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/cm². 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/cm² 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 Effect and Bioactivity of Innovative and Currently Used Intracanal Medicaments in Regenerative Endodontics

IntroductionThe purpose of this study was to determine the antibacterial effect and bioactivity of triple antibiotic paste (TAP), calcium hydroxide (Ca[OH]₂), and calcium hypochlorite (Ca[OCl]₂).MethodsRoot canals were infected with 3-week-old Enterococcus faecalis biofilm and then medicated for 7 days with TAP, Ca(OH)₂, or Ca(OCl)₂ (n = 10/group). Untreated and uninfected canals were used as positive and negative controls. The antibacterial effect was determined using colony-forming units and a Live/Dead bacterial viability kit. Dental pulp stem cells were seeded on medicated dentin surfaces for 7 days. Sodium thiosulfate and various concentrations of ascorbic acid (1%, 5%, and 10%) were also used to neutralize the samples treated with Ca(OCl)₂ before cell seeding (n = 3 in triplicate). Cell viability and morphology were evaluated using a viability assay and Live/Dead cell analysis. Alkaline phosphatase (ALP) activity was also measured to determine the cells’ mineralization activity.ResultsAll medicaments decreased the initial bacterial load (P < .05). The highest bacterial reduction in the main canal and dentinal tubules was observed in the Ca(OCl)₂ group (P < .05). TAP- or Ca(OH)₂-treated dentin surface improved cell viability and ALP activity compared with the untreated dentin surface (P < .05), whereas Ca(OCl)₂ decreased cell viability and ALP activity (P < .05). Ten percent ascorbic acid neutralized the effect of Ca(OCl)₂ on the treated dentin surface, showing higher cell viability (P < .05) and similar ALP activity with the untreated dentin surface and the other groups (P > .05).ConclusionsCa(OCl)₂ medication improved root canal disinfection against E. faecalis biofilm compared with TAP and Ca(OH)₂. The adverse effects caused by Ca(OCl)₂ on cell viability and mineralization activity can be neutralized with 10% ascorbic acid.     

Scanning electron microscopy evaluation of the hard tissue barrier after pulp capping with calcium hydroxide,mineral trioxide aggregate (MTA) or ProRoot MTA

The aim of this study was to investigate the morphology and localisation of calcium hydroxide‐ and mineral trioxide aggregate (MTA)‐induced hard tissue barriers after pulpotomy in dogs' teeth. Pulpotomies were performed on maxillary and mandibular premolars of five dogs. The teeth were assigned into three groups according to the pulp‐capping agent used. The pulpal wounds were capped with calcium hydroxide (Ca(OH)₂– control), MTA or ProRoot MTA, and the cavities were restored with amalgam. After a 90‐day follow‐up period, the dogs were euthanised and the teeth were examined under scanning electron microscopy (SEM). An image‐processing and analysis software was used to delimit the perimeters of the root canal area and the hard tissue barrier to determine the percentage of root canal obliteration. SEM data were used to assess the morphology, localisation and extension of the reparative hard tissue barriers. ProRoot MTA was statistically different from MTA and Ca(OH)₂ (P < 0.05) regarding tissue barrier morphology. Localisation data showed that ProRoot MTA was significantly different from Ca(OH)₂ (P < 0.05) and similar to MTA (P > 0.01; P > 0.05). No statistically significant difference (P > 0.01; P > 0.05) was observed between MTA and Ca(OH)₂. A larger number of complete (centroperipheral) hard tissue barriers with predominance of dentinal tubules was observed to the ProRoot MTA when compared with the Ca(OH)₂ group.     

Efficacy of self‐adjusting file,XP‐endo finisher and passive ultrasonic irrigation on the removal of calcium hydroxide paste from an artificial standardized groove

The purpose of this study was to compare the effectiveness of self‐adjusting file (SAF), XP‐endo finisher (XP), passive ultrasonic irrigation (PUI) and conventional syringe and needle irrigation (SNI) in the removal of Ca(OH)₂ from an artificial groove. Eighty mandibular incisors with single oval canals were prepared to size 40/0.04 and split longitudinally. A standardised groove was prepared in the apical third and filled with Ca(OH)₂. The root halves were reassembled and divided into two control groups (n = 4) and four experimental groups (n = 18) according to the removal methods used. The amount of residual Ca(OH)₂ was evaluated using a four‐grade scoring system. The differences among the groups were analysed using the Kruskal–Wallis test (P < 0.05). SAF, XP and PUI removed significantly more Ca(OH)₂ than SNI (P < 0.001), although there were no significant differences among them (P = 0.209). None of the tested methods could completely clean Ca(OH)₂ from the groove.     

