Human osteoblast damage after antiseptic treatment

Abstract

Purpose: Antiseptics are powerful medical agents used for wound treatment and decontamination and have a high potential for defeating joint infections in septic surgery. Both chlorhexidine and polyhexanide are frequently used in clinical practice and have a broad antimicrobial range, but their effect on human osteoblasts has not been sufficiently studied. Our objective was to investigate the toxic effects of polyhexanide and chlorhexidine on human osteoblasts in vitro to evaluate their clinical applicability in septic surgery.

Methods: We isolated and cultivated human osteoblasts in vitro and assayed the toxic effects of chlorhexidine 0.1% and polyhexanide 0.04%, concentrations commonly applied in clinical practice. Toxicity analysis was performed by visualisation of cell structure, lactate dehydrogenase (LDH) activity and evaluation of vital cells. Toxicity was evaluated by microscopic inspection of cell morphology, trypan blue staining and determination of LDH release.

Results: Damaged cell structure could be shown by microscopy. Both antiseptics promoted LDH activity after incubation with osteoblasts. The evaluation of vital osteoblasts showed a significant decrease of vital cells.

Conclusions: Both antiseptics induced significant cell death of osteoblasts at optimum exposure. We therefore recommend cautious use of polyhexanide and chlorhexidine in septic surgery to avoid severe osteoblast toxicity.

Fig. 1

Light microscopy. Damage to osteoblasts after 5 min of antiseptic incubation as revealed by light microscopy. a Control osteoblasts treated with phosphate-buffered saline (PBS). b Osteoblasts treated with Triton X 100, positive control. c Chlorhexidine 0.1 %. d Polyhexanide 0.04 %

Fig. 2

Trypan blue. Trypan blue staining of defective osteoblasts after antiseptic treatment for 1 and 10 min. a Untreated osteoblasts (control). b Chlorhexidine 1 min. c Polyhexanide 1 min. d Chlorhexidine 10 min. e Polyhexanide 10 min

Fig. 3

Lactate dehydrogenase (LDH) activity in the supernatant of osteoblasts treated with antiseptics. Osteoblasts were incubated with chlorhexidine 0.1 % and polyhexanide 0.04 % for 1, 5 and 10 min following enzyme-linked immunosorbent assay (ELISA) analysis of LDH activity in the supernatant, n = 4. Values given ± standard error of mean (SEM). Nonparametric Wilcoxon matched-pairs test. Average of four independently performed experiments. *p < 0.05

Fig. 4

CASY cell counting. Vital cell counts after antiseptic treatment in per cent of control vital count. Untreated osteoblasts are shown compared with osteoblasts treated with chlorhexidine or polyhexanide for 1, 5 and 10 min. 100 % = mean of vital cells in control (CTR) = 17.505 cells. Values given as mean ± standard error of mean (SEM). Comparison of multiple variables according to Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001