José Flávio Affonso de Almeida

Concentration-dependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation.

INTRODUCTION Intracanal disinfection is a crucial step in regenerative endodontic procedures. Most published cases suggest the use of sodium hypochlorite (NaOCl) as the primary irrigant. However, the effect of clinically used concentrations of NaOCl on the survival and differentiation of stem cells is largely unknown. In this study, we tested the effect of various concentrations of NaOCl on the stem cells of the apical papilla (SCAPs) survival and dentin sialophosphoprotein (DSPP) expression. METHODS Standardized root canals were created in extracted human teeth and irrigated with NaOCl (0.5%, 1.5%, 3%, or 6%) followed by 17% EDTA or sterile saline. SCAPs in a hyaluronic acid-based scaffold were seeded into the canals and cultured for 7 days. Next, viable cells were quantified using a luminescence assay, and DSPP expression was evaluated using quantitative real-time polymerase chain reaction. RESULTS There was a significant reduction in survival and DSPP expression in the group treated with 6% NaOCl compared with the untreated control group. Comparable survival was observed in the groups treated with the lower concentrations of NaOCl, but greater DSPP expression was observed in the 1.5% NaOCl group. In addition, 17% EDTA resulted in increased survival and DSPP expression partially reversing the deleterious effects of NaOCl. CONCLUSIONS Collectively, the results suggest that dentin conditioning with high concentrations of NaOCl has a profound negative effect on the survival and differentiation of SCAPs. However, this effect can be prevented with the use of 1.5% NaOCl followed by 17% EDTA. The inclusion of this irrigation regimen might be beneficial in regenerative endodontic procedures.

Chlorhexidine in Endodontics

Chemical auxiliary substances (CAS) are essential for a successful disinfection and cleanness of the root canals, being used during the instrumentation and if necessary, as antimicrobial intracanal medicaments. Different CAS have been proposed and used, among which sodium hypochlorite (NaOCl), chlorhexidine (CHX), 17% EDTA, citric acid, MTAD and 37% phosphoric acid solution. CHX has been used in Endodontics as an irrigating substance or intracanal medicament, as it possesses a wide range of antimicrobial activity, substantivity (residual antimicrobial activity), lower cytotoxicity than NaOCl whilst demonstrating efficient clinical performance, lubricating properties, rheological action (present in the gel presentation, keeping the debris in suspension); it inhibits metalloproteinase, is chemically stable, does not stain cloths, it is odorless, water soluble, among other properties. CHX has been recommended as an alternative to NaOCl, especially in cases of open apex, root resorption, foramen enlargement and root perforation, due to its biocompatibility, or in cases of allergy related to bleaching solutions. The aim of this paper is to review CHXs general use in the medical field and in dentistry; its chemical structure, presentation form and storage; mechanism of action; antimicrobial activity including substantivity, effects on biofilms and endotoxins, effects on coronal and apical microbial microleakage; tissue dissolution ability; interaction with endodontic irrigants; effects on dentin bonding, metalloproteinases and collagen fibrils; its use as intracanal medicament and diffusion into the dentinal tubules; its use as disinfectant agent of obturation cones; other uses in the endodontic therapy; and possible adverse effects, cytotoxicity and genotoxicity.

Structural analysis of bovine root dentin after use of different endodontics auxiliary chemical substances.

INTRODUCTION The use of chemical substances in endodontics during chemical-mechanical preparation may alter the structure of dentin, mainly collagen, which contributes considerably to the mechanical properties of this structure. This study evaluated the effect of different auxiliary chemical substances during endodontic treatment in root canal walls by implementing polarized light microscopy (PLM) and scanning electron microscopy (SEM). METHODS Sixty bovine incisors were randomly divided into six groups (n = 10): G1, 5.25% NaOCl + 17% EDTA; G2, 2% chlorhexidine (CHX) gel; G3, 5.25% NaOCl; G4, 17% EDTA; G5, 2% CHX + 17% EDTA; and G6, 0.9% sodium chloride solution (control). Teeth were chemomechanically prepared and fractured into two fragments. One fragment was used for PLM, and the other was used for SEM analysis. RESULTS Groups treated with 5.25% NaOCl, whether associated or not associated to 17% EDTA, presented a significantly different birefringence pattern compared with the control group. The results revealed that groups irrigated with 2% CHX and EDTA, independently or combined, did not present any significant differences compared with the control group. SEM analysis showed structural alterations of the root canal inorganic matrix for all groups that used 17% EDTA. CONCLUSIONS It is possible to conclude that 5.25% NaOCl, whether associated or not associated with 17% EDTA, causes alterations in the dentin collagen. The obtained SEM images revealed demineralization areas for groups that used 17% EDTA alone or associated with other substances.

