Antibacterial effect of low-temperature plasma on Enterococcus faecalis in dentinal tubules in vitro / 北京大学学报(医学版)

OBJECTIVE@#To construct a model of Enterococcus faecalis (E. faecalis) infection in dentinal tubules by gradient centrifugation and to evaluate the antibacterial effect of low-temperature plasma on E. faecalis in dentinal tubules.@*METHODS@#Standard dentin blocks of 4 mm×4 mm×2 mm size were prepared from single root canal isolated teeth without caries, placed in the E. faecalis bacterial solution, centrifuged in gradient and incubated for 24 h to establish the model of dentinal tubule infection with E. faecalis. The twenty dentin blocks of were divided into five groups, low-temperature plasma jet treatment for 0, 5 and 10 min, calcium hydroxide paste sealing for 7 d and 2% chlorhexidine gel sealing for 7 d. Scanning electron microscopy and confocal laser scanning microscope were used to assess the infection in the dentinal tubules and the antibacterial effect of low-temperature plasma.@*RESULTS@#The results of scanning electron microscopy and confocal laser scanning microscopy showed that after 24 h of incubation by gradient centrifugation, E. faecalis could fully enter the dentinal tubules to a depth of more than 600μm indicating that this method was time-saving and efficient and could successfully construct a model of E. faecalis infection in dentinal tubules. Low-temperature plasma could enter the dentinal tubules and play a role, the structure of E. faecalis was still intact after 5 min of low-temperature plasma treatment, with no obvious damage, and after 10 min of low-temperature plasma treatment, the surface morphology of E. faecalis was crumpled and deformed, the cell wall was seriously collapsed, and the normal physiological morphology was damaged indicating that the majority of E. faecalis was killed in the dentinal tubules. The antibacterial effect of low-temperature plasma treatment for 10 min exceeded that of the calcium hydroxide paste sealing for 7 d and the 2% chlorhexidine gel sealing for 7 d. These two chemicals had difficulty entering deep into the dentinal tubules, and therefore only had a few of antibacterial effect on the bacterial biofilm on the root canal wall, and there was also no significant damage to the E. faecalis bacterial structure.@*CONCLUSION@#Gradient centrifugation could establish the model of E. faecalis dentin infection successfully. Low-temperature plasma treatment for 10 min could kill E. faecalis in dentinal tubules effectively, which is superior to the calcium hydroxide paste sealing for 7 d and the 2% chlorhexidine gel sealing for 7 d.

Effect of a calcium hydroxide-based intracanal medicament containing N-2-methyl pyrrolidone as a vehicle against Enterococcus faecalis biofilm

Abstract This study investigated the effect of a calcium hydroxide (CH) paste (CleaniCal®) containing N-2-methyl pyrrolidone (NMP) as a vehicle on Enterococcus faecalis (E. faecalis) biofilms compared with other products containing saline (Calasept Plus™) or propylene glycol (PG) (Calcipex II®). Methodology Standardized bovine root canal specimens were used. The antibacterial effects were measured by colony-forming unit counting. The thickness of bacterial microcolonies and exopolysaccharides was assessed using confocal laser scanning microscopy. Morphological features of the biofilms were observed using field-emission scanning electron microscopy (FE-SEM). Bovine tooth blocks covered with nail polish were immersed into the vehicles and dispelling was observed. The data were analyzed using one-way analysis of variance and Tukey tests (p<0.05). Results CleaniCal® showed the highest antibacterial activity, followed by Calcipex II® (p<0.05). Moreover, NMP showed a higher antibacterial effect compared with PG (p<0.05). The thickness of bacteria and EPS in the CleaniCal® group was significantly lower than that of other materials tested (p<0.05). FE-SEM images showed the specimens treated with Calasept Plus™ were covered with biofilms, whereas the specimens treated with other medicaments were not. Notably, the specimen treated with CleaniCal® was cleaner than the one treated with Calcipex II®. Furthermore, the nail polish on the bovine tooth block immersed in NMP was completely dispelled. Conclusions CleaniCal® performed better than Calasept Plus™ and Calcipex II® in the removal efficacy of E. faecalis biofilms. The results suggest the effect might be due to the potent dissolving effect of NMP on organic substances.

