Annie Shrestha

Nanoparticulates for antibiofilm treatment and effect of aging on its antibacterial activity.

INTRODUCTION Issues pertaining to the effective elimination of bacterial biofilm and disruption of biofilm structure still remains in endodontic disinfection. Nanoparticulates such as chitosan (CS-np) and zinc oxide (ZnO-np) are known to possess significant antibacterial properties. This study aimed to test (1) the efficacy of CS-np and ZnO-np in disinfecting and disrupting biofilm bacteria and (2) the long-term efficacy of these nanoparticulates following aging. METHODS Enterococcus faecalis (ATCC & OG1RF) in planktonic and biofilm forms were treated with different concentrations of CS-np and ZnO-np. The treated bacteria were quantified by using microbiologic methods. The biofilm viability and structure after nanoparticulate treatment were assessed by using confocal laser scanning microscopy. The effect of aging by using sterile saliva and phosphate-buffered saline on the antibacterial properties of the nanoparticulates was also determined. RESULTS The rate of bacterial killing by the nanoparticulates depended on the concentration and time of interaction. Total elimination of planktonic bacteria was observed in contrast to the biofilm bacteria, which survived even after 72 hours. The confocal microscopy images showed predominantly dead bacterial cells and significant reduction in the thickness of biofilm (P < .01) after nanoparticulate treatment in both groups. Both CS-np and ZnO-np were found to retain their antibacterial properties after aging for 90 days. CONCLUSIONS The present study highlighted the efficacy of the nanoparticulates to reduce biofilm bacteria, disrupt biofilm structure, and retain the antibacterial property even after aging. CS-np and ZnO-np present a potential approach in biofilm disinfection.

Photoactivated rose bengal functionalized chitosan nanoparticles produce antibacterial/biofilm activity and stabilize dentin-collagen

UNLABELLED Treatment of infected teeth presents two major challenges: persistence of the bacterial-biofilm within root canals after treatment and compromised structural integrity of the dentin hard-tissue. In this study bioactive polymeric chitosan nanoparticles functionalized with rose-bengal, CSRBnp were developed to produce antibiofilm effects as well as stabilize structural-integrity by photocrosslinking dentin-collagen. CSRBnp were less toxic to fibroblasts and had significant antibacterial activity even in the presence of bovine serum albumin. CSRBnp exerted antibacterial mechanism by adhering to bacterial cell surface, permeabilizing the membrane and lysing the cells subsequent to photodynamic treatment. Photoactivated CSRBnp resulted in reduced viability of Enterococcus faecalis biofilms and disruption of biofilm structure. Incorporation of CSRBnp and photocrosslinking significantly improved resistance to degradation and mechanical strength of dentin-collagen (P<0.05). The functionalized chitosan nanoparticles provided a single-step treatment of infected root dentin by combining the properties of chitosan and that of photosensitizer to eliminate bacterial-biofilms and stabilize dentin-matrix. FROM THE CLINICAL EDITOR In this study, bioactive polymeric chitosan nanoparticles functionalized with rose-bengal (a photosensitizer), CSRBnp were developed to produce antibiofilm effects as well as stabilize structural-integrity of dental root dentin by photocrosslinking dentin-collagen, leading to efficient elimination of bacterial-biofilms and stabilization of dentin-matrix.

The effect of tissue inhibitors on the antibacterial activity of chitosan nanoparticles and photodynamic therapy.

INTRODUCTION Newer antibacterial alternatives such as chitosan nanoparticles (CSnps) and photodynamic therapy (PDT) have been investigated to achieve effective root canal disinfection. The current study aims to assess the effect of various tissue inhibitors such as dentin, dentin matrix, pulp tissue, bacterial lipopolysaccharides (LPSs), and bovine serum albumin (BSA) on the antibacterial activity of CSnps and PDT. METHODS The antibacterial effect of CSnps and PDT using photosensitizers, rose bengal (RB), and methylene blue (MB) were tested on planktonic Enterococcus faecalis American Type Culture Collection 29212 with or without pretreatment using different tissue inhibitors for an hour. Bacterial survival was assessed after 1, 8, and 24 hours of incubation with CSnps and after PDT using RB and MB. RESULTS Pulp and BSA inhibited the antibacterial effect of CSnps significantly (P < .05). The antibacterial effect of CSnps was not affected by dentin, dentin matrix, or LPSs. The antibacterial activity of PDT using MB and RB was inhibited in a decreasing order by dentin matrix, BSA, pulp, dentin, and LPSs (P < .05). The effect of tissue inhibitors was higher in the case of PDT with RB. Depending on the antibacterial mechanism of CSnps and PDT, different inhibitory patterns were observed with different tissue inhibitors. CONCLUSIONS The tissue inhibitors existing within the root canal affected the antibacterial activity of CSnps and PDT at varying degrees. Further research is required to enhance their antimicrobial efficacy in an endodontic environment.

