Wnt-Responsive Odontoblasts Secrete New Dentin after Superficial Tooth Injury.

The objective of our experiments was to identify new therapeutic strategies to stimulate dentin formation in an adult tooth. To address this objective, we evaluated dentin production in 2 acute trauma models: one involving a pulp exposure and the other involving a superficial dentin injury. Molecular, cellular, and histologic analyses revealed that in response to a severe injury, where the pulp is exposed to the oral cavity, cell death is rampant and the repair response initiates from surviving pulp cells and, to a lesser extent, surviving odontoblasts. When an injury is superficial, as in the case of a dentin injury model, then disturbances are largely confined to pulp tissue immediately underneath the damaged dentin tubules. We found that the pulp remained vital and innervated; primary odontoblasts upregulated HIF1α; and the rate of mineralization was significantly increased. A tamoxifen-inducible Axin2CreERT2/+; R26R mTmG/+ reporter strain was then used to demonstrate that a population of long-lived Wnt-responsive odontoblasts, which secreted dentin throughout the life of the animal, were responsible for depositing new dentin in response to a superficial injury. Amplifying Wnt signaling in the pulp stimulates dentin secretion, and in the dentin injury model, we show that a liposomal formulation of human WNT3A protein passes through dentinal tubules and is capable of upregulating Wnt signaling in the pulp. These data provide strong proof of concept for a therapeutic pulp-capping material to stimulate Wnt signaling in odontoblasts and thus improve the pulp repair response.

Osseous characteristics of mice lacking cannabinoid receptor 2 after pulp exposure.

INTRODUCTION: Endogenous cannabinoid compounds are involved in many physiological processes, including bone metabolism. Cannabinoid receptor 2 (CB2) plays a role in modulating bone density, but published research results are conflicting. Furthermore, the specific role of CB2 in inflammation-induced bone resorption and craniofacial bone density has not been reported. The objective of this study was to assess the role of CB2 in dental pulp exposure-induced periapical bone loss and mandibular bone density. METHODS: Adult female wild-type (WT) and CB2 homozygous knockout (KO) mice were used. Pulp exposures were created unilaterally in the mandibular first molars, and the pulp was left exposed to the oral cavity to induce periapical lesion formation. Mandibles were harvested 26 days after pulp exposure. Mandibular bone mineral density and periapical lesion volume were assessed using micro-computed tomographic imaging. RESULTS: Periapical lesion volume measured on the mesial root of the pulp-exposed first molar was significantly less in CB2 KO than WT mice (P < .05). No significant difference was detected between KO and WT mice in the size of the PDL space measured on the mesial root of the contralateral intact first molar. CB2 KO mice exhibited greater mandibular bone density than WT mice (P < .05). CONCLUSIONS: CB2 plays a role in mandibular bone metabolism. Increased bone density in CB2 KO mice may contribute to the smaller periapical lesion size observed after pulp exposure in KO compared with WT mice. Additional experiments are needed to further elucidate the function of CB2 and clinical implications of cannabinoids on bone and periapical pathosis.

Expression of CX3CL1 and its receptor, CX3CR1, in the development of periapical lesions.

