Immunofluorescent localization of myosin, alpha-actinin and tropomyosin in odontoblast microfilament bundles of the rat incisor

The localization of alpha-actinin, tropomyosin, myosin and actin in odontoblasts was examined by fluorescence microscopy using well characterized antibodies and rhodamine-phalloidin. All the reagents labeled the distal end of the cell body in the form of an oval ring with a preferential axis along the tooth axis. This ring was often interrupted. In conventional electron microscopy, microfilament bundles with periodical dense spots were running along the tooth axis at the level of the distal end of the cell body. The periodicity was about 0.6-1.0 microns. It may be possible that this dynamic structure functions to keep odontoblasts in a layer by contracting in an isometric form.     

Leptin and vascular endothelial growth factor regulate angiogenesis in tooth germs

Leptin, a 16 kDa non-glycolated polypeptide of 146 amino acids produced by the ob gene, has a variety of physiological roles not only in lipid metabolism, hematopoiesis, thermogenesis and ovarian function, but also in angiogenesis. This study focuses to investigate the possibility that leptin, as an angiogenic factor, may regulate the angiogenesis during tooth development. We firstly studied the expression of leptin and vascular endothelial growth factor (VEGF) during tooth development immunohistochemically. This investigation revealed that leptin is expressed in ameloblasts, odontoblasts, dental papilla cells and stratum intermedium cells. This expression pattern was similar to that of VEGF, one of the most potent angiogenic factors. Interestingly, more leptin-positive cells were observed in the upper third portion of dental papilla, which is closest to odontoblastic layer, compared to middle and lower thirds. Moreover, in the dental papilla, more CD31 and/or CD34-positive vascular endothelial cells were observed in the vicinity of ameloblasts and odontoblasts expressing leptin and VEGF. These findings strongly suggest that ameloblasts, odontoblasts and dental papilla cells induce the angiogenesis in tooth germs by secretion of leptin as well as VEGF.     

The enzyme histochemistry of developing odontoblasts in cattle, pigs and horses

Synopsis The histochemical distribution of some hydrolytic and oxidative enzymes in developing odontoblasts and subodontoblasts in cattle, pigs and horses has been observed in cryostat sections of teeth that have been decalcified with neutral EDTA.Undifferentiated dental epithelium and immature odontoblasts of the bell stage tooth germ showed lower levels of enzymatic activity as compared with the well-developed tooth germ.When the dentine matrix began to form, the young odontoblasts appeared to have a significantly positive reaction for acid phosphatase, and gradually other enzymes developed an activity at the top of the cusp.Odontoblasts as well as subodontoblastic-rich cells showed strong enzymatic activities for hydrolytic and oxidative enzymes, that is, they were strongly reactive for alkaline and acid phosphatase and lactate and malate dehydrogenases, and moderately reactive for other oxidative enzyme systems.It is suggested that the subodontoblastic layer is concerned with the biosynthesis of dentinal matrix as well as with the odontoblasts themselves.     

Succinate dehydrogenase activity in the developing molar of the hairless mouse Mus musculus

Synopsis Succinate dehydrogenase activity in the odontogenic tissues of the hairless mouse (hr/hr) has been studied from the initiation of the dental lamina through apposition. Enzyme activities were designated as negative, slight, moderate and strong as a function of intensity of the reaction product. Enzyme levels in the odontogenic tissues increased with advancing tooth morphogenesis. Greatest activity was observed in the ameloblastic layer which peaked on the fourth to sixth postnatal days. This cell layer displayed higher enzyme activity than the ectomesenchymally-derived odontoblasts. Succinate dehydrogenase activity appeared to be related to the degree of differentiation and functional competence on the odontogenic tissues of the hairless mouse.     