Antimicrobial effect of grape seed extract as a potential intracanal medicament combined with Nd:YAG laser

This study evaluated the antimicrobial efficacy of grape seed extract medicament combined with Nd:YAG laser, against Enterococcus faecalis, Staphylococcus aureus and Candida albicans biofilms. Root canals infected with 4-week-old biofilms were divided into five groups (n = 11): calcium hydroxide, 6.5% GSE, Nd:YAG laser (1064 nm, 1.5 w, 15 Hz and 100 mj) and 6.5% GSE followed by Nd:YAG laser and normal saline (control). Dentin chips were collected using Gates–Glidden and cultured to obtain colony-forming units. Statistical analysis was performed using Kruskal–Wallis and Mann–Whitney U test. GSE showed higher antibacterial activity against all species investigated compared to Ca(OH)₂. However, the lowest microbial count was obtained using a combination of GSE and Nd:YAG laser (p < 0.001). No significant difference in the susceptibility of tested pathogens to each of the protocols was observed (p > 0.05). Application of Nd:YAG laser following GSE medicament is efficient against endodontic biofilms; also, GSE can be considered as an alternative to Ca(OH)₂ dressing.     

Characterisation of the efficacy of endodontic medications using a three‐dimensional fluorescent tooth model: An ex vivo study

The purpose of this study was to establish a three‐dimensional fluorescent tooth model to investigate bacterial viability against intra‐canal medicaments across the thickness and surface of root dentine. Dental microbial biofilms (Enterococcus faecalis and Streptococcus mutans) were established on the external root surface and bacterial kill was monitored over time against intra‐canal medicament (Ca(OH)₂) using fluorescent microscopy in conjunction with BacLight SYTO9 and propidium iodide stains. An Olympus digital camera fitted to SZX16 fluorescent microscope captured images of bacterial cells in biofilms on the external root surface. Viability of biofilm was measured by calculating the total pixel area of green (viable bacteria) and red (non‐viable bacteria) for each image using ImageJ® software. All data generated were assessed for normality and then analysed using a Mann–Whitney t‐test. The viability of S. mutans biofilm following Ca(OH)₂ treatment showed a significant decline compared with the untreated group (P = 0.0418). No significant difference was seen for E. faecalis biofilm between the Ca(OH)₂ and untreated groups indicating Ca(OH)₂ medicament is ineffective against E. faecalis biofilm. This novel three‐dimensional fluorescent biofilm model provides a new clinically relevant tool for testing of medicaments against dental biofilms.     

Effectiveness of Antibiotic Medicaments against Biofilm Formation of Enterococcus faecalis and Porphyromonas gingivalis

IntroductionIn this study we compared the antibacterial effect of triple antibiotic paste (TAP), double antibiotic paste (DAP), and calcium hydroxide [Ca(OH)₂] against Enterococcus faecalis and Porphyromonas gingivalis biofilm.MethodsThe minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and biofilm formation were measured by using microtiter plate methods. The 2 bacteria were treated with different dilutions of TAP, DAP, and Ca(OH)₂ solutions. The turbidities of the bacterial cultures in the microtiter plate were measured by optical density at 490 nm by using a spectrophotometer. Data were analyzed by 2-way analysis of variance (α = 0.05).ResultsFor TAP, the MIC and MBIC values were 0.003 mg/mL for E. faecalis and 0.006 mg/mL for P. gingivalis. The MBC values for TAP were 0.3 mg/mL for both bacteria. The MIC and MBIC values for DAP were 0.001 mg/mL for E. faecalis and P. gingivalis. The MBC values for DAP were 0.14 mg/mL for both bacteria. Biofilm formation of the 2 bacteria was significantly decreased with TAP and DAP at all tested dilutions (P < .0001) compared with control groups; however, TAP and DAP biofilm formations were not significantly different from each other. Ca(OH)₂ significantly decreased bacterial biofilm formation compared with the control, but it was significantly less than TAP and DAP (P < .05).ConclusionsBoth TAP and DAP were more effective than Ca(OH)₂ against E. faecalis and P. gingivalis. DAP can be considered an effective and comparable antibacterial substitute for TAP.     

