Residual antibiofilm effects of various concentrations of double antibiotic paste used during regenerative endodontics after different application times

ObjectiveWe investigated the residual antibiofilm effects of different concentrations of double antibiotic paste (DAP) applied on radicular dentin for 1 or 4 weeks.DesignDentin samples were prepared (n = 120), sterilized and pretreated for 1 or 4 weeks with the clinically used concentration of DAP (500 mg/mL), low concentrations of DAP (1, 5 or 50 mg/mL) loaded into a methylcellulose system, calcium hydroxide (Ca(OH)₂), or placebo paste. After the assigned treatment time, treatment pastes were rinsed off and the samples were kept independently in phosphate buffered saline for 3 weeks. Pretreated dentin samples were then inoculated with Enterococcus faecalis and bacterial biofilms were allowed to grow for an additional 3 weeks. Biofilms were then retrieved from dentin using biofilm disruption assays, diluted, spiral plated, and quantified. Fisher’s Exact and Wilcoxon rank sum tests were used for statistical comparisons (α=0.05).ResultsDentin pretreatment for 4 weeks with 5, 50 or 500 mg/mL of DAP demonstrated significantly higher residual antibiofilm effects and complete eradication of E. faecalis biofilms in comparison to a 1 week pretreatment with similar concentrations. However, dentin pretreated with 1 mg/mL of DAP or Ca(OH)₂ did not provide a substantial residual antibiofilm effect regardless of the application time.ConclusionsDentin pretreatment with 5 mg/mL of DAP or higher for 4 weeks induced significantly higher residual antibiofilm effects in comparison to a 1 week pretreatment with the same concentrations.     

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.     

Effect of Dentin Conditioning with Intracanal Medicaments on Survival of Stem Cells of Apical Papilla

Introduction

Regenerative endodontics is a valuable treatment modality for immature teeth with pulpal necrosis. A common feature in regenerative cases is the use of intracanal medicaments. Although these medicaments are chosen because of their antibacterial properties, their enduring effect on dentin (conditioning) and the subsequent impact on stem cell survival has never been evaluated. In this study, we hypothesized that triple antibiotic paste (TAP), double antibiotic paste (DAP), or Ca(OH)₂ has an indirect adverse effect on the survival of stem cells of apical papilla (SCAP) by dentin conditioning.

Methods

Human dentin disks were created with a standardized root canal diameter of 3.2 mm. The disks were then exposed to either TAP or DAP (at concentrations of 1 mg/mL or 1000 mg/mL), Ca(OH)₂ (Ultracal), or Hank's balanced salt solution for 7 or 28 days. Next, the medicaments were removed with copious irrigation, followed by placement of SCAP in a Matrigel scaffold in the lumen of the disks. The bioengineered constructs were cultured for 7 days, followed by determination of cellular viability by using the CellTiter-Glo luminescence assay. Data were analyzed using 1-way analysis of variance with Bonferroni post hoc test.

Results

Exposure of dentin to TAP or DAP at 1000 mg/mL resulted in no viable SCAP, whereas the use of these medicaments at 1 mg/mL had no adverse effect on cell viability. In contrast, Ca(OH)₂ treatment significantly increased SCAP survival and proliferation when compared with the control group.

Conclusions

Dentin conditioning with TAP and DAP at commonly used clinical concentration (approximately 1000 mg/mL) alters dentin in such a way as to prevent SCAP survival. This lethal indirect effect of both TAP and DAP can be largely avoided if these medicaments are used at the 1 mg/mL concentration. Conversely, dentin conditioning with Ca(OH)₂ promotes SCAP survival and proliferation.     

