Capacity of dental pulp differentiation after tooth transplantation

Under pathological conditions, dental pulp elaborates both bone and dentin matrix in which the contribution of periodontal tissue cannot be excluded. This study has aimed to clarify the capability of dental pulp to deposit bone matrix in an auto-graft experiment by using (1) immunohistochemistry for 5-bromo-2′-deoxyuridine (BrdU) and nestin and (2) histochemistry for tartrate-resistant acid phosphatase (TRAP). Following the extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately transplanted into the sublingual region. On Days 5–7, tubular dentin formation commenced next to the pre-existing dentin at the pulp horn in which nestin-positive odontoblast-like cells were arranged. Up until Day 14, bone-like tissue formation occurred in the pulp chamber in which intense TRAP-positive cells appeared. These results suggest that odontoblast- and osteoblast-lineage cells reside in the dental pulp. Overall, specific dental pulp regeneration should provide fundamental knowledge for the realization of human tooth regeneration in the near future.This work was supported in part by a grant from MEXT to promote the 2001-multidisciplinary research project (in 2001–2005), KAKENHI (B) (no. 16390523 to H.O.) from MEXT, and the Japan-Korea Joint Research Project from JSPS and KOSEF (no. F01-2005-000-10212-0).     

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. This work was supported in part by KAKENHI (B) from MEXT, Japan (no. 16390523 to H.O.) and by the Japan-Korea Joint Research Project from JSPS and KOSEF.     

The relationship between odontoblasts and pulp capillaries in the process of enamel- and cementum-related dentin formation in rat incisors

Summary The relationship between odontoblasts and pulp capillaries in the process of dentinogenesis was studied in rat lower incisors, both on the labial and lingual sides, using light and transmission electron microscopy. The odontoblasts showed remarkable differences from the apical to the incisal end. Near the apical end of the tooth, immature odontoblasts, which were thought to be involved in the formation of the mantle dentin, were arranged in a single layer, and continuous capillaries were located just beneath the odontoblasts. In the middle of the tooth, mature odontoblasts with highly developed cell organelles and notable processes formed a pseudostratified layer; fenestrated capillaries were found between these cells close to the predentin. The height of the odontoblast layer and the rate of dentin deposition on the labial (enamel-related) side was significantly greater than that on the lingual (cementum-related) side. Near the incisal end, cementum-related odontoblasts gradually decreased in height and number to become post-odontoblasts that produced atubular dentin; continuous capillaries were located subjacent to the post-odontoblasts. On the labial (enamel-related) side, however, odontoblasts retained their pseudostratification; fenestrated capillaries were still observed in the odontoblast layer. No atubular dentin was formed on the labial side.     

Cell dynamics in the pulpal healing process following cavity preparation in rat molars

Odontoblast-lineage cells acquire heat-shock protein (HSP)-25-immunoreactivity (IR) after they complete their cell division, suggesting that this protein acts as a switch between cell proliferation and differentiation during tooth development. However, there are few available data concerning the relationship between cell proliferation and differentiation following cavity preparation. The present study aims to clarify the expression of HSP-25 in the odontoblast-lineage cells with their proliferative activity after cavity preparation by immunocytochemistry for HSP-25 and cell proliferation assay using 5-bromo-2'-deoxyuridine (BrdU) labeling. In untreated control teeth, intense HSP-25-IR was found in odontoblasts and some subodontoblastic mesenchymal cells. Cavity preparation caused the destruction of odontoblasts and the disappearance of HSP-25-IR was conspicuous at the affected site, although some cells retained HSP-25-IR and subsequently most of them disappeared from the pulp-dentin border by postoperative day 1. Contrary, some subodontoblastic mesenchymal cells with weak HSP-25-IR began to take the place of degenerated cells, although no proliferative activity was recognizable in the dental pulp. Interestingly, proliferative cells in the dental pulp significantly increased in number on day 2 when the newly differentiating cells already arranged along the pulp-dentin border, and continued their proliferative activity in the wide range of the pulp tissue until day 5. These findings indicate that progenitor cells equipped in the subodontoblastic layer firstly migrate and differentiate into new odontoblast-like cells to compensate for the loss of the odontoblast layer, and subsequently the reorganization of dental pulp was completed by active proliferation of the mesenchymal cells occurring in a wide range of pulp tissue.     

