Rat Molar Pulp Research Articles

Proliferation of rat molar pulp cells after direct pulp capping with dentine adhesive and calcium hydroxide

The aim was to evaluate the proliferation of pulp cells 1, 3 and 7days after direct pulp capping with the dentine adhesive Gluma Comfort Bond (GCB) and to compare it with calcium hydroxide (Ca(OH)(2)). An occlusal cavity was prepared in 72 molar teeth of 36 Wistar rats. Then GCB or Ca(OH)(2) was placed on the exposed pulp. All cavities were restored with composite. After 1, 3 and 7days, the animals were sacrificed. One hour prior sacrification, 5-bromo-2'-desoxyuridine (BrdU) was injected into the intraperitoneal cavity for immunohistological analysis of 18 animals. BrdU was incorporated into the DNA to tag proliferating cells using an antibody staining. Three animals served as controls and were not further treated. The number of the tagged cells was statistically analysed by comparing the results of the three groups. In 18 rats, routine histological analysis was performed in order to evaluate the pulp tissue for bacterial infection, inflammatory cells and necrosis. The marked cells were identified as fibroblasts, endothelial cells (after 1, 3 and 7days) and Höhl cells (after 7days). One day after capping, significantly more cells were stained in the GCB than in the Ca(OH)(2) group (p < 0.05). After 3days, significantly more cells were stained in the GCB than in the Ca(OH)(2) and the control group (p < 0.016). Direct contact of GCB with pulp tissue leads to an increased formation of granulation tissue (fibroblasts, endothelial cells) because of an inflammatory reaction. This may be explained by missing antibacterial effect and foreign body reactions. Also, GCB may have a negative effect on Höhl cells.

Possibility of application of calcium carbonate in pulpotomy of rat molars

We investigated restitution processes in mechanically exposed rat molar pulp during pulpotomy with calcium carbonate (CC). The results of the CC treatment were then compared with Calvital ® -containing calcium hydroxide (CH). Micro-computerized tomography (micro-CT), hematoxylin and eosin (H&E) staining and immunoreactivity for nestin, dentin matrix protein-1 (DMP-1) and osteopontin (OPN) were also analyzed. The increment of dentinlike calcified tissue in the pulp was observed by micro-CT. Both CC and CH groups induced pulpotomy resulted in changes associated with inflammation followed by progressive odontoblasts differentiation, dentin matrix secretion and dentin-like bridge formation. Necrotic layer formation and moderate to severe inflammation occurred during the early stages in the CH group. Necrotic layer formation was not observed in the CC group and only associated with mild to moderate inflammation. Immunoreactivity of nestin was observed earlier in the CC group than the CH group. In the CC group, immunoreactivity of DMP-1 was identified beneath the amputated site after 7 days, before increasing until 28 days, and immunoreactivity of OPN was observed in the dentin-like bridge at 28 days, which was also similar to the CH group. These findings suggested that the primary processes of reparative dentinogenesis after pulpotomy with CC may involve natural pulpal wound-healing mechanisms that are similar to the restitution processes observed during pulpotomy with CH. However, CC may prove to be less irritation and more calcified tissue formation than traditional CH-based materials when used as a pulpotomy agent.

Rat molar teeth as a study model for direct pulp capping research in dentistry

The aim of this review is to evaluate the suitability of rat molar teeth in preclinical evaluation of medical devices for direct pulp capping. The ISO standard 7405 states clearly that only non-rodent mammals are suitable species for animal research in dentistry. Furthermore, without clear justification a considerable number of researchers previously rejected results of animal experiments concerning preclinical evaluation of the biocompatibility of dental materials undertaken in rat molar teeth. However, in the past 50 years about 70 studies have been published using rat molar teeth in order to evaluate direct pulp capping, pulpotomies and tissue reactions after pulp exposure. Numerous studies showed that the healing of rat molar pulp tissue after direct pulp capping is histologically comparable with humans and other animal species pulp tissue. Rat molar teeth, including pulp tissue, can be seen anatomically, histologically, biologically, and physiologically as miniature human molar teeth. Hence, the essential biological reactions of the pulp tissue and the interaction during the different stages of wound healing of rat molar teeth are comparable with that of other mammals. Rat molar teeth are a valid study model in order to provide valuable data concerning pulp tissue reaction after direct pulp capping and related questions in dentistry. Therefore, the use of rats may significantly reduce the number of currently used higher animals in research. Tests in higher developed animals should be limited to experiments which clarify inconsistent results. However, some technical difficulties, like the small size of rat molar teeth must be dealt with before undertaking any research.

