Preparation Of Bioactive Titanium Surface By Acid And Heat Treatments After Alkali Treatment And The Research Of Its Bioactivity In Vitro

Objective In this study, acid and heat treatments after alkali treatment was applied to build up a bioactive layer with nano-network structure on the surface of pure titanium. Mouse bone marrow stromal cells(BMSCs) were cultured with treated titanium plates to evaluate the effect of modified titanium surface on the biological activity of BMSCs in vitro.Material and methods Titanium plates with thickness of 0.25 mm were polished with silicon carbide sandpaper(800 mesh to 7000 mesh) to achieve the mirror effect, and washed sequentially with acetone, ethanol and ultra pure water for 15 min respectively in an ultrasonic cleaner and then dried overnight in an oven at 60 ℃. Afterwards, the polished titanium plates were soaked in a 5mol/L Na OH solution at 60 ℃for 24 h, followed by gently washed with ultra pure water and dried in an oven. Then the alkali treated titanium plates were soaked in a 5mmol/L HCL solution at 40 ℃ for 24 h, and then gently washed with ultra pure water and dried in an oven. Then the samples were heated at 600 ℃, maintained for 1h, then cooled naturally to room temperature in the furnace. The surface morphology and elemental composition have been analyzed by Field emission scanning electron microscope(FE-SEM) and X-ray energy dispersive spectroscopy(EDS), respectively. BMSCs and modified titanium plates were co-cultured to evaluate the effect of modified bioactive layer on the cell adhesion, proliferation and differentiation.Results FE-SEM showed that the surface of polished titanium(PT) was quite rough with clearly visible scratches, and a fine network structure of nanometer-scale was generated on the surface by alkali and heat treatment(AH), and the structure remained essentially unchanged by alkali-acid-heat treatment(AAH). The result of EDS shows the element on the surface of the polished titanium is Ti and Si, and the surface of the AH group contain Ti, Si, Na and O elements, meanwhile, the surface of the AAH group contain Ti, Si and O elements. The growth of BMSCs on the surface of the PT group was general, less pseudopodia and limited spreading. The cells on the surface of the AH group spread well, with fusiform shape, meanwhile,some cell pseudopodia extend into the nano-network structure. The cells on the surface of the AAH group can also spread well, with fusiform shape. Compared with the AH group, the cells on the surface of AAH group spread largely, with more tentacles. The cells on the surface of specimen on 1st, 3rd, 5th day after inoculation showed growth movement in proliferative activity. There was no statistical significance on the 1st day. However, compared with the PT group, the differences in the proliferative activity in AH group and AAH group were statistically significant on the 3rd and 5th day(P<0.05). Compared with the AH group, the differences in the proliferative activity in AAH group were statistically significant on the 3rd and 5th day(P<0.05). Compared with the PT group, the differences in AKP activity in AH group and AAH group were statistically significant on the 5th and 7th day(P<0.05). Compared with the AH group, the differences in the AKP activity in AAH group were statistically significant on the 7th day(P<0.05).Conclusion The experiment of cell culture in vitro confirms that the bioactive layer with a fine nano-network structure on the surface of pure titanium by alkali-acid-heat treatment can promote cell adhesion, proliferation and differentiation, showing good biocompatibility. Therefore, it is expected to be applied to the surface modification in the future.