Abstract
Summary Statement
Description of an original in vitro protocol for assessing combined bacteria and cell competitive adherence on the surface of biomaterials of medical interest
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.
Methods
An in vitro study was performed using as a substrate a surface of chemically polished Ti6Al4V (CP) alloy ELI grade, according to the standard ASTM F136-02. For bacterial adhesion studies we used S. aureus 15981 and S. epidermidis ATCC 35984 strains. Bacteria were inoculated in tryptic soy broth with 5% CO2. After overnight culture, bacteria were centrifuged for 10 min at 3500g at 22°C. Supernatant was discarded and the pellet was washed × 3 with sterile PBS. Bacteria were then suspended and diluted in PBS to obtain a 108 CFU/mL concentration. Serial 1:10 dilutions were then made in order to obtain different bacterial concentrations (107, 106, 105, 104, 103 CFU/mL). Osteoblastic MC3T3-E1 cells at of 100.000 cells/mL were mixed (1:1, v/v) with the different bacterial dilutions in a total volume of 2 ml of α-MEM. A bacterial control (1 mL of 108 CFU/mL of bacteria + 1 mL of PBS), a cellular control (1 mL of 100.000 cells/mL + 1 mL of PBS) and a negative control (2 mL of PBS) were also included. A total of 9 CP specimens were covered with the different dilutions tested, and incubated for 6 h at 37°C to allow adhesion in a static model. After incubation, samples were washed three times with PBS, and fixed to the CP surface with MicroFIX spray during 1 minute. After that, they were washed again with sterile water, stained for 2 min with acridine orange, and rinsed with sterile water. Eight photographs were made for each sample at 40 × magnification to study the bacterial adhesion, and at 20X magnification to study the cellular adhesion, by using an ultraviolet microscope.
Results and Discussion
Bacterial adhesion was observed in the specimens inoculated with 108, 107, 106 CFU/mL, but not at lower dilutions. Cellular adhesion was observed for all samples. Differences in the cellular morphology were observed when the bacterial concentration decrease, probably because bacterial adhesion has a negative influence in the cellular adhesion to the surface. This protocol can be useful for the evaluation of the bacterial and cellular adherence process on modified biomaterials.
Conclusion
These results demonstrate that bacteria cannot colonise cell-covered material at a certain breakpoint.