Formation of Dentinal Bridge on Surface of Regenerated Dental Pulp in Dentin Defects by Controlled Release of Fibroblast Growth Factor–2 From Gelatin Hydrogels

Introduction Pulp regeneration therapy is important to overcome the limitations of conventional therapy to induce reparative dentinogenesis. In the present study, we examined the effects of controlled release of different dosages of fibroblast growth factor–2 (FGF-2) from gelatin hydrogels to regenerate the dentin-pulp complex. Methods After the amputation of dental pulp of rat molars, gelatin hydrogels incorporating various dosages of FGF-2 were individually implanted into dentin defects above the sites of the amputated pulps. Histologic changes as well as the expression of dentin matrix protein–1 (DMP-1) and nestin in the dentin defect area above the amputated pulp were analyzed. Results We found that controlled release of high doses of FGF-2 from gelatin hydrogels induced DMP-1–positive calcified particles in the proliferating pulp, whereas a moderate dose of FGF-2 induced DMP-1–positive dentinal bridge on the surface of the proliferating pulp. These findings indicate that the dosage of released FGF-2 has an influence on the structure of calcified tissue regenerated in dentin defects. In addition, pulp cells near calcified tissues regenerated in dentin defects were nestin-negative, suggesting that the calcified tissues might be osteodentin. Conclusions Our results showed that the dentin regeneration on amputated pulp, not reparative dentin formation toward amputated pulp, can be regulated by adjusting the dosage of FGF-2 incorporated in biodegradable gelatin hydrogels.

Histochemistry of nerve fibres double labelled with anti-TRPV2 antibodies and sensory nerve marker AM1-43 in the dental pulp of rat molars

AM1-43 can label sensory nerve fibres and sensory neurons. Permeation of non-selective cation channels of the nerve cell membrane is suggested to be the mechanism responsible for labelling. To identify these channels, two candidates, TRPV1 and TRPV2 were examined by immunocytochemistry in the dental pulp and trigeminal ganglion of rats injected with AM1-43. A part of AM1-43-labelled nerve fibres was also positive for anti-TRPV2 antibody but negative for anti-TRPV1 antibody in the dental pulp. In the trigeminal ganglion, a part of the neuron showed both bright AM1-43 labelling and anti-TRPV2 immunolabelling, but neurons double labelled with AM1-43 and TRPV1 were rare. These results suggest that TRPV2 channels, but not TRPV1 channels, contribute to the fluorescent labelling of AM1-43 in the dental pulp.

Short-term effects of amelogenin gene splice products A+4 and A-4 implanted in the exposed rat molar pulp

In order to study the short-time effects of two bioactive low-molecular amelogenins A+4 and A-4, half-moon cavities were prepared in the mesial aspect of the first maxillary molars, and after pulp exposure, agarose beads alone (controls) or beads soaked in A+4 or A-4 (experimental) were implanted into the pulp. After 1, 3 or 7 days, the rats were killed and the teeth studied by immunohistochemistry. Cell proliferation was studied by PCNA labeling, positive at 3 days, but decreasing at day 7 for A+4, whilst constantly high between 3 and 7 days for A-4. The differentiation toward the osteo/odontoblast lineage shown by RP59 labeling was more apparent for A-4 compared with A+4. Osteopontin-positive cells were alike at days 3 and 7 for A-4. In contrast, for A+4, the weak labeling detected at day 3 became stronger at day 7. Dentin sialoprotein (DSP), an in vivo odontoblast marker, was not detectable until day 7 where a few cells became DSP positive after A-4 stimulation, but not for A+4. These results suggest that A +/- 4 promote the proliferation of some pulp cells. Some of them further differentiate into osteoblast-like progenitors, the effects being more precocious for A-4 (day 3) compared with A+4 (day 7). The present data suggest that A +/- 4 promote early recruitment of osteogenic progenitors, and evidence functional differences between A+4 and A-4.

