Lipopolysaccharide-Induced TRPA1 Upregulation in Trigeminal Neurons is Dependent on TLR4 and Vesicular Exocytosis.

Pain from bacterial infection was believed to be the consequence of inflammation induced by bacterial products. However recent studies have shown that bacterial products can directly activate sensory neurons and induce pain. The mechanisms by which bacteria induce pain are poorly understood, but toll-like receptor (TLR)4 and transient receptor potential A1 (TRPA1) receptors are likely important integrators of pain signaling induced by bacteria. Using male and female mice we show that sensory neuron activation by bacterial lipopolysaccharides (LPS) is mediated by both TRPA1 and TLR4 and involves the mobilization of extracellular and intracellular calcium. We also show that LPS induces neuronal sensitization in a process dependent on TLR4 receptors. Moreover, we show that TLR4 and TRPA1 are both involved in sensory neurons response to LPS stimulation. Activation of TLR4 in a subset of sensory neurons induces TRPA1 upregulation at the cell membrane through vesicular exocytosis, contributing to the initiation of neuronal sensitization and pain. Collectively these data highlight the importance of sensory neurons to pathogen detection, and their activation by bacterial products like LPS as potentially important to early immune and nociceptive responses.SIGNIFICANCE STATEMENT Bacterial infections are often painful and the recent discovery that bacteria can directly stimulate sensory neurons leading to pain sensation and modulation of immune system have highlighted the importance of nervous system in the response to bacterial infection. Here, we showed that lipopolysaccharide, a major bacterial by-product, requires both toll-like receptor (TLR)4 and transient receptor potential A1 (TRPA1) receptors for neuronal activation and acute spontaneous pain, but only TLR4 mediates sensory neurons sensitization. Moreover, we showed for the first time that TLR4 sensitize sensory neurons through a rapid upregulation of TRPA1 via vesicular exocytosis. Our data highlight the importance of sensory neurons to pathogen detection and suggests that TLR4 would be a potential therapeutic target to modulate early stage of bacteria-induced pain and immune response.

Excess Growth Hormone Triggers Inflammation-Associated Arthropathy, Subchondral Bone Loss, and Arthralgia.

Growth hormone (GH) is a key mediator of skeletal growth. In humans, excess GH secretion due to pituitary adenoma, seen in patients with acromegaly, results in severe arthropathies. This study investigated the effects of long-term excess GH on the knee joint tissues. One year-old wild-type (WT) and bovine GH (bGH) transgenic mice were used as a model for excess GH. bGH mice showed increased sensitivity to mechanical and thermal stimuli, compared with WT mice. Micro-computed tomography analyses of the distal femur subchondral bone revealed significant reductions in trabecular thickness and significantly reduced bone mineral density of the tibial subchondral bone-plate associated with increased osteoclast activity in both male and female bGH compared with WT mice. bGH mice showed severe loss of matrix from the articular cartilage, osteophytosis, synovitis, and ectopic chondrogenesis. Articular cartilage loss in the bGH mice was associated with elevated markers of inflammation and chondrocyte hypertrophy. Finally, hyperplasia of synovial cells was associated with increased expression of Ki-67 and diminished p53 levels in the synovium of bGH mice. Unlike the low-grade inflammation seen in primary osteoarthritis, arthropathy caused by excess GH affects all joint tissues and triggers severe inflammatory response. Data from this study suggest that treatment of acromegalic arthropathy should involve inhibition of ectopic chondrogenesis and chondrocyte hypertrophy.

Neuronal-immune axis alters pain and sensory afferent damage during dental pulp injury.

ABSTRACT: Dental pulp tissue is densely innervated by afferent fibers of the trigeminal ganglion. When bacteria cause dental decay near the pulpal tissue, a strong neuronal and immune response occurs, creating pulpitis, which is associated with severe pain and pulp tissue damage. Neuroimmune interactions have the potential to modulate both the pain and pathological outcome of pulpitis. We first investigated the role of the neuropeptide calcitonin gene-related peptide (CGRP), released from peptidergic sensory afferents, in dental pain and immune responses by using Calca knockout (Calca -/- ) and wild-type (Calca +/+ ) mice, in a model of pulpitis by creating a mechanical exposure of the dental pulp horn. We found that the neuropeptide CGRP, facilitated the recruitment of myeloid cells into the pulp while also increasing spontaneous pain-like behavior 20% to 25% at an early time point. Moreover, when we depleted neutrophils and monocytes, we found that there was 20% to 30% more sensory afferent loss and increased presence of bacteria in deeper parts of the tissue, whereas there was a significant reduction in mechanical pain response scores compared with the control group at a later time point. Overall, we showed that there is a crosstalk between peptidergic neurons and neutrophils in the pulp, modulating the pain and inflammatory outcomes of the disease.