AIM: To investigate the expression of CX3CL1 and its receptor, CX3CR1, in the development of periapical lesions induced in rats and explore the possible role of these substances in the pathogenesis of periapical lesions. METHODOLOGY: Periapical lesions in mandibular first molar teeth were established in 30 rats following pulp exposure to the oral environment. The animals were killed 0, 7, 14, 21, 28 and 42 days after lesion induction. The development of periapical lesions was investigated by histological and enzyme histochemical examination. The distributions of CX3CL1 and CX3CR1 in the periapical tissue were examined by immunohistochemistry and immunofluorescence staining. Osteoclasts and CX3CL1-positive cells were counted in each specimen. The data were then analysed by one-way anova using the SPSS 13.0 statistical package. RESULTS: The lesions expanded from days 0 to 14 and stabilized thereafter. Where expansion of the periapical lesion was most evident, numerous CX3CL1-positive cells were observed around the apical foramen and adjacent periapical areas. From days 21 to 42, subjacent connective tissues presented intense CX3CL1 immunostaining in inflammatory cells with different morphologies, such as round, oval and fibroblastic. The number of CX3CL1-positive cells increased from days 7 to 28, but decreased on day 42 post-operation. Double immunofluorescence staining showed CX3CL1- and CX3CR1-positive cells around periapical lesions surrounding the apical foramen. Most CX3CL1-positive cells were mononuclear in nature, which suggests the presence of macrophages, infiltrating neutrophils and lymphocytes, and overlapped with CX3CR1-positive cells. CONCLUSIONS: CX3CL1 and CX3CR1 are related to the development of periapical lesions. The chemokine and its receptor may be involved in the progression of tissue destruction, including bone resorption, during periapical inflammation.

Leptin receptor is up-regulated in inflamed human dental pulp.

INTRODUCTION: After leptin receptor (LEPR) identification in hematopoietic, immune system, and other tissues, a role for leptin regulating inflammation and immune response has been accepted. This study aims to describe the possible expression of LEPR in healthy human dental pulp and to compare it with LEPR expression in inflamed human dental pulp. METHODS: Twenty-one pulp samples were obtained from freshly extracted caries-free and restoration-free human third molars. In 7 third molars (inflamed pulp group), inflammation was experimentally induced before extraction. Pulp samples were processed, and LEPR expression was determined by quantitative real-time polymerase chain reaction, and the amount of LEPR protein was analyzed by immunoblot. RESULTS: All healthy and inflamed dental pulp samples expressed LEPR. Western blot analysis of human dental pulp revealed the presence of a protein with an apparent molecular weight of approximately 120 kDa, which corresponds to the estimated molecular weight of LEPR. The expression of LEPR mRNA was confirmed by quantitative real-time polymerase chain reaction analysis, and the size of the amplified fragment (338 base pairs for LEPR and 194 base pairs for cyclophilin) was assessed by agarose gel electrophoresis. The relative amount of LEPR in inflamed pulps was approximately 50% higher than in healthy pulps (P < .05). CONCLUSIONS: The presence of LEPR in human dental pulp tissues has been demonstrated for the first time. The up-regulation of LEPR expression in inflamed pulp samples suggests that leptin can play a role in inflammatory and local immune responses in human dental pulp.

Leptin expression in healthy and inflamed human dental pulp.

AIM: To investigate the expression of leptin in healthy and inflamed human dental pulp. METHODOLOGY: Twenty-one pulp samples were obtained from freshly caries- and restoration-free extracted human third molars. In seven-third molars (inflamed pulp group), inflammation was induced prior to extraction. Pulp samples were processed, and leptin expression was determined by quantitative real-time PCR (qRT-PCR) and the amount of leptin by immunoblot. RESULTS: All healthy and inflamed dental pulp samples expressed leptin. Western blot analysis revealed the presence of a protein with an apparent molecular weight of ~16 kDa in human dental pulp, which corresponds to the estimated molecular weight of leptin. The expression of leptin mRNA in dental pulp was confirmed by qRT-PCR analysis, and the size of the amplified fragments (296 bp for leptin and 194 bp for cyclophilin) was confirmed by agarose gel electrophoresis. The expression of leptin in the inflamed pulp group was significantly greater (P < 0.05) than in healthy teeth. The relative amount of leptin in inflamed pulps was almost twice than in healthy pulps. CONCLUSIONS: For the first time, the presence of leptin in human dental pulp tissues has been demonstrated. The upregulation of leptin expression in inflamed pulp samples suggests that leptin can play a role in pulpal inflammatory and immune responses.

Vascular endothelial growth factors and receptors are up-regulated during development of apical periodontitis.

