Disturbed bone remodelling activity varies in different stages of experimental, gradually progressive apical periodontitis in rats.

Bone remodelling keeps going through the lifespan of human by bone formation and bone resorption. In the craniofacial region, mandibles act as the main force for biting and chewing, and also become susceptible to a common bone-loss disease, namely, apical periodontitis, once infected dental pulp is not treated timely, during which bone resorption occurs from the apical foramen to the apical bone area. Although conventional root canal treatment (RCT) can remove the most of the infection, chronical apical periodontitis due to incomplete removal of dental pulp and subsequent microleakage will become refractory and more challenging, and this process has scarcely been specifically studied as a bone remodelling issue in rat models. Therefore, to study chronical and refractory apical periodontitis owing to incomplete cleaning of infected dental pulp and microleackage in vivo, we establish a modified rat model of gradually progressive apical periodontitis by sealing residual necrotic dental pulp and introducing limited saliva, which simulates gradually progressive apical periodontitis, as observed in the clinical treatment of chronical and refractory apical periodontitis. We show that bone-loss is inevitable and progressive in this case of apical periodontitis, which confirms again that complete and sound root canal treatment is crucial to halt the progression of chronical and refractory apical periodontitis and promote bone formation. Interestingly, bone remodelling was enhanced at the initial stage of apical periodontitis in this model while reduced with a high osteoblast number afterwards, as shown by the time course study of the modified model. Suggesting that the pathological apical microenvironment reserve its hard tissue formation ability to some degree but in a disturbed manner. Hopefully, our findings can provide insights for future bone regenerative treatment for apical periodontitis-associated bone loss.

Investigations on the adhesion of new composites for restoring cervical lesions using energy dispersive X-ray analysis and scanning electron microscopy.

Restoration of noncarious cervical lesions with resin composites is one of the treatment options, but the retention of the restorations located at the crown-root junction is still a cause of clinical concern. The aim of this study was to evaluate the adhesive properties of three experimental resin composites and two commercial materials used to restore cavities prepared on extracted teeth as well as to determine the relative elemental composition of these materials. We tested the null hypothesis, which considered that the adhesive behaviours of different resin composites did not differ. The microleakage test using tracers showed that all tested materials exhibited some degree of dentinal microleakage, although they all had good dentinal adhesion. The results failed to reject the null hypothesis. The scanning electron microscopy revealed completely adapted adhesive interfaces underneath the restorations along with well-developed hybrid layers depending on the adhesive system. Energy dispersive X-ray analysis analyses showed that the restorative materials have similar chemical compositions, with some differences between the samples from the same material. The results support the implementation of experimental resins in clinical settings.

Dental abnormalities in individuals with pathogenic germline variation in DICER1.

Pathogenic germline variation in the microRNA processing gene DICER1 gives rise to an autosomal dominant, tumor-predisposition disorder. Conditional deletion of Dicer1 in murine dental epithelium shows that it controls tooth patterning, size, number, and shape. The human dental phenotype of people with germline pathogenic variation in DICER1 is unknown. DICER1-carriers (n = 57) and family controls (n = 55) were evaluated at the NIH Clinical Center dental clinic as part of a comprehensive medical evaluation. Digital panoramic radiographs, bite-wing radiographs, and oral photographs were collected. A single observer, blind to DICER1 status, reviewed the dental records and determined the presence or absence of 11 dental characteristics as described in the clinic notes, radiographs, or oral photographs. Subjective phenotypes were reviewed on radiographs by two examiners (blind to DICER1 status) for the presence or absence of the dental characteristics to reduce inconsistencies. By simple association, bulbous crown, periodontitis, and taurodontism were all significant (p < .05). Logistic regression with chi-square maximum likelihood estimates showed that bulbous crown and periodontitis remained significant. Recognition of these phenotypes may aid identification of individuals and families at risk for DICER1-associated neoplasms. These findings may also guide dental care for individuals with germline DICER1 pathogenic variation.

