Real-time Quantitative Polymerase Chain Reaction and Culture Analyses of Enterococcus faecalis in Root Canals
Christine Sedgley, PhD, Aaron Nagel, BSc, Gunnar Dahlén, PhD, Claes Reit, PhD, Anders Molander, PhD
Reports on the prevalence of Enterococcus faecalis in root canals vary considerably, potentially because of variations in clinical sampling and sample analysis methods. This study compared culture and real-time quantitative polymerase chain reaction (qPCR) to detect and quantify E. faecalis in the same root canal sample. Consecutive root canal samples obtained from primary infection (n = 40) and retreatment (n = 48) cases were divided into two equal aliquots that were independently analyzed using culture and qPCR by investigators blinded to the analysis results of the other sample. E. faecalis was detected in 10.2% and 79.5% of samples by culture and qPCR, respectively (p < 0.0001; McNemar’s test). E. faecalis was detected in more retreatment than primary infection samples (89.6% versus 67.5%; p = 0.01, Fisher’s exact test). qPCR reported a significantly higher prevalence of E. faecalis in endodontic samples than culture techniques.
The Pulpal Origin of Immunoglobulins in Dentin Beneath Caries: An Immunohistochemical Study
Chin-Lo Hahn, PhD, DDS, A.M. Best, PhD
Immunoglobulins localized in uninfected dentin beneath caries are thought to be protective, but their origin remains controversial. We reasoned that the localization and dominance of serum IgG1 would support the pulpal origin of the immunoglobulins while a predominance of secretory component (SC) bearing IgA1 and IgA2 would support their salivary origin. The prevalence and staining intensity of IgG1, IgA1, IgA2, IgM, and SC in uninfected dentinal tubules beneath shallow, deep caries, and noncaries teeth were examined immunohistologically. SC was only localized in caries, and IgG1 was the predominant subclass in uninfected dentinal tubules beneath shallow and deep caries, followed by IgA1. In noncaries teeth, IgG1 was localized on the pulpal end. The intensity of IgG1 was significantly higher than either IgA1 or IgA2 in both shallow and deep caries. Our data support the serum origin of immunoglobulins in uninfected dentin beneath caries.
Quantification of Lactate-Dehydrogenase and Cell Viability in Postmortem Human Dental Pulp
Javier Caviedes-Bucheli, DDS, MSc, Nuvia Avendaño, DDS, Rhina Gutierrez, DDS, Sandra Hernández, DDS, Gloria Cristina Moreno, DDS, MSc, María Consuelo Romero, MSc, Hugo Roberto Muñoz, DDS, MSc
Understanding pulp repair and regeneration requires being familiar with this tissue’s behavior under extreme conditions, such as postmortem state where an abrupt interruption of tissue blood supply occurs. The purpose of this study was to quantify cell viability and the amount of lactate-dehydrogenase (LDH) expressed in human pulp tissue 6, 12, and 24 hours postmortem to establish how long dental pulp remains viable after death. Pulp samples were obtained from 14 unidentified corpses of people who had received lethal injuries in car accidents or from gunshot wounds; they had at least three caries- and restoration-free incisors. Half of each sample was used for determining cell viability at three different time intervals. The rest of each sample was used for quantifying LDH expression at the same time intervals. Another 14 pulp samples were obtained from live patients’ healthy premolars where extraction was indicated for orthodontic reasons to assess normal LDH value in pulp tissue. The results showed cell viability decreasing from 89 to 68 to 41% measured 6, 12, and 24 hours postmortem, respectively. LDH expression in healthy pulps was 246 U/mg pulp weight. Expression increased after death from 249 U/mg at 6 hours to 337 U/mg at 12 hours. LDH expression decreased to 131 U/mg 24 hours postmortem. These findings are valuable in understanding dental pulp survival capability under extreme conditions that may have important clinical significance in terms of repair and regeneration.