INTRODUCTION: Apical periodontitis is a common inflammatory disease caused by persistent root canal infection and is characterized by bone resorption. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) have been described in many pathologic and inflammatory conditions, but their involvement in the development of apical periodontitis has not been thoroughly investigated. The aim of this study was to quantify gene expression and localize VEGF-A, VEGF-C, and VEGF-D and VEGFR-2 and VEGFR-3 in a rat model of apical periodontitis. METHODS: Molar pulps were unilaterally exposed to the oral cavity for 10 or 21 days. Jaw sections were used for localization of VEGFs and VEGFRs with immunohistochemistry and identification of cells with double immunofluorescence. Gene expression analysis for VEGF-A, VEGF-C, and VEGFR-3 of periapical tissues was performed with quantitative real-time polymerase chain reaction. RESULTS: All investigated factors and receptors were expressed immunohistochemically in blood vessels at the periodontal ligament of control teeth and were up-regulated during lesion development. In apical lesions, macrophages and neutrophils expressed all studied factors and receptors, with macrophages being an important source of VEGF-C and VEGF-D. Osteoclasts expressed VEGFR-2 and VEGFR-3, and the latter was also identified in fibroblast-like cells in the lesions. VEGF-A and VEGFR-3 gene expression was up-regulated at days 10 and 21 (P < .05). CONCLUSIONS: The current findings indicate that the VEGF family and receptors are involved in vascular remodeling and immune functions during disease development. The presence of VEGFR-2 and VEGFR-3 on osteoclasts indicates that bone resorbing activity is influenced by VEGFs.

Mechanotransducers in rat pulpal afferents.

The hydrodynamic theory suggests that pain associated with stimulation of a sensitive tooth ultimately involves mechanotransduction as a consequence of fluid movement within exposed dentinal tubules. To determine whether putative mechanotransducers could underlie mechanotransduction in pulpal afferents, we used a single-cell PCR approach to screen retrogradely labeled pulpal afferents. The presence of mRNA encoding BNC-1, ASIC3, TRPV4, TRPA1, the alpha, beta, and gamma subunits of ENaC, and the two pore K+ channels (TREK1, TREK2) and TRAAK were screened in pulpal neurons from rats with and without pulpal inflammation. ASIC3, TRPA1, TREK1, and TREK2 were present in approximately 67%, 64%, 14%, and 10% of pulpal neurons, respectively. There was no detectable influence of inflammation on the proportion of neurons expressing these mechanotransducers. Given that the majority of pulpal afferents express ASIC3 and TRPA1, our results raise the possibility that these channels may be novel targets for the treatment of dentin sensitivity.

Localized increases in corticotropin-releasing factor receptors in pulp after dental injury.

Corticotropin-releasing factor (CRF) binds to membrane-bound CRF receptors (CRF-Rs). Among the actions mediated by activated CRF-Rs is beta-endorphin (END) release from immune cells, increasing peripheral antinociception. For assessment of inflammatory regulation of CRF-R expression, rats underwent pulp exposure of left, first mandibular molars and recovered for 6 days. Control pulpal tissue consisted of contralateral, uninjured molars and left, first mandibular molars of uninjured animals. Pulp tissue specimens were incubated with antibodies directed against CRF-R (both isoforms), neurofilament, CD45, and END. We observed (1) increases in pulp CRF-R immunoreactivity after injury, (2) increased CRF-R immunoreactivity expressed in 3 distinct zones in relation to the injury, and (3) increased CD45 and END immunoreactivity in regions surrounding the pulpal abscess. CRF-Rs might provide an additional target for novel analgesics to treat pulpal pain.

Hyperpolarization-activated channels in trigeminal ganglia innervating healthy and pulp-exposed teeth.

