Odontoblast response to cavity preparation with Er:YAG laser in rat molars: an immunohistochemical study.

This study aimed to examine the dynamics of odontoblast-lineage cells following cavity preparation with erbium:yttrium-aluminum-garnet (Er:YAG) laser in rat molars. Cavity preparation was made with Er:YAG laser in the mesial surface of the maxillary left first molar of 8-week-old Wistar rats. Contralateral first molar served as unirradiated control. Immediately, 6 and 12 h and 1, 2, 3, 5 and 7 days after the lasing (n = 5, each), specimens were collected and processed for immunohistochemistry for heat-shock protein (HSP)-25 and nestin as markers for odontoblast-lineage cells. Cell proliferation assay using bromodeoxyuridine (BrdU) labeling was also performed. Unirradiated teeth showed HSP-25- and nestin-immunoreactivity in odontoblasts. At 6-12 h after irradiation, the odontoblastic layer was disorganized and some of odontoblasts lost the immunoreactivity to HSP-25 and nestin. At 1-2 days, however, HSP-25- and nestin-immunoreactivities in the odontoblast layer showed a noticeable recovery, resulting in the rearrangement of odontoblast-like cells intensely immunoreactive to HSP-25 and nestin at 3-7 days. BrdU-positive cells showed a significant increase at 2 days (P < 0.05 vs. immediate previous time point; one-way analysis of variance and Scheffé post hoc test), peaked at 3 days and then decreased significantly (P < 0.05). It was concluded that under the present experimental condition in rat molars, cavity preparation with Er:YAG laser induced mild and reversible damage to odontoblasts. The reparative process was characterized by the rearrangement of HSP-25- and nestin-immunoreactive odontoblast-like cells, which took place subsequent to the odontoblastic layer disorganization with partial loss of these immunoreactivities.

GaAlAs laser irradiation induces active tertiary dentin formation after pulpal apoptosis and cell proliferation in rat molars.

INTRODUCTION: This study aimed to clarify pulpal responses to gallium-aluminum-arsenide (GaAlAs) laser irradiation. METHODS: Maxillary first molars of 8-week-old rats were irradiated at an output power of 0.5 or 1.5 W for 180 seconds, and the samples were collected at intervals of 0 to 14 days. The demineralized paraffin sections were processed for immunohistochemistry for heat-shock protein (HSP)-25 and nestin in addition to cell proliferation assay using bromodeoxyuridine (BrdU) labeling and apoptosis assay using deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL). RESULTS: Intense HSP-25 and nestin immunoreactivities in the odontoblast layer were weakened immediately after 0.5-W irradiation and recovered on day 1, resulting in slight tertiary dentin formation by day 14. On the contrary, 1.5-W irradiation immediately induced the loss of HSP-25 and nestin-immunoreactivities in the odontoblast layer. On day 1, numerous TUNEL-positive cells appeared in a degenerative zone that was surrounded by intense HSP-25 immunoreactivity. BrdU-positive cells occurred within the intensely HSP-25-immunopositive areas during days 2 through 5, whereas TUNEL-positive cells gradually decreased in number by day 5. HSP-25- and nestin-positive odontoblast-like cells were arranged along the pulp-dentin border by day 7, resulting in remarkable tertiary dentin formation on day 14. CONCLUSIONS: The output energy determined pulpal healing patterns after GaAlAs laser irradiation; the higher energy induced the apoptosis in the affected dental pulp including odontoblasts followed by active cell proliferation in the intense HSP-25-immunoreactive areas surrounding the degenerative tissue, resulting in abundant tertiary dentin formation. Thus, the optimal GaAlAs laser irradiation elicited intentional tertiary dentin formation in the dental pulp.

Mapping of BrdU label-retaining dental pulp cells in growing teeth and their regenerative capacity after injuries.

Recent studies have demonstrated that human dental pulp contains adult stem cells. A pulse of the thymidine analog BrdU given to young animals at the optimal time could clarify where slow-cycling long-term label-retaining cells (LRCs), putative adult stem cells, reside in the pulp tissue. This study focuses on the mapping of LRCs in growing teeth and their regenerative capacity after tooth injuries. Two to seven peritoneal injections of BrdU into pregnant Wistar rats revealed slow-cycling long-term dense LRCs in the mature tissues of born animals. Numerous dense LRCs were postnatally decreased in number and reached a plateau at 4 weeks after birth when they mainly resided in the center of the dental pulp, associating with blood vessels. Mature dental pulp cells were stained with Hoechst 33342 and sorted into (<0.76%) side population cells using FACS, which included dense LRCs. Some dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 or CD146. Tooth injuries caused degeneration of the odontoblast layer, and newly differentiated odontoblast-like cells contained LRCs. Thus, dense LRCs in mature pulp tissues were supposed to be dental pulp stem cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells. The present study proposes the new hypothesis that both granular and dense LRCs are equipped in the dental pulp and that the dense LRCs with proliferative capacity play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with the granular LRCs.