Antibiofilm Efficacy of Silver Nanoparticles Alone or Mixed with Calcium Hydroxide as Intracanal Medicaments: An Ex-Vivo Analysis

IntroductionThis study aimed to determine the most effective antibiofilm concentration of silver nanoparticles (AgNPs) alone or in combination with calcium hydroxide (Ca[OH]₂) against Fusobacterium nucleatum biofilm.MethodsThe minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AgNPs alone or with Ca(OH)₂ were determined. Dentin slices were sterilized and inoculated with F. nucleatum for 3 weeks to establish a biofilm. Samples were randomly assigned to determine the MIC and MBC for AgNPs alone or mixed with Ca(OH)₂. A higher concentration of AgNPs for both preparations was also used. Triple antibiotic paste, Ca(OH)₂, and saline were used as controls. Specimens in each group were subdivided over 2 observation periods: 7 and 14 days. At the end of each period, specimens were analyzed with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to determine the metabolic activity. Also, samples from each group were assessed by scanning electron microscopy.ResultsThe MIC and MBC of AgNPs alone against F. nucleatum coincided at 0.04%. The combination of AgNPs + Ca(OH)₂ exhibited a lower MIC and MBC of 0.03%. MTT analysis showed a significant reduction in bacterial viability in all groups compared with negative controls (P < .05). A more substantial reduction in bacterial cells was observed with increasing concentrations of AgNPs at both periods. The combination (AgNPs [0.06%] + Ca[OH]₂) was the most potent against F. nucleatum.ConclusionsThe findings demonstrated that combining AgNPs with Ca(OH)₂ was more effective on the F. nucleatum biofilm than either material alone, suggesting a combined effect.     

The Radiopacity and Antimicrobial Properties of Different Radiopaque Double Antibiotic Pastes Used in Regenerative Endodontics

Introduction

We evaluated the radiopacity and antibacterial properties of various concentrations of double antibiotic paste (DAP) containing barium sulfate (BaSO₄) or zirconium oxide (ZrO₂) radiopaque agents.

Effects of Intracanal Antimicrobials on Viability and Differentiation of Stem Cells From the Apical Papilla: An In Vitro Study

BackgroundRecent studies have indicated that intracanal antimicrobials used to disinfect the root canal in regenerative endodontic therapies (RETs) may be cytotoxic to stem cells from the apical papilla (SCAP), leading to inconsistent treatment outcomes. However, the effects of intracanal antimicrobial agents on the odontogenic differentiation capacity of SCAP at sub-lethal concentrations have not been investigated. The aim of this study was to determine the effects of intracanal antimicrobials on SCAP viability and odontogenic differentiation capacity using a clinically relevant concentration range (0.1–0.8 mg/mL).MethodsImmature human third molars were collected from 71 patients and the apical papillae were harvested to form single-cell suspensions. The cytotoxic effects of intracanal antimicrobials including double antibiotic paste (DAP), triple or modified-triple antibiotic paste (TAP or MTAP), and calcium hydroxide (Ca(OH)₂) on STRO-1⁺ SCAP were assessed using AlamarBlue and Live/Dead assays after exposing cells to treatment groups for 7 days at 0.1 to 0.8 mg/mL. The odontogenic differentiation potential of STRO-1⁺ SCAP was evaluated by immunocytochemistry staining of dentin matrix protein-1 and dentin sialophosphoprotein expression.ResultsAll concentrations of TAP significantly reduced STRO-1⁺ SCAP viability and odontogenic differentiation (P < .001), whereas no DAP concentrations were significantly cytotoxic. Ca(OH)₂ and MTAP concentrations below 0.4 mg/mL and 0.2 mg/mL, respectively, did not significantly reduce viability. The DAP, MTAP, and Ca(OH)₂ did not significantly impact the odontogenic differentiation capacity of STRO-1⁺ SCAP.ConclusionThe varying effects of intracanal antimicrobials on STRO-1⁺ SCAP in vitro suggest amendments to the current root canal disinfection protocol may improve the success of RETs.     

The antimicrobial properties,cytotoxicity, and differentiation potential of double antibiotic intracanal medicaments loaded into hydrogel system

Objective

This study evaluated the antimicrobial properties, cytotoxicity, and mineralization potential of methylcellulose hydrogels loaded with low concentrations of double antibiotic pastes (DAP).

