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Although a big deal of dental research is being focused to the understanding of early stages of tooth development, a huge gap exist on our knowledge on how the dental hard tissues are formed and how this process is controlled daily in order to produce very complex and diverse tooth shapes adapted for specific functions. Emerging evidence suggests that clock genes, a family of genes that controls circadian functions within our bodies, regulate also dental mineralized tissues formation. Enamel formation, for example, is subjected to rhythmical molecular signals that occur on short (24h) periods and control the secretion and maturation of the enamel matrix. Accordingly, gene expression and ameloblast functions are also tightly modulated in regular daily intervals. This review summarizes the current knowledge on the circadian controls of dental mineralized tissues development with a special emphasis on amelogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Classic tissue recombination studies have demonstrated that, in the early developing mouse tooth germ, the odontogenic potential, known as the tooth-inductive capability, resides initially in the dental epithelium and then shifts to the dental mesenchyme. However, it remains unknown if human embryonic dental tissues also acquire such odontogenic potential. Here we present evidence that human embryonic dental tissues indeed possess similar tooth-inductive capability. We found that human dental epithelium from the cap stage but not the bell stage was able to induce tooth formation when confronted with human embryonic lip mesenchyme. In contrast, human dental mesenchyme from the bell stage but not the cap stage could induce mouse embryonic second-arch epithelium as well as human keratinocyte stem cells, to become enamel-secreting ameloblasts. We showed that neither post-natal human dental pulp stem cells (DPSCs) nor stem cells from human exfoliated deciduous teeth (SHED) possess odontogenic potential or are odontogenic-competent. Our results demonstrate a conservation of odontogenic potential in mouse and human dental tissues during early tooth development, and will have an implication in the future generation of stem-cell-based bioengineered human replacement teeth.

In order to gain insight into possible cellular functions of the prion protein (PrP) during normal development, the expression of Prnp (encoding the PrP) and the distribution of the PrP were studied in murine tooth germs. Expression of Prnp in the mouse first molar tooth germ was highly dynamic, increasing several-fold during the secretory phase of odontogenesis, exhibiting a time-course of expression similar to that of genes coding for other extracellular proteins [e.g. enamel matrix proteins (Amelx, Ambn, Enam), Aplp1, Clstn1, and Clu]. Western blot analysis suggested that the amounts of PrP and amyloid beta (A4) precursor-like protein 1 (APLP1) in the tooth germ followed time-courses similar to those of the corresponding mRNAs. Immunohistochemical studies of the distribution of PrP in murine molar and incisor tooth germs at embryonic day (E)18.5 suggested that this protein was located in the cervical loop, outer enamel epithelium, pre-ameloblasts, and dental papilla. Different degrees of immunolabelling of pre-ameloblasts on the mesial and distal aspects of a lower molar cusp may be related to different enamel configurations on the two aspects. It is concluded that the dynamic patterns of expression of Prnp, and of distribution of PrP, suggest that PrP may have functions during secretory odontogenesis, perhaps in relation to amelogenesis., (© 2010 Eur J Oral Sci.)

Granulin epithelin precursor (GEP) is a new growth factor that functions in brain development, chondrogenesis, tissue regeneration, tumorigenesis, and inflammation. The goal of this study was to study whether GEP was critical for odontogenesis and amelogenesis both in vivo and in vitro. The in situ hybridization and immunohistochemistry data showed that GEP was expressed in both odontoblast and ameloblast cells postnatally. Knockdown of GEP by crossing U6-ploxPneo-GEP and Sox2-Cre transgenic mice led to a reduction of dentin thickness, an increase in predentin thickness, and a reduction in mineral content in enamel. The in vitro application of recombinant GEP up-regulated molecular markers important for odontogenesis (DMP1, DSPP, and ALP) and amelogenesis (ameloblastin, amelogenin and enamelin). In conclusion, both the in vivo and the in vivo data support an important role of GEP in tooth formation during postnatal development.

The implantation of cultured dental cell-cell re-associations allows for the reproduction of fully formed teeth, crown morphogenesis, epithelial histogenesis, mineralized dentin and enamel deposition, and root-periodontium development. Since vascularization is critical for organogenesis and tissue engineering, this work aimed to study: (a) blood vessel formation during tooth development, (b) the fate of blood vessels in cultured teeth and re-associations, and (c) vascularization after in vivo implantation. Ex vivo, blood vessels developed in the dental mesenchyme from the cap to bell stages and in the enamel organ, shortly before ameloblast differentiation. In cultured teeth and re-associations, blood-vessel-like structures remained in the peridental mesenchyme, but never developed into dental tissues. After implantation, both teeth and re-associations became revascularized, although later in the case of the re-associations. In implanted re-associations, newly formed blood vessels originated from the host, allowing for their survival, and affording conditions organ growth, mineralization, and enamel secretion.

