Bone Growth Stimulated By Silicon-incorporated TiO₂ Nanotubes On Titanium Surface In Vivo

Part I Preparation and Characterization of silicon-incorporated Ti O₂ nanotubes on titanium surfaceObjective: To prepare Si-incorporated TiO₂ nanotubes films on titanium surface by anodization and silicon plasma immersion ion implantation and detect the characterization of the novel film.Method: Ti O₂ nanotubes were prepared by anodization and Si-incorporated Ti O₂ nanotubes were produced by Si-PIII with a filtered cathodic arc plasma source.The surface morphologies both films were observed by scanning electron microscopy?SEM?.The elemental compositions of both films were measured by X-ray photoelectron spectroscopy?XPS?.The wettability of the samples was illustrated by water contact angle measurement using a camera system.The surface energy was determined with one non-polar liquid and two polar liquids.Result: The Si-incorporated Ti O₂ nanotubes were prepared successfully and the concentration of Si was about 0.51wt%.There was no difference in topography between the films,which showed that,the surface topography was not altered by the addition of Si.The contact angle was 35.89±1.38°on Ti O₂-NTs films and?11.25±0.88°on Si-Ti O₂-NTs films,which indicated that the SiTi O₂-NTs films show the better wettability with a lower contact angle.The corresponding surface energy of Si-Ti O₂-NTs films was increased than that of Ti O₂-NTs films.Conclusion: The surface topography and phase composition were not altered apparently after the doping of Si,but chemical composition of the Si-Ti O₂-NTs film surface was changed.Water contact angle of the surface decreases and shows better hydrophilicity with the doping of silicon.The surface energy of Si-Ti O₂-NTs films was improved.SiTi O₂-NTs film with strong bond might possess significantly enhanced bioactivity and provides potential options for surface modification of implants in the future.The SiTi O₂-NTs film is worth further consideration for orthopedic implant applications.Part II The influence of silicon-incorporated Ti O₂ nanotubes on the adhesion and activity of OsteoblastsObjective: To study the influences of silicon-incorporated Ti O₂ nanotubes on adhesion of MC3T3-E1 cells.Method: Si-Ti O₂-NTs film served as experimental group;Ti O₂-NTs and Ti film served as control group.MC3T3-E1 cells were seeded on Ti?Ti O₂-NTs and Si-Ti O₂-NTs film surfaces at the same density.After 1 d,3 d,5 d of cultivation,samples were collected for detection.MTS was used to measure the number of cell attachment;At 12 h,actin stress fibers and nucleus were double labeled with fluorescent dye to observe the actin cytoskeleton.At 12 h,vinculin,actin stress fibers and nucleus were labeled in triple with fluorescent dye to observe the focal adhesion and actin cytoskeleton.After 1 d,3 d and 5 d,cell spreading was detected by SEM;Result: The result of MTS showed that after 1 and 3d of culture,most cells attached onto both SiTi O₂-NTs and Ti O₂-NTs films,and no significant difference of the number of cells was observed.After 5d of culture,most cells attached onto Si-Ti O₂-NTs films than Ti O₂-NTs films?p<0.05?.After 12 h of culture,better cell morphology of cells attached on SiTi O₂-NTs films and Ti O₂-NTs films than Ti films.The actin filaments seemed to be better developed within MC3T3-E1 cells on Si-Ti O₂-NTs substrates than on Ti O₂-NTs substrates.SEM images of MC3T3-E1 pre-osteoblastic cells after 1 day of cultivation,cells on the Ti showed a flattened morphology;meanwhile these on Ti O₂-NTs and Si-Ti O₂-NTs were round and irregular in shape.Moreover,finger-like filopodia extensions could be observed on the Si-Ti O₂-NTs.At day 3,cells on the Ti were elongated and flattened morphology.Filopodia extensions could also be observed on the Ti O₂-NTs,and an increased filopodia activity had been shown on the Si-Ti O₂-NTs,of which cells began to merge.At day 5,all three surfaces of samples were covered with cells.From the images,much more mesh-like filopodia were appeared on Ti O₂-NTs than Ti,while many filamentous network structures with excellent intercellular attachment could be found on Si-Ti O₂-NTs.Conclusion: At day 5,cells on Si-Ti O₂-NTs were more than Ti O₂-NTs significantly,indicating an enhanced cell proliferation after Si incorporation Moreover,cells on the Si-incorporated nanotubes