Pivotal Role of Tenascin-W (-N) in Postnatal Incisor Growth and Periodontal Ligament Remodeling.

The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. In situ hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.

The Role of Transient Receptor Potential (TRP) Channels in the Transduction of Dental Pain.

Dental pain is a common health problem that negatively impacts the activities of daily living. Dentine hypersensitivity and pulpitis-associated pain are among the most common types of dental pain. Patients with these conditions feel pain upon exposure of the affected tooth to various external stimuli. However, the molecular mechanisms underlying dental pain, especially the transduction of external stimuli to electrical signals in the nerve, remain unclear. Numerous ion channels and receptors localized in the dental primary afferent neurons (DPAs) and odontoblasts have been implicated in the transduction of dental pain, and functional expression of various polymodal transient receptor potential (TRP) channels has been detected in DPAs and odontoblasts. External stimuli-induced dentinal tubular fluid movement can activate TRP channels on DPAs and odontoblasts. The odontoblasts can in turn activate the DPAs by paracrine signaling through ATP and glutamate release. In pulpitis, inflammatory mediators may sensitize the DPAs. They could also induce post-translational modifications of TRP channels, increase trafficking of these channels to nerve terminals, and increase the sensitivity of these channels to stimuli. Additionally, in caries-induced pulpitis, bacterial products can directly activate TRP channels on DPAs. In this review, we provide an overview of the TRP channels expressed in the various tooth structures, and we discuss their involvement in the development of dental pain.

Activation of trigeminal ganglion satellite glial cells in CFA-induced tooth pulp pain in rats.

This study further investigated the mechanisms underlying the rat model of tooth pulp inflammatory pain elicited by complete Freund's adjuvant (CFA), in comparison to other pulpitis models. Pulps of the left maxillary first molars were accessed. In the CFA group, the pulps were exposed, and CFA application was followed by dental sealing. In the open group, the pulps were left exposed to the oral cavity. For the closed group, the pulps were exposed, and the teeth were immediately sealed. Naïve rats were used as negative controls. Several parameters were evaluated at 1, 2, 3 and 8 days. There was no statistical significant difference among the groups when body weight variation, food or water consumption were compared. Analysis of serum cytokines (IL-1ß, TNF or IL-6) or differential blood cell counts did not reveal any evidence of systemic inflammation. The CFA group displayed a significant reduction in the locomotor activity (at 1 and 3 days), associated with an increased activation of satellite glial cells in the ipsilateral trigeminal ganglion (TG; for up to 8 days). Amygdala astrocyte activation was unaffected in any experimental groups. We provide novel evidence indicating that CFA-induced pulp inflammation impaired the locomotor activity, with persistent activation of ipsilateral TG satellite cells surrounding sensory neurons, without any evidence of systemic inflammation or amygdala astrogliosis.

Conditional TNF-α Overexpression in the Tooth and Alveolar Bone Results in Painful Pulpitis and Osteitis.

Tumor necrosis factor-α (TNF-α) is a proalgesic cytokine that is commonly expressed following tissue injury. TNF-α expression not only promotes inflammation but can also lead to pain hypersensitivity in nociceptors. With the established link between TNF-α and inflammatory pain, we identified its increased expression in the teeth of patients affected with caries and pulpitis. We generated a transgenic mouse model (TNF-α(glo)) that could be used to conditionally overexpress TNF-α. These mice were bred with a dentin matrix protein 1 (DMP1)-Cre line for overexpression of TNF-α in both the tooth pulp and bone to study oral pain that would result from subsequent development of pulpitis and bone loss. The resulting DMP1/TNF-α(glo) mice show inflammation in the tooth pulp that resembles pulpitis while also displaying periodontal bone loss. Inflammatory infiltrates and enlarged blood vessels were observed in the tooth pulp. Pulpitis and osteitis affected the nociceptive neurons innervating the orofacial region by causing increased expression of inflammatory cytokines within the trigeminal ganglia. With this new mouse model morphologically mimicking pulpitis and osteitis, we tested it for signs of oral pain with an oral function assay (dolognawmeter). This assay/device records the time required by a mouse to complete a discrete gnawing task. The duration of gnawing required by the DMP1/TNF-α(glo) mice to complete the task was greater than that for the controls; extended gnaw time in a dolognawmeter indicates reduced orofacial function. With the DMP1/TNF-α(glo) mice, we have shown that TNF-α expression alone can produce inflammation similar to pulpitis and osteitis and that this mouse model can be used to study dental inflammatory pain.

