Examining the Effects of Asiaticoside on Dental Pulp Stem Cell Viability and Proliferation: A Promising Approach to Root Canal Treatment.

AIM: This study aims to evaluate the impact of asiaticoside (AC) on the viability and proliferation of dental pulp stem cells (DPSCs), considering the known negative effects of routinely used intracanal medicaments. This evaluation will be compared with the outcomes from using traditional intracanal medicaments, specifically triple antibiotic paste (TAP) and calcium hydroxide [Ca(OH)2]. MATERIALS AND METHODS: The DPSCs were obtained from the third molars of an adult donor. The application of flow cytometry was employed to do a phenotypic analysis on DPSCs using CD90, CD73, CD105, CD34, CD14, and CD45 antibodies. The methylthiazol tetrazolium (MTT) assay was employed to assess cellular viability. The cells were treated with different concentrations of TAP and Ca(OH)2 (5, 2.5, 1, 0.5, and 0.25 mg/mL), along with AC (100, 50, 25, 12.5, and 6.25 µM). A cell proliferation rate was performed at 3, 5, and 7 days. RESULTS: The characterization of DPSCs was conducted by flow cytometry analysis, which verified the presence of mesenchymal cell surface antigen molecules (CD105, CD73, and CD90) and demonstrated the absence of hematopoietic markers (CD34, CD45, and CD14). Cells treated with concentrations over 0.5 mg/mL of TAP and Ca(OH)2 showed a notable reduction in cell viability in comparison to the untreated cells (p < 0.05). Additionally, the cells treated with different concentrations of AC 12.5, 6.25, 25, and 50 µM did not differ significantly from the untreated cells (p > 0.05). Nevertheless, cells treated with concentrations of 100 µM showed a significant reduction in viability compared to the untreated cells (p < 0.05). After a period of 7 days, it was noted that cells exposed to three different concentrations of AC (50, 25, and 12.5 µM) had a notable rise in cell density in comparison to TAP and Ca(OH)2 (p < 0.05). Furthermore, cells that were exposed to a concentration of 12.5 µM exhibited the highest cell density. CONCLUSION: The cellular viability of the AC-treated cells was superior to that of the TAP and Ca(OH)2-treated cells. Moreover, the AC with a concentration of 12.5 µM had the highest degree of proliferation. CLINICAL SIGNIFICANCE: This study underscores the importance of evaluating alternative root canal medicaments and their effects on DPSCs' growth and vitality. The findings on AC, particularly its influence on the survival and proliferation of DPSCs, offer valuable insights for its probable use as an intracanal medication. This research contributes to the ongoing efforts to identify safer and more effective intracanal treatments, which are crucial for enhancing patient outcomes in endodontic procedures. How to cite this article: Alazemi MJ, Badawi MF, Elbeltagy MG, et al. Examining the Effects of Asiaticoside on Dental Pulp Stem Cell Viability and Proliferation: A Promising Approach to Root Canal Treatment. J Contemp Dent Pract 2024;25(2):118-127.

The impact of methylene blue photosensitizer, aPDT and a calcium hydroxide-based paste on the physicochemical and mechanical characteristics of root canal dentin and the bonding interface of fiberglass posts.

PURPOSE: To investigate the influence of methylene blue (MB)-mediated antimicrobial photodynamic therapy (aPDT) and calcium hydroxide (CH) medication on the mechanical characteristics, degree of conversion (DC), quantification, and volume of gaps at the adhesive interface of glass fiber posts (GFPs) luted to distinct thirds of root canal dentin. Additionally, the microhardness (MH), elastic modulus (Eit), morphology, and chemical structure of the intraradicular dentin were assessed. MATERIALS AND METHODS: 6 experimental groups were formed by sorting 102 bovine incisors. Canals receiving deionized water irrigation as a negative control; canals receiving deionized water irrigation and filled with CH as a positive control; groups treated with CH + MB at 50 and 100 mg/L without irradiation; and groups treated with CH + MB at 50 and 100 mg/L irradiated by red laser for 60 s (660 nm; 100 mW; 6.5 J; 72 J/cm2). MH, Eit, and DC properties were evaluated for both the resin cement layer and root dentin substrate (n = 8). Volume and quantification of gaps at the bonding interface (n = 6), and dentin morphology and chemical content were investigated (n = 3). Data were analyzed using a repeated-measures 2-way ANOVA followed by Tukey post hoc analysis (α = 0.05). RESULTS: The distinct intraradicular thirds and treatment with MB-mediated aPDT, whether activated or not, in combination with CH, had a significant impact on the mechanical characteristics of the root dentin. This effect was also observed in the MH, Eit, DC, quantification, and volume of gaps at the luting interface (P < .05). In general, a higher concentration of MB, whether activated by a red laser or not, led to lower values in the mechanical properties of the root dentin, as well as in MH, Eit, and DC at the adhesive interface (P < .05). Additionally, these groups exhibited higher values for quantification and volume of gaps at the luting substrate (P < .05). Scanning electron micrographs and energy dispersive X-ray spectra showed qualitative similarity among all groups, except for the negative experimental control group. CONCLUSIONS: MB-mediated aPDT at 50 mg/L, in combination with CH, demonstrated favorable physico-chemical and mechanical characteristics in intraradicular dentin, along with satisfactory mechanical features and the adhesive interface integrity for GFPs at all intraradicular depths. CLINICAL SIGNIFICANCE: MB-mediated aPDT at a concentration of 50 mg/L combined to CH medication represents a suitable choice for photosensitization in the context of intracanal disinfection following the biomechanical procedure and prior to luting of intraradicular restorations.