In vitro study of the inactivation by dentine of some endodontic medicaments and their bases

Background: The aim of this study was to investigate the antimicrobial effect of endodontic medicaments and their bases in the presence of dentine powder. Methods: The medicaments tested were Ledermix paste, Pulpdent paste, a 50:50 combination of the Pulpdent:Ledermix and their bases. The test organism was Enterococcus faecalis ATCC 29212. The presence or absence of dentine was examined as well as the effect of autoclaving dentine. Serial dilutions of samples at 1 hour, 1 day and 3 days were used for colony counting. The effects of dentine powder on pH for saturated Ca(OH)₂ solution and Pulpdent paste at 1 hour and 24 hours were also measured. Results: Pulpdent and the 50:50 combination of Pulpdent:Ledermix completely inhibited the growth of E. faecalis from 1 hour onwards, and these results were not affected by the presence/absence of dentine powder, pre‐incubation period, timing of autoclaving, or exposure time. Saturated solutions of Ca(OH)₂ are prone to inactivation by dentine powder unlike Pulpdent paste. Ledermix paste took 3 days to exert a significant effect on the growth of E. faecalis. Conclusions: In this laboratory study, both Pulpdent and the 50:50 mixture of Pulpdent with Ledermix were effective medicaments against E. faecalis in the presence of dentine powder.     

Comparative assessment of time-related bioactive glass and calcium hydroxide effects on mechanical properties of human root dentin

Abstract – Suspensions of micro‐ or nanoparticulate SiO₂‐Na₂O‐CaO‐P₂O₅ bioactive glasses could potentially be used as dressings in traumatized front teeth with open apices as an alternative to Ca(OH)₂. These materials have a disinfecting capacity similar to Ca(OH)₂, but bear the advantage of bioactivity. However, because bioactive glasses initially act as alkaline biocides just as Ca(OH)₂ does, they may also negatively affect mechanical dentin properties over time. This was assessed in the current study using standardized human root dentin bars. Specimens were immersed in 1:20 (wt vol^?1) suspensions of nanometric bioactive glass 45S5 or calcium hydroxide for 1, 10, or 30 days. Control specimens were immersed in pure saline for 30 days (n = 20 per group). Subsequently, modulus of elasticity (E) and flexural strength (FS) of the specimens were determined. Results were compared between groups using one‐way anova and Scheffé’s post‐hoc test. Ca(OH)₂ caused a significant (P < 0.001) 35% drop in mean flexural strength values compared to the control treatment after 10 days. No further change was observed between 10 days and 30 days. Bioactive glass caused a 20% drop in mean flexural strength as compared to the control after 10 days. However, this difference did not reach statistical significance (P > 0.05). No effects of either material on dentin modulus of elasticity values were observed. It was concluded that the calcium hydroxide suspension affected the dentin more than the bioactive glass counterpart; however, the effect was self‐limiting and probably restricted to superficial dentin layers, as suggested by the mere decrease in flexural strength but not in modulus of elasticity values.     

Antimicrobial effectiveness of grape seed extract against Enterococcus faecalis biofilm: A Confocal Laser Scanning Microscopy analysis

This study evaluated the antimicrobial effectiveness of 6.5% Vitis vinifera grape seed extract (GSE) against Enterococcus faecalis biofilm using confocal laser scanning microscopy (CLSM). Saline solution (SS), 5.25% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) were used for comparison. Dentin discs were inoculated with E. faecalis strain establishing a 3‐week‐old biofilm. Discs (n = 10) were exposed to 5.25% NaOCl, 2% CHX, 6.5% GSE and SS (negative control) for 10 min. Discs were stained with the fluorescent LIVE/DEAD‐BacLight? dye and analysed using CLSM. The proportion of dead cells in biofilm was analysed using one‐way anova and Tukey tests (P < 0.05). A higher proportion of dead cells was found in GSE group compared with CHX and SS (P < 0.05). NaOCl group was associated with the highest proportion of dead cells (P < 0.05). GSE presented antimicrobial activity against E. faecalis; however, NaOCl was the most effective irrigant solution. GSE was more effective than CHX and SS.     