Differential NMR spectroscopy reactions of anterior/posterior and right/left insular subdivisions due to acute dental pain.

OBJECTIVES: The insular cortex has an important role within the cerebral pain circuitry. The aim of this study was to measure metabolic alterations by MR spectroscopy due to experimentally induced trigeminal pain in the anterior/posterior and right/left insular subdivisions. METHODS: Sixteen male volunteers were investigated using magnetic resonance (MR) spectroscopy before, during and after experimentally induced dental pain. Biphasic bipolar electric current pulses of 1 ms duration were administered based on the subjectively determined pain threshold. Volunteers were instructed to rate every stimulus using a MR compatible rating scale. RESULTS: Due to the pain stimulation a significant absolute increase in glutamate (Glu, F = 6.1; P = 0.001), glutamine (Gln, F = 11.2; P = 0.001) as well as glutamate/glutamine (Glx, F = 17.7; P = 0.001) were observed, whereas myo-inositol (mI, F = 9.5;P = 0.001) showed a significant drop. Additionally, these metabolites showed a significant effect towards lateralisation, meaning that metabolic concentration differed either in left or right insular subdivision. Creatine demonstrated also an absolute significant decrease during stimulation (F = 2.8; P = 0.022) with a significant anterior-posterior difference (F = 40.7; P = 0.001). CONCLUSIONS: Results confirm that the insular cortex is a metabolically high active region in pain processing within the brain. Quantitative metabolic changes show that there is a distinct but locally diverse neurometabolic activity under acute pain. The known cytoarchitectonic subdivisions show different metabolic reactions and give new insights into pain-processing physiology.

[Expression of Nav1.8 in human dental pulp].

OBJECTIVE: To investigate the relationship between dental pain and Nav1.8 expression level by detecting the expression of voltage-gated sodium channel Nav1.8 in normal human dental pulps and painful pulp tissues. METHODS: Immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of Nav1.8 in normal human dental pulp and painful dental pulp. RESULTS: Nav1.8 expressed in dental pulps and the expression level of Nav1.8 increased significantly in painful dental pulps in comparison with normal dental tissues. The immunohistochemistry results revealed that Nav1.8 expression level in painful dental issue was 0.547 ± 0.049 in relative intensity,and in normal dental issue 0.356 ± 0.058 (P < 0.05). Western blotting showed similar results of 0.234 ± 0.030 vs 0.108 ± 0.012. RT-PCR results indicated that Nav1.8 mRNA expression level in painful dental issue was 7.130 ± 2.471 and in normal dental issue was 1.024 ± 0.295 (P < 0.05). CONCLUSIONS: The expression level of Nav1.8 increased significantly in painful dental pulp tissue, suggesting that Nav1.8 may play an important role in the development and transmission of dental pain.

Systemic pregabalin attenuates sensorimotor responses and medullary glutamate release in inflammatory tooth pain model.

Our previous studies have demonstrated that application of inflammatory irritant mustard oil (MO) to the tooth pulp induces medullary glutamate release and central sensitization in the rat medullary dorsal horn (MDH), as well as nociceptive sensorimotor responses in craniofacial muscles in rats. There is recent evidence that anticonvulsant drugs such as pregabalin that influence glutamatergic neurotransmission are effective in several pain states. The aim of this study was to examine whether systemic administration of pregabalin attenuated glutamate release in the medulla as well as these nociceptive effects reflected in increased electromyographic (EMG) activity induced by MO application to the tooth pulp. Male adult rats were anesthetized with isofluorane (1.0-1.2%), and jaw and tongue muscle EMG activities were recorded by needle electrodes inserted bilaterally into masseter and anterior digastric muscles and into the genioglossus muscle, and also the medullary release of glutamate was assessed by in vivo microdialysis. Pregabalin or vehicle control (isotonic saline) was administered 30 min before the pulpal application of MO or vehicle control (mineral oil). Application of mineral oil to the maxillary first molar tooth pulp produced no change in baseline EMG activity and glutamate release. However, application of MO to the pulp significantly increased both the medullary release of glutamate and EMG activity in the jaw and tongue muscles for several minutes. In contrast, pre-medication with pregabalin, but not vehicle control, significantly and dose-dependently attenuated the medullary glutamate release and EMG activity in these muscles after MO application to the tooth pulp (analysis of variance (ANOVA), p<0.05). These results suggest that pregabalin may attenuate the medullary release of glutamate and associated nociceptive sensorimotor responses in this acute inflammatory pulpal pain model, and that it may prove useful for the treatment of orofacial inflammatory pain states.