Root canal dressings for revascularization influence in vitro mineralization of apical papilla cells

Abstract Endodontic revascularization is based on cell recruitment into the necrotic root canal of immature teeth after chemical disinfection. The clinical outcome depends on the ability of surviving cells from the apical tissue to differentiate and promote hard tissue deposition inside the dentinal walls. Objective To investigate the effect of calcium hydroxide (CH) and modified triple antibiotic paste (mTAP - ciprofloxacin, metronidazole and cefaclor) on the viability and mineralization potential of apical papilla cells (APC) in vitro . Material and Methods APC cultures were kept in contact with CH or mTAP (250-1000 µg/mL) for 5 days, after which cell viability was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Next, APCs were subjected to CH or mTAP at 250 µg/mL for 5 days before inducing the differentiation assay. After 14 and 21 days, calcium deposition was assessed by the Alizarin Red S staining method, followed by elution and quantification using spectrophotometry. Data were analyzed using ANOVA followed by Tukey post hoc test. Results CH induced cell proliferation, whereas mTAP showed significant cytotoxicity at all concentrations tested. APC treated with CH demonstrated improved mineralization capacity at 14 days, while, for mTAP, significant reduction on the mineralization rate was observed for both experimental periods (14 and 21 days). Conclusion Our findings showed that CH induces cell proliferation and improves early mineralization, whereas mTAP was found cytotoxic and reduced the mineralization potential in vitro of APCs.

Cytotoxicity and biocompatibility of a new bioceramic endodontic sealer containing calcium hydroxide

Abstract: This study evaluated the cytotoxicity and biocompatibility of a new bioceramic endodontic sealer (i.e., Sealer Plus BC) in comparison with those of MTA Fillapex and AH Plus. L929 fibroblasts were cultured and Alamar Blue was used to evaluate cell viability of diluted extracts (1:50, 1:100, and 1:200) from each sealer at 24 h. Polyethylene tubes that were filled with material or empty (as a control) were implanted in the subcutaneous tissue of rats. The rats were killed after 7 and 30 d (n = 8), and the tubes were removed for histological analysis. Parametric data was analyzed using a one-way ANOVA test, and nonparametric data was analyzed via the Kruskal-Wallis test followed by the Dunn test (p < 0.05). A reduction in cell viability was observed in the extracts that were more diluted for Sealer Plus BC when compared to that of Control and AH Plus (p < 0.05). However, the 1:50 dilution of the Sealer Plus BC was similar to that of the Control (p > 0.05). Conversely, more diluted extracts of MTA Fillapex (1:200) and AH Plus (1:100 and 1:200) were similar to the Control (p > 0.05). Histological analysis performed at 7 d did not indicate any significant difference between tissue response for all materials, and the fibrous capsule was thick (p > 0.05). At 30 d, Sealer Plus BC was similar to the Control (p > 0.05) and MTA Fillapex and AH Plus exhibited greater inflammation than the Control (p < 0.05). The fibrous capsule was thin for the Control and for most specimens of Sealer Plus BC and AH Plus. Thus, Sealer Plus BC is biocompatible when compared to MTA Fillapex and AH Plus, and it is less cytotoxic when less-diluted extracts are used.

Removal and killing of multispecies endodontic biofilms by N-acetylcysteine

ABSTRACT Removal of bacterial biofilm from the root canal system is essential for the management of endodontic disease. Here we evaluated the antibacterial effect of N-acetylcysteine (NAC), a potent antioxidant and mucolytic agent, against mature multispecies endodontic biofilms consisting of Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans and Enterococcus faecalis on sterile human dentin blocks. The biofilms were exposed to NAC (25, 50 and 100 mg/mL), saturated calcium hydroxide or 2% chlorhexidine solution for 7 days, then examined by scanning electron microscopy. The biofilm viability was measured by viable cell counts and ATP-bioluminescence assay. NAC showed greater efficacy in biofilm cell removal and killing than the other root canal medicaments. Furthermore, 100 mg/mL NAC disrupted the mature multispecies endodontic biofilms completely. These results demonstrate the potential use of NAC in root canal treatment.

Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth

Abstract Objective: The aim of the study was to evaluate the effects of the capping materials mineral trioxide aggregate (MTA), calcium hydroxide (CH) and BiodentineTM (BD) on stem cells from human exfoliated deciduous teeth (SHED) in vitro. Material and Methods: SHED were cultured for 1 - 7 days in medium conditioned by incubation with MTA, BD or CH (1 mg/mL), and tested for viability (MTT assay) and proliferation (SRB assay). Also, the migration of serum-starved SHED towards conditioned media was assayed in companion plates, with 8 μm-pore-sized membranes, for 24 h. Gene expression of dentin matrix protein-1 (DMP-1) was evaluated by reverse-transcription polymerase chain reaction. Regular culture medium with 10% FBS (without conditioning) and culture medium supplemented with 20% FBS were used as controls. Results: MTA, CH and BD conditioned media maintained cell viability and allowed continuous SHED proliferation, with CH conditioned medium causing the highest positive effect on proliferation at the end of the treatment period (compared with BD and MTA) (p<0.05). In contrast, we observed increased SHED migration towards BD and MTA conditioned media (compared with CH) (p<0.05). A greater amount of DMP-1 gene was expressed in MTA group compared with the other groups from day 7 up to day 21. Conclusion: Our results show that the three capping materials are biocompatible, maintain viability and stimulate proliferation, migration and differentiation in a key dental stem cell population.

Do different strains of E. faecalis have the same behavior towards intracanal medications in in vitro research?

Abstract: The aim of this study was to evaluate the antimicrobial action of different endodontic pastes against Enterococcus faecalis ATCC 29212, isolated from the urinary tract, and compare the action with E. faecalis ATCC 4083, isolated from the root canal. For this purpose, dentin blocks were infected for 21 days with both bacteria at different time-intervals to ensure there would be no cross contamination. After this period, blocks were immersed in the test medications for 7 days, according to the following groups: CH/S, CH/P, CH/CMCP, CH/CHX, CH/DAP and TAP. Images of the samples were captured with a confocal microscope and the percentage of live cells was computed by means of the Bioimage program. The ATCC 29212 strain was shown to be more resistant to CH/SS, Calen, CH/DAP, and TAP than the ATCC 4083 strain. The antimicrobial action of the medications against each strain were divergent concerning the order of susceptibility. The authors concluded that the strains behaved in a different manner: in general, those extracted from the urinary tract were more resistant to the tested medications. Therefore, when E. faecalis must be used for in vitro research in endodontics, we suggest the use of ATCC 4083 strain to obtain results that are closer to the clinical reality.

Immediate tooth replantation: root canal filling for delayed initiation of endodontic treatment

Abstract The aim of this study is to evaluate the action of paramonochlorophenol associated with Furacin followed by calcium hydroxide (CH) dressing in the control of inflammatory root resorption in cases of immediate tooth replantation with delayed endodontic treatment. A total of 28 incisors of 3 male dogs were extracted and replanted after 15 minutes, and randomly divided into 3 groups: Group I (n = 8) - endodontic treatment was performed before the extraction and replantation; Group II (n = 10) - endodontic treatment was performed 30 days after replantation and the root canal was filled with CH dressing; Group III (n = 10) - endodontic treatment was performed 30 days after replantation and root canals received temporary medication of paramonochlorophenol-Furacin followed by CH dressing. The animals were euthanized 90 days after replantation. The histomorphological events analyzed at the epithelial reattachment site were the intensity and extent of acute and chronic inflammatory processes, periodontal ligament (PDL) organization, the intensity and extent of acute and chronic inflammatory processes in the PDL space, root resorption, bone tissue, and ankylosis. Data were submitted to the Wilcoxon Signed Ranks Test for group comparison (α = 5%). In Groups I, II and III the periodontal ligament was regenerated and most of the resorption areas were repaired by newly formed cementum. The depth and extent of root resorption were significantly higher in Group II than in Group III. The use of paramonochlorophenol-furacin followed by CH dressing was more effective in controlling inflammatory root resorption after immediate tooth replantation.

