Molar Tooth Pulp Research Articles

Histological Evaluation of A unilateral Critical-Sized Mandibular Defect Reconstruction using human dental pulp stem cells by Light Microscope and Real-Time Quantitative PCR

Objective: To evaluate the osteogenic differentiation potential of human dental pulp stem cells (DPSCs) isolated from the dental pulp of third molar teeth in vitro cultures, and to evaluate the bone regenerative capacity of human dental pulp stem cells (DPSCs) when transplanted into a unilateral critical-sized bone defect in the mandibular bone in vivo after receiving a hydroxyapatite matrix and polylactic-polyglycolic acid (HA/PLGA) scaffold. Material and methods: A total of 18 mandibular defects were made, and three groups (each n = 6) were created. The first group: the transplanted DPSCs implanted in the critical-sized bone defect after receiving (HA/PLGA) scaffold. The second group received only (HA/PLGA) scaffold. The third group, which served as the control, had a critical-sized defect left empty. After characterization, Von Kossa [VK] and Alizarin red staining were employed to identify differentiated osteoblasts at the 14th and 21st days, and histological analyses, as well as polymerase chain reactions (PCR), were also used. Results: It showed that DPSCs had high proliferation potential and typical fibroblastic shape. Additionally, osteogenic differentiation of DPSCs was validated by morphological alterations, histological examination, and the expression of lineage-specific genes confirmed osteogenic differentiation of DPSCs. Conclusion: High proliferation potential and the capacity to differentiate into osteoblasts are two characteristics of DPSCs taken from impacted third molars.

Inhibition of the Nav1.7 Channel in the Trigeminal Ganglion Relieves Pulpitis Inflammatory Pain.

Pulpitis causes significant changes in the peripheral nervous system, which induce hyperalgesia. However, the relationship between neuronal activity and Nav1.7 expression following pulpal noxious pain has not yet been investigated in the trigeminal ganglion (TG). The aim of our study was to verify whether experimentally induced pulpitis activates the expression of Nav1.7 peripherally and the neuronal activities of the TGs can be affected by Nav1.7 channel inhibition. Acute pulpitis was induced through allyl isothiocyanate (AITC) application to the rat maxillary molar tooth pulp. Three days after AITC application, abnormal pain behaviors were recorded, and the rats were euthanized to allow for immunohistochemical, optical imaging, and western blot analyses of the Nav1.7 expression in the TG. A significant increase in AITC-induced pain-like behaviors and histological evidence of pulpitis were observed. In addition, histological and western blot data showed that Nav1.7 expressions in the TGs were significantly higher in the AITC group than in the naive and saline group rats. Optical imaging showed that the AITC group showed higher neuronal activity after electrical stimulation of the TGs. Additionally, treatment of ProTxII, selective Nav1.7 blocker, on to the TGs in the AITC group effectively suppressed the hyperpolarized activity after electrical stimulation. These findings indicate that the inhibition of the Nav1.7 channel could modulate nociceptive signal processing in the TG following pulp inflammation.

Evaluation of human pulp tissue response following direct pulp capping with a self-etching adhesive system containing MDPB.

This study evaluated the human pulp tissue response following direct pulp capping with Clearfil Protect Bond (CPB) self-etching adhesive containing an antibacterial monomer MDPB. The pulps of third molar teeth were exposed by the removal of carious tissue. In an experimental group, CPB was applied to the exposed pulp and dentin. In the control groups, Clearfil SE Bond (CSE) or calcium hydroxide-based cement (CH) was applied to the exposed pulp surfaces. All teeth were filled with resin composite, extracted after 90 days, and the pulp responses were histologically analyzed. No severe inflammation or soft tissue disorganization was observed in CPB and CH groups. CSE group exhibited a disorganized odontoblastic layer and severe inflammatory infiltration. No hard tissue formation was observed in CSE group, and CH formed more of a hard tissue formation than CPB. CPB induced an acceptable healing response when directly applied to exposed pulps with bacterial contamination.

Vesicular glutamate transporter-immunopositive axons that coexpress neuropeptides in the rat and human dental pulp.

