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.     

Odontoblasts: the cells forming and maintaining dentine

Odontoblasts are tall columnar cells located at the periphery of the dental pulp. They derive from ectomesenchymal cells originated by migration of neural crest cells during the early craniofacial development. Odontoblasts form the dentine, a collagen-based mineralized tissue, through secretion of its collagenous and noncollagenous organic matrix components and by control the mineralization process. A conspicuous cell process arises from the cell body of odontoblasts and penetrates into the mineralized dentine. After dentinogenesis, odontoblasts deposit new layers of dentine throughout life and might also form a type of reactionary/reparative dentine in response to dental caries and other external factors may affect teeth.     

Rehardening of caries-like lesions in root surfaces by saliva substitutes

Objective: This study sought to evaluate whether root dentine caries‐like lesions could be remineralised by saliva substitutes. Methods: Root dentine slabs (3 × 3 × 2 mm) were cut from bovine incisors, ground flat, polished and pre‐tested for Knoop microhardness (KHN) at five locations spaced 500 μm apart and 500 μm from the left edge of each sectioned piece. After 60 out of the 100 slabs had been selected based upon their KHN values, specimens were coated with wax except for their outer surface. Specimens were then cycled through a highly cariogenic challenge model to induce caries‐like lesions, whose formation was confirmed by KHN measurements located 500 μm from the right edge of the specimen. According to a randomised complete block design, the experimental units (n = 15) were exposed to 1.5 ml of saliva substitutes, based on either mucin (MC) or carboxymethylcellulose (CM), to natural human saliva (HS) or to 100% relative humidity (RH) over 20 days. Remineralisation was verified by KHN measurements located 1000 μm apart from the right edge of the specimen. Results: Analysis of variance indicated a significant (p < 0.0001) difference among the KHN values attained by the carious root dentine after exposure to the remineralising agents. Tukey's test ascertained that remineralisation was greatest with MC, intermediate with CM and least with HS, but rehardening did not reach the pre‐caries lesion formation values. Conclusion: Saliva substitutes may provide partial remineralisation to preformed caries‐like lesions in root dentine.     

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.     

Effect of glypican‐1 gene on the pulp cells during the reparative dentine process

GPC‐1 (glypican‐1) is a cell surface heparan sulfate proteoglycan that acts as a co‐receptor for heparin‐binding growth factors and members of the TGF‐β (transforming growth factor beta‐1) family. The function of cell‐surface proteoglycans in the reparative dentine process has been under investigation. Gpc‐1 was detected with similar frequency as tgf‐β1 in the cDNA library using mRNA from the odontoblast‐like cell‐enriched pulp of rat incisors. The aim of this study was to test our hypothesis that gpc‐1 may be related to reparative dentine formation. We examined the expression of this gene during the reparative dentine process, as well as the effect of gpc‐1 on odontoblast‐like cell differentiation using siRNA (small interfering RNA) to down‐regulate gpc‐1 expression. Immunohistological examination showed that GPC‐1 was expressed in pulp cells entrapped by fibrodentine and odontoblast‐like cells as well as TGF‐β1. The mRNAs for gpc‐1, ‐3 and ‐4, except for gpc‐2, were expressed during odontoblast‐like cell differentiation in pulp cells. The relative levels of gpc‐1 mRNA were increased prior to the differentiation stages and were decreased during the secretory and maturation stages of pulp cells. Down‐regulation of gpc‐1 expression resulted in a 3.9‐fold increase in tgf‐β1 expression in pulp cells and a 0.3‐fold decrease in dspp (dentine sialophosphoprotein) expression compared with control. These results suggested that gpc‐1 and tgfβ‐1 expression are necessary for the onset of differentiation, but should be down‐regulated before other molecules are implicated in the formation of reparative dentine. In conclusion, gpc‐1 expression in odontoblast‐like cells is associated with the early differentiation but not with the formation of reparative dentine.     

Foreign body giant cells and osteoclasts are TRAP positive,have podosome‐belts and both require OC‐STAMP for cell fusion

Macrophages have the ability to fuse and form multinucleated giant cells such as Osteoclast (OCs) and FBGCs. Osteoclast stimulatory transmembrane protein (OC‐STAMP) is an important cell surface protein involved in the formation of OCs. This study sought to determine if OC‐STAMP also regulates formation of FBGCs using expression analysis and subsequent inhibition studies. qPCR and Western blot analysis showed that OC‐STAMP expression is significantly higher in FBGCs compared to control monocytes (P < 0.05). Four days following cell culture, OCs were positive for TRAP and F‐actin ring formation, but FBGCs were not. In contrast, FBGCs were positive for TRAP and showed podosome belts comprised of F‐actin on Day 8. FBGCs were subsequently plated onto dentine, but despite presenting some morphologic features of OCs (OC‐STAMP expression, TRAP reactivity, and podosome belts) they failed to resorb bone. To evaluate a role for OC‐STAMP in FBGCs, we inhibited this cell surface protein with anti‐OC‐STAMP antibody and observed that cell fusion and podosome belt formation was inhibited in both OCs and FBGCs. Our data support the hypothesis that OC‐STAMP is a regulatory molecule for FBGCs; and that they are functionally distinct from OCs, despite similarities in gene expression profile, podosome belt formation, and TRAP expression. J. Cell. Biochem. 114: 1772–1778, 2013. © 2013 Wiley Periodicals, Inc.     

