| Objective Osseointegration between all pure titanium implants and around bone interface is the key for the success of the implant restoration. This study aims first to form double honeycomb Ti O2 nanotubes on pure titanium surface by virtue of two-step anodization method, then to treat double honeycomb Ti O2 nanotubes with polydopamine, which can act as intermediate layer coupling agent and further outward graft RGD peptides. Eventually, Ti O2 nanotubes-polydopamine-RGD peptide bioactive layers were built on pure titanium surface after above three-step treatment. Mouse bone marrow stromal cells(BMSCs) were cultured with treated pure titanium plates in vitro to evaluate the impact of modified titanium surface on the adhesion, proliferation and differentiation of BMSCs, to provide new ideas for the titanium implant surface biological modification.Material and methods 1,Titanium plates pretreatment and Ti O2 nanotubes preparation: Titanium foils with thickness of 0.25 mm were machined to diameter 12 mm titanium plates. Each titanium plates should be polished by metallographic sandpaper(800 mesh to 7000 mesh) to achieve the mirror effect, and then be cleaned ultrasonically in the acetone, ethanol and deionized water successively for 20 min respectively, followed by them drying in nitrogen flow. Afterwards, titanium plates were immersed into ethylene glycol electrolyte containing 88 mmol/L ammonium fluoride with titanium plates as the anode and graphite as cathode. Anodic oxidation was divided into two steps: first, energization at 60 voltages for 2.5 h, then remove the titanium plates into deionized water for ultrasonic vibration to remove the oxide film formed by anodic oxidation, followed by them drying in nitrogen flow; second, energization at 12 voltages for 40 min, then remove the titanium plates into deionized water for ultrasonic vibration until the solution clarification, followed by them drying in nitrogen flow. 2, Dopamine modification and RGD peptide loading: Titanium plates modified by electrochemistry process were immersed into 50 ml, 2 mg/ml dopamine solution for 12 h, keeping away from light and unobstructed airflow around. The modified titanium plates were then removed out of electrolyzer and were washed repeatedly with deionized water to remove residual unpolymerized dopamine, drying at room temperature. The above-mentioned titanium plates were then immersed in 30 ml, 200 mg/ml RGD peptide solution on a shaker at 37℃ for 12 h. The modified titanium plates were then removed out of electrolyzer and were washed repeatedly with deionized water to remove residual unbound peptides, drying at room temperature. 3, Experimental grouping design: The polished titanium plates were classified as smooth group; the titanium plates treated by anodization were classified as Ti O2 nanotube group; the titanium plates modified by dopamine soak treatment on the basis of Ti O2 nanotubes were classified as dopamine group; the titanium plates conjugated with RGD peptide grafted with dopamine were classified as RGD peptide group. 4, Analysis of surface morphology and elemental composition: Field emission scanning electron microscopy(FESEM) and X-ray photoelectron spectroscopy(XPS) were used to analyze surface morphology and elemental composition of titanium plates in each group. 5, Cytologic evaluation in vitro: BMSCs and modified titanium plates were co-cultured to evaluate the effect of Ti O2 nanotubes-polydopamine-RGD peptide active layer on the adhesion, proliferation and adhesion of BMSCs.Results 1, FESEM showed that the surface of titanium plates in smooth group was quite rough with clearly visible scratches; honeycomb porous Ti O2 nanotubes were seen on the surface of Ti O2 nanotube group and the outer tube is hexagonal and the diameter of inner and outer tube was about 20 nm and160nm, respectively; the diameter of the nanotubes in dopamine group was partially narrowed, further completely closed; scattered granular RGD peptides were visibly seen on the surface of titanium plates in RGD peptide group, partially into nanotubes. 2, Cell adhesion: The growth of BMSCs on the surface of smooth group was general, fat fusiform shape and limited spreading; the cells in Ti O2 nanotube group spread well but with few cell tentacles; the cells in dopamine group and RGD peptide group spread very well, thin fusiform shape and many cell tentacles and contacts between cells in RGD peptide group were more than that in dopamine group. 3, Cell proliferation: The proliferation vitality of BMSCs presented an increasing trend on the 1st day from smooth group to RGD peptide group, but the difference was not statistically significant. Compared with the smooth group, the differences in increasing proliferation activity in Ti O2 nanotubes group, dopamine group and RGD peptide group were statistically significant on the 3rd and 5th day(P<0.05). Compared with Ti O2 nanotubes group and dopamine group respectively, the differences in increasing proliferative activity in RGD peptide group were statistically significant on the 3rd and 5th day(P<0.05). 4, Cell differentiation: The AKP vitality of BMSCs presented an increasing trend on the 3rd and 5th day from smooth group to RGD peptide group, but the difference was not statistically significant. Compared with smooth group, the differences in increasing AKP activity in Ti O2 nanotube group, dopamine group and RGD peptide group were statistically significant on the 7th day, respectively(P<0.05). Compared with Ti O2 nanotube group and dopamine group respectively, the differences in increasing proliferative activity in RGD peptide group were statistically significant on the 7th day(P<0.05).Conclusion A Ti O2 nanotubes-polydopamine-RGD bioactive layer was successfully constructed on the surface of pure titanium. Cell culture in vitro showed that the bioactive layer was conducive to cell adhesion, proliferation and differentiation, showing good biocompatibility. The method of titanium surface functionalization modification is expected to improve titanium implants and also to improve osseointegration around titanium implants,which still needs animal studies for further validation. |
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