Antimicrobial action of intracanal medicaments on the external root surface

OBJECTIVES To assess in vitro the antimicrobial action of intracanal medicaments in the external root surface, with or without of cementum. METHODS After the placement of intracanal medicaments, the coronal and apical openings were sealed. The antimicrobial action of 2% chlorhexidine gel (2%CHX); calcium hydroxide+2% chlorhexidine gel (CH+2%CHX); calcium hydroxide+2% chlorhexidine gel+zinc oxide (CH+2%CHX+ZnO); calcium hydroxide+saline solution (CH+SS) was evaluated against Enterococcus faecalis, Candida albicans, Actinomyces viscosus and Porphyromonas gingivalis. The samples were placed over the agar, and the zones of inhibition of microbial growth were measured after the incubation period. RESULTS The antimicrobial effects of the medicaments could be ranked from strongest to weakest as follows: 2%CHX, CH+2%CHX, CH+2%CHX+ZnO, CH+SS. No differences among groups were verified in the presence or absence of cementum. CONCLUSIONS 2%CHX containing medicaments diffused into the dentin and reached the outer surface, exerting antimicrobial action.

Characterization of a stem cell of apical papilla cell line: Effect of passage on cellular phenotype

INTRODUCTION Stem cells of the apical papilla (SCAP) have been identified as an important population of mesenchymal stem cells (MSCs) in regenerative endodontics. Preclinical studies that evaluate the various aspects of the regenerative process must use fully characterized MSCs. The phenotype of these cells when maintained in culture is crucial for the translational applicability of these studies. Thus, in this study, we aimed to characterize a SCAP cell line that preferentially expressed and maintained the required MSCs markers in culture, namely the RP-89 cell line. METHODS Apical papillae from extracted mandibular third molars from a single donor were processed for immunohistochemistry, cell culture, and RT-PCR. SCAP were successfully cultured and maintained in culture for up to 20 passages. The expression of MSC-related molecular markers was analyzed by laser confocal microscopy, flow cytometry, and real-time RT-PCR arrays. RESULTS Cells within the apical papillae tissue had a widespread expression of CD90, whereas the expression of CD105 and CD73 was compartmentalized to the blood vessels and periphery, respectively. The RP-89 cell line population coexpressed CD73, CD90, and CD105 in all passages evaluated. There was a dramatic change in gene expression when cells were cultured and maintained in culture with the up-regulation of MSCs markers, inhibitors of differentiation, and stemness markers. Conversely, genes involved in the differentiation of MSCs were suppressed. CONCLUSIONS Collectively, these results highlight the need to use fully characterized cell lines in regenerative studies and provide the foundational knowledge of gene expression modulation that occurs in cultured SCAP.

Pulp Revascularization after Root Canal Decontamination with Calcium Hydroxide and 2% Chlorhexidine Gel

INTRODUCTION Pulp revascularization may be considered a promising alternative for necrotic immature teeth. Many studies have accomplished passive decontamination associated with an antibiotic paste. To date, there is no report evaluating calcium hydroxide associated with 2% chlorhexidine gel for revascularization therapy. The aim of this case report was to describe a new proposal for pulp revascularization with mechanical decontamination and intracanal medication composed of calcium hydroxide and 2% chlorhexidine gel. METHODS The patient, a 9-year-old girl, suffered an intrusion associated with pulp exposure caused by an enamel-dentin fracture in her maxillary left central incisor. After diagnosis, treatment consisted of revascularization therapy with gentle manual instrumentation of the cervical and medium thirds of the root in addition to intracanal medication with calcium hydroxide and 2% chlorhexidine gel for 21 days. In the second session, a blood clot was stimulated up to the cervical third of the root canal. Mineral trioxide aggregate (MTA; Angelus, Londrina, Paraná, Brazil) was used for cervical sealing of the canal. Coronal sealing was performed with temporary filling material and composite resin. RESULTS During the follow-up period, the root canal space showed a progressive decrease in width, mineralized tissue deposition on root canal walls, and apical closure. A cone-beam computed tomography scan taken at the 2-year follow-up confirmed these findings and did not show complete root canal calcification. CONCLUSIONS This new proposal for revascularization therapy with 2% chlorhexidine gel may be used for the treatment of necrotic immature root canals.

Influence of Chlorhexidine and Ethanol on the Bond Strength and Durability of the Adhesion of the Fiber Posts to Root Dentin Using a Total Etching Adhesive System

INTRODUCTION The aim of this study was to investigate the effects of pretreatment of gel chlorhexidine (CHX) and ethanol (EtOH) on the bond strength and durability of the adhesion of the fiber post relined with resin composite to the root dentin using a total etch adhesive system. METHODS Forty bovine incisor roots were divided into four groups after phosphoric acid etching: irrigation with physiologic solution (control), 5 minutes with CHX, 1 minute with EtOH, and 5 minutes with chlorhexidine followed by 1 minute with EtOH. Fiber posts relined with resin composite were cemented with either RelyX ARC (3M ESPE, St Paul, MN) and a total etch adhesive system Scotchbond Multi-Purpose (3M ESPE). Each group was randomly divided into two subgroups: 24 hours of storage and 12 months of storage. All roots were sectioned transversely, and the push-out test was performed. Failure modes were observed, and the bond strength means were analyzed by analysis of variance and the Tukey test (α = 0.05). RESULTS CHX irrigation resulted in homogeneous bond strength values at 24 hours and 12 months of storage (P < .05). A significant bond strength decrease was noticed after 12 months of storage when irrigations were performed with physiologic solution and EtOH application only or associated with CHX (P < .05). CONCLUSIONS The use of CHX pretreatment could preserve the bond strength of the fiber post relined with resin composite to root dentin for 12 months. The use of EtOH and CHX followed by EtOH did not preserve the bond strength of the total etch adhesive system Scotchbond Multi-Purpose.