Radiographic Evaluation of Regenerative Endodontic Procedures and Apexification Treatments with the Assessment of External Root Resorption.

INTRODUCTION: This multi-centered cohort study evaluated the radiographic outcomes of regenerative endodontic procedures (REPs) and apexification treatments (APEX) of immature teeth with endodontic disease. MATERIALS AND METHODS: This cohort study included a retrospective record review and prospective data collection of pediatric patients with teeth treated with REPs or APEX between 2005-2014. Data including the presence of a periapical lesion, external root resorption (ERR), obliteration, apical hard tissue, apical closure, intracanal calcifications, and radiographic root area (RRA) change based on measurements were collected/measured from radiographic images. Univariate and multivariate analyses were conducted. RESULTS: The cohort included 190 subjects (204 teeth (92 REPs; 112 APEX)). The frequency of pre-treatment periapical pathology was similar between cases in which the clinical treatment failed versus successful treatment cases. However, the frequency of pre-treatment ERR was higher in failed cases than in successful cases (p=0.007). The mean RRA change was greater than twenty percent in 21% of the REPs cases. In traumatized teeth, REP treatment resulted in less hard tissue formation than other endodontic disease etiologies measured by RRA (p=0.001). 53% of cases with ERR (16/30) showed signs of healing/arrest and were mostly treated with REPs (11/16). CONCLUSIONS: The presence of ERR negatively affected the treatment outcome. There was significant variability in RRA change in REPs. Signs of healing/arrest of the resorptive lesion were radiographically visible in many cases treated with REPs.

Dynamics of Innate Immune Response in Bacteria-Induced Mouse Model of Pulpitis.

INTRODUCTION: During pulpitis, as bacteria penetrate deeper into the dentin and pulp tissue, a pulpal innate immune response is initiated. However, the kinetics of the immune response, how this relates to bacterial infiltration during pulpitis and an understanding of the types of immune cells in the pulp is limited. METHODS: Dental pulp exposure in the molars of mice was used as an animal model of pulpitis. To investigate the kinetics of immune response, pulp tissue was collected from permanent molars at different time points after injury (baseline, day 1, and day 7). Flow cytometry analysis of CD45+ leukocytes, including macrophages, neutrophils monocytes, and T cells, was performed. 16S in situ hybridization captured bacterial invasion of the pulp, and immunohistochemistry for F4/80 investigated spatial and morphological changes of macrophages during pulpitis. Data were analyzed using two-way ANOVA with Tukey's multiple comparisons. RESULTS: Bacteria mostly remained close to the injury site, with some expansion towards noninjured pulp horns. We found that F4/80+ macrophages were the primary immune cell population in the healthy pulp. Upon injury, CD11b + Ly6Ghigh neutrophils and CD11b + Ly6GintLy6Cint monocytes constituted 70-90% of all immune populations up to 7 days after injury. Even though there was a slight increase in T cells at day 7, myeloid cells remained the main drivers of the immune response during the seven-day time period. CONCLUSIONS: As bacteria proliferate within the pulp chamber, innate immune cells, including macrophages, neutrophils, and monocytes, predominate as the major immune populations, with some signs of transitioning to an adaptive immune response.

Elevated Cytokine Levels in Gingival Crevicular Fluid of Teeth with Apical Periodontitis.