The role of angiogenesis and pulpal healing in tooth replantation and allograft transplantation

Tooth transplantation is one of the treatment options for extracted teeth that can be considered before dental implantation. Although the success rate of tooth transplantation is lower than that of implantation, tooth replantation and transplantation have the great advantage of using natural teeth. Tooth replantation might be considered a promising option in some cases. In present study, the expression patterns of revascularization and pulpal healing, which are the most important for the pulp viability, were analyzed after tooth replantation and allograft in mice. The inflammatory response and root dentin resorption were observed and not different between replantation and allograft in initiation of healing process. However, bonelike tissue formation, pulp revascularization and pulp healing were faster in replantation. The difference of healing patterns between tooth replantation and allograft found in present study will be helpful to select the treatment option and to understand healing mechanism.     

Semaphorin 3A controls timing and patterning of the dental pulp innervation

Timing and patterning of dental pulp innervation are strictly spatio-temporally regulated but it is still not known how they are controlled at molecular level. We analyzed postnatal innervation of the dental pulp in the mandibular first molar of mice deficient for Semaphorin 3A (Sema3A) axon repellant molecule. Immunohistochemical localization of nerve fibers on serial sections covering the whole tooth germs using anti-peripherin antibody revealed that nerve fibers were prematurely present within the preodontoblast layer next to the inner enamel epithelium already at PN0 in Sema3A^−/− mice. In contrast, in the wild-type (Sema3A^+/+) mice nerve fibers were seen in the pulp only after enamel formation at PN3. The nerves in Sema3A^−/− pulp were notably defasciculated and thinner compared to that of Sema3A^+/+ mice. A premature formation of an abnormal, enlarged nerve plexus with a high number of arborizations was apparent in the pulp–dentin border target area in Sema3A^−/− already at PN2 whereas in the wild-type mice the first sign of plexus formation was seen at PN7. The expression of mRNAs for Ngf, Gdnf and Ncam neuroregulatory molecules in mandibular molar as well as receptors for neurotrophic factors and class 3 semaphorins including Sema3A (TrkA, p75, TrkB, TrkC, Ret, Npn1, Npn2, PlxA4) in trigeminal ganglia were not altered in the Sema3A^−/− mice. Collectively, this data show that Sema3A serves an essential role in molar tooth pulp innervation controlling the timing of nerve fiber penetration into the pulp, their patterning and the formation of nerve plexus at pulp–dentin border area, and provide further support for the hypothesis that tooth innervation is regulated by the coordinated activity of locally expressed neuroregulatory molecules exerting positive and negative influences on growing dental nerve fibers.     

Responses of immunocompetent cells in the dental pulp to replantation during the regeneration process in rat molars

Responses of immunocompetent cells to tooth replantation during the regeneration process of the dental pulp in rat molars were investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6 antibody), monocyte/macrophage lineage cells (ED1 antibody) and protein gene product 9.5 (PGP 9.5), as well as by histochemical reaction for periodic acid-Schiff (PAS). Tooth replantation caused an increase in both the number of OX6- and ED1-positive cells and their immunointensity in the replanted pulp, but almost all PGP 9.5-immunoreactive nerves diminished in the initial stages. By postoperative day 3, many OX6- and ED -immunopositive cells had accumulated along the pulp-dentin border to extend their cytoplasmic processes into the dentinal tubules in successful cases. Once reparative dentin formation had begun after postoperative day 7, OX6- and ED1-immmunopositive cells became scattered in the odontoblast layer, while reinnervation was found in the coronal pulp. The temporal appearance of these immunocompetent cells at the pulp-dentin border suggests their participation in odontoblast differentiation as well as in initial defense reactions during the pulpal regeneration process. On postoperative day 14, the replanted pulp showed three regeneration patterns: (1) reparative dentin, (2) bone-like tissue formation, and (3) an intermediate form between these. In all cases, PAS-reactive cells such as polymorphonuclear leukocytes (PML) and mesenchymal cells occurred in the pulp space. However, the prolonged stagnation of inflammatory cells was also discernible in the latter two cases. Thus, the findings on PAS reaction suggest that the migration of the dental follicle-derived cells into the pulp space and the subsequent total death of the proper pulpal cells are decisive factors for eliciting bone-like tissue formation in the replanted pulp.     

