A micro-computed tomography study of canal configuration of multiple-canalled mesiobuccal root of maxillary first molar.

OBJECTIVES: Detailed information of complex anatomical configuration of mesiobuccal (MB) root is essential for successful endodontic treatment in maxillary first molars. The aims of this study were to investigate the configuration types present in multiple-canalled MB roots of maxillary first molars using micro-computed tomography (µCT) and to evaluate whether further modification to current configuration classifications are needed for in-depth morphology study of MB root canal system. MATERIALS AND METHODS: One hundred and fifty-four extracted human maxillary first molar MB roots were scanned by µCT (Skyscan) and their canals were reconstructed by 3D modeling software. Root canal configurations were categorized according to the classifications proposed by Weine and Vertucci. Canal configurations that did not fit into both classifications were categorized as non-classifiable. RESULTS: One hundred and thirteen (73.4 %) MB roots had multiple canals. The most predominant canal configuration was Weine type III (two orifices and two foramens). Thirty-three (29.2 %) and 20 (17.7 %) MB roots had non-classifiable configuration types that could not be classified by the Weine and Vertucci classification, respectively. Three configurations (types 1-3, 2-3-2-3-2, and 2-3-4-3-2) were first reported in maxillary first molar MB roots. CONCLUSIONS: The present µCT study provided an in-depth analysis of canal configurations of the MB roots of maxillary first molar and suggests that additional modification of current configuration classifications may be needed to more accurately reflect the morphology configurations of MB roots. CLINICAL RELEVANCE: Clinicians should consider the complex canal configurations of the maxillary first molar MB roots during surgical or nonsurgical endodontic procedures.

Defining Endodontic Residents' Clinical Experiences: A National Survey.

Endodontic residency programs across the U.S. vary in the exposure they provide to residents in procedures, protocols, and equipment. Having information on the range of clinical experiences provided in programs would be useful for program directors and for applicants who are seeking the best fit for their residency. The aim of this study was to collect information from residents in U.S. endodontic residency programs about the procedures and equipment they experienced in their programs. In January 2018, a 14-question survey was emailed to all 437 endodontic residents with an email address in the 2016-17 American Association of Endodontists Membership Directory. Survey items asked about the number of endodontic procedures, techniques employed, and products used in residents' programs. A total of 133 endodontic residents responded to all or part of the survey, for a 30% response rate. The majority reported completing 151-250 nonsurgical root canals, 26-50 nonsurgical retreatments, 0-10 surgical retreatments, and 0-10 regenerative endodontic procedures during their residencies. All respondents said they used a surgical operating microscope (SOM), and 82% reported using a multi-file rotary system for nonsurgical procedures. Respondents reported that the main instruments they used were Dentsply Sirona file systems, and the most commonly used obturation technique was warm vertical compaction/condensation, reported by 92% of respondents. These endodontic residents reported being exposed to a variety of procedures, products, and protocols during their residency. Based on information they provided, prospective endodontic residency applicants can expect to use the SOM for treatment, to gain extensive experience in primary nonsurgical endodontic treatment, and to not perform endodontic surgery during their first year of postgraduate training.

Quantitative analysis of osteoclast-specific gene markers stimulated by lipopolysaccharide.

Lipopolysaccharide (LPS) in the outer layers of Gram-negative bacteria plays an important role in initiating and sustaining periapical lesions. To understand the mechanisms of osteoclastic bone resorption in periapical lesions induced by LPS, we stimulated osteoclast precursors, RAW 264.7 cells with LPS. LPS stimulated osteoclastogenesis when osteoclast precursors were primed with activator for NF-kappaB ligand (RANKL) as little as 24 h. By employing real-time PCR analysis, we have confirmed that osteoclast-like cells stimulated by LPS express high level of osteoclast-specific gene markers such as TRAP, cathepsin K, and calcitonin receptor. These results suggest that bone-resportive action by LPS is partially independent of RANKL.

Effects of root-end filling materials on fibroblasts and macrophages in vitro.