Enamel and dentine ultrastructure in the early Jurassic therian Kuehneotherium

Three fragmentary molars of Kuehneotherium from Wales were progressively abraded by grinding and studied by scanning electron microscopy. A fourth molar was reduced to small blocks, thin sections of which were examined by transmission electron microscopy. Enamel showed a 'preprismatic' pattern, consisting of columns of crystals disposed as pinnae but not separated by an interprismatic substance. No enamel tubules were observed. Incremental lines were present. Inner dentine was permeated by numerous tubules not surrounded by peritubular dentine but a peripheral dentine layer adjacent to the enamel-dentine junction was atubular. Calcified deposits were occasionally observed in the lumen of dentine tubules, some of which were interpreted here as dentinal sclerosis consecutive to masticatory wear.     

Acid phosphatase in the Golgi apparatus of cells forming extracellular matrix of hard tissues

Summary Histochemical studies using cryostat sections of fixed rodent fetal and newborn tissues indicated that acid phosphatase (APase) staining of the Golgi apparatus (GA) of cells secreting matrix for hard tissue formation was a general phenomenon. The enzyme was chiefly observed in the GA of tall secretory ameloblasts involved in enamel formation and in the GA of odontoblasts forming dentine; lysosome-like granules reactive for this enzyme were also observed in these cells. Activity was also intense in the GA and lysosomes of osteoblasts involved in intramembranous and endochondral bone formation.High levels of APase in the GA of extracellular matrix-forming cells appeared to correlate with secretory activity. The GA of most other cells, even chondroblasts forming cartilage matrix, had much less marked APase activity. Contrary to previous suggestions, it appears that APase may have a more direct role in osteogenesis than the osteolytic or resorptive action usually cited.This investigation was supported by PHS Research Grant No. DE 02668 from the National Institute of Dental Research and in part by General Research Support Grant No. RR 5333 from the General Research Support Branch of the National Institutes of Health.The authors gratefully acknowledge the excellent technical assistance of Dorothy H. Clapp and Peggy E. Yates.     

Evidence for tight junctions between odontoblasts in the rat incisor

Odontoblasts are known to be involved in the process of dentinogenesis but it is not clear whether substances may also be deposited in predentine and dentine by passing between these cells. Although tight junctions have been described, it is not clear if they are macular or "leaky" as opposed to continuous or "tight". In this study use has been made of the permeability of fenestrated capillaries amongst the odontoblasts to deposit the penetrative tracer lanthanum in the interodontoblastic space. This was done by perfusion of anaesthetized rats with physiological solutions containing lanthanum nitrate at 37 degrees C. Immersion fixation of transverse segments of mandibular incisors and examination with an electron microscope showed that lanthanum could permeate 40-50 microns between the odontoblasts to reach the peripheral pulp. Towards the predentine, often less than 10 microns from the capillaries, its progress was abruptly and completely halted by the junctions at the apical ends of the odontoblast cell bodies. Lanthanum was not found in the predentine. The mature secretory odontoblasts in the rat incisor have therefore been shown to be joined by continuous tight junctions. In the process of dentinogenesis this means that all substances deposited in predentine and dentine must arrive by passing through the odontoblasts.     

The effect of caffeine,different fixation regimes and low temperature on microtubules in the cells of higher plants

Caffeine, (1:3:7-tri-methyl-xanthine), either as a prefixation treatment or included with glutaralde-hyde as the primary fixative, destroys or disorganises the microtubules associated with the formation of secondary walls in fibres from the flowering stem of the grass Lolium temulentum L. There is no observable effect of caffeine treatment on the microtubules associated with primary wall formation in collenchyma and young fibres from L. temulentum or in root cap cells of Zea mays L. and Phaseolus vulgaris L. The microtubules associated with primary wall formation are destroyed by cold treatment but not those associated with secondary wall formation. Tannic acid included in the fixative shows the microtubules associated with secondary wall formation in fibres of L. temulentum to be composed of 13 subunits. Treatment with lanthanum hydroxide does not stain the core or the halo of the microtubules.Abbreviation PIPES Piperazine N-N- bis 2 ethanol sulphonic acid The Grassland Research Institute is financed through the Agricultural Research Council     