Pulpal regeneration following allogenic tooth transplantation into mouse maxilla.

Autogenic tooth transplantation is now a common procedure in dentistry for replacing a missing tooth. However, there are many difficulties in clinical application of allogenic tooth transplantation because of immunological rejection. This study aims to clarify pulpal regeneration following allogenic tooth transplantation into the mouse maxilla by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and nestin, and by the histochemistry for tartrate-resistant acid phosphatase (TRAP). The upper right first molar (M1) of 2-week-old mice was extracted and allografted in the original socket in both the littermate and non-littermate after the extraction of M1. Tooth transplantation weakened the nestin-positive reactions in the pulp tissue that had shown immunoreactivity for nestin before operation. On postoperative Days 5-7, tertiary dentin formation commenced next to the preexisting dentin where nestin-positive odontoblast-like cells were arranged in all cases of the littermate group until Day 14, except for one case showing immunological rejection in the pulp chamber. In the non-littermate group, bone-like tissue formation occurred in the pulp chamber in addition to tertiary dentin formation until Day 14. The rate of tertiary dentin was 38%, and the rate of the mixed form of dentin and bone-like tissue formation was 23% (the remainder was immunological rejection). Interestingly, the periodontal tissue recovered even in the case of immunological rejection in which the pulp chamber was replaced by sparse connective tissue. These results suggest that the selection of littermate or non-littermate is decisive for the survival of odontoblast-lineage cells and that the immunological rejection does not influence the periodontal regeneration.

Capacity of dental pulp differentiation in mouse molars as demonstrated by allogenic tooth transplantation.

Dental pulp elaborates both bone and dentin under pathological conditions such as tooth replantation/transplantation. This study aims to clarify the capability of dental pulp to elaborate bone tissue in addition to dentin by allogenic tooth transplantation using immunohistochemistry and histochemistry. After extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately allografted into the sublingual region in a littermate. In addition, we studied the contribution of donor and host cells to the regenerated pulp tissue using a combination of allogenic tooth transplantation and lacZ transgenic ROSA26 mice. On Days 5-7, tubular dentin formation started next to the preexisting dentin at the pulp horn where nestin-positive odontoblast-like cells were arranged. Until Day 14, bone-like tissue formation occurred in the pulp chamber, where intense tartrate-resistant acid phosphatase-positive cells appeared. Furthermore, allogenic transplantation using ROSA26 mice clearly showed that both donor and host cells differentiated into osteoblast-like cells with the assistance of osteoclast-lineage cells, whereas newly differentiated odontoblasts were exclusively derived from donor cells. These results suggest that the odontoblast and osteoblast lineage cells reside in the dental pulp and that both donor and host cells contribute to bone-like tissue formation in the regenerated pulp tissue.

Histological and elemental analyses of impaired bone mineralization in klotho-deficient mice.

