SCF/C-Kit Signaling Induces Self-Renewal of Dental Pulp Stem Cells.

INTRODUCTION: The maintenance of a stem cell pool is imperative to enable healing processes in the dental pulp tissue throughout life. As such, knowing mechanisms underlying stem cell self-renewal is critical to understand pulp pathophysiology and pulp regeneration. The purpose of this study was to evaluate the impact of stem cell factor (SCF) signaling through its receptor tyrosine kinase (c-Kit) on the self-renewal of human dental pulp stem cells (hDPSCs). METHODS: The hDPSCs were stably transduced with lentiviral vectors expressing shRNA-c-Kit or vector control. The impact of the SCF/c-Kit axis on hDPSC self-renewal was evaluated by using a pulpsphere assay in low attachment conditions and by evaluating the expression of polycomb complex protein Bmi-1 (master regulator of self-renewal) by Western blot and flow cytometry. RESULTS: The c-Kit-silenced hDPSCs formed fewer pulpspheres when compared with hDPSCs transduced with control vector (P < .05). Evaluation of pulpsphere morphology revealed the presence of 3 distinct sphere types, ie, holospheres, merospheres, and paraspheres. Although c-Kit silencing decreased the number of holospheres compared with control cells (P < .05), it had no effect on the number of merospheres and paraspheres. Recombinant human stem cell factor (rhSCF) increased the number of holospheres (P < .05) and induced dose-dependent Bmi-1 expression in hDPSCs. As expected, the inductive capacity of rhSCF on Bmi-1 expression and fraction of Bmi-1-positive cells was inhibited when we silenced c-Kit in hDPSCs. CONCLUSIONS: These results unveiled the role of SCF/c-Kit signaling on the self-renewal of hDPSCs and suggested that this pathway enables long-term maintenance of stem cell pools in human dental pulps.

Early life metal dysregulation in amyotrophic lateral sclerosis.

OBJECTIVE: Deficiencies and excess of essential elements and toxic metals are implicated in amyotrophic lateral sclerosis (ALS), but the age when metal dysregulation appears remains unknown. This study aims to determine whether metal uptake is dysregulated during childhood in individuals eventually diagnosed with ALS. METHODS: Laser ablation-inductively coupled plasma-mass spectrometry was used to obtain time series data of metal uptake using biomarkers in teeth from autopsies or dental extractions of ALS (n = 36) and control (n = 31) participants. Covariate data included sex, smoking, occupational exposures, and ALS family history. Case-control differences were identified in temporal profiles of metal uptake for individual metals using distributed lag models. Weighted quantile sum (WQS) regression was used for metals mixture analyses. Similar analyses were performed on an ALS mouse model to further verify the relevance of dysregulation of metals in ALS. RESULTS: Metal levels were higher in cases than in controls: 1.49 times for chromium (1.11-1.82; at 15 years), 1.82 times for manganese (1.34-2.46; at birth), 1.65 times for nickel (1.22-2.01; at 8 years), 2.46 times for tin (1.65-3.30; at 2 years), and 2.46 times for zinc (1.49-3.67; at 6 years). Co-exposure to 11 elements indicated that childhood metal dysregulation was associated with ALS. The mixture contribution of metals to disease outcome was likewise apparent in tooth biomarkers of an ALS mouse model, and differences in metal distribution were evident in ALS mouse brains compared to brains from littermate controls. INTERPRETATION: Overall, our study reveals direct evidence that altered metal uptake during specific early life time windows is associated with adult-onset ALS.

Dysregulated biodynamics in metabolic attractor systems precede the emergence of amyotrophic lateral sclerosis.