Formation of Dentin-like Particles in Dentin Defects above Exposed Pulp by Controlled Release of Fibroblast Growth Factor 2 from Gelatin Hydrogels

The induction of dentin formation on exposed dental pulp is a major challenge in research on the regeneration of the dentin-pulp complex. We examined the effects of fibroblast growth factor 2 (FGF2), which was delivered in either a collagen sponge (noncontrolled release) or incorporated into gelatin hydrogels (controlled release), on the formation of dentin in exposed rat molar pulps. During the early phase of pulp wound healing, pulp cell proliferation and invasion of vessels into dentin defects above exposed pulp were induced in both groups. In the late phase, the induction of dentin formation was distinctly different between the 2 types of FGF2 release. The noncontrolled release of free FGF2 from collagen sponge induced excessive reparative dentin formation in the residual dental pulp, although dentin defects were not noted. In contrast, controlled release of FGF2 from gelatin hydrogels induced the formation of dentin-like particles with dentin defects above exposed pulp. These results suggest the possibility of a novel therapeutic approach for dentin-pulp complex by controlled release of bioactive FGF2.

Dentonin, a MEPE Fragment, Initiates Pulp-healing Response to Injury

Phosphorylated extracellular matrix proteins, including matrix extracellular phosphoprotein (MEPE), are involved in the formation and mineralization of dental tissues. In this study, we evaluated the potential of Dentonin, a synthetic peptide derived from MEPE, to promote the formation of reparative dentin. Agarose beads, either soaked with Dentonin or unloaded, were implanted into the pulps of rat molars, and examined 8, 15, and 30 days after treatment. At day 8, Dentonin promoted the proliferation of pulp cells, as visualized by PCNA-labeling. RP59-positive osteoblast progenitors were located around the Dentonin-soaked beads. PCNA- and RP59-labeling were decreased at day 15, while osteopontin, weakly labeled at day 8, was increased at 15 days, but dentin sialoprotein was undetectable at any time. At 8 days, precocious reparative dentin formation occurred in pulps containing Dentonin-soaked beads, with formation slowing after 15 days. These results suggest that Dentonin affects primarily the initial cascade of events leading to pulp healing.

Laser capture microdissection of rat periodontal ligament for gene analysis

The periodontal ligament (PDL) is a connective tissue interposed between two hard tissues, viz., the root of a tooth and the alveolar bone, which makes it difficult to obtain directly. In the study reported here, PDL, subgingival connective tissue and pulp of rat molars were extracted directly by laser capture microdissection and the gene expression of TGF-β1 on the microdissected PDL was examined. The maxillae of rats were dissected and rapidly immersed in isopentane cooled with liquid nitrogen. Serial frontal sections of the rat first molar area were used for immunohistochemistry and for laser capture microdissection to localize the TGF-β1 gene. Gene expression and immunohistochemical localization of TGF-β1 also were examined in the pulp and subgingival connective tissues. TGF-β1 was located immunohistochemically in the fibroblasts in the PDL. A considerable amount of RNA was obtained by laser capture microdissection of these three tissues for analysis of gene expression. The reverse transcription- polymerase chain reaction demonstrated that glyceraldehyde 3-phosphate dehydrogenase was amplified in all three tissues, and that TGF-β1 was detected in the PDL. Laser capture microdissection makes it possible to analyze the gene expression of PDL and expression of TGF-β1in the PDL suggests that this gene could function in maintaining PDL.

Immunocytochemical evidence that most sensory neurons of the rat molar pulp express receptors for both glial cell line-derived neurotrophic factor and nerve growth factor

Most pulpal afferent neurons have cytochemical features in common with the class of nociceptors that express neuropeptides and respond to NGF, while very few bind the plant lectin IB4, a widely used marker for the class of nociceptors that respond to the GDNF family of neurotrophic factors. The present study was undertaken to determine whether the GDNF receptor, GFRalpha-1, is expressed by pulpal afferents, and, further, to determine whether tooth injury evokes changes in expression of the GDNF and NGF receptors among pulpal afferents. The tracer, fluoro-gold (FG), was applied to shallow cavities in dentin of first and second maxillary molars. After 4 weeks, the molars of one side received a test injury consisting of a deeper cavity that exposed pulp horns. Animals were perfusion fixed 2 days later, and sections of the trigeminal ganglia were double immunostained with combinations of antibodies against GFRalpha-1, and TrkA. Under control conditions, GFRalpha-1 immunostaining was observed in 72% of neurons that projected to the molar pulp, TrkA in 78%, and immunostaining for both receptors was observed in 65% of pulpal afferents. Tooth injury evoked up-regulation of GFRalpha-1 expression (to 93%) and a slight down-regulation of TrkA expression (67%) among tooth afferents, while there was no discernable change in the proportion of pulpal afferents that expressed both TrkA and GFRalpha-1 (to 61%).