Fracture-toughening mechanisms responsible for differences in work to fracture of hydrated and dehydrated dentine

This study investigates the nature of deformation and differences in the mechanisms of fracture and properties of dentine where there has been a loss of moisture, as may occur with removal of the pulp in the endodontic treatment of teeth. Controlled fracture toughness testing was conducted on bovine teeth to determine the influence of hydration on the work of fracture of dentine. Significant differences (p<0.01) were observed between the fracture toughness of hydrated (554+/-27.7J/m2) and dehydrated (113+/-17.8J/m2) dentine. Observations of the crack tip region during crack extension revealed extensive ligament formation occurred behind the crack tip. These ligaments provide considerable stability to the crack by significantly increasing the work of fracture, thereby acting as a fracture-toughening mechanism. Micro-cracking, reported as a fracture-toughening mechanism in bone, is also clearly seen. A zone of in-elastic deformation may occur as hydrated specimens revealed upon crack extension, a region about the tip that appeared to suck water into the structure and to exude water behind the crack tip. In dehydrated dentine, no in-elastic zone was observed. Micro-cracking is present though the cracks are smaller, straighter and with less opening than hydrated dentine. Only limited ligament formation just behind the crack tip was observed. These differences resulted in a significantly lower work of fracture with unstable brittle fracture characteristics. Based on these results, several fracture-toughening mechanisms were identified in dentine, with micro-cracking not considered the most important. These findings may be relevant for bone, a similar mineralised hydrated tissue.     

Immunocytochemical localization of choline acetyltransferase and muscarinic ACh receptors in the antenna during development of the sphinx moth <Emphasis Type="Italic">Manduca sexta</Emphasis>

Immunocytochemistry with monoclonal antibodies was used to investigate the locations of muscarinic acetylcholine receptors (mAChR) and choline acetyltransferase (ChAT) in sections of the developing antennae of the moth Manduca sexta. The results were correlated with a previous morphological investigation in the developing antennae which allowed us to locate different cell types at various stages of development. Our findings indicated that the muscarinic cholinergic system was not restricted to the sensory neurons but was also present in glial and epidermal cells. By day 4–5 of adult development, immunoreactivity against both antibodies was present in the axons of the antennal nerve, and more intense labeling was present in sections from older pupae. At days 4–9, the cell bodies of the sensory neurons in the basal part of the epidermis were also intensely immunolabeled by the anti-mAChR antibody. In mature flagella, large numbers of cells, some with processes into hairs, were strongly labeled by both antibodies. Antennal glial cells were intensely immunolabeled with both antibodies by days 4–5, but in later stages, it was not possible to discriminate between glial and neural staining. At days 4–9, we observed a distinctly labeled layer of epidermal cells close to the developing cuticle. The expression of both ChAT and mAChRs by neurons in moth antennae may allow the regulation of excitability by endogenous ACh. Cholinergic communication between neurons and glia may be part of the system that guides axon elongation during development. The cholinergic system in the apical part of the developing epidermis could be involved in cuticle formation.This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Foundation for Innovation, and the Nova Scotia Research and Innovation Trust to P.H.T. and a NSERC postdoctoral fellowship to J.C.     

Waste-reducing cultivation of Pleurotus ostreatus and Pleurotus pulmonarius on coffee pulp: changes in the production of some lignocellulolytic enzymes

Fruiting body production for one strain of Pleurotus ostreatus and three strains of P. pulmonarius was evaluated on coffee pulp pasteurized at 80 °C for 1 h. Based upon three harvests per strain, the single P. ostreatus line was found to display a 40-day culture cycle, whereas the three P. pulmonarius strains completed their cycles after more than 50 days of incubation. These time periods were notably shorter than those observed in previous studies using other growth substrates. Nevertheless, yields expressed as biological efficiencies were not significantly different among strains, fluctuating between 125 and 138%. Extracellular enzymatic activity was also monitored for P. ostreatus and P. pulmonarius (one strain only). To do this, samples of mycelium-bearing substrate were taken every 4 days throughout the incubation period. Care was taken to represent all developmental stages, including primordial and fruiting bodies. Samples were either lyophilized and then analysed or, in some cases, analysed immediately without lyophilization. Hydrolase activity (i.e. endoglucanase (CMC) and cellobiohydrolase (CBH)) was found to depend on developmental stage, showing peak production during fruiting body formation. On the other hand, oxidase activity-(i.e. laccase (LAC) and Mn-peroxidase (MnP)) was associated with phenol degradation. Nevertheless, in the case of oxidases developmental timing differences were also observed. Specifically, LAC activity was detected as early as 8 days after inoculation in non-lyophilized samples, whereas MnP appeared near the end of the incubation period. No LAC activity was observed in lyophilized samples. This study concludes that coffee pulp might be successfully employed in the cultivation of mushrooms, not only because important extracellular enzymes are produced by mushrooms when grown upon this substrate, but also because the abbreviated cultivation cycle associated with this medium favours commercial processes. Commercialization might be further improved if strains specifically adapted to this novel substrate are selected.     