Effect of EGCG-based paste as intracanal dressing, in MMPs 2 and 9 expression in dog's periapical lesions.

High expression of MMP-2 and MMP-9 in periapical lesions plays an important role in the degradation of the extracellular matrix. This study aimed to investigate the effect of epigallocatechin-3-gallate (EGCG)-based endodontic paste as an intracanal dressing on the expression of MMP-2 and MMP-9 in periapical lesions. Periapical lesions were experimentally induced in 35 mature beagle dog premolars randomly divided into healthy teeth, untreated periapical lesions, periapical lesions treated in a single session (control groups), and periapical lesions treated in two sessions with EGCG or calcium hydroxide-based pastes (experimental groups). After 120 days, specimens were obtained for histopathologic and immunofluorescence analyses to assess the expression of MMP-2 and MMP-9. The statistical analysis was performed using a p-value of 0.05. Endodontic treatment in two sessions using medication with EGCG and calcium hydroxide-based pastes provided similar repair of the apical and periapical tissues and neoformation of periodontal ligament fibers, cementum, and alveolar bone (p>0.05). The experimental groups treated in two sessions with both medications presented expression of MMP-2 and MMP-9 similar to that in healthy teeth (p>0.05), and significantly lower than teeth treated in a single session or untreated periapical lesions (p <0.001). Expression of MMP-2 and MMP-9 was observed in the cytoplasm of fibroblasts, osteoblasts, cementoblasts, cementocytes, and vascular endothelium. The use of EGCG-based endodontic paste reduced the expression of MMP-2 and MMP-9 and allowed repair of periapical lesions, similar to calcium hydroxide-based paste, and superior to treatment performed in a single session.

Antibacterial biofilm efficacy of calcium hydroxide loaded on Gum Arabic nanocarrier: an in-vitro study.

BACKGROUND: An innovative intracanal medication formulation was introduced in the current study to improve the calcium hydroxide (Ca(OH)2) therapeutic capability against resistant Enterococcus faecalis (E. faecalis) biofilm. This in-vitro study aimed to prepare, characterize, and evaluate the antibacterial efficiency of Ca(OH)2 loaded on Gum Arabic (GA) nanocarrier (Ca(OH)2-GA NPs) and to compare this efficiency with conventional Ca(OH)2, Ca(OH)2 nanoparticles (NPs), GA, and GA NPs. MATERIALS AND METHODS: The prepared nanoparticle formulations for the tested medications were characterized using Transmission Electron Microscope (TEM) and Fourier-Transform Infrared Spectroscopy (FTIR). 141 human mandibular premolars were selected, and their root canals were prepared. Twenty-one roots were then sectioned into 42 tooth slices. All prepared root canals (n = 120) and teeth slices (n = 42) were divided into six groups according to the intracanal medication used. E. faecalis was inoculated in the samples for 21 days to form biofilms, and then the corresponding medications were applied for 7 days. After medication application, the residual E. faecalis bacteria were assessed using CFU, Q-PCR, and SEM. Additionally, the effect of Ca(OH)2-GA NPs on E. faecalis biofilm genes (agg, ace, and efaA) was investigated using RT-PCR. Data were statistically analyzed at a 0.05 level of significance. RESULTS: The synthesis of NPs was confirmed using TEM. The results of the FTIR proved that the Ca(OH)2 was successfully encapsulated in the GA NPs. Ca(OH)2-GA NPs caused a significant reduction in the E. faecalis biofilm gene expression when compared to the control (p < 0.001). There were significant differences in the E. faecalis CFU mean count and CT mean values between the tested groups (p < 0.001) except between the Ca(OH)2 and GA CFU mean count. Ca(OH)2-GA NPs showed the least statistical E. faecalis mean count among other groups. SEM observation showed that E. faecalis biofilm was diminished in all treatment groups, especially in the Ca(OH)2-GA NPS group when compared to the control group. CONCLUSIONS: Ca(OH)2 and GA nanoparticles demonstrate superior anti-E. faecalis activity when compared to their conventional counterparts. Ca(OH)2-GA NPs showed the best antibacterial efficacy in treating E. faecalis biofilm. The tested NP formulations could be considered as promising intracanal medications.