Expression of Set-α during Morphogenesis of Mouse Lower First Molar

The detailed in situ expression pattern of the Set-α gene has been studied. Previously we showed that Set-α is a differentially expressed gene in the embryonic mouse mandible at day 10.5 (E10.5) gestational age. Cells expressing Set-α were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5. At E12, they were slightly aggregated in an area where tooth germ of the lower first molar is estimated to be formed. At E13.5, Set-α was strongly expressed in the tooth germ. At the cap stage, Set-α was expressed in the enamel organ and dental papilla. At the bell stage, Set-α was distinctly expressed in the inner enamel epithelial and dental papilla cells facing the inner enamel epithelial layer, which were intended to differentiate into ameloblasts and odontoblasts, respectively. Interestingly, Set-α was also expressed in several embryonic craniofacial tissues derived from the ectoderm. This study is the first report that Set-α is distinctly expressed in the developing tooth germ, and suggests that Set-α plays an important role in both the initiation and the growth of the tooth germ, as well as in the differentiation of ameloblasts and odontoblasts.     

Apoptosis in developmental and repair-related human tooth remodeling: a view from the inside

Apoptosis is a key phenomenon in the regulation of the life span of odontoblasts, which are responsible for dentin matrix production of the teeth. The mechanism controlling odontoblasts loss in developing, normal, and injured human teeth is largely unknown. A possible correlation between apoptosis and dental pulp volume reduction was examined. Histomorphometric analysis was performed on intact 10 to 14 year-old premolars to follow dentin deposition and evaluate the total number of odontoblasts. Apoptosis in growing healthy teeth as well as in mature irritated human teeth was determined using a modified TUNEL technique and an anti-caspase-3 antibody. In intact growing teeth, the sequential rearrangement of odontoblasts into a multi-layer structure during tooth crown formation was correlated with an apoptotic wave that leads to the massive elimination of odontoblasts. These data suggest that apoptosis, coincident with dentin deposition changes, plays a role in tooth maturation and homeostasis. Massive apoptotic events were observed after dentin irritation. In carious and injured teeth, apoptosis was detected in cells surrounding the lesion sites, as well as in mono-nucleated cells nearby the injury. These results indicate that apoptosis is a part of the mechanism that regulate human dental pulp chamber remodeling during tooth development and pathology.     

Voltage-gated sodium channels confer excitability to human odontoblasts: possible role in tooth pain transmission

Odontoblasts are responsible for the dentin formation. They are suspected to play a role in tooth pain transmission as sensor cells because of their close relationship with nerve, but this role has never been evidenced. We demonstrate here that human odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na(+) currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts express neuronal isoforms of alpha2 and beta2 subunits of sodium channels. Co-cultures of odontoblasts with trigeminal neurons indicate a clustering of alpha2 and beta2 sodium channel subunits and, at the sites of cell-cell contact, a co-localization of odontoblasts beta2 subunits with peripherin. In vivo, sodium channels are expressed in odontoblasts. Ankyrin(G) and beta2 co-localize, suggesting a link for signal transduction between axons and odontoblasts. Evidence for excitable properties of odontoblasts and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts may operate as sensor cells that initiate tooth pain transmission.     

Immuno-electron-microscopic localization of laminin and collagen type IV in normal and denervated tooth pulp of the cat

Summary The distribution of laminin-like immunoreactivity in adult normal and denervated cat mandibular tooth pulps was studied by the use of fluorescence microscopy and pre-embedding immunogold electron microscopy. Immunoreactivity to collagen IV was also assessed in order to distinguish basement membranes. In normal pulps, light-microscope laminin-like immunoreactivity was strong along blood vessels and Schwann cell sheaths, and a faint immunoreactivity was seen also in the odontoblast layer. Electron microscopy confirmed the laminin-like immunoreactivity of endothelial and Schwann cell basement membranes at all pulpal levels. In the odontoblast layer and the predentine, nerve-like structures lacking basement membranes but possessing strong membrane laminin-like immunoreactivity were encountered. In addition, a clear-cut laminin-like immunoreactivity of plasma membranes of the somata and processes of odontoblasts was seen. Observations on denervated pulps as well as pulps in which nerve regeneration had taken place did not reveal any changes in the pattern of laminin-immunoreactivity in basement membranes or odontoblasts. Distribution of collagen IV-like immunoreactivity was very similar to laminin-like immunoreactivity in basement membranes of blood vessels and Schwann cells, and appeared unaffected by denervation. The odontoblasts and nerve-like profiles in the odontoblast layer were devoid of collagen IV-like immunoreactivity. We propose that odontoblast-associated laminin could be of significance as guidance for regenerating terminal pulpal nerve fibers to appropriate targets.     