Nav1.7 via Promotion of ERK in the Trigeminal Ganglion Plays an Important Role in the Induction of Pulpitis Inflammatory Pain.

The trigeminal ganglion (TG) refers to sensory neurons bodies that innervate the spinal cord and peripheral axons that innervate teeth. The tetrodotoxin-sensitive sodium (NA) channels (Nav1.7) play important roles in the pathophysiology of pain. In this study, we investigated the TG expression of Nav1.7 and extracellular signal-regulated kinase (ERK) in a rat model of pulpitis to explore the correlation between these channels and inflammatory pain. Pulpitis was confirmed by hematoxylin-eosin staining. In this study, we demonstrated that the reflex of rats to mechanical stimulation increases after pulp exposure and that the exposed rat molar pulp can upregulate the expression of Nav1.7 and ERK in the rat TG. Three days after rat pulp exposure, the expression levels of the two ion channels in the TG increased. TG target injection of PF04856264, a Nav1.7 inhibitor, dose-dependently increased the mechanical pain threshold and was able to inhibit ERK expression. TG target injection of PD98059, an ERK inhibitor, dose-dependently increased the mechanical pain threshold. These factors simultaneously resulted in the highest production. In this study, with the established link to inflammatory pain, we found that Nav1.7 and ERK both play important roles in the induction of inflammatory pain caused by pulpitis. We also found a correlation between the expression levels of Nav1.7 and ERK and the degree of inflammatory pain. Furthermore, ERK signaling pathways were promoted by the Nav1.7 in TG after pulpitis.

Does Achievement of Hemostasis After Pulp Exposure Provide an Accurate Assessment of Pulp Inflammation?

PURPOSE: The purpose of this study was to determine in primary molars with carious exposures whether hemostasis at the exposure site and pulp orifice reflected inflammatory status of the pulp at the canal orifice based on cytokine levels. METHODS: Forty mandibular primary molars with deep caries were included in the study. Teeth were divided into two groups: group A had teeth where hemostasis at the exposure site was achieved within five minutes, and group B had teeth where hemostasis at the exposure site could not be achieved within five minutes. Blood samples were harvested from the exposure sites and canal orifices. Cytokine levels for IL-1ß, IL-2, IL-6, IL-8, IL-10, TNF-α, and PGE2 were measured using ELISA for all sample sites. RESULTS: The IL-6 levels at the exposure sites were found to be significantly higher in group A when compared to group B, but there was no statistically significant differences in any of the cytokine levels at the canal orifices between the two groups. CONCLUSIONS: Controlling bleeding at the exposure site or canal orifices does not provide accurate assessment of inflammation at the canal orifice and may be misleading for diagnosing vital pulp treatment in primary teeth with a carious pulp exposure.

WNT10B mutations associated with isolated dental anomalies.

Isolated hypodontia is the most common human malformation. It is caused by heterozygous variants in various genes, with heterozygous WNT10A variants being the most common cause. WNT10A and WNT10B are paralogs that likely evolved from a common ancestral gene after its duplication. Recently, an association of WNT10B variants with oligodontia (severe tooth agenesis) has been reported. We performed mutational analysis in our cohort of 256 unrelated Thai families with various kinds of isolated dental anomalies. In 7 families afflicted with dental anomalies we detected 4 heterozygous missense variants in WNT10B. We performed whole exome sequencing in the patients who had WNT10B mutations and found no mutations in other known hypodontia-associated genes, including WNT10A, MSX1, PAX9, EDA, AXIN2, EDAR, EDARADD, LPR6, TFAP2B, LPR6, NEMO, KRT17, and GREM2. Our findings indicate that the variants c.475G>C [p.(Ala159Pro)], found in 4 families, and c.1052G>A [p.(Arg351His)], found in 1 family, are most probably causative. They also show that WNT10B variants are associated not only with oligodontia and isolated tooth agenesis, but also with microdontia, short tooth roots, dental pulp stones, and taurodontism.