A novel model for testing the efficiency of removal of calcium hydroxide from complex root canal anatomies

The purpose of this study was to evaluate the efficacy of several irrigation protocols in the removal of calcium hydroxide [Ca(OH)₂] from simulated internal root resorption cavities in a complex root canal anatomy model. The 20° to 35° curved mesiobuccal roots of 94 maxillary molars were sectioned longitudinally; internal resorption cavities were prepared in the apical third of the canal walls. Calcium hydroxide was placed into the cavities and the root halves reassembled. Four teeth were used as controls, and 90 teeth were randomly divided into six experimental groups (n = 15), according to the irrigation protocols used: syringe irrigation; H₂O₂ (HP); Navitip FX; Vibringe‐syringe; Vibringe‐NaviTip FX; ultrasonically activated irrigation (UAI) using an ultrasonic K‐file. In the HP group, 2.5% NaOCl and 3% H₂O₂ were used, while 2.5% NaOCl and 17% EDTA were used in the remaining groups. Stereomicroscope images and radiographs were used to measure the remaining Ca(OH)₂. The model proved to be suitable for simulating complex anatomy. Positive correlation was found between stereomicroscope and radiographic analyses (P < 0.05). UAI removed significantly more Ca(OH)₂ than the other experimental groups (P < 0.05). The HP group was the least efficient protocol (P < 0.05). It would appear that a reliable model has been developed that simulates complex root canal anatomy. Irrigant activation protocols enhanced Ca(OH)₂ removal.     

Does Calcium Hydroxide Reduce Endotoxins in Infected Root Canals? Systematic Review and Meta-analysis

IntroductionThe purpose of this study was to evaluate the potential of endotoxin reduction by comparing the number of lipopolysaccharides (LPSs) before and after the use of calcium hydroxide (Ca[OH]₂) as intracanal medication (ICM).MethodsSearches were performed up to June 2020. Clinical and experimental studies comparing the amount of LPSs before and after the use of Ca(OH)₂ as ICM in infected root canals were included. Risks of bias assessment and data extraction were performed. Meta-analysis was conducted by subgrouping according to Ca(OH)₂, the presence of an antimicrobial substance (AS), irrigant solution during chemomechanical preparation (CMP), and the incidence of LPS reduction. The certainty of evidence was determined by the Grading of Recommendations Assessment, Development and Evaluation approach.ResultsNine studies were included in the qualitative synthesis and 7 in the meta-analysis. Three articles had low risk of bias (RB), 1 had moderate RB, 2 had high RB, and 3 “some concerns.” Overall, Ca(OH)₂, with or without AS, reduced mean LPSs before CMP (standardized mean difference [SMD] = −1.087 [confidence interval {CI}, −1.453 to −0.721], P < .001, I² = 58.7%) and after CMP (SMD = −0.919 [CI, −1.156 to −0.682], P < .001, I² = 24.7%). Considering the irrigant solutions, the overall results showed a reduction before (SMD = −1.053 [CI, −1.311 to −0.795], P < .001, I² = 58.7%) and after CMP (SMD = −0.938 [CI, −1.147 to −0.729], P < .001, I² = 24,6%). Analyses presented very low certainty of evidence. The incidence of LPS reduction was 98.9% and 61.7% for Ca(OH)₂ with and without AS, respectively.ConclusionsCa(OH)₂ reduces endotoxin levels when used as ICM but is unable to eliminate LPSs completely independent of the irrigating solution used with very low certainty of evidence.     

