Modification of titanium surfaces to enhance bacteriostatic properties

摘要

Introduction: Infections caused by bacterial biofilm on dental implants affect a considerable amount of patients. Anodic oxidation and physical vapor deposition are current methods in modifying the surface of implants in order to make them more resistant to bacterial biofilm formation. An easier and economically attractive method is soaking the surface in hydrogen peroxide (H2O2), sodium hydroxide (NaOH) and calcium dihyroxide (Ca(OH)2). This method is hypothesized to not only provide antibacterial activity, but also preserve the bioactive properties and biocompatibility of the surface. The aim of this study was to determine which surface treatment provides the best antibacterial effect, as well as examine if the addition of calcium ions results in additional bioactivity. Method: Discs of grade 2 Ti were punched into circular coins with diameter 9 mm and 1 mm thickness. The coins were sequentially sonicated for 15 min in acetone, ethanol and dH_2O, before being immersed in H2O2 for 1 h at 90°C. Subsequently the coins were soaked in NaOH for 15 min. The coins were divided in six test groups where three groups were further soaked in Ca(OH)2 for 15 min and then either heated at 200°C for 1 h (Ti200_Ca), autoclaved at 125°C for 1 h (TiAuto_Ca), or simply kept at room temperature for 1 h (Ti25_Ca). The remaining three groups received the same final heat treatment, but without the soaking in Ca(OH)2 (T1200 TiAuto and Ti25, respectively). The coins were then immersed in Dulbecco's PBS enriched with Ca2+- and Mg2+-ions in an incubator at 37°C for 7 days. The coin surfaces were examined for hydroxyapatite (HA) in a LEO 1550 scanning electron microscope. The coins were checked for H2O2 release by studying the degradation of the organic dye rhodamine B. Two cell lines; MC3T3 murine preosteoblasts and human dermal fibroblasts (hDF) were seeded onto the coins. Cell viability was measured after 3 days using the Alamar Blue assay. Results: The test groups soaked in Ca(OH)2 showed a higher degree of HA formation compared to the test groups not soaked in Ca(OH)2, see Fig. 1. Treatment in room temperature showed better HA formation than 200°C and autoclaving. The rhodamine B degradation test showed that the test groups Ti200_Ca, TiAuto_Ca, T125 and Ti200 showed approximately 30 % degradation after 7 days. The hDF cells had no observed changes in cell viability as compared to the control after 3 days. The MC3T3s, however, had greater proliferation on the modified coins, compared to the unmodified Ti. Discussion and conclusions: Calcium ion addition increased the bioactivity, providing more available sites for phosphate to bind to calcium. Preliminary tests with rhodamine B suggest an antibacterial activity of the modified surfaces. Future studies will be conducted to further investigate this potential effect with bacteria.