Evolutionarily conserved mechanisms maintain homeostasis of essential elements, and are believed to be highly time-variant. However, current approaches measure elemental biomarkers at a few discrete time-points, ignoring complex higher-order dynamical features. To study dynamical properties of elemental homeostasis, we apply laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) to tooth samples to generate 500 temporally sequential measurements of elemental concentrations from birth to 10 years. We applied dynamical system and Information Theory-based analyses to reveal the longest-known attractor system in mammalian biology underlying the metabolism of nutrient elements, and identify distinct and consistent transitions between stable and unstable states throughout development. Extending these dynamical features to disease prediction, we find that attractor topography of nutrient metabolism is altered in amyotrophic lateral sclerosis (ALS), as early as childhood, suggesting these pathways are involved in disease risk. Mechanistic analysis was undertaken in a transgenic mouse model of ALS, where we find similar marked disruptions in elemental attractor systems as in humans. Our results demonstrate the application of a phenomological analysis of dynamical systems underlying elemental metabolism, and emphasize the utility of these measures in characterizing risk of disease.

Deciphering Endodontic Microbial Communities by Next-generation Sequencing.

INTRODUCTION: Biofilms are present in more than 70% of endodontically diseased teeth. Through the advancements in the next-generation sequencing (NGS) technologies, microbiome research has granted a deeper analysis of the microbial communities living in human hosts. Here, we reviewed previous studies that used NGS to profile the microbial communities of root canals. METHODS: A total of 12 peer-reviewed articles from PubMed were identified and critically reviewed. The study criteria were as follows: NGS platforms, sequenced bacterial hypervariable regions, teeth diagnosis with available patient information, sample characteristics, collection method, and microbial signatures. RESULTS: The most common NGS platforms used were 454 pyrosequencing (Roche Diagnostic Corporation, Risch-Rotkreuz, Switzerland) and Illumina-based technology (Illumina Inc, San Diego, CA). The hypervariable regions sequenced were between the V1 and V6 regions. The patient and sample population ranged from ages 12-76 years and asymptomatic and symptomatic teeth diagnosed with pulp necrosis with or without apical periodontitis. Microbial sampling was conducted directly from the infected pulp or the extracted teeth. The most abundant phyla were Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Fusobacteria. The most frequently detected genera were Prevotella, Fusobacterium, Porphyromonas, Parvimonas, and Streptococcus. Other notable microbial signatures at different taxa levels were identified but were widely variable between studies. CONCLUSIONS: Technologies based on high-throughput 16S ribosomal RNA NGS can aid in deciphering the complex bacterial communities of root canal biofilms. Thus far, only a few studies have been published with relatively small sample sizes, variable sample collection protocols, and community analyses methods. Future larger clinical studies are essential with validated standardized protocols for improved understanding of the pathogenic nature of bacterial biofilm communities in root canals.

Efficacy of Articaine versus Lidocaine in Supplemental Infiltration for Mandibular First versus Second Molars with Irreversible Pulpitis: A Prospective, Randomized, Double-blind Clinical Trial.

INTRODUCTION: Profound pulpal anesthesia is difficult to achieve in mandibular molars with irreversible pulpitis (IP). However, there are no published randomized controlled clinical trials comparing the success of supplemental buccal infiltration (BI) in mandibular first versus second molars with IP. The purpose of this prospective, randomized, double-blind study was to compare the efficacy of 4% articaine with 2% lidocaine for supplemental BIs in mandibular first versus second molars with IP after a failed inferior alveolar nerve block (IANB). This study's sample was combined with data from a previous trial. METHODS: One hundred ninety-nine emergency subjects diagnosed with IP of a mandibular molar were selected and received an IANB with 4% articaine. Subjects who failed to achieve profound pulpal anesthesia, determined by a positive response to cold or pain upon access, randomly received 4% articaine or 2% lidocaine as a supplemental BI. Endodontic access was begun 5 minutes after infiltration. Success was defined as less than mild pain during endodontic access and instrumentation on the Heft-Parker visual analog scale. RESULTS: There was a 25% IANB success rate with 4% articaine. The success rate for articaine supplemental BI in first molars was 61% versus 63% for second molars (P > .05). The success of lidocaine in first molars was 66%, but for second molars it was 32% (P = .004). CONCLUSIONS: The success rate for IANB with 4% articaine was 25%. Articaine and lidocaine had similar success rates for supplemental infiltration in first molars, whereas articaine was significantly more successful for second molars. However, because BI often did not provide profound pulpal anesthesia, additional techniques including intraosseous anesthesia may still be required.

