Gentamicin-Coated Hydroxyapatite in Prevention of Biofilm Formation in Bone Tissue

Biofilm is a multilayered complex microorganism, which attaches on any surface and is typically more resistant to the host immune response and routine antibiotic therapy. In order to limit biofilm formation, biomaterials loaded with suitable antibiotics can be used as a preventative measure. The biomaterial hydroxyapatite (HA) is an osteoconductive space filler and is produced locally by Malaysia Nuclear Agency. In this study, HA coated with the antibiotic gentamicin was explored whether it can reduce or remove biofilm formation. To assess IC50 values of gentamicin-coated HA, 108 CFU/ml of Staphylococcus aureus (ATCC 12600) and Pseudomonas aeruginosa were cultured for 48 hours in a 96-well plate for biofilm formation. MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide) assays were performed to determine the effect of various doses of gentamicin (0 mg/ml, 0.02 mg/ml, 0.04 mg/ml, 0.06 mg/ml, 0.08 mg/ml, 0.1 mg/ml and 10 mg/ml) coated on a constant number of HA particles on viability of S. aureus and P. aeruginosa biofilm. It was demonstrated that the IC50 values of gentamicin-coated HA were 0.1 mg/ml for S. aureus and 5 mg/ml for P. aeruginosa biofilm. Fluorescence staining with acridine orange and propidium iodide (AOPI) was also conducted to visualize viability of the biofilm. Accordingly, the doses of 0.1 mg/ml and 5 mg/ml for S. aureus and P. aeruginosa biofilm respectively decreased cell viability, as cells showed higher PI staining. In an attempt to determine the possible cytotoxic effects of gentamicin-coated HA on human cells, the human osteoblast cell line (NHOst, Lonza) was co-cultured with the doses of gentamicin (0 mg/ml, 0.1 mg/ml, 1 mg/ml and 10 mg/ml) coated on HA particles as tested above for biofilm cytotoxicity. Cell viability of osteblasts decreased with increasing doses of gentamicin when assessed at 72 hours using MTT assay (for example, 10 mg/ml gentamicin-coated HA reduced osteoblast cell viability to 75%). The efficacy of gentamicin-coated HA was also tested in vivo. A Teflon catheter was used to create catheter-associated biofilm segments for in vivo implantation. Catheter-associated biofilm were examined with scanning electron microscope (SEM) to confirm S. aureus biofilm formation. The catheter-associated biofilm was then implanted subcutaneously into the right flank of Sprague Dawley rats. Rats were sacrificed after 7 days post-implantation and the catheters were removed and assessed for bacteria count. This study showed that the gentamicin-coated HA significantly reduced S. aureus bacteri count from 14.12 ± 1.09 log10 CFU/ml to 4.61 ± 0.49 log10 CFU/ml (p≤0.05). To investigate the structure of biofilm formation in vivo post-implantation, tissues immediately surrounding the implanted catheter was histologically assessed using haematoxylin and eosin (H&E) staining. The result obtained from H&E staining showed no inflammatory cells or tissue damage was observed. Thus, this study showed that gentamicin-coated HA is effective in reducing biofilm viability without causing overt toxicity to human osteoblasts in vitro or inflammation when implanted in skin.

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