Experimental determination of the periodicity of incremental features in enamel

Vital labelling of hard tissues was used to examine the periodicity of features of dental enamel microstructure. Fluorescent labels were administered pre- and postnatally to developing macaques (Macaca nemestrina), which were identified histologically in dentine and related to accentuated lines in enamel, allowing for counts of features within known-period intervals. This study demonstrates that cross-striations represent a daily rhythm in enamel secretion, and suggests that intradian lines are the result of a similar 12-h rhythm. Retzius lines were found to have a regular periodicity within individual dentitions, and laminations appear to represent a daily rhythm that also shows 12-h subdivisions. The inclusion of intradian lines and laminations represents the first empirical evidence for their periodicities in primates; these features frequently complicate precise measurements of secretion rate and Retzius line periodicity, which are necessary for determination of crown formation time. The biological basis of incremental feature formation is not completely understood; long-period features may result from interactions between short-period rhythms, although this does not explain the known range of Retzius line periodicities within humans or among primates. Studies of the genetic, neurological and hormonal basis of incremental feature formation are needed to provide more insight into their physiological and structural basis.     

Characterization of short-period and long-period incremental markings in porcine enamel and dentine—Results of a fluorochrome labelling study in wild boar and domestic pigs

Mammalian dental hard tissues exhibit incremental markings that reflect the periodic variation of appositional growth rates. In order to use these markings to characterize dental growth processes and to infer life-history traits, an unequivocal identification of their periodicities is required. We performed a fluorochrome labelling study on forming enamel and dentine in molar teeth of wild boar and domestic pigs to establish the periodicity and temporal correspondence of incremental markings in enamel and dentine. The dominant incremental markings in enamel (laminations) and dentine (von Ebner lines) recorded in the pig teeth are of a daily nature. In addition, long-period incremental markings with a periodicity of 2 days were recorded in enamel (striae of Retzius) and dentine (Andresen lines). The 2-day growth rhythm was also expressed at the lateral crown surface, as evidenced by the pattern of perikymata. In enamel, also markings with a sub-daily periodicity, representing an ultradian growth rhythm, were observed. Our study provides experimental evidence for the periodicity of incremental markings in porcine enamel and dentine. The findings correct previous misconceptions on incremental markings in dental hard tissues of pigs and other ungulates that had led to erroneous conclusions regarding crown formation parameters.     

Reconstructing impairment of secretory ameloblast function in porcine teeth by analysis of morphological alterations in dental enamel

We studied the relationship between the macroscopic appearance of hypoplastic defects in the dental enamel of wild boar and domestic pigs, and microstructural enamel changes, at both the light and the scanning electron microscopic levels. Deviations from normal enamel microstructure were used to reconstruct the functional and related morphological changes of the secretory ameloblasts caused by the action of stress factors during amelogenesis. The deduced reaction pattern of the secretory ameloblasts can be grouped in a sequence of increasingly severe impairments of cell function. The reactions ranged from a slight enhancement of the periodicity of enamel matrix secretion, over a temporary reduction in the amount of secreted enamel matrix, with reduction of the distal portion of the Tomes' process, to either a temporary or a definite cessation of matrix formation. The results demonstrate that analysis of structural changes in dental enamel allows a detailed reconstruction of the reaction of secretory ameloblasts to stress events, enabling an assessment of duration and intensity of these events. Analysing the deviations from normal enamel microstructure provides a deeper insight into the cellular changes underlying the formation of hypoplastic enamel defects than can be achieved by mere inspection of tooth surface characteristics alone.     

Immunolocalization of enamel matrix protein-like proteins in the tooth enameloid of spotted gar,Lepisosteus oculatus,an actinopterygian bony fish

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.     

Circadian rhythms in mitotic activity and 3H-thymidine uptake in the duodenum: Effect of isoproterenol on the mitotic rhythm

Mitotic activity in the duodenum of the rat and mouse exhibits a circadian periodicity with a peak in the rat between 1200 and 1500 hours and a sustained trough between 1800 and 0600. Scintillation counts revealed a similar rhythm in the total uptake of ³H-thymidine by the rat duodenum with a sustained but fluctuating crest occurring between 0800 and 1800 and a trough between 1900 and 0100. In the mouse the peak mitotic activity occurred at 0900 and the trough at 1700. Isoproterenol completely abolishes the rhythm in mitosis in mouse duodenum, when injected exactly 28 hours previous to sacrifice. The results are discussed in relation to reports that deny a circadian rhythm in mitotic activity in the duodenum.     