Histochemical and immunocytochemical study of the migration of neurons from the rat olfactory placode

Immunocytochemical and histochemical methods have been used to describe the neuronal population migrating from the rat olfactory placode and to analyze the spatio-temporal evolution of this neuronal migration during development. Several neuronal markers, such as binding to the lectin Ulex europaeus (UEA I) and the presence of neuron-specific enolase (NSE), olfactory marker protein (OMP), and luteinizing hormone-releasing hormone (LHRH), have been tested in order to determine whether migrating neurons originate from both the medial and the lateral parts of the placode and whether they all express LHRH. Our data show that a large population of differentiated migrating neurons can be identified with an antibody against NSE from the 14th day of gestation and with UEA I one day later. Migrating neurons are closely associated with both the vomeronasal axon fascicles emerging from the medial pit and the olfactory axons originating from the lateral pit. However, the neuron migration from the lateral pit appears to be more discrete than that from the medial pit. No LHRH immunoreactivity has been detected among neurons migrating from the lateral pit. Some neurons accompanying the olfactory axon fascicles exhibit a high level of maturation as shown by their OMP-positivity. Numerous neurons positive for both NSE and UEA I have also been observed within the presumptive olfactory nerve layer in early embryonic stages.     

Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.     

Relationship between enamel formation and eruption rate in rat mandibular incisors

Summary The relationship between the formation of dental enamel and tooth eruption was investigated. Rat mandibular incisor eruption rate was accelerated by maintaining incisors out of occlusion. Rate of eruption, enamel thickness, secretory zone length and matrix breakdown were measured. Eruption rate increased by 120% in experimental teeth but enamel secretion increased by only 90%. There were no obvious differences between control and experimental teeth in final enamel thickness or in the molecular weight distribution of the enamel matrix proteins.     

Platelet-derived growth factor exerts disparate effects on odontoblast differentiation depending on the dimers in rat dental pulp cells

Platelet-derived growth factor (PDGF) has recently been demonstrated to control the expression of alkaline phosphatase and proteoglycan synthesis of odontoblastic cells in dental pulp tissues. Although PDGF appears to be closely related to dentinogenesis, much about the mode of action of PDGF on odontoblast differentiation remains unclear. In this study, we examined the effects of three PDGF dimers (PDGF AA, AB, and BB) on odontoblastic differentiation of dental pulp cells in long-term mineralized cultures. Dental pulp cells isolated from rat lower incisors were continuously treated with each of PDGF AA, AB, and BB in separate cultures for 20 days. The three PDGF dimers suppressed alkaline phosphatase activity, osteocalcin and calcium content, and the formation of dentin-like nodules. The expression of mRNA for dentin sialoprotein (DSP) in the cells was inhibited by PDGF AA treatment, whereas PDGF AB and BB treatment stimulated the expression of DSP, even though the dentin-like nodule formation was inhibited. Although the effects of PDGF on odontoblastic differentiation varied among the dimers, the cells expressed both PDGF and receptors, whose quantities were similar. These results suggest that PDGF exerts diverse effects on odontoblastic differentiation depending on its dimeric form. These in vitro findings explain, at least in part, the in vivo action of PDGF in dentinogenesis during the repair process of damaged dental pulp.This work was supported in part by grants-in-aid from the Ministry of Science, Education, and Culture of Japan     

Effects of alendronate on tooth eruption and molar root formation in young growing rats