AIM: To use immunocytochemistry for determining the expression of HCN1, HCN2 and HCN3 (three subunits of the hyperpolarization-activated cyclic nucleotide-gated current channel) in rodent trigeminal ganglia (TG) that innervate healthy teeth and determine if expression of HCN subunits is increased in TG following pulp exposure. METHODOLOGY: Pulps were exposed in right maxillary incisors of male Sprague-Dawley rats. After fixation, TG were removed, cryostat sectioned, and immunocytochemistry was utilized to study the expression of HCN1-3 subunits. Immunoreactivity of individual neurons from the maxillary region of the TG was determined with ImageJ software. Differences in the number immunopositive neurons amongst groups were tested for statistical significance with either a Yates or Pearson's chi-square or Fisher's exact probability tests depending on neuron sample size. Differences in the intensity of immunoreactivity between groups were tested for statistical significance with a Student's t-test. RESULTS: The majority of TG neurons were immunopositive for HCN1-3. Moreover, statistically significant increases in the number of TG neurons immunopositive for HCN1 and the intensity of HCN1-3 immunoreactivity were observed within hours of exposing the tooth pulp. CONCLUSIONS: HCN1-3 expression, as determined by immunocytochemistry, is increased within hours after injury. Given that I(h) can facilitate neuronal excitability, results of the current study suggest that antagonists to HCN1-3 subunits could work as analgesics in the alleviation of orofacial pain.

Immunohistochemical expression of fibronectin and tenascin after direct pulp capping with calcium hydroxide.

The aim of this study was to investigate the expression of 2 extracellullar matrix glycoproteins, fibronectin (FNC) and tenascin (TNC), following direct pulp capping with calcium hydroxide (CH). Third molars scheduled for extraction were used. Standardized class I cavities with pulp exposures were prepared. After control of bleeding, CH powder was applied in the exposure sites, which were covered with CH cement (Dycal; Dentsply) and the cavities were filled with zinc oxide-eugenol cement. Three teeth were extracted at each post-treatment period (1, 7, 14, and 30 days). Demineralized and paraffin-embedded specimens were stained for histologic technique (hematoxylin-eosin) and for immunohistochemical analysis. Anti-TNC and anti-FNC monoclonal antibodies were used with the streptavidin-biotin complex method. Generally, similar patterns of immunohistochemical expression were observed for TNC and FNC in the pulp tissue as a whole. In the exposure site, TNC immunostaining increased over time, exhibiting a thicker immunostaining pattern within 30 days. The imunohistochemical technique showed expression of both glycoproteins during pulp healing process.

Quantification of neuropeptides (calcitonin gene-related peptide, substance P, neurokinin A, neuropeptide Y and vasoactive intestinal polypeptide) expressed in healthy and inflamed human dental pulp.

AIM: To quantify the expression of calcitonin gene-related peptide (CGRP), substance P (SP), neurokinin A (NKA), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) in healthy and inflamed human dental pulp tissue. METHODOLOGY: Six pulp samples were obtained from teeth having a clinical diagnosis of acute irreversible pulpitis. Another 12 pulp samples were obtained from premolars where extraction was indicated for orthodontic purposes. In six of these premolar teeth inflammation was induced by mechanical pulp exposure prior to sample collection. All samples were processed and 125I-labelled; neuropeptides were quantified by competition assays. ANOVA and Mann-Whitney's (post hoc) tests were used to establish statistically significant differences between the groups. RESULTS: Expression of five neuropeptides was found in all human pulp samples. Statistical analysis revealed a significantly higher (P < 0.05) expression of CGRP, SP, NKA and NPY in both inflammatory conditions compared with healthy pulp control values. VIP expression remained stable during the inflammatory conditions. CONCLUSION: Expression of CGRP, SP and NKA released from C-fibres and NPY released from sympathetic fibres is significantly higher in the inflamed human pulp compared with healthy pulp. Expression of VIP released from parasympathetic fibres is not increased during the inflammatory conditions of human dental pulp.

Adhesive resin and the hydrophilic monomer HEMA induce VEGF expression on dental pulp cells and macrophages.