Background: To analyze the distribution pattern of claudins 1, 4, 5, 7 and occludin in benign and malignant ameloblastomas and developing human teeth., Methods: Paraffin-embedded tissue specimens of 25 benign and four malignant ameloblastomas and two developing human teeth were examined immunohistochemically using antibodies against claudins 1, 4, 5, 7 and occludin., Results: In ameloblastomas strongest expression was seen for claudins 1 and 7 while claudin 4 was expressed less frequently. Claudin 5 and occludin were seen only in a minority of cases. There were no evident differences in the expression of claudins or occludin neither between different histologic subtypes of ameloblastomas nor between benign or malignant cases. The strongest expression for claudins was present in the central stellatum reticulum-like cells surrounding the microcysts and in the areas with squamous differentiation of the ameloblastomas. In developing teeth both claudin 1 and 7 stained strongly in the enamel epithelium, ameloblasts, and enamel matrix, but staining for claudin 4 was relatively weak. Claudin 5 was preferentially expressed only in vessels, and occludin staining ranged from negative to weak in ameloblastomas and teeth germs., Conclusion: There were no clear differences in the expression levels between benign and malignant ameloblastic tumors. The overexpression of claudins in the areas with microcyst formation may indicate their attempt to maintain the interepithelial cohesion of the cells. The strong immunoreactivity of ameloblasts and newly synthesized enamel matrix for claudins 1 and 7 indicates that they may be involved in cell signaling influencing enamel formation.

Objectives: The aim of this study is to clarify the functional significance of heat-shock protein (HSP)-25 during tooth development., Design: We compared the expression of HSP-25 in the dental epithelial and mesenchymal cells with their proliferative activity during odontogenesis in rat molars on postnatal days 1-100 by immunohistochemistry using anti-HSP-25 and anti-5-bromo-2'-deoxyuridine (BrdU) for cell proliferation assay., Results: On day 1, BrdU-immunoreactive cells were densely located in the inner enamel epithelium in the cervical loop and intercusped areas and the dental pulp adjacent to them, whereas HSP-25-immunoractivity (IR) was restricted to the cusped area where odontoblasts and ameloblasts had already differentiated. Subsequently, BrdU-IR shifted in the apical direction to be localized around Hertwig's epithelial root sheath during days 5-30, never overlapping with concomitantly apically-shifted HSP-25-IR. On days 60-100, BrdU-immunoreactive cells were hardly recognizable in the dental pulp, where HSP-25-IR was exclusively localized in the odontoblast layer. Furthermore, the odontoblast- and ameloblast-lineage cells exhibited two steps in the expression of HSP-25 throughout the postnatal stages: first, dental epithelial and pulpal mesenchymal cells showed a weak IR for HSP-25 after the cessation of their proliferative activity, and subsequently odontoblasts and ameloblasts consistently expressed an intense HSP-25-IR., Conclusion: Odontoblast- and ameloblast-lineage cells acquire HSP-25-IR after they complete their cell division, suggesting that this protein acts as a switch between cell proliferation and differentiation during tooth development. The consistent expression of HSP-25-IR in the formative cells may be involved in the maintenance of their functional integrity.

Organotypic cultures of embryonic mouse tooth germs were used to investigate the developmental expression and roles of MMPs in the formation and mineralization of dentin and enamel matrices. The spatially and temporally regulated expression of MMP-2, MMP-9 and MMP-20 in developing mouse tooth germs in vivo was maintained in culture. The inhibition of metalloproteinases activity by marimastat altered the morphogenesis and mineralization of the tooth germs associated with an inhibition of the activation of both MMP-20 and MMP-2. MMP inhibition deregulated the molecular processing of two major dental matrix proteins, amelogenin and dentin sialoprotein (DSP). This coincided with their accumulation and the loss of their normal distribution within the extracellular matrix, resulting in a defective mineralization of dentin and enamel matrices. These findings demonstrate the critical role of MMPs in the processing and maturation of the dental matrix.