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro

Summary Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyperor hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.     

Finspine morphogenesis in squalid and heterodontid sharks

Modern selachian finspines develop within a single, enlarged dermal papilla much earlier than any other papillary derived structure, and rapidly enlarge beyond the confines of the papilla. Within this, the spine primordium differentiates into two parts (mantle and trunk). Both components elongate basally and become mineralized by odontoblasts, but only the mantle has an enameloid layer. Mantle and trunk are reunited secondarily by dentine. The finspine vascular supply becomes blocked in adults, possibly to avoid loss of body fluids should the finspine (which is not shed) be damaged. Finspines probably have both defensive (perhaps mainly in juveniles) and locomotory (unctions. Odontocytes sometimes occur in the finspine base of Squalus , although the rest of the spine consists of acellular metadentine. The innermost dentine layers are centripetally deposited, resembling lamellar orthodentine of a tooth pulp. Over this is a continuous layer of centrifugally deposited lamellar dentine. Apically there is another centripetal layer (the mantle), which occurs only on certain parts of the spine. Enamelled tubercles can occur in Squalus , as a result of scleroblast disorganization, not as a result of secondary coalescence.     

Bone morphogenetic proteins in dentin regeneration for potential use in endodontic therapy

The human dentition is indispensable for nutrition and physiology. The teeth have evolved for mastication of food. Caries is a common dental problem in which the dentin matrix is damaged. When the caries is deep and the dental pulp is exposed, the pulp has to be removed in many cases, resulting ultimately in loss of the tooth. Therefore, the regeneration of dentin-pulp complex is the long-term goal of operative dentistry and endodontics. The key elements of dentin regeneration are stem cells, morphogens such as bone morphogenetic proteins (BMPs) and a scaffold of extracellular matrix. The dental pulp has stem/progenitor cells that have the potential to differentiate into dentin-forming odontoblasts in response to BMPs. Pulpal wound healing consists of stem/progenitor cells release from dental pulp niche after noxious stimuli such as caries, migration to the injured site, proliferation and differentiation into odontoblasts. There are two main strategies for pulp therapy to regenerate dentin: (1) in vivo method of enhancing the natural healing potential of pulp tissue by application of BMP proteins or BMP genes, (2) ex vivo method of isolation of stem/progenitor cells, differentiation with BMP proteins or BMP genes and transplantation to the tooth. This review summarizes recent advances in application of BMPs for dentin regeneration and possible use in endodotic therapy.     

Bono1: a gene associated with regions of deposition of bone and dentine

We have examined the mRNA expression pattern of the murine expressed sequence tag (EST) clone in embryonic and early postnatal mice. Expression was strongly and specifically localised to developing bones and odontoblasts in teeth, therefore we have named this gene Bono1 (Bone and odontoblasts). Bono1, which has human, rat and chicken orthologues designated as FKSG28 was expressed in most ossification regions of the head including calvarial bones, skull and jaws. Expression was localised to osteoblasts derived from both intramembraneous and endochondral ossification processes. Comparative analysis of the expression of Bono1 in the mandible with Bone sialoprotein (BSP), a marker of advanced osteoblastogenesis, revealed that Bono1 expression starts later in the osteoblast cell lineage than BSP. In the tooth, Bono1 was localised in secretory odontoblasts. This expression was complementary to BSP, which was only present in early pre-odontoblasts. In secretory odontoblasts, Bono1 was shown to be co-expressed with Dentin sialophosphoprotein (DSPP). In summary, Bono1 was expressed in functional osteoblasts and odontoblasts and was associated with regions of matrix mineralization.     