Antimicrobial effect of Pentaclethra Macroloba plant extract against Enterococcus Faecalis.

This study evaluated the antimicrobial efficacy of a new intracanal drug based on Pentaclethra macroloba extract, a plant of Amazonian origin, against Enterococcus faecalis using macrodilution test and intratubular evaluation with Confocal Laser Scanning Microscopy (CLSM). The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the pure extract of Pentaclethra macroloba andits association with calcium hydroxide and ultracall were determined. Then, thirty-three dentin cylinders were prepared and inoculated with E. faecalis, to evaluate the antimicrobial effect of the medications on the dentinal tubules with CLSM. The data was analyzed using the Kruskal-Wallis and Dunn tests. The extract in association with calcium hydroxide showed a lower CBM, and in the intratubular test all tested medications were effective against E. faecalis (P >0.05). The new intracanal drug based on P. macroloba extract has an antimicrobial effect against E. faecalis and further studies are needed for its clinical use.

Ion release, biocompatibility, and bioactivity of resin-modified calcium hydroxide cavity liners.

BACKGROUND: The placement of liners near the pulp area is essential for therapeutic effects and maintaining pulp health while stimulating the formation of tertiary dentin. This in vitro study aimed to evaluate the calcium release, pH, biocompatibility, solubility, and bioactivity of three resin-modified calcium hydroxide cavity liners. METHODS: The disc specimens of each cavity liner were prepared using polyethylene molds of 7 mm in diameter and 2 mm in height (n = 10). Three light-cure liners evaluated include Ultra-Blend Plus (UB), Base-it (BI), and Master Dent (MD). The samples were then immersed in flasks containing 10 mL of distilled water. Calcium ion release, pH, and solubility were evaluated in two weeks of incubation. The cytotoxicity of extracts adjacent to the specimens was evaluated by MTT assay using NIH/3T3 cells after 1, 3, and 7 days of incubation. The ability to induce the nucleation of calcium phosphates (CaPs) after 28-day immersion in a simulated body fluid was investigated by SEM-EDX analysis. Statistical analysis was performed using ANOVA, Kruskal-Wallis, and repeated measures tests at the significant level of 0.05. RESULTS: There was a significant difference in the release of calcium ions among the three liners investigated on days 1, 7, and 14 (p < 0.05). UB liners exhibited a significantly higher amount of calcium release than the other two liners, followed by BI, and MD. On day 1, there was no significant difference in the average pH among the three liners. However, after day 7, the MD liner showed a significant decrease in pH compared to the other two liners. BI liner demonstrated the highest level of biocompatibility, followed by the MD and UB liners. UB showed a high calcium release, solubility with no alkalizing activity, and the formation of more mature Ca-rich apatite deposits than the other two liners. CONCLUSION: Based on the results of this study, the cavity liner material's performance is material dependent. It can impact ion release, biocompatibility, and bioactivity which are important factors to consider in clinical practice. Further studies are needed to investigate the long-term effects of different liner materials on oral tissues.

Comparative evaluation of antimicrobial efficacy of different combinations of calcium hydroxide against Enterococcus faecalis.

BACKGROUND: The study aims to compare the synergistic antibacterial efficacy of different combinations of calcium hydroxide as an intracanal medicament against E. faecalis. MATERIAL AND METHODS: The current study included four hundred extracted human permanent mandibular premolar teeth. After complete chemo-mechanical preparation, the middle third of the root was sectioned using a rotary diamond disc and a total of 400 samples were obtained. The specimens were inoculated with E. faecalis for 21 days. After that, specimens were divided into five groups (n = 80) based on materials used for the disinfection of samples: Group I, calcium hydroxide alone; Group II, calcium hydroxide + 2% chlorhexidine gel; Group III, calcium hydroxide + 2% chitosan gel; Group IV, calcium hydroxide + 0.02% silver nanoparticle gel; Group V, calcium hydroxide + Bioactive glass S53P4. Dentin shavings from the apical third were obtained from the inner third of dentin were obtained using gates glidden no.1 to the apical depth, followed by no.2, 3, 4 and 5 analyzed for E. faecalis using the culture method. One-way analysis of variance (ANOVA) was used for data analysis, followed by post-hoc Tukey's test for multiple comparisons of means to check the difference in bacterial inhibition between the groups. RESULTS: ANOVA results revealed a significant reduction of bacterial counts in all the groups compared (p < 0.001). Intergroup comparison showed maximum bacterial reduction (p < 0.001) with calcium hydroxide + bioactive glass S53P4 compared with other groups. CONCLUSION: Synergistic effect of calcium hydroxide showed better bacterial reduction compared to calcium hydroxide alone. Among the combinations evaluated, calcium hydroxide with bioactive glass, found to be most effective compared to other groups.