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis by inducing endothelial cell proliferation, migration and survival. Direct pulp capping with an adhesive resin system was shown to induce local increase in blood vessel density and lack of dentin bridging. However, the mechanisms involved in the increase in blood vessel density observed near the pulp exposures capped with an adhesive resin are largely unknown. OBJECTIVES.: To investigate the effect of an adhesive resin or one of its hydrophilic monomers (HEMA), in the expression of VEGF by pulp cells. METHODS.: Mouse odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD-21), gingival fibroblasts, and macrophages were exposed to SingleBond (3M) or to 0-1000nM HEMA. VEGF expression was evaluated by ELISA and semi-quantitative RT-PCR. RESULTS AND SIGNIFICANCE.: VEGF expression was upregulated in MDPC-23 cells exposed to HEMA (p<0.001) or to SingleBond (p<0.018), and in macrophages exposed to HEMA (p<0.001) or SingleBond (p=0.001). In contrast, VEGF expression remained unchanged in undifferentiated pulp cells (OD-21), or fibroblasts exposed to either HEMA or Single Bond (p>0.05). Treatment with SingleBond or HEMA did not affect VEGF expression at the mRNA level of any cell type evaluated here, suggesting that the induction of VEGF expression in these cells is regulated primarily at the post-transcriptional level. These findings suggest that VEGF is involved in the regulation of pulp neovascularization observed in response to the application of adhesive resins at site of pulp exposure.

Effects of pre-emptive morphine, ibuprofen or local anesthetic on fos expression in the spinal trigeminal nucleus following tooth pulp exposure in the rat.

In the present study, we used Fos expression as an index of nociceptive input to the spinal trigeminal nucleus after exposure of the coronal pulp tissue of maxillary right first molars and examined the effects of pretreatment with an opioid, a nonsteroidal anti-inflammatory drug or a local anesthetic before pulp exposure. Exposure of the tooth pulp produced a significant increase in Fos-like immunoreactivity in the superficial laminae of subnucleus caudalis; pretreatment with a control infiltration injection of saline directly above the maxillary molar 30 min before pulp exposure had no effect on Fos expression. Pretreatment with morphine 30 min before pulp exposure dose-dependently (2.5, 5, and 10 mg/kg subcutaneously) reduced Fos expression in subnucleus caudalis whereas pretreatment with ibuprofen (10-100 mg/kg subcutaneously) did not significantly affect Fos expression. Local anesthetic pretreatment was effective in reducing Fos expression only for the long acting bupivacaine; lidocaine without and with epinephrine (1:100,000) failed to significantly affect Fos expression. These results suggest that pre-emptive opioid treatment can decrease postoperative central nervous system changes associated with tooth pulp injury, and therefore, may decrease postoperative pain. Given the effects of local anesthetic on Fos expression, a combination of long acting local anesthetic with pre-emptive opioid would likely be most efficacious in decreasing postoperative dental pain.

Patterns of fluoro-gold entry into rat molar enamel, dentin, and pulp.

Permeabilities of enamel and dentin are not fully understood despite their importance for caries, restorative materials, and pulp-dentin-enamel interactions. We have found that Fluoro-Gold is useful for examining tooth permeability, and we designed studies to test the effects of aging, injury, neural function, and dentinal repair on its influx into vital rat teeth. We used fluorescence microscopy and immunocytochemistry to show that Fluoro-Gold rapidly penetrates enamel, the dentin-enamel junction, and outer dentinal acellular tubules, and then concentrates in odontoblasts, where it remains for weeks. As predicted, influx was greatest in immature teeth, and formation of reparative dentin impeded it. We expected that denervation would disrupt influx, because of neural regulation of dentinal fluid movement, but it did not. Damage to odontoblasts under injured dentin caused increased influx and efflux of Fluoro-Gold. Analysis of our data suggests that permeabilities of enamel and dentin to Fluoro-Gold are age-related, inter-dependent, and regulated by odontoblasts.

