Anil Kishen

An Investigation on the Antibacterial and Antibiofilm Efficacy of Cationic Nanoparticulates for Root Canal Disinfection

This study aimed to investigate the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection. Experiments were performed in two stages. In stage 1, experiments were conducted to examine the physical properties of three types of nanoparticulates. The antibacterial properties of nanoparticulates alone and nanoparticulates mixed with zinc oxide-eugenol-based sealer were studied. In stage 2, the ability of nanoparticulates-treated dentin to prevent bacterial adherence was examined. Zinc oxide nanoparticulates, chitosan nanoparticulates, a mixture of zinc oxide and chitosan nanoparticulates, and zinc oxide nanoparticulates with multilayered coating of chitosan were tested. This study showed that the incorporation of nanoparticulates did not alter the flow characteristics of sealer but improved the direct antibacterial property and the ability to leach out antibacterial components. There was a significant reduction in the adherence of Enterococcus faecalis to nanoparticulates-treated dentin (p < 0.05). These experiments highlighted the potential advantage of nanoparticulates in root canal disinfection.

Uptake pathways of anionic and cationic photosensitizers into bacteria

The effect of divalent cations (calcium and magnesium) and a permeabilizing agent (EDTA) on the uptake of a cationic photosensitizer (PS), methylene blue (MB), and two anionic PSs, rose bengal (RB) and indocyanine green (ICG), by Gram-positive Enterococcus faecalis and Gram-negative Actinobacillus actinomycetemcomitans was examined. The possible roles of multidrug efflux pumps and protein transporters in photosensitizer uptake were assessed in E. faecalis cells by studies using an efflux pump inhibitor (verapamil) and trypsin treatment respectively. Divalent cations enhanced the uptake and photodynamic inactivation potential of both RB and ICG in E. faecalis and A. actinomycetemcomitans, while they decreased the uptake and bacterial killing by MB. Verapamil increased the uptake of RB (possibly due to efflux pump inhibition), whereas trypsin treatment resulted in significant decrease in RB and ICG uptake. The results suggested that the uptake of anionic PSs by bacterial cells may be mediated through a combination of electrostatic charge interaction and by protein transporters, while the uptake of cationic PSs, as previously reported, is mediated by electrostatic interactions and self promoted uptake pathways.

Influence of Irrigation Regimens on the Adherence of Enterococcus faecalis to Root Canal Dentin

Enterococcus faecalis is frequently associated with post-treatment endodontic infections. Because adherence of bacteria to a substrate is the earliest stage in biofilm formation, eliciting the factors that links adherence of this bacterium to dentin would help in understanding its association with treatment-failed root canals. This investigation aimed to study the effects of endodontic irrigants on the adherence of E. faecalis to dentin. The bacteria adherence assay was conducted by using fluorescence microscopy, and the adhesion force was measured by using atomic force microscopy. There were significant increases in adherence and adhesion force after irrigation of dentin with ethylenediaminetetraacetic acid (EDTA), whereas sodium hypochlorite (NaOCl) reduced it. With the use of chlorhexidine (CHX), the force of adhesion increased, but the adherence assay showed a reduction in the number of adhering bacteria. The irrigation regimen of EDTA, NaOCl, and CHX resulted in the least number of adhering E. faecalis cells. This study highlighted that chemicals that alter the physicochemical properties of dentin will influence the nature of adherence, adhesion force, and subsequent biofilm formation of E. faecalis to dentin.

Photophysical, photochemical, and photobiological characterization of methylene blue formulations for light-activated root canal disinfection