To examine the type of vesicular glutamate transporter (VGLUT)-immunopositive (+) axons that coexpress neuropeptides in the rat and human dental pulp, which may help understand peripheral mechanism of pulpal inflammatory pain in rats and humans. The trigeminal ganglia (TG) and the dental pulp of the maxillary molar teeth from three male Sprague-Dawley rats weighing 300-330g and dental pulps of three healthy human (male) maxillary premolar teeth from three 16 to 28-year-old patients extracted for orthodontic treatment were used. The type of VGLUT+axons that coexpress substance P (SP)- and/or calcitonin gene-related peptide (CGRP) and parvalbumin in the rat TG and in the axons of the rat and the human dental pulp was examined by double fluorescence immunohistochemistry and quantitative analysis. Results were analyzed using one-way anova and the Kruskal-Wallis test. SP and CGRP were expressed in many human VGLUT1+pulpal axons but not in the rat VGLUT1+TG neurons and pulpal axons (P<0.05). SP and CGRP were expressed in a considerable number of human VGLUT2+pulpal axons and also in many rat TG neurons and pulpal axons. The fraction of VGLUT1+axons expressing parvalbumin was about three times higher in the rat than in the human dental pulp (P<0.05). These findings suggest that the types of VGLUT+axons, which release neuropeptides, may be different between the rat and the human dental pulp, raising a possibility that peripheral mechanism of pulpal inflammatory pain may be different between rats and humans.

Role of macrophage-mediated Toll-like receptor 4\u2013interleukin-1R signaling in ectopic tongue pain associated with tooth pulp inflammation

BackgroundThe existence of referred pain and ectopic paresthesia caused by tooth pulp inflammation may make definitive diagnosis difficult and cause misdiagnosis or mistreatment; thus, elucidation of that molecular mechanism is urgent. In the present study, we investigated the mechanisms underlying ectopic pain, especially tongue hyperalgesia, after tooth pulp inflammation.MethodsA rat model with mandibular first molar tooth pulp exposure was employed. Tooth pulp exposure-induced heat and mechanical-evoked tongue hypersensitivity was measured, and immunohistochemical staining for Iba1, a marker of active macrophages, IL-1β, IL-1 type I receptor (IL-1RΙ), and toll-like receptor 4 in the trigeminal ganglion was performed. In addition, we investigated the effects of injections of liposomal clodronate Clophosome-A (LCCA), a selective macrophage depletion agent, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS, a toll-like receptor 4 antagonist), IL-1β, or heat shock protein 70 (Hsp70, a selective agonist of toll-like receptor 4), to examine changes in tongue hypersensitivity and in the regulation of IL-1RΙ, toll-like receptor 4, and transient receptor potential vanilloid 1 (TRPV1) biosynthesis.ResultsAt day 1 after tooth pulp exposure, obvious tooth pulp inflammation was observed. Tooth pulp exposure-induced heat and mechanical tongue hypersensitivity was observed from days 1 to 3 after tooth pulp exposure. The production of IL-1β in activated macrophages and toll-like receptor 4 and IL-1RΙ expression were significantly increased in trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of LCCA significantly suppressed tongue hypersensitivity; however, toll-like receptor 4 and IL-1RΙ expression in trigeminal ganglion neurons innervating the tongue was not significantly altered. Intra-trigeminal ganglion injection of LPS-RS significantly suppressed tongue hypersensitivity and reduced IL-1RΙ expression in the trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of recombinant Hsp70 significantly promoted tongue hypersensitivity and increased IL-1RI expression in trigeminal ganglion neurons innervating the tongue in naive rats. Furthermore, intra-trigeminal ganglion injection of recombinant IL-1β led to tongue hypersensitivity and enhanced TRPV1 expression in trigeminal ganglion neurons innervating the tongue in naive rats.ConclusionsThe present findings suggest that the neuron-macrophage interaction mediated by toll-like receptor 4 and IL-1RI activation in trigeminal ganglion neurons affects the pathogenesis of abnormal tongue pain following tooth pulp inflammation via IL-1RI and TRPV1 signaling in the trigeminal ganglion. Further research may contribute to the establishment of new therapeutic and diagnostic methods.

Investigation of the effect of a time delay on the characteristics and survival of dental pulp stem cells from extracted teeth

ObjectivesThis study investigates the post-extraction storage period of human dental pulp stem cells (hDPSCs) for stem cell banking by investigating the viability, function, mineralization, and gene expression of hDPSCs isolated from extracted teeth after 1 h, 6 h and 24 h post-tooth extraction. DesignhDPSCs were extracted from the pulp of impacted third molar teeth after 1 h, 6 h, and 24 h after extraction. The mesenchymal stem cell (MSCs) properties of three groups of cells were analyzed using flow cytometry. Cell morphology and proliferation were analyzed using a light microscope and an MTT assay. The viability, function, mineralization, and gene expression of hDPSCs of 1 h, 6 h, and 24 h groups were also assessed. ResultsThe delayed harvesting of hDPSCs for 1, 6 or 24 h caused a 31 % reduction in mineral nodule formation and a reduction in the gene expression of osteocalcin (OCN) and vascular endothelial growth factor-alpha (VEGFA). However, the 1, 6 or 24 h, time delay had little effect on MTT cell proliferation, cell viability or morphology. The delayed of harvesting of hDPSCs for 1, 6 or 24 h also had little effect on the expression of MSCs positive (CD44, CD106, CD90) or negative surface markers (CD45 and CD11b). ConclusionsOur results suggest that a 24 h delay in harvesting hDPSCs from extracted teeth can reduce their mineralization and gene activity but does not markedly reduce survival. Quicker hDPSCs harvesting is likely to yield more useful hDPSCs for experimentation and clinical treatment.