Peripheral mechanisms of dental pain: the role of substance P.

Current evidence supports the central role of neuropeptides in the molecular mechanisms underlying dental pain. In particular, substance P, a neuropeptide produced in neuron cell bodies localised in dorsal root and trigeminal ganglia, contributes to the transmission and maintenance of noxious stimuli and inflammatory processes. The major role of substance P in the onset of dental pain and inflammation is increasingly being recognised. Well-grounded experimental and clinical observations have documented an increase in substance P concentration in patients affected by caries, pulpitis, or granulomas and in those undergoing standard orthodontic or orthodontic/dental care procedures. This paper focuses on the role of substance P in the induction and maintenance of inflammation and dental pain, in order to define future lines of research for the evaluation of therapeutic strategies aimed at modulating the complex effects of this mediator in oral tissues.

Modeling the onset and offset of dental pain relief by ibuprofen.

Onset and offset of dental pain relief by ibuprofen following third molar extraction were modeled in a randomized, double-blind, placebo-controlled, parallel-group, 8-hour study of patients receiving either a novel effervescent ibuprofen tablet (400 mg; N = 30), standard ibuprofen tablets (Nurofen(®) 2 × 200 mg; N = 22), or placebo (N = 37). An Emax model was fit to pain relief scores. Linear hazard models were used to analyze the time to first perceptible relief (TFPR), the time to meaningful pain relief (TMPR), and time to remedication (REMD). Nomograms were created to correlate TFPR, TMPR, and REMD with different ibuprofen pharmacokinetic profiles. Effervescent ibuprofen was absorbed rapidly with 95% completion within 15 minutes. Maximum pain relief score by ibuprofen was 1.8 units greater than placebo, with an EC50 (effect-site) for ibuprofen concentration of 10.2 µg·mL(-1). The likelihood to achieve TFPR and TMPR was doubled for every 10 µg·mL(-1) increase in ibuprofen plasma concentration. REMD risk decreased 40-fold as the categorical pain relief score increased from 0 to 3. Rapid absorption of ibuprofen effervescent resulted in an earlier TFPR and TMPR, and a lower REMD rate than standard ibuprofen. The nomograms may be useful in predicting the onset and offset of new faster acting ibuprofen formulations, based on pharmacokinetic profiles.

Expression of ecto-ATPase NTPDase2 in human dental pulp.

Dental pulpal nerve fibers express ionotropic adenosine triphosphate (ATP) receptors, suggesting that ATP signaling participates in the process of dental nociception. In this study, we investigated if the principal enzymes responsible for extracellular ATP hydrolysis, namely, nucleoside triphosphate diphosphohydrolases (NTPDases), are present in human dental pulp. Immunohistochemical and immunofluorescence experiments showed that NTPDase2 was predominantly expressed in pulpal nerve bundles, Raschkow's nerve plexus, and in the odontoblast layer. NTPDase2 was expressed in pulpal Schwann cells, with processes accompanying the nerve fibers and projecting into the odontoblast layer. Odontoblasts expressed the gap junction protein, connexin43, which can form transmembrane hemichannels for ATP release. NTPDase2 was localized close to connexin43 within the odontoblast layer. These findings provide evidence for the existence of an apparatus for ATP release and degradation in human dental pulp, consistent with the involvement of ATP signaling in the process of dentin sensitivity and dental pain.

Matrix metalloproteinase-8 and substance P levels in gingival crevicular fluid during endodontic treatment of painful, nonvital teeth.

OBJECTIVE: The aim of this study was to investigate levels of matrix metalloproteinase-8 (MMP-8) and substance P (SP) in gingival crevicular fluid (GCF) during root canal treatment (RCT) of nonvital teeth. STUDY DESIGN: Patients scheduled for nonsurgical RCT were prospectively selected; all patients provided informed consent. GCF samples were collected from teeth scheduled for RCT and their contralateral teeth across 3 different time periods. MMP-8 and SP levels were measured using enzyme-linked immunosorbent assay (ELISA). Data were analyzed using a mixed model analysis and the Pearson correlation analysis. RESULTS: Patients' subjective pain levels were significantly related to both MMP-8 and SP levels. MMP-8 and SP levels in GCF were decreased during RCT, and they showed a positive correlation with each other (P < .05). CONCLUSIONS: This study demonstrated that periradicular inflammation of endodontic origin can elevate SP and MMP-8 levels in GCF.

