Nanofilms of adsorbed thymol formed on titanium surfaces for biomedical applications. Antimicrobial activity and biocompatibility

Abstract

Titanium (Ti) and its alloys are widely used in the construction of permanent orthopedic and cardiovascular implants. However, one of the most frequent causes of failures are bacterial infections by Staphylococcus aureus. This is aggravated by the abusive use of antibiotics that generate microbial resistance to conventional therapies. As a consequence, new antimicrobial nanotechnologies (AMN) emerge as promising alternatives to prevent prosthetic infections. The aim of this work was to evaluate the antimicrobial effect of an innovative AMN: thymol (TOH, phenolic phytocompound) nanofilms adsorbed on Ti (NPTOH-Ti) against S. aureus. The biocompatibility was also determined using preosteoblast cells (MC3T3-E1). To that end, 1 cm diameter grade 2 Ti discs were used and TOH was adsorbed onto their surface by 2 h immersion in 0.1 M TOH acid solution. NPTOH-Ti was detected by infrared spectroscopy (FTIR-ATR). The antibiofilm activity of NPTOH-Ti and Ti (control) was determined by immersing the metal discs in a suspension of S. aureus (108 bacteria/ml) for 3 h. Subsequently, the number of bacteria adhered on the discs was caunted after sonication by colony forming unit (CFU). In addition, Live/Dead (Invitrogen) staining was used to determine if the adhered bacteria were alive or dead. Finally, biocompatibility of NPTOH-Ti and Ti was assessed by staining the preosteoblast cells with acridine orange. The results showed that NPTOH-Ti has effective anti-biofilm properties. On the one hand, viable bacteria were not observed by the plating count method and Live/Dead staining exhibited only dead (red) bacteria on the surface. On the other hand, control Ti revealed 4 ± 0.5 x105 adhered bacteria that were mostly (95 %) alive (green). In addition, NPTOH-Ti and Ti showed similar cell adhesion and growth (107 ± 12 and 100 ± 16 % respectively; p>0.05). It was concluded that NPTOH-Ti are biocompatible and have anti-biofilm properties which make them promising to prevent prosthetic infections.