S100 protein-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

S100-immunoreactivity (ir) was examined in tooth pulp primary neurons of the rat. An immunofluorescence method demonstrated that the molar tooth pulp contained S100-immunoreactive (ir) nerve fibers. In the root pulp, pulp horn and roof of the pulp chamber, S100-ir smooth and varicose fibers ramified and formed subodontoblastic nerve plexuses. All the fibers became varicose at the base of the odontoblastic layer and extended to the odontoblastic layer. Some varicose endings could be traced into the dentin. The trigeminal neurons retrogradely labeled with fluorogold (FG) from the first and second maxillary molar tooth pulps exhibited S100- and parvalbumin-ir. Approximately 60% and 24% of the labeled cells were ir for S100 and parvalbumin, respectively. Virtually all parvalbumin-ir FG-labeled cells showed S100-ir, while 40% of S100-ir ones coexpressed parvalbumin-ir. An immunoelectron microscopic method revealed that all myelinated axons and half of the unmyelinated axons in the root pulp contained S100-ir. In the odontoblastic layer, predentin and dentin, S100-ir neurites lost the Schwann cell ensheathment and made close contact with cell bodies and processes of odontoblasts. The odontoblastic layer also contained parvalbumin-ir neurites. These neurites were devoid of the Schwann cell ensheathment and in close apposition to cell bodies and processes of odontoblasts. S100-ir pulpal axons seemed to be insensitive to repeated neonatal capsaicin treatment. This study suggests that S100-ir tooth pulp primary neurons are mostly myelinated and that S100-ir unmyelinated axons in the root pulp are preterminal segments of myelinated stem axons.     

Transforming Growth Factor-β Isoform Expression in Mature Human Healthy and Carious Molar Teeth

Transforming growth factor (TGF)-β isoforms have been implicated in cellular signalling during tooth development and repair, but little is known of their cellular localisation or distribution within the dental tissues in the mature tooth. This study investigated the presence of TGF-β1, β2 and β3 isoforms in tissues of sound and carious human molar teeth, to understand better the expression of TGF-βs during health and disease. In healthy tissues, odontoblasts, cells of the cell rich layer, pulpal fibroblasts and endothelial cells were stained to varying degrees for all isoforms, with TGF-β3 showing the greatest intensity and TGF-β1 the weakest intensity. Similar patterns of staining were observed in carious teeth; however, TGF-β1 showed significantly increased staining intensity within odontoblasts and pulpal cells of carious teeth (p<0.001). Biochemical analysis showed greater amounts of TGF-β1 in tertiary dentine than in primary dentine samples. The expression of TGF-βs in odontoblasts and the increased presence of TGF-β1 in tertiary dentine suggest that these isoforms may be important in odontoblast behaviour and the modulation of the tissue response to injury.     

Postcanine microstructure in Cricodon metabolus,a Middle Triassic gomphodont cynodont from south‐eastern Africa

Cricodon metabolus is a trirachodontid cynodont from the Anisian (Middle Triassic) of eastern and southern Africa. It has labiolingually expanded (gomphodont) postcanines but also a sectorial tooth in the last postcanine locus. In this paper, we examine the crown microstructure of isolated sectorial and gomphodont postcanines belonging to the holotype specimen of this taxon using scanning electron microscopy. The enamel of both teeth is prismless and composed of discontinuous columnar divergence units, supporting the consistent presence of synapsid columnar enamel in cynognathians. Abundant tubules and numerous irregularly spaced incremental lines are also visible in the enamel and dentine layers in each tooth. This study reveals that the enamel thickness varies along the tooth row in Cricodon as the enamel layer of the gomphodont postcanines is 11.5 times thicker than that of the sectorial crown. It is likely that this difference reflects occlusal stresses and fewer replacements in gomphodont postcanines relative to sectorial teeth. Approximately 100 incremental growth lines of von Ebner are present in the dentine layer, indicating that the deposition of the dentine by odontoblasts occurred for three months before the animal's death.     