Interleukin-1 α Alters the Expression of Matrix Metalloproteinases and Collagen Degradation by Pulp Fibroblasts
Kessiri Wisithphrom, DDS, Peter E. Murray, PhD, L. Jack Windsor, PhD
Matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs) have been suggested to play a role in dental pulp destruction. This study examined the effects of interleukin (IL)-1 α on pulp fibroblasts. The ability of these cells to degrade collagen was determined with or without IL-1 α utilizing a cell-mediated collagen degradation assay. Reverse transcriptase-polymerase chain reaction was utilized to examine the mRNA expression of multiple MMPs and TIMPs with and without IL-1 α, while Western blot analyses and zymography were utilized to examine their protein expression. The collagen degradation mediated by these cells was stimulated by IL-1 α and inhibited by MMP inhibitors. IL-1 α increased the mRNA expression of MMP-1 and MMP-3, as well as induced MMP-7. Western blot analyses confirmed these results. IL-1 α increased the secreted protein level of TIMP-1, while only slightly affected the level of TIMP-2. These results suggest that IL-1 α can induce pulp destruction by differentially regulating MMPs and TIMPs.
Comparison of the Physical and Mechanical Properties of MTA and Portland Cement
Intekhab Islam, MSc, Hui Kheng Chng, MDSc, Adrian U. Jin Yap, PhD
This study evaluated and compared the pH, radiopacity, setting time, solubility, dimensional change, and compressive strength of ProRoot MTA (PMTA), ProRoot MTA (tooth colored formula) (WMTA), white Portland cement (WP), and ordinary Portland cement (OP). The results showed that PMTA and Portland cement have very similar physical properties. However, the radiopacity of Portland cement is much lower than that of PMTA. The compressive strength of PMTA was greater than Portland cement at 28 days. The major constituent of PMTA is Portland cement. Given the low cost of Portland cement and similar properties when compared to PMTA, it is reasonable to consider Portland cement as a possible substitute for PMTA in endodontic applications. However, industrially manufactured Portland cement is not approved currently for use in the United States and therefore no clinical recommendation can be made for its use in the human body. Further in vitro and in vivo tests, especially with regards its biocompatibility, should be conducted to ascertain if it meets the FDA requirements for use as a medical device.
Effects of pH on Mineralization Ability of Human Dental Pulp Cells
Tatsu Okabe, DDS, Maki Sakamoto, DDS, PhD, Hitomi Takeuchi, BD, Kiyoshi Matsushima, DDS, PhD
The purpose of this study was to investigate the effect of alkaline pH on calcification in human dental pulp (HDP) cells. HDP cells were cultured in pH 7.8 conditioned medium, and alkaline phosphatase (ALP) activity was measured. The ALP activity was higher in the pH 7.8 conditioned medium group than in the pH 7.2 conditioned medium group. Expression of mRNAs for bone morphogenetic protein (BMP)-2 was measured by the RT-PCR technique. The expression of BMP-2 in the pH 7.8 groups was greater than that in the pH 7.2 group. Furthermore, we determined Calcified nodule formation by von Kossa staining. The number of calcified nodules was increased in the pH 7.8 conditioned medium. These results suggest that HDP cell mineralization was enhanced in alkaline pH (pH 7.8) conditioned medium.
Residual Dentin Thickness in Bifurcated Maxillary Premolars After Root Canal and Dowel Space Preparation
Alexander Katz, DMD, Silvia Wasenstein-Kohn, DMD, Aviad Tamse, DMD, Ofer Zuckerman, DMD
Residual dentin thickness in 25 extracted bifurcated maxillary premolars after canal and Parapost preparation was assessed using acrylic muffle, after embedding in polyester resin and sectioned horizontally. After each procedure, the residual dentin thickness was compared to the original dentin thickness at eight aspects in three root levels. Residual dentin thickness measurements were calculated using photographs and a Digitizer. In the lingual aspect, the original dentin thickness averaged 0.99 mm (coronal slice) and 0.78 mm (middle slice). Dentin removal after dowel preparation was 31%. Three-way ANOVA with repeated measures was used. The difference in residual dentin thickness was highly significant regarding procedure (canal and dowel preparation, p < 0.0001), slice (middle, coronal, p < 0.0001), and the eight aspects (p < 0.0001). This in vitro study emphasizes the minimal dentin width of the buccal root, especially towards the bifurcation. Thus, dowels should be avoided in this root.