Novel strategies enhancing endodontic disinfection: Antibacterial biodegradable calcium hydroxide nanoparticles in an ex vivo model.

Due to the high failure rates associated to endodontic disinfection, this study aimed to investigate the antibacterial properties of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with Ca(OH)2 for endodontic disinfection procedures. Ca(OH)2 NPs production and physicochemical characterization were carried out as well as multiple antibacterial tests using three bacterial strains and an ex vivo model of endodontic infection with extracted human teeth. Agar diffusion test and broth dilution determined the inhibition growth zones (n = 5) and the minimal inhibitory concentration (MIC, n = 5), respectively. Cell viability was assessed using Live/Dead staining with confocal microscopy (n = 5). Data was analysed using ANOVA followed by post-hoc analysis. After 24 h of incubation, Ca(OH)2 NPs demonstrated a MIC of 10 µg/mL for Porphyromonas gingivalis (p < 0.001) and Enterococcus faecalis and 5 µg/mL for Fusobacterium nucleatum (p < 0.001). Although the agar diffusion test did not exhibit any inhibition area for Ca(OH)2 nor for Ca(OH)2 NPs, this was probably due to the buffering effect of the agar medium. However, the antibacterial capacity was confirmed in an ex vivo model, where instrumentalized teeth were infected with Enterococcus Faecalis and treated after 28 days of culture. A significant reduction in bacterial metabolic activity was confirmed for Ca(OH)2 NPs (40 % reduction with a single dose) and confirmed by Live/Dead staining. In conclusion, Ca(OH)2-loaded PLGA NPs present promising antibacterial efficacy for endodontic disinfection procedures.

Evaluation of antimicrobial efficacy of ethanolic extract of Cuminium Cyminium as intracanal medicament on common bacteria of endodontic infections: An in vitro study.

Context: Antimicrobial intracanal medicaments play a vital role in successful outcome of any endodontic procedure. One such plant extract Cuminium cyminium, as intracanal medicaments needs to be researched. Aims: The purpose of this study was in vitro assessment of the antibacterial activity of ethanol extract of C. Cyminium in comparison to Calcium hydroxide (Ca[OH]2) as intracanal medicament against the pathogens of endodontic infection, at an interval 1 h, 24 h, 48 h, and 72 h. Settings and Design: The study was conducted in the central research laboratory of our institute. Freshly prepared C. cyminium extract was procured from AYUSH approved laboratory and direct contact test (DCT) was utilized, which is based on turbidometric determination of microbial growth in a 96-well microplate, carrying 6 times for each bacteria. Methodology: Three groups were assigned for each material in a 96 microwell plate for DCT. Bacterial growth kinetics was monitored at intervals of 1 h, 24 h, 48 h, and 72 h using spectrophotometer at 595 nm. The optical density of T2 (Test group), P2 (Positive control), and N2 (Negative control) was considered. Statistical Analysis Used: After compiling the data, based on the normality of data, further statistical analysis was conducted using Kolmogorov-Smirnov test, Paired t-test, and pairwise comparisons by Turkey's multiple post hoc procedures. The level of statistical significance was set at P = 0.05. Results: The comparison of mean optical density values of C. cyminium in comparison with Ca(OH)2 against the microorganisms of endodontic origin showed a statically significant decrease in bacterial viability at the end of 24 h, 48 h, and 72 h. Conclusion: Based on the results of the study, it can be concluded that C. cyminium has significant antibacterial action against endodontic origin, at interval of 24 h, 48 h, and 72 h.

The Effect of Calcium hydroxide, Glass Ionomer and light cured resin modified calcium silicate on viability, proliferation and differentiation of stem cells from human exfoliated deciduous teeth.

BACKGROUND: Vital pulp therapy, based on the use of stem cells, has promising research and therapeutic applications in dentistry. It is essential to understand the direct effect of capping materials on the dental pulp stem cells of primary teeth, which contribute to the healing powers of the tooth. The aim of this study is to evaluate the effect of different capping materials (Calcium Hydroxide (DyCal®) - Glass Ionomer (Fuji IX®) and light-cured resin modified calcium silicate (TheraCal LC®)) on the viability, proliferation, and differentiation of stem cells from human exfoliated deciduous teeth (SHEDs). METHODS: SHEDs were isolated from extracted primary teeth, then divided into four groups and each of the capping materials were applied to the stem cells as follows: group I the controls, group II with Ca(OH)2, group III with the GIC, and group IV with the Theracal LC. For all groups assessment of viability and proliferation rate was done using the MTT cell proliferation assay. Also, Differentiation was evaluated by measuring the gene expression of Alkaline phosphatase enzyme activity (ALP) and Dentin matrix protein-1 (DMP1) through quantitative real-time PCR. Morphological assessment was conducted using Alizarin Red S staining. All evaluations were performed after 7 and 14 days of culture. RESULTS: TheraCal LC showed the highest values of proliferation, which was significant only compared to the control group after 2 weeks (p = 0.012). After one week, TheraCal LC showed the highest significant values of ALP and DMP1 compared to all other groups (p < 0.001). CONCLUSION: The three materials under study are biocompatible, maintain viability, and stimulate the proliferation and differentiation of SHEDs. However, TheraCal LC allows better proliferation of SHEDs than Dycal Ca(OH)2 and Fuji IX GIC.