Combined Effect of a Mixture of Silver Nanoparticles and Calcium Hydroxide against Enterococcus faecalis Biofilm

IntroductionThe aim of this study was to evaluate the antibiofilm effectiveness of calcium hydroxide (Ca[OH]₂) mixed with 0.02% silver nanoparticles (AgNPs) in comparison with 1 mg/mL triple antibiotic paste (TAP), Ca(OH)₂, and 0.02% AgNPs against Enterococcus faecalis using confocal laser scanning microscopy.MethodsNinety dentin disks were prepared, sterilized, and inoculated with E. faecalis to establish a 3-week-old biofilm model. The samples received 1 mg/mL TAP, a mixture of Ca(OH)₂ + 0.02% AgNPs, Ca(OH)₂, or 0.02% AgNPs (n = 20/group). Specimens in each group were equally subdivided into 2 groups and incubated for 2 and 4 weeks. Untreated dentin disks (n = 10) were exposed to sterile saline solution and acted as a positive control. Sterile dentin disks (n = 10) were incubated anaerobically in brain-heart infusion broth and served as a negative control. At the end of each observation period, the specimens were stained with LIVE/DEAD BacLight dye (Molecular Probes, Eugene, OR) and analyzed with confocal laser scanning microscopy to determine the proportion of dead cells in the biofilm. Statistical analysis was performed using the generalized linear model repeated measure and Tukey tests (P < .05).ResultsA significantly greater proportion of dead cells was observed in the samples treated with 1 mg/mL TAP (90.39% and 99.41%) and a mixture of Ca(OH)₂ + AgNPs (90.85% and 98.49%) than those in the samples treated with Ca(OH)₂ (76.14% and 91.71%) and AgNPs (62.83% and 88.07%) at 2 and 4 weeks, respectively. A significant difference in the antibiofilm effectiveness was observed among the groups (P < .05), except for 1 mg/mL TAP and the mixture of Ca(OH)₂ + AgNPs (P > .05). All medicaments showed a significant difference in antibiofilm efficacy at the 2 time points.ConclusionsThe mixture of Ca(OH)₂ + AgNPs showed a high antibiofilm effect and was not significantly different from 1 mg/mL TAP. Furthermore, long-term contact between intracanal medicaments and bacterial cells achieved significant antibiofilm efficacy.     

Viscoelastic and chemical properties of dentine after different exposure times to sodium hypochlorite,ethylenediaminetetraacetic acid and calcium hydroxide

This study aims to evaluate the viscoelastic and chemical properties of dentine after different durations of exposure to 5.25% NaOCl, 17% EDTA and Ca(OH)₂ solutions, and NaOCl in alternating combination with EDTA. Standard dentine bars were randomly assigned to: (i) formal‐saline control‐1; (ii) NaOCl; (iii) EDTA; (iv) NaOCl/EDTA; (v) formal‐saline control‐2; (vi) Ca(OH)₂ pH 12.6; and (vii) Ca(OH)₂ pH 9.8. Groups 1‐–4 underwent 10 min cycles of soaking and dynamic mechanical analysis up to 120 min. Groups 5–7 underwent similar tests at days 7, 14, 28 and 84. FTIR spectra of dentine discs exposed to the same regimens assessed surface chemistry. NaOCl or Ca(OH)₂ (pH 12.6) solutions reduced the organic (N‐H[1], N‐H[3], C=0) peak components of dentine. This study demonstrated that accumulative damage of dentine could be facilitated by alternated exposure to NaOCl and EDTA. Exposure of dentine to Ca(OH)₂ (pH12.6) for 7 days reduced viscous behaviour, inferring increased potential for fatigue failure.     

Antibacterial activity of Propolis versus conventional endodontic disinfectants against Enterococcus faecalis in infected dentinal tubules

Introduction

The antibacterial activity against Enterococcus faecalis of 2 propolis samples was investigated in a dentin block model, and their effectiveness was compared with that of established endodontic disinfectants, chlorhexidine (CHX) and calcium hydroxide [Ca(OH)₂].

Methods

Standardized dentin blocks were infected with E. faecalis ATCC 29212. The root canal space was filled with one of the ethanolic extracts of propolis (Artvin or Tekirda? mix [TM]), CHX 2%, Ca(OH)₂, or ethanol or phosphate-buffered saline for control. Canal dentin was sampled after 1 or 7 days by using a standard-size bur. The dentinal shavings were vortexed vigorously in phosphate-buffered saline, and aliquots were cultured on tryptone soy agar plates. Colonies were counted after 2 days of incubation. Statistical significance was set to 5%.

Results

All experimental agents significantly reduced the number of the cultivable bacteria. CHX was the most potent disinfectant at both times. Compared with the ethanol control, no significant reduction in the number of colonies was found for the propolis extracts at day 1; however, significant reduction was found at day 7. The 2 propolis samples were statistically similar to each other and to Ca(OH)₂, but the TM sample was also similar to CHX at day 7. This has been linked to the greater concentration of flavonoids, a group of antibacterially active compounds, in the TM sample as determined by gas chromatography-mass spectrometry analysis.

