Rapid Bacterial Detection during Endodontic Treatment.

Bacteria present in the root canal (RC) space following an RC treatment (RCT) can lead to persistent infections, resulting in treatment failure and the need for reintervention or extraction. Currently, there are no standardized methods in use to clinically detect bacterial presence within RC spaces. The use of paper point sampling and fluorescence staining was shown to be a rapid method, able to detect residual bacteria following treatment. The study demonstrated that Calcein acetoxymethyl (AM) proved to be a suitable dye for detecting vital bacteria within mature endodontic biofilms, with an improved sensitivity over colony-forming unit counting in a stressed biofilm model. Furthermore, in a clinical trial with primary RCTs, 53 infected teeth were sampled in vivo, and increased detection of vital cells was found when compared with colony-forming unit counting, highlighting the sensitivity of the technique in detecting low cell numbers. By combining fluorescent staining and microspectroscopy with software-based spectral analysis, successful detection of vital cells from RCs was possible after 5 min of Calcein AM incubation. Application of this technology during RCT has the potential to reduce persistent infections through vital cell detection and additional treatment. Furthermore, this technique could be applied to antimicrobial research and disinfection control in clinical settings ( ClinicalTrials.gov NCT03055975).

Evaluation of dental adhesive systems incorporating an antibacterial monomer eugenyl methacrylate (EgMA) for endodontic restorations.

OBJECTIVE: The purpose of this study was to incorporate EgMA, an antibacterial monomer into two commercial dental adhesive systems for their application in endodontic restoration with the aim to disinfect the root canal space before curing and to inhibit bacterial growth on their surfaces after being cured. METHODS: EgMA monomer was added at 20%wt. into the formulation of the single-component self-etch, Clearfil Universal Bond™ (CUB) and into the catalyst and the adhesive components of the total-etch Adper Scotchbond-multipurpose™ (SBMP) adhesive systems. The degree of conversion (DC) was calculated from FTIR spectra, glass transition temperature (Tg) determined by DSC, water sorption and solubility were measured gravimetrically, and surface free energy (SFE) via contact angle measurements. The bonding performance to coronal and middle root canal dentin was assessed through push-out bond strength after filling the canals with a composite core material and the surface integrity was observed using SEM and confocal laser scanning microscopy (CLSM). The standard agar diffusion test (ADT) was used to identify the sensitivity of three endodontically pathogenic bacteria, Enterococcus faecalis, Streptococcus mutans and Propionibacterium acnes to uncured EgMA modified adhesives. Multispecies biofilm model from these strains was grown on the disc surface of cured adhesives and investigated using quantitative microbial culture and CLSM with live/dead staining. MTT assay was also used to determine the cytotoxicity of these adhesives. RESULTS: The incorporation of EgMA lowered polymerization exotherm and enhanced the hydrophobic character of these adhesives, without changing the DC and Tg in comparison to the controls (without EgMA). The total push-out bond strengths of the EgMA-containing adhesives were not significantly different from those of the controls (p>0.05). The modification of self-etch adhesive system enhanced the bond strength in the middle region of the roots canal. SEM of debonded specimens and CLSM examination showed the integrity of the resin-dentin interfaces. For all three bacteria tested, the sizes of the inhibition zones produced by uncured EgMA modified adhesives were significantly greater (p<0.05) than those of the controls. The results of biofilm inhibition tests showed less CFU for total bacteria on bonding agents with EgMA compared to the control materials (p<0.05). The modification at 20% monomer concentration had no adverse effects on cytocompatibility of both adhesives tested. SIGNIFICANCE: The inclusion of EgMA endows dental adhesives with effective antibacterial effects without influencing their curing properties, bonding ability to root canal dentin, and cytotoxicity against human gingival fibroblasts, indicating the usefulness of their application in endodontic restorations.

Influence of a polymerizable eugenol derivative on the antibacterial activity and wettability of a resin composite for intracanal post cementation and core build-up restoration.