The klotho gene-deficient mouse is known as an animal model for an accelerated gerontic state, mimicking osteoporosis, skin atrophy, ectopic calcification, and gonadal dysplasia. To elucidate the influence of klotho deficiency on bone mineralization, we examined the ultrastructures of osteoblasts and bone matrices in addition to performing the elemental mapping of calcium, phosphorus, and magnesium in the bone. Under anesthesia, 4- and 5-week-old klotho-deficient mice (klotho(-/-)mice) and their wild-type littermates were perfused with either 4% paraformaldehyde for light microscopic observation or 4% paraformaldehyde and 0.0125% glutaraldehyde for electron microscopic observation. The femurs and tibiae were processed for both observations. Paraffin sections were subject to alkaline phosphatase and tartrate resistant acid phosphatase histochemistry. Semithin and ultrathin sections obtained from epoxy resin-embedded specimens were used for detecting mineralization - according to von Kossa's staining method - and for elemental mapping by electron probe micro-analyzer, respectively. Alkaline phosphatase-positive plump osteoblasts adjacent to the growth plate normally developed cell organelles in the klotho(-/-)metaphyses. This, however, contrasted with the flattened osteoblasts covering the metaphyseal trabeculae and accompanied by small tartrate resistant acid phosphatase-positive osteoclasts. The wild-type mice displayed the mineralized matrix at the zone of hypertrophic chondrocyte of the growth plate and well-mineralized metaphyseal trabeculae parallel to the longitudinal axis of the bone. Alternatively, the klotho(-/-)mice demonstrated a thick mineralized matrix from the proliferative zone of the growth plate as well as the large non-mineralized area in the metaphyseal trabeculae. Consistently, electron probe micro-analysis verified sporadic distributions of higher or lower concentrations of calcium and phosphorus in each trabecule of the klotho(-/-)mice. The distribution of magnesium, however, was almost uniform. Under transmission electron microscopy, osteoblasts on the metaphyseal trabeculae displayed less-developed cell organelles in the klotho(-/-)mice. Thus, the klotho deficiency appears not only to reduce osteoblastic population, but also to disturb bone mineralization.

Involvement of the klotho protein in dentin formation and mineralization.

Klotho-deficient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho-deficient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion influences tooth formation. The present study aimed to elucidate the altered histology of incisors of klotho-deficient mice-comparing the findings with those from their wild-type littermates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein-1 (DMP-1), terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho-deficient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin-predentinal border at the labial region. Several odontoblast-like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP-1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast-like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast-like cells and pulpal cells in the klotho-deficient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho-deficient dentin, except for the dentin around abnormal odontoblast-like cells. These findings suggest the involvement of the klotho gene in dentinogenesis and its mineralization.

The relationship between the cusp pattern and plural stem cell compartments in Guinea pig cheek teeth by chasing BrdU-labeling.

Continuously growing rodent incisors have a special epithelial structure for maintaining adult stem cells that shows a bulbous epithelial protrusion at the apical end and is referred to as an "apical bud". Guinea pig cheek teeth (premolars and molars), also continuously growing teeth, have a complex crown shape consisting of plural cusps. The present study clarifies the existence of apical buds in guinea pig premolars/molars as it examines the relationship between the crown shape and the orientation of the apical buds by micro-computed tomography (micro-CT) and immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU). One premolar and three molar teeth in each side of the maxillae and mandibles assumed characteristic features: each horizontally-sectioned tooth showing a complex zigzag shape was composed of a core of dentin covered by a layer of enamel on all axial surfaces except the buccal of the uppers and the lingual of the lowers. Furthermore, four bulbous epithelial protrusions--including the stellate reticulum--were recognized in the apical end of each tooth, where slow-cycling long-term label-retaining cells resided 20 days after a peritoneal injection of BrdU. These data indicate that guinea pig premolars/molars have four apical buds where the epithelial adult stem cells reside. In contrast, rodent incisors, which show a single cone appearance, are covered by enamel on the labial side and possess only one apical bud. The results of this study suggest that plural apical buds, being arranged bucco-lingually and mesio-distally, produce the crown mold in a zigzag fashion.

Influence of extended operation time and of occlusal force on determination of pulpal healing pattern in replanted mouse molars.

The mechanism regulating the divergent healing processes following tooth replantation is unclear. This study clarifies the relationship between the healing pattern, the time taken for tooth replantation, and the influence of occlusal force. We investigated the pulpal healing process after tooth replantation by immunohistochemistry for 5-bromo-2'-deoxyuridine and nestin and by histochemistry for tartrate-resistant acid phosphatase. The upper right first molar of 3-week-old mice was extracted and repositioned in the original socket immediately or 30 min to 6 h after the operation. We divided the animals into a non-occluded group in which the lower right first molar was extracted and an occluded group without extraction of the counterpart tooth. In control teeth (upper left first molar), the periphery of the coronal dental pulp showed intense nestin-positive reaction. Tooth replantation weakened the nestin-positive reaction in the pulp tissue. On postoperative days 5-7, tubular dentin formation commenced next to preexisting dentin in which nestin-positive odontoblast-like cells were arranged in successful cases. In other cases, bone-like tissue formation occurred in the pulp chamber until day 14. The ratio of tertiary dentin formation was significantly higher in the non-occluded group. The intentionally prolonged time for the completion of tooth replantation induced bone-like tissue formation, expanded inflammatory reaction, or fibrous tissue formation in pulp tissue. Thus, the lack of a proper oxygenated medium is probably decisive for the survival of odontoblast-lineage cells, and occlusal force during and/or after operation worsens the fate of these cells.