The Effects of Calcium Silicate- and Calcium Hydroxide-based Root Canal Sealers on Postoperative Pain: A Randomized Clinical Trial.

INTRODUCTION: This study aimed to compare the postoperative pain level changes resulting from using calcium silicate- (EndoSeal MTA) and calcium hydroxide-based (Sealapex) root canal sealers in mandibular first and second molar teeth with symptomatic apical periodontitis. METHODS: A total of 60 patients with symptomatic apical periodontitis in their lower molar teeth were randomly allocated into 2 groups according to sealer type (n = 30). Demographic data, including gender, age, and smoking habit, and preoperative pain measures were recorded. Root canal treatments were performed in a single visit. Postoperative pain measurements and analgesic intake were measured at 6, 12, 24, and 48 hours and after 3, 5, and 7 days using the visual analog scale. The data were statistically analyzed using a chi-squared test (to compare gender, age, smoking habit, analgesic intake, and sealer extrusion), the Mann-Whitney U test (to compare pain levels), Friedman tests (for the evaluations of the reduction in pain levels over time), and Spearman's correlation test (to analyze the relationships of age, gender, smoking habit factors with postoperative pain) (P = .05). RESULTS: The statistical analysis showed no significant differences between the groups in postoperative pain and analgesic intake at any of the time intervals evaluated (P > .05). CONCLUSIONS: Patients treated with calcium silicate- and calcium hydroxide-based root canal sealers experienced similar postoperative pain and no statistically significant differences were observed in analgesic intake.

Evaluation of the biological effect of mineral trioxide aggregate in inflamed pulp-in vivo analysis.

The health of dental pulp tissue is critical to maintaining normal tooth function from the eruption of permanent teeth to the formation of the apex. The study evaluated the inflamed pulp response to the mineral trioxide aggregate (MTA) after direct pulp capping with the mechanical pulp exposure in rats' incisor. Forty-eight mandibular central incisors of twenty-four Sprague-Dawley rats which were prepared with the cavities of one mm diameter, and the pulp exposures were randomly assigned into two groups: MTA group and calcium hydroxide (Ca(OH)2) group. The direct pulp capping was performed after three days and samples histological observations conduction within eight weeks. In both MTA and Ca(OH)2 groups, dentin -like structures were observed in the pulp tissues of some teeth. The number of teeth with reparative tissue in MTA group was statistically significantly higher than that in Ca(OH)2 group (p = 0.041). Inflammatory cell infiltration was found in the crown pulp tissues in two groups, and no statistical difference was observed between the two groups (p = 0.243). Pulp necrosis occurred in both groups, and there was no statistical difference between the two groups (p = 0.622). The results in this paper suggest that MTA promotes direct pulp capping and hence has certain potential clinical applications value in the treatments for the preservation of inflamed pulp.

Direct and residual antimicrobial effect of 2% chlorhexidine gel, double antibiotic paste and chitosan- chlorhexidine nanoparticles as intracanal medicaments against Enterococcus faecalis and Candida albicans in primary molars: an in-vitro study.