Apical Microleakage in Root Canals Obturated With 2 Different Endodontic Sealer Systems in Canine Teeth of Dogs.

An apical dye leakage test was utilized to compare 2 endodontic sealer systems commonly used in veterinary endodontic treatment. Rotary instrumentation followed by injection of 2 different sealers (GuttaFlow 2 and AH Plus) and a single master cone obturation technique were compared. Following obturation and restoration, specimens were immersed in India ink for 48 hours. A tooth mineral clearing technique was utilized to render the tooth samples transparent. Subsequently, the apices were evaluated for prevalence and magnitude of apical dye penetration under magnification. Statistical analysis of the results revealed no significant differences in the prevalence or magnitude of apical dye penetration between groups.

A scoping review of root canal revascularization: relevant aspects for clinical success and tissue formation.

The aim of this scoping study was to evaluate the survival rate and nature of tissue formed inside root canals of human immature permanent teeth with necrotic pulps (NIPT) under root canal revascularization (RCR). The search was performed in SciVerse Scopus®, PubMed/MEDLINE, Web of Science®, BIREME and in the grey literature up to November 2015. The keywords were selected using MeSH terms and DECs. Two independent reviewers scrutinized the records obtained considering specific inclusion criteria. The included studies were evaluated in accordance with a modified Arksey and O' Malley's framework. From 375 studies that were evaluated, 75 were included. A total of 367 NIPT were submitted to RCR, from which only 21 needed further endodontic treatment. The weighted mean follow-up time was 17.6 months. The data were derived mainly from case reports (69%) or small case series (15%). NaOCl [0.5-6%] was applied as the disinfecting solution in almost all studies. Triple antibiotic paste was as effective as Ca(OH)2 as on intracanal medicament. De novo tissue was cementum and poorly mineralized bone positive to bone sialoprotein (BSP) but negative to dentine sialoprotein (DSP). Failures were associated mainly with reinfection of the root canal. The majority of included studies reported a significant increase in both root length and width. However, as most of these data came from case reports, they must be interpreted with care, as most were focused on treatment successes (not failures). Therefore, well-designed randomized controlled trials comparing RCR with available apexification treatments are needed to address this gap in the literature.

Tissue characterization following revascularization of immature dog teeth using different disinfection pastes.

Revascularization of immature teeth with necrotic pulps traditionally involves the use of triple antibiotic paste, which may sometimes lead to undesirable complications. The objective of this study was to assess tissue repair in immature dog teeth with apical periodontitis subjected to revascularization, comparing two different pastes used for root canal disinfection. Apical periodontitis was induced in 30 dog premolars. Teeth were randomly divided into three experimental groups: root canals filled with triple antibiotic paste (n = 10); root canals filled with 1% propolis paste (n = 10); and no medication (n = 10). An additional group (n = 10, no intervention) was used as control. After 7 months, the jaws were histologically evaluated for the following variables: newly formed mineralized tissue (present/absent); vital tissue in the canal space (absent/periodontal ligament-like/pulp-like); apical extension of root (present/absent); and severity of inflammatory process (absent/mild/moderate/severe). There were no statistically significant differences among the experimental groups in new mineralized tissue formation and apical root development. The formation of vital tissue in the canal space, in turn, was statistically different between the triple paste and propolis groups: vital tissues were present in all revascularized teeth disinfected with propolis paste (100%), compared to 71% of those disinfected with the triple paste. Severity of inflammatory process was different between the triple paste and no medication groups. The new tissues formed onto canal walls and in the root canal space showed characteristics of cementum and periodontal ligament, respectively. Propolis may have some advantages over the triple paste for the revascularization of immature teeth.

Life cycles of traumatized teeth: long-term observations from a cohort of dental trauma victims.