Materials and methods

The direct and residual antibacterial effects of 1, 5, and 10 mg/mL of DAP loaded into hydrogels as well as calcium hydroxide (Ca(OH)₂) were tested against single-species biofilms of Enterococcus faecalis and dual-species biofilms (Enterococcus faecalis and Prevotella intermedia). The effects of DAP hydrogels on proliferation and mineralization of dental pulp stem cells (DPSC) were tested using MTT assays, alkaline phosphate activity (ALP), and alizarin red staining. Fisher’s exact tests, Wilcoxon rank sum tests, and one-way ANOVA were used for statistical analyses (α = 0.05).

Results

All tested concentrations of DAP hydrogels as well as Ca(OH)₂ demonstrated significant direct antibacterial effects against single- and dual-species biofilms. However, only 5 and 10 mg/mL of DAP hydrogels exhibited significant residual antibacterial effects against both types of tested biofilms. Only 1 mg/mL of DAP hydrogels did not have significant negative effects on DPSC viability, ALP activity, and mineralization nodule formation. However, 5 and 10 mg/mL of DAP hydrogels caused significant negative effects on cytotoxicity and mineralization nodule formation of DPSC.

Conclusions

Hydrogels containing 1 mg/mL DAP offered significant direct antibacterial effects against single- and dual-species biofilms without causing significant negative effects on viability, ALP activity, and mineralization nodule formation of DPSC.

Clinical relevance

The methylcellulose-based hydrogel proposed in this study can be used clinically as a biocompatible system to deliver controlled low concentrations of DAP.

     

Antimicrobial efficacy of clindamycin and triple antibiotic paste as root canal medicaments on tubular infection: An in vitro study

This study compared the antibacterial effect of 2% clindamycin and 2% and 100% concentration of triple antibiotic paste (TAP) on an Enterococcus faecalis biofilm. Dentinal tubules of 100 root specimens were infected and randomly assigned to five groups. A total of 1000 mg mL^?1 of TAP, 20 mg mL^?1 of TAP and clindamycin, calcium hydroxide or methylcellulose (control) were placed in the root canal for 1 week. After treatment, dentine shavings were collected from 200 and 400 μm dentine depth and the number of colony‐forming units (CFU) per mg was determined. Reduction in viable bacteria in first three groups was significantly better than calcium hydroxide and control groups. However, the antimicrobial effectiveness among these three groups was not significantly different from each other. There was no significant difference between data at 200 and 400 μm in all groups except the Ca(OH)₂ group. The antibiofilm effect of clindamycin was comparable with TAP, so it may be used instead of TAP.     

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.     

Antibacterial Efficacy and Discoloration Potential of Endodontic Topical Antibiotics

Introduction

The optimal concentration for the use of endodontic topical antibiotics is not known. The aims of this study were to determine the minimum bactericidal concentrations (MBCs) and minimum inhibitory concentrations (MICs) of metronidazole, ciprofloxacin, minocycline, Augmentin (GlaxoSmithKline, Research Triangle Park, NC), and tigecycline against common endodontic pathogens and to evaluate ex vivo the antibacterial efficacy and discoloration effect of triple antibiotic paste (TAP), Augmentin, and tigecycline at different concentrations using a slow-release hydrogel scaffold.

Antibacterial TAP-mimic electrospun polymer scaffold: effects on P. gingivalis-infected dentin biofilm

Objectives

This study sought to investigate, in vitro, the effects of a recently developed triple antibiotic paste (TAP)-mimic polymer nanofibrous scaffold against Porphyromonas gingivalis-infected dentin biofilm.

Materials and methods

Dentin specimens (4 × 4 × 1 mm³) were prepared from human canines. The specimens were sterilized, inoculated with P. gingivalis (ATCC 33277), and incubated for 1 week to allow for biofilm formation. Infected dentin specimens were exposed for 3 days to the following treatments: antibiotic-free polydioxanone scaffold (PDS, control), PDS + 25 wt% TAP [25 mg of each antibiotic (metronidazole, ciprofloxacin, and minocycline) per mL of the PDS polymer solution], or a saturated TAP-based solution (50 mg of each antibiotic per mL of saline solution). In order to serve as the negative control, infected dentin specimens were left untreated (bacteria only). To determine the antimicrobial efficacy of the TAP-mimic scaffold, a colony-forming unit (CFU) per milliliter (n = 10/group) measurement was performed. Furthermore, additional specimens (n = 2/group) were prepared to qualitatively study biofilm inhibition via scanning electron microscopy (SEM). Statistics were performed, and significance was set at the 5 % level.