Tissue-specific modification of treatment strategy is proposed to increase the antimicrobial activity of light-activated therapy (LAT) for root canal disinfection. Methylene blue (MB) dissolved in different formulations: water, 70% glycerol, 70% poly ethylene glycol (PEG), and a mixture of glycerol:ethanol:water (30:20:50) (MIX), is analyzed for photophysical, photochemical, and photobiological characteristics. Aggregation of MB molecules, as evident from monomer to dimer ratio, depends on the molar concentrations of MB, which is significantly higher in water compared to other formulations. MIX-based MB formulation effectively penetrates the dentinal tubules. Although, the affinity of MB for Enterococcus faecalis (gram positive) and Actinomycetes actinomycetemcomitans (gram negative) was found to be high in the water-based formulation, followed by MIX, the MIX-based formulation significantly enhanced the model substrate photooxidation and singlet oxygen generation compared to MB dissolved in other formulations. Finally, the efficacy of LAT is evaluated on biofilms produced by both organisms under in vitro and ex vivo conditions. A dual-stage approach that applies a photosensitization medium and an irradiation medium separately is tested. The MIX-based photosensitization medium in combination with dual-stage approach demonstrates thorough disinfection of the root canal with bacterial biofilms. This method will have potential application for root canal disinfection.

A strain gauge and photoelastic analysis of in vivo strain and in vitro stress distribution in human dental supporting structures

Strain gauge and photoelastic experiments have been workhorses of experimental stress analysis for over 50 years. In this study, both were used to analyse the nature of stress distribution from the tooth root surface to the supporting alveolar bone. Such studies help in understanding the behaviour of dental supporting structures under physiological function. In the strain gauge experiment, the mechanical strains were measured on the supporting bone surface and the root surface of the tooth under applied bite force. It was found that higher strains were distributed along the cervical region of the supporting bone and the root surface. The photoelastic study was also done to evaluate the stress distribution pattern from the root surface to the supporting bone under clinical conditions. The stress patterns were found to decrease from the cervical to the apical region of the root surface. These studies highlight the role of the periodontium in stress distribution and bone remodelling.

Experimental studies on the nature of property gradients in the human dentine

We conducted an investigation into the nature of dentine mineralization and mechanical property gradients with the aid of experimental techniques such as the fluoroscopic X-ray microanalysis and instrumented microindentation, respectively. It was found that the tooth adapts to a complex structure with significant gradients in properties. We observed a significant correlation between the degree of mineralization within the dentine and the mechanical properties. The natural gradation in mechanical properties is explained by the stress analysis within anatomical-sized tooth specimens done using digital photoelasticity. These results are explained within the context of the functional requirements that are imposed on the tooth. This study highlights tooth structure as a biologically adapted, functionally graded material.

Efflux Pump Inhibitor Potentiates Antimicrobial Photodynamic Inactivation of Enterococcus faecalis Biofilm

Microbial biofilm architecture contains numerous protective features, including extracellular polymeric material that render biofilms impermeable to conventional antimicrobial agents. This study evaluated the efficacy of antimicrobial photodynamic inactivation (aPDI) of Enterococcus faecalis biofilms. The ability of a cationic, phenothiazinium photosensitizer, methylene blue (MB) and an anionic, xanthene photosensitizer, rose bengal (RB) to inactivate biofilms of E. faecalis (OG1RF and FA 2‐2) and disrupt the biofilm structure was evaluated. Bacterial cells were tested as planktonic suspensions, intact biofilms and biofilm‐derived suspensions obtained by the mechanical disruption of biofilms. The role of a specific microbial efflux pump inhibitor (EPI), verapamil hydrochloride in the MB‐mediated aPDI of E. faecalis biofilms was also investigated. The results showed that E. faecalis biofilms exhibited significantly higher resistance to aPDI when compared with E. faecalis in suspension (Pu2003<u20030.001). aPDI with cationic MB produced superior inactivation of E. faecalis strains in a biofilm along with significant destruction of biofilm structure when compared with anionic RB (Pu2003<u20030.05). The ability to inactivate biofilm bacteria was further enhanced when the EPI was used with MB (Pu2003<u20030.001). These experiments demonstrated the advantage of a cationic phenothiazinium photosensitizer combined with an EPI to inactivate biofilm bacteria and disrupt biofilm structure.

