Antimicrobial Efficacy of Triple Antibiotic-eluting Polymer Nanofibers against Multispecies Biofilm

Abstract

The elimination of microbial flora in cases of immature permanent teeth with necrotic pulp is both key and a challenging goal for the long-term success of regenerative therapy. Recent research has focused on the development of cell-friendly intracanal drug delivery systems. This in vitro study aimed to investigate the antimicrobial action of 3-dimensional (3D) tubular-shaped triple antibiotic-eluting nanofibrous constructs against a multispecies biofilm on human dentin. Polydioxanone polymer solutions, antibiotic-free or incorporated with metronidazole, ciprofloxacin, and minocycline, were electrospun into 3D tubular-shaped constructs. A multispecies biofilm consisting of Actinomyces naeslundii, Streptococcus sanguinis, and Enterococcus faecalis was forced inside the dentinal tubules via centrifugation in a dentin slice in vitro model. The infected specimens were exposed to 2 experimental groups (ie, 3D tubular-shaped triple antibiotic-eluting constructs and triple antibiotic paste [TAP]) and 2 control groups (7-day biofilm untreated and antibiotic-free 3D tubular-shaped constructs). Biofilm elimination was quantitatively analyzed with confocal laser scanning microscopy. Confocal laser scanning microscopic (CLSM) analysis showed a dense population of viable (green) bacteria adhered to dentin and penetrated into the dentinal tubules. Upon 3D tubular-shaped triple antibiotic-eluting nanofibrous construct exposure, nearly complete elimination of viable bacteria on the dentin surface and inside the dentinal tubules was shown in the CLSM images, which was similar (P < .05) to the bacterial death promoted by the TAP group but significantly greater when compared with both the antibiotic-free 3D tubular-shaped constructs and the control (saline). The proposed 3D tubular-shaped antibiotic-eluting construct showed pronounced antimicrobial effects against the multispecies biofilm tested and therefore holds significant clinical potential as a disinfection strategy before regenerative endodontics.

Keywords: Antibiotic; bacteria; disinfection; electrospinning; nanofibers; pulp; regeneration; root canal; scaffold; stem cells.

Figure 1

Representative SEM micrograph suggesting the presence of the 3 distinct bacterial morphologies corresponding to E. faecalis (white arrow), S. sanguinis (black arrow), and A. naeslundii (yellow arrow), all of which make up the multispecies biofilm.

Figure 2

(A) CLSM macrophotographs of 7-day multispecies biofilm (positive control) growth inside dentinal tubules. (B) Infected dentin treated with 3D tubular-shaped antibiotic-free nanofibrous constructs. Infected dentin treated for 7 days (C) 3D tubular-shaped triple antibiotic-eluting nanofibrous constructs (D) TAP. (E) CLSM images collected from inner root canal walls with a mosaic technique, allowing for deeper analysis in sequential illumination mode. (F) Table illustrating the median percentage of DEAD and LIVE bacterial cells for each group, demonstrating that 3D tubular-shaped triple antibiotic-eluting nanofibrous constructs eliminated almost all live cells, not differing from TAP paste (p>0.05). Median values, followed by distinct uppercase letters (comparing bacterial viability within each group), denote statistical difference.

Figure 3

Summary of future perspectives for the clinical use of the proposed 3D tubular-shaped triple antibiotic-eluting nanofibrous constructs in regenerative endodontics. (A) Synthesis of 3D tubular-shaped constructs incorporated with metronidazole (MET), ciprofloxacin (CIP), and minocycline (MINO); (B) Triple antibiotic-eluting nanofibers’ antimicrobial activity against bacterial biofilms and associated dental pulp stem cells’ (DPSCs) compatibility (31); (C) Schematic representation of an immature tooth associated with periapical lesion and placement of the proposed 3D tubular-shaped triple antibiotic-eluting construct to act as a localized drug delivery system (I); Evoked Bleeding — Blood clot evoked from the periapical tissue to the root canal space using a pre-curved K file (II); and Injectable Scaffold Strategy — with growth factors and/or stem cells (III).