In this study, we tested the hypothesis that a microbubble containing photosensitizer when activated with light
would enable comprehensive disinfection of bacterial biofilms in infected root dentin by antimicrobial
photodynamic therapy (APDT). Experiments were conducted in two stages. In the stage-1, microbubble
containing photosensitizing formulation was tested for its photochemical properties. In the stage-2, the
efficacy of microbubble containing photosensitizing formulation was tested on in vitro infected root canal
model, developed with monospecies biofilm models of Enterococcus faecalis on root dentin substrate. The
findings from this study showed that the microbubble containing photosensitizing formulation was overall the
most effective formulation for photooxidation, generation of singlet oxygen, and in disinfecting the biofilm
bacteria in the infected root canal model. This modified photosensitizing formulation will have potential
advantages in eliminating bacterial biofilms from infected root dentin.
A photosensitizer formulation and strategy was developed based on the photophysical, photochemical and
photobiological characteristics of methylene blue (MB) for the disinfection of root canal using light activated therapy.
Disinfection of matured <i>E. faecalis</i> biofilms on root canal dentine was tried with the newly developed 'Advanced Non-
Invasive Light Activated Disinfection' (ANILAD), conventional photodynamic therapy, and conventional root canal
therapy alone or in combination with ANILAD. The results showed that, although complete disinfection of nonmatured
biofilm is possible by ANILAD alone, a combination of conventional root canal treatment (RCT) with ANILAD
could achieve significantly higher bacterial killing (6log<sub>10</sub>-7log<sub>10</sub> bacterial reduction) compared to any other tested treatment in matured biofilm (p<0.001).
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.
This investigation studies the influence of cations on photosensitizer uptake by Enterococcus faecalis (gram positive)
and <i>Actinobacillus actinomycetemcomitans </i>(gram negative). Methods- The uptake of Methylene blue (MB) and
Indocyanine Green (ICG), by bacteria were studied under the influence of divalent cations (CaCl<sub>2</sub> & MgCl<sub>2</sub>) and EDTA.
Further, <i>E. faecalis </i>cells subjected to trypsinisation and calcium channel blocker (verapamil) were also analysed for MB
and ICG uptake inorder to understand the mechanism of photosensitizer uptake. Results- Uptake of ICG was enhanced
in the presence of divalent cations in <i>E. faecalis </i>and <i>A. actinomycetemcomitans</i>. Treating cells with EDTA had no
significant effect on the photosensitizer uptake, although the highest concentration tested showed an enhancement of
uptake. In contrast to ICG, MB showed a decreased uptake by bacterial cells on subjecting them to divalent cations and
EDTA. Calcium channel blocker had no significant inhibitory effect on photosensitizers uptake. However, trypsin
treatment resulted in significant reduction of ICG uptake. The result suggested that ICG uptake by bacteria is mediated
through specific transporter protein while MB is associated with the outer surface structures of bacterial cells.
Bacterial interaction with host tissues plays a major role in the cause and persistence of diseases. It has been confirmed by different clinical investigations that <i>Enterococcus faecalis</i> resist root canal treatment and commonly persist in tooth with post treatment infection. The purpose of this study is to apply different microscopic techniques to study the dynamics of the <i>E. faecalis </i>biofilm on root-canal-dentine tissues. <b>Method</b><b>-</b> Ten intact non-carious human maxillary molars were prepared and incubated with bacterium in nutrient media under anaerobic condition for 16 weeks. Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray microanalysis (EDX), Fluorescents microscopy Light microscopy and Laser Confocal Scanning Microscopy (LCSM) were carried out to characterize the ultrastructure of biofilm. In addition Fourier Transfer Infra Red Spectroscopy (FTIR) and Von-Kossa staining and Fluorescent microscopy were also carried out to confirm the biochemical characteristics of the biofilm structure. <b>Result-</b> The mature biofilm formed on the root-canal wall showed a honey-comb like structure with viable cells bacterial cells inside. The EDX and FTIR analysis showed a significant increase in the levels of Calcium (Ca) and Phosphorus (P) and evidence of biomineralization of the matured biofilm.