OBJECTIVE: The aim of this study was to investigate the effects of 4 root-end filling materials (mineral trioxide aggregate [MTA], intermediate restorative material [IRM], amalgam, and Retroplast) on cell growth, cell morphology, and cytokine (interleukin [IL]1beta and IL-6) production in mouse fibroblasts and macrophages. STUDY DESIGN: Millipore culture plate inserts with freshly mixed or set root-end filling material were placed into 6-well cell culture plates with already attached mouse fibroblasts or macrophages. Cells cultured with only the Millipore culture plate inserts served as a control. After a 3-day incubation, cell morphology was examined, and the total cell number per well was counted and analyzed by using 1-way analysis of variance. For cytokine assay, mouse macrophages were incubated in 24-well flat-bottom plates with set root-end filling material disks in the bottom. Cells cultured without the material disks served as negative controls, and cells cultured with lipopolysaccharides served as positive controls. After 24-hour incubation, culture media were collected for cytokine assay by using enzyme-linked immunosorbent assay. RESULTS: All root-end filling materials inhibited the cell growth of mouse fibroblasts and macrophages. There was no growth in the originally seeded cells in the fresh IRM, the fresh Retroplast, and the set IRM group. There was no difference between MTA and amalgam for cell growth either in the fresh material groups or in the set material groups. The total cell number in the set Retroplast group was significantly less than that in the set MTA group. Morphologically, MTA was characterized by denatured medium proteins and dead cells adjacent to the material, which were observed only in the fresh MTA group. There was no detected cytokine production in any of the tested material groups. CONCLUSION: All root-end filling materials inhibited cell growth, and none induced IL-1beta and IL-6 production.

Direct contact between enamel matrix derivative (EMD) and osteoblasts is not required for EMD-induced cell proliferation.

OBJECTIVE: The purpose of this study was to determine whether the effect of enamel matrix derivative (EMD) on osteoblast proliferation is dependent on direct contact between EMD and the cells. STUDY DESIGN: MC3T3-E1 cells were seeded onto 6-well culture plates at an initial density of 5000/cm(2) in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS). Serum was removed from the culture medium after 24 hours with or without the addition of EMD. Four groups were evaluated: group 1, DMEM only; group 2, DMEM with 100 microg/mL EMD directly added to the culture medium; group 3, DMEM with a culture plate insert (30-mm diameter; 0.4-microm pore size) only; group 4, DMEM with 100 microg/mL EMD added onto a culture plate insert. The porous membrane of the insert prevented direct contact between EMD and the cells. After 3-day incubation, cell morphology was examined and the total cell number per well was counted and analyzed using 1-way ANOVA. RESULTS: EMD formed precipitated aggregates on the membrane of the culture insert with the same appearance as when it was added directly to the medium. The culture plate insert alone did not cause any changes in cell morphology or proliferation. The addition of EMD significantly increased cell number regardless the presence of the culture plate insert. CONCLUSION: This study suggests that direct contact between EMD and osteoblasts is not required to induce cell proliferation. Soluble peptides released from EMD may contribute to the stimulating effects of EMD on cell proliferation.

Emdogain promotes osteoblast proliferation and differentiation and stimulates osteoprotegerin expression.

PURPOSE: The purpose of this study was to investigate the effects of EMD on the growth and differentiation of osteoblastic cells (MC3T3-E1) and on the expression of osteoprotegerin (OPG), a key cytokine that inhibits osteoclastogenesis and osteoclast function. STUDY DESIGN: MC3T3-E1 cells were treated with 100 microg/mL EMD in serum-free medium for 1, 2, 3, 5, and 7 days, or in 2% fetal bovine serum (FBS) for 3 weeks. Cells incubated without EMD served as negative control. At the end of each incubation period, cell numbers were counted and total cellular mRNA was extracted. Northern blot analysis and RT-PCR were performed to determine the mRNA levels of core binding factor alpha (Cbfa1), collagen alpha1 (I), bone sialoprotein (BSP), osteocalcin (OC), insulin-like growth factor I (IGF-I), and OPG. Alkaline phosphatase (ALP) activity was also determined and compared between treatment and control groups. RESULTS: A marked increase in cell numbers was observed in EMD-treated groups from day 2 to day 7 (P < .01). mRNA expression of collagen alpha1 (I), BSP, OC, OPG, and IGF-I were up-regulated in cells treated with EMD. ALP activity was significantly increased by EMD treatment after 3-week culture under differentiating conditions (P < .05). The expression of Cbfa1 was not affected by EMD treatment from day 1 to day 5; the levels were elevated after culturing for 3 weeks in EMD-treated cells. CONCLUSIONS: EMD promotes both proliferation and differentiation of MC3T3-E1 cells and indirectly inhibits osteoclastogenesis and osteoclast function by stimulating the expression of OPG.