Toll-Like Receptor 4 in the Rat Caudal Medulla Mediates Tooth Pulp Inflammatory Pain.

The aims of this study were to investigate if Toll-like receptor 4 (TLR4) is expressed in the medullary dorsal horn (MDH) and if medullary application of a TLR4 antagonist (lipopolysaccharides from Rhodobacter sphaeroides, LPS-RS) can attenuate changes in nociceptive sensorimotor responses or TLR4 expression that might be evoked by mustard oil (MO) application to the right maxillary first molar tooth pulp. Of 41 adult male Sprague-Dawley rats used in the study, 23 received intrathecal application of the TLR4 antagonist LPS-RS (25 μg/10 μl; LPS-RS group) or isotonic saline (10 μl; vehicle control group) 10 min before pulpal application of MO (95%; 0.2 μl). Bilateral electromyographic (EMG) activities of the anterior digastric and masseter muscles were recorded continuously before and until 15 min after the MO application to the pulp. In 6 of these 23 rats and an additional 18 rats, the caudal medulla containing the ipsilateral and contralateral MDH was removed after euthanasia for subsequent Western Blot analysis of TLR4 expression in LPS-RS (n = 8) and vehicle (n = 8) groups and a naïve group (n = 8). The % change from baseline in the MO-evoked EMG activities within the anterior digastric muscles were significantly smaller in the LPS-RS group than the control group (two-way ANOVA, post hoc Bonferroni, P < 0.0001). Western Blot analysis revealed similar levels of TLR4 expression in the caudal medulla of the naïve, vehicle and LPS-RS groups. These novel findings suggest that TLR4 signaling in the caudal medulla may mediate MO-induced acute dental inflammatory pain in rats.

Influence of low-level laser therapy on inflammation, collagen fiber maturation, and tertiary dentin deposition in the pulp of bleached teeth.

We evaluated the effects of low-level laser therapy (LLLT) using an infrared laser (IRL) and a red laser (RL) on the pulp of molar teeth in rats after dental bleaching to assess inflammation, collagen fiber maturation, and tertiary dentin formation. Eighty Wistar rats (Rattus norvegicus, albinus) were randomly divided into eight groups with 10 hemimaxillae in each of the following: control; bleached (Ble, 35% hydrogen peroxide [H2O2]); Ble-1IRL and Ble-1RL (one IRL [808nm, 30s, 3J] or RL [660nm, 15s, 1.5J] application immediately after H2O2); Ble-3IRL and Ble-3RL (three [immediately, 24h, and 48h] IRL or RL applications after H2O2); and 3IRL and 3RL (three IRL or RL applications without bleaching). The rats were euthanized after 2 and 30days for histological evaluation of inflammation (hematoxylin-eosin) and maturation of collagen fibers (picrosirius red). Additionally, the dentin deposition in the specimens obtained at 30days was quantified via microtomography of the pulp chamber volume. Statistical analyses were performed (P < 0.05). Initially, severe damages to the pulp were observed in the Ble and Ble-1RL groups. Ble-1IRL and Ble-3RL groups showed lower inflammation. The bleached groups had a greater amount of mature collagen fibers than the control group. The Ble-3IRL group had a greater number of immature fibers than the Ble group. At 30days, there was an absence of inflammation and equal proportion of mature and immature collagen fibers. All bleached groups showed a reduction in the volume of the pulp chamber. Three consecutive applications of RL and one IRL application can minimize damage to the pulp of bleached teeth, whereas three IRL applications can minimize pulp fibrosis. However, LLLT did not prevent deposition of tertiary dentin. This study describes LLLT protocols capable of minimizing inflammation and maturation of collagen fibers in pulp tissue after dental bleaching. However, the protocols proved insufficient for reducing the formation of tertiary dentin in bleached teeth.