Uso de hidróxido de cálcio e MTA na odontolgia: conceitos, fundamentos e aplicação clínica / Use of calcium hydroxide and mta in dentistry: concepts, rationele and clinical practice

Introdução: atualmente os produtos à base de hidróxido de cálcio são amplamente utilizados e difundidos na odontologia em várias situações clínicas, desde capeador em exposição pulpar à pulpotomias, em virtude de suas propriedades físicas, mecânicas, do baixo custo e de seu próprio mecanismo de ação, representando assim, o material mais próximo do ideal. Objetivo: descrever os conceitos, os fundamentos e a aplicação clínica do Hidróxido de Cálcio e do MTA, por meio de uma revisão da literatura. Material e Métodos: foi realizada uma revisão da literatura, por meio de busca bibliográfica nas seguintes bases de pesquisa online: Lilacs, Scielo, PubMED/Medline e Bireme, com uso dos descritores: hidróxido de cálcio (calcium hydroxide); hidróxido de cálcio e MTA (calcium hydroxide and MTA) e MTA, rastreando artigos relevantes publicados entre o período de 2010 a 2015. Resultados e Discussão: materiais a base de hidróxido de cálcio, são biocompatíveis e apresentam propriedades antimicrobianas, anti-inflamatórias, estimulantes da formação de dentina esclerosada, de tecido ósseo mineralizado além de proteger a polpa contra estímulos termoelétricos e tóxicos, provenientes de alguns materiais restauradores, mantendo assim, a integridade pulpar. São amplamente utilizados para: proteção pulpar, pulpotomias, cimentação protética, forramento cavitário, apicificação e em casos de reabsorção radicular. As formas de apresentação desses materiais pode ser produtos na forma de pó, ou na forma de pastas, autoativadas ou fotoativadas. O Agregado Trióxido Mineral (MTA) apresenta muitas propriedades coincidentes com o hidróxido de cálcio, mas uma das suas principais desvantagens é seu alto custo, o que inviabiliza o rotineiro na clínica, esse material é apresentado na forma de pó e líquido, o qual é composto apenas por água destilada. Conclusão: Diante das várias opções de produtos presentes no mercado, recomenda-se ao profissional a escolha do melhor material para cada caso, incluindo seguir protocolos adequados durante a aplicação dos materiais para alcançar resultados clínicos satisfatórios.

Introduction: products of calcium hydroxide are largely used in a variety of dental clinical condition due to its physical and mechanical properties and its low cost as well as its rationelle. In this contexto, it representes a material close to the ideal. Objective: to describe the concepts, fundamentals and clinical application of Calcium Hydroxide and MTA, through a review of the literature. Materials and Methods: a review of the literature was carried out by means of bibliographic search in the following online databases: Lilacs, Scielo, PubMED/Medline and Bireme, using the descriptors: calcium hydroxide; Calcium hydroxide and MTA, and MTA, tracking relevant articles published between the period 2010-2015. Results and Discussion: materials based on calcium hydroxide, are biocompatible and exhibit antimicrobial, anti-inflammatory, stimulating properties of the formation of sclerosed dentin, of mineralized bone tissue, besides protecting the pulp against thermoelectric and toxic stimuli from some restorative materials, thus maintaining pulp integrity. They are widely used for: pulp protection, pulpotomies, prosthetic cementation, cavity lining, apicification and in cases of root resorption. The forms of presentation of such materials may be products in the form of powder, or in the form of pastes, either self-activating or photoactivated. The Mineral Trioxide Aggregate (MTA) has many properties that coincide with calcium hydroxide, but one of its main disadvantages is its high cost, which makes the routine in the clinic unfeasible. This material is presented in the form of powder and liquid, which is Composed only of distilled water. Conclusion: in view of the various product options present in the market, the professional is advised to choose the best material for each case, including following appropriate protocols during the application of the materials to achieve satisfactory clinical results.

Calcium hydroxide associated with a new vehicle: Psidium cattleianum leaf extracts. Tissue response evaluation

Abstract The aim of this study was to evaluate edemogenic activity and subcutaneous inflammatory reaction induced by Psidium cattleianum leaf extracts associated with Ca(OH)2. Thirty male Wistar rats, split equally into three groups [aqueous extract + Ca(OH)2; ethanolic extract + Ca(OH)2; and propylene glycol + Ca(OH)2], were assessed every 3 h or 6 h (five animals in each period). Under general anesthesia, 0.2 mL of 1% Evans blue per 100 g of body weight was injected into the penile vein and each combination to be evaluated was subcutaneously injected into the dorsal region 30 min thereafter. Edemogenic activity was analyzed by spectrophotometry (λ=630 nm). For inflammatory reaction analysis, 50 rats received four polyethylene tubes (three experimental groups) and an empty tube (control group). The assessments were made at 7, 15, 30, 60, and 90 days, followed by hematoxylin-eosin staining and by the assignment of scores for evaluation of tissue response intensity. Ethanolic extract + Ca(OH)2 yielded the largest edemogenic activity at 3 h. Intergroup differences at 6 h were not significant. The histological analysis showed progressive repair over time (p<0.05) and aqueous and ethanolic extracts produced similar responses to those of the control and Ca(OH)2 + propylene glycol groups. Psidium cattleianum leaf extracts used as Ca(OH)2 vehicles evoked similar tissue response when compared to Ca(OH)2 associated with propylene glycol.