Delta (δ) opioid receptors in small and medium-sized trigeminal neurons supporting the dental pulp of rats

The control of pain perception is a challenge in clinical dentistry, most prominent during tooth pulp inflammation. The tooth pulp is a well-defined target, and is densely supplied by a sensory trigeminal innervation. Opioids are signaling molecules that are suggested to participate in pain perception. Here we analysed the presence of delta opioid receptor (DOR) in trigeminal neurons innervating the tooth pulp of rat molars. Immunohistochemical and ultrastructural analysis revealed that DOR was identified in peripheral nerves in the molar dental pulp, both in the root and the coronal pulpal parts, with branching in the highly innervated subodontoblast layer. DOR was localised in about one third of all the trigeminal dental neurons, identified by means of retrograde neuronal transport of fluorogold (FG) from the dental pulp. Of the DOR-labeled neurons, nearly all were small and medium-sized (147.5-1,810.2 microm(2), mean 749.1 +/- 327.3 microm(2)). Confocal microscopy confirmed that DOR-immunoreactivity was distributed as granules in the neuronal cytoplasm. Approximately 70% of the DOR-immunoreactive neurons were also immunopositive for vanilloid receptor 1 (TRPV1). Ultrastructural analysis demonstrated DOR-immunoreactivity in the unmyelinated and in some of the myelinated nerve fibers in the dental pulp. These results indicate that DOR may influence the function in a subset of small and medium-sized trigeminal sensory neurons supporting the tooth, which are mainly known for their ability to mediate nociceptive stimuli. Agonists, acting on DOR, may thus have an influence on a subpopulation of nociceptive neurons supporting the rat tooth.

A Real Time Quantitative PCR Analysis and Correlation of COX-1 and COX-2 Enzymes in Inflamed Dental Pulps Following Administration of Three Different NSAIDs

Dental pain is encountered daily by clinicians. Nonsteroidal anti-inflammatory drugs (NSAIDs) commonly used for pain management are traditionally cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitors, and more recently selective COX-2 inhibitors. This study was designed to identify and quantify COX-1 and COX-2 gene expression level in inflamed rat molar pulps after administration of three NSAIDs: Celebrex, Vioxx, and Advil. Fifty male Wistar rats had their first and second molar pulps exposed and sealed with Cavit for 4 days. Rats were randomly divided into the three drug groups and two control groups. RNA was isolated from the rat pulps. Real Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction assay, a relatively new PCR technique, was used to quantify COX-1 and COX-2 mRNA. Statistical analysis demonstrated no significant differences in COX-1 and COX-2 levels among the drug groups. However, Vioxx and Advil significantly reduced COX-2 expression levels compared to inflamed (positive control) pulps (p < 0.05).

Porcine enamel matrix derivative enhances the formation of reparative dentine and dentine bridges during wound healing of amputated rat molars.

We examined the biological effects of porcine enamel matrix derivative (EMD; Emdogain) on the formation of reparative dentine and dentine bridges in rat molars after pulp amputation. The pulp chambers of upper molars of Wistar rats were perforated and the amputated pulp surfaces were directly capped with either EMD or its carrier propylene glycol alginate (PGA) as control. The cavities were then restored with glass-ionomer cement. On post-amputation days 4-30, the dissected maxillae were examined by light and electron microscopy. In PGA-capped pulp, reparative dentine had been formed over the dentine walls under the prepared cavity on day 7 post-amputation and its thickness extended until day 30. On day 30, as well as reparative dentine formation, diffuse calcification had occurred beneath the amputated wound surfaces. Dentine bridge formation under the amputated coronal pulp surface was observed in 18.2% of amputated pulp on day 30. In EMD-capped pulp, reparative dentine had already been formed by odontoblast-like cells over the dentine walls, already on day 4 post-amputation, and its thickness extended until day 30. The Ca and P weight % and Ca/P ratio of reparative dentine matrix were similar to those of pre-existing dentine matrix, and these values were not different between PGA and EMD-capped pulp. Dentine bridge formation was observed in 27.3% of EMD-capped pulp on day 30. Our results suggest that EMD enhances the formation of both reparative dentine and dentine bridges during wound healing of amputated rat molar pulp.