Cadherins are cell adhesion molecules that are critical for tissue development. In this report, we identified members of the cadherin family cadherin-related neuronal receptors (CNRs) 1 and 5 expressed in rat incisors by the differential display method. Quantitative RT-PCR revealed that CNR1 mRNA is expressed in the secretory stage but reduced in the early-maturation stage, while CNR5 mRNA is expressed in both these stages. In situ hybridization showed that strong expression of CNR1 is strong in the secretory stage, but reduced in the early phase and diminished in the late phase of the early-maturation stage. CNR5 mRNA is expressed almost at the same levels in the secretory and in the early phase of the early-maturation stages but is absent in the late phase of the early-maturation stage. Both CNR1 and 5 mRNA are continuously expressed in odontoblasts. Immunohistology showed that CNR proteins are expressed in the secretory and early-maturation stages of ameloblasts, but no protein expression at the late-maturation stage was observed. CNR proteins were continuously expressed in odontoblasts. We found that recombinant CNR1 binds dental epithelial and mesenchymal cells through N-terminal domain EC1 in vitro. These results suggest that CNR1 and CNR5 may play an important role in enamel and dentin formation, probably through cell-cell and/or cell-matrix interactions.

The present immunocytochemical study reports on the expression of heat-shock protein (Hsp) 25 during odontogenesis in rat molars from postnatal 1 to 100 days. Hsp 25 immunoreactivity (IR) appeared in the immature dental mesenchymal cells and the differentiating and differentiated odontoblasts. At 30 days, the coronal odontoblasts retained intense Hsp25-IR, whereas the odontoblasts in the root and floor pulp were initially weak or negative but increased in IR in the later stages, indicating that the expression of Hsp 25 reflects the differentiation status of odontoblasts. During amelogenesis, the secretory ameloblasts were Hsp 25 immunopositive and the enamel free area (EFA) cells showed intense Hsp 25-IR when they developed a ruffled border. Ruffle-ended ameloblasts (RA) also consistently showed intense Hsp 25-IR, but smooth ended ameloblasts (SA) showed weak IR. These data suggest that Hsp 25 is related to the formation and maintenance of the ruffled border of RA and EFA cells.

Bax and Bcl-2 are members of a family of intracellular, membrane-associated proteins that regulate programmed cell death. It has been suggested that, when Bax predominates, programmed cell death is accelerated and the apoptosis inhibitory activity of Bcl-2 is suppressed. The present study was undertaken to immunohistochemically (IHC) localize Bax and Bcl-2 in the cells of the enamel organ during amelogenesis in rat molars. Also, apoptotic cells were detected by TUNEL staining. The IHC intense localization of Bcl-2 and light staining for Bax in the pre-ameloblasts suggest that apoptosis is inhibited in the proliferating pre-ameloblasts. This is consistent with an absence of TUNEL staining for apoptosis in these cells. However, in the late secretory and transition ameloblasts, and adjacent stratum intermedium, evidence of apoptosis of the ameloblasts was observed. Bax and Bcl-2 were co-localized in the proximal ends of late secretory, transition and early maturation-stage ameloblasts, but immunoreactivity for Bax markedly increased in the proximal ends of late secretory and transition ameloblasts, while the Bcl-2 staining appeared to be lighter. This suggests that Bax antagonized Bcl-2 function, limiting the ability of Bcl-2 to prolong cell survival. In the early maturation stage, Bax staining faded while the immunoreactivity for Bcl-2 increased. Evidence of distinct apoptosis was reduced in the early maturation stage ameloblasts. When related to the occurrence of apoptosis during amelogenesis, the relative intensity of expression of Bax and Bcl-2 changed in a pattern consistent with that observed in other cell lines. This indicates that these proteins play essential roles in the process of amelogenesis, as predicted by their proposed mechanisms of action in the control of apoptosis.

Light and electron microscopic investigations of lectin binding patterns in rat tooth germs were undertaken in order to elucidate glycoconjugate localization in cells of the reduced enamel epithelia and their derivatives. It was found that Ulex europaeus agglutinin (UEA-1), peanut agglutinin (PNA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) exhibited variable reactivity patterns with different epithelia. UEA-1 was reactive with cells of the stratum intermedium and stellate reticulum in the tooth germ but unreactive with ameloblasts, outer enamel epithelial cells, and junctional epithelium at later stages. Reaction patterns of PNA in these cells differed from those of UEA-1. Results indicated that inner and outer cells of the reduced enamel epithelium are heterogeneous with regard to lectin binding patterns.

