Localization of perlecan and heparanase in Hertwig's epithelial root sheath during root formation in mouse molars.

During cementogenesis, dental follicular cells penetrate the ruptured Hertwig's epithelial root sheath (HERS) and differentiate into cementoblasts. Mechanisms involved in basement membrane degradation during this process have not been clarified. Perlecan, a heparan sulfate (HS) proteoglycan, is a component of all basement membranes. Degradation of HS of perlecan by heparanase cleavage affects a variety of biological processes. We elucidated immunolocalization of perlecan and heparanase in developing murine molars to clarify their roles in cementoblast differentiation. At the initial stage of root formation, perlecan immunoreactivity was detected on the basement membrane of HERS. Weak heparanase immunoreactivity was detected in HERS cells. HERS showed intense staining for heparanase as root formation progressed. In contrast, labeling for perlecan disappeared from the basement membrane facing the dental follicle, and weak immunoreactivity for perlecan was detected on the inner side of the basement membrane of HERS. These findings suggest that perlecan removal is an important step for root and periodontal tissue formation. Heparanase secreted by the cells of HERS may contribute to root formation by degrading perlecan in the dental basement membrane.     

Quiescent epithelial cell rests of Malassez can differentiate into ameloblast-like cells

Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. After completion of crown formation, HERS are converted from cervical loop cells, which have the potential to generate enamel for tooth crown formation. Cervical loop cells have the potential to differentiate into ameloblasts. Generally, no new ameloblasts can be generated from HERS, however this study demonstrated that subcultured ERM can differentiate into ameloblast-like cells and generate enamel-like tissues in combination with dental pulp cells at the crown formation stage. Porcine ERM were obtained from periodontal ligament tissue by explant culture and were subcultured with non-serum medium. Thereafter, subcultured ERM were expanded on 3T3-J2 feeder cell layers until the tenth passage. The in vitro mRNA expression pattern of the subcultured ERM after four passages was found to be different from that of enamel organ epithelial cells and oral gingival epithelial cells after the fourth passage using the same expansion technique. When subcultured ERM were combined with subcultured dental pulp cells, ERM expressed cytokeratin14 and amelogenin proteins in vitro. In addition, subcultured ERM combined with primary dental pulp cells seeded onto scaffolds showed enamel-like tissues at 8 weeks post-transplantation. Moreover, positive staining for amelogenin was observed in the enamel-like tissues, indicating the presence of well-developed ameloblasts in the implants. These results suggest that ERM can differentiate into ameloblast-like cells.     

Ameloblastin in Hertwig’s Epithelial Root Sheath Regulates Tooth Root Formation and Development

Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig’s epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-2′-deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21^Cip1 and p27^Kip1 was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.     

Possible involvement of maspin in tooth development

Maspin is a 42 kDa serine protease inhibitor that possesses tumor suppressive and anti-angiogenic activities. Despite of a huge amount of data concerning the expression pattern of maspin in various tissues and its relevance to the biological properties of a variety of human cancer cells, little is known on the maspin expression in skeletal and tooth tissues. Recently, we reported that maspin may play an important role in extracellular matrix formation in bone by enhancing the accumulation of latent TGF-β in the extracellular matrix. This study was performed to elucidate the possible role of maspin in tooth development. First, an immunohistochemical analysis for human tooth germs at the late bell stage showed the expression of maspin by active ameloblasts and odontoblasts that were forming enamel and dentin, respectively. During rat tooth development, maspin expression was observed for the first time in inner and outer enamel epithelial cells and dental papilla cells at early bell stage. The neutralizing anti-maspin antibody inhibited the proper dental tissue formation in organ cultures of mandibular first molars obtained from 21-day-old rat embryos. In addition, the proliferation of HAT-7 cells, a rat odontogenic epithelial cell line, and human dental papilla cells were suppressed in a dose-dependent manner with anti-maspin antibody. Moreover, RT-PCR analysis showed that the expression of mRNA for tooth-related genes including dentin matrix protein 1, dentin sialophosphoprotein and osteopontin in human dental papilla cells was inhibited when treated with anti-maspin antibody. These findings suggest that maspin expressed in ameloblasts and odontoblasts plays an important physiological role in tooth development through the regulation of matrix formation in dental tissues.     

