Cytotoxicity evaluation of chlorhexidine gluconate on human fibroblasts, myoblasts, and osteoblasts

Abstract

Introduction: Chlorhexidine gluconate (CHX) is widely used as a preoperative surgical skin-preparation solution and intra-wound irrigation agent, with excellent efficacy against wide variety of bacteria. The cytotoxic effect of CHX on local proliferating cells following orthopaedic procedures is largely undescribed. Our aim was to investigate the in vitro effects of CHX on primary fibroblasts, myoblasts, and osteoblasts. Methods: Cells were exposed to CHX dilutions (0%, 0.002%, 0.02%, 0.2%, and 2%) for either a 1, 2, or 3-minute duration. Cell survival was measured using a cytotoxicity assay (Cell Counting Kit-8). Cell migration was measured using a scratch assay: a "scratch" was made in a cell monolayer following CHX exposure, and time to closure of the scratch was measured. Results: All cells exposed to CHX dilutions of ≥ 0.02% for any exposure duration had cell survival rates of less than 6% relative to untreated controls (p < 0.001). Cells exposed to CHX dilution of 0.002% all had significantly lower survival rates relative to control (p < 0.01) with the exception of 1-minute exposure to fibroblasts, which showed 96.4% cell survival (p = 0.78). Scratch defect closure was seen in < 24 hours in all control conditions. However, cells exposed to CHX dilutions ≥ 0.02% had scratch defects that remained open indefinitely. Conclusions: The clinically used concentration of CHX (2%) permanently halts cell migration and significantly reduces survival of in vitro fibroblasts, myoblasts, and osteoblasts. Further in vivo studies are required to examine and optimize CHX safety and efficacy when applied near open incisions or intra-wound application.

Keywords: chlorhexidine; cytotoxicity; fibroblasts; myoblasts; osteoblasts.

Figure 1

A-C: Percent survival of cells 48 hours after exposure to different concentrations of chlorhexidine gluconate for 1, 2, and 3-minute durations. A) Fibroblast B) Myoblast C) Osteoblast

Figure 1

A-C: Percent survival of cells 48 hours after exposure to different concentrations of chlorhexidine gluconate for 1, 2, and 3-minute durations. A) Fibroblast B) Myoblast C) Osteoblast

Figure 1

A-C: Percent survival of cells 48 hours after exposure to different concentrations of chlorhexidine gluconate for 1, 2, and 3-minute durations. A) Fibroblast B) Myoblast C) Osteoblast

Figure 2

A-C. Scratch test results. At Time 0, scratch defects were initiated in monolayer cell cultures and exposed to either control conditions or the clinically used concentration of 2% CHX for 3 minutes. 48 hours later, photos were taken of the scratch defects to observe defect closure. White arrows demonstrate the width of scratch defect. Closure of the scratch defect is seen at 48 hours following exposure to control conditions for all cell types. Open scratch defects are seen at 48 hours following exposure to 2% CHX for 3 minutes. A) Fibroblasts, B) Myoblasts, C) Osteoblasts

Figure 2

A-C. Scratch test results. At Time 0, scratch defects were initiated in monolayer cell cultures and exposed to either control conditions or the clinically used concentration of 2% CHX for 3 minutes. 48 hours later, photos were taken of the scratch defects to observe defect closure. White arrows demonstrate the width of scratch defect. Closure of the scratch defect is seen at 48 hours following exposure to control conditions for all cell types. Open scratch defects are seen at 48 hours following exposure to 2% CHX for 3 minutes. A) Fibroblasts, B) Myoblasts, C) Osteoblasts

Figure 2

A-C. Scratch test results. At Time 0, scratch defects were initiated in monolayer cell cultures and exposed to either control conditions or the clinically used concentration of 2% CHX for 3 minutes. 48 hours later, photos were taken of the scratch defects to observe defect closure. White arrows demonstrate the width of scratch defect. Closure of the scratch defect is seen at 48 hours following exposure to control conditions for all cell types. Open scratch defects are seen at 48 hours following exposure to 2% CHX for 3 minutes. A) Fibroblasts, B) Myoblasts, C) Osteoblasts