This review documents recent insights into the roles of growth hormone and insulin-like growth factor-I during tooth formation. Hereditarily growth hormone-deficient Lewis dwarf rats and hypophysectomized rats have been used to document the influence of growth hormone on growth of the rat incisor and molar teeth in vivo. Cell population studies using bromodeoxyuridine labeling have shown that growth hormone administration to dwarf rats affects odontogenic cell proliferation in the incisor teeth. Immunohistochemistry, employing well-characterized monoclonal antibodies directed against the hormone, its binding protein/receptor, the growth factor and its receptor, has enabled the location of these proteins to be mapped in the ontogenic sequences of ameloblasts, odontoblasts and cementoblasts. This mapping is consistent with the concept that differentiating odontogenic cells are targets for the hormone and that insulin-like growth factor I is implicated as a secondary messenger in the same differentiating cell populations. The content of predentine and precementum matrices proteoglycans appears to be growth hormone-dependent. The proteoglycans implicated so far are rich in chondroitin sulphate and thus they may also be insulin-like growth factor I (sulphation factor)-dependent. Thus matrix synthesis may be what is principally affected by growth hormone in odontogenesis although no evidence of an effect on enamel matrix synthesis or proteoglycan content has yet been documented.

While the precise mechanism of tooth eruption remains unknown it has been established that for tooth eruption to occur a functioning dental follicle is essential and normal bone resorption is required. In the osteopetrotic microphthalmic (mi) mouse, teeth fail to erupt and development is affected. In the present study, an organ culture technique was established to culture successfully lower first molars from normal and affected mi mice so as to determine if developmental defects were intrinsic to the dental tissue or whether such defects were a result of local environmental factors. Tissue was cultured for up to 13 days and development assessed morphologically at varying time intervals using standard light microscopy. Teeth developed similarly in both animal groups studied. The only variation in the appearance of cultured tissue occurred in the cervical loop region with more curling evident in dental tissue from the affected animals. The results of this study therefore showed that dental tissue from both normal and affected mi mice can be cultured for up to 13 days and that there is no difference in their development. It would appear that these teeth have the same ability to develop and consequently erupt, however the local environment influences tooth development and alters the eruption potential.

The study provides a survey of shark tooth morphogenesis based on SEM and EDX analyses of whole tooth families in six shark species. The teeth, demonstrating different stages of development, were acid-etched and coated with palladium. Calcium content was determined semi-quantitatively by using the palladium coating as an internal standard. Due to the rapid development of the enameloid, all major events took place in the two or three youngest teeth of a tooth family. Enameloid appeared to develop as a transformation of the peripheral part of the dental papilla. Mineralization started immediately. Based on morphological criteria the middle zone of the enameloid was established at an early stage, excluding the possibility of an unambiguous centrifugal or centripetal direction of growth. Substantial mineral increase first occurred in the middle zone, spreading from the tooth tip toward the base. Dentin formed after the enameloid was completely established. Dentin formation started basally as a direct prolongation of the enameloid cap, then spreading toward the tooth tip, first along the edges. It is concluded that shark enameloid has a mesenchymal background, but a role played by the inner dental epithelium can not be excluded.

Cytokeratin expression by the developing human enamel organ between the 10th and the 23rd gestational week was studied by indirect immunofluorescence microscopy technique using a panel of 15 monoclonal antibodies. The results showed that five antibodies (RKSE 60, Kk 8-60, EE 21-6, 6B10 and 1 C-7) were never reactive, that five antibodies (RCK 102, 42.39.13.1, Ks 19 and Pan 1-8) were always positive and that five antibodies (KB 37, RPN 11-62, Ks 13.1, Ks 8-12 and Ks 18.174) obtained or increased their positivity between weeks 12 and 13. It was concluded that a switch in cytokeratin expression occurred around the 12th-13th weeks. No further important change could be noticed after this period. So it is suggested that final cell differentiation was initiated at weeks 12-13.

With the invagination of the deep surface of the tooth buds of mouse embryos (15 days of gestation), heparan sulfate in the region of the lamina lucida of the dental basement membrane began to disappear. Subsequently, as the outline of the future dentino-enamel junction, which elaborates the molar cusp pattern, was formed (16-17 days of gestation), heparan sulfate disappeared not only in the lamina lucida but also at the external side of the lamina densa. However, prior to or during predentin matrix deposition (18 days of gestation), heparan sulfate reappeared in the dental basement membrane.

Oral mandibular epithelium determines the position and number of tooth organs. Cranial neural crest-derived ectomesenchyme provides inductive signals for (i) tooth shape, and (ii) sequential determination and expression of several different epithelial-derived enamel proteins. Inductive signalling follows a precise sequence, can be expressed in serumless, chemically-defined medium in vitro, and appears to be highly conserved during vertebrate evolution.