Apoptosis of the reduced enamel epithelium and its implications for bone resorption during tooth eruption

Bone remodeling, the selective deposition and resorption of bone, is an important cause of tooth eruption. During tooth eruption, reduced enamel epithelia of the enamel organ interact with follicle cells to recruit osteoclasts for bone remodeling. However, little is known about the relationship between cellular activity of reduced enamel epithelium and bone resorption during tooth eruption. The purpose of this study was to investigate the effect of apoptosis in reduced enamel epithelium on osteoclastogenesis and its implications for bone resorption. We have analyzed erupting mandibular molars in mice by TdT-mediated dUTP-biotin nick end labeling assay, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry. TRAP-positive cells were detected in the osteoclasts near both the buccal and lingual sides of tooth socket at postnatal day 0 (PN0). They significantly increased until PN3 and decreased thereafter as the tooth erupted. Interestingly, apoptosis was barely detected in the reduced enamel epithelium at PN3 but clearly at PN7. A few apoptotic cells were also investigated within the dental follicle surrounding developing tooth at PN7 and PN10. We observed apoptotic osteoblast-lineage cells along the inner margin of alveolar bone facing the buccal cusp and at the base of the bony crypt at PN3 decreasing until PN10. In contrast, expression levels of bone sialoprotein increased at PN10 compared to levels at PN3. These results suggest that apoptosis of reduced enamel epithelium resulted in a reduction of osteoclast activity and of bone resorption mediated by dental follicle during tooth eruption.     

Disturbed tooth germ development in the absence of MINT in the cultured mouse mandibular explants

The Msx2-interacting nuclear target protein (MINT) is a nuclear matrix protein that regulates the development of many tissues. However, little is known regarding the role of MINT in tooth development. In this study, we prepared polyclonal antibodies against MINT, and found that that MINT was expressed in different cells at each stage of tooth germ development by immunohistochemistry. The role of MINT in tooth development was further illustrated by the misshapen and severely hypoplastic tooth organ in the cultured mandibular explants of MINT deficient mice. From the initiation to cap stage, the differences between mutants and wild-type molars were more and more distinguished histologically. In the MINT-deficient mandibular explants, the tooth germ was reduced in the overall size and lacked enamel knot, with abnormal dental lamina and collapsed stellate reticulum. Furthermore, the BrdU incorporation experiment showed that the proliferation activity was significantly reduced in MINT-deficient dental epithelium. Our results suggest that MINT plays an important role in tooth development, in particular, epithelial morphogenesis.     

The Ca2+-binding protein calretinin is selectively enriched in a subpopulation of the epithelial rests of Malassez

During tooth development, the inner and outer enamel epithelia fuse by mitotic activity to produce a bilayered epithelial sheath termed Hertwig’s epithelial root sheath (HERS). The epithelial rests of Malassez (ERM) are the developmental residues of HERS and remain in the adult periodontal ligament (PDL). Although the cellular regulation of the Ca²⁺-binding proteins parvalbumin, calbindin-D28k, and calretinin has been reported in the inner and outer enamel epithelia during tooth development, an involvement of Ca²⁺-binding proteins in the ERM has not so far been characterized. Among the three Ca²⁺-binding proteins tested (calbindin D28k, parvalbumin, calretinin), we have only been able to detect calretinin in a subpopulation of adult rat molar ERM, by using quantitative immunohistochemical and confocal immunofluorescence techniques. TrkA (a marker for ERM) is present in numerous epithelial cell clusters, whereas calretinin has been localized in the cytosol and perinuclear region of a subpopulation of TrkA-positive cells. We conclude that, in inner and outer enamel epithelial cells, Ca²⁺ is regulated by calbindin, parvalbumin, and calretinin during tooth development, whereas in the ERM of adult PDL, Ca²⁺ is regulated only by calretinin. The expression of Ca²⁺-binding proteins is restricted in a developmental manner in the ERM.     