Mineral Trioxide Aggregate and Diluted Formocresol Pulpotomy: Prospective and Retrospective Study Outcomes.

Mineral Trioxide Aggregate (MTA) has been used in many endodontic procedures with success. We hypothesized that MTA as a pulpotomy medicament elicits outcomes no different than that of the diluted formocresol (DFC). PURPOSE: The purpose of this study was to compare the outcomes of grey MTA and DFC in primary molar pulpotomies at a teaching institution and a pediatric dental practice. METHODS: At the teaching institution, 206 primary molars of 122 children were enrolled. At 48-months, 20 teeth treated with MTA and 25 teeth treated with DFC, were available for evaluation. At the private practice, dental records of 245 primary molars of 68 patients were available for evaluation. RESULTS: At 48 months, the results from both sites showed a radiographic success rate of 80 percent for DFC and 95 percent for MTA. The odds of radiographic failure were not affected by study sites. The Cox-regression analysis revealed that patient's age at the time of pulpotomy impacted the "hazard of exfoliation." Each year following the completion of DFC or MTA pulpotomy, there is a 4.6-times-more-likely chance for early exfoliation of the pulpotomized tooth. CONCLUSION: Grey MTA is an acceptable alternative for primary molar pulpotomies.

Clinical Evidence for Regenerative Endodontic Procedures: Immediate versus Delayed Induction?

Clinicians face many challenges when treating immature permanent teeth in young patients. Immediate blood clot induction can be a successful option as described by some case reports. No experimental studies or clinical trials have addressed this question. We have designed a clinical trial in which we hypothesized that there is no difference in success between immediate or delayed induction protocols. After confirmation of pulpal necrosis, patients were randomized. In the delayed group, 15 teeth were treated following the American Association of Endodontists guidelines, and calcium hydroxide was used as the intracanal medication. In the immediate group, 13 teeth had a blood clot inducted at the first appointment. The teeth were evaluated after 1, 3, and 12 months. Three independent evaluators assessed the periapical healing. The Pearson chi-square test or the Fisher exact test was used to compare the success rates between the 2 groups. Currently, of the 25 recruited patients (28 teeth), 19 have completed their 12-month follow-up. The group with delayed induction had a 71% success rate, and the group with immediate induction had a 33% success rate. In most cases (79%), trauma was the etiology. All successful cases started at stage 9 of root development (Nolla), and the majority showed healing type 2. Determination of the stage of root formation and etiology are possible critical factors for any therapeutic decision. In summary, it is early to conclude or suggest any of the protocols. Clearly, much more data are needed before sample size requirements can be met.

In Patients With Symptomatic Irreversible Pulpitis, Articaine is 3.6 Times More Efficacious Than Lidocaine in Achieving Anesthetic Success When Used for Supplementary Infiltration After Mandibular Block Anesthesia.

ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION: Does articaine provide an advantage over lidocaine in patients with symptomatic irreversible pulpitis? A systematic review and meta-analysis. Kung J, McDonagh M, Sedgley CM. J Endod 2015; 41(11):1784-94. SOURCE OF FUNDING: The study was supported by the OHSU Department of Endodontology Les Morgan Endowment Fund and a resident research grant from the American Association of Endodontists Foundation TYPE OF STUDY/DESIGN: Systematic review with meta-analysis of data.

White mineral trioxide aggregate induces migration and proliferation of stem cells from the apical papilla.

