| BackgroundAs we known that the surface characteristics of biomedical implants play a key role in cell or extracellular matrix interactions with the biomedical implants and eventual osseointegration. In tissue engineering and biomaterial field, the interaction between cells and microtopographies has been extensively studied, and some micronscale surface topographies and related technologies have already been adopted on commercial implant products. It has been revealed that microtopographies can improve bone-to-implant contact by such means as mechanical interlocking and promote osteoblast functions by these microtopographies. Some micronscale surface topographies have already been used on commercial implant products and good clinical effect has been obtained, for example sandblasting and large-grits etching (SLA) treated titanium implants. Nevertheless, micronscale surfaces inhibit osteoblast proliferation although they are positive in promoting osteoblast differentiation, resulting in a smaller bone mass accumulated compared to a smooth surface. With the progress of surface treatment technology, researcher can get different nanotopographies. The interactions between cells and nanotopographies are of increasing interest as a nanostructures may be more efficient in promoting cell functions. Including titania nanotubes have attracted much attention. This is because titania nanotubes can be fabricated easily with precisely controlled diameters and lengths by tailoring the process parameters. Such surfaces can almost ideally be used as nanoscale spacing models for size-dependent cellular response. Some studies reported that titania nanotubes with the suitable tube dimensions have been detected to improve bone cell functions, although there is still some controversy. These nanotubes can also serve as carriers for drugs for instance growth factors, antibacterial agents and genes and show good prospects in bone implant applications.As above, different group have different opinion about titania nanotubes influence cell functions. So how to precisely use titania nanotube with different diameter it is a chanllenge for us. Especially for dental implant, which kind of size is best for osteoblast, and little is known about the osseointegration effects of titania nanotubes in vivo. These questions need further research.From the biomimetics standpoint, natural tissues are hierarchical structures assembled in a highly organized way composed of nano-, micro-, and macro-scale building blocks, of which bone tissues constitute a good example. Bone tissues are composed of nanostructures including non-collageneous organic proteins, fibrillar collagen and hydroxyapatite crystals, microstructures including lamellae, osteons and Haversian systems, as well as macrostructures such as cancellous and cortical bones. So a hybrid structure composed of micro-and nanoscale components may offer a more favorable surface topography for cell functions as it can better imitate the structure of the natural extracellular matrix.Despite these advances, little is known about the hybrid structure used in biomedical domain, Kubo and coworkers produced a micropit-and-nanonodule hybrid titania topography on the titanium surface which improved the attachment, spreading, adhesion, proliferation, and differentiation of osteoblasts. Tan et al. have developed micro-and nanoscale structures by depositing nanostructured hydroxyapatite on a microscale architecture created by microfabrication technology and these structures enhances some selective cellular functions. In our earlier research, we get a microscale surface by SLA, nanotube with different diameters were impose on this microscale surfaces by anodization, then we can get a hybrid structure, but the studies about this structure is absent. Whether further enhancement of cellular behaviors and responses can be obtained by the addition of nanotubes to microstructures without sacrificing the established advantages of microstructures, and what size range of nanostructures when resided with microstructures is most effective in creating a bioactive environment, are largely unknownIn this study we fabricate a micronscale surface by SLA, and then nanotubes with different diameter were imposed on this microrough surface, we hope this hybrid surface to overcome the shortcomings of the two surfaces and combination with the advantages of both, control different cellular functions by different nanotubes, and improve the osseointegration.Objective:1. To investigated the effect of different sizes of TiO2nanonodules on their in vitro bioactivity.2. To investigated what size range of nanotubes was imposed on the microstructures surface is most effective in creating a bioactive environment in vivo.Methods:1. Specimen preparationCommercially pure titanium discs,15mm in diameter and1.5mm in thickness, were prepared, and treated by SLA to get microrough surface.Then nanotubes with different diameters(30nm、50nm、80nm) were imposed on this microrough surface get the hybrid surface.2. Surface characterizationThe surface morphology of the discs was examined with field emission scanning electron microscopy (FE-SEM)(Quanta400, FEI, USA) and Optical Profiler (Expert, BMT, German). The wettability of the discs was determined by means of an automatic contact angle measuring device (OCA15,Dataphysics, German).3. Protein adsorption assayBovine serum albumin was used as model protein. The protein concentration was quantified by means of a microplate reader at562nm. The amount of protein adsorbed by the specimens was determined from the difference in protein concentration between the later and initial protein solution.4. Osteoblastic cell cultureMG63osteoblast-like cells were used in these experiments. The cells were seeds onto the tested discs in24-well plates at a density of2.4×104cell/cm2.Initial attachment of cells was evaluated by measuring the amount of cells attached to titanium substrates after30min,1h and2h of