Tooth eruption consists of the movement of teeth from the bony crypt in which they initiate their development to the occlusal plane in the oral cavity. Interactions between the tooth germ and its surrounding alveolar bone occur in order to offer spatial conditions for its development and eruption. This involves bone remodeling during which resoption is a key event. Bisphosphonates are a group of drugs that interfere with the resorption of mineralized tissues. With the purpose of investigating the effects of sodium alendronate (a potent bisphosphonate inhibitor of osteoclast activity) on alveolar bone during tooth development and eruption, we gave newborn rats daily doses of this drug for 4, 14, and 30 days. Samples of the maxillary alveolar process containing the tooth germs were processed for light, transmission, and scanning electron microscopy and were also submitted to tartrate-resistant acid phosphatase histochemistry and high-resolution colloidal-gold immunolabeling for osteopontin. Inhibition of osteoclast activity by sodium alendronate caused the absence of tooth eruption. The lack of alveolar bone remodeling resulted in primary bone with the presence of latent osteoclasts and abundant osteopontin at the interfibrillar regions. The developing bone trabeculae invaded the dental follicle and reached the molar tooth germs, provoking deformities in enamel surfaces. No root formation was observed. These findings suggested that alendronate effectively inhibited tooth eruption by interfering with the activation of osteoclasts, which remained in a latent stage. This work was supported by grants from Fapesp (04/05831-9 and 06/60094-5) and CNPq (Brazil).     

Temporospatial localization of acetylcholinesterase activity in the dental epithelium during mouse tooth development

Acetylcholinesterase (AChE), a principal modulator of cholinergic neurotransmission, also has been demonstrated to be involved in the morphogenetic processes of neuronal and non-neuronal tissues. This study shows that AChE exhibits temporospatial activity in the dental epithelium of the developing mouse tooth. To identify the AChE activity in the mouse tooth during development, we performed enzyme histochemistry on the mouse embryos from embryonic day 13 (E13) to E18 and on the incisors and molars of the neonatal mouse at 10 days after birth (P10). In the developing molars of mouse embryos, AChE activity was not found in the dental epithelium at E13 (bud stage). AChE activity first appeared in the developing cervical loops of the enamel organ at E14 (cap stage), but was not found in the enamel knot. At E18 (bell stage), AChE activity was localized in the inner enamel epithelium except the cervical-loop area. In the incisors and molars of neonatal mice (P10), AChE activity was localized in the inner enamel epithelium of the cervical-loop and enamel-free area. Overall, AChE activity was localized in the differentiating dental epithelium while the activity of butyrylcholinesterse, another cholinesterase, was located primarily in the cells of the dental follicle. The results suggest that AChE may play a role in the histo- and cytodifferentiation of dental epithelium during tooth development.     

Convergence of nociceptive information from temporomandibular joint and tooth pulp afferents on C1 spinal neurons in the rat

The aim of the present study was to test the hypothesis that there is a convergence of afferent inputs from the temporomandibular joint (TMJ) on C1 spinal neurons responding to electrical stimulation of the tooth pulp (TP). In 14 pentobarbital anesthetized rats, the extracellular single unit activity of 31 C1 spinal neurons and the amplitude in a digastric muscle electromyogram (n = 31) increased proportionally during 1.0-3.5 times the threshold for the jaw-opening reflex (JOR). Of 31 C1 spinal neurons responsive to TP afferents, 28 (approximately 90%) were also excited by electrical stimulation of the ipsilateral TMJ capsule. All neurons tested were divided into three categories of nociceptive specific, wide dynamic range and non-responsive as to their responsiveness to mechanical stimuli (pin prick and touch) of the somatic receptive field (skin of the face, neck, jaw and upper forearm) and TMJ capsule. Nineteen (68%) of 28 C1 spinal neurons received nociceptive information from C fibers of the TMJ capsule. These results suggest that there is a convergence of noxious information from the TMJ and TP afferents on the same C1 spinal neurons, which importantly contribute to pain perception from the TMJ region.     

Culture and characterization of dental follicle cells from rat molars

Summary Because the dental follicle is necessary for the eruption of teeth of limited eruption, it was the objective of this study to determine if the cells of the follicle could be cultured in vitro. To achieve this, dental follicles and associated enamel organs were dissected from the first and second mandibular molars of 6–7-day-old rats (secretory stage of amelogenesis), and then cultured in a medium that promotes fibroblast growth — the predominant cell type of the dental follicle. The cultured cells grew to confluency and were kept through 3 passages before experimentation. The cultured cells were fibroblastic in shape, elongate with processes, and transmission electron microscopy revealed that they contained an abundant rough endoplasmic reticulum, but did not form desmosomes. Immunofluorescent staining for anti-vimentin showed that all the cells stained and electron-microscopic immunogold labeling indicated that the antibody was associated with intermediate filaments. As revealed by SDS-polyacrylamide gel electrophoresis and Western blotting, the cultured cells synthesized and secreted the extracellular matrix molecules fibronectin and procollagens. Subsequent immunofluorescence staining of permeabilized and non-permeabilized cells confirmed the presence of fibronectin and type I collagen both intra- and extracellularly. Thus, based on all the above characteristics, the cultured cells appeared to be fibroblasts derived from the dental follicle, although a few of the fibroblasts may be derived from undifferentiated mesenchymal cells interposed between the alveolar bone and follicle. Experiments now can be conducted to determine how these cultured cells respond directly to growth factors that alter the rates of tooth eruption.     