Runx2条件性敲除小鼠釉质缺陷的部分挽救

该研究旨在探讨外源性Runx2过表达对小鼠成釉细胞Runx2敲除导致的釉质缺陷的挽救作用。采用免疫组化验证Runx2在Runx2条件性敲除且人源性Runx2过表达小鼠成釉细胞中的表达。HE染色观察成熟期成釉细胞形态及釉质基质蛋白残余。用体视显微镜和扫描电镜观察小鼠牙齿表面形态和釉柱结构。结果显示,RUNX2蛋白在出生后10天龄Tg;cKO小鼠成熟早期成釉细胞中成功表达。15天龄Tg;cKO小鼠与cKO小鼠相比,成熟晚期成釉细胞形态及排列未见明显改善,但釉质基质蛋白残余量明显减少。3月龄Tg;cKO小鼠与cKO小鼠相比,釉质磨耗减轻,釉柱间孔隙减少,釉柱排列更规则。该研究结果表明,人源性Runx2过表达可部分挽救小鼠成釉细胞Runx2敲除导致的釉质缺陷。     

Unicuspid and bicuspid tooth crown formation in squamates

The molecular and developmental factors that regulate tooth morphogenesis in nonmammalian species, such as snakes and lizards, have received relatively little attention compared to mammals. Here we describe the development of unicuspid and bicuspid teeth in squamate species. The simple, cone-shaped tooth crown of the bearded dragon and ball python is established at cap stage and fixed in shape by the differentiation of cells and the secretion of dental matrices. Enamel production, as demonstrated by amelogenin expression, occurs relatively earlier in squamate teeth than in mouse molars. We suggest that the early differentiation in squamate unicuspid teeth at cap stage correlates with a more rudimentary tooth crown shape. The leopard gecko can form a bicuspid tooth crown despite the early onset of differentiation. Cusp formation in the gecko does not occur by the folding of the inner enamel epithelium, as in the mouse molar, but by the differential secretion of enamel. Ameloblasts forming the enamel epithelial bulge, a central swelling of cells in the inner enamel epithelium, secrete amelogenin at cap stage, but cease to do so by bell stage. Meanwhile, other ameloblasts in the inner enamel epithelium continue to secrete enamel, forming cusp tips on either side of the bulge. Bulge cells specifically express the gene Bmp2, which we suggest serves as a pro-differentiation signal for cells of the gecko enamel organ. In this regard, the enamel epithelial bulge of the gecko may be more functionally analogous to the secondary enamel knot of mammals than the primary enamel knot.     

The primary enamel knot determines the position of the first buccal cusp in developing mice molars

The enamel knot (EK), which is located in the center of bud and cap stage tooth germs, is a transitory cluster of non-dividing epithelial cells. The EK acts as a signaling center that provides positional information for tooth morphogenesis and regulates the growth of tooth cusps by inducing secondary EKs. The morphological, cellular, and molecular events leading to the relationship between the primary and secondary EKs have not been described clearly. This study investigated the relationship between the primary and secondary EKs in the maxillary and mandibular first molars of mice. The location of the primary EK and secondary EKs was investigated by chasing Fgf4 expression patterns in tooth germ at some intervals of in vitro culture, and the relationship between the primary EK and secondary EK was examined by tracing the primary EK cells in the E13.5 tooth germs which were frontally half sliced to expose the primary EK. After 48 hr, the primary EK cells in the sliced tooth germs were located on the buccal secondary EKs, which correspond to the future paracone in maxilla and protoconid in mandible. The Bmp4 expression in buccal part of the dental mesenchyme might be related with the lower growth in buccal epithelium than in lingual epithelium, and the Msx2 expressing area in epithelium was overlapped with the enamel cord (or septum) and cell dense area. The enamel cord might connect the primary EK with enamel navel to fix the location of the primary EK in the buccal side during the cap to bell stages. Overall, these results suggest that primary EK cells strictly contribute to form the paracone or protoconid, which are the main cusps of the tooth in the maxilla or mandible.     

