Antimicrobial effect of silver-embedded titania nanotubular implant surface against Porphyromonas gingivalis

Objectives: To fabricate and characterize silver and non-­silver embedded titania nanotubes surfaces (TiNT­Ag and TiNT) and to investigate their effect on Porphyromonas gingivalis biofilm attachment. Methods: TiNT and TiNT­Ag (0, 5 10, and 30 seconds reverse polarity) surfaces were fabricated on commercially pure Ti (CP­Ti) sheet by using a two step anodization procedure in an electrolyte composed of 80 % 1,2-­propane diol and 20% deionized water (v/v) with or without AgF (0.66%, w/v). For surface characterization, the analyses utilized were fourier transform infrared (FTIR) spectroscopy, energy­ dispersive X­ray (EDX) spectroscopy, field emission scanning electron microscopy (FESEM), white light interferometry (WLI), and water contact angle (WCA) analysis. For biofilm attachment, TiNT and TiNT­Ag surfaces were inoculated with P. gingivalis 33277 and incubated anaerobically at 37oC. After 12 hr, samples were stained with crystal violet and biofilm attachment was determined based on the amount of dye absorption (OD550nm). All assays were performed independently 3 times in triplicate. Data were analyzed by one­-way ANOVA and Tukey’s HSD test (α = 0.05).
 Results: FTIR analysis showed characteristic absorption peaks at 2200 ­ 2800 cm­1 for Ag+ and Ti+ interaction with CO2 and 500­1100 cm­1 for Ti­O and Ag­O bond stretch. FESEM revealed uniform titania nanotubes on surfaces with diameters ranging 80 – 100 nm. EDX spectra showed the presence of silver along with other known elements including titanium, oxygen, carbon and fluoride. The Ra and WCA at Day 0 of the CP-Ti, TiNT, TiNT-Ag-0, TiNT-Ag-5, TiNT-Ag-10, and TiNT-Ag-30 were 0.10±0.01 μm, 9.84±0.25 μm, 0.31±0.02 μm, 0.30±0.02 μm, 0.29±0.02 μm, and 0.37±0.03 μm, and 7.4 ± 2.4°, 8.05 ± 2.2°, 8.95 ± 4.7°, and 6.25°± 1.518°, respectively. Statistically significant reduction (>87%) in P. gingivalis attachment was noted on all TiNT­Ag surfaces compared with the non­-silver embedded TiNT surfaces (P<0.05). No significant differences in bacterial attachment were detected among the various TiNT­Ag groups. FESEM and live/dead stain of the inoculated surfaces confirmed the presence of bacteria on the surfaces and reduction of bacterial attachment on the silver surfaces. Conclusions: Silver-­embedded titania nanotubes were successfully fabricated on Ti surface by using the two step anodization procedure. Titanium nanotubular surfaces have increased surface wettability and surface roughness compared to commercially pure titanium surfaces. The incorporation of silver into the surfaces resulted in a more clinically desirable dental implant surface in terms of surface wettability, surface roughness, and uniform surface morphology. The surface architecture and characteristics of TiNT and TiNT-Ag were stable over 26 days. The titania nanotubular surface may have potential for improved bone response and ongoing tissue health. These surfaces suppressed the in vitro formation and attachment of P. gingivalis biofilm. The antimicrobial properties of silver-­embedded titania nanotubes surfaces may contribute to the reduction of bacterial colonization, reduced inflammation, reduced clinical peri-implant complication, and improved ongoing hard and soft tissue peri-implant health.