Effect of ultrasonic streaming on intra-dentinal disinfection and penetration of calcium hydroxide paste in endodontic treatment

ABSTRACT Objective The antimicrobial effect of ultrasonic agitation of calcium hydroxide (CH) pastes in infected bovine dentin and their penetrability were evaluated using confocal laser scanning microscopy (CLSM) and microbiological culture. Material and Methods Fifty-two bovine teeth were infected with Enterococcus faecalis using a new contamination protocol; then they received CH paste and were divided into groups with or without ultrasound. Ultrasonic agitation was conducted for 1 min with a plain point insert. After 15 d, the CLSM analyzed the viable and dead bacteria with Live and Dead assay. The dentinal wall debris was collected by burs, and the colony forming units (CFU/mL) were counted. The penetrability of the paste inside dentinal tubules was tested using the B-rodamine dye. Results The calcium hydroxide paste showed better results with the use of ultrasonic agitation (p<0.05). Conclusion The ultrasonic agitation of CH paste increased its antimicrobial action and was responsible for intradentinal penetration with the fulfilment of the tubules.

Exopolysaccharide dispelled by calcium hydroxide with volatile vehicles related to bactericidal effect for root canal medication

ABSTRACT Objective: Enterococcus faecalis is the dominant microbial species responsible for persistent apical periodontitis with ability to deeply penetrate into the dentin. Exopolysaccharides (EPS) contribute to the pathogenicity and antibiotic resistance of E. faecalis. Our aim was to investigate the antimicrobial activity of calcium hydroxide (CH), camphorated parachlorophenol (CMCP), and chlorhexidine (CHX) against E. faecalis in dentinal tubules. Material and Methods: Decoronated single-canal human teeth and semicylindrical dentin blocks were incubated with E. faecalis for 3 weeks. Samples were randomly assigned to six medication groups for 1 week (n=10 per group): CH + 40% glycerin-water solution (1:1, wt/vol); CMCP; 2% CHX; CH + CMCP (1:1, wt/vol); CH + CMCP (2:3, wt/vol); and saline. Bacterial samples were collected and assayed for colony-forming units. After dentin blocks were split longitudinally, confocal laser scanning microscopy was used to assess the proportion of viable bacteria and EPS production in dentin. Results: CMCP exhibited the best antimicrobial activity, while CH was the least sensitive against E. faecalis (p<0.05). CHX showed similar antimicrobial properties to CH + CMCP (1:1, wt/vol) (p>0.05). CH combined with CMCP inhibited EPS synthesis by E. faecalis, which sensitized biofilms to antibacterial substances. Moreover, increasing concentrations of CMCP decreased EPS matrix formation, which effectively sensitized biofilms to disinfection agents. Conclusion: The EPS matrix dispelled by CH paste with CMCP may be related to its bactericidal effect; the visualization and analysis of EPS formation and microbial colonization in dentin may be a useful approach to verify medicaments for antimicrobial therapy.

Avaliação da atividade antibacteriana do hidróxido de cálcio associado a diferentes veículos em blocos de dentina contaminados por Enterococcus faecalis / Evaluation of the antibacterial activity of calcium hydroxide associated different vehicles in dentin blocks contaminated with Enterococcus faecalis