Life cycles of dental trauma victims can provide important clinical information, especially when viewed over many years. In this first series of life cycles, the pulp and periodontal responses to traumatic injuries of four patients are documented over periods varying from 26 to 51 years. The dynamics of pulp survival following an intrusive luxation and two avulsions are followed, with particular reference to pulp canal calcification to which a new term, root canal stenosis, has been proposed. The life cycles include the successful management of inflammatory root resorption in a replanted tooth with an open apex contrasting with the early prophylactic endodontic treatment of two replanted teeth in a patient with mature apices. The long-term development of invasive cervical resorption in one of the patient's life cycle highlights the importance of ongoing follow-up examinations for dental trauma victims.

Combined effects of photodynamic therapy and irrigants in disinfection of root canals.

In this study, the combined effects of photodynamic therapy and irrigants in eradicating common endodontic pathogens are evaluated. Roots of 80 extracted single rooted teeth are divided into 2 groups (1) mechanical flushing; (2) antibacterial irrigation. After cleaning and shaping, they are inoculated with either (A) Streptococcus mutans or (B) Enterococcus faecalis and incubated. They are again subdivided and either only irrigated or irrigated and lased. Dentin shavings are taken from root canal walls and cultured. Statistical analysis using One-Way ANOVA and Post-hoc tests are done. The combination eradicated both bacteria. Antibacterial irrigants controlled S. mutans better than PDT (p = 0.041). The combination of PDT and antibacterial irrigation proposed in this study can be used in all primary cases for thorough and reliable disinfection of root canals but may be highly effective in resistant cases like endodontic failures, as E. faecalis is prevalent in such cases.

Tissue characterization following revascularization of immature dog teeth using different disinfection pastes

Abstract Revascularization of immature teeth with necrotic pulps traditionally involves the use of triple antibiotic paste, which may sometimes lead to undesirable complications. The objective of this study was to assess tissue repair in immature dog teeth with apical periodontitis subjected to revascularization, comparing two different pastes used for root canal disinfection. Apical periodontitis was induced in 30 dog premolars. Teeth were randomly divided into three experimental groups: root canals filled with triple antibiotic paste (n = 10); root canals filled with 1% propolis paste (n = 10); and no medication (n = 10). An additional group (n = 10, no intervention) was used as control. After 7 months, the jaws were histologically evaluated for the following variables: newly formed mineralized tissue (present/absent); vital tissue in the canal space (absent/periodontal ligament-like/pulp-like); apical extension of root (present/absent); and severity of inflammatory process (absent/mild/moderate/severe). There were no statistically significant differences among the experimental groups in new mineralized tissue formation and apical root development. The formation of vital tissue in the canal space, in turn, was statistically different between the triple paste and propolis groups: vital tissues were present in all revascularized teeth disinfected with propolis paste (100%), compared to 71% of those disinfected with the triple paste. Severity of inflammatory process was different between the triple paste and no medication groups. The new tissues formed onto canal walls and in the root canal space showed characteristics of cementum and periodontal ligament, respectively. Propolis may have some advantages over the triple paste for the revascularization of immature teeth.

Evaluation of Cold Plasma Treatment and Safety in Disinfecting 3-week Root Canal Enterococcus faecalis Biofilm In Vitro.