Conclusions

The antimicrobial activity of the propolis samples tested in this study was between Ca(OH)₂ and CHX. Both propolis samples were antimicrobially effective; however, their activity did not exceed CHX.     

Effect of calcium hydroxide-based materials on periapical tissue healing and orthodontic root resorption of endodontically treated teeth in dogs

Abstract – This study evaluated periapical tissue healing and orthodontic root resorption of endodontically treated teeth sealed with calcium hydroxide in dogs. The sample consisted of three contralateral pairs of maxillary incisors and two contralateral pairs of mandibular incisors in each of two dogs using a split mouth design. After biomechanical preparation of the teeth in the first group (n = 10), a Ca(OH)₂ dressing was placed for 14 days before root canal filling with Ca(OH)₂‐based sealer (Sealapex) and gutta‐percha points. In the second group (n = 10), root canals were obturated immediately after the mechanical preparation with gutta‐percha points and zinc oxide and eugenol (ZOE)‐based sealer (Endofill). After completion of endodontic treatment, the teeth were moved with an orthodontic appliance with a calibrated force of 200 g, reactivated every 21 days. After 105 days, the animals were killed and the teeth were removed upon completion of active treatment, without a period of recovery, and prepared for histomorphological analysis. All sections of each tooth were graded subjectively on a scale from one to four to obtain the average of the 16 histomorphological parameters analysed. Evaluation of the differences between the two treatment protocols was made with Mann–Whitney U‐test. It was observed that the teeth treated with Ca(OH)₂‐based materials provided better outcomes (P = 5%), with complete repair of all root resorption areas, high rate of biological closure of the main canal and apical accessory canals by newly formed cementum, less intense and extensive chronic inflammatory infiltrate, and better organization of the periodontal ligament. Under the tested conditions, Ca(OH)₂‐based materials had a favourable action on periapical tissue healing and repair of orthodontic root resorption in endodontically treated dogs’ teeth.     

The effect of short-term calcium hydroxide treatment on dentin bond strengths to composite resin

Abstract – The purpose of this study was to evaluate the influence of calcium hydroxide (Ca(OH)₂) treatment on the bond strengths to dentin of a resin‐based composite material. Two distinct dental adhesives, Prime & Bond (P&B) NT and Single Bond (SB) were used. One hundred and twenty‐seven bovine incisors were mounted in acrylic, ground flat to expose middle dentin, polished to 600‐grit, and randomly assigned to 10 groups: (i) 0 days, no Ca(OH)₂, P&B NT; (ii) 7 days, no Ca(OH)₂, P&B NT; (iii) 7 days, Ca(OH)₂, P&B NT; (iv) 30 days, no Ca(OH)₂, P&B NT; (v) 30 days, Ca(OH)₂, P&B NT; (vi) 0 days, no Ca(OH)₂, SB; (vii) 7 days, no Ca(OH)₂, SB; (viii) 7 days, Ca(OH)₂, SB; (ix) 30 days, no Ca(OH)₂, SB; (x) 30 days, Ca(OH)₂, SB. All specimens were stored in an incubator at 37°C. Specimens were then retrieved and the dentin surface was rinsed with air/water spray from a triple syringe. Dentin was etched with 37% phosphoric acid (PA) for 15 s, rinsed, blot dried, and coated with adhesive according to manufacturer's instructions. The adhesive was light cured for 10 s, and Filtek Z250 was applied to the surface with a #5 gelatin capsule and light cured for 160 s. Shear bond strengths were measured for each specimen in an Instron unit at a cross‐head speed of 0.5 mm min^?1. The data were subjected to anova and Duncan post‐hoc tests. Mean shear bond strengths for P&B NT ranged from 8.02 to 11.79 MPa. There was no significant difference between P&B NT groups treated with or without Ca(OH)₂ at any time interval. Mean shear bond strengths for SB ranged from 14.70 to 19.54 MPa. No significant difference was found between SB groups treated with or without Ca(OH)₂ at 7 days. At 30 days, SB with Ca(OH)₂ was significantly higher than SB without Ca(OH)₂. Short‐term Ca(OH)₂ treatment had no detrimental effect on dentin bond strengths to the ethanol or acetone‐based adhesive resin systems tested.     