Effects of removing residual peroxide and other oxygen radicals on the shear bond strength and failure modes at resin-tooth interface after tooth bleaching.

PURPOSE: To investigate the effects of removing residual peroxide on the bond strength and the failure mode at the interface of resin-based composite and enamel after tooth bleaching. METHODS: Standard-sized light-cured resin cylinders were formed on, and bonded to the flattened bleached enamel surfaces of 60 human canine and premolars which had previously been subjected to three different surface treatments for 3 minutes: (1) catalase; (2) 70% ethanol; (3) sprayed water. For each experimental group (n=12), non-bleached teeth and 2-week post-bleached teeth without any surface treatment were used for negative and positive control respectively. Specimens were thermocycled, tested in shear until failure, and the results statistically analyzed. All fractured specimens were examined by scanning electron microscopy. RESULTS: Pretreatment of bleached surface with the catalase and the ethanol prior to bonding significantly improved the composite-enamel bond strength compared to the water-sprayed group (P< 0.05). However, the bond strength level of the ethanol group did not return to the level recorded for the non-bleached negative control group. Scanning electron microscopic examination of randomly selected, fractured specimens indicated that the peroxide-induced reduction in bond strength was related to alterations in both attachment-surface area at the resin-enamel interface and resin quality.

Biologic activity of porphyromonas endodontalis complex lipids.

INTRODUCTION: Periapical infections secondary to pulpal necrosis are associated with bacterial contamination of the pulp. Porphyromonas endodontalis, a gram-negative organism, is considered to be a pulpal pathogen. P. gingivalis is phylogenetically related to P. endodontalis and synthesizes several classes of novel complex lipids that possess biological activity, including the capacity to promote osteoclastogenesis and osteoclast activation. The purpose of this study was to extract and characterize constituent lipids of P. endodontalis and evaluate their capacity to promote proinflammatory secretory responses in the macrophage cell line, RAW 264.7, as well as their capacity to promote osteoclastogenesis and inhibit osteoblast activity. METHODS: Constituent lipids of both organisms were fractionated by high-performance liquid chromatography and were structurally characterized using electrospray mass spectrometry or electrospray-mass spectrometry/mass spectrometry. The virulence potential of P. endodontalis lipids was then compared with known biologically active lipids isolated from P. gingivalis. RESULTS: P. endodontalis total lipids were shown to promote tumor necrosis factor alpha secretion from RAW 264.7 cells, and the serine lipid fraction appeared to account for the majority of this effect. P. endodontalis lipid preparations also increased osteoclast formation from RAW 264.7 cells, but osteoblast differentiation in culture was inhibited and appeared to be dependent on Toll-like receptor 2 expression. CONCLUSIONS: These effects underscore the importance of P. endodontalis lipids in promoting inflammatory and bone cell activation processes that could lead to periapical pathology.

Three-dimensional analysis of root canal curvature and direction of maxillary lateral incisors by using cone-beam computed tomography.

INTRODUCTION: Root canal curvature can affect the technical quality of endodontic treatment. Prior studies measured canal curvature mainly by 2-dimensional radiography. The aim of this study was to measure the 3-dimensional (3D) root canal curvature and canal direction of maxillary lateral incisors by using cone-beam computed tomography (CBCT) and mathematical modeling. METHODS: The CBCT images of 186 maxillary lateral incisors from 110 patients were used to measure 3D root canal curvature by using V-works and kappa software. In addition, the direction of each root canal was determined by measuring the orientation of the apical one-third with respect to the coronal two-thirds. RESULTS: All 186 maxillary lateral incisors were found to have canal curvature that was mainly oriented in the disto-palatal direction. The point of maximum curvature was located 0.5 mm from the root apex. CONCLUSIONS: Maxillary lateral incisors have 3D canal curvature that is maximal near the root apex, oriented in the disto-palatal direction. These CBCT analyses provide valuable information for root canal instrumentation of maxillary lateral incisors.