Results

Both the TAP-mimic scaffold and the positive control (TAP solution) led to complete bacterial elimination, differing statistically (p < 0.05) from the negative control group (bacteria only). No statistical differences were observed for CFU per milliliter data between antibiotic-free scaffolds (2.7 log₁₀ CFU/mL) and the negative control (5.9 log₁₀ CFU/mL).

Conclusions

The obtained data revealed significant antimicrobial properties of the novel PDS-based TAP-mimic scaffold against an established P. gingivalis-infected dentin biofilm.

Clinical relevance

Collectively, the data suggest that the proposed nanofibrous scaffold might be used as an alternative to the advocated clinical gold standard (i.e., TAP) for intracanal disinfection prior to regenerative endodontics.

     

De novo synthetic short antimicrobial peptides against cariogenic bacteria

ObjectiveAntimicrobial peptides (AMPs) have shown the ability to inhibit planktonic bacteria and biofilms. The objectives of this study were to de novo design and synthesize a series of cationic, amphipathic α-helical AMPs that would be shorter, less cytotoxic, and more potent than existing AMPs against cariogenic bacteria.DesignThree short AMPs (GH8, GLLWHLLH-NH₂; GH12, GLLWHLLHHLLH-NH₂; and GH16, GLLWHLLHHLLHLLHH-NH₂) were designed, synthesized and characterized structurally. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against eight major cariogenic bacteria were tested to select the most promising peptide. Scanning electron microscopy (SEM) was used to observe the bacterial membrane after treatment with selected peptides. The bactericidal kinetics, effects on biofilm and cytotoxity were further investigated.ResultsOf the three AMPs, GH12 had the most balanced structural parameters and a high content of α-helical structure. GH12 had a MIC of 4.0-8.0 μg/mL and MBC of 8.0-32.0 μg/mL. The corresponding values for the other two AMPs were 2- to 64- fold higher. In time-kill assays, GH12 killed all bacterial strains within 60 min at 4- fold MBC. SEM observed lysis and pore formation of the cytomembrane after treatment with GH12. 8.0 μg/mL GH12 inhibited Streptococcus mutans biofilm formation. Confocal laser scanning microscopy showed that GH12 effectively reduced the biomass of 1-day-old S. mutans biofilm. Cytotoxicity assays indicated that GH12 showed little toxic effect on the viability of human gingival fibroblasts.ConclusionThese results indicate that GH12 shows antimicrobial activity against cariogenic bacteria and biofilms in vitro.     

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.     

Susceptibility of Porphyromonas gingivalis in biofilms to amoxicillin,doxycycline and metronidazole

The susceptibility of Porphyromonas gingivalis to amoxicillin, doxycycline and metronidazole was determined by a standardized method taking into account the biofilm mode of growth of subgingival bacteria. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 48‐h biofilms of P. gingivalis established on membrane filters in a Modified Robbins Device were determined by agar dilution. The results were compared to (i) conventional MIC determinations, (ii) the susceptibility of planktonic cultures with cell numbers equal to those of the biofilms, and (iii) results for detached biofilm cells. The MICs of the biofilms of the six reference strains and clinical isolates containing 10^7?8 cells/filter were much higher than the conventional MIC values. However, the MIC of planktonic cultures of equal cell numbers also increased, indicating that an inoculum effect is part of the explanation of the increased resistance of biofilms. Still, the MBCs of biofilms were 2–8 times, and those for doxycycline up to 64 times, greater than the MBC values for planktonic cultures.     

