Cellular and tissue compatibility of quaternary ammonium silane for endodontic applications

Quaternary ammonium silane (K21) is an innovative antibacterial agent with prospective endodontic applications. This study assessed the cytocompatibility of K21 in comparison to sodium hypochlorite (NaOCl) and chlorhexidine (CHX). Murine macrophages (RAW 264.7) and human periodontal ligament stem cells (hPDLSCs) were subjected to saline (S ct ), 2% chlorhexidine (CHX), 6% sodium hypochlorite (NaOCl), 0.5% K21, and 1% K21. Cell viability was evaluated using MTT and Alamar Blue assays at 1 min, 5 min, 15 min, 30 min, 1 h, and 4 h. Mitochondrial staining intensity was assessed with MitoTracker Green (MTG) fluorescence as a surrogate marker of mitochondrial mass integrity. Cellular structures were analysed utilising scanning and transmission electron microscopy (SEM/TEM). Relative gene expression (IL-1β, TNF-α, TGF-β, VEGF-α) was measured by real-time PCR after macrophage polarisation. Biocompatibility was additionally assessed using a rat subcutaneous implantation model, with histological analysis performed at 2 h, 2 days, and 2 weeks. K21 exhibited significantly improved cell viability compared with 6% NaOCl, and a comparable or modestly improved viability profile compared with 2% CHX, in both macrophages and hPDLSCs across the time intervals tested (p < 0.05). Mitochondrial imaging revealed better preserved MTG staining intensity in K21-treated cells compared to NaOCl and CHX groups. SEM and TEM showed largely maintained cellular features with K21; these were the morphological observations and do not by themselves confirm cellular function. Gene expression analysis demonstrated lower fold-induction of pro-inflammatory markers (IL-1β, TNF-α) and higher fold-induction of angiogenic mediators (TGF-β, VEGF-A) relative to 6%NaOCl and 2%CHX. I n vivo , K21 elicited only mild, temporary inflammatory reactions, with swift tissue normalisation in contrast to the moderate to severe inflammation associated with NaOCl. Within the limitations of this preclinical cytocompatibility evaluation, 0.5% and 1% K21 demonstrated favourable cytocompatibility relative to 6% NaOCl and comparable or slightly better performance than 2% CHX; however these findings do not establish clinical efficacy or applicability, which would require dedicated orthotopic and clinical investigation.