows better hydrophilicity with the doping of silicon. The surface energy ofSi-TiO₂films was improved. Si-TiO₂film with strong bond might possess significantly enhancedbioactivity and provides potential options for surface modification of implants in the future. The Si-TiO₂ film is worth further consideration for orthopedic implant applications. Partâ…¡The influence of silicon-doped TiO₂film prepared bycathodic arc deposition on osteoblastic adhesionObjective: To study the influences of Si-TiO₂film by cathodic arc deposition onadhesion of osteoblast-like cells. Method: Si-TiO₂film served as experimental group; TiO₂film servedas control group. MG63cells were seeded on TiO₂and Si-TiO₂film surfaces at the same density. After1,4,12,24h of cultivation, samples were collected for detection. CCK8was used to measure the numberof cell attachment; At4,12and24h, cell spreading was detected by SEM; At2and12h, actin stressfibers and nucleus were double labeled with fluorescent dye to observe the actin cytoskeleton. At12and24h, vinculin, actin stress fibers and nucleus were labeled in triple with fluorescent dye to observe thefocal adhesion and actin cytoskeleton. Result: The result of CCK8showed that after1and4h of culture,most cells attached onto both films, and no significant difference of the number of cells was observed.After12and24h of culture, the number of cells attached on Si-TiO₂film surfaces was larger than thoseon TiO₂film surfaces （P <0.05）. After2h of culture, no significant difference of actin stress fibers wasobserved. When the incubation time was increased to12and24h, actin stress fibers and vinculinimmunofluorescence on Si-TiO₂films were significantly stronger than those on TiO₂films. After4h ofculture, no significant difference of cellular morphology was observed on both films. The SEMobserved that cellular filopodia spreaded better on Si-TiO₂film surfaces than those on TiO₂filmsurfaces after12h of culture. After24h of culture, the percentages of cellular coverage area on Si-TiO₂film are larger than those on TiO₂film （P <0.05）. Conclusion: Cells cultured on Si-TiO₂films bycathodic arc deposition exhibited stronger F-actin and vinculin, which promoted cytoskeletonreorganization and the formation of focal adhesion. The novel Si-TiO₂film by cathodic arc depositionwas able to promote adhesion behavior of MG63cells. Part â…¢ The influence of silicon-doped TiO₂film prepared by cathodicarc deposition on osteoblastic proliferation and apoptosisObjective: To study the influences of Si-TiO₂film by cathodic arc deposition onproliferation and apoptosis of osteoblast-like cells. Method: Si-TiO₂film served as experimental group;TiO₂film served as control group. MG63cells were seeded on TiO₂and Si-TiO₂film surfaces at thesame density, and cells were cultured on both film surfaces at1,3and5days. After1and5days ofcultivation, samples were collected for detection. Cell morphologies were observed by SEM. Thepercentages of viable and apoptotic cells were determined from the plotting pattern of the cellularpopulation by a flow cytometry analysis. After1,3and5days of cultivation, CCK8assay was used todetect the number of cell proliferation. Result: At1and5days, the result of SEM showed that cellularnumber on Si-TiO₂film surfaces was much more than that on TiO₂film surfaces; Flowcytometricanalysis showed that no significant difference of the percentages of vital and apoptotic cells was foundamong MG63cells cultured on the TiO₂and Si-TiO₂film surfaces （P>0.05）. A quicker increase of theCCK-8value of cells cultured on the Si-TiO₂film surfaces was noticed when compared with the TiO₂film surfaces at1,3and5days （P <0.05）. Conclusion: MG63cells on the Si-TiO₂film surfacesdisplayed better proliferation than those on the TiO₂film surfaces through the increase of cellularnumber. Moreover, no significant difference of the percentage of apoptotic cells was found betweencells cultured on both film surfaces. Therefore, Si-TiO₂film may be a good bioactive coating. Part â…£ The influence of Si-TiO₂film prepared by cathodic arcdeposition on osteoblastic differentiationObjective: To study the influences of Si-TiO₂film by cathodic arc deposition ondifferentiation of osteoblast-like cells. Method: Si-TiO₂film served as experimental group; TiO₂filmserved as control group. MG63cells were seeded on TiO₂and Si-TiO₂film surfaces at the same density,and cells were cultured on both film surfaces. After1,3,5days