Comparison of acetaminophen, ibuprofen, and nabumetone therapy in rats with pulpal pathosis.

The purpose of this study was to determine if daily treatment with acetaminophen, ibuprofen, or nabumetone would alter prostaglandin E2 (PGE2)/leukotriene B4 (LTB4) synthesis in exposed, infected dental pulp and periradicular tissue in rats. Dental pulp of the bilateral first and second mandibular molars were exposed for 15 or 24 days. Rats were divided into four groups. A daily pharmacological oral dose of one of the medications or the suspending solution alone was administered to the designated group. Eicosanoids were extracted from pulpal and periradicular tissues and assayed. PGE2 was significantly elevated in pulp-exposed, nontreated rats and was significantly reduced in the ibuprofen- and nabumetone-treated groups. LTB4 was significantly increased in all pulp-exposed groups at 15 days when compared with control nonexposed groups. Results showed that only ibuprofen reduced LTB4 in the exposed dental pulp at 24 days, although it did not do so at 15 days. Repetitive treatment with acetaminophen did not suppress PGE2/LTB4 in pulp-exposed molars.

Pulpal exposure alters neuropeptide levels in inflamed dental pulp and trigeminal ganglia: evaluation of axonal transport.

Dental pulp is richly innervated with neurons containing calcitonin gene-related peptide (CGRP) or substance P (SP). Prior studies have demonstrated that inflammation alters these pulpal neuropeptides. In this study, we used a radioimmunoassay to evaluate the specificity of this response and the contribution of axonal transport. Rat mandibular molars were exposed and immunoreactive CGRP (iCGRP) and immunoreactive SP were measured. At 7 to 14 days after exposure, both pulpal iCGRP (73%) and immunoreactive SP (135%) displayed peak increases above control levels. This response was somatotopically restricted, with no changes observed in contralateral (intact) molars, or in ipsilateral mandibular molars after exposure of maxillary molars. Transection of the inferior alveolar nerve on day 13 significantly reduced pulpal levels of iCGRP on day 14. Collectively, these studies indicate that pulpal inflammation evokes a selective alteration in neuropeptide levels, due at least in part to alterations in transport or synthesis of neuropeptides in the trigeminal ganglion.

Effect of inflammation on the delivery of drugs to dental pulp.

Although numerous studies have evaluated the effects of drugs on inflammation, comparatively few studies have evaluated the effects of inflammation on drugs. In this study, we have evaluated whether pulpal inflammation alters the delivery of flurbiprofen or Evan's blue, two agents that bind with high affinity to plasma proteins. The results indicate that pulpal inflammation alters the delivery of these agents to inflamed molars, that activation of capsaicin-sensitive nerves increases pulpal content of protein-bound agents, and that reduced pH increases free drug concentrations of flurbiprofen. Thus, alterations in both plasma extravasation and tissue pH seem to be relevant factors regulating the delivery and bioavailability of this nonsteroidal anti-inflammatory drug to dental pulp. Because many drugs used in endodontics (e.g. nonsteroidal anti-inflammatory drugs, clindamycin, bupivacaine, etc.) are heavily bound to plasma proteins, it is likely that the status of pulpal inflammation is a contributing factor in modifying the pharmacological efficacy of these agents.

Pathogenesis of induced rat periapical lesions.