Polycationic Chitosan-Conjugated Photosensitizer for Antibacterial Photodynamic Therapy†

The complex nature of bacterial cell membrane and structure of biofilm has challenged the efficacy of antimicrobial photodynamic therapy. This study was aimed to synthesize a polycationic chitosan‐conjugated rose bengal (CSRB) photosensitizer and test its antibiofilm efficacy on Enterococcus faecalis (gram positive) and Pseudomonas aeruginosa (gram negative) using photodynamic therapy. During experiments, CSRB was tested along with an anionic photosensitizer rose bengal (RB) and a cationic photosensitizer methylene blue (MB) for uptake and killing efficacy on 7‐day‐old E. faecalis and P. aeruginosa biofilms. Microbiological culture based analysis was used to analyze the cell viability, while laser scanning confocal microscopy (LSCM) was used to examine the structure of biofilm. The synthesized CSRB showed absorbance spectrum similar to the RB. The concentration of CSRB uptaken by both the bacterial biofilms was significantly higher than that of RB and MB (P < 0.05). Photoactivation resulted in significantly higher elimination of both bacterial biofilms sensitized with CSRB than RB and MB. The structure of biofilm under LSCM was found to be disrupted following CSRB treatment. The present study highlighted the importance of inherent cell membrane permeabilizing effect of chitosan and increased cell/biofilm uptake of conjugated photosensitizer to produce significant antibiofilm efficacy during photodynamic therapy.

Characterization of a Conjugate between Rose Bengal and Chitosan for Targeted Antibiofilm and Tissue Stabilization Effects as a Potential Treatment of Infected Dentin

ABSTRACT Bacterial biofilms and dentin structural changes are some of the major challenges in the management of infected dentin tissue. This study characterized a photosensitizer-conjugated chitosan with enhanced photodynamic efficacy against dental biofilms, as well as the ability to reinforce the postinfected dentin matrix in order to improve its mechanical and chemical stability. Rose Bengal-conjugated chitosan (CSRB) was synthesized using a chemical cross-linking method and characterized for photophysical, photobiological, and cytotoxicity properties. Its potential as an antibacterial and matrix-reinforcing agent on dentin collagen was also evaluated. Enterococcus faecalis as planktonic and in vitro biofilms was treated with CSRB and photodynamically activated with 5 to 60 J/cm2 green light. Dentin collagen was used for the CSRB cross-linking experiments and evaluated for chemical changes, resistance to enzymatic degradation, and mechanical properties. CSRB was a photosensitizer with efficient singlet oxygen yield. In vitro photoactivation gave higher fibroblast cell survival than did RB alone. CSRB showed significant antibiofilm photoinactivation (P < 0.01). The CSRB-cross-linked dentin collagen showed higher resistance to collagenase degradation and superior mechanical properties (P < 0.05). In summary, the photoactivated CSRB particles synthesized in this study may be a synergistic multifunctional treatment approach with lower cytotoxicity and effective antibiofilm activity as well as the ability to reinforce the dentin collagen to enhance resistance to degradation and improve mechanical properties. This may be a targeted treatment strategy to deal with infected dentin hard tissues in a clinical scenario, where both disinfection and structural integrity need to be addressed concomitantly.