Ultrastructural and lanthanum tracer examination of rapidly resorbing rat alveolar bone suggests that osteoclasts internalize dying bone cells

Glutaraldehyde-formaldehyde fixed undecalcified alveolar bone from 7-day-old rats was prepared for light and electron microscopy. Colloidal lanthanum was used as an ultrastructural tracer, and both random and semi-serial sections were examined. Lanthanum penetrated the infoldings of the ruffled border and some nearby vacuoles and vesicles. The majority of vacuoles and vesicles were lanthanum-free. Some osteoclast profiles contained a large vacuole with a cell enclosed in its interior. The enclosed cell exhibited an irregular nucleus containing condensed peripheral chromatin, intact cytoplasmic organelles, conspicuous rough endoplasmic reticulum and large blebs on the cell surface. These features are characteristic of osteoblasts or bone-lining cells or immature osteocytes which may be undergoing apoptosis or necrosis. The observation of remnants of cellular structures within internalized osteoclast vacuoles, together with the above results, suggests that osteoclasts engulf and probably degrade dying osteoblasts/bone-lining cells or immature osteocytes. Received: 29 April 1997 / Accepted: 20 February 1998     

Inhibition of connexin 43 expression and function in cultured rat dental pulp cells by antisense oligonucleotide

Connexins are gap-junction proteins forming hexameric structures in the plasma membranes of adjacent cells, thereby creating intercellular channels. Connexin 43 (CX43) is expressed in pulp tissue. However, its function in dental pulp tissue has yet to be fully investigated. We have employed antisense oligonucleotides (AS) against rat CX43 to study the role of CX43 in dental pulp cells. Cultured dental pulp cells were treated with AS or sense (S) oligonucleotides. The number of cells in the AS-treated groups was approximately 1.3-fold that in the S-treated controls. Growth rates were significantly different between the AS- and S-treated groups at 48 h (P < 0.01). An alkaline phosphatase assay revealed that AS-treated pulp cells dramatically decreased at 48 h after AS incorporation, whereas S-treated pulp cells showed no marked changes. Western blot analysis revealed that heat-shock protein 25 was highly expressed in S-treated cells but was only weakly expressed in AS-treated cells at 48 h. Furthermore, AS-treated cells highly expressed CX45, whereas S-treated cells exhibited high expression of CX32. These results suggest that CX43 is involved in cell growth, mineralization, and differentiation to odontoblasts in rat pulp cells, and that CX43 plays the opposite role to that of CX45.     

The immunocytochemical localization of angiotensinogen in the rat ovary

Summary The present study examined the presence and cellular distribution of angiotensinogen, the precursor to the angiotensin peptides, in the ovary of the normal cycling rat by immunocytochemistry. Angiotensinogen staining was present in the granulosa cells of maturing follicles and to a lesser extent in those undergoing atresia. Staining was not seen in the granulosa cells of primordial or early primary follicles. In maturing follicles intense staining for angiotensinogen was confined to the antral cell layers, cells of the cumulus oophorus and in the follicular fluid. Strong immunostaining was also seen in the germinal epithelium covering the ovary. Lighter angiotensinogen staining was observed in some parts of the cortical and medullary stroma and occasionally in corpora lutea. No variation in the intensity or pattern of angiotensinogen staining was observed throughout the estrous cycle. Comparison of the distribution of angiotensinogen with the previously described localization of renin, AII, angiotensin converting enzyme and AII receptors, suggests that there are a number of intra-ovarian sites at which AII could be produced.