Studies of the mechanisms of pathogenesis of periapical lesions were undertaken using a rat model of surgical pulp exposure. In this model, periapical lesions develop rapidly between days 0 and 15 (active phase) and more slowly thereafter (chronic phase). A Gram-negative anaerobic flora, similar to that seen in human beings, are quickly established. Lesions contain a mixed inflammatory cell infiltrate consisting of T cells, neutrophils, B cells, macrophages, and plasma cells. Helper T cells predominate during the active phase, whereas suppressor T cells are more frequent in the chronic phase. Extracts of periapical lesions contain bone-resorbing activity, the highest levels of which are present when lesions are actively expanding. Most bone-resorbing activity is mediated by the cytokine interleukin-1 alpha, as determined by biochemical criteria and antibody neutralization studies. Prostaglandin2 accounts for 10% to 15% of resorptive activity. Cells that express interleukin-1 alpha were identified in pulp beginning on day 2 after exposure and in periapical tissue beginning on day 7, as determined by in situ hybridization and immunostaining. Macrophages, fibroblasts, neutrophils, and osteoclasts were positive for interleukin-1 alpha mRNA and protein. Cells that express tumor necrosis factor alpha were also detected, whereas cells expressing interleukin-1 beta or tumor necrosis factor beta were absent. Finally, periapical bone destruction was inhibited by 60% by treatment with interleukin-1 receptor antagonist. These studies establish a key role for interleukin-1 alpha in the pathogenesis of periapical lesions in the rat model.

Analysis of low affinity nerve growth factor receptor during pulpal healing and regeneration of myelinated and unmyelinated axons in replanted teeth.

Nerve regeneration was examined in rat molars that were briefly extracted and then replanted in the socket for 1-90 days. Immunocytochemistry was used to evaluate neural and nonneural immunoreactivity (IR) for low affinity nerve growth factor receptor (p75-NGFR) and for laminin and calcitonin gene-related peptide (CGRP). Three different types of pulpal response to replantation were found. Type I: Some replanted teeth had mild injury and still contained coronal odontoblasts and associated fibroblasts that retained p75-NGFR-IR; they continued regular dentin formation and had excellent reinnervation. Type II: Teeth with intermediate injury lost most or all of the coronal pulp tissue, but they regenerated odontoblast-like cells that formed irregular dentin, they had numerous dispersed p75-NGFR-IR fibroblasts in crown pulp during early regeneration, and they had excellent reinnervation. Type III: Severely injured teeth lost their original pulp; they filled with dense connective tissue and bone and had poor reinnervation. After Type I or II injury the Schwann cells around degenerating myelinated and unmyelinated axons had increased expression of p75-NGFR by 1-3 days. By 7-10 days those Schwann cells had formed hollow tubes (bands of Bungner) along the degenerating axon tracks. They maintained their increased p75-NGFR-IR during and after regeneration of unmyelinated axons, whereas Schwann cells involved in remyelination lost p75-NGFR-IR at that stage. The number of CGRP-IR axons in the regenerating pulp increased from 7 to 90 days. Laminin-IR increased in all replanted teeth at 3-10 days and only returned to normal patterns in teeth with Type I or Type II response at 20-90 days. The special p75-NGFR-IR of pulpal fibroblasts of adult rat molars did not usually persist in regenerated, reinnervated pulp. The extensive depletion of fibroblast p75-NGFR-IR and the continuing enhanced p75-NGFR-IR in unmyelinated nerve fibers at 90 days show that altered growth factor conditions characterize regenerated pulp of replanted teeth.

Effect of injury on pulpal levels of immunoreactive substance P and immunoreactive calcitonin gene-related peptide.

Neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P are present in dental pulp in relatively high concentrations. Previous studies have demonstrated that the staining density of immunoreactive CGRP (iCGRP) changes in dental pulp after tissue injury. This study evaluated injury-related changes in levels of both immunoreactive CGRP (iCGRP) and immunoreactive substance P (iSP) in dental pulp using radioimmunoassays. After pulpal exposure, iSP levels decreased to about 10% of baseline values, while iCGRP levels decreased to about 45% of baseline measures. After dentin exposure with acid etch, iSP levels decreased to about 10 to 20% of baseline measures, while iCGRP levels decreased to 60% of baseline values. For both forms of injury, iSP decreased to a greater extent than did iCGRP levels. Collectively, these findings indicate that pulpal neuropeptides undergo dynamic, injury-specific, and peptide-specific responses following trauma to dental pulp.