Outcome of Regenerative Endodontic Procedures in Nonvital Immature Permanent Teeth Using 2 Intracanal Medications: A Prospective Randomized Clinical Study

IntroductionThis randomized clinical trial aimed to assess and compare the long-term clinical and radiographic outcomes of regenerative endodontic procedures (REPs) in treating nonvital immature permanent teeth using 2 intracanal medicaments.MethodsForty-five patients yielding a total of 50 anterior and posterior nonvital immature teeth were randomly divided into 2 groups. REPs using either nonsetting calcium hydroxide (Ca[OH]₂) (n = 25) or modified triple antibiotic paste (TAP) (n = 25) as intracanal medicaments were performed. NeoMTA Plus (Avalon Biomed Inc) was applied for coronal sealing. Cases were followed up clinically and radiographically for 36 months. The survival rate, success rate, and clinical outcome measures were analyzed. Preoperative and recall radiographs were evaluated for dimensional changes in root length, dentin thickness, apical diameter, and periapical radiolucency.ResultsAt the 36-month follow-up, the success and survival rates were 81.6% and 100%, respectively, in which 79.4% of cases showed complete resolution of periapical radiolucency with no significant difference between the nonsetting Ca(OH)₂ and modified TAP groups (P > .050). The cumulative changes in root length, root dentin thickness, and apical diameter throughout the study period were observed in 47.9%, 77.1%, and 89.6% of cases, respectively, with no significant differences between groups (P ≥ .39). Intracanal calcifications were detected in 60% of cases with no significant difference between groups (P = .77).ConclusionsREPs, using either nonsetting Ca(OH)₂ or modified TAP as the intracanal medicament, exhibited high success and survival rates over a follow-up period of 36 months with equally favorable clinical and radiographic outcome data.     

Antibacterial Efficacy of Triple Antibiotic Medication With Macrogol (3Mix-MP), Traditional Triple Antibiotic Paste,Calcium Hydroxide,and Ethanol Extract of Propolis: An Intratubular Dentin Ex Vivo Confocal Laser Scanning Microscopic Study

IntroductionThis study aimed to assess the effectiveness of antibacterial activity of medications used in regenerative endodontic treatment.MethodsSixty-seven dentin cylinders of single-rooted teeth were contaminated with a culture of Enterococcus faecalis (ATCC 29212; American Type Culture Collection, Manassas, VA) for 5 days. Samples were divided into 1 control group and the following experimental groups according to the medication applied: traditional triple antibiotic paste (TAP), clindamycin-modified TAP (mTAP), triple antibiotic medication with macrogol (3Mix-MP), clindamycin-modified 3Mix-MP (m3Mix-MP), calcium hydroxide (CH), and ethanol extract of propolis (EEP). After 14 days, the medications were removed, and the samples were submitted to confocal laser scanning microscopic analysis to quantify the percentage of viable bacteria. The distribution of data was confirmed by the Shapiro-Wilk test. The Kruskal-Wallis and Dunn tests were used for intergroup comparisons, and the Wilcoxon test was used for comparison between superficial and deep antibacterial efficacy for the same medication. The level of significance was set at P < .05.Results3Mix-MP and m3Mix-MP presented significantly higher antibacterial efficacy compared with the other tested medications (P < .05), except for mTAP. mTAP was more effective than TAP (P < .05). The antibacterial efficacy of EEP and CH did not differ significantly from TAP and mTAP (P > .05). All medications showed effective antibacterial action compared with the control group (P < .05).Conclusions3Mix-MP and m3Mix-MP, which present extremely high concentrations of antibiotics (1500 mg/mL), were not more effective than mTAP at the concentration recommended by the American Association of Endodontists (5 mg/mL). Moreover, CH and EEP were as effective as TAP and mTAP.     