INTRODUCTION: Biomarkers assayed from gingival crevicular fluid (GCF) are a potential tool for endodontic diagnosis and for monitoring treatment response. This cross-sectional study measured cytokines in GCF from teeth with apical periodontitis and evaluated their relationship with preoperative pain and other clinical findings. METHODS: Participants presenting for root-end resection surgery due to apical periodontitis diagnosis (n = 56) underwent standardized clinical testing and completed preoperative questionnaires. GCF from diseased and control teeth were collected, processed, and analyzed. Mann-Whitney U and Wilcoxon tests were used to examine the cytokine levels in diseased compared to healthy control teeth. We also assessed the relationship of cytokine levels with clinical findings. RESULTS: Interleukins (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-γ, and tumor necrosis factor-⍺ (TNF-⍺) were detected in GCF. TNF-⍺ levels were significantly higher in GCF collected from diseased versus control teeth (P = .02) and increased IL-1ß levels in diseased teeth were detected (P = .06). Lower IL-10 levels were observed in teeth with a sinus tract and/or swelling compared to teeth without a sinus tract and/or swelling (P = .08). Cytokine levels did not clearly relate to the presence of pain. CONCLUSIONS: Elevated levels of proinflammatory cytokines, including TNF-⍺ and IL1- ß, were detected in GCF from diseased teeth compared to the healthy controls. Additional studies are needed to further investigate the utility of these biomarkers for objectively evaluating periradicular pathology.

The value of the International Association for the Study of Pain to career development: perspectives of trainee and early career members.

ABSTRACT: Supporting its young members has been a key priority of the International Association for the Study of Pain (IASP) for the past 5 decades. The IASP, along with its federations, chapters, and special interest groups, has provided benefits to its trainee and early career members for their career development. This article summarizes various key IASP initiatives and benefits offered to IASP members and how these benefits have positively impacted their careers, including examples from the authors of this article. Suggestions are made for future directions that the IASP could implement to enhance the value provided to its trainee and early career members, which will in turn contribute to IASP achieving its mission to stimulate and support the study of pain and to translate that knowledge into improved pain relief worldwide.

Contribution of Endodontic Pathology to Persistent Orofacial Pain: A Case Report.

Identifying the etiology and correct diagnoses for long-standing orofacial pain can be very challenging, especially in patients who have both odontogenic and nonodontogenic pain. This case report describes the successful management of a complex case of chronic orofacial pain in a patient with nonodontogenic chronic pain conditions and a maxillary molar tooth with persistent periapical pathology after endodontic treatment. The debilitating orofacial pain began after initial nonsurgical root canal treatment of the maxillary molar 3 years before presenting to our clinic. The initial clinical and radiographic assessment by our multidisciplinary team found that there were potentially both peripheral endodontic pathology and central pain mechanisms contributing to the long-standing pain. The diagnosis was shared with the patient's neurologist, who prescribed gabapentin, a centrally acting analgesic, and partial pain reduction was achieved. The odontogenic component of the orofacial pain was then addressed, by treating the persistent periapical infection and buccal bone fenestration of the roots of the maxillary molar. Treatments included both nonsurgical retreatment and surgical endodontic therapy (including root resection, root-end preparation, and retrofilling), and each significantly improved the patient's ongoing orofacial pain. After the successful endodontic treatments, the patient reported minimal pain and normal oral function. The case report highlights the importance of systematically treating endodontic pathology in a patient with long-standing orofacial pain, with both odontogenic and nonodontogenic components.

Outcomes of referrals from endodontic to orofacial pain specialists: A retrospective cohort study.

OBJECTIVES: Diagnosis and treatment of non-odontogenic pain is challenging for endodontists. The purpose of the study was to investigate the outcomes of referrals to orofacial pain specialists made for patients with suspected non-odontogenic pain, after evaluation and/or treatment by an endodontist. MATERIALS AND METHODS: A retrospective review of dental records was conducted for 60 patients referred from a postgraduate endodontic clinic to an orofacial pain clinic. Patient demographics, pain history, endodontic, and orofacial pain diagnoses were collected. Number of visits, length of treatment, and treatments prescribed were recorded. For analysis of outcomes, data pertinent to resolution/persistence of symptoms and patient compliance were analyzed. RESULTS: Thirty-five patients were included in the study. The most frequent pulpal and periapical diagnoses were previously treated (62%) and symptomatic apical periodontitis (72%), respectively. The most common orofacial pain diagnosis was temporomandibular disorder. The average time spent to diagnose and treat the pain was 17 months. Pain reduction varied and was documented for 51% of patients. Indications of non-compliance with orofacial pain appointments and treatments were documented for 66% of patients. CONCLUSIONS: Non-odontogenic pain diagnosis and treatment are challenging. Patients may have an increased predilection for developing persistent pain after endodontic treatment and/or have an undiagnosed, chronic orofacial pain condition as a true source of their chief complaint. It may be helpful for endodontists to set expectations of typical treatment times/plans when referring patients for evaluation and treatment of non-odontogenic pain.