showed the best filopodia activities,indicating this hydrophilic surface was beneficial for cell adhesion.The novel Si-Ti O₂-NTs film was able to promote adhesion behavior of MC3T3-E1 cells.Part III The influence of silicon-incorporated Ti O₂ nanotubes on the mineralization and osteogenic genes of OsteoblastsObjective: To study the influences of silicon-incorporated Ti O₂ nanotubes on the mineralization and osteogenic genes of Osteoblasts Method: Si-Ti O₂-NTs film served as experimental group;Ti O₂-NTs film and Ti served as control group.MC3T3-E1 cells were seeded on Si-Ti O₂-NTs,Ti O₂-NTs film and Ti film surfaces at the same density,and cells were cultured on three film surfaces.After 1 week of cultivation,samples cultured on three film surfaces were collected for detection of Col-I?ALP?Runx2?OCN?OPN of MC3T3-E1 cells by real-time quantitative PCR.MC3T3-E1 preosteoblasts were harvested at Week 1,2,and 3,protein expressions of Runx2,ALP and Col-I in cells cultured on both film surfaces were measured by western blot analysis.After osteogenic induction for 3 and 4 weeks,extracellular matrix?ECM?mineralization of cultured cells on the samples was evaluated by Alizarin Red S staining.Result: the expression of all above genes in the cells cultured on Si-Ti O₂-NTs for 7 days was significantly stronger than that of cells on Ti and Ti O₂-NTs substrates?p <0.01?.Therefore,the osteogenic activity of cells was improved when they were cultured on Si-Ti O₂-NTs compared to both Ti and Ti O₂-NTs.An enhanced secretion of Runx2 was observed on Si-Ti O₂-NTs compared to Ti O₂-NTs and Ti at week 1 and 3,and levels of ALP expression were obviously increased on Si-Ti O₂-NTs than Ti O₂-NTs at week 1.The protein expression of Col-I was shown with no significant difference among these samples.Compared to Ti disks,cells on Ti O₂-NTs and Si-Ti O₂-NTs showed an elevated calcium mineral nodule formation at both 3 and 4 weeks?p<0.05?.Moreover,the deposited calcium mineral on Si-incorporated samples exhibited significantly improved than those on nanotubes at 4 weeks?p < 0.05?.Conclusion: Compared with Ti O₂-NTs substrates,the Si-Ti O₂-NTs can up-regulate the expression of osteogenic genes and Runx2,ALP protein expression.Si-Ti O₂-NTs can increase the level of osteoblast extracellular matrix mineralization Si-Ti O₂-NTs film was able to promote differentiation of MC3T3-E1 cells,which indicated that Si-Ti O₂-NTs film may be also a favorable bioactive biomaterial.Part? The signal transduction passway mediating osteoblastic activity on Si-Ti O₂ film prepared by cathodic arc depositionObjective: To study the effects of Si-incorporated Ti O₂ nanotubes films on osteogenesis by various biological characterization techniques in vivo.Method: Implantations were divided into three groups as follows: Ti?pure titanium screws?,Ti O₂-NTs?titanium screws modified by Ti O₂ nanotubes?,and Si-Ti O₂-NTs?titanium screws modified by Si-incorporated Ti O₂ nanoubtes?.Each rat received one implant in each femur.Pull-out tests were performed for all implants at 2,4 and 6 weeks after surgery.The bone-screw interface and trabecular micro-structure of specimens were analyzed by micro-CT and histological.Result: The peak pull-out forces increased approximate 18% and 21% on Si-implanted samples than nanotubes at week 2 and 4,respectively?P<0.05?.At week 6,there were no statistically differences among the three kinds of surfaces.Compared to Ti screws,%OI was increased about 65% and 54%,and the bone volume ratio?BV/TV?about 4.2-fold and 2.4-fold,the mean trabecular thickness?Tb.Th?about 88% and 86%,the mean trabecular number?Tb.N?about 1.3-fold and 12%,as well as decreased the mean trabecular separation?Tb.Sp?about 65% and 54% on Si-Ti O₂-NTs and Ti O₂-NTs,respectively.Extensive bone apposition could be found between the screw threads at the spongy level on the Si-Ti O₂-NTs and Ti O₂-NTs.And more bone contacts was appeared on the internal part of the Si-Ti O₂-NTs screw compared to the Ti O₂-NTs,indicating a new bone trabecular formation on the Si-Ti O₂-NTs screw implant surface.Conclusion: In vivo osseointegration studies revealed that early-stage new bone formation and the bone contact ratio were promoted on titanium screws modified by Si-incorporated Ti O₂ nanotubes films,indicating the ability to accelerate osseointegration of Si-incorporated films.