Immunohistochemical localization of LIM mineralization protein 1 during mouse molar development

LIM mineralization protein 1 (LMP-1) is an essential positive regulator of osteoblast differentiation, maturation and bone formation. Our previous investigations on the distribution of LMP-1 in mature human teeth indicated that LMP-1 might play a role in the odontoblast differentiation and dentin matrix mineralization. The aim of the present study was to use immunohistochemistry to determine the expression of LMP-1 during tooth development in mouse molars. In embryonic and postnatal Kunming mice, LMP-1 protein was expressed during molar development, but the expression levels and patterns differed at various developmental stages. At embryonic day 13.5 (E13.5), LMP-1 was found in the enamel organ. At E14.5, LMP-1 was detected in the entire enamel organ and in the underlying mesenchyme. At E16.5, LMP-1 was observed in the inner and outer enamel epithelium and the stratum intermedium. The expression also converged at the cusps in the dental papilla. At E18.5 and postnatal day 2.5 (P2.5), LMP-1 was restricted to the stratum intermedium, in differentiating dental papilla cells at cusps, while it disappeared in terminal differentiated ameloblasts and odontoblasts. At P13.5, no positive staining was detected in the odontoblasts or in the dental pulp cells. Therefore, LMP-1 showed spatiotemporal expression patterns during molar development and might participate in molar crown morphogenesis and odontoblast differentiation at late molar development.     

Comparison of tryptophan-labeled constituents of developing rodent molar enamel matrix, non-enamel occlusal cusp, Hertwig's epithelial root sheath, and presumptive root furcation regions: light microscopic autoradiography

During the process of organogenesis involving the developing rodent molar and incisor tooth organs, novel gene products termed enamel proteins are expressed by ectodermally-derived enamel organ epithelia at precise times and positions within the course of morphogenesis. The present studies were designed to identify the relative distribution of tryptophan-labeled, non-collagenous, epithelial-derived proteins associated with rat maxillary first molar crown (M') and initial root formation. Our experimental strategy was to utilize semi-quantitative autoradiography methods to compare and contrast the distribution of silver grains resulting from tryptophan incorporation into developing postnatal pups associated with enamel matrix, non-enamel occlusal cusp, Hertwig's Epithelial Root Sheath (HERS), and presumptive root furcation regions of M'. Five-day-old Wistar rats were injected with 14C-labeled tryptophan. Four animals were sacrificed at 15 minutes and then at 1, 2, 4, and 24 hour intervals following the administration of this essential aromatic amino acid. Following fixation and subsequent processing for autoradiography, semiquantitative analyses were performed of the silver grain distribution localized within selected regions of the developing M' tooth organs. All enamel organ epithelia were found to incorporate tryptophan and silver grains were identified (above background) in the extracellular matrices (ECM) of the enamel matrix, non-enamel occlusal cusp adjacent to the inner enamel epithelia, and the ECM (2-4, micron) adjacent to presumptive root furcation and HERS regions. Tryptophan incorporation was not significant in the odontoblasts or dentine ECM of the crown or forming presumptive root regions. These results support the hypothesis that inner enamel epithelia associated with rat molar crown formation, as well as HERS, synthesize tryptophan-labeled, non-collagenous, ECM molecules. We speculate that HERS participates in root development by possibly producing non-collagenous proteins for intermediate cementum formation.     

Constitutively active PTH/PTHrP receptor in odontoblasts alters odontoblast and ameloblast function and maturation

Parathyroid hormone (PTH)-related protein (PTH-rP) is an important autocrine/paracrine attenuator of programmed cell differentiation whose expression is restricted to the epithelial layer in tooth development. The PTH/PTHrP receptor (PPR) mRNA in contrast is detected in the dental papilla, suggesting that PTHrP and the PPR may modulate epithelial-mesenchymal interactions. To explore the possible interactions, we studied the previously described transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells. These transgenic mice have a vivid postnatal bone and tooth phenotype, with normal tooth eruption but abnormal, widened crowns. Transgene mRNA expression was first detected at birth in the dental papilla and, at 1 week postnatally, in odontoblasts. There was no transgene expression in ameloblasts or in other epithelial structures. Prenatally, transgenic molars and incisors revealed no remarkable change. By the age of 1 week, the dental papilla was widened, with disorganization of the odontoblastic layer and decreased dentin matrix. In addition, the number of cusps was abnormally increased, the ameloblastic layer disorganized, and enamel matrix decreased. Odontoblastic and, surprisingly, ameloblastic cytodifferentiation was impaired, as shown by in situ hybridization and electron microscopy. Interestingly, ameloblastic expression of Sonic Hedgehog, a major determinant of ameloblastic cytodifferentiation, was dramatically altered in the transgenic molars. These data suggest that odontoblastic activation of the PPR may play an important role in terminal odontoblastic and, indirectly, ameloblastic cytodifferentiation, and describe a useful model to study how this novel action of the PPR may modulate mesenchymal/epithelial interactions at later stages of tooth morphogenesis and development.     