INTRODUCTION: Regenerative endodontic protocols recommend white mineral trioxide aggregate (WMTA) as a capping material because of its osteoinductive properties. Stem cells from the apical papilla (SCAP) are presumed to be involved in this regenerative process, but the effects of WMTA on SCAP are largely unknown. Our hypothesis was that WMTA induces proliferation and migration of SCAP. METHODS: Here we used an unsorted population of SCAP (passages 3-5) characterized by high CD24, CD146, and Stro-1 expression. The effect of WMTA on SCAP migration was assessed by using transwells, and its effect on proliferation was determined by the WST-1 assay. Fetal bovine serum (FBS) and calcium chloride-enriched medium were used as positive controls. RESULTS: The SCAP analyzed here showed a low percentage of STRO-1+ and CD24+ cells. Both set and unset WMTA significantly increased the short-term migration of SCAP after 6 hours (P < .05), whereas calcium chloride-enriched medium did after 24 hours of exposure. Set WMTA significantly increased proliferation on days 1-5, whereas calcium-enriched medium showed a significant increase on day 7, with a significant reduction on proliferation afterwards. SCAP migration and proliferation were significantly and steadily induced by the presence of 2% and 10% FBS. CONCLUSIONS: Collectively, these data demonstrate that WMTA induced an early short-term migration and proliferation of a mixed population of stem cells from apical papilla as compared with a later and longer-term induction by calcium chloride or FBS.

Efficacy of articaine versus lidocaine as a supplemental buccal infiltration in mandibular molars with irreversible pulpitis: a prospective, randomized, double-blind study.

INTRODUCTION: Profound pulpal anesthesia in mandibular molars with irreversible pulpitis (IP) is often difficult to obtain and often requires supplemental injections after an ineffective inferior alveolar nerve block (IANB). The purpose of this prospective, randomized, double-blind study was to compare the efficacy of 4% articaine with 2% lidocaine for supplemental buccal infiltrations (BIs) after an ineffective IANB in mandibular molars with IP. In addition, the use of articaine for IANB and intraosseous injections was investigated. METHODS: One hundred emergency patients diagnosed with IP of a mandibular molar were selected and received an IANB with 4% articaine. All injections were 1.7 mL with 1:100,000 epinephrine. All patients reported profound lip numbness after IANB. Patients with ineffective IANB (positive pulpal response to cold or pain on access) randomly received 4% articaine or 2% lidocaine as a supplemental BI. Endodontic access was initiated 5 minutes after deposition of the infiltration solution. Success was defined as no pain or no more than mild pain during endodontic access and instrumentation as measured on a visual analogue scale. RESULTS: Seventy-four patients failed to achieve pulpal anesthesia after IANB with 4% articaine, resulting in IANB success rate of 26%. Success rates for supplemental BIs were 62% for articaine and 37% for lidocaine (P < .05). This effect was most pronounced in second molars (P < .05). CONCLUSIONS: Supplemental BI with articaine was significantly more effective than lidocaine. The IANB success rate of 4% articaine confirmed published data.

Comparison of mineral trioxide aggregate and diluted formocresol in pulpotomized human primary molars: 42-month follow-up and survival analysis.

PURPOSE: The purpose of this study was to test the hypothesis that there is no significant difference in the clinical and radiographic outcomes of diluted formocresol (DFC) compared to gray mineral trioxide aggregate (GMTA) pulpotomy in human primary molars. METHODS: A total of 152 children with 252 primary molars met selection criteria. Of those, 119 and 133 teeth were randomly assigned to the GMTA and DFC groups, respectively. Periapical radiographs, taken pre- and/or postoperatively and at each 6-month follow-up, were digitized and evaluated by three blinded and calibrated examiners. RESULTS: Over a 42-month period, a total of 865 clinical and radiographic evaluations were conducted. There was no significant difference in clinical success, with the cumulative proportion of GMTA-treated teeth surviving at 0.98 vs DFC-treated teeth at 0.95 (P>.05). Radiographic success, however, was significantly greater for GMTA vs DFC, with the cumulative proportion of GMTA-treated teeth surviving at 0.90 vs DFC-treated teeth at 0.47 (P<.001). Overall, DFC-treated teeth were 5.1 times more likely to fail than GMTA-treated teeth. Radiographic pathologies were observed more frequently in the DFC-treated teeth (P<.05). CONCLUSION: Gray mineral trioxide aggregate can be considered an acceptable replacement for diluted formocresol when used as a medicament for primary molar pulpotomies.