Usability of dental pulp visibility and tooth coronal index in digital panoramic radiography in age estimation in the forensic medicine.

Age estimation has a great importance due to legal requirements. The aim of our study was to determine the applicability of two different methods in age estimation; one of them based on the calculation of the visibility of the root pulp of mandibular third molar teeth and the other based on the calculation of the tooth coronal index (TCI) in the mandibular first and second molar teeth in the 9059 digital orthopantomogram of people aged between 15 and 40 in Bursa. In the first method in which the visibility of the fully mineralized root pulp of the mandibular third molar teeth was evaluated in 4 stages; the stages 0, 1, 2, and 3 were observed regardless of sex at the earliest 17.2; 19.1; 20.1, and 25.1 years, respectively. In the second method in which TCI was regressed on chronological age using measuring crown and coronal pulp cavity heights, the most accurate age estimation model based on simple linear regression for all cases without any sex difference was found to be with right first molar tooth (SEE ±7304 years) and the most accurate age model based on multiple regression model was found to be with bilateral first molar teeth (SEE ±7413 years). In conclusion, we believe that the root pulp visibility of the third molar teeth can be applied safely for stages 1, 2, and 3 at 16, 17, and 21 years of ages, respectively. A correlation was also found between TCI and age, and our findings have shown that both methods are available for forensic purposes.

Osteogenic potential of human dental pulp-derived mesenchymal stem cells in bone regeneration of rabbit

Background/aim Human dental pulp-derived mesenchymal stem cells (hDP-MSCs) offer a promising source of progenitor cells for regenerative medicine and bone tissue engineering. Cranial defects are common complications that can arise secondary to trauma, surgery, or infection. This study aimed to evaluate the osteogenic differentiation potential of hMSCs isolated from dental pulp of third molar teeth in vitro cultures and the bone regenerative capacity of hDP-MSCs transplanted into induced temporomandibular joint (TMJ) defect in rabbits. Patients and methods hDP-MSCs were isolated from third molar teeth and cultured. Alizarin staining was performed to assess the osteogenic differentiation at the 14th and 28th days. The therapeutic potential of hDP-MSCs in craniofacial bone defects was investigated in the left side of the rabbits’ TMJ. The transplanted cells involved three groups: the osteogenic differentiated DP-MSCs (O), undifferentiated MSCs (M), and control group (cell-free matrix) (C). Cells were loaded on gel foam. Eighteen rabbits were used and sacrificed at subsequent three time points, 4, 6, and 9 weeks, after transplantation, with six rabbits/each time point and two rabbits/each cell group. Histopathological studies were applied to evaluate the healing potential of hDP-MSCs in the induced rabbit TMJ defect. Results hDP-MSCs showed a high proliferative potential and osteogenic differentiation in vitro. Histological results demonstrated a timely correlated mandibular defects’ repair in all the experimental groups, including the control group, with more enhanced bone healing effect for the osteogenic differentiated DP-MSCs. Conclusion hDP-MSCs possess high proliferation capacity and osteogenic differentiation potential in vitro. Histological observations revealed the osteogenic differentiated DP-MSCs have higher bone healing potential than the undifferentiated DP-MSCs at 9 weeks after transplantation, and gel foam promotes bone formation in the control group. The bone regenerative potential of osteogenic differentiated DP-MSCs revealed a significant capacity when implanted in rabbit TMJ defect. Hence, hDP-MSCs could be a promising source for craniofacial bone regeneration.

In vitro assessment of alendronate toxic and apoptotic effects on human dental pulp stem cells.

Objective(s):Osteonecrosis of the jaw, as an exposed necrotic bone in the oral cavity, is one of the adverse effects of bisphosphonates, which have an affinity for bone minerals. This study investigates the cytotoxic effects of alendronate (ALN) as a nitrogen-containing bisphosphonate, on human dental pulp stem cells (hDPSCs). Materials and Methods:The mesenchymal stem cells (MSCs), obtained from third molar tooth pulps were characterized by immunophenotyping assay in order to identify surface markers to evaluate their expression. To detect multipotency hDPSCs, they were differentiate into osteocytes and adipocytes. Cell proliferation was measured by MTT assay. PI staining of DNA fragmentation by flowcytometry (sub-G1 peak) was performed for determination of apoptotic cells and Bax, Bcl-2, and cleaved caspase 3 expressions. Protein expression was detected by Western blotting. Results:As the results revealed, ALN decreased viable cells (in 0.8–100 µM) after 72 hr and 168 hr (P<0.001), significantly. ALN could lower cell proliferation in hDPSCs in a concentration and time-dependent manner. Sub-G1 peak as an indicator of flowcytometry histogram of treated cells by ALN, showed apoptosis was involved in ALN-induced cytotoxicity. Expressions of cleaved caspase 3 and Bax protein, as pro-apoptotic proteins, were increased and Bcl-2 protein as anti-apoptotic protein was decreased in response to increases in the concentration of ALN (0.8–25 µM).Conclusion:Long-term effects of ALN on cell proliferation and apoptosis in hDPSCs can result in either initiation or potentiation of ALN-induced osteonecrosis.