Do Mid‐Crown Enamel Formation Front Angles Reflect Factors Linked to the Pace of Primate Growth and Development?

Enamel formation front (EFF) angles represent the leading edge of enamel matrix secretion at particular points in time. These angles are influenced by rates of enamel extension (the rates at which tooth crowns grow in height), rates of enamel matrix secretion and the angles that prisms make with the enamel‐dentine junction. Previous research suggests, but has not yet established, that these angles reflect aspects of primate biology related to their pace of growth and development, most notably brain and body size. The present study tested this possibility on histological sections using phylogenetically‐controlled and Bonferroni‐corrected analyses spanning a broad taxonomic range. Ten species were represented in the analysis of anterior teeth; 17 in the analysis of posterior (postcanine) teeth (with varying sample sizes). Also, tested was the relationship of EFF angles to striae of Retzius periodicity (long period growth rhythms in enamel) and degree of folivory, as both factors are related to primate developmental rates. Finally, several analyses were conducted to investigate whether tooth size (operationalized as EDJ length) might mediate these relationships. Central results are as follows: (1) Relationships between EFF angles and brain weight (anterior teeth) and between EFF angles and body mass (anterior and posterior teeth) are statistically significant and (2) Mid‐crown EFF angles are not statistically significantly related to EDJ lengths. These results suggest that tooth size does not mediate relationships between EFF angles and brain weight/body mass and are discussed with respect to underlying enamel growth variables (especially rates of enamel extension and secretion). Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:125–139, 2018. © 2017 Wiley Periodicals, Inc.     

Review: Extracellular Matrix Regulates Tooth Morphogenesis

Mineralized tissues are unique in that they use proteins to attract and organize calcium and phosphate ions into a structured mineral phase, thus precise knowledge of the expression and extracellular distribution of matrix proteins is very important to understand their function. Tooth development is regulated by sequential and reciprocal interactions between neural crest-derived mesenchymal cells and the oral environment. However, the precise molecular mechanisms that mediate interactions between epithelium and mesenchymal cells are not clear, although basement membrane (BM) components have been shown to play important roles in these regulatory events. In addition, the extracellular matrix layer, whose main components are laminin, collagen IV, nidogen, and sulfated proteoglycan, and the BM layer are both considered to be involved with cell proliferation and differentiation. During tooth morphogenesis, extracellular matrices are dramatically changed. Further, the BM components, laminin and collagen IV support dental epithelium; however, in the late stage, they begin the processes of enamel matrix secretion and calcification, after which the BM structure between the dental epithelium and mesenchyme disappears. In addition, tooth abnormalities associated with several kinds of human diseases that cause mutations in the extracellular matrix, as well as the molecular mechanisms of the basement membrane and enamel matrix during tooth morphogenesis, are not clearly understood. In our review, we discuss the role of the extracellular matrix, with focus on the BM and enamel matrix during tooth morphogenesis.     

Knife chatter during thin sectioning of rat incisor enamel can cause periodicities resembling cross-striations

Cross-striations are traditionally associated with the enamel rods in many species including man. Although these striations are obvious with light microscopy, their exact nature has been difficult to determine with the transmission electron microscope on thin sections of enamel. Thin section microscopy either reveals no structures that can be called cross-striations, or shows periodic light and dark bands across the rods. Superficially, these bands resemble chatter artifact. To test this possibility, rat incisor enamel was used because cross-striations have not been demonstrated on these enamel rods. Thin sections were prepared of enamel blocks oriented in various ways with respect to the cutting edge of the diamond knife. The sections showed either uniform enamel or light and dark bands over rod profiles or interrod enamel. Since these bands could be produced artifactually it is concluded that similar bands seen on enamel rods of other species may also be artifacts.     