Histological and immunohistochemical changes in the submandibular gland in klotho-deficient mice.

The submandibular gland (SMG) has been regarded as an age-stable organ in spite of reports on its structural changes with aging. Although the klotho gene is involved in aging, little information is available regarding its effects on morphological changes of SMGs. The present study examined the histological and immunohistochemical features of SMGs in klotho-deficient mice--which are well-established aging models--by immunohistochemical and histochemical techniques. Five kinds of cellular markers--against NGF, EGF, Mnand Cu/Zn-SOD, and RITC-conjugated phalloidin--were used for the identification of cell types. In klotho-deficient mice, the SMGs lost their granular ducts and each lobe diminished. The granular duct showed strong immunoreactivities for NGF and EGF in the wild-type mice, but the NGF- and EGF-immunopositive ducts decreased in number remarkably in klotho-deficient mice. Interestingly, instead of a loss of the granular duct, the striated duct located on the distal portion in the homozygous mice came to show NGF- and EGF-immunoreactions. Neither Mn- and Cu/Zn-SOD immunoreactivities in the duct system nor the phalloidin- reaction in the myoepithelial cells differed between the wild-type and klotho-deficient mice. Our findings suggest that the klotho gene inhibited the differentiation of the granular duct from the striated duct due to the repression and/or down-regulation of sexual and growth hormones.

Platelet-rich plasma provides nucleus for mineralization in cultures of partially differentiated periodontal ligament cells.

Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous studies, we demonstrated that PRP mimics TGF-beta1 to modulate proliferation in a cell type- specific manner, that fibrin clot formation by PRP upregulates type I collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells but failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.

Platelet-rich plasma combined with a porous hydroxyapatite graft for the treatment of intrabony periodontal defects in humans: a comparative controlled clinical study.

BACKGROUND: The aim of the present controlled clinical study was to compare platelet-rich plasma (PRP) combined with a biodegradable ceramic, porous hydroxyapatite (HA) with a mixture of HA and saline in the treatment of human intrabony defects. METHODS: Seventy interproximal intrabony osseous defects in 70 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. Thirty-five subjects each were randomly assigned to either the test group (PRP and HA) or control group (HA with saline). Clinical and radiographic measurements were determined at baseline and the 12-month evaluation. RESULTS: When compared to baseline, the 12-month results indicated that, while both treatment modalities resulted in significant changes in all clinical parameters (gingival index, bleeding on probing, probing depth, clinical attachment level, and intrabony defect fill; P <0.001), the test group exhibited statistically significant changes compared to the control sites in probing depth reduction: 4.7 +/- 1.6 mm versus 3.7 +/- 2.0 mm (P <0.05); clinical attachment gain: 3.4 +/- 1.7 mm versus 2.0 +/- 1.2 mm (P <0.001); and vertical relative attachment gain: 70.3% +/- 23.4% versus 45.5% +/- 29.4% (P <0.001). CONCLUSION: Treatment with a combination of PRP and HA compared to HA with saline led to a significantly more favorable clinical improvement in intrabony periodontal defects.

Histological evidence of the altered distribution of osteocytes and bone matrix synthesis in klotho-deficient mice.

Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.

Immunohistochemical localization of periostin in tooth and its surrounding tissues in mouse mandibles during development.

Previous reports have shown expression of immunoreactivity for periostin, originally identified as osteoblast-specific factor-2, in the periosteum and periodontal ligament. However, the developmental changes in its expression and the detailed immunolocalization have remained veiled. The present study was undertaken to examine the spatiotemporal expression of this protein in teeth and their associated tissues of mice during development at light and electron microscopic levels. In tooth germs at cap stage, periostin immunoreactivity was recognizable in the interface between inner enamel epithelium and preodontoblasts as well as in the mesenchymal tissues around cervical loop. Dental follicles around tooth germs at bell stage localized periostin immunopositivity in addition to the immunopositive areas observed in cap-staged tooth germs, although the functional significance of periostin has remained unclear in tooth development. Furthermore, periostin immunoreactivity was also found in the alveolar bone surface. In the incisors of both 7- and 21-day-old mice, immunoreaction for periostin was discernible in the lingual periodontal ligament and labial fibrous tissue adjacent to the papillary layer. After postnatal day 7, immunoreaction for periostin came to be restricted to the fibrous bundles in the periodontal ligament in accordance with the organization of the periodontal fibers, indicating its localization matched the morphogenesis of the periodontal ligament. Immunoelectron microscopic observation of the mature periodontal ligament verified the localization of periostin between the cytoplasmic processes of periodontal fibroblasts and cementoblasts and the adjacent collagen fibrils. Our findings suggest that periostin is involved at the sites of the cell-to-matrix interaction, serving as adhesive equipment for bearing mechanical forces, including occlusal force and tooth eruption.