BACKGROUND: Thorough disinfection of root canals in primary molars may be complicated by the complex root canal morphology. This in-vitro study aimed to compare direct and residual antimicrobial effect of 2% chlorhexidine (CHX) gel, 500 mg/ml double antibiotic paste (DAP) and chitosan-chlorhexidine nanoparticles (CS-CHX NPs) as intracanal medicaments against Enterococcus faecalis (E. faecalis) and Candida albicans (C. albicans) in primary molars. METHODS: Mesial roots of 63 mandibular second primary molars were infected with E. faecalis and C. albicans. Teeth were divided into 9 groups: Ia: (CS-CHX NPs), IIa: (CHX), IIIa: (DAP), IVa: chitosan nanoparticles (CSNPs) in which medicaments were placed for 3 days, groups Ib: (CS-CHX NPs), IIb: CHX, IIIb: (DAP), IVb: (CSNPs) in which medicaments were placed for 7 days, and Group V (control): teeth were infected, irrigated with saline, and sampled 3- and 7-days post-infection. Microbiological samples were obtained after infection, 3, and 7 days after medicament placement and 7 days after medicament removal for both time points. One-way ANOVA, Tukey's post hoc test and paired t-test were used at p < 0.05. RESULTS: CS-CHX NPs had the highest anticandidal effect which was comparable to CHX and significantly higher than other medicaments (p < 0.001) at both time points. CS-CHX NPs had the highest effect against E. faecalis which was comparable to DAP and significantly higher than other medicaments (p < 0.001) at 3 days. All medicaments showed similar effect against E. faecalis after 7 days. The 7-days placement significantly increased the antimicrobial effect against both micro-organisms in all groups, except CS-CHX NPs which showed an insignificant increase. CS-CHX NPs showed the highest residual effect against both micro-organisms that increased with 7-days placement. CONCLUSION: CSNPs and CHX combination showed a synergistic effect against both micro-organisms. CS-CHX NPs displayed a higher effect at a shorter period compared to other medicaments, yet its residual effect was higher with 7-days placement.

Biological investigation of resinous endodontic sealers containing calcium hydroxide.

The purpose of this study was to evaluate, in vivo, the biocompatibility, biomineralization, collagen maturation and the in vitro antibacterial and cytotoxicity of resinous endodontic sealers containing calcium hydroxide. Forty rats were implanted with polyethylene tubes containing Sealer 26, Sealer Plus, Dia-ProSeal and an empty tube, examined after 7, 15, 30 and 60 days. Antimicrobial activity was evaluated against Enterococcus faecalis by Agar Diffusion Test (ADT) through inhibition zones. For cytotoxicity, undifferentiated pulp cells (OD-21) were cultured and assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, exposed to dilution of serial extracts at 6, 24, 48h. Cytotoxicity was analyzed by two-way ANOVA and Bonferroni correction. Kruskal-Wallis test followed by Dunn test was performed for nonparametric data (p<0.05). MTT assay revealed cell proliferation affected by sealers extract in all periods (p<0.0001), except for Dia-Proseal and Sealer Plus ⅛ dilution. Subcutaneous analysis showed at day 7th moderate inflammatory infiltration. After 30 days, Sealer 26 still showed moderate inflammatory infiltrate compared to mild inflammation from control and Dia-ProSeal (p = 0.006). At day 60th, all groups showed similar mild inflammatory infiltrate (p>0.05). Sealer 26 induced more biomineralization than other sealers in all periods. At 7 and 15 days, all sealers had significant percentage of immature collagen fibers. After 60 days Sealer 26 showed more mature fibers compared to other sealers (p<0.001). All sealers had a smaller zone of inhibition than chlorhexidine, but with no significant difference among any group (p>0.05). All sealers showed satisfactory biological responses with in vitro/in vivo biocompatibility and antimicrobial activity against planktonic bacteria. Sealer 26 induced more biomineralization than Sealer Plus and Dia-ProSeal.

Evaluation of Antibacterial Efficacy of Two Commercially Available Probiotics as Intracanal Medicament against Enterococcus faecalis: An In Vitro Study.

AIM: This study was performed to evaluate the antibacterial efficacy of two commercially available probiotics (BIFILAC and VSL 3) as intracanal medicament against Enterococcus faecalis in endodontic therapy. MATERIALS AND METHODS: Microorganisms from commercially available probiotics (BIFILAC and VSL 3) were extracted via the manufacturer's recommendations and mixed by weight. About 30 microliters were then placed on sterile discs. The pathogenic test organism was E. faecalis set to a 1 McFarland standard challenge. A two-probiotic disc template on blood agar plates was inoculated with E. faecalis and incubated at 37°C for 48 hours and 1 week respectively. Phase-1 of the study was conducted by a disc diffusion assay test to evaluate zones of inhibition (ZOI) in millimeters (mm). Phase-2 was conducted by mixing 9 mL of 30% poloxamer 407 and MRS broth in a test tube, together with the two probiotic mixtures and E. faecalis, set at a 2 McFarland standard. Serial dilutions up to 108 were done and the mixture was placed inside root canals and incubated at 37ºC for 36 hours and evaluated for colony-forming unit (CFU)/mL counts. RESULTS: The results of phase-1 showed that probiotics Lactobacillus rhamnosus and Bifidobacterium species are effective in fighting against E. faecalis with the acceptable zone of inhibition. The results of phase-2 showed that both the probiotics are effective against E. faecalis with a reduction in the number of CFU after probiotic usage. CONCLUSION: Commercially available probiotics can be used effectively as an intracanal medicament to fight against E. faecalis, Poloxamer 407 is a promising vehicle for delivering probiotics inside the root canal system. Further in vitro and in vivo studies are needed to determine the full potential of "Bacteriotherapy" with an application of probiotics. CLINICAL SIGNIFICANCE: If probiotics are proved to be an effective intracanal medicament against E.faecalis they can be used as an alternative to calcium hydroxide as intracanal medicament with no side effects to the host.

