Effectiveness of Hypochlorous Acid to Reduce the Biofilms on Titanium Alloy Surfaces in Vitro

Abstract

Chemotherapeutic agents have been used as an adjunct to mechanical debridement for peri-implantitis treatment. The present in vitro study evaluated and compared the effectiveness of hypochlorous acid (HOCl), sodium hypochlorite (NaOCl), and chlorhexidine (CHX) at eliminating Gram-negative (E. coli and P. gingivalis) and Gram-positive (E. faecalis and S. sanguinis) bacteria. The effect of irrigating volume and exposure time on the antimicrobial efficacy of HOCl was evaluated, and a durability analysis was completed. Live/dead staining, morphology observation, alamarBlue assay, and lipopolysaccharide (LPS) detection were examined on grit-blasted and biofilm-contaminated titanium alloy discs after treatment with the three chemotherapeutic agents. The results indicated that HOCl exhibited better antibacterial efficacy with increasing irrigating volumes. HOCl achieved greater antibacterial efficacy as treatment time was increased. A decrease in antimicrobial effectiveness was observed when HOCl was unsealed and left in contact with the air. All the irrigants showed antibacterial activity and killed the majority of bacteria on the titanium alloy surfaces of biofilm-contaminated implants. Moreover, HOCl significantly lowered the LPS concentration of P. gingivalis when compared with NaOCl and CHX. Thus, a HOCl antiseptic may be effective for cleaning biofilm-contaminated implant surfaces.

Keywords: antimicrobial activity; antiseptics; dental implant; hypochlorite acid.

Figure 1

The antimicrobial effect of hypochlorous acid (HOCl) at different volume ratios and treatment times on (a) E. coli; (b) P. gingivalis; (c) E. faecalis; and (d) S. sanguinis. The results were reported in absorbance units. HOCl showed volume-dependent antibacterial ability against all four species. The antibacterial efficacy of HOCl was also time-dependent.

Figure 2

The changes in the antimicrobial effectiveness of HOCl as a function of contact time with air on (a) E. coli; (b) P. gingivalis; and (c) E. faecalis. The volume ratio of HOCl to bacterial suspension was 4:1. The results were reported in absorbance units. The duration of air contact adversely affected the antimicrobial activity of HOCl; however, the treatment duration did not have a significant effect.

Figure 3

Viability staining of (a) E. coli exposed to (b) HOCl, (c) sodium hypochlorite (NaOCl), and (d) chlorhexidine (CHX). Viable bacteria are labeled green, and dead bacteria are labeled red. Fewer viable E. coli bacteria were found after treatment with the three irrigants.

Figure 4

Viability staining of (a) P. gingivalis exposed to (b) HOCl, (c) NaOCl, and (d) CHX. Viable bacteria are labeled green, and dead bacteria are labeled red. The three irrigants induced significant reductions in bacterial numbers when compared with the control.

Figure 5

Viability staining of (a) E. faecalis exposed to (b) HOCl, (c) NaOCl, and (d) CHX. Viable bacteria are labeled green, and dead bacteria are labeled red. The dead bacteria in the irrigant-treated groups made up a greater proportion of the total bacteria on the titanium alloy surfaces when compared with the control.

Figure 6

Viability staining of (a) S. sanguinis exposed to (b) HOCl, (c) NaOCl, and (d) CHX. Viable bacteria are labeled green, and dead bacteria are labeled red. An increased amount of dead bacteria was observed after irrigant treatment.

Figure 7

Scanning electron micrographs of (a) the four bacterial species exposed to (b) HOCl, (c) NaOCl, and (d) CHX showed that the three irrigants appreciably reduced the number of bacteria. Almost a complete removal of the bacteria adhered to the surface was observed after treatment with NaOCl and CHX. Scale bar, 1 μm.

Figure 8

Antimicrobial effectiveness of HOCl, NaOCl, and CHX against E. coli, P. gingivalis, E. faecalis, and S. sanguinis on titanium alloy surfaces after culture for 24 h. The data were presented as absorbance units. Notably, all the irrigants significantly eliminated bacterial adhesion.

Figure 9

Residual lipopolysaccharide (LPS) levels from E. coli and P. gingivalis on titanium alloy surfaces after HOCl, NaOCl, and CHX treatment for 60 s. There was no significant difference in the residual LPS levels from E. coli. However, treatment with HOCl caused a significant decrease in the LPS from P. gingivalis when compared with NaOCl and CHX.