Comparison of gray mineral trioxide aggregate and diluted formocresol in pulpotomized primary molars: a 6- to 24-month observation.

PURPOSE: The purpose of this multisite, multioperator, prospective, randomized, controlled clinical trial was to evaluate 2-year outcomes of diluted formocresol (DFC) compared to gray mineral trioxide aggregate (GMTA) as pulpotomy medicaments. METHODS: Following the standard pulpotomy procedure, the pulp stumps of 252 primary molars in 168 healthy children were randomly covered with GMTA or DFC. Pulp chambers were filled with Intermediate Restorative Material (IRM(®)) and teeth were restored with stainless steel crowns. At each follow-up appointment, the clinical status of the treated tooth was assessed and radiographs were taken. A total of 694 clinical and radiographic evaluations were analyzed. RESULTS: Gender, study site, arch type, and tooth type did not influence treatment outcome. At the combined 6- to 24-month follow-up, clinical success in the DFC group was no different than for the GMTA group. Radiographically, a significantly lower success rate was found in the DFC group vs the MTA group at all time points (P<.01). Dentin bridge formation was observed at a significantly higher frequency among the GMTA group (P<.01), while internal root resorption was observed at a higher frequency in the DFC group (P<.01). CONCLUSION: At the combined 6- to 24-month follow-up, gray mineral trioxide aggregate demonstrated significantly better radiographic outcomes vs diluted formocresol as pulpotomy medicaments.

Dental stem cells and their sources.

The search for more accessible mesenchymal stem cells than those found in bone marrow has propelled interest in dental tissues. Human dental stem/progenitor cells (collectively termed dental stem cells [DSCs]) that have been isolated and characterized include dental pulp stem cells, stem cells from exfoliated deciduous teeth, stem cells from apical papilla, periodontal ligament stem cells, and dental follicle progenitor cells. Common characteristics of these cell populations are the capacity for self-renewal and the ability to differentiate into multiple lineages. In vitro and animal studies have shown that DSCs can differentiate into osseous, odontogenic, adipose, endothelial, and neural-like tissues.

Regenerative endodontics in light of the stem cell paradigm.

Stem cells play a critical role in development and in tissue regeneration. The dental pulp contains a small sub-population of stem cells that are involved in the response of the pulp to caries progression. Specifically, stem cells replace odontoblasts that have undergone cell death as a consequence of the cariogenic challenge. Stem cells also secrete factors that have the potential to enhance pulp vascularisation and provide the oxygen and nutrients required for the dentinogenic response that is typically observed in teeth with deep caries. However, the same angiogenic factors that are required for dentine regeneration may ultimately contribute to the demise of the pulp by enhancing vascular permeability and interstitial pressure. Recent studies focused on the biology of dental pulp stem cells revealed that the multipotency and angiogenic capacity of these cells could be exploited therapeutically in dental pulp tissue engineering. Collectively, these findings suggest new treatment paradigms in the field of endodontics. The goal of this review is to discuss the potential impact of dental pulp stem cells to regenerative endodontics.

Comparing gray mineral trioxide aggregate and diluted formocresol in pulpotomized human primary molars.