Introdução: A infecção endodôntica ocasionada por Enterococcus faecalis é um problema sério no tratamento de dentes comprometidos. É de preocupação do cirurgião dentista um completo saneamento do sistema de canais radiculares pela aplicação de medicação antisséptica entre sessões. Dentre estas medicações, a pasta de hidróxido de cálcio tem sido associada a diferentes veículos para potencializar sua ação. Objetivos: Diante disto, este estudo objetivou avaliar e comparar a eficiência da atividade antimicrobiana in vitro de diferentes pastas de hidróxido de cálcio frente ao E. faecalis. Material e Métodos: Os testes foram executados em 49 blocos de dentina infectados com E. faecalis e tratados com pastas de hidróxido de cálcio em diferentes veículos por uma semana. A eficiência das pastas foi avaliada pela microscopia confocal de varredura a laser. Para comparação entre as pastas foi empregado o teste de Kruskal-Wallis e pelo teste de Dunn para comparações individuais com nível de significância estabelecido em 5%. Resultados: A aplicação das diferentes pastas proporcionou uma significativa alteração na proporção de bactérias viáveis e não viáveis encontradas no biovolume celular total dos blocos de dentina. Conclusão: A pasta que revelou melhor desempenho antimicrobiano foi aquela cujo veículo foi água destilada. A pasta de hidróxido de cálcio associada ao extrato propilenoglicólico de guaçatonga não apresentou desempenho antimicrobiano sobre células de E. faecalis. (AU)

Introduction: The endodontic infection caused by Enterococcus faecalis is a serious problem in the treatment of compromised teeth. It is a concern to the dental surgeon a complete sanitation of the root canal system by applying antiseptic medication between sessions. Among these medications, the calcium hydroxide paste has been linked to different vehicles to enhance its action. Objectives: Thus, this study aimed to evaluate and compare the efficiency of in vitro antimicrobial activity of different pastes of calcium hydroxide against E. faecalis. Material and Methods: Tests were performed in 49 blocks dentin infected with E. faecalis and treated with calcium hydroxide pastes in different vehicles for a week. The efficiency of the pastes was evaluated by confocal laser scanning. The Kruskal-Wallis test was used to compare the pastes and the Dunn test was used for individual comparisons with a significance level set at 5%. Results: The application of different pastes provided a significan change in the proportion of viable and non-viable bacteria found in the total cell biovolume of the blocks of dentin. Conclusion: The paste that revealed the best antimicrobial performance was the one whose vehicle was distilled water. The calcium hydroxide paste associated with the extract of guaçatonga in propylene glycol showed no antimicrobial performance on cells of E. faecalis. (AU)

Influence of diabetes mellitus on the mineralization ability of two endodontic materials

Abstract The aim of this study was to evaluate the influence of diabetes mellituson tissue response and mineralization ability of Sealapex®and MTA Fillapex® sealers. Twenty-four Wistar rats were divided into two groups: diabetic and non-diabetic. The materials were placed in polyethylene tubes and implanted into dorsal connective tissue of rats for 7 and 30 days. Six animals from each group received injection of calcein, alizarin, and oxytetracycline on days 7, 14, and 21, respectively. The animals were killed after 7 and 30 days and specimens were prepared for histologic analysis by staining with hematoxylin and eosin or Von Kossa or left unstained for polarized light or fluorescence microscopy. On day 7, inflammatory reactions were characterized. Moderate inflammatory responses were observed for all groups and on day 30, a mild inflammatory response against MTA Fillapex® and a moderate inflammatory response against Sealapex® were observed. Von Kossa-positive structures were observed in response to both materials and birefringent structures were observed upon polarized light analysis; these had no relation to the diabetic condition (p > 0.05). The fluorescence intensity was unaffected in diabetic rats (p > 0.05). In conclusion, diabetes mellitus did not influence the tissue response or mineralization stimulated by Sealapex® or MTA Fillapex®.

Immunohistochemical expression of TGF-ß1 and osteonectin in engineered and Ca(OH)2-repaired human pulp tissues

Abstract The aim of the present study was to evaluate the expression of transforming growth factor-β1 (TGF-β1) and osteonectin (ON) in pulp-like tissues developed by tissue engineering and to compare it with the expression of these proteins in pulps treated with Ca(OH)2 therapy. Tooth slices were obtained from non-carious human third molars under sterile procedures. The residual periodontal and pulp soft tissues were removed. Empty pulp spaces of the tooth slice were filled with sodium chloride particles (250-425 µm). PLLA solubilized in 5% chloroform was applied over the salt particles. The tooth slice/scaffold (TS/S) set was stored overnight and then rinsed thoroughly to wash out the salt. Scaffolds were previously sterilized with ethanol (100-70°) and washed with phosphate-buffered saline (PBS). TS/S was treated with 10% EDTA and seeded with dental pulp stem cells (DPSC). Then, TS/S was implanted into the dorsum of immunodeficient mice for 28 days. Human third molars previously treated with Ca(OH)2 for 90 days were also evaluated. Samples were prepared and submitted to histological and immunohistochemical (with anti-TGF-β1, 1:100 and anti-ON, 1:350) analyses. After 28 days, TS/S showed morphological characteristics similar to those observed in dental pulp treated with Ca(OH)2. Ca(OH)2-treated pulps showed the usual repaired pulp characteristics. In TS/S, newly formed tissues and pre-dentin was colored, which elucidated the expression of TGF-β1 and ON. Immunohistochemistry staining of Ca(OH)2-treated pulps showed the same expression patterns. The extracellular matrix displayed a fibrillar pattern under both conditions. Regenerative events in the pulp seem to follow a similar pattern of TGF-β1 and ON expression as the repair processes.