Platelet-rich plasma contains high levels of platelet-derived growth factor and transforming growth factor-beta and modulates the proliferation of periodontally related cells in vitro.

BACKGROUND: Platelet-rich plasma (PRP) is a fraction of plasma, in which platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are thought to be concentrated. It is plausible that topically-applied PRP up-regulates cellular activity and subsequently promotes periodontal regeneration in vivo. However, the concentrations of these growth factors in PRP have not been specifically determined and the biological effects of PRP at the cellular and molecular levels have not been determined. METHODS: PRP obtained from 20 healthy subjects was prepared from plasma by centrifugation. These PRP preparations were immediately subjected to an evaluation for PDGF-AB and TGF-beta1 using enzyme-linked immunosorbent assay (ELISA) kits. The biological effects of the PRP preparations were evaluated on osteoblastic, epithelial, fibroblastic, and periodontal ligament cells. Cellular mitogenic activity was evaluated by counting cell numbers or evaluating 5-bromodeoxyuridine (BrdU) incorporation. Expression of alkaline phosphatase (ALP) was immunocytochemically evaluated. RESULTS: In the PRP preparations, platelets were concentrated up to 70.9 x 10(4) cells/microl (283.4% of the unconcentrated plasma). The levels of PDGF-AB and TGF-beta1 were also concentrated up to 182.0 ng/ml (440.6%) and 140.9 ng/ml (346.6%), respectively. Scatter plots revealed significant correlations between platelet counts and levels of these growth factors. PRP stimulated osteoblastic DNA synthesis and cell division (138% of control), with simultaneous down-regulation of ALP, but suppressed epithelial cell division (80% of control). PRP also stimulated DNA synthesis in gingival fibroblasts and periodontal ligament cells. CONCLUSIONS: These data demonstrated that both PDGF-AB and TGF-beta1 were highly concentrated in the PRP preparations. It is suggested PRP modulates cell proliferation in a cell type-specific manner similar to what has been observed with TGF-beta1. Since synchronized behavior of related cell types is thought to be required for successful periodontal regeneration, it is further suggested these cell type-specific actions may be beneficial for periodontal regenerative therapy.

Dental neuroplasticity, neuro-pulpal interactions, and nerve regeneration.

This review covers current information about the ability of dental nerves to regenerate and the role of tooth pulp in recruitment of regenerating nerve fibers. In addition, the participation of dental nerves in pulpal injury responses and healing is discussed, especially concerning pulp regeneration and reinnervation after tooth replantation. The complex innervation of teeth is highly asymmetric and guided towards specific microenvironments along blood vessels or in the crown pulp and dentin. Pulpal products such as nerve growth factor are distributed in the same asymmetric gradients as the dentinal sensory innervation, suggesting regulation and recruitment of those nerve fibers by those specific factors. The nerve fibers have important effects on pulpal blood flow and inflammation, while their sprouting and cytochemical changes after tooth injury are in response to altered pulpal cytochemistry. Thus, their pattern and neuropeptide intensity are indicators of pulp status, while their local actions continually affect that status. When denervated teeth are injured, either by pulp exposure on the occlusal surface or by replantation, they have more pulpal necrosis than occurs for innervated teeth. However, small pulp exposures on the side of denervated crowns or larger lesions in germ-free animals can heal well, showing the value of postoperative protection from occlusal trauma or from infection. Current ideas about dental neuroplasticity, neuro-pulpal interactions, and nerve regeneration are related to the overall topics of tooth biomimetics and pulp/dentin regeneration.

Expression of galanin receptor-1 (GALR1) in the rat trigeminal ganglia and molar teeth.

The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immunoreactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion, the immunoreactivity for GALR1 was recognizable in about 30% of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells. In the dental pulp, a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor, and suggests the existence of nociceptive primary afferents functioning as autocrine cells.