Role of medullary astroglial glutamine synthesis in tooth pulp hypersensitivity associated with frequent masseter muscle contraction.

BackgroundThe mechanisms underlying tooth pulp hypersensitivity associated with masseter muscle hyperalgesia remain largely underinvestigated. In the present study, we aimed to determine whether masseter muscle contraction induced by daily electrical stimulation influences the mechanical head-withdrawal threshold and genioglossus electromyography activity caused by the application of capsaicin to the upper first molar tooth pulp. We further investigated whether astroglial glutamine synthesis is involved in first molar tooth pulp hypersensitivity associated with masseter muscle contraction.MethodsThe first molar tooth pulp was treated with capsaicin or vehicle in masseter muscle contraction or sham rats, following which the astroglial glutamine synthetase inhibitor methionine sulfoximine or Phosphate buffered saline (PBS) was applied. Astroglial activation was assessed via immunohistochemistry.ResultsThe mechanical head-withdrawal threshold of the ipsilateral masseter muscle was significantly decreased in masseter muscle contraction rats than in sham rats. Genioglossus electromyography activity was significantly higher in masseter muscle contraction rats than sham rats. Glial fibrillary acidic protein-immunoreactive cell density was significantly higher in masseter muscle contraction rats than in sham rats. Administration of methionine sulfoximine induced no significant changes in the density of glial fibrillary acidic protein-immunoreactive cells relative to PBS treatment. However, mechanical head-withdrawal threshold was significantly higher in masseter muscle contraction rats than PBS-treated rats after methionine sulfoximine administration. Genioglossus electromyography activity following first molar tooth pulp capsaicin treatment was significantly lower in methionine sulfoximine-treated rats than in PBS-treated rats. In the ipsilateral region, the total number of phosphorylated extracellular signal-regulated protein kinase immunoreactive cells in the medullary dorsal horn was significantly smaller upon first molar tooth pulp capsaicin application in methionine sulfoximine-treated rats than in PBS-treated rats.ConclusionsOur results suggest that masseter muscle contraction induces astroglial activation, and that this activation spreads from caudal to the obex in the medullary dorsal horn, resulting in enhanced neuronal excitability associated with astroglial glutamine synthesis in medullary dorsal horn neurons receiving inputs from the tooth pulp. These findings provide significant insight into the mechanisms underlying tooth pulp hypersensitivity associated with masseter muscle contraction.

Role of Neuron-Glial Interaction Mediated by IL-1β in Ectopic Tooth Pain

Although many reports have demonstrated that ectopic pain develops in the orofacial region following tooth pulp inflammation, which often causes misdiagnosis and inappropriate treatment for patients with pulpitis, the precise mechanism remains unknown. In the present study, we hypothesized that the functional interaction between satellite glial cells and neurons mediated by interleukin 1β (IL-1β) in the trigeminal ganglion (TG) is involved in ectopic orofacial pain associated with tooth pulp inflammation. The digastric muscle electromyogram (D-EMG) activity elicited by capsaicin administration into the maxillary second molar tooth pulp was analyzed to evaluate the noxious reflex and was significantly increased in rats with inflammation of the maxillary first molar (M1) versus rats injected with saline. A significant increase in the expression of connexin43 (Cx43), a gap junction containing protein, was observed in activated satellite glial cells surrounding second molar–innervating neurons in the TG after M1 pulpitis. Daily administration of Gap26, a Cx43 mimetic peptide and inhibitor, in the TG significantly suppressed the enhancement of capsaicin-induced D-EMG activity and the percentage of Fluoro-Gold (FG)–labeled cells encircled by glial fibrillary acid protein–immunoreactive (IR) + Cx43-IR cells after M1 pulp inflammation (P < 0.01). The percentage of FG-labeled cells encircled by glial fibrillary acid protein–IR + IL-1β-IR cells, IL-1 type I receptor–IR cells labeled with FG, and TRPV1-IR cells labeled with FG significantly increased after M1 pulp inflammation (P < 0.01). Daily administration of IL-1ra, an IL-1 receptor antagonist, into the TG significantly reduced the enhancement of capsaicin-induced D-EMG activity and the percentage of TRPV1-IR neurons labeled with FG after M1 pulp inflammation (P < 0.01). The present findings suggest that satellite glial cell is activated in the TG via activated gap junctions composed of Cx43 following tooth pulp inflammation, which leads to the hyperactivation of remote neurons via IL-1β mechanisms and results in ectopic tooth pulp pain in the adjacent tooth.