Microbial analysis in primary and persistent endodontic infections by using pyrosequencing.

INTRODUCTION: The aim of this study was to investigate the bacterial community profile of intracanal microbiota in primary and persistent endodontic infections associated with asymptomatic chronic apical periodontitis by using GS-FLX Titanium pyrosequencing. The null hypothesis was that there is no difference in diversity of overall bacterial community profiles between primary and persistent infections. METHODS: Pyrosequencing analysis from 10 untreated and 8 root-filled samples was conducted. RESULTS: Analysis from 18 samples yielded total of 124,767 16S rRNA gene sequences (with a mean of 6932 reads per sample) that were taxonomically assigned into 803 operational taxonomic units (3% distinction), 148 genera, and 10 phyla including unclassified. Bacteroidetes was the most abundant phylum in both primary and persistent infections. There were no significant differences in bacterial diversity between the 2 infection groups (P > .05). The bacterial community profile that was based on dendrogram showed that bacterial population in both infections was not significantly different in their structure and composition (P > .05). CONCLUSIONS: The present pyrosequencing study demonstrates that persistent infections have as diverse bacterial community as primary infections.

Effect of selected accelerants on setting time and biocompatibility of mineral trioxide aggregate (MTA).

OBJECTIVE: Various additives have been suggested to be used with mineral trioxide aggregate (MTA) to improve its handling characteristics. The purpose of this study was to evaluate the effects of various additives on setting time and cell attachment on gray MTA (GMTA). STUDY DESIGN: Single-rooted caries-free teeth were split, and dentin disks with class I cavity were made and filled with test and control materials. Setting time was measured using Gilmore apparatus. Mouse MC3T3-E1 osteoblasts and L929 mouse fibroblasts were grown on dentin and GMTA disks. Cell attachment was examined under fluorescent microscope. RESULTS: Adding KY liquid, CaCl(2), and NaOCl to GMTA improved the handling properties and decreased setting time. Osteoblasts and fibroblasts attached and spread on GMTA mixed with additives in a manner similar to GMTA mixed with water. CONCLUSIONS: The various additives could be possible substitutes to water to decrease MTA setting time. MTA is biocompatible when mixed with the various additives.

Cytotoxicity evaluation of Gutta Flow and Endo Sequence BC sealers.

OBJECTIVE: This study evaluated the cytotoxicity of GuttaFlow and EndoSequence BC sealers and compared them with AH Plus and Tubli-Seal sealers. STUDY DESIGN: Samples (0.5 mg) of freshly mixed or set BC, GuttaFlow, AH Plus, and Tubli-Seal sealers were eluted with 300, 600, and 1,000 µL cell culture medium for 24 and 72 hours. L929 cells were seeded into 96-well plates at 3 × 10(4) cells/well and cultured with 100 µL eluate from each eluate group. Cells cultured only with culture medium served as control. After 24 hours' incubation, the cytotoxicity was evaluated by MTT assay. Cell viability was calculated as the percentage of the control group, and the results were analyzed with a one-way analysis of variance. RESULTS: For the freshly mixed sealer, cell viability in the AH Plus group was less than in all of the other 3 sealer groups. The Tubli-Seal sealer group had less cell viability than the EndoSequence BC and GuttaFlow sealer groups. For the set sealer, the Tubli-Seal and AH Plus groups had less cell viability than the EndoSequence BC and GuttaFlow sealer groups. There was no cell viability difference between the EndoSequence BC and GuttaFlow sealer groups in the either freshly mixed or set sealer group. CONCLUSIONS: The GuttaFlow and EndoSequence BC sealers have lower cytotoxicity than the AH Plus and Tubli-Seal sealers.

Cytotoxicity evaluation of endosequence root repair material.