Biocompatibility and biomineralization assessment of bioceramic-, epoxy-, and calcium hydroxide-based sealers

Abstract Obturation of the root canal system aims to fill empty spaces, promoting hermetic sealing and preventing bacterial activity in periapical tissues. This should provide optimal conditions for repair, stimulating the process of biomineralization. An endodontic sealer should be biocompatible once it is in direct contact with periapical tissues. The aim of this study was to evaluate the rat subcutaneous tissue response to implanted polyethylene tubes filled with Smartpaste Bio, Acroseal, and Sealapex and investigate mineralization ability of these endodontic sealers. Forty Wistar rats were assigned to the three sealers groups and control group, (n = 10 animals/group) and received subcutaneous implants containing the test sealers, and the control group were implanted with empty tubes. After days 7, 15, 30, and 60, animals were euthanized and polyethylene tubes were removed with the surrounding tissues. Inflammatory infiltrate and thickness of the fibrous capsule were histologically evaluated. Mineralization was analyzed by Von Kossa staining and polarized light. Data were tabulated and analyzed via Kruskal-Wallis and Dunn's test. All tested materials induced a moderate inflammatory reaction in the initial periods. Smartpaste Bio induced the mildest inflammatory reactions after day 15. No difference was observed among groups after days 30 or 60. Von Kossa-positive staining and birefringent structures observed under polarized light revealed a larger mineralization area in Sealapex-treated animals followed by Smartpaste Bio-treated animals. At the end of the experiment, all tested sealers were found to be biocompatible. All sealers induced biomineralization, except Acroseal, which induced a mild tissue reaction.

Antimicrobial action of calcium hydroxide-based endodontic sealers after setting, against E. faecalis biofilm

Abstract Enterococcus faecalis are gram positive bacteria that can mostly resist endodontic therapy, inducing persistent infection in the root canal system. Endodontic sealers with antimicrobial activity may help eliminate residual microorganisms that survive endodontic treatment. The present study aimed at comparing the antimicrobial activity of Acroseal, Sealapex and AH Plus endodontic sealers in an in vitro biofilm model. Bovine dentin specimens (144) were prepared, and twelve blocks for each sealer and each experimental time point (2, 7 and 14 days) were placed and left in contact with plates containing inoculum of E. faecalis (ATCC 51299), to induce biofilm formation. After 14 days, the samples were transferred to another plate with test sealers and kept at 37°C and 5% CO2 for 2, 7 and 14 days. The specimens without sealers were used as a control for each period. The samples were agitated in a sonicator after each experiment. The suspensions were agitated in a vortex mixer, serially diluted in saline, and triple plated onto m-Enterococcus agar. Colonyforming units were counted, and the data were statistically analyzed using ANOVA, Shapiro-Wilk and Kruskal-Wallis one-way tests (p < 0.05) to determine antimicrobial potential. Sealapex showed significant differences at all the experimental time points, in comparison with all the other groups. AH Plus and Acroseal showed antimicrobial activity only on the 14th experimental day. Neither of the sealers tested were able to completely eliminate the biofilm. Sealapex showed the highest antimicrobial activity in all the experimental periods. The antimicrobial activity of all the sealers analyzed increased over time.