incubation. The adherent cells were fixed with4%paraformaldehyde and then stained with the fluorescent dye Hoechst (nucleolus, Blue color; Sigma). Cell adhesion was evaluated by counting the number of stained nuclei on each sheet.FESEM was used to analyze the cell morphology3h and6h after the MG63cells had been seeded onto the different discs.After1h、3h and6h incubation, specimens stained with fluorescent dye rhodamine phalloidin. Fluorescence microscope was used to examine cell morphology and cytoskeletal arrangement of the osteoblasts seeds onto titanium surfaces.Cell proliferation was determined in terms of cell density on culture days1、3、5and7using MTS based colorimetry. The amount of formazan product was measured using a microplate reader at490nm.The expressions of osteogenisis-related genes were evaluated using the real-time polymerase chain reaction (Real-time PCR). The MG63cells were seeded with2×104cells/disc and cultured for7and14days. Expressions of osteogenesis-related genes including runt-related transcription factor2(RUNX2), ALP and osteocalcin (OCN) were quantified using Real-time PCR.5. Animals and surgical proceduresEight beagle dogs were used as experimental animal. Four different surfaces implants were installed into each tibia by planned sequence, beagle dogs were sacrified at2,4weeks after the operation. The tibiaes with implants were harvested for histological analysis.6. Histological analysisBone-implant contact (BIC) and new bone area (BA) were calculated to estimate the ability of osseointegration about different groups.Statistics analysisThe experimental data are expressed as mean±standard deviation. A one-way ANOVA was used to assess the statistical significance of the results between groups. All statistical analyses were performed using SPSS13.0software. P<0.05was considered stastically significant and P<0.01was considered to be highly stastically significant.Results:1. By SLA we get a microrough surface. Nanotubes with different diameters(30nm、50nm、80nm) were imposed on this microrough surface by tailoring the process parameters and get the hybrid surface.2. Contact angle measurement indicated that the surface of SLA group exhibited hydrophilic property with a contact angle of11°. But other three groups with a contact angle of0°Show a superhydrophilic property.3. The protein adsorption rates of different groups are presented as a percentage relative to the total amount incubated for different times. SLA+30nm group had the highest protein adsorption rate of all the groups regardless of incubation time. The protein adsorptive rate of SLA+30nm group reached18%after one hour of incubation, and approximately38%after incubation for6hours. SLA group showed the lowest protein absorption rate.4. Initial cell adhesion on fifferent groups was estimated by counting the number of MG63cell nuclei stained with Hoechst dye. The numbers of cells adherent on the SLA surface were significantly smaller than those adherent to other group. The numbers of cells adherent to SLA+30nm is the highest were about72%and90%after30min and2h, respectively. SLA group showed the lowest initial cell adhesion rate at different incubation times. Cell adhesion on the SLA+50nm surface is obviously higher than that on the SLA+80nm surfaces with a statistical difference.5. Cell proliferation on the specimens during the first7days of incubation has been assessed. SLA+30nm group have the highest OD valves at four different incubation times. There is no obvious difference among other samples.6. After3h and6h of incubation, FE-SEM was used to examine the morphology of MG63cells cultured on four different groups. At the time point of3h the shape of MG63cell are irregular, some filopodia begin stretching out except SLA group. After6h of incubation, the MG63cells attached and spread well on the surfaces of all four different groups, especially on the surface of SLA+80nm group, with a lot of lamellipodia and filopodia stretching out. In contrast, on the surface of SLA group cell just stretching out few filopodia. Fluorescence microscope was also used to examine cell cytoskeletal arrangement, and get the same resule with FE-SEM.7. The gene expressions on the different group surfaces have been quantified using Real-time PCR. In general, gene expressions on the different surfaces show a time dependent pattern. After7days of incubation, for the genes ALP and OC, the group of SLA+80nm group induces the highest expression followed by the SLA+50nm group surface. But the gene Runx2SLA+50nm group induces the highest expression followed by the SLA+80nm group. SLA+30nm group always exhibits the lowest levels. After culturing for14days, the group of SLA+80nm group yields highest expressions for ALP and OC. The gene Runx2SLA+50nm group still induces the highest expression and followed by the SLA+80nm group.8. The implants and osseointegration of peri-implant were showed in the undecalcified sections with Toluidine blue stain. At2weeks after implantation, SLA+80nm group implants showed significantly increased BIC compared to other groups(P<0.01), followed by SLA+50nm group、SLA+30nm group and SLA group. After4weeks implantation, SLA+80nm group still keep the highest expression. Coincident with BIC SLA+80nm group showed the highest expression of BA both at2and4weeks.Conclusion1. Hierarchical hybrid micro/nano-textured surface topographies are produced on titanium using common and easy sandblasting and large-grits etching combined with anodization.2. The micro/nano-textured surface structures to induce different biological reactions from MG63cells according different diameter nanotubes. Nanotubes with smaller diameters induces higher initial cell adhesion and proliferation, but larger diameter nanotubes induces higher osteogenesis-related gene expressions. 3. The group of SLA+80nm surface which could greatest improve titanium implant osseointegration at an early stage of bone development. |
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