Patient Centered Outcomes among a Cohort Receiving Regenerative Endodontic Procedures or Apexification Treatments.

INTRODUCTION: This multicentered cohort study evaluated factors associated with patient-centered outcomes of immature permanent teeth that received regenerative endodontic procedures (REPs) or apexification treatment (APEX). METHODS: A record review identified teeth treated with REPs or APEX between September 2005 and December 2014. Data regarding treatment and patient-centered outcomes were extracted from records with a 3-month minimum recall. When possible, participants presented for an in-person prospective research visit. Patient-centered success was defined as an asymptomatic, functional tooth not requiring further endodontic or surgical intervention after completion of the original treatment during the study observation. Risk ratios and adjusted and unadjusted Cox proportional hazard ratios were calculated. RESULTS: The analytic cohort of 187 individuals included 211 teeth (93 REPs and 118 APEX) with an average follow-up of 32 months. Most cases were successful (81% REPs and 92% APEX) and survived the observation period (96% REPs and 97% APEX). The success rate of REPs was lower than APEX and decreased more rapidly over time. Cox regression analysis demonstrated that when controlling for other variables, the association between treatment type and outcome is not significant. Preoperative infection, teeth with more immature roots, and REP treatment are potentially important predictors. Among teeth receiving REPs, a lower failure rate was observed for teeth that received multiantibiotic paste (3/43) compared with calcium hydroxide (11/45). CONCLUSIONS: Teeth receiving REPs required clinical intervention earlier than teeth that received APEX treatment, although a preoperative abscess and more immature root also affected this outcome. Using multiantibiotic paste versus calcium hydroxide in REPs may improve success.

Associations between Pain Severity, Clinical Findings, and Endodontic Disease: A Cross-Sectional Study.

INTRODUCTION: Thorough pain assessment and thermal and mechanical testing are the primary diagnostic tools used to assess the status of pulp and periapical tissues in teeth with potential endodontic pathology. This study evaluated predictors of acute odontogenic pain to better understand the relationship between endodontic pain, clinical testing, endodontic disease, and diagnoses. METHODS: Participants (N = 228) presenting with acute odontogenic pain underwent standardized clinical testing and reported their pain intensity. Univariate and multiple regression analyses were performed to evaluate the predictors of acute endodontic pain. Chi-square tests with Bonferroni adjustments were conducted to measure the frequency of endodontic diagnostic test findings and clinical observations in patients with different pulpal diagnoses. RESULTS: A negative response to cold stimulation on the causative tooth and percussion hypersensitivity on the healthy adjacent tooth were the strongest predictors of higher levels of acute endodontic pain. Percussion hypersensitivity on the healthy adjacent tooth was present in a quarter of the cohort and was reported with equal frequency in teeth diagnosed with irreversible pulpitis, necrotic pulp, and previously initiated/treated teeth. Although painful percussion on the causative tooth was more frequently reported in teeth diagnosed with necrotic pulp, painful palpation was more frequently reported on teeth diagnosed with previously initiated/treated teeth. CONCLUSIONS: Percussion hypersensitivity on the healthy adjacent tooth may reveal a lowered pain threshold and heightened pain sensitization. It is also possible that the 2 commonly performed mechanical sensory tests, percussion and palpation hypersensitivity, may detect different aspects of endodontic pathophysiology and pain processing.

Effects of Calcitonin Gene-related Peptide on Dental Pulp Stem Cell Viability, Proliferation, and Differentiation.

INTRODUCTION: Pulpitis is an inflammation of dental pulp caused by bacterial proliferation near or within pulpal tissues. In advanced stages, when the inflammation is associated with pulp necrosis, pulp preservation is dependent on dental pulp stem cells (DPSCs) that can differentiate into odontoblastlike cells and produce reparative dentin. In this study, we evaluated the influence of sensory neurons through calcitonin gene-related peptide (CGRP) on DPSC viability and proliferation and the ability of DPSCs to differentiate into mineralizing cells. METHODS: Commercially available DPSCs were treated with varying doses of CGRP, and metabolic activity, viability, proliferation, and cell death were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays, trypan blue staining, 5-bromo-2'-deoxyuridine cell proliferation assay, and caspase-3 staining, respectively. DPSC differentiation was assessed with alizarin red staining and by quantifying messenger RNA expression of odontoblast makers. RESULTS: CGRP induced a dose-dependent decrease of DPSC metabolic activity that was prevented by the CGRP receptor antagonist CGRP 8-37. The decrease in the proportion of live cells induced by CGRP is associated with a decrease of cell proliferation but not with caspase-3-dependent apoptosis. Interestingly, dexamethasone-induced DPSC differentiation into mineralizing cells was neither inhibited nor enhanced by CGRP treatment. CONCLUSIONS: The neuropeptide CGRP has an inhibitory effect on DPSC proliferation but does not enhance or inhibit the differentiation of DPSCs into mineralizing cells. This suggests that CGRP might negatively influence the ability of DPSCs to contribute to regenerative or tissue repair processes.