Antibiofilm Efficacy of Photosensitizer-functionalized Bioactive Nanoparticles on Multispecies Biofilm

INTRODUCTION Newer disinfection strategies based on antibacterial nanoparticles and photodynamic therapy (PDT) aim to eliminate residual biofilm bacteria during root canal treatment. The aim of the current study was to test the newly developed rose bengal-functionalized chitosan nanoparticles (CSRBnps) for their interaction/uptake with monospecies bacteria/biofilm and assess their antibiofilm efficacy on a multispecies biofilm model in vitro. METHODS The interaction of CSRBnps with bacterial cells was conducted using atomic force microscopy. Their membrane-damaging effect was determined by measuring the absorbance at 260 nm (OD260nm) using Enterococcus faecalis. The penetration of CSRBnps into E. faecalis biofilms was evaluated using confocal laser scanning microscopy (CLSM). Multispecies biofilms of Streptococcus oralis, Prevotella intermedia, and Actinomyces naeslundii were grown on dentin sections for 21 days to assess the antibiofilm efficacy. The biofilms were subjected to PDT (60 J/cm(2)) using CSRBnps and rose bengal. The treated/untreated biofilms were examined under scanning electron microscopy and CLSM. RESULTS The CSRBnps synthesized were 60 ± 20 nm and showed absorption spectra similar to rose bengal. Atomic force microscopy showed adherence of CSRBnps to bacteria, roughening of cell surface, and cell disruption after PDT. CSRBnp treatment resulted in significantly increased bacterial membrane damage (P < .05). CSRBnps exhibited deeper penetration into the biofilm structure. Scanning electron microscopy and CLSM confirmed the complete disruption of multispecies biofilm with a reduction in viable bacteria and biofilm thickness (P < .05). CONCLUSIONS These novel photosensitizer functionalized bioactive nanoparticles with increased affinity to bacterial cell membrane, higher penetration into biofilm structure, and enhanced ability to eliminate clinically relevant multispecies bacterial biofilm present a potential antibiofilm agent for root canal disinfection.

Role of Efflux Pump Inhibitors on the Antibiofilm Efficacy of Calcium Hydroxide, Chitosan Nanoparticles, and Light-activated Disinfection

INTRODUCTION The purpose of this study was to evaluate the role of efflux pumps in altering the susceptibility of Enterococcus faecalis biofilms to calcium hydroxide (Ca(OH)(2)), chitosan nanoparticles, and light-activated disinfection (LAD). METHODS E. faecalis as 4-day-old biofilms and biofilm-derived cells were tested with aqueous Ca(OH)(2) in concentrations of 25%, 50%, and 100%; chitosan nanoparticles in concentrations of 10 and 20 mg/mL (3, 12, and 24 hours); and methylene blue (MB) mediated LAD at an energy dose range of 2-40 J/cm(2). An efflux pump inhibitor (EPI) was incorporated into all 3 modalities of treatment. The antimicrobial activity was assessed by determining the colony-forming units. RESULTS E. faecalis biofilms, in contrast to the biofilm-derived cells, were found to persist even after 24-hour treatment with different concentrations of Ca(OH)(2) and chitosan nanoparticles. LAD at an energy dose of 40 J/cm(2) completely inactivated 4-day-old E. faecalis biofilms. The addition of EPI improved the antibiofilm efficacy of Ca(OH)(2) at lower concentrations (P < .001) and LAD (P < .001). EPI did not influence the antibiofilm effect of chitosan nanoparticles and Ca(OH)(2) at higher concentrations. CONCLUSIONS E. faecalis biofilms were more susceptible to killing by LAD, when compared with the tested concentrations of Ca(OH)(2) and chitosan nanoparticles. The effect of EPI was more significant with LAD, when compared with Ca(OH)(2) and chitosan nanoparticles. This study highlighted the role of biofilm matrix in providing resistance to antimicrobials.

Antibacterial Properties Associated with Chitosan Nanoparticle Treatment on Root Dentin and 2 Types of Endodontic Sealers