Mucopolysaccharides in odontogenesis

Summary Localization of sulfomucopolysaccharides in developing teeth of Swiss albino mice was detected by S³⁵ autoradiography and histochemistry.A positive correlation was found to exist between autoradiographic and histochemical data with regard to the localization of sulfomucopolysaccharides. Autoradiography, however, revealed some sites of localization which were not detectable by histochemistry, namely, the odontoblasts and stratum intermedium.Fetuses which received the isotope via maternal injection at the cap stage of tooth development and were sacrificed after 2 hours of isotope action displayed rapid incorporation of the isotope in the components of the dental papilla. In the enamel organ, however, only moderate activity was recorded. When the time interval between injection and sacrifice of the experimental animals was increased to 20 hours, intense activity was observed in the enamel organ. With progressively longer intervals between injection and sacrifice, S³⁵ was demonstrable first in odontoblasts and later in the predentin. This occurred as a band or active zone which migrated toward the dentino-enamel junction. With the increasing intervals between injection and sacrifice, first the odontoblasts were active, then predentin was active while the odontoblasts became reduced in activity, after which the dentin matrix gained activity while the predentin decreased somewhat in activity. This pattern is consistent with appositional growth. A linear band of activity was not observed in the enamel matrix; rather, the activity was present as a diffuse stippling over a relatively large area of the matrix. The sulfomucopolysaccharide which existed in dentin matrix was postulated to have originated from the cells of both the odontoblastic layer and the dental papilla.Supported by PHS Grant No 2800-02, Tooth Germ Development, National Institute of Dental Research, National Institutes of Health.     

Accelerated dentinogenesis by inhibiting the mitochondrial fission factor,dynamin related protein 1

Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth.     

Dentin sialoprotein: biosynthesis and developmental appearance in rat tooth germs in comparison with amelogenins,osteocalcin and colagen type-I

A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with ¹⁴C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction.     

Differential expression of decorin and biglycan genes during mouse tooth development.

Small leucine-rich proteoglycans (SLRPs) have a number of biological functions and some of them are thought to regulate collagen mineralizaton in bone and tooth. We have previously identified and immunolocalized two members of the SLRPs family, decorin and biglycan, in bovine tooth/periodontium. To investigate their potential roles in tooth development, we examined the mRNA expression patterns of decorin, biglycan and type I collagen in newborn (day 19) mice tooth germs by in situ hybridization. At this developmental stage, the first maxillary and mandibular molars include stages before and after secretion of the predentin matrix, respectively. The expression of decorin mRNA coincided with that of type I collagen mRNA and was mostly observed in secretory odontoblasts, while the biglycan mRNA was expressed throughout the tooth germ, including pre-secretory odontoblasts/ameloblasts, dental papilla and stellate reticulum. However, its signal in secretory odontoblasts was not as evident as that of decorin. In mandibular incisors, where a significant amount of predentin matrix and a small amount of enamel matrix were already secreted, a similar differential expression pattern was observed. In secretory ameloblasts the biglycan mRNA expression was apparent, while that of decorin was not. These differential expression patterns suggest the distinct roles of biglycan and decorin in the process of tooth development.     

On the origin of ganoine: histological and ultrastructural data on the experimental regeneration of the scales of Calamoichthys calabaricus (Osteichthyes, Brachyopterygii, Polypteridae)

In order to understand the process of ganoine formation on the ganoid scales, scale regeneration has been studied to overcome the lack of a growth series of scale ontogeny. Seven stages of ganoid scale regeneration have been defined over a period of five months in the polypterid fish Calamoichthys calabaricus. The study has been carried out using transmission electron microscopic techniques. After wound healing and differentiation of the osseous basal plate, a layer of vascular dentin is deposited at the upper surface of the basal plate owing to the presence there of odontoblasts closely applied to the dentin. When these cells move away, a close contact is then established between the stratified epidermis and the regenerating scale. Numerous alterations of the epidermal-dermal boundary occur until its disappearance and a thick layer of pre-ganoine is formed. This layer is progressively mineralized; and finally an organic intermediate layer differentiates between the ganoine, which is a hyper-mineralized tissue, and the overlying epidermis. This ultrastructural study demonstrates rather unequivocally the involvement of the inner epidermal layer (IEL) in the appearance and growth of the ganoine. It is suggested that these epidermal cells can be compared functionally to the inner dental epithelium (IDE) described during mammal tooth morphogenesis. Consequently, our results allow us to propose that ganoine can be identified as true enamel, although additional data are necessary to analyze the proteinaceous component or its organic matrix.