Lipopolysaccharide-induced pulpitis up-regulates TRPV1 in trigeminal ganglia.

Tooth pain often accompanies pulpitis. Accumulation of lipopolysaccharides (LPS), a product of Gram-negative bacteria, is associated with painful clinical symptoms. However, the mechanisms underlying LPS-induced tooth pain are not clearly understood. TRPV1 is a capsaicin- and heat-gated nociceptive ion channel implicated in thermosensation and hyperalgesia under inflammation or injury. Although TRPV1 is expressed in pulpal afferents, it is not known whether the application of LPS to teeth modulates TRPV1 in trigeminal nociceptors. By assessing the levels of protein and transcript of TRPV1 in mouse trigeminal ganglia, we demonstrate that dentinal application of LPS increases the expression of TRPV1. Our results suggest that the up-regulation of TRPV1 in trigeminal nociceptors following bacterial infection could contribute to hyperalgesia under pulpitis conditions.

Cannabinoid receptor CB1-immunoreactive nerve fibres in painful and non-painful human tooth pulp.

The cannabinoid receptor CB1 is involved in modulation of neuronal hypersensitivity and pain. The aim of this study was to evaluate CB1 receptor levels for the first time in dental pain. A total of 19 patients due for molar extraction were divided into two groups, those with existing dental pain (n=9), and those with no history of pain (n=10). Immunohistochemistry and computer image analysis was used to evaluate CB1-positive nerve fibres in tooth pulp, with neurofilament-immunostaining as a structural nerve marker. CB1-immunoreactive nerve fibres were scattered throughout the tooth pulp and often seen in nerve bundles, but the fibres did not penetrate the subodontoblastic layer. There was no statistically significant change in the CB1 nerve fibre percentage area in the painful group compared to the non-painful group (p=0.146); the neurofilament fibres were significantly reduced in the painful group compared to the controls (p=0.028), but there was no difference in the ratio of CB1 to neurofilaments between the two groups. Thus, CB1 expression is maintained by nerve fibres in painful human dental pulp, and peripherally-restricted CB1 agonists currently in development may advance the treatment of dental pain.

Pulpitis increases the proportion of atypical nodes of Ranvier in human dental pulp axons without a change in Nav1.6 sodium channel expression.

Studies show a change in sodium channel (NaCh) expression after inflammatory lesions, and this change is implicated in the generation of pain states. We are using the extracted human tooth to study NaCh expression and here examine the expression of the major NaCh isoform located at nodes of Ranvier, Na(v)1.6, in normal and painful samples. Pulpal sections were double-labeled with human-specific Na(v)1.6 antibody and caspr antibody (paranodal protein to identify nodes). Confocal microscopy was used to obtain a z-series of optically-sectioned images of axon bundles surrounded by inflammatory cells in painful samples and of similar regions within the coronal pulp of normal samples. Nodes contained within these images were classified as typical or atypical as based on caspr staining relationships, and NIH ImageJ software was used to quantify the size and immunofluorescence staining intensity of Na(v)1.6 accumulations at these nodal sites. Results show no significant difference in the size or immunofluorescence staining intensity of Na(v)1.6 nodal accumulations located at either typical or atypical nodal sites (heminodes and split nodes) within axons in normal samples when compared to painful samples (n=9/each group). In contrast, there was a highly significant decrease in the proportion of typical nodal sites and an increase in atypical nodal sites in painful samples when compared to normal samples. The unchanged expression of Na(v)1.6 contrasts to our previous finding that showed an increased expression of Na(v)1.7 at both typical and atypical nodal sites within painful samples. Together, these findings suggest there is not a simple replacement of one isoform with another, but rather an increased co-expression of multiple isoforms at both intact and remodeling/demyelinating (atypical) nodal sites within the painful dental pulp. The resultant heterogeneous population of isoforms may produce unique axonal excitability properties that could contribute to spontaneous pain sensations that are common in toothache.

Estimation of salivary cortisol in children with rampant caries.