INTRODUCTION The aim of this study was to evaluate the efficacy of carboxymethyl chitosan (CMCS) and chitosan nanoparticles (CNps) to inactivate bacteria and prevent biofilm formation at sealer-dentin interfaces. METHODS The study was divided into 3 stages: first stage, the experiment was conducted to analyze the antibacterial properties of CMCS in different formulations against biofilms; second stage, direct-contact and membrane-restricted methods were used to evaluate the antibacterial properties of an epoxy resin (ThermaSeal Plus; Dentsply Tulsa Dental, Tulsa, OK) and calcium silicate (MTA Fillapex; Angelus SA, Londrina, PR, Brazil) based-sealers with or without CNps; and third stage, biofilm formation at the sealer dentin interfaces of root dentin treated with CMCS and filled with gutta-percha and CNp incorporated sealer were analyzed after 1- and 4-week aging periods. The samples were treated and filled as follows: (1) distilled water: unaltered sealer (control group), (2) CMCS: sealer+CNps (CMCS group), and (3) CMCS/rose bengal: sealer+CNps (CMCS/RB group). Enterococcus faecalis was used to infect all the samples. Microbiological and microscopic analyses were used to assess the antibacterial characteristics. RESULTS CMCS-based treatments effectively killed bacteria adherent on root dentin (P < .05). The addition of CNps to ThermaSeal enhanced its antibacterial ability by direct-contact and membrane-restricted tests (P < .05). The CNp incorporation significantly increased the antibacterial efficacy of root canal sealers even after a 4-week aging time (P < .05). CONCLUSIONS This study highlighted the ability of CMCS to disinfect root canal dentin and inhibit bacterial adhesion. CNps in root canal sealers are capable of maintaining their antibacterial activity even after prolonged aging.

Antibacterial Efficacy of Photosensitizer Functionalized Biopolymeric Nanoparticles in the Presence of Tissue Inhibitors in Root Canal

INTRODUCTION Application of antibacterial nanoparticles to improve root canal disinfection has received strong interest recently. The current study aims to assess the antibacterial effect of a novel photosensitizer (rose bengal functionalized chitosan nanoparticles [CSRBnp]) to eliminate bacteria in the presence of various root canal constituents that are known to inhibit the antibacterial efficacy of root canal disinfectants. METHODS The synthesized CSRBnp were evaluated for size, charge, and singlet oxygen release. The antibacterial effect of CSRBnp was tested on planktonic Enterococcus faecalis with or without pretreatment by using different inhibiting agents such as dentin, dentin-matrix, pulp tissue, bacterial lipopolysaccharides, and bovine serum albumin (BSA). Bacterial survival was assessed in a time-dependent manner. The antibacterial effects after photodynamic activation on CSRBnp, a cationic photosensitizer (methylene blue), and an anionic photosensitizer (rose bengal [RB]) in the presence of inhibitors were also evaluated. RESULTS CSRBnp were 60 ± 20 nm in size and showed reduced rate of singlet oxygen release as compared with methylene blue and RB. Pulp and BSA inhibited the antibacterial effect of CSRBnp (without photoactivation) significantly (P < .05) even after 24 hours of interaction. In case of photodynamic therapy, the pulp and BSA significantly inhibited the antibacterial activity of all 3 photosensitizers. CSRBnp showed residual effect and completely eliminated the bacteria after 24 hours of interaction after photodynamic therapy. CONCLUSIONS The inherent antibacterial activity of polycationic chitosan nanoparticles and the singlet oxygen released after photoactivation of RB synergistically provided CSRBnp the potential to achieve significant antibacterial efficacy even in the presence of tissue inhibitors within root canals.

Antibacterial Nanoparticles in Endodontics: A Review

INTRODUCTION A major challenge in root canal treatment is the inability of the current cleaning and shaping procedures to eliminate bacterial biofilms surviving within the anatomic complexities and uninstrumented portions of the root canal system. METHODS Nanoparticles with their enhanced and unique physicochemical properties, such as ultrasmall sizes, large surface area/mass ratio, and increased chemical reactivity, have led research toward new prospects of treating and preventing dental infections. This article presents a comprehensive review on the scientific knowledge that is available on the application of antibacterial nanoparticles in endodontics. RESULTS The application of nanoparticles in the form of solutions for irrigation, medication, and as an additive within sealers/restorative materials has been evaluated to primarily improve the antibiofilm efficacy in root canal and restorative treatments. In addition, antibiotic or photosensitizer functionalized nanoparticles have been proposed recently to provide more potent antibacterial efficacy. CONCLUSIONS The increasing interest in this field warrants sound research based on scientific and clinical collaborations to emphasize the near future potential of nanoparticles in clinical endodontics.