Copal bone cement is more effective in preventing biofilm formation than Palacos R-G

Abstract

Bone cements loaded with combinations of antibiotics are assumed more effective in preventing infection than bone cements with gentamicin as a single drug. Moreover, loading with an additional antibiotic may increase interconnectivity between antibiotic particles to enhance release. We hypothesize addition of clindamycin to a gentamicin-loaded cement yields higher antibiotic release and causes larger inhibition zones against clinical isolates grown on agar and stronger biofilm inhibition. Antibiotic release after 672 hours from Copal bone cement was more extensive (65% of the clindamycin and 41% of the gentamicin incorporated) than from Palacos R-G (4% of the gentamicin incorporated). The higher antibiotic release from Copal resulted in a stronger and more prolonged inhibition of bacterial growth on agar. Bacterial colony counting and confocal laser scanning microscopy of biofilms grown on the bone cements suggest antibiotic release reduced bacterial viability, most notably close to the cement surface. The gentamicin-sensitive Staphylococcus aureus formed gentamicin-resistant small colony variants on Palacos R-G and therefore Copal more effectively decreased biofilm formation than Palacos R-G.

Fig. 1

Cumulative gentamicin and clindamycin concentrations in 20 mL phosphate-buffered saline as a function of time after release from Palacos R-G (open symbols) and Copal (closed symbols) bone cement are shown. The triangles denote gentamicin release, whereas the squares indicate clindamycin release. The error bars denote the average standard deviation over three experiments performed per group. Copal increased antibiotic release compared with Palacos R-G.

Fig. 2A–C

Confocal laser scanning microscopy images of biofilms on bone cement discs after LIVE/DEAD Baclight and Calcofluor staining are shown. The live bacteria are green, dead bacteria are red, and extracellular polysaccharides are blue. Overlay projections show the 7-day Staphylococcus aureus biofilms on (A) Palacos R, (B), Palacos R-G, and (C) Copal bone cement. The projection image (square image, x–y plane, 375 μm × 375 μm) includes all the slices in an image stack. The rectangular micrographs on the sides represent the x–z plane and y–z optical cross sections through the thickness of the biofilms (the bottom of each biofilm is indicated B [bone cement] in each cross section). Bar = 10 μm. Copal reduced biofilm to a level that no or only a few bacteria were visible on confocal laser scanning microscopy.