OBJECTIVE: The purpose of this study was to evaluate the cytotoxicity of EndoSequence Root Repair Material (Brasseler USA, Savannah, GA) and compare it with gray and white MTA. STUDY DESIGN: Samples of 2 mg freshly mixed or set gray MTA (GMTA), white MTA (WMTA), EndoSequence Root Repair Material (ERRM), and AH26 were eluted with 300, 600, and 1,000 microL cell culture medium for 24 and 72 hours. L929 cells were seeded into 96-well plates at 3 x 10(4) cells/well and incubated with 100 microL elute from each elute group. Cells cultured only with culture medium served as negative control. AH26 was used as positive control. After 24 hours' incubation, cell cytotoxicity was evaluated by MTT assay. Cell viability was calculated as percentage of the control group. The results were analyzed with 1-way analysis of variance. RESULTS: For both set and fresh samples, there were no significant cell viability differences among GMTA, WMTA, and ERRM. Cell viability in the AH26 group was less than in all of the other 3 materials. CONCLUSION: This study suggests that ERRM may have cell viability similar to GMTA and WMTA in both set and fresh conditions.

Evaluation of cytotoxicity of MTA employing various additives.

OBJECTIVE: Various additives have been suggested to be used with MTA to improve its handling characteristics. The objective of this study was to evaluate the effects of various additives on cytotoxicity of MTA. STUDY DESIGN: Freshly mixed or set MTA pellet (1-mm diameter and 1-mm high cylinder) was prepared by mixing MTA with various additives. Additives tested included water, saline, 2% lidocaine, 5% CaCl(2), 3% NaOCl gel, and K-Y liquid. L929 cells were seeded into 96-well plates at 3 x 10(4) cells per well and incubated with MTA pellets for 24 hours, 48 hours, and 72 hours. Cells without treatment served as a control group. Cell viability was evaluated by MTT assay and calculated as the percentage of the control group. The results were analyzed with 1-way ANOVA. RESULTS: For the set MTA, there were no significant cell viability differences (P > .05) among the various additives at each tested time. For the freshly mixed MTA, 3% NaOCl gel has lower cell viability (P < .05) than all the other groups. The cell viability of 3% NaOCl gel group was 29% to 50%. Gray and white MTA have similar results. CONCLUSION: This study shows that the various additives have no effect on the cytotoxicity of MTA when it becomes set. Also, all the tested additives, except 3% NaOCl gel, had no effect on the cytotoxicity of MTA when it was freshly mixed. The cytotoxicity of 3% NaOCl gel probably has no clinical significance considering there is still 29% to 50% of cell viability after cells were treated with MTA pellet in a 0.32-cm(2) culture well. MTA is biocompatible when mixed with the various additives.

Cytotoxicity evaluation of Activ GP and Resilon sealers in vitro.

OBJECTIVE: This study is to evaluate the cytotoxicity of Activ GP and RealSeal sealers in a cell culture system in vitro, and to compare them with traditional AH 26 and Kerr sealers. STUDY DESIGN: Samples of 0.5 mg freshly mixed or set RealSeal, Activ GP, AH 26, and Kerr sealers were eluted with 200, 400, 800, and 1,200 microL cell culture medium for 24 and 72 hours. L929 cells were seeded into 96-well plates at 3 x 10(4) cells/well and cultured with 100 microL eluate from each eluate group. Cells cultured with culture medium only served as a control. After 24 hours' incubation the cytotoxicity was evaluated by MTT assay. Cell viability was calculated as the percentage of the control group, and the results were analyzed with 1-way analysis of variance. RESULTS: For the freshly mixed sealer, cell viability in the AH 26 group was less than in all of the other 3 sealer groups. The Kerr sealer group had greater cell viability than RealSeal and Activ GP groups. For the set sealer, cell viability in the AH 26 group was greater than in all of the other 3 groups. Cell viability in the RealSeal group was less than in the Kerr and Activ GP groups. CONCLUSION: Freshly mixed RealSeal and Activ GP sealers have lower cytotoxicity than AH 26 sealer and more cytotoxicity than Kerr sealer. When sealers are set, RealSeal sealer has more cytotoxicity than AH 26 and Kerr sealer. Activ GP sealer has more cytotoxicity than AH 26 and is similar to Kerr sealer.

A rapid nondestructive method for root dentin moisture measurements: in vitro pilot study.