Capillary Flow Porometry to Assess the Seal Provided by Root-End Filling Materials in a Standardized and Reproducible Way
Mieke A.A. De Bruyne, DDS, Roger J.E. De Bruyne, MSc, PhD, Roeland J.G. De Moor, DDS, MSc, PhD
This in vitro study evaluated the root-end sealing ability of gutta-percha + AH26 (GP), Ketac-Fil, Fuji IX (FIX), tooth-colored MTA (MTA), IRM, Ketac-Fil + conditioner (Ketac-FilC), and Fuji IX + conditioner (FIXC). A total of 140 standardized bovine root sections were divided into seven groups, filled with the mentioned root-end filling materials, and, at 48 h, submitted to capillary flow porometry to assess minimum, mean flow and maximum pore diameters. Results were statistically analyzed using nonparametric Kruskal-Wallis and Dunn tests. Level of significance was set at 0.05. Using the Kruskal-Wallis tests we found that there was no significant difference between the minimum pore diameters of the different materials, but significant differences between the mean flow (p < 0.001) and maximum (p < 0.001) pore diameters could be demonstrated. For the mean flow pore diameters, there was a significant difference between FIX and all other materials, between Ketac-Fil and IRM and between Ketac-FilC and IRM. Concerning maximum pore diameters, there was a significant difference between FIX and all other materials, between Ketac-Fil and MTA, GP and IRM, FIXC and IRM, and Ketac-FilC and IRM. The data showed that each sample had leaked. Glass ionomer cements leaked more than other materials, although dentin conditioning diminished the maximum through pore diameters. This maximum pore diameter, which corresponds to the largest leak in the sample, together with the size of bacteria and their metabolites, will be indicative of the eventual leakage along the root-end filling materials.
Effects of Gray and White Mineral Trioxide Aggregate on the Proliferation of Oral Keratinocytes and Cementoblasts
Tiina Oviir, DDS, MS, Dustin Pagoria, Gabriela Ibarra, DDS, MS, Werner Geurtsen, DDS, PhD
Mineral trioxide aggregate (MTA) is used as a repair material and may directly contact cells from different cell lineages. The purpose of this study was to assess cell proliferation of immortalized Murine cementoblasts (OCCM.30) and immortalized keratinocytes (OKF6/TERT1) on gray MTA (GMTA) and white MTA (WMTA) with the DNA intercalating dye Hoechst 33342. Cells were grown for 72 hours on GMTA or WMTA that had been cured for 24 hours or 12 days. WMTA significantly (p < 0.001) increased the proliferation of OCCM.30 cementoblasts compared to control and OKF6/TERT1 keratinocytes. Both cell types grew significantly (p < 0.001) better on the surface of WMTA compared to GMTA. In addition, both cell types showed significantly (p < 0.005) higher proliferation when grown on 12-day-cured GMTA compared to 24-hour-cured GMTA.
Antibacterial Properties of Temporary Filling Materials
Hagay Slutzky, DMD, I. Slutzky-Goldberg, DMD, E.I. Weiss, DMD, S. Matalon, DMD
The purpose of this study was to investigate the antibacterial properties of temporary fillings. The direct contact test (DCT) was used to evaluate the antibacterial properties of Revoltek LC, Tempit, Systemp inlay, and IRM. These were tested in contact with Streptococcus mutans and Enterococcus faecalis. The materials were examined immediately after setting, 1, 7, 14, and 30 days after aging in phosphate buffered saline (PBS). Statistical analysis included two-way ANOVA, one-way ANOVA, and Tukey multiple comparison. Systemp inlay, Tempit, and IRM exhibited antibacterial properties when in contact with S. mutans for at least 7 days, Tempit and IRM sustained this ability for at least 14 days. When in contact with E. faecalis Tempit and IRM were antibacterial immediately after setting, IRM sustained this ability for at least 1 day. Our study suggests that the difference in temporary filling materials may influence which microorganism will be able to invade the root canal system.