Sensory testing associates with pain quality descriptors during acute dental pain.

BACKGROUND: Pain descriptors capture the multidimensional nature of pain and can elucidate underlying pathophysiological mechanisms. This study determined whether the pain descriptors chosen by subjects experiencing acute dental pain associate with the outcomes of two commonly performed dental sensory tests. The goal of the study is to clarify whether pain descriptors are useful in discriminating the underlying biological processes contributing to dental pain. METHODS: Participants (n = 228) presenting with acute toothache underwent standardized clinical dental sensory testing and described their pain in reference to 22 pain quality descriptors. Univariate and two-way ANOVA determined the relationship between groups defined by cold detection (positive or negative) and percussion hypersensitivity (painful or not) on the affected tooth, and pain descriptor reporting. RESULTS: Subjects experiencing painful toothache most frequently reported evoked pain to temperature and chewing, and pain descriptors such as "throbbing" and "aching." They also reported neuropathic pain descriptors such as "tingling" and "electric shock." Subjects who detected a cold stimulus (thermal) on the affected tooth, frequently reported high intensity paroxysmal shooting pain compared to those that did not detect cold. By contrast, patients with percussion (mechanical) hypersensitivity on the affected tooth, reported higher levels of global pain intensity at rest and in function, and reported significantly higher intensity "radiating" and "throbbing" pain, than subjects with non-painful percussion. CONCLUSIONS: The reporting of neuropathic pain descriptors by subjects experiencing acute toothache was more frequent than expected, suggesting that neuropathic mechanisms could contribute to typical toothache pain. Subjects experiencing toothache with mechanical hypersensitivity experience more intense pain overall. SIGNIFICANCE: In subjects experiencing acute toothache, specific pain descriptors associate with the responses to routine clinical sensory tests performed on the injured tooth. The frequent reporting of neuropathic pain descriptors suggests that neuropathic mechanisms could create a diagnostic challenge to differentiate toothache from intraoral neuropathic conditions. Persons experiencing toothache with mechanical hypersensitivity experience more intense pain overall, suggesting patients with this clinical feature will have distinct clinical pain management needs.

TRPM8 and TRPA1 do not contribute to dental pulp sensitivity to cold.

Sensory neurons innervating the dental pulp have unique morphological and functional characteristics compared to neurons innervating other tissues. Stimulation of dental pulp afferents whatever the modality or intensity of the stimulus, even light mechanical stimulation that would not activate nociceptors in other tissues, produces an intense pain. These specific sensory characteristics could involve receptors of the Transient Receptor Potential channels (TRP) family. In this study, we compared the expression of the cold sensitive receptors TRPM8 and TRPA1 in trigeminal ganglion neurons innervating the dental pulp, the skin of the cheek or the buccal mucosa and we evaluated the involvement of these receptors in dental pulp sensitivity to cold. We showed a similar expression of TRPM8, TRPA1 and CGRP in sensory neurons innervating the dental pulp, the skin or the mucosa. Moreover, we demonstrated that noxious cold stimulation of the tooth induced an overexpression of cFos in the trigeminal nucleus that was not prevented by the genetic deletion of TRPM8 or the administration of the TRPA1 antagonist HC030031. These data suggest that the unique sensory characteristics of the dental pulp are independent to TRPM8 and TRPA1 receptors expression and functionality.

Variability in Capsaicin-stimulated Calcitonin Gene-related Peptide Release from Human Dental Pulp.