Decreased face primary motor cortex (face-M1) excitability induced by noxious stimulation of the rat molar tooth pulp is dependent on the functional integrity of medullary astrocytes.

We have recently shown that application of the small-fiber excitant and inflammatory irritant mustard oil (MO) to the rat molar tooth pulp can decrease face-M1 excitability, but increase the excitability of trigeminal medullary dorsal horn (MDH) nociceptive neurons and that application of the astrocytic inhibitor methionine sulfoximine (MSO) to the face-M1 or MDH can attenuate the MO-induced changes. The present study aimed to determine whether medullary MSO application could modulate the MO-induced decreased face-M1 excitability. Under ketamine general anesthesia, electromyographic (EMG) electrodes were implanted into the right anterior digastric (RAD, jaw-opening muscle) of adult male Sprague-Dawley rats. A microelectrode was positioned at a low-threshold (≤30 μA) site in the left face-M1. Then MO (n = 16) or control-solution (n = 16) was applied to the previously exposed molar tooth pulp, and intracortical microstimulation threshold intensities for evoking RAD EMG activities were monitored for 15 min. MSO (0.1 mM, n = 8) or phosphate-buffered saline (PBS, n = 8) was then applied to the MDH and RAD thresholds monitored every 15 min for 120 min. Statistics used ANOVA followed by post hoc Bonferroni as appropriate (p < 0.05). As compared to baseline, RAD thresholds significantly increased (i.e., decreased excitability) within 1 min (26.3 ± 7.9%, p = 0.007) and peaked at 15 min following pulpal MO application (49.9 ± 5.7%, p < 0.001) but not following control-solution. Following MSO (but not PBS) application to the medulla, RAD thresholds significantly decreased within 15 min (26.5 ± 3.0%, p = 0.05) and at 60 min approached 6.3 ± 2.4%, of baseline values (p = 0.1). These novel findings suggest that clinically related motor disturbances arising from dental pain may involve decreased face-M1 excitability that is modulated by medullary astrocytes.

Comparison of speed of action and injection discomfort of 4% articaine and 2% mepivacaine for pulpal anesthesia in mandibular teeth: A randomized, double-blind cross-over trial.

Objective:To compare the injection pain and speed of local anesthetic effect induced by tissue infiltration of mepivacaine 2% with epinephrine 1:100,000 versus articaine 4% with epinephrine 1:100,000 in securing mandibular first molar pulp anesthesia.Materials and Methods:Totally, 25 patients were recruited in a crossover, randomized, double-blind study. Each subject received injections of mepivacaine 2% with epinephrine 1:100,000 as inferior alveolar nerve block (IANB) supplemented with either articaine 4% with epinephrine 1:100,000 (septocaine) or mepivacaine 2% buccal infiltration (BI) injection on two visits. The time of first numbness to associated lip, tongue and tooth was recorded by asking the participant directly and using electrical pulp tester. Anesthetic success was considered when two consecutive maximal stimulation on pulp testing readings without sensation. The patients rated the pain of infiltration using a 100 mm visual analog scale immediately after receiving each injection. The pain scores were compared using the paired t-test.Results:There were significant differences in the meantime of first numbness to associated lip and tooth of volunteers between mepivacaine and articaine BI groups P = 0.03 and 0.002. Volunteers in articaine group recorded earlier lip and teeth numbness than those in mepivacaine group. There were significant differences between the mean pain scores for volunteers in the post IANB and postbuccal injection groups (t-test: P <0.001). Mepivacaine IANB injection was significantly more painful than articaine/mepivacaine buccal injection.Conclusions:About 4% articaine was faster than 2% mepivacaine (both with 1:100,000 adrenaline) in anesthetizing the pulps of lower molar teeth after BIs. Earlier lip and teeth numbness were recorded in articaine group. Articaine and mepivacaine BIs were more comfortable than mepivacaine IANB injections.

Decreased face primary motor cortex (face-M1) excitability induced by noxious stimulation of the rat molar tooth pulp is dependent on the functional integrity of face-M1 astrocytes.