INTRODUCTION: Although endodontic infection is caused by multi-bacteria species, Enterococcus faecalis is usually isolated in chronic apical periodontitis. The aim of this study was to evaluate the effectiveness and mechanical safety of cold plasma therapy in disinfecting 3-week E. faecalis biofilms. METHODS: Teeth with 3-week E. faecalis biofilm were treated with AC argon/oxygen (Ar/O2) cold plasma for various treatment times and compared with those treated with Ca(OH)2, 2% chlorhexidine gel, and Ca(OH)2/chlorhexidine for a week. Antimicrobial efficacy was assessed by colony-forming unit method. Scanning electron microscopy was used to assess the morphologic changes of E. faecalis biofilm by plasma. Confocal laser scanning microscopy was used to confirm the viability of the biofilm after the plasma treatment. Microhardness and roughness changes of root canal dentin caused by plasma were verified with Vickers Hardness Tester and 3D Profile Measurement Laser Microscope, respectively. RESULTS: There were no detectable live bacteria after 12 minutes of cold plasma treatment. This was further confirmed by scanning electron microscopy and confocal laser scanning microscopy results. Microhardness and roughness of root canal dentin showed no significant difference after plasma treatment. CONCLUSIONS: Atmospheric pressure cold plasma is an effective therapy in endodontics for its strong sterilization effect on fully matured biofilm within a few minutes. Meanwhile, it has an accepted mechanical safety for its low temperature and not affecting the microhardness and roughness of root canal dentin significantly.

Effect of a zirconia primer on the push-out bond strength of zirconia ceramic posts to root canal dentin.

STATEMENT OF PROBLEM: The retention of zirconia ceramic posts to root canal dentin with resin-based luting cements is relatively poor. PURPOSE: The purpose of this in vitro study was to evaluate the effect of a new zirconia primer, a mixture of organophosphate and carboxylic acid monomers, on the push-out bond strength of zirconia posts to root canal dentin. MATERIAL AND METHODS: The root canals of 40 extracted human maxillary central incisors were endodontically treated and the post spaces were prepared. Zirconia posts were luted with 2 different resin luting agents (Panavia F and Clearfil SA luting cements) with and without the zirconia primer (Z-Prime Plus; Bisco). Three segments, each 2 mm high, were cut perpendicular to the post from each root. Bond strength was determined by pushing out the post with a universal testing machine. Three-way ANOVA and the Tukey HSD test was used to assess the effects of the zirconia primer, the 2 different resin luting cements, and different thirds of the root canal (α=.05). RESULTS: The zirconia primer significantly increased the push-out bond strength of zirconia posts to root canal dentin. Clearfil SA luting cement provided significantly higher bond strength than did Panavia F. For all experimental groups combined, bond strength decreased from the coronal to the apical section. CONCLUSIONS: A zirconia primer based on organophosphate/carboxylic acid monomers increased the bond strength of zirconia posts to root canal dentin bonded with both resin luting cements.

Evaluation of the Effect of Enterococcus faecalis Biofilm on the 2% Chlorhexidine Substantivity: An In Vitro Study.

INTRODUCTION: The aim of this study was to correlate the bacterial viability and the presence of 2% chlorhexidine (CHX) solution on dentin by means of confocal laser scanning microscopy and high-performance liquid chromatography for 48 hours, 7 days, and 30 days. METHODS: One hundred twenty-three extracted human teeth were used. Samples were divided into 4 groups according to the solution (CHX or saline) and the presence of Enterococus faecalis biofilm. Samples were kept in contact with 5 mL of the solution for 5 minutes. Each group was divided into 3 subgroups according to the evaluation period (n = 10). Statistical analysis was performed by using the Kruskal-Wallis test, the Mann-Whitney U test (P < .05), and the Spearman rank correlation coefficient (P < .01). RESULTS: There was a negative correlation between the percentage of live cells and the amount of remaining CHX (P = .000). CHX significantly reduced the percentage of viable cells compared with saline after 48 hours (P = .007). Differences were maintained in the 7-day evaluation period (P = .001). After 30 days, the CHX group presented an increase of viable cells, thereby becoming similar to saline (P = .623). Simultaneously, the remaining CHX was significantly reduced in the 30-day specimens (P = .000). CONCLUSIONS: The results of this study indicate that 2% CHX solution was detected for 48 hours and 7 days with a low percentage of viable cells. The presence of microorganisms on human dentin did not affect 2% CHX maintenance.

Antibacterial Properties Associated with Chitosan Nanoparticle Treatment on Root Dentin and 2 Types of Endodontic Sealers.