INTRODUCTION: The unique innervation and anatomic features of dental pulp contribute to the remarkable finding that any physical stimulation of pulpal tissue is painful. Furthermore, when pathological processes such as caries affect teeth and produce inflammation of the pulp, the pain experienced can be quite intense and debilitating. To better understand these underlying neurobiological mechanisms and identify novel analgesic targets for pulpally derived pain, we have developed a powerful ex vivo model using human tooth slices. METHODS: Noncarious, freshly extracted teeth were collected and sectioned longitudinally into 1-mm-thick slices containing both dental pulp and the surrounding mineralized tissues. Tooth slices from 36 patients were exposed to 60 µmol/L capsaicin to stimulate the release of calcitonin gene-related peptide (CGRP) from nerve terminals in the pulp. Patient factors were analyzed for their effects on capsaicin-stimulated CGRP release using a mixed model analysis of variance. RESULTS: Approximately one third of the variability observed in capsaicin-evoked CGRP release was attributable to differences between individuals. In terms of individual factors, there was no effect of anesthesia type, sex, or age on capsaicin-stimulated CGRP release. Using a within-subject study design, a significant effect of capsaicin on CGRP release was observed. CONCLUSIONS: Capsaicin-stimulated CGRP release from dental pulp is highly variable between individuals. A within-subject study design improves the variability and maximizes the potential of this powerful translational model to test the efficacy of novel pharmacotherapeutic agents on human peripheral nociceptors.

Management of Teeth with Persistent Apical Periodontitis after Root Canal Treatment Using Regenerative Endodontic Therapy.

Regenerative endodontic therapy (RET) is currently used to treat immature teeth with necrotic pulp and/or apical periodontitis. However, recently RET has been used to treat mature teeth with necrotic pulp and/or apical periodontitis and resulted in regression of clinical signs and/or symptoms and resolution of apical periodontitis. The purpose of this case report was to describe the potential of using RET to treat 2 mature teeth with persistent apical periodontitis after root canal therapy using RET. Two male patients, one 26-year old and another 12-year old, presented for retreatment of persistent apical periodontitis after root canal treatment of 2 mature teeth (#9 and #19). The gutta-percha fillings in the canals of teeth #9 and #19 were removed with Carvene gutta-percha solvent (Prevest DenPro, Jammu, India) and ProTaper Universal rotary retreatment files (Dentsply Maillefer, Ballaigues, Switzerland). The canals of both teeth were further chemomechanically debrided with rotary retreatment files and copious amounts of sodium hypochlorite irrigation and dressed with Metapaste (Meta Biomed, Chungbuk, Korea). RET was performed on teeth #9 and #19. Periapical bleeding was provoked into the disinfected root canals. The blood clots were covered with mineral trioxide aggregate plugs, and the access cavities were restored with intermediate restorative material. Teeth #9 and #19 showed regression of clinical signs and/or symptoms and healing of apical periodontitis after 13-month and 14-month follow-ups, respectively. Tooth #9 revealed narrowing of the canal space and apical closure by deposition of hard tissue. RET has the potential to be used to retreat teeth with persistent apical periodontitis after root canal therapy.

Clinical and radiographic outcomes of traumatized immature permanent necrotic teeth after revascularization/revitalization therapy.

INTRODUCTION: Revascularization treatment is rapidly becoming an accepted treatment alternative for the management of endodontic pathology in immature permanent teeth with necrotic dental pulps. However, the success and timing of clinical resolution of symptoms, and radiographic outcomes of interest, such as continued hard tissue deposition within the root, are largely unknown. METHODS: In this prospective cohort study, 20 teeth were treated with a standardized revascularization treatment protocol and monitored for clinical and radiographic changes for 1 year. Standardized radiographs were collected at regular intervals, and radiographic changes were quantified. RESULTS: All 20 treated teeth survived during the 12-month follow-up period, and all 20 also met the clinical criteria for success at 12 months. As a group, the treated teeth showed a statistically significant increase in radiographic root width and length and a decrease in apical diameter, although the changes in many cases were quite small (such that the clinical significance is unclear). The within-case percent change in apical diameter after 3 months was 16% and had increased to 79% by 12 months, with 55% (11/20) showing complete apical closure. The within-case percent change in root length averaged less than 1% at 3 months and increased to 5% at 12 months. The within-case percent change in root thickness averaged 3% at 3 months and 21% at 12 months. CONCLUSIONS: Although clinical success was highly predictable with this procedure, clinically meaningful radiographic root thickening and lengthening are less predictable after 1-year of follow-up. Apical closure is the most consistent radiographic finding.

