In-vitro testing of biofilm formation on infected bone grafts and bone substitutes

Background: Bacteria form biofilms on the surface of orthopaedic devices, causing persistent infections. Monitoring biofilm formation on bone grafts and bone substitutes is challenging due to heterogeneous surface characteristics. We analyzed various bone grafts and bone substitutes regarding their propensity for in-vitro biofilm formation caused by S. aureus and S. epidermidis. Methods: Beta-tricalciumphosphate (b-TCP, ChronOsTM), processed human spongiosa (TutoplastTM) and PMMA (PalacosTM) were investigated. PE was added as a growth control. As test strains S. aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984) were used. Test materials were incubated with 105 cfu/ml. After 24 h, test materials were removed and washed, followed by a standardised sonication protocol. The resulting sonication fluid was plated and bacterial counts were enumerated and expressed as cfu/sample. Sonicated samples were transferred to a microcalorimeter (TA Instrument) and heat flow monitored over a 24 h period with a precision of 0.0001°C and a sensitiviy of 200 μW. Experiments were performed in triplicates to calculate the mean ± SD. One-way ANOVA analysis was used for statistical analysis. Results: Bacterial counts (log10 cfu/sample) were highest on b-TCP (S. aureus 7.67 ± 0.17; S. epidermidis 8.14 ± 0.05) while bacterial density (log10 cfu/surface) was highest on PMMA (S. aureus 6.12 ± 0.2, S. epidermidis 7.65 ± 0.13). Detection time for S. aureus biofilms was shorter for the porous materials (b-TCP and Tutoplast, p <0.001) compared to the smooth materials (PMMA and PE) with no differences between b-TCP and TutoplastTM (p >0.05) or PMMA and PE (p >0.05). In contrast, for S. epidermidis biofilms the detection time was different (p <0.001) between all materials except between Tutoplast and PE (p >0.05). Conclusion: Our results demonstrate biofilm formation with both strains on all tested materials. Microcalorimetry was able to detect quantitatively the amount of biofilm. Further studies are needed to see whether calorimetry is a suitable tool also to monitor approaches to prevent and treat infections associated with bone grafts and bone substitutes.