Effects Of Micro-arc Oxidation-hydrothermal TLM Alloy On The Biological Behaviors Of Human Gingival Fibroblast

In recent years, a new type of near beta TLM titanium alloy had been developedby the Northwest Research Institute. While good implant materials required abiological activity, which contributed to the early adhesion, proliferation,differentiation, more protein secretion in periodontal tissue and bone tissue cells, thusmaking the surface modification of the implant had being a hot topic.Beside good osseointegration, peri-implant soft tissue healthy had played a vitalrole in maintaining the long-time success of dental implants as well. The peri-implantjunctional epithelium was attached to the implant surface by hemidesmosomes andbasal plate which were similar to natural teeth. The combination of implant andconnective tissue was controversial. Therefore, as the main cell in gingival connectivetissue, the study of gingival fibroblasts on the biological behavior of microarcoxidation hydrothermal treatment of TLM titanium alloy surface was guided to thefurther clinical application.Objective：TLM alloy surface bulid by Micro-arc Oxidation-hydrothermal technology wasformed a ceramic coating which contains TiO₂and HA. Of this part was to evaluatethe effect of surface modification on the behaviors of human gingival fibroblast, andto explore the relationship between the material and the surrounding soft tissues. Thuspreliminary investigation was studied in the influence of LPS on the secretion of IL-6and MMP-1by HGF which was cultured in vitro. Early inflammatory response invitro to a MAO-HS surface of TLM alloy was evaluated, which providedexperimental basis for future clinical application. Methods:With the help of micro-arc oxidation hydrothermal treatment technology, thesurface of TLM alloy had formed a layer of rough and porous structure, with a certainthickness and strength of TiO₂ceramic coating. And through hydrothermal treatment,nanoscale HA oxide film was prepared on the TiO₂ceramic oxide layer foundation.The surface topography, roughness, phase structure and wettability were detected byfield emission scanning electron microscopy, X-ray diffraction analysis, energyspectrum analysis, and the hydrophilic experiment.Primary cultured human gingival fibroblasts were cultured on theTLM-MAO-HS and smooth TLM alloy surfaces. The proliferation of HGF and earlyadhesion and the spread of cell morphology were measured by CCK8, immunofluore-scence microscopy, confocal laser scanning microscopy.The secretion of IL-6、MMP-1of HGF cultured on different surfaces weredetected by ELISA under the LPS induced.Results:（1） A TiO₂/HA ceramic oxide layer was formed on the surface of the titaniumalloy by micro-arc oxidation and hydrothermal treatment，which was observed that avolcano and holes of2-5μm diameter by SEM and nanometer needle-shaped crystalswhich were hydroxyapatite including calcium and phosphorus ratio was1.58uniformed distribution on the surface. The roughness of TLM-MAO-HS alloy was1.59±0.056μm by the surface roughness measurement which was belong to microngrade. Surface wetting angle of MAO-HS titanium alloy was superior to smoothgroup.（2） Early cell adhesion: Cells were cultured on the TLM-MAO-HS andsmooth TLM alloy surface after2h,4h,8h, and the numbers of cells attached to thesurfaces were quantified using the DAPI assay, which was counted by thefluorescence microscope. Cultured after4h、8h, the numbers of cells in the MAO-HSgroup were much more than smooth group, and there was a statistical signification （P<0.05）. Cell morphology: Observed the morphological spread of cells cultured onthe MAO-HS and smooth TLM alloy surface by CLSM after24h, it was found thatHGF was a spindle growth in the MAO-HS group which spread area was larger than the smooth group, and the intracellular actin staining was cleared. Unlike MAO-HSgroup, HGF grew along polishing lines in smooth group which was a slender spindle.After6h, it was observed that cells wered rounded but not fully extended byScanning electron microscope. While cells in MAO-HS group were extendedfilopodia and pseudo. After24h cells wered spreading in smooth group, while thespread of HGF was larger in MAO-HS group. Cell proliferation: The cellproliferation in the MAO-HS group was detected by CCK8after HGF was culturedon the titanium surface after1d,3d,5d,7d. There was no difference within eachgroup after1d. But the cell proliferation in MAO-HS group after3d,5d,7d washigher than the other, which has significance in statistics.（3） The secretion of IL-6、MMP-1of HGF cultured on different surfaces weredetected by ELISA under the LPS induced after6h、24h、48h. Results showed thatthe expression of IL-6secreted by HGF plated in MAO-HS group after6h was morethan smooth group, but without statistical significance. But after24h、48h, therewas a statistical significance （P <0.05）. To detect the secretion of MMP-1, it wasresearched that the expression secreted by HGF in TLM-MAO-HS after6h、24h、48h was more than other groups, which was no statistical significance.Conclusions:Using microarc oxidation hydrothermal treatment technology，a micron porous,rough surface surface is formed on the near beta TLM titanium alloy, which isdistributed uniformly by HA on the ceramic oxide layer surface. The Ca/P ratio is1.58. TLM-MAO-HS alloy can promote the adhesion, spreading of HGF andproliferation. TLM-MAO-HS titanium alloy do not significantly increase the earlyinflammatory response in vitro.