Acute inflammatory dental pain is a prevalent condition often associated with limited jaw movements. Mustard oil (MO, a small-fiber excitant/inflammatory irritant) application to the rat molar tooth pulp induces increased excitability (i.e., central sensitization) of trigeminal medullary dorsal horn (MDH) nociceptive neurons that can be modulated by MDH application of the astrocytic inhibitor methionine sulfoximine (MSO). The objectives of the study were to determine whether MO application to the rat right maxillary first molar tooth pulp affects left face-M1 excitability manifested as altered intracortical microstimulation thresholds for evoking electromyographic activity in the right anterior digastric (RAD, jaw-opening muscle), and whether MSO application to face-M1 can modulate this MO effect. Under Ketamine general anesthesia, Sprague-Dawley male rats had a microelectrode positioned at a low-threshold (≤30 μA) face-M1 site. Then MO (n = 16) or control solution (n = 16) was applied to the previously exposed tooth pulp, and RAD threshold was monitored for 15 min. MSO (0.1 mM, n = 8) or saline (n = 8) was then applied to the face-M1, and RAD thresholds were monitored every 15 min for 120 min. ANOVA followed by post hoc Bonferroni was used to analyze data (p < 0.05). Within 15 min of MO (but not control) pulp application, RAD thresholds increased significantly (p < 0.001) as compared to baseline. One hour following MSO (but not saline) application to the face-M1, RAD thresholds decreased significantly (p = 0.005) toward baseline. These novel findings suggest that acute inflammatory dental pain is associated with decreased face-M1 excitability that may be dependent on the functional integrity of face-M1 astrocytes and related to mechanisms underlying limited jaw movements in acute orofacial pain conditions.

Involvement of trigeminal transition zone and laminated subnucleus caudalis in masseter muscle hypersensitivity associated with tooth inflammation.

A rat model of pulpitis/periapical periodontitis was used to study mechanisms underlying extraterritorial enhancement of masseter response associated with tooth inflammation. Periapical bone loss gradually increased and peaked at 6 weeks after complete Freund’s adjuvant (CFA) application to the upper molar tooth pulp (M1). On day 3, the number of Fos-immunoreactive (IR) cells was significantly larger in M1 CFA rats compared with M1 vehicle (veh) rats in the trigeminal subnucleus interpolaris/caudalis transition zone (Vi/Vc). The number of Fos-IR cells was significantly larger in M1 CFA and masseter (Mass) capsaicin applied (M1 CFA/Mass cap) rats compared with M1 veh/Mass veh rats in the contralateral Vc and Vi/Vc. The number of phosphorylated extracellular signal-regulated kinase (pERK)-IR cells was significantly larger in M1 CFA/Mass cap and M1 veh/Mass cap rats compared to Mass-vehicle applied rats with M1 vehicle or CFA in the Vi/Vc. Pulpal CFA application caused significant increase in the number of Fos-IR cells in the Vi/Vc but not Vc on week 6. The number of pERK-IR cells was significantly lager in the rats with capsaicin application to the Mass compared to Mass-vehicle treated rats after pulpal CFA- or vehicle-application. However, capsaicin application to the Mass did not further affect the number of Fos-IR cells in the Vi/Vc in pulpal CFA-applied rats. The digastric electromyographic (d-EMG) activity after Mass-capsaicin application was significantly increased on day 3 and lasted longer at 6 weeks after pulpal CFA application, and these increase and duration were significantly attenuated by i.t. PD98059, a MEK1 inhibitor. These findings suggest that Vi/Vc and Vc neuronal excitation is involved in the facilitation of extraterritorial hyperalgesia for Mass primed with periapical periodontitis or acute pulpal-inflammation. Furthermore, phosphorylation of ERK in the Vi/Vc and Vc play pivotal roles in masseter hyperalgesia after pulpitis or periapical periodontitis.

Hypoxic condition promotes differentiation and mineralization of dental pulp cells in vivo

To investigate the behaviour of dental pulp cells under hypoxic conditions in vivo using an experimental animal model. A mini-screw was inserted into the inferior dental nerve canal of rats to arrest the blood supply, which resulted in a reduced oxygen level in the dental pulps of molar teeth used for the experimental group. The decrease in blood supply was evaluated by injected India ink in transparent specimens. The hypoxia marker hypoxyprobe-1 was investigated by immunohistochemical staining. The mRNA expressions of ATP-binding cassette transporter (ABC) G2 (ABCG2) which is a marker for the capacity to excrete metabolites and for stem-like cells as well as dentine sialophosphoprotein (DSPP) and osteocalcin (OCN) which are markers for mineralization were evaluated by RT-PCR. Protein was evaluated by immunohistochemical staining using ABCG2, dentine sialoprotein (DSP) and OCN. The evaluation of India ink indicated a decreased blood supply in the transparent specimens, and hypoxyprobe-1 immunohistochemical staining showed positive expression. ABCG2, DSPP and OCN mRNA expressions increased at 7 and 14 days. Immunohistochemically, ABCG2, DSP and OCN-positive cells were localized in the odontoblastic layer. Hypoxic conditions promoted mineralization and differentiation of dental pulp cells of the odontoblastic layer.