Sodium chloride and potassium sorbate: a synergistic combination against Enterococcus faecalis biofilms: an in vitro study

Incomplete disinfection of the root canal system is a major cause of post‐treatment disease. This study aimed to investigate the disinfecting property of organic acid salts and sodium chloride (NaCl), in a double‐hurdle strategy, on Enterococcus faecalis biofilms. First of all, the high‐throughput resazurin metabolism assay (RMA) was used to test a range of organic acid salts. Then, to gain more insight into the efficacy of sorbate salt solutions, 48‐h E. faecalis biofilms were evaluated in colony‐forming unit (CFU) assays. Chlorhexidine (CHX) and calcium hydroxide [Ca(OH)₂] were tested in parallel as controls. Sorbate salt produced the largest and most significant reduction of fluorescence intensity in the RMA assay. Neither NaCl nor potassium sorbate (KS) alone induced a clinically relevant reduction of CFU counts after 1 h. Surprisingly, the combination of the two in a single solution had a synergistic effect on the inactivation of E. faecalis. Potassium sorbate amplified the efficacy of NaCl. Of the salts tested, NaCl with KS eradicated E. faecalis biofilms within 1 h. This study showed that the double‐hurdle strategy indeed leads to synergistic efficacy and is a possible next step in the complete disinfection of endodontic infections.     

Proline‐Rich Peptide Mimics Effects of Enamel Matrix Derivative on Rat Oral Mucosa Incisional Wound Healing

Background: Proline‐rich peptides have been shown to promote periodontal regeneration. However, their effect on soft tissue wound healing has not yet been investigated. The aim of this study is to evaluate the effect of enamel matrix derivative (EMD), tyrosine‐rich amelogenin peptide (TRAP), and a synthetic proline‐rich peptide (P2) on acute wound healing after a full‐thickness flap procedure in an incisional rat model. Methods: This experimental study has a split‐mouth, randomized, placebo‐controlled design. Test and control wounds were created on the palatal mucosa of 54 Sprague‐Dawley rats. Wounds were histologically processed, and reepithelialization, leukocyte infiltration, and angiogenesis were assessed at days 1, 3, and 7 post‐surgery. Results: EMD and P2 significantly promoted early wound closure at day 1 (P <0.001 and P = 0.004, respectively). EMD maintained a significant acceleration of reepithelialization at day 3 (P = 0.004). Wounds treated by EMD and P2 showed increased angiogenesis during the first 3 days of healing (P = 0.03 and 0.001, respectively). Leukocyte infiltration was decreased in EMD‐treated wounds at day 1 (P = 0.03), and P2 and TRAP induced a similar effect at days 3 (P = 0.002 and P <0.0001, respectively) and 7 (P = 0.005 and P <0.001). Conclusion: EMD and P2 promoted reepithelialization and neovascularization in full‐thickness surgical wounds on rat oral mucosa.     

Effectiveness of calcium and sodium hypochlorite in association with reciprocating instrumentation on decontamination of root canals infected with Enterococcus faecalis

The aim of this study was to evaluate the antimicrobial activity of sodium and calcium hypochlorite utilising reciprocating instrumentation. Sixty root canals were inoculated with E. faecalis for 14 days. Samples were divided into six groups according to decontamination protocol: G1: no treatment, G2: distilled water, G3: 2.5% sodium hypochlorite, G4: 2.5% calcium hypochlorite, G5: 5.25% sodium hypochlorite and G6: 5.25% calcium hypochlorite. Instrumentation was performed with Wave One reciprocating system (Dentsply Sirona Endodontics, York, PA, USA) in groups G2 to G6. Colony‐forming units (CFUs) counting was performed and the data were subjected to Anova and Tukey (α  =  0.05). Group 1 and 2 showed the highest mean contamination, with a significant difference between them (P < 0.05). Groups 3, 4, 5 and 6 showed the lowest contamination means with no significant difference between them (P < 0.05). Sodium and calcium hypochlorite, in association with reciprocating instrumentation, can be an effective decontamination protocol in root canals infected with E. faecalis.