Influence of Photosensitizer Solvent on the Mechanisms of Photoactivated Killing of Enterococcus faecalis

This study evaluated the mechanisms involved and the influence of photosensitizer solvent in the killing of Enterococcus faecalis using photodynamic therapy (PDT). Enterococcus faecalis cells incubated with 100u2003μm methylene blue dissolved in water and in MIX (a mixture of glycerol:ethanol:water) were irradiated with 664u2003nm diode laser (63.69u2003Ju2003cm−2). The effect of PDT on the viability of bacteria, and the functional integrity of cell wall, chromosomal DNA and membrane proteins were analyzed. The bactericidal action of PDT was significantly higher when a MIX‐based photosensitizer solvent was used (Pu2003<u20030.001). Fluorimetric and fluorescence microscopy‐based analysis showed the functional impairment of E. faecalis cell wall which was significantly higher when a MIX‐based photosensitizer solvent was used (Pu2003<u20030.001). PDT with MIX‐based photosensitizer solvent showed extensive damage to chromosomal DNA. However, both PDT conditions showed similar trend in the degradation of membrane proteins, although cross‐linked proteins were evident only in PDT conducted with MIX‐based photosensitizer solvent. The findings from our study showed that PDT destroyed the functional integrity of cell wall, DNA and membrane proteins of E. faecalis. The degrees of damage on these targets were influenced by the photosensitizer solvent used during PDT.

Delivery of Antibacterial Nanoparticles into Dentinal Tubules Using High-intensity Focused Ultrasound

INTRODUCTIONnHigh-intensity focused ultrasound (HIFU) produces collapsing cavitation bubbles. This study aims to investigate the efficacy of collapsing cavitation bubbles to deliver antibacterial nanoparticles into dentinal tubules to improve root canal disinfection.nnnMETHODSnIn stage 1, experiments were performed to characterize the efficacy of collapsing cavitation bubbles to deliver the miniature plaster beads into a tubular channel model. In stage 2, experiments were conducted on root-dentin blocks to test the efficacy of HIFU applied at 27 kHz for 2 minutes to deliver antibacterial nanoparticles into dentinal tubules. After the stage 2 experiment, the samples were sectioned and analyzed using field-emission scanning electron microscopy and energy dispersive X-ray analysis.nnnRESULTSnThe stage 1 experiment showed that collapsing cavitation bubbles using HIFU delivered plaster beads along the entire length of the tubular channel. It was observed from the stage 2 experiments that the diffusion of fluids alone was not able to deliver antibacterial nanoparticles into dentinal tubules. The collapsing cavitation bubbles treatment using HIFU resulted in significant penetration up to 1,000 microm of antibacterial nanoparticles into the dentinal tubules. The statistical analysis showed a highly significant difference in the depth of penetration of antibacterial nanoparticles between the two groups (<0.005).nnnCONCLUSIONnThe cavitation bubbles produced using HIFU can be used as a potential method to deliver antibacterial nanoparticles into the dentinal tubules to enhance root canal disinfection.

Augmenting the antibiofilm efficacy of advanced noninvasive light activated disinfection with emulsified oxidizer and oxygen carrier.

In this study, we tested the hypothesis that the inclusion of an oxidizer and oxygen carrier in the photosensitization formulation would facilitate comprehensive disinfection of matured endodontic biofilm by light-activated disinfection (LAD). Photosensitizing formulations containing methylene blue (MB) and an oxygen carrier alone (perfluorodecahydronaphthalene) (PF1) or in combination with oxidizer (H(2)O(2)) (PF2) or their emulsions formed with triton-X100 (Bio-Rad Laboratories, Hercules, CA) in different proportions (PF3 and PF4) were tested for photochemical properties and damage to the biofilm structure using confocal laser scanning microscopy. Conventional chemomechanical preparation, LAD using MB in water, and LAD using MB in emulsion (PF4) were also conducted on 10-week-old Enterococcus faecalis biofilm within root canals. MB in emulsion (PF4) was overall the most effective photosensitizer formulation for photooxidation, generation of singlet oxygen (p = 0.001), and in disinfecting biofilm bacteria. Advanced noninvasive LAD using a photosensitizer formulation containing oxidizer and oxygen carrier disrupted the biofilm matrix and facilitated comprehensive inactivation of biofilm bacteria. This modified photosensitizer formulation will have potential advantages in endodontic disinfection.