Methods

We describe a method for the study of bacterial adherence in the presence of preosteoblastic cells. For this purpose we mixed different concentrations of bacterial cells from collection and clinical strains of staphylococci isolated from implant-related infections with preosteoblastic cells, and analysed the minimal concentration of bacteria able to colonise the surface of the material with image analysis.

Objectives

Biomaterial-related infections are a major clinical problem. The pathogenesis of this syndrome has been described as a competitive adherence between bacteria and human cells in the so-called “race for the surface” theory. The aim of this study is to develop an in vitro protocol method that can be used to investigate this competence and its implications in the development of materials aimed to be used in orthopedic surgery.

MATERIAL AND METHODS

A 18mm diameter rod of Ti–6Al–4V alloy ELI grade according to the standard ASTMF136-02 supplied by SURGIVAL was cut into 2 mm thick disk specimens, ground through successive grades of SiC paper to 1200 grade, degreased with a conventional detergent and rinsed in tap water followed by deionised water. The specimens were then chemically polished (CP).

The disks were anodized only on one side by using a two electrode cell in a suitable electrolyte. TiO₂ barrier layers, without fluoride (BL), were produced by anodizing in 1 M H₂SO₄ at 15 mA cm-2 to 90 V, reaching 200 nm of thickness.

Fluoride barrier layers (FBL) were produced in an electrolyte containing 1 M NH₄H₂PO₄ and 0.15 M NH₄F, at constant voltage controlled at 20 V for 120 min at 20°C; the thickness of the layer is 140 nm.

Laboratory biofilm-forming strains of Staphylococcus aureus 15981 [6] and Staphylococcus epidermidis ATCC 35984 were used in adherence studies, which were performed using the protocol by Kinnari et al [7]. Photographs obtained were studied by ImageJ software. Statistical analysis was performed by EPI-INFO software. The experiments were performed in triplicates