Nano eggshell-based slurry as a direct pulp-capping material: In vitro characterization and histopathological assessment in an experimental animal model.

AIM: Pulp vitality is essential for tooth integrity. Following pulp exposure, choosing a suitable pulp-capping material is crucial to maintain pulp vitality. However, the reparative dentine bridge created by calcium hydroxide (Ca(OH)2 ) is generally porous and incomplete. The aim of the current study is to assess the in vitro and in vivo bioactivities of nano eggshell-based slurry (NES), using NES as a direct pulp-capping material, compared with Ca(OH)2 in rabbit animal model. METHODOLOGY: Nano eggshell powder (NE) was characterized for particle morphology, chemical composition and ion release. In vitro bioactivity was tested by immersion in simulated body fluid (SBF) for 7 days. For histopathological evaluation, 36 adult New Zealand rabbits (72 pulp exposures) were divided into nine groups (n = 8) according to the pulp-capping material (NES, Ca(OH)2 and no capping as negative control group) and the animals were sacrificed after 7, 14 or 28 days. The pulps of the two lower central incisors were exposed and then directly capped by Ca(OH)2 or NES or left untreated. The cavities were then sealed with glass ionomer cement. Teeth were collected for histopathological evaluation using an optical microscope. Pulp haemorrhage, inflammation, fibrosis and calcific bridge formation were assessed. Results were statistically analysed using anova and Tukey's tests. RESULTS: Nano eggshell particles were spherical with a 20 nm diameter and were composed mainly of calcite. Statistical analysis showed that there was a significant increase in the release of all investigated ions between days 1 and 28, except for copper. NES group showed a significantly higher release of all elements as compared to Ca(OH)2 . Environmental scanning electron microscope micrographs of NES incubated for 7 days in SBF showed the formation of HAp with a Ca/P ratio (1.686). For histopathological evaluation, the difference between groups was statistically significant. At day 28, 75% of the pulps of the Ca(OH)2 group showed mild calcific bridge in comparison with 100% moderate calcific bridge in the NES group. The NES group showed significantly less inflammation at days 7 and 28, and higher fibrosis at day 7 compared with Ca(OH)2 . CONCLUSIONS: Nano eggshell-based slurry represents a promising novel direct pulp-capping material with favourable pulp tissue response.

Effect of different final irrigation agitation techniques on root canal dentin tubule penetration of nanoparticle calcium hydroxide dressing.

This study is aimed to assess the efficacy of different final irrigation agitation techniques on dentin tubule penetration of nanoparticle calcium hydroxide (NCH) dressing. Ninety-six extracted upper incisors were shaped up to #40file. Then, Four experimental groups were formed according to the final irrigation procedure: conventional needle irrigation (CNI), manual dynamic agitation (MDA), sonic agitation (SA) and ultrasonic irrigant agitation (UIA). According to the intracanal drug used, these groups were divided into two subgroups: calcium hydroxide (CH) and NCH. Prepared CH preparations were labelled with Rhodamine B, and CH or NCH were placed in root canals. In the UIA group, both CH and NCH had the highest penetration depth and percentage compared to the other groups (p < 0.05). The penetration depth and percentage of NCH in the UIA and SA groups were significantly higher than in the CH groups (p < 0.05). UIA is more effective than other groups in increasing dentinal tubule penetration of CH and NCH.

Antibacterial effectiveness of multi-strain probiotics supernatants intracanal medication on Enterococcus faecalis biofilm in a tooth model.