OBJECTIVES: Eugenol has been used in dentistry due to its ability to inhibit the growth of a range of microorganisms, including facultative anaerobes commonly isolated from infected root canals. The aim of this study was to evaluate the antibacterial activity of the experimental composites containing eugenyl methacrylate monomer (EgMA), a polymeric derivative of eugenol, against a range of oral bacteria, commonly associated with failure of coronal and endodontic restorations. In vitro composite behavior and wettability were also studied in conjunction with their antibacterial activity. METHODS: EgMA monomer (5 and 10% by weight) was added into BisGMA/TEGDMA resin based formulations with filler mixtures of hydroxyapatite (HA) and zirconium oxide ZrO2. The antibacterial activity of the experimental composites against Enterococcus faecalis, Streptococcus mutans and Propionibacterium acnes were evaluated by direct contact test and compared with composite formulation without inclusion of EgMA. To clarify the antibacterial mode of action, agar diffusion test (ADT) was also performed. Water sorption, solubility, diffusion coefficient, contact angle and surface free energy as complementary clinically relevant properties were determined. RESULTS: Water sorption and wettability studies showed reduction of water uptake and surface free energy values with increasing content of EgMA monomer, resulting in significant increase in the hydrophobicity of the composites. No inhibition zones were detected in any of the composites tested against the three bacteria employed as expected, due to the absence of any leachable antibacterial agent. The covalently anchored EgMA monomer with the composite surface exhibited an effective bacteriostatic activity by reducing the number of CFUs of the three species of bacteria tested with no significant dependence on the concentration of EgMA at 5 and 10% by weight. The surface antibacterial activity R of the experimental composites were different against the three tested species with values in the range 2.7-6.1 following the order E. faecalis

Synergistic effect of 2% chlorhexidine combined with proteolytic enzymes on biofilm disruption and killing.

AIM: To investigate the dynamics of a disinfection regimen using 1% trypsin and 1% proteinase K in combination with 2% chlorhexidine (with or without ultrasonics) using a nutrient-stressed endodontic multispecies model biofilm. METHODOLOGY: Nutrient-stressed biofilms (Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis, Streptococcus mitis and Enterococcus faecalis OMGS 3202) were grown in prepared root canals of single-rooted teeth. The treatment groups included 1% trypsin and 2% chlorhexidine (CHX), 1% proteinase K and 2% CHX (with and without ultrasonics). 2% CHX was the positive control and untreated group, and sterile saline (with and without ultrasonics) was the negative control. The biofilms were investigated using confocal laser scanning microscopy (CLSM) with live/dead staining and quantitative microbial culture. RESULTS: The trypsin and CHX group with ultrasonics was significantly more effective in reducing viable counts and the substratum coverage than those of all other groups (P < 0.05). The viable counts of the proteinase K and CHX group used with (4.26 ± 0.58 log10  cfu mL(-1) ) or without ultrasonics (5.05 ± 1.36 log10  cfu mL(-1) ) were significantly reduced (P < 0.05) as compared with the untreated control (7.67 ± 0.84 log10  cfu mL(-1) ) and saline groups used with (6.57 ± 0.73 log10  cfu mL(-1) ) and without ultrasonics (6.74 ± 0.10 log10  cfu mL(-1) ). The CHX group was less effective in biofilm disruption compared to when used in combination with trypsin and proteinase K. CONCLUSION: The trypsin and CHX group with ultrasonics was significantly more effective at reducing bacterial viable counts and disrupting biofilm.

The effectiveness of enzymic irrigation in removing a nutrient-stressed endodontic multispecies biofilm.

AIM: To establish a nutrient-stressed multispecies model biofilm and investigate the dynamics of biofilm killing and disruption by 1% trypsin and 1% proteinase K with or without ultrasonic activation. METHODOLOGY: Nutrient-stressed biofilms (Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis, Streptococcus mitis and Enterococcus faecalis OMGS 3202) were grown on hydroxyapatite discs and in prepared root canals of single-rooted teeth in modified fluid universal medium. The treatment groups included trypsin, proteinase K, 0.2% chlorhexidine gluconate and 1% sodium hypochlorite (NaOCl) (with and without ultrasonics). NaOCl and chlorhexidine were the positive controls and untreated group, and sterile saline was the negative control. The biofilms were investigated using confocal laser scanning microscopy (CLSM) with live/dead staining and quantitative microbial culture. RESULTS: Nutrient stress in the multispecies biofilm was apparent as the medium pH became alkaline, glucose was absent, and serum proteins were degraded in the supernatant. The CLSM showed the percentage reduction in viable bacteria at the biofilm surface level due to nutrient starvation. On the disc model, trypsin and proteinase K were effective in killing bacteria; their aerobic viable counts were significantly lower (P < 0.01) than the negative control and chlorhexidine. NaOCl was the most effective agent (P < 0.001). In the tooth model, when compared to saline, trypsin with ultrasonics caused significant killing both aerobically and anaerobically (P < 0.05). Chlorhexidine (1.46 ± 0.42), trypsin (3.56 ± 1.18) and proteinase K (4.2 ± 1.01) with ultrasonics were significantly effective (P < 0.05) in reducing the substratum coverage as compared to saline with ultrasonics (12% ± 4.9). CONCLUSION: Trypsin with ultrasonic activation has a biofilm killing and disrupting potential.