Effects Of Different Titanium Alloy (Ti6Al4V) Surface Topographies On The Biological Behavior Of Human Gingival Epithelia

Objective：The success of dental implantation is not only depended onthe osseointegration, but also afavorable biological barrier betweenimplant and soft tissue. The topography of implant neck surface is one ofthe most important factors which can affect the soft tissue attachment.The purpose of this study is to evaluate the effect of three differenttitanium alloy surface characteristics on the biological behavior ofepithelia, in order to find the most proper surface which can promote theproliferation and attachment of the epithelia and enhance the soft tissueintegration, meanwhile, provide a guidance for the designation of theimplant neck part.Methods: Three different titanium alloy surface topographies werecreated by machining(M),direct current electrochemical etching(DC) anddirect and alternating current electrochemical etching(DAC) respectively.Their surface topographies were investigated by using field emissionscanning electron microscopy(SEM). Roughnesses were measured bylaser scanning confocal microscope. Through these methods we ensurethat the surface topographies were the same with previous experiments.Epithelial cells were co-cultured with these titanium alloys. Theproliferation of cells was measured using MTT assay, and cellmorphology were observed by SEM.Results: First, three different surface structures are obtained through three different methods. The results of SEM observation were as follows:The machined group had a relatively smooth surface with isotropicgrooves, the direct current electrochemical etching group had amicro-scale rough surface with micron holes and the alter currentelectrochemical etching group had a micro-submicro hierarchicalstructure.Second, the MTT assay shown that cells on all surfaces increased in atime-dependent manner in culture. At24h the OD value of M group wastwo-fold of DC and DAC groups, and had statistical significances. TheOD values of DC and DAC surfaces were similar to the M surface at the48h,72h and96h, and had no statistical significances among three groups.Cells on the M group disks showed the lowest rate, with approximately1.4-fold,2.4-fold and3.3-fold in proliferation at48h,72h and96h,relative to cell numbers at24h. Cells on the DC group disks showed thehighest rate compared to all other surfaces, with an approximately2.6-fold,4.5-fold and6.4-fold increase in proliferation at48h,72h and96h, relative to cell numbers at24h.Third, the observations of cell morphology were as followed: at24h hecells on the machined surface were similar to hemisphere, spread not verywell numerous microvilli and retained a slight swelling. Cells on the DCsurface were spread well with irregular shape, and had strongpseudopodia than the machined surface adhering to the pore. High-magnification micrographs of DC group surface showed cellspseudopodia were spanning over ridges and combined with each other.Cells morphology on DAC group surfaces were similar with DC group,high-magnification micrographs of DAC group surface revealed that thepseudopodia were adhering intimately to the submicron topography. At72h, cells on M group were more extended with polygon-shape anddiameter of12~14μm. The morphology changed from hemisphere to flat,and substantial binding to the disk surface. Compared with24h,microvilli of epithelia were bolder than24h, and cells connected to eachother. Cells on the DC surface were spread well with irregular shape likestars with diameter of6~8μm, and extend strong pseudopodia in alldirections. High-magnification micrographs of DC group surface showedcells pseudopodia were adhering to the pore, microvilli of pseudopodiaintimately adhering to the suface. Cells morphology on DAC groupsurfaces were similar with DC group with star-like and irregular shape.The surfaces were covered by the extracellular matrix. Highmagnification micrographs of DAC group surface revealed that thepseudopodia were adhering intimately to the submicron topography.Conclusion: The DC surface obtained by direct current electrochemicaletching and DAC surface obtained by direct and alternating currentelectrochemical etching had a good biocompatibility, which can promotethe proliferation of the epithelia cells. They could form strong adhesive with epithelia cells. This cell behavior may play a key role in maintainingthe peri-implant oral mucosal tissue barrier.