| ObjectiveOur study focused on improving soft tissue healing and inhibiting bacteria invasion in transmucosal part of dental implant to make sure the long-term survival of dental implant.Besides,we hoped to identify an optimal set of dimension and antibacterial coating for microgroove surface,the mechanism for "contact guidance" and search for surface material that has admirable biocompatibility,superior exposure "contact guidance" activity and antibacterial properties for transmucosal part of dental implantMethodsPART 1:Microgroove titanium surfaces were fabricated by photolithography with parallel grooves:15?m,30?m or 60?m in width and 5?m or 10?m in depth.The groups that used different microgroove surfaces were denoted as T15/5,T15/10,T30/5,T30/10,T60/5,and T60/10.Smooth titanium surfaces(TO)were used as controls.Physicochemical properties such as surface topography,microroughness and hydrophilicity were detected.HGFs from primary culture were cultured on the topographically modified surfaces.Morphology of "contact guidance" was analyzed using CLSM and SEM.Differences in cell proliferation and cell cycle progression were analyzed and compared using CCK-8 and Flow cytometry.Expression level of focal adhesion(FAs)related structural proteins(Intergrin a5,Intergrin ?1,vinculin)were compared among different groups by Real-time PCR and western blotting.The difference in activation of "contact guidance" related proteins(p-FAK and GTP-RhoA/ROCK)on different surfaces were also compared.The optimal set of dimensions for microgrooves was determined according to the comprehensive results.The mechanism for "contact guidance" and crosstalk between different signal pathways were elucidated using related inhibitors that inhibit signal protein activation.PART 2:Depending on the optimal dimensions for microgroove selected in PART 1,TiN and Ag coatings were deposited onto microgroove surface by magnetron sputtering.Smooth titanium surface(Ti-S)was used as the controls.The coatings applied in the experimental groups were antibacterial Ag-S and TiN-S coatings for smooth surfaces and antibacterial Ti-MG,Ag-MG and TiN-MG coatings for groove surfaces.The morphology of microgroove,morphology and roughness of nanocoatings,hydrophilicity,elemental analysis and release of Ag+ from Ag coatings were detected to characterize physicochemical properties.The differences regarding biocompatibility,"contact guidance" and antibacterial effect against Porphyromonas gingivalis(Pg)for different antibacterial coating were compared.Results1.Photolithography successfully fabricated titanium surfaces with different micro-groove morphology.Microgrooves in all the groups were arranged in typical anisotropy parallel.The microroughness for all the microgrooves was higher than that for the TO surface,with the highest microroughness showed in T60/10 microgroove and lowest in the T15/5 microgroove.Furthermore,T60/5 microgroove had the highest surface hydrophilicity.However,T15/10 microgroove had the highest surface hydrophobicity,even higher than TO surface.2.Different groove surfaces induced different Cell proliferation and cell cycle progression.Cell adhesion significantly increased at early incubation and cell proliferation was significantly improved on T60/10 microgroove surface,Besides,the cells proliferated for the longest period,with cells in cell cycle of phase S occupied the highest percentage.3.Surfaces of microgrooves with different dimensions achieved "contact guidance"for the cultured HGF,but with varied degree.In term of cell lineage consistency,T60 microgrooves ranked the first and T15 microgrooves ranked the last.Highest cytoskeleton deformation rate was observed on the T60/10 surface.4.Focal adhesion related structural proteins(Intergrin a5,Intergrin ?1,vinculin)expressed highest on T60/10 microgroove surfaces.5.Surfaces of microgrooves with T60 width had the poorest plasma FN absorption but expressed the highest cellular FN.6.For expression of signal proteins related to "contact guidance"(p-FAK and GTP-RhoA/ROCK),T60/10 group had the highest level.Difference in expression of p-FAK between T30 group and T10 group was not significant.T15 group had higher expression level of GTP-RhoA compared to T30 group,but with lower expression level of ROCK.7.T60/10 appears to be optimal for the transmucosal part of the dental implant.The inhibition of P-FAK didn't induce significantly effect on Cell lineage consistency and GTP-RhoA activation didn't be affected.Respectively inhibited RhoA-GTP/ROCK induce significantly effect on Cell lineage consistency,ROCK inhibition resulted in most effective in disturbed“contact guidance",RhoA-GTP inhibition made also reduced the FAK phosphorylation.8.Ag/TiN antibacterial nanocoatings were successfully deposited on smooth and T60/10 microgroove surfaces by magnetron sputtering technology,which not only maintained the smoothness and micro-morphology but also endowed the surfaces with new physicochemical properties.TiN-MG coating showed the smallest nanoroughness,however Ag coating showed the highest.Furthermore,TiN-MG had the highest surface hydrophilicity compared with Ag-S that had the highest hydrophobicity.XPS analysis revealed interstitial nitrogen and oxygen on the TiN coating.For Ag coating,lowest oxygen concentration caused the decrease of hydrophilicity.The quantity of Ag+ released from microgroove surface was higher than that from the smooth surfaces.The release of Ag+ was all higher than 0.1ppb within a continuous 7 days.9.Different coatings affected cell cycle progression and cell proliferation.Cell proliferation and percentage of S phase cells on Ag coating were less than that on other coatings.Cell proliferation and percentage of S phase(cell cycle)cells were highest on TiN-MG coatings.Difference in expression of focal adhension related structural proteins among different coatings were significant.Expression of intergrin a5,Intergrin ?1,Vinculin and FN was lowest on Ag coating.But TiN-MG coating enjoyed the highest expression.Ag-MG and TiN-MG coatings successfully guided the cell to arrange linearly along the microgrooves and the difference in linearity consistency rate was not significant.p-FAK and ROCK expressed higher on all the coatings when compared to those on the smooth surface.ROCK expression did not change with chemical composition on the surface.However,expression of p-FAK was lower on Ag-MG coating when compared with Ti-MG and TiN-MG coatings.10.Ag coating had the strongest antibacterial activity,followed by TiN coating.Under the same coating,microgroove morphology did not increase the amount of bacterial adhesion.Conclusions1.Among all the anisotropic microgroove surfaces,T60/10 surface showed highest hydrophilicity.T60/10 surface benefited cell proliferation and cell cycle progression the most,promoted "contact guidance" the most,and enjoyed the highest cell lineage consistency and cytoskeleton deformation rate.Furthermore,expression of focal adhesion related structural proteins and "contact guidance" related signal proteins were also the highest.Although with lower plasma FN absorption compared to other surfaces,T60/10 surface had higher cellular FN,which functionally compensated plasma FN.A microgroove with width of 60?m and depth of 10?m appears to be optimal for the transmucosal part of the dental implant.2.Using the magnetron sputtering successfully achieved both microgroove morphology(T60/10),which has the function of antibacterial nanocoating,Ag-MG surface which showed poorer in cell compatibility but highest in antimicrobial properties.However TiN-MG has certain antibacterial performance,optimal cell biocompatibility and keep the induction“contact guidance " for HGF.So TiN nanocoating is recommended with T60/10 microgrooves for transmucosal part of dental implant. |
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