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.

Expression of adenosine triphosphate P2X3 receptors in rat molar pulp and trigeminal ganglia.

The objective of this study was to investigate the expression of adenosine triphosphate P2X3 receptor subunits in rat molar pulp and trigeminal ganglia and their relationship to substance P. Rat molar pulp and trigeminal ganglia were fixed and sectioned. Double immunostaining with anti-P2X3 and anti-substance P were used to localize P2X3 and substance P expression simultaneously with different stains. P2X3 immunoreactivity (IR) fibers were present in root and coronal pulp. P2X3-IR fibers formed subodontoblastic plexus and advanced into the predentin and dentin. P2X3-IR neurons were localized in small and medium-sized cells in trigeminal ganglia. Colocalization of P2X3 and substance P was not found in either molar pulp or trigeminal ganglia. The results of this study indicated that adenosine triphosphate in pulp tissues may stimulate a subpopulation of non-substance P trigeminal afferent fibers through activation of P2X3 receptors.

Immunodetection of Osteopontin at Sites of Resorption in the Pulp of Rat Molars

Osteopontin (OPN) has been proposed to act as a substrate for osteoclast adhesion during bone resorption. The aim of the present study was to examine the presence and distribution of OPN at sites of resorption in traumatized radicular pulp. The upper first molars of 6-week-old male Sprague-Dawley rats were luxated and then repositioned in the original sockets. The animals were sacrificed by intracardiac perfusion at 10 and 14 days after tooth reimplantation. The teeth were decalcified in EDTA and then processed for embedding in paraffin for histochemistry or LR White resin for immunocytochemistry. Odontoclasts were identified by their multinucleated morphology and expression of tartrate-resistant acid phosphatase (TRAP). Osteopontin was immunolocalized using postembedding colloidal gold labeling with a chicken egg yolk anti-rat OPN antibody. After reimplantation of the teeth, TRAP-positive cells were present along the pulp dentin wall. Osteopontin was not consistently detected at exposed predentin/dentin surfaces. However, gold particles were often found at the margin of resorption lacunae. Labeling was also seen over the Golgi region and cytoplasmic vesicles of odontoclasts and of neutrophils and fibroblast-like cells. The results suggest that accumulation of OPN at the predentin/dentin surface is not a prerequisite for adhesion of odontoclasts to the wall substance and that recruited odontoclasts produce OPN locally to mediate cell and/or matrix events within the resorption lacuna.

Age-related changes in the immunoreactivity of the monocyte/macrophage system in rat molar pulp after cavity preparation.

Our objective was to compare the response of the monocyte/macrophage system of dental pulp to cavity preparation in aged rats (12 and 18 months old) with that seen in young adult rats (3 and 6 months old). Cavities were prepared on the upper first molars, and the lower first molars served as intact controls. Specimens were collected at 1 day after cavity preparation, and cryostat sections were made. Accumulation of OX6+ antigen-presenting cells along the pulp-dentin border and a marked increase in cell size ED2+ resident macrophages were noted in both young adult and aged rats after cavity preparation. In both cases, the number of ED1+ cells increased significantly after cavity preparation because of infiltrating monocytes. These findings suggest that the pulpal defense reaction of the monocyte/macrophage system to cavity preparation in aged rats does not differ markedly from that in young adult rats.