Review: extracellular matrix regulates tooth morphogenesis

Mineralized tissues are unique in that they use proteins to attract and organize calcium and phosphate ions into a structured mineral phase, thus precise knowledge of the expression and extracellular distribution of matrix proteins is very important to understand their function. Tooth development is regulated by sequential and reciprocal interactions between neural crest-derived mesenchymal cells and the oral environment. However, the precise molecular mechanisms that mediate interactions between epithelium and mesenchymal cells are not clear, although basement membrane (BM) components have been shown to play important roles in these regulatory events. In addition, the extracellular matrix layer, whose main components are laminin, collagen IV, nidogen, and sulfated proteoglycan, and the BM layer are both considered to be involved with cell proliferation and differentiation. During tooth morphogenesis, extracellular matrices are dramatically changed. Further, the BM components, laminin and collagen IV support dental epithelium; however, in the late stage, they begin the processes of enamel matrix secretion and calcification, after which the BM structure between the dental epithelium and mesenchyme disappears. In addition, tooth abnormalities associated with several kinds of human diseases that cause mutations in the extracellular matrix, as well as the molecular mechanisms of the basement membrane and enamel matrix during tooth morphogenesis, are not clearly understood. In our review, we discuss the role of the extracellular matrix, with focus on the BM and enamel matrix during tooth morphogenesis.     

Immunohistochemical localization of connexin 43 in the developing tooth germ of rat

Distribution of gap junction protein in maxillary tooth germs of 1-day-old rats was examined by immunohistochemistry, using an affinity-purified antibody specific to residues 360–376 of rat connexin (CX) 43. In 1-day-old rats, the maxillary second molar formed the shape of the cusp, but neither dentine nor enamel was formed between the cells of the dental papilla and the inner enamel epithelium. In the tooth germ, CX 43 was expressed in the cells of the stratum intermedium and the inner enamel epithelium. Labelling in the stratum inter-medium was extensive and showed an increasing gradient from peripheral to cuspal regions. CX 43 detected in the inner enamel epithelium was at cell surfaces facing the interface between the dental papilla and the inner enamel epithelium. The cells of the dental papilla and the inner enamel epithelium began differentiation as odontoblasts and secretory ameloblasts respectively, in the cusps of the first molars, where predentine and dentine were formed but enamel matrix was not secreted. CX 43 was present in the stratum intermedium, inner enamel epithelium, preodontoblasts, odontoblasts and subodontoblasts. The incisors showed the most advanced stage of development, where the enamel matrix and calcified dentine were formed in the labial part of the teeth. The CX 43 epitope was seen in the stratum intermedium, inner enamel epithelium, preameloblasts, preodontoblasts, odontoblasts, and subodontoblasts. Immunolabelling was more extensive in the stratum intermedium and subodontoblasts than in preameloblasts, preodontoblasts, and odontoblasts. The immunolabelling in preameloblasts and preodontoblasts was accumulated at cell surfaces facing the predentine. Further, the labelling in preameloblasts and preodontoblasts disappeared or was reduced at the initiation of enamel matrix secretion and calcification of dentine matrix.The present results suggest that gap junctional cell communication has important roles in tooth development. Further, the extensive CX 43 expression in the stratum intermedium and the subodontoblast layer suggests that gap junctions have an important role in amelogenesis and dentinogenesis.     

Microfibrils in glomerulopathies of children: An ultrastructural study

Extracellular microfibrils were found in 47 per cent. of 360 renal biopsies performed in children with a variety of nephropathies. They were observed in the mesangial matrix and basal lamina. They are 17 to 35 nm in diameter and a few show cross-striations with a periodicity of about 10 nm. They are different from fibrin or interstitial collagen. Their close association with thinner filaments in the mesangial matrix and basal lamina suggests that they are derived from such filaments.     