INTRODUCTION: The aim of this study was to evaluate the efficacy of carboxymethyl chitosan (CMCS) and chitosan nanoparticles (CNps) to inactivate bacteria and prevent biofilm formation at sealer-dentin interfaces. METHODS: The study was divided into 3 stages: first stage, the experiment was conducted to analyze the antibacterial properties of CMCS in different formulations against biofilms; second stage, direct-contact and membrane-restricted methods were used to evaluate the antibacterial properties of an epoxy resin (ThermaSeal Plus; Dentsply Tulsa Dental, Tulsa, OK) and calcium silicate (MTA Fillapex; Angelus SA, Londrina, PR, Brazil) based-sealers with or without CNps; and third stage, biofilm formation at the sealer dentin interfaces of root dentin treated with CMCS and filled with gutta-percha and CNp incorporated sealer were analyzed after 1- and 4-week aging periods. The samples were treated and filled as follows: (1) distilled water: unaltered sealer (control group), (2) CMCS: sealer+CNps (CMCS group), and (3) CMCS/rose bengal: sealer+CNps (CMCS/RB group). Enterococcus faecalis was used to infect all the samples. Microbiological and microscopic analyses were used to assess the antibacterial characteristics. RESULTS: CMCS-based treatments effectively killed bacteria adherent on root dentin (P < .05). The addition of CNps to ThermaSeal enhanced its antibacterial ability by direct-contact and membrane-restricted tests (P < .05). The CNp incorporation significantly increased the antibacterial efficacy of root canal sealers even after a 4-week aging time (P < .05). CONCLUSIONS: This study highlighted the ability of CMCS to disinfect root canal dentin and inhibit bacterial adhesion. CNps in root canal sealers are capable of maintaining their antibacterial activity even after prolonged aging.

Neurotoxicity of various root canal sealers on rat sciatic nerve: an electrophysiologic and histopathologic study.

OBJECTIVES: The aim of this study was to compare the neurotoxicity of various root canal sealers on rat sciatic nerve by electrophysiologic and histopathologic analyses. MATERIALS AND METHODS: A total of 40 male rats were randomly divided into five groups: Control, AH Plus, GuttaFlow, Sealapex and Smartpastebio. Sciatic nerves of the rats were uncovered using the surgical procedures, and the prepared sealers were then applied on nerves with a polyethylene tube vehicle for 15 days. Nerve potentials were recorded at initial exposure, 5, 30 and 120 min (early phase), and 15 days (late phase) by an electrophysiologic analysis system for all groups. The obtained measurements were then used to calculate the nerve conduction velocities (NCV). Subsequently, all rats were sacrificed, and their sciatic nerves were removed for histopathologic analysis. Statistical analysis was performed using the Kruskal-Wallis and Mann-Whitney U tests for intergroup variables and the Friedman and Wilcoxon test for intragroup variables. Statistical significance was set at P < 0.05. RESULTS: There was no significant difference between early and late phase results in the control group. This group showed little or no lasting damage to nerve tissue. All sealers decreased the NCV in the early phase time periods, but this decrease was only statistically significant in the AH Plus group at 120-min time period (P < 0.0125). During the late phase, the AH Plus and GuttaFlow groups almost reached initial NCV values, and it was lower than the initial values in the Sealapex and Smartpastebio groups. However, this decrease was not statistically significant. When intergroup comparisons were performed, statistically significant differences occurred at 30 min in the Sealapex group and 120 min in the AH Plus group compared with the control group (P < 0.0125). All sealers induced neurotoxicity as a result of degenerative and inflammatory responses of nerve tissue in histologic analysis. Histologic analysis revealed Sealapex and GuttaFlow to be the most and least neurotoxic, respectively. CONCLUSIONS: All tested root canal sealers exhibited a variable degree of neurotoxicity depending on their chemical compositions. CLINICAL RELEVANCE: Apical extrusion of endodontic filling materials may cause undesired consequences, such as inflammation and severe neurotoxic damage; therefore, extrusion factor plays an important role during the root canal treatment.