Dentin moisture content is important in adhesive bonding and structural strength research; however, there is no rapid method available to assess dentin moisture without sample destruction. This study examined the use of a digital grain moisture meter to measure root dentin moisture in vitro. Extracted mandibular single-rooted teeth were sectioned at the CEJ. The moisture of the root dentin was measured at 6 measuring modes for different grains and repeated 5 times. Dentin weight changes before and after drying were measured to obtain control values. The control values were compared with machine readings. In conclusion, (1) each nondestructive measurement took less than 30 seconds, (2) 24 hours of storage at 37 degrees C and 100% humidity did not restore dentin moisture, and (3) 5 grain modes had a high validity and could be used for dentin moisture measurements.

Particle size and shape of calcium hydroxide.

The aim of this study was to examine the particle length, width, perimeter, and aspect ratio of calcium hydroxide powder using a flow particle image analyzer (FPIA). Five sample groups each with 10 mg of calcium hydroxide were mixed with 15 mL of alcohol and sonicated. Digital images of the particle samples were taken using the FPIA and analyzed with a one-way analysis of variance. The overall averages +/- standard deviation among the five groups for particle length (microm), width (microm), perimeter (microm), and aspect ratio were 2.255 +/- 1.994, 1.620 +/- 1.464, 6.699 +/- 5.598, and 0.737 +/- 0.149, respectively. No statistical significance was observed among the groups for all parameters. When the total of 46,818 particles from all five groups were classified into the five length categories of 0.5-microm increments, there were significant differences in width, perimeter, and aspect ratio (all p values <0.0001). In conclusion, calcium hydroxide particles have a size and shape that may allow direct penetration into open dentin tubules.

Cytotoxicity evaluation of Activ GP and Resilon cones in vitro.

OBJECTIVE: This study was done to evaluate the cytotoxicity of Activ GP and Resilon cones in an in vitro cell culture system. STUDY DESIGN: Gutta-percha (GP), Activ GP, and Resilon cones were tested in this study. L929 cells were seeded into 96-well plates at 3 x 10(4) cells/well. In one set of experiments, 2-mm segments cut from the tip of GP and Resilon cones were placed into the cell culture wells and incubated for 1, 2, and 3 days. In another set of experiments, 2 20-mm segments of GP, Activ GP, and Resilon cones were incubated in 2 mL cell culture medium for 1 week. Then 100 microL elutes were tested for 24 and 48 h. Cell viability was evaluated by MTT assay. Data were analyzed using 1-way analysis of variance. RESULTS: When GP, Activ GP, and Resilon segments were placed into cell cultures, cell viability in the Resilon group was significantly greater than in the GP and Activ GP groups at any test time. There was no cell viability difference between the Activ GP and GP groups. When the elutes of GP, Activ GP, and Resilon was placed into cell cultures, the results were the same as using segments of the tested material. The cytotoxicity of GP and Activ GP is greater than that of the Resilon cone. There was no cell viability difference between Activ GP and regular GP. CONCLUSION: Resilon has better biocompatibility than regular GP and Activ GP cones. The cytotoxicity of Activ GP is similar to that of regular GP.

Stimulation of cytokines in osteoblasts cultured on enamel matrix derivative.

OBJECTIVE: The purpose of this study was to evaluate the influence of enamel matrix derivative (EMD) on the release of transforming growth factor beta 1 (TGF-beta1), interleukin-6 (IL-6), insulin-like growth factor I (IGF-I), bone morphogenetic protein 2 (BMP-2), and osteoprotegerin (OPG) in human and mouse osteoblasts. STUDY DESIGN: Human MG-63 and mouse MC3T3-E1 cells were seeded onto 6-well culture plates at an initial density of 5,000/cm(2) and grown in Dulbecco's eagle medium (DMEM) with 10% fetal bovine serum for 24 h. Then cells were cultured either with 100 microg/mL EMD added to DMEM or with DMEM only. After 2, 5, and 9 days' incubation the culture medium was collected and analyzed by enzyme-linked immunosorbent analysis. Data were analyzed using Student t test. RESULTS: The EMD treatment significantly increased the production of IL-6 and TGF-beta1 (P < .05) at all time points. The release of OPG was also increased in mouse osteoblasts (P < .05). IGF-I and BMP-2 were not detected in both control and EMD-treated groups. CONCLUSION: This study suggests that the stimulatory effects of EMD on tissue regeneration are mediated by the up-regulation of local mediators released by osteoblasts.