Effect of Chlorhexidine and Ethanol on the Durability of the Adhesion of the Fiber Post Relined with Resin Composite to the Root Canal

INTRODUCTION The aim of this study was to investigate the effects of pretreatment of gel chlorhexidine and ethanol on the bond strength and durability of the adhesion of the fiber post relined with resin composite to the root dentin. METHODS Sixty bovine incisor roots were divided into four groups: irrigation with physiologic solution (control), 5 minutes with chlorhexidine, 1 minute with ethanol, and 5 minutes with chlorhexidine followed by 1 minute with ethanol. Fiber posts relined with resin composite were cemented with RelyX ARC (3M ESPE, St Paul, MN) and a self-etching adhesive system Clearfil SE Bond (Kuraray, Kurashiki, Japan). Each group was randomly divided into three subgroups: 24 hours of water storage, 12 months of water storage, and 12 months of oil storage. All roots were sectioned transversely in the coronal, middle, and apical regions, producing 1-mm thick slices, and the push-out test was performed. Statistical analysis was performed using analysis of variance and the Tukey test for post hoc comparisons (α = 0.05). RESULTS Immediate groups showed similar bond strength values with or without chlorhexidine and/or ethanol pretreatment (P > .05). A significant decrease in the bond strength in the control group was observed after 12 months of storage in water and oil (P < .05). The use of chlorhexidine- and/or ethanol-preserved bond strength in the groups stored in water and oil for 12 months (P < .05). CONCLUSIONS Chlorhexidine and/or ethanol pretreatment were capable of preserving the bond strength of the fiber post relined with resin composite to root dentin for 12 months.

Clinical investigation of the efficacy of chemomechanical preparation with rotary nickel-titanium files for removal of endotoxin from primarily infected root canals.

INTRODUCTION This clinical study was conducted to investigate the ability of chemomechanical preparation with 2.5% NaOCl + 17% ethylenediaminetetraacetic acid (EDTA) and rotary nickel-titanium (NiTi) system in removing endotoxin from primary root canal infection with apical periodontitis. METHODS Twenty-one root canals with necrotic pulps were selected. Samples were collected before (s1) and after chemomechanical preparation (s2). The limulus amebocyte lysate (LAL) assay was used to quantify endotoxins. RESULTS The LAL assay indicated that endotoxins were present in 100% of the root canals investigated (19/19) before (s1) and after chemomechanical preparation (s2). Analyses of the quantitative data revealed that the endotoxin content was significantly reduced at s2 (98.06%) compared with that at s1 (P < .05). CONCLUSIONS Our findings indicate that chemomechanical preparation with 2.5% NaOCl + 17% EDTA and rotary NiTi files was effective in reducing endotoxin load in the root canal infection from primarily infected teeth with apical periodontitis.

Microbial Evaluation of Traumatized Teeth Treated with Triple Antibiotic Paste or Calcium Hydroxide with 2% Chlorhexidine Gel in Pulp Revascularization

INTRODUCTION Revascularization outcome depends on microbial elimination because apical repair will not happen in the presence of infected tissues. This study evaluated the microbial composition of traumatized immature teeth and assessed their reduction during different stages of the revascularization procedures performed with 2 intracanal medicaments. METHODS Fifteen patients (7-17 years old) with immature teeth were submitted to the revascularization procedures; they were divided into 2 groups according to the intracanal medicament used: TAP group (n = 7), medicated with a triple antibiotic paste, and CHP group (n = 8), dressed with calcium hydroxide + 2% chlorhexidine gel. Samples were taken before any treatment (S1), after irrigation with 6% NaOCl (S2), after irrigation with 2% chlorhexidine (S3), after intracanal dressing (S4), and after 17% EDTA irrigation (S5). Cultivable bacteria recovered from the 5 stages were counted and identified by means of polymerase chain reaction assay (16S rRNA). RESULTS Both groups had colony-forming unit counts significantly reduced after S2 (P < .05); however, no significant difference was found between the irrigants (S2 and S3, P = .99). No difference in bacteria counts was found between the intracanal medicaments used (P = .95). The most prevalent bacteria detected were Actinomyces naeslundii (66.67%), followed by Porphyromonas endodontalis, Parvimonas micra, and Fusobacterium nucleatum, which were detected in 33.34% of the root canals. An average of 2.13 species per canal was found, and no statistical correlation was observed between bacterial species and clinical/radiographic features. CONCLUSIONS The microbial profile of infected immature teeth is similar to that of primarily infected permanent teeth. The greatest bacterial reduction was promoted by the irrigation solutions. The revascularization protocols that used the tested intracanal medicaments were efficient in reducing viable bacteria in necrotic immature teeth.