of cultivation, samples cultured on bothfilm surfaces were collected for detection of ALP activity. COL I of MG63cells was stained and visualized by immunofluorescence microscopy after3and5days of cultivation. The expression levelsof the two differentiation-specific genes including COL I and OC were measured by real-timequantitative PCR after1,3and5days of cultivation. Immunoblotting test was used to detect COL Iprotein expression level between MG63cells cultured on both film surfaces after1,3and5days ofcultivation. Result: As time progressed, ALP activity was continuously increasing in both groups. After1day of culture, no significant difference of ALP activity was observed between MG63cells culturedon both film surfaces （P>0.05）. But the ALP activity of MG63cells on the Si-TiO₂film surfaces wassignificantly higher compared to those on the TiO₂film surfaces after3and5days of cultivationrespectively （P <0.05）. Immunofluorescence microscopy observed that widespread and abundant COL Iwas formed in cells cultured on the Si-TiO₂film surfaces after3and5days of culture. However, sparseand deficient COL I was observed in those cultured on the TiO₂film surfaces at the same time ofculture. After1day of culture, no significant difference of COL I and OC gene expression levels byreal-time quantitative PCR was observed between MG63cells cultured on both film surfaces （P>0.05）.After the osteoblasts were cultured for3and5days, higher COL I and OC gene expression levels incells were exhibited on the Si-TiO₂film surfaces than those on the TiO₂film surfaces （P <0.05）. COL Iprotein expression levels by western blot analysis in cells cultured on the TiO₂and Si-TiO₂film surfacesfor1day was not statistically different （P>0.05）. However, when the incubation time was increased to3and5days, the COL I protein expression in the osteoblasts cultured on the Si-TiO₂film surfacesobviously mounted （P <0.05）. Conclusion: On the Si-TiO₂film by cathodic arc deposition, MG63osteoblast-like cells displayed better differentiation through the increase of ALP viability, up-regulationexpression levels of OC gene, and COL I gene and protein. Si-TiO₂film was able to promotedifferentiation of MG63cells, which indicated that Si-TiO₂film may be also a favorable bioactivebiomaterial. Part â…¤ The signal transduction passway mediating osteoblastic activityon Si-TiO film prepared by cathodic arc depositionObjective: To study the possible signal transduction pathway which mediates enhancedosteoblast-like cell adhesiveness on Si-TiO₂film by cathodic arc deposition. Method: Si-TiO₂filmserved as experimental group; TiO₂film served as control group. MG63cells were seeded on TiO₂andSi-TiO₂film surfaces at the same density, and cells were cultured on both film surfaces. After1,4,12and24h of cultivation, samples were collected for detection. Gene expressions of integrin β1and FAKin cells cultured on both film surfaces were measured by real-time quantitative PCR. Proteinexpressions of FAK and the phosphorylation of FAK in cells cultured on both film surfaces weremeasured by western blot analysis. Result: After1h of cultivation, no difference in the gene expressionof integrinβ1and FAK was detected between MG63cells cultured on both film surfaces.（P>0.05）.However, after4h of cultivation, though no difference in the gene expression of integrinβ1wasdetected between MG63cells cultured on both film surfaces （P>0.05）; FAK gene expression wasup-regulated in cells on the Si-TiO₂films than that on the TiO₂films （P <0.05）. When cells werecultured on both films for12h and24h, higher integrinβ1and FAK gene expressions were detected incells on the Si-TiO₂films （P <0.05）. No statistically different of FAK and P-FAK protein expressionswas detected between MG63cells cultured on both film surfaces after4h of cultivation （P>0.05）.However, when the incubation time was increased to12h and24h, the protein expressions of FAK andP-FAK in MG63cells cultured on the Si-TiO₂film surfaces increased dramatically and expressedsignificantly than those cultured on the TiO₂film surfaces （P <0.05）. Conclusion: It was proved thatSi-TiO₂films were able to promote MG63cells’ adhesiveness compared to the pure TiO₂films. Theeffect of the Si-TiO₂film on cytoskeleton and focal adhesion was regulated by integrin β1and thephosphorylation of FAK. The enhanced cell adhesion may partly be mediated by integrin β1and FAKsignal transduction pathway.