Characterization of stem cells from the pulp of unerupted third molar tooth.

Dental pulp stem cells (DPSCs) are the most diagnosed type of stem cells isolated from dental tissues. Previous studies demonstrate that tissues in earlier stages of development could be better stem cell resources for tissue engineering. In this study, aiming at finding younger stem cell resources, we chose the pulp of human unerupted third molar teeth when the crown was completely formed and the roots had not begun their development, Nolla's 6 th developmental stage (N6 th ). Surgical removal of the third molar was performed by aseptic technique with minimal trauma. The tissues were digested enzymatically and the resulted single cells were cultured. Immunophenotypic characterization of the cells was done via immunocytochemistry, immunofluorescence, and flow cytometry assays. Adipogenic and osteogenic differentiation potential of these cells was examined and confirmed by histochemical staining and reverse transcription-polymerase chain reaction analysis. This study is descriptive. N6 th -unerupted dental pulp cultured cells expressed DPSC markers: Vimentin, CD73, CD90, CD105, CD166, CD44, CD146, and STRO-1, but did not express hematopoietic cell markers: CD14, CD34, CD45, HLA-DR and were also negative for dentin sialoprotein negative showing an undifferentiated preodontogenic state. Adipocytes differentiated from N6 th -DPSCs were positively stained with Oil-Red-O and expressed both early and late adipocyte specific genes. Formation of Alizarin-red positive condensed calcium-phosphate nodules accompanied by strong expression of two osteogenic mRNAs, exhibited osteogenic differentiation. Based on the results of this study, we suggest that N6 th -DMSCs are a viable choice for cryo-banking and future usage in regenerative therapies; however, more investigations are necessary before clinical application can commence.

Toll-like receptor 4 signaling in trigeminal ganglion neurons contributes tongue-referred pain associated with tooth pulp inflammation

BackgroundThe purpose of the present study is to evaluate the mechanisms underlying tongue-referred pain associated with tooth pulp inflammation.MethodUsing mechanical and temperature stimulation following dental surgery, we have demonstrated that dental inflammation and hyperalgesia correlates with increased immunohistochemical staining of neurons for TLR4 and HSP70.ResultsMechanical or heat hyperalgesia significantly enhanced in the ipsilateral tongue at 1 to 9 days after complete Freund’s adjuvant (CFA) application to the left lower molar tooth pulp compared with that of sham-treated or vehicle-applied rats. The number of fluorogold (FG)-labeled TLR4-immunoreactive (IR) cells was significantly larger in CFA-applied rats compared with sham-treated or vehicle-applied rats to the molar tooth. The number of heat shock protein (Hsp) 70-IR neurons in trigeminal ganglion (TG) was significantly increased on day 3 after CFA application compared with sham-treated or vehicle-applied rats to the molar tooth. About 9.2% of TG neurons were labeled with DiI applied to the molar tooth and FG injected into the tongue, and 15.4% of TG neurons were labeled with FG injected into the tongue and Alexa-labeled Hsp70-IR applied to the tooth. Three days after Hsp70 or lipopolysaccharide (LPS) application to the tooth in naive rats, mechanical or heat hyperalgesia was significantly enhanced compared with that of saline-applied rats. Following successive LPS-RS, an antagonist of TLR4, administration to the TG for 3 days, the enhanced mechanical or heat hyperalgesia was significantly reversed compared with that of saline-injected rats. Noxious mechanical responses of TG neurons innervating the tongue were significantly higher in CFA-applied rats compare with sham rats to the tooth. Hsp70 mRNA levels of the tooth pulp and TG were not different between CFA-applied rats and sham rats.ConclusionsThe present findings indicate that Hsp70 transported from the tooth pulp to TG neurons or expressed in TG neurons is released from TG neurons innervating inflamed tooth pulp, and is taken by TG neurons innervating the tongue, suggesting that the Hsp70-TLR4 signaling in TG plays a pivotal role in tongue-referred pain associated with tooth pulp inflammation.