Dental profession is largely challenged with rampant caries and probable predisposing factors for the same have been evaluated for ages. There is considerable evidence that emotional disturbances may be a causative factor in some cases of rampant caries. Thus the objective of the present study was to estimate the levels of salivary cortisol in children with active rampant caries before and after caries control using electrochemiluminescence assay. A total of 60 children between the age group of 5 to 10 years of both sexes were selected for the study. A questionnaire was also included to evaluate predisposing factors for caries. The results showed that there was an increase in salivary cortisol levels in children with rampant caries, the level decreased gradually when observed for a period of three months following dental treatment which was statistically significant.

Gene expression analysis of acutely traumatized pulps.

INTRODUCTION: Vertical root fracture of vital teeth is a relatively rare occurrence. To address early molecular biologic events that take place in the pulp of such cases, we measured mRNA expression levels of selected molecules related to nociception, bacterial pattern recognition, and hard tissue repair/mineralization. METHODS: Three extracted roots obtained from vital molars diagnosed as vertical root fracture were used. The samples were first analyzed with light and transmission electron microscopy. Then mRNA expression in the apical (root fractured) and coronal portions of the pulp was analyzed by using reverse transcription-polymerase chain reaction (PCR) or real-time PCR after laser capture microdissection. RESULTS: In all the samples, cracks and vital pulp tissue, but not signs of infection and inflammation, were recognized in the apical portion of the fractured root. The gene expression analysis showed that mRNAs of pattern recognition receptors (CD14, Toll-like receptor 2, and Toll-like receptor 4) and neurokinin-1 receptor were equally expressed in both regions of the pulp. On the other hand, mRNA expression levels of hard tissue-associated proteins (osteopontin, osteonectin, and osteocalcin) and calcium channel voltage-dependent alpha 2 delta subunit 1 (CACNA2D1) in the apical portion of the pulp tissue and periodontal ligaments were significantly up-regulated, as compared with those in the coronal portion of the pulp. CONCLUSIONS: In the traumatized apical pulp of vertically root-fractured vital teeth, elevated mRNA expression of CACNA2D1, a nociception-related molecule, and proteins related to hard tissue repair/mineralization occurs under noninfectious condition.

Changes in proteinase-activated receptor 2 expression in the human tooth pulp in relation to caries and pain.

AIMS: To investigate the presence of proteinase-activated receptor 2 (PAR2) in the human tooth pulp and to determine whether there are any changes in receptor expression with caries and pain. METHODS: Forty-four mandibular first permanent molars were collected from children (n = 36, mean age 9.96 years +/- 2.11) requiring dental extractions under general anesthesia. Teeth were categorized as either intact (n = 22) or carious (n = 22). Carious teeth were further subdivided into asymptomatic (n = 10) and painful (n = 12). The coronal pulp was removed and processed for indirect immunofluorescence by using antibodies raised against PAR2 and double labeled with either a neuronal marker (protein gene product 9.5) or both a smooth muscle cell (aSMA) and endothelial (UEIL) marker, in order to examine PAR2 presence in both neuronal and vascular tissue. In addition, hemotoxylin and eosin staining was performed to identify pulpal fibroblasts. RESULTS: PAR2 expression was found to be present in pulpal nerve fibers, vascular tissue, and pulpal fibroblasts. PAR2 neuronal expression was not affected by the presence of caries (P > .05) but was significantly less in carious painful teeth than in carious asymptomatic teeth (P < .05). No changes in vascular PAR2 expression were found (P > .05); however, the number of PAR2-labeled fibroblast-like cells per mm2 was significantly greater in carious teeth (P < .05). CONCLUSION: These findings indicate that PAR2 receptors and changes in their level of expression may have relevance and clinical importance in nociception.

Theophylline attenuates hippocampal blood flow responses induced by tooth pulp stimulation in rats.

In this study, we performed tests to determine whether tooth pulp stimulation (TPS) increases hippocampal blood flow (HBF), and if so, to investigate whether the increase in HBF is mediated via the activation of adenosine receptors. We measured HBF in urethane-anesthetized rats using laser Doppler flowmetry (LDF) and examined the effect of theophylline, a nonselective adenosine receptor antagonist, on TPS-induced HBF responses. TPS increased HBF, and its response was significantly attenuated by the intraperitoneal administration of theophylline (20 mg/kg). These results suggest that the HBF response induced by TPS may be, at least in part, produced through adenosine receptors.