Ultrastructure of Smear Layer-Covered Intraradicular Dentin After Irrigation with BioPure MTAD
Franklin R. Tay, BDSc (Hons), PhD, David H. Pashley, DMD, PhD, Robert J. Loushine, DDS, Michael D. Doyle, DDS, W. Trent Gillespie, DMD, MPH, R. Norman Weller, DMD, MS, Nigel M. King, BDS, MS, PhD
The structure of mechanically instrumented intraradicular dentin after irrigation with NaOCl as the initial rinse and BioPure MTAD as the final rinse were examined from the coronal, middle, and apical parts of root canal walls using transmission electron microscopy. Sterile distilled water and EDTA as final rinses were employed as the respective positive and negative controls under the same experimental conditions. There were 2 to 5 μm thick smear layers produced on mechanically instrumented root canal walls that were completely removed by EDTA and BioPure MTAD under agitation. Both irrigants created a zone of demineralized collagen matrices in eroded dentin and around the dentinal tubules, with the mildly acidic BioPure MTAD being more aggressive than EDTA. These demineralized dentin zones create the opportunity for dentin hybridization by infiltration of hydrophilic adhesives/sealers. However, the potential consequences of compaction of hydrophobic sealers against air-dried, collapsed collagen matrices, and hydrolytic degradation of incompletely infiltrated matrices remain unresolved.
A Comparative Photoelastic Stress Analysis of Internal Root Stresses between RC Prep and Saline When Applied to the Profile/GT Rotary Instrumentation System
Darin N. Anderson, DMD, Anthony P. Joyce, DDS, Steven Roberts, DDS, Royce Runner
The purpose of this study was to measure and compare the amount of stress produced by GT ProFile (GT) and ProFile (PF) rotary files lubricated with either RC Prep (RCP) or saline (S) on the walls of prepared simulated canals. Twenty-four 10-mm long canals with a 10 mm radius of curvature were prepared in PL-2 photoelastic acrylic blocks which were lubricated with either RCP or S. The files were inserted into a Nouvag handpiece mounted to an Instron 4502 universal testing machine and lowered into the canal at a rate of 60 mm/min and a rotational speed of 300 RPM. The following four GTs and four PFs were sequentially introduced into predetermined depths of each canal: 20/.10, 20/.08, 20/.06, 20/.04, 40/.04, 35/.04, 30/.04, and 25/.04. Total areas of stress created by each file were captured by digital video images and measured using Image J software. Statistical analysis of the data was performed using a two-way ANOVA (p < 0.05, n = 24). The GT/RCP group demonstrated a significant average of 64.22% less stress than GT/S group. The PF (25, 30, and 35)/ RCP groups demonstrated a significant average of 27.02% less stress than the same S groups. PF (40) produced a negligible amount of stress regardless of the lubricant type. The results reveal that using RCP as a lubricant when instrumenting curved canals with GT and PF results in less stress when compared to saline.
Sealing Ability of Three Materials in the Orifice of Root Canal Systems Obturated With Gutta-Percha
Stephen Jenkins, DDS, James Kulild, DDS, MS, Karen Williams, RDH, MS, PhD, William Lyons, Charles Lee, DDS
There were 130 single roots randomly assigned to one of 12 experimental or two control groups. Forty specimens each were sealed with 1, 2, 3, or 4 mm of Cavit, ProRoot MTA, or Tetric. After creation of a uniform orifice diameter, the smear layer was removed and the canal systems obturated using warm lateral compaction of gutta-percha (GP). GP was removed to the experimental depth, experimental materials placed in the orifice, and roots submerged in India ink in a vacuum flask. Specimens were demineralized and leakage measured using a 10× stereomicroscope and graded for depth of leakage by one calibrated, blinded rater. There was no significant interaction (p > 0.05) between test materials and orifice depths, nor main effect of orifice depth (p > 0.05). However, there was a statistically significant main effect of test materials with Tetric demonstrating a significantly better seal than Pro Root or Cavit (p < 0.0001) irrespective of orifice depth.
Conventional and Surgical Endodontic Retreatment of A Maxillary First Molar: Unusual Anatomy
I.-Ping Chen, DDS, Bekir Karabucak, DMD, MS
The prognosis for endodontic treatment in teeth exhibiting a complex anatomy is unfavorable if clinicians fail to recognize extra root canals. This case demonstrates the presence of a second canal in the disto-buccal root of a maxillary right first molar. Conventional retreatment followed by endodontic surgery was performed and a 1-year postoperative radiograph shows apparent periapical healing.