Differential repair responses in the coronal and radicular areas of the exposed rat molar pulp induced by recombinant human bone morphogenetic protein 7 (osteogenic protein 1)

Bone morphogenetic protein 7 (BMP 7), also termed osteogenic protein 1, a member of the transforming growth-factor superfamily, was examined for its efficacy in inducing reparative dentinogenesis in the exposed pulps of rat molars. To determine if the reaction was dose-dependent, collagen pellets containing 1, 3 or 10 μg of recombinant BMP 7 were inserted in intentionally perforated pulps (10–12 pulps per group) in the deepest part of half-moon class V-like cavities cut in the mesial aspect of upper first molars. As controls, the collagen carrier (CC group) alone and calcium hydroxide (Ca group) were used as capping agents. All cavities were then restored with a glass-ionomer cement. Half of the animals were killed after 8 days and the other half after 28 days, by intracardiac perfusion of fixative. The molars were processed for histological evaluation by light microscopy. No difference in effect could be detected between the three concentrations of BMP 7 groups at either time interval. After 8 days, all groups showed varying inflammation, from mild of severe, and the Ca group demonstrated early formation of a reparative dentine bridge. At 28 days the CC group displayed irregular osteodentine formation, leaving some unmineralized areas at the exposure site and interglobular unmineralized areas containing pulp remnants. In the Ca-treated pulps, the initial formation of thick reparative osteodentine bridges that sealed more or less completely the pulp perforation was followed, in the deeper part, by irregular tubular dentine. In most BMP 7-treated specimens, the initial inflammation has resolved at 8 days and at 28 days heterogeneous mineralization or osteodentine filled the mesial coronal pulp. They also had complete filling of the radicular pulp by homogenous mineralization in the mesial root; this reaction was found in 11 teeth in the BMP 7 group, one tooth in the CC group an none of the Ca group. These results emphasize the biological differences the coronal and radicular parts of the pulp, and the potential of bioactive molecules such as BMP 7 to provide an a alternative conventional endodontic treatments.

The Effect of Unilateral Sympathectomy and Cavity Preparation on Peptidergic Nerves and Cells in Rat Dental Pulp

Recent evidence suggests interactions between primary afferent nociceptors and postganglionic sympathetic efferents in the pathogenesis of inflammation. The effect of unilateral removal of the superior cervical ganglion on the innervation pattern of nerve fibers immunoreactive (IR) to calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY), as well as the occurrence of cells in the injured and uninjured rat molar pulp, was investigated. Light microscopic immunocytochemistry demonstrated that the molar pulps contralateral to the sympathectomy contained a NPY-IR nerve fiber network more dense and heavily stained than unoperated control rats. The NPY-IR fibers showed, however, no sprouting after deep cavity preparation. There was no compensatory increase in CGRP- and SP-IR nerve fibers in the dental pulp after unilateral sympathectomy, although a significant increase in cells IR to CGRP and SP was found in the ipsilateral trigeminal ganglion. Unilateral sympathectomy induced a significant increase in cell density both in the inflamed and in the uninflamed dental pulp bilaterally. Our results demonstrate, for the first time, a trophic effect of the sympathetic nerves on cells in the dental pulp, indicating that an imbalance of sympathetic nerves may induce inflammation and pain in teeth.

Occurrence and Histopathologic Features of Odontoclastic Resorption on the Internal Dentin Wall after Replantation of Rat Molars.

The etiology, pathogenesis and prognosis of internal root resorption in human dentition remain to be better understood. The present study aimed to gain better insight into the pathways by which multinucleated odontoclasts are recruited and activated on the dentin wall, giving rise to resorption lacunae in the disrupted pulp environment after tooth replantation. The upper first molars were luxated and then repositioned in the original socket. Post-treatment events were monitored over 28 days, and tissue specimens were prepared at time intervals. Histologic examination of the mesial and disto-palatal roots of the replanted first molars verified the occurrence of resorption lacunae accompanying TRAP-positive multinuclear cells on the internal dentin wall around day 7 after tooth replantation. Ninety-nine resorption lacunae were detected for the histologically examined 46 roots. These lacunae were classified into A and B types, which appeared to correspond to surface and inflammatory types of external root resorption, respectively. Key events leading to the establishment of odontoclastic resorption of the pulp-side dentin wall were the replacement of the innate pulp tissue with the ingrowth of granulation tissues and the assembly of a new cell population comprising macrophages and fibroblastic stromal cells. The overall results support the hypothesis that the innate pulp of rat molars possesses in potency inhibitory functions against undesirable resorption activities, but after impairment of the innate pulp environment after tooth replantation, a new paradigm of cell-matrices and cell-cell interactions may act cooperatively to induce clastic activities on the internal predentin/dentin wall.