Dynamic assembly of tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin during mouse tooth development

Tight junctions might play a role during tissue morphogenesis and cell differentiation. In order to address these questions, we have studied the distribution pattern of the tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin in the developing mouse tooth as a model. A specific temporal and spatial distribution of tight junction-associated proteins during tooth development was observed. ZO-1 appeared discontinuously in the cell membrane of enamel organ and dental mesenchyme cells. However, endothelial cells of the dental mesenchyme capillaries displayed a continuous fluorescence at the cell membrane. Inner dental epithelium first showed an evident signal for ZO-1 at the basal pole of the cells at bud/cap stage, but ZO-1 was accumulated at the basal and apical pole of preameloblast/ameloblasts at late bell stage. Surprisingly, in the incisor ZO-1 decreased as the inner dental epithelium differentiated, and was re-expressed in secretory and mature ameloblasts. On the contrary, ZO-2 was confined to continuous cell-cell contacts of the enamel organ in both molars and incisors. The lateral cell membrane of inner dental epithelial cells was specifically ZO-2 labeled. However, ZO-3 was expressed in oral epithelium whereas dental embryo tissues were negative. In addition, occludin was hardly detected in dental tissues at the early stage of tooth development, but was distributed continuously at the cell membrane of endothelial cells of ED19.5 dental mesenchyme. In incisors, occludin was detected at the cell membrane of the secretory pole of ameloblasts. The occurrence and relation during tooth development of tight junction proteins ZO-1, ZO-2 and occludin, but not ZO-3, suggests a combinatory assembly in tooth morphogenesis and cell differentiation.     

Physiological implications of DLX homeoproteins in enamel formation

Tooth development is a complex process including successive stages of initiation, morphogenesis, and histogenesis. The role of the Dlx family of homeobox genes during the early stages of tooth development has been widely analyzed, while little data has been reported on their role in dental histogenesis. The expression pattern of Dlx2 has been described in the mouse incisor; an inverse linear relationship exists between the level of Dlx2 expression and enamel thickness, suggesting a role for Dlx2 in regulation of ameloblast differentiation and activity. In vitro data have revealed that DLX homeoproteins are able to regulate the expression of matrix proteins such as osteocalcin. The aim of the present study was to analyze the expression and function of Dlx genes during amelogenesis. Analysis of Dlx2/LacZ transgenic reporter mice, Dlx2 and Dlx1/Dlx2 null mutant mice, identified spatial variations in Dlx2 expression within molar tooth germs and suggests a role for Dlx2 in the organization of preameloblastic cells as a palisade in the labial region of molars. Later, during the secretory and maturation stages of amelogenesis, the expression pattern in molars was found to be similar to that described in incisors. The expression patterns of the other Dlx genes were examined in incisors and compared to Dlx2. Within the ameloblasts Dlx3 and Dlx6 are expressed constantly throughout presecretory, secretory, and maturation stages; during the secretory phase when Dlx2 is transitorily switched off, Dlx1 expression is upregulated. These data suggest a role for DLX homeoproteins in the morphological control of enamel. Sequence analysis of the amelogenin gene promoter revealed five potential responsive elements for DLX proteins that are shown to be functional for DLX2. Regulation of amelogenin in ameloblasts may be one method by which DLX homeoproteins may control enamel formation. To conclude, this study establishes supplementary functions of Dlx family members during tooth development: the participation in establishment of dental epithelial functional organization and the control of enamel morphogenesis via regulation of amelogenin expression.