PURPOSE: The purpose of this multisite, multioperator, prospective, randomized, controlled clinical trial was to evaluate the 6-month outcomes of diluted formocresol (DFC) compared to gray mineral trioxide aggregate (GMTA) as pulpotomy medicament. METHODS: Determined by a power analysis, 252 molars of 152 children were recruited. The teeth were randomly assigned to receive GMTA or DFC. At the 6-month follow-up, 118 children with 203 treated teeth were evaluated. RESULTS: Four blinded and calibrated evaluators scored each radiograph for pathologies. Clinical success was similar for DFC (97%) and GMTA (100%), (P<.09). Radiographic success differed significantly (P<.04) for DFC (86%) and GMTA (95%). Pulp canal obliteration was radiographically observed in 25% of the DFC group and in 37% of the GMTA group (P=.07). Dentin bridging was observed in 22% of the GMTA group but was not found in the DFC group (P<.01). CONCLUSION: Teeth treated with GMTA showed more favorable radiographic outcomes than DFC at 6 months post-treatment.

TLR4 mediates LPS-induced VEGF expression in odontoblasts.

Lipopolysaccharide (LPS) from gram-negative bacteria cell walls such as Prevotella intermedia and Escherichia coli induce vascular endothelial growth factor (VEGF) expression in odontoblasts, but not in undifferentiated dental pulp cells. CD14 and TLR4 are responsible for LPS signaling in macrophages, but their expression levels and function in dental pulp cells are unknown. We showed here that murine odontoblast-like cells (MDPC-23) express CD14 and TLR4 by immunohistochemistry and flow cytometry. In contrast, undifferentiated dental pulp cells (OD-21) presented low or no expression of these two receptors. MDPC-23 cells showed CD14 and TLR4 up-regulation upon exposure to LPS, as determined by real time PCR. Dominant negative murine TLR4 (DN-mTLR4) transfected MDPC-23 cells did not show upregulated VEGF expression in response to LPS stimulation. These results demonstrate that odontoblast-like cells express CD14 and TLR4, and that LPS-induced VEGF expression is mediated, at least in part, by TLR4 signaling.

Effect of ProRoot MTA on pulp cell apoptosis and proliferation in vitro.

ProRoot Mineral Trioxide Aggregate (MTA) has been indicated as a pulp capping material. The purpose of this study was to evaluate the effect of tooth-colored (white) MTA on pulp cell apoptosis and cell cycle. Mouse odontoblast-like cells (MDPC-23) and undifferentiated pulp cells (OD-21) were exposed to 0 to 100 mg MTA for 24 h. Propidium iodide staining followed by flow cytometry demonstrated that MTA did not induce apoptosis of MDPC-23 or OD-21 (p > 0.05). Cell cycle analysis showed that MTA induced a modest (but significant) increase in the percentage of MDPC-23 in the S and G2 phases, and OD-21 in the S phase of cell cycle, as compared to untreated controls (p

Effect of lipopolysaccharides on vascular endothelial growth factor expression in mouse pulp cells and macrophages.

Vascular endothelial growth factor (VEGF), a potent pro-angiogenic factor, might regulate the neovascularization observed in the pulp of teeth with deep caries. The purpose of this in vitro study was to evaluate the effect of bacterial lipopolysaccharides (LPS) on VEGF expression in dental pulp cells. Mouse odontoblast-like cells (MDPC-23) or undifferentiated pulp cells (OD-21) were exposed to 0-20 microg ml-1Escherichia coli LPS or 0-80 microg ml-1Prevotella intermedia LPS. As controls, mouse macrophages or gingival fibroblasts were exposed to LPS, since these cells are known to secrete VEGF. The VEGF expression was evaluated by reverse transcriptase polymerase chain reaction or enzyme-linked immunosorbent assay. The baseline expression levels of VEGF protein were higher in MDPC-23 and OD-21 than in fibroblasts or macrophages. Vascular endothelial growth factor protein expression was upregulated in MDPC-23 and macrophages exposed to E. coli LPS, but not in OD-21 cells or fibroblasts. Higher concentrations of P. intermedia LPS were required to induce VEGF expression in MDPC-23 cells. Treatment with LPS did not affect VEGF expression at the mRNA level in any of the cells evaluated. These results demonstrate that bacterial LPS upregulates VEGF expression in odontoblast-like cells and macrophages, and suggest that the regulation of VEGF expression occurs primarily at a post-transcriptional level.