Quaternary ammonium silane (k21) based intracanal medicament triggers biofilm destruction

Abstract

Background: Compare antimicrobial efficacy of a quarternary ammonium silane (QAS)/k21 as an intracanal medicament against E. faecalis and C. albicans biofilms formed on root dentin.

Methodology: Dentin blocks were sterilized and E. faecalis and C. albicans microbial colonies were counted for colony-forming-units against 2%k21, 2%CHX and Ca(OH)2 medicaments. Biofilm colonies after 7 days on dentin were analysed using confocal laser scanning microscopy with live/dead bacterial viability staining. TEM was done to study dentin collagen matrix. Dentin discs from 3rd day and 7th day well plate was used for Raman spectra and observed under fluorescent-microscope. Docking studies were carried out on MMP-2 S1 binding-domain with k21.

Results: There was reduction of E. faecalis/C. albicans when k21, chlorhexidine and calcium hydroxide were used with highest percentage in 2%k21 treated specimens. 2%k21 showed dense and regular collagen network with intact cross-banding and decreased Raman intensity for 2%k21 on 3rd day. NaOCl + k21 showed least adherence, whereas saline groups showed highest adherence of E. faecalis and C. albicans to root-canal dentin. Alizarin red staining of hDPSCs revealed calcium deposition in all groups with significant difference seen amongst 2%k21 groups. MMP-2 ligand binding was seen accurately indicating possible target sites for k21 intervention.

Conclusion: 2%k21 can be considered as alternative intracanal medicament.

Keywords: Calcium hydroxide; Chlorhexidine; Dentine; Intracanal medicament; Quaternary ammonium silane.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1

Representative confocal images of E. faecalis biofilms of different groups stained using live/dead bac light bacterial viability kit; (A, B) control after 3 days; (C) control after 7 days; (D) 2%CHX after 7 days; (E) 41% Ca(OH)2 after 7 days; (F) 2%k21 after 3 days; and (G) 2%k21 after 7 days. Excitation was performed at λ = 514 nm. Green indicates a high level of bacterial viability in control specimens. Most of the red fluorescence in 2%k21 specimens indicating dead cell population. Scale bar = 50 μm

Fig. 2

Representative confocal images of Candida albicans (A, B) control group cells (in green) and (C) 2%CHX treated biofilms. (D) Specimens treated with 41% Ca(OH)2; (E) Structural destruction of Candida albicans micro-colonies after treatment with 2%k21 disinfectant. (F) degraded collagen fibers seen in after enzymatic degradation showing presence of denatured collagen fibers; TEM showed non-intact collagen fibres suggesting a significant degradation effect of bacterial biofilm in the control group. The collagen fibres were thinner and irregular in contour as compared to the medicament-treated specimens. (G) 2%CHX; (H) 41% Ca(OH)2; (I) 2%k21 where an increased number of fibrils seen in a relatively uniform manner. (G): Specimen treated by 2% chlorhexidine and (H) specimen treated by 41% calcium hydroxide both showing improved collagen fibrils compared to control. However, both 2%CHX (G) and calcium hydroxide (F) showed isolated fibrils and irregular arrangement of substructure. (I) Specimens treated by 2% k21 showing increased number of collagen fibrils and well demarcated collagen cross banding pattern. This could be due to the ability of 2%k21 as an inhibitor to MMP and cysteines cathepsins leading to increased resistance of dentine collagen against degradation and ingress by bacteria leading to better preservation of the collagen fibrils framework

Fig. 3

Raman spectra of E. faecalis biofilms on dentin disc specimens treated with different intracanal medicaments protocol. (a) Significant spectral differences of control and treated specimens can be seen in the 480–490 cm−1 region after normalization (region highlighted yellow). The Raman intensity on the y-axis represented the amount of amylopectin exopolysaccharide detected in an arbitrary unit, (3) represented the Raman analysis from dentin disc samples sent on the 3rd day while (7) represented samples from 7th day. In the 2%k21 case, the Raman fit was applied to the spectrum. While calculating, the weak curved sections, it was subtracted from the main spectrum producing the flattest baseline for 2%k21 at the same carbohydrate fingerprint region. The two spectra fall on the red, green, grey and blue regions belonging to the Raman intensity created by dentin disc specimens in control group, 2%CHX, Ca(OH)2 and 2%k21 respectively in A; and red, green, grey, and blue control group, 2%CHX, Ca(OH)2 and 2%k21 respectively in B. Raman peak acquired in same region during calibration process with silicon wafer indicated with black arrow showing remarkable difference between testing peaks and experimental groups. (b) Raman shift from 598–588 cm−1 presented the Raman effect caused by β-galactosidase. The β-COC ring deformations are the mode of vibrations due to the strong coupling due to the glycosidic ring skeletal deformations of the carbohydrate. The phospho-β-galactosidase is found in bacterial cytoplasm

Fig. 4

Alizarin red staining of human dental pulp stem cells (hDPSCs). Mineralized nodules as seen in light micrographs of human dental pulpal stem cell specimens treated with (A) no treatment as control; (B) 2%CHX; (C) 41% Ca(OH)2; (D) 2%k21. Lesser staining was observed after 3 days as compared to the 2%k21 specimens. Positive dark red staining shows presence of calcium deposits i.e. mineralization in the tested human dental pulpal stem cells.. Scale bar = 50 μm

Fig. 5

Mineralized nodule production by hDPSCs cells after application of intracanal medicaments on root dentine discs. Values are mean ±  standard deviation (n = 5). Groups identified by different symbols are not statistically different (p > 0.05)

Fig. 6

(a, b) The binding poses of quaternary ammonium compound in MMP-2 in both (a) 3D and (b) 2D. (c) MMP-2 is seen posed with K-21 chains up to quaternary nitrogen and associated with the silanol