Comparative Study of Antiseptic Toxicity on Basal Keratinocytes, Transformed Human Keratinocytes and Fibroblasts

Abstract

The cytotoxic effects of a range of antiseptic agents were examined on cultured human fibroblasts and basal keratinocytes and compared to those on a transformed keratinocyte line (SVK 14 cells). Cells were exposed to chlorhexidine, hydrogen peroxide and sodium hypochlorite for 15 min and cell viability was assessed 24 h later with a colorimetric assay which utilizes the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). At concentrations recommended for wound cleansing all agents produced 100% killing of all cell types. The results show that fibroblasts and keratinocytes, cells which are fundamental to the wound healing process are equally sensitive to the effects of the antiseptic agents tested, and are highly susceptible to the effects of free-chlorine containing agents. These observations are of particular importance to the use of cultured keratinocytes (culture grafts) to enhance wound healing; the application of antiseptics to such wounds is contraindicated. All three cell types tested showed similar susceptibilities to the agents tested. These findings suggest that the transformed cell line, which has the advantage of immortality and ready availability, can replace fibroblasts and keratinocytes in studies designed to investigate the adverse effects of antiseptic agents in vitro. Comparison of the ED₅o concentration for each agent on all cell types to the standard use concentration produced a ranking order of toxicity which showed chlorhexidine to be the least toxic agent and sodium hypochlorite the most. These findings are in agreement with recent in vivo studies on wound healing which have shown that chlorhexidine does not inhibit wound healing but that sodium hypochlorite inhibits both the formation of granulation tissue and re-epithelialization.