Analysis of growth and stable isotopes in teeth of male Australian fur seals reveals interannual variability in prey resources

To detect and monitor long‐term ecosystem responses to environmental variability, managers must utilize reliable and quantitative techniques to predict future ecosystem responses. Canine teeth from 67 male Australian fur seals (aged 2–19 yr), collected at Seal Rocks, between 1967 and 1976, were measured for relative growth within the dentine growth layer groups (GLGs), as an index of body growth. Fluctuations in relative growth were apparent during 1956–1971, suggesting interannual variation in prey resources within Bass Strait. These were positively correlated with the Southern Oscillation Index and negatively with the Indian Ocean Subtropical Dipole, both on a 2 yr lag. The observed delay may reflect the time required for the nutrient cascade to filter through to the predominantly benthic prey of Australian fur seals. Stable isotope analysis (δ¹⁵N/δ¹³C) was also used to investigate whether fluctuations in growth were associated with differences in diet. Relative growth was found to be negatively correlated with δ¹⁵N, suggesting years of greater resource availability may be associated with individuals consuming proportionally more prey biomass of lower isotopic value. This study demonstrates that fluctuations in the dentine GLGs of male Australian fur seals are related to environmental parameters, suggesting variation in body growth is mediated by changes in prey resources.     

Quantitative immunohistochemical evidence of a functional gradient of chondroitin 4-sulphate/dermatan sulphate, developmentally regulated in the predentine of rat incisor

A quantitative examination was carried out on the early and mature stages of dentinogenesis in the rat incisor, using a post-embedding immunogold labelling with an anti-chondroitin 4 sulphate/dermatan sulphate antibody (2B6). At a very early stage of predentine formation, before polarizing odontoblasts have established junctional complexes, immunolabelling was weak. In contrast, when polarized odontoblasts established distal junctional complexes, immunolabelling in predentine was uniform and threefold denser than in initial predentine. The same gold particle density was found in the non-mineralized mantle dentine. During circumpulpal dentine formation, a gradient was seen in predentine, a larger number of gold particles being scored in the proximal zone compared with the distal region adjacent to the mineralization front. In circumpulpal dentine, some labelling was found within the lumen of the tubules and in the bordering dentine around the tubules. A few particles were also detected in intertubular matrix after demineralization. Together, these data provide evidence for a developmentally regulated gradient during the transition between mantle and circumpulpal dentine, and also in a more mature part of the tooth, a functional gradient that probably plays a role in the process of mineralization. © 1998 Chapman & Hall     

Localization and synthesis of type III collagen and fibronectin in human reparative dentine

Summary The injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue.Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine.Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.     

Localization and synthesis of type III collagen and fibronectin in human reparative dentine. Immunoperoxidase and immunogold staining

The injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue. Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine. Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.     

Development of larval and transformed teeth in Ambystoma mexicanum (Urodela, Amphibia): an ultrastructural study

Odontogenesis of early larval non-pedicellate teeth, late larval teeth with a more or less distinct dividing zone and fully transformed pedicellate teeth in Ambystoma mexicanum (Urodela) was studied to obtain insights into the development of differently structured teeth in lower vertebrates. Using transmission electron microscopy we investigated five developmental stages: (1) papilla; (2) bell stage (secretion of the matrix begins); (3) primordium (mineralization and activity of ameloblasts starts); (4) replacement tooth (young, old); and (5) established, functional tooth. Development of the differently structured teeth is largely identical in the first three stages. Mineralization takes place in apico-basal direction up to the (prospective) pedicel (early and some late larvae) or up to the zone that divides the late larval and transformed tooth in pedicel and dentine shaft (pedicellate condition). Mineralization starts directly at the collagen and by means of matrix vesicles. First odontoblasts develop small processes that extend to the basal lamina of the inner epithelial layer of the enamel organ. The processes are small and lack organelles in early larval teeth, but become larger, arborescent, and contain some organelles in late larval and transformed teeth. The processes are surrounded by unmineralized matrix (predentine). Odontoblasts at the basis of the teeth, at the pedicel, and in the zone of division do not develop significant cytoplasmic processes that extend into the matrix. Cells of the inner enamel epithelium differentiate to ameloblasts that secrete the enamel. In the early larval tooth they show an extensive basal labyrinth that becomes regressive when the enamel layer is completed. In late larval and transformed teeth, however, a large cavity arises between the basal ruffled border of ameloblasts and their basal lamina. This cavity appears to mediate amelogenesis. A small apical zone in early, but not in late larval teeth directly below the thin enamel layer consists of enameloid and is free of dentine channels.