The Effect Of Different Pure Titanium Surface Treatment On The Proliferation Of Human Gingival Fibroblasts

Objective: Periimplant soft tissue attachment which called〝cuff〞form a firm biological containment barrier. The establishment of a firm functional periimplant soft tissue barrier is considered to be important to protect the implant's interface from invasion of bacteria, reduce marginal bone loss after implant. It is one of the fundamental factors to maintain the implant in long-term stability. Reasonable implant neck design shoud be benefit to the formation of periimplant soft tissue barrier for the success rate of plant improved. The purpose of this study was to find a proper surface treatment for soft tissue attachment by comparing the effect of three different pure titanium surface treatments on the proliferation of human gingival fibroblasts in vitro, and provide evidence for the design-modified of implant cervical part in clinic.Methods:1 Preparation and grouping of pure titanium discsTwenty-four slices pure titanium discs were prepared with 10mm in diameter, 1mm thickness and 0.8μm roughness, divided into 3 groups, and 8 slices each.Group A (TiO₂ particle blasted and HCl/H₂SO₄ etched group): titanium discs were blasted TiO₂ particle with 5Kpa atmospheric pressure and etched by HCl/H₂SO₄ mixed liquor.Group B (Al₂O₃ particle blasted and HCl/H₂SO₄ etched group): titanium discs were blasted Al₂O₃ particle with 5Kpa atmospheric pressure and etched by HCl/H₂SO₄ mixed liquor.Group C ( smooth group): the surface without any treatment.2 The surfaces morphology of three groups by SEMThree slices titanium discs obtained respectively from three groups were observed by SEM.3 Cultivation and identification of human gingival fibroblastsSpecimens of healthy gingival tissue without inflammation and hyperplasia were obtained from the exelcymosis of impacted tooth in clinic with average age from 18 to 25 years. Primary culture of human gingival fibroblasts was performed by using the tissue culture method. At firstly, epithelial cell confounding initially, adherence method was utilized repeatedly to depurate the gingival fibroblasts. In order to identify the cultured cells, immunocytochemical stains against antibodies Vimentin, Cytokeratin (CKs) andαstriated muscle actin staining were performed. Morphology of the cells of fourth passage was observed by HE staining.4 the cultivation of HGFs on different treated pure titanium surfaceThe fourth passage of fine growing HGFs were dispensed into unicell suspension with the density of 4×105 piece/ml and respectively vaccinated on different titanium surfaces of the three groups as well as the controlled slips surfaces. Then attachment and proliferation of human gingival fibroblasts was examined by Methylthiazoletrazolium (MTT) assay after 24 hours and the morphology and arrangement was observed by scanning electron microscope (SEM) after 3 days.5 The morphology and and arrangement of HGFs on different treatment titanium surfaces observed by SEM6 The cytoactive of HGFs on three groups of titanium discs by MTT assayThe mean absorbance values ( x±SD) of different titanium surfaces obtained by MTT assay received statistical analysis.7 Statistical analysisThe absorbance value that was determination by MTT assay was analyzed with SPSS 13.0 statistical soft. The comparison among three groups used one-way ANOVA test of multiple independent samples when gauss distribution and homoscedasticity corresponded, P<0.05 indicated results had significant differences, and the comparison between two groups used SNK test. On the contrary, the comparison among three groups used Kruskal-Wallis H test of multiple independent samples, P<0.05 indicated results had significant differences, and the comparison between two groups used Wilcoxon signed-rank test.Results:1 The surfaces morphology of three groups by SEMThe surface morphology of TiO₂ blasted and etched titanium disc and Al₂O₃ blasted and etched titanium disc were similar, which formed the first and second grade of pouches. superficialis pouches were saw on smooth titanium discs surfaces.2 The identification of HGFsUnder the inverted microscope, cells emigrated from the isolated tissue were stellate or spindle-shaped, radiating from the tissue edge. The cells possesed the characteristic that turgor vitalis of soma, pellucid cytoplasm and limpid nucleolus were observed by HE staining. Cells by immunohisto- chemical staining showed that: anti-vimentin staining was positive, the positive parts in the cytoplasm brown-stain showing. Anti-cytokeratin and anti-αstriated muscle actin staining were negative, proved that cells derived from mesoderm without confounding myogenic or epithelial cells, consistent with biological characteristic of fibroblast-like cells. The human gingival fibroblasts (HGFs) were cultured successfully in vitro.3 The morphology and and arrangement of HGFs on different treatment titanium surfaces observed by SEMCompare with the smooth surface without treated group, the attached cells were more on the pure titanium surface with particles sandblasted and HCl/H₂SO₄ etched treatment groups significantly.The cells on the smooth surface without treatment were thiner compared with the other two groups. The cells did not spread very well, same similar to round even, which had brevis processus pseudopodia and less extracellular matrix secreted. However the cells on the Al₂O₃ particle blasted and HCl/H₂SO₄ acid-etched surface were intensively distributed as well as the TiO₂ particle blasted and HCl/H₂SO₄ acid-etched surface. The cells of turgor vitalis spread well and had longer processus pseudopodia than the smooth surface with some pseudopodia putting in the pore, some pseudopodia could form three-dimension structure suspended in midair. More extracellular matrix secreted on the slightly rough surface. As comparison, the cells on the slide were thin and flat, fusiform shape with less pseudopodia expanded. It demonstrated that cell attachment was significantly stronger on the rough surface than on the smooth surfaces.4 Statistical analysisComparisons of the mean absorbance value ( x±SD) of HGFs cultivated on three groups for 24 hours by MTT assay showed that: the mean absorbance value of group A was 1.122±0.026, the mean absorbance value of group B was 0.772±0.079, the mean absorbance value of group C was 0.305±0.035. Treated with SPSS 13.0 statistical soft, the comparison among three groups used one-way ANOVA test of multiple independent samples, P <0.05. It indicated results had significant differences. The comparison between every two groups used SNK test had differences as well.Conclusion:1 All of three different suface treatment pure titanium have fine biological property human and gingival fibroblasts on them growth well.2 Grit-blasting and double acid-etching treatment is profit to adhesion of human gingival fibroblast and TiO₂ particles sandblasted and HCl/H₂SO₄ etched treatment better.