A standardized novel method to measure radiographic root changes after endodontic therapy in immature teeth.

INTRODUCTION: Outcome studies of endodontic treatment of necrotic immature permanent teeth rely on radiographic measures as surrogates of whether the treatment achieved regeneration/revascularization/revitalization. An increase in radiographic root length and/or width is thought to result in a better long-term prognosis for the tooth. In this study, a method to measure radiographic outcomes of endodontic therapies on immature teeth was developed and validated. METHODS: A standardized protocol was developed for measuring the entire area of the root of immature teeth. The radiographic root area (RRA) measurement accounts for the entire surface area of the root as observed on a periapical radiograph. Reviewers were given instructions on how to measure RRA, and they completed measurements on a set of standardized radiographs. RESULTS: The intraclass correlation between the 4 reviewers was 0.9945, suggesting a high concordance among reviewers. There was no effect of the reviewer on the measured RRA values. High concordance was also observed when 1 rater repeated the measurements, with an intraclass correlation value of 0.9995. There was no significant difference in RRA values measured at the 2 sessions by the same rater. Furthermore, significant differences in RRA were detectable between clinical cases that showed obvious continued root development and cases that did not demonstrate discernible root development. CONCLUSIONS: These results suggest that RRA is a valid measure to assess radiographic outcomes in endodontically treated immature teeth, and RRA should be useful in future clinical studies of regenerative endodontic outcomes.

Histologic study of a human immature permanent premolar with chronic apical abscess after revascularization/revitalization.

INTRODUCTION: Histologic studies of teeth from animal models of revascularization/revitalization are available; however, specimens from human studies are lacking. The nature of tissues formed in the canal of human revascularized/revitalized teeth was not well established. METHODS: An immature mandibular premolar with infected necrotic pulp and a chronic apical abscess was treated with revascularization/revitalization procedures. At both the 18-month and 2-year follow-up visits, radiographic examination showed complete resolution of the periapical lesion, narrowing of the root apex without root lengthening, and minimal thickening of the canal walls. The revascularized/revitalized tooth was removed because of orthodontic treatment and processed for histologic examination. RESULTS: The large canal space of revascularized/revitalized tooth was not empty and filled with fibrous connective tissue. The apical closure was caused by cementum deposition without dentin. Some cementum-like tissue was formed on the canal dentin walls. Inflammatory cells were observed in the coronal and middle third of revascularized/revitalized tissue. CONCLUSIONS: In the present case, the tissue formed in the canal of a human revascularized/revitalized tooth was soft connective tissue similar to that in the periodontal ligament and cementum-like or bone-like hard tissue, which is comparable with the histology observed in the canals of teeth from animal models of revascularization/revitalization.

Histologic and histobacteriologic observations of failed revascularization/revitalization therapy: a case report.

INTRODUCTION: Mechanical debridement plays an important role in eliminating intracanal bacteria, such as biofilm on the canal walls and bacteria in the dentinal tubules. Mechanical debridement is not recommended for root canal disinfection in revascularization/revitalization therapy. Here we report a failed revascularization/revitalization case, which could be due to inadequate root canal disinfection without mechanical removal of biofilm and bacteria in dentinal tubules. METHODS: A 6-year-old boy had a traumatic injury to tooth #9, which was avulsed and replanted within 40 minutes. The tooth subsequently developed a local swelling in the periapical area. The patient was referred to the Postgraduate Endodontic Clinic for revascularization/revitalization therapy on tooth #9. The treated tooth remained asymptomatic for 16 months and then developed pain and local periapical swelling. The oral surgeon extracted the revascularized/revitalized tooth. On request, the extracted tooth was processed for histologic and histobacteriologic examination. RESULTS: The tissue in the canal was completely destroyed. Most bacteria were observed in the apical portion and not in the coronal portion of the canal and formed biofilm on the canal walls and penetrated into the dentinal tubules. CONCLUSIONS: On the basis of histobacteriologic observations, the failure of revascularized/revitalized tooth could be due to inadequate root canal disinfection without mechanical debridement. It may be important to perform mechanical debridement as part of the revascularization/revitalization therapy to disrupt the biofilm on the canal walls and remove bacteria in the dentinal tubules because revascularization/revitalization therapy is able to increase thickening of the canal walls.