High molecular weight hyaluronic acid reduces the growth and biofilm formation of the oral pathogen Porphyromonas gingivalis

BackgroundPorphyromonas gingivalis (P. gingivalis) is viewed as a keystone microorganism in the pathogenesis of periodontal and peri-implant diseases. Hyaluronic acid (HA) is believed to exert antimicrobial activity. The aim of this study is to assess the in-vitro growth and biofilm formation of P. gingivalis under HA and compare the effect of HA to that of azithromycin (AZM) and chlorhexidine (CHX).Materials and methodsIn each material, the minimum inhibitory concentration (MIC), 50% MIC, 25% MIC, and 12.5% MIC were tested. The growth of P. gingivalis was evaluated by absorbance spectrophotometry after 48 h. A biofilm inhibition assay was performed on a 72-hour culture by washing planktonic bacterial cells, fixing and staining adherent cells, and measuring the variation in stain concentrations relative to the untreated control using absorbance spectrophotometry.ResultsThe results show that the overall growth of P. gingivalis after 48 h was 0.048 ± 0.030, 0.008 ± 0.013, and 0.073 ± 0.071 under HA, AZM, and CHX, respectively, while the untreated control reached 0.236 ± 0.039. HA was also able to significantly reduce the biofilm formation of P. gingivalis by 64.30 % ± 22.39, while AZM and CHX reduced biofilm formation by 91.16 %±12.58 and 88.35 %±17.11, respectively.ConclusionsHigh molecular-weight HA significantly inhibited the growth of P. gingivalis. The overall effect of HA on the growth of P. gingivalis was similar to that of CHX but less than that of AZM. HA was also able to significantly reduce the biofilm formation of P. gingivalis. However, the ability of HA to prevent the biofilm formation of P. gingivalis was generally less than that of both AZM and CHX.     

Comparison of bond strength of self-etch adhesive to pulp chamber dentin after placement of calcium hydroxide and various antibiotic pastes

Abstract

Objective. The aim of this study was to evaluate the effect of calcium hydroxide, double antibiotic paste (DAP) and triple antibiotic paste (TAP) with minocycline, cefaclor and amoxicillin on the micro tensile bond strength (µTBS) of self-etch adhesive to pulp chamber dentin. Materials and methods. Sixty mandibular first molars were cut horizontally and randomly divided into a control group and five experimental groups, which received an intra-canal dressing, as follows: calcium hydroxide, DAP, TAP with minocycline, TAP with cefaclor and TAP with amoxicillin. After storing the specimens for 4 weeks, the medicaments were removed by irrigation with 10 mL each of the following solution: 2.5% NaOCl, 17% EDTA and distilled water. A self-etch adhesive (Clearfil S3 Bond, Okayama, Japan) was applied and composite resin (Clearfil Majesty Posterior, Kuraray Medical Inc., Japan) was placed into the cavity. A µTBS test was performed on each specimen using a universal test machine. Results. The DAP reduced the µTBS of self-etch adhesive compared to the control group, calcium hydroxide and TAP with minocycline and with cefaclor (p < 0.05). However, the other medicaments did not result in a decreased µTBS of self-etch adhesive to pulp chamber dentin as compared to the control group (p > 0.05). Conclusions. The use of DAP resulted in a reduced µTBS of self-etch adhesive to pulp chamber dentin.     

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.     

Antimicrobial and antibiofilm action of Casbane Diterpene from Croton nepetaefolius against oral bacteria

ObjectiveThe antibacterial activity of Casbane Diterpene (CD) was evaluated in vitro against Streptococcus oralis, S. mutans, S. salivarius, S. sobrinus, S. mitis and S. sanguinis. The viability of planktonic cells was analysed by susceptibility tests (MIC and MBC) and antibiofilm action was assayed.MethodsThe minimal inhibitory and bactericidal concentrations (MIC and MBC) of oral Streptococcus were evaluated through microdilution tests. To assay antibiofilm activity, biofilms were generated on 96-wells polystyrene plates under the presence of CD and quantified by a crystal violet technique and colonies forming units counting.ResultsThe CD isolated from Croton nepetaefolius showed antimicrobial effect on planktonic forms and biofilms of oral pathogens, with MIC values of 62.5 μg/mL against Streptococcus oralis and values between 125 and 500 μg/mL against S. mutans, S. salivarius, S. sobrinus, S. mitis and S. sanguinis. CD showed an inhibitory effect on S. mutans biofilm formation at 250 μg/mL, and a decrease on viable cell of 94.28% compared to the normal biofilm growth.ConclusionsThe compound CD can be considered as a promising molecule for the treatment against oral pathogens responsible for dental biofilm.