On the dynamics of root canal infections-what we understand and what we don't.

Infections of the root canal space and their sequelae can be extremely painful and potentially dangerous, yet they do not necessarily have to be. Chronic, asymptomatic inflammatory lesions around the apex of a tooth with a necrotic dental pulp or an insufficient root canal treatment can develop unnoticed by the patient, and remain so for years. The course of disease is modulated by both the virulence of the microbiota established in the root canal space and the capacity of the immune system to curb the infection. To both ends, highly convincing investigations to help us understand when and why the tissues around an endodontically involved tooth become acutely inflamed are missing. We will discuss how recent advances in molecular identification of microorganisms have altered our understanding of root canal infections, and which information is currently missing to link clinical experience with observations from experimental research.

Middle mesial canals in mandibular molars: incidence and related factors.

INTRODUCTION: Although the internal anatomy of mandibular molars has been extensively studied, information about middle mesial (MM) canals is limited. The primary aim of this retrospective study was to evaluate the incidence of MM canals in mandibular first and second molars. The secondary aim was to correlate the incidence of MM canals with variables of molar type, sex, age, ethnicity, and presence of a second distal canal. METHODS: All mature permanent first and second mandibular molars treated from August 2012 to May 2014 were included in the analysis. After completion of root canal instrumentation in all main canals, the clinician inspected the isthmus area of the mesial root using the dental operating microscope. If there was a catch point in this area with a file or explorer, the operator spent more time attempting to negotiate an MM canal. RESULTS: Seventy-five mandibular first and second molars were treated during the specified period. Fifteen (20%) teeth had negotiable MM canals. The incidence of MM canals was 32.1% in patients ≤ 20 years old, 23.8% in patients 21-40 years old, and 3.8% in patients > 40 years. Analysis of data revealed a significant difference in the distribution of MM canals among different age groups (P < .05). The differences in the distribution of MM canals based on sex, ethnicity, molar type, and presence of a second distal canal were not significant. CONCLUSIONS: The incidence of negotiable MM canals overall and their frequency of identification in younger patients were higher than in previous reports.

Prevalence of middle mesial canals in mandibular molars after guided troughing under high magnification: an in vivo investigation.

INTRODUCTION: A limited number of in vivo studies have discussed the prevalence of middle mesial canals in root canal systems of mandibular molars. The reported results have varied between 1% and 25%, with no detailed description of the depth and direction of troughing needed to identify such small canal orifices. The objective of the present study was to determine (1) the prevalence of a middle mesial canal before and after troughing by using a standardized troughing technique, (2) the pathway of the middle mesial canal in relation to the mesiobuccal (MB) and mesiolingual (ML) canals, and (3) its correlation with the patient's age. METHODS: Ninety-one mandibular molars from 87 patients were included in this study. The patient's age and tooth number were recorded. After access cavity preparation, a standardized troughing technique was performed between MB and ML canals to search for a middle mesial canal by using a dental operating microscope. If a middle mesial canal was located, it was recorded as separate or as joining the MB or the ML canals. Results were statistically analyzed by using Z test and logistic regression. RESULTS: A middle mesial canal was found in 42 of 91 mandibular molars (46.2%). Six middle mesial canals were located after conventional access preparation (6.6%). The other 36 were located after standardized troughing (39.6%). The results were statistically significant (P < .001). There was a higher tendency to locate the middle mesial canal in second molars (60%) versus first molars (37.5%). Younger patients had a significantly higher incidence of a middle mesial canal (P = .004). CONCLUSIONS: The middle mesial canal was present in 46.2% of mandibular molars. High magnification, troughing, and patient's age appeared to be determining factors in accessing the middle mesial canal.