Analysis of the reaction of subcutaneous tissues in rats and the antimicrobial activity of calcium hydroxide paste used in association with different substances

The aim of this study was to evaluate the subcutaneous tissue response in rats and the antimicrobial activity of intracanal calcium hydroxide dressings mixed with different substances against E. faecalis. Fifty four rats were divided into three experimental groups according to the vehicle in the calcium hydroxide treatment: 0.4% chlorohexidine in propylene glycol (PG),Casearia sylvestris Sw in PG and calcium hydroxide+PG (control group). The pastes were placed into polyethylene tubes and implanted into the subcutaneous tissue. After 7, 14 and 30 days, the samples were processed and histologically evaluated (hematoxylin and eosin). The tissue surface in contact with the material was analyzed, and the quantitative analysis determined the volume density occupied by the inflammatory infiltrate (giant cells, polymorphonuclear cells and mononuclear cells), fibroblasts, collagen fibers and blood vessels. For the antimicrobial analysis, 20 dentin blocks infected with E. faecalis were treated with calcium hydroxide pastes in different vehicles; 0.4% chlorhexidine in PG, PG, extract fromCasearia sylvestris Sw in PG and a positive control (infection and without medication) for 7 days. The efficiency of the pastes was evaluated by the live/dead technique and confocal microscopy. The results showed that 0.4% chlorhexidine induced a higher inflammatory response than the other groups. The Casearia sylvestris Sw extract showed satisfactory results in relation to the intensity of the inflammatory response. In the microbiological test, there were no statistical differences between the evaluated intracanal dressings and the percentage of bacterial viability was between 33 and 42%. The control group showed an 86% viability. Antimicrobial components such as chlorhexidine or Casearia sylvestris Sw did not improve the antimicrobial activity against E. faecalis in comparison to the calcium hydroxide+PG treatment. In addition, the incorporation of chlorhexidine in the calcium hydroxide paste promoted the highest inflammatory response.

Effects of the association of antifungal drugs on the antimicrobial action of endodontic sealers

This in vitro study aimed to determine the susceptibility of oral specimens and ATCC lineages of Candida albicans for five endodontic sealers, which were pure and associated with two antifungal drugs, and to analyze their effect on the physical properties. For this purpose, 30 lineages of C. albicans, collected from the oral cavity of patients assisted at the endodontics clinic of the Universidade Sagrado Coração, were analyzed. Yeasts susceptibility to the sealers was tested by diffusion on agar plates. Physical properties were evaluated according to the ADA specification no. 57. The pure versions of the Sealer 26, AH Plus, Endofill, Fillapex, and Sealapex demonstrated antifungal activity, with Endofill presenting the greatest inhibition zones. All cements, except for Endofill, had their antifungal actions enhanced by addition of ketoconazole and fluconazole (p < 0.05), and the AH Plus presented the best antifungal activity. The addition of antifungal drugs did not interfere with the setting time and flowability of the sealers. It was concluded that the addition of antifungals to endodontic sealers enhanced the antimicrobial action of most cements tested without altering their physical properties.

Antimicrobial efficacy of the EndoVac system plus PDT against intracanal Candida albicans: an ex vivo study

This study evaluated the ex vivoantimicrobial efficacy of the EndoVac system and the photodynamic therapy (PDT) associated with chemomechanical debridement (CMD) and intracanal medication on Candida albicans. Seventy-eight sterile premolars were contaminated withC. albicans (ATCC 21433) for 30 days. The teeth were randomly assigned into four groups: Control (CMD with conventional irrigation); Endovac (CMD with EndoVac system); PDT (CMD with conventional irrigation and PDT); and Endovac + PDT (CMD with EndoVac and PDT). After the therapies, intracanal dressing (calcium hydroxide) was applied to all teeth for seven days. Samples were obtained before (T1) and after the therapeutic procedures (T2), and after intracanal medication (T3), plated onto BHI agar and incubated (37°C, 48 h) to determine the colony-forming units (CFU)/mL. The overall mean level ofC. albicans at baseline was relatively high (1.85 x 106 ± 2.7 x 106 CFU mL-1). A significant reduction of C. albicans(p < 0.05) was observed over time (T1 to T2 and T1 to T3) in all groups. An additional significant reduction from T2 to T3 was observed only in the Endovac group (p < 0.05). No differences in mean reduction of C. albicans were observed among groups. However, the Endovac group presented the lowest mean counts of C. albicans at T3, whereas the PDT group had the highest counts of this microorganism (p < 0.05). The EndoVac system of irrigation/aspiration associated with CMD was the most effective therapeutic protocol for reducing intracanal levels of C. albicans. PDT showed a very limited efficacy against this species.