BACKGROUND: To assess the antibacterial activity of multi-strain probiotics supernatants (MSP); Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus acidophilus as an intracanal medication on Enterococcus faecalis (E. faecalis) biofilm in a tooth model. METHODS: Sixty extracted human single-rooted teeth with single canals were instrumented, sterilized, and inoculated with E. faecalis. After 21 days of incubation, four specimens were randomly selected to validate the biofilm formation by scanning electron microscope (SEM). The remaining specimens were randomly divided (n = 14), according to the intracanal medication (ICM) received into: Ca(OH)2: calcium hydroxide paste (35% Ultra Cal XS Ca(OH)2), Probiotics supernatants: MSP in poloxamer gel vehicle Poloxamer: poloxamer gel vehicle and, Control: E. faecalis biofilm only. The tested groups were further subdivided into two equal subgroups (n = 7) according to the incubation period (24 h and 7 days). Shaved dentin chips were obtained and collected by H-files and paper points, respectively for bacterial culture. The antibacterial activity was assessed after each incubation period quantitatively and qualitatively using bacterial colony-forming units per milliliter (CFUs/ml) and SEM, respectively. RESULTS: The lowest CFUs/ml was found in Ca (OH)2 with a significant difference compared to other groups after 24 h. After 7 days, a similar outcome was found with a further significant reduction of CFUs/ml in all groups with no statistical difference between Ca(OH)2 and probiotics supernatants groups. Ca (OH)2 and Probiotics supernatants groups showed a significant (p < 0.05) percentage of overall bacterial reduction (100.00 ± 0.00% and 70.30 ± 12.95%, respectively) compared to poloxamer and control groups (27.80 ± 14.45 and 28.29 ± 19.79). SEM images showed a bacteria-free state in the Ca(OH)2 group after 7 days while few bacteria were found in the probiotics supernatants group. An extensive invasion of bacteria was found in poloxamer and controls groups. CONCLUSION: MSP has a potential antibacterial effect on E. faecalis growth closely similar to the routinely used Ca (OH)2.

Synthesis and characterization of calcium-releasing elastomeric resin-based endodontic sealers.

OBJECTIVES: To evaluate the incorporation of halloysite nanotubes (HNTs) loaded with one of two calcium sources (i.e., calcium hydroxide/CaOH2 or beta-tricalcium phosphate/ß-TCP) on the physicochemical and biological properties of an experimental resin-based dual-cured endodontic sealer. MATERIALS AND METHODS: HNTs were encapsulated with CaOH2 or ß-TCP at 10 wt.%. HNTs containing CaOH2 or ß-TCP were added into the experimental sealers at 50 wt.%. The control sealers were the calcium-free HNT-modified resin-based experimental sealer and AH Plus™, a commercially available endodontic sealer. Degree of conversion, setting time, flow, film thickness, radiopacity, dimensional stability, and calcium ions release were determined. Antibiofilm properties and cytocompatibility of the formulated sealers and commercial control were also evaluated. One and two-way ANOVA analysis followed by Tukey's post hoc test was conducted to evaluate the effect of the independent variable on the evaluated properties. RESULTS: FTIR confirmed the encapsulation of calcium sources into HNTs. Regarding flow and film thickness, the values obtained from these sealers were in accordance with the specifications provided by ISO 6876. For radiopacity, AH Plus™ achieved the highest radiopacity (p<0.05). Among the experimental formulations, all experimental HNT-containing compositions exhibited values below 3 mm Al. The experimental sealers showed greater dimensional changes when compared to the commercial (AH Plus™) control. The release of calcium ions was observed for the HNT_CaOH2 and HNT_ß-TCP sealers without statistical differences. Experimental sealers containing HNT_CaOH2 and HNT_ß-TCP significantly reduced the CFU/mL count and showed cell compatibility. CONCLUSIONS: The findings of this study demonstrate that the incorporation of HNT_CaOH2 or HNT_ß-TCP into resin-based experimental sealers promoted antimicrobial effects and gradual calcium release without impairing cytocompatibility or physicochemical properties of the sealers. Still, an adjustment to reach the minimal radiopacity established by ISO 6876 is needed. CLINICAL RELEVANCE: The experimental resin-based sealers seemed to be an alternative for endodontics. The incorporation of calcium sources exerts promising antimicrobial effects while displaying low cell toxicity.

Mechanism of action of Bioactive Endodontic Materials.

A continuous search for bioactive materials capable of supporting the replacement of damaged pulp tissue, with effective sealing potential and biocompatibility, has represented the attention of studies over the last decades. This study involves a narrative review of the literature developed by searching representative research in PUBMED/MEDLINE and searches in textbooks associated with the mechanism of action of bioactive materials (calcium hydroxide, mineral trioxide aggregate (MTA), and calcium silicate cements). The reflective analysis of the particularities of the chemical elements of these materials, considering the tissue and antibacterial mechanism of action, allows a better understanding of the characteristics and similarities in their tissue responses. Calcium hydroxide paste remains the antibacterial substance of choice as intracanal dressing for the treatment of root canal system infections. Calcium silicate cements, including MTA, show a favorable biological response with the stimulation of mineralized tissue deposition in sealed areas when in contact with connective tissue. This is due to the similarity between the chemical elements, especially ionic dissociation, the potential stimulation of enzymes in tissues, and the contribution towards an alkaline environment due to the pH of these materials. The behavior of bioactive materials, especially MTA and the new calcium silicate cements in the biological sealing activity, has been shown to be effective. Contemporary endodontics has access to bioactive materials with similar properties, which can stimulate a biological seal in lateral and furcation root perforations, root-end fillings and root fillings, pulp capping, pulpotomy, apexification, and regenerative endodontic procedures, in addition to other clinical conditions.