The central clock controls the daily rhythm of <Emphasis Type="Italic">Aqp5</Emphasis> expression in salivary glands

Salivary secretion displays day–night variations that are controlled by the circadian clock. The central clock in the suprachiasmatic nucleus (SCN) regulates daily physiological rhythms by prompting peripheral oscillators to adjust to changing environments. Aquaporin 5 (Aqp5) is known to play a key role in salivary secretion, but the association between Aqp5 and the circadian rhythm is poorly understood. The aim of our study was to evaluate whether Aqp5 expression in submandibular glands (SMGs) is driven by the central clock in the SCN or by autonomous oscillations. We observed circadian oscillations in the activity of period circadian protein homolog 2 and luciferase fusion protein (PER2::LUC) in cultured SMGs with periodicity depending on core clock genes. A daily rhythm was detected in the expression profiles of Aqp5 in SMGs in vivo. In cultured SMGs ex vivo, clock genes showed distinct circadian rhythms, whereas Aqp5 expression did not. These data indicate that daily Aqp5 expression in the mouse SMG is driven by the central clock in the SCN.     

Slow oscillation circuit of the intergeniculate leaflet

The slow oscillation circuit of the intergeniculate leaflet seems to constitute a natural basic rhythm of the neuronal mechanism of mammalian biological clock. The results of studies conducted so far indicate that photic information flowing from ganglion cells of the retina is necessary for its generation. On the other hand, this circuit is maintained thanks to the oscillatory activity of GABAergic interneurons, the majority of which build up this structure, mainly in combination with neuropeptide Y and enkephalins. The activity of non-specific projections of the brain, whose terminals are present in the intergeniculate leaflet, modulates the slow oscillation circuit of the leaflet neurons, without changing its oscillatory pattern, though. Our hypothesis predicts a role of the oscillatory activity of intergeniculate leaflet neurons in facilitation the secretion of neuropeptides and neurohormones present in the very elements making up the mechanism of mammalian biological clock and structures linked to it. This constitutes a kind of functional junction between the central mechanism of mammalian biological clock with an ultradian rhythm and its peripheral clocks whose rhythm is circadian.     

Essential roles of ameloblastin in maintaining ameloblast differentiation and enamel formation

During tooth development, dental epithelial cells interact with extracellular matrix components, such as the basement membrane and enamel matrix. Ameloblastin, an enamel matrix protein, plays a crucial role in maintaining the ameloblast differentiation state and is essential for enamel formation. Ameloblastin-null mice developed severe enamel hypoplasia. In mutant mice, dental epithelial cells started to differentiate into ameloblasts, but ameloblasts soon lost cell polarity, proliferated, and formed multiple cell layers, indicative of some aspects of preameloblast phenotypes. In addition, the expression of amelogenin, another component of the enamel matrix, was specifically reduced in mutant ameloblasts. More than 20% of amelobastin-null mice developed odontogenic tumors. We also found that recombinant ameloblastin specifically bound to ameloblasts and inhibited proliferation of dental epithelial cells. These results suggest that ameloblastin is an important regulator to maintain the differentiation state of ameloblasts.     

Proteomics and genetics of dental enamel

The initiation of enamel crystals at the dentino-enamel junction is associated with the expression of dentin sialophosphoprotein (DSPP, a gene normally linked with dentin formation), three 'structural' enamel proteins--amelogenin (AMELX), enamelin (ENAM), and ameloblastin (AMBN)--and a matrix metalloproteinase, enamelysin (MMP20). Enamel formation proceeds with the steady elongation of the enamel crystals at a mineralization front just beneath the ameloblast distal membrane, where these proteins are secreted. As the crystal ribbons lengthen, enamelysin processes the secreted proteins. Some of the cleavage products accumulate in the matrix, others are reabsorbed back into the ameloblast. Once crystal elongation is complete and the enamel layer reaches its final thickness, kallikrein 4 (KLK4) facilitates the breakdown and reabsorption of accumulated enamel matrix proteins. The importance of the extracellular matrix proteins to proper tooth development is best illustrated by the dramatic dental phenotypes observed in the targeted knockouts of enamel matrix genes in mice (Dspp, Amelx, Ambn, Mmp20) and in human kindreds with defined mutations in the genes (DSPP, AMELX, ENAM, MMP20, KLK4) encoding these matrix proteins. However, ablation studies alone cannot give specific mechanistic information on how enamel matrix proteins combine to catalyze the formation of enamel crystals. The best approach for determining the molecular mechanism of dental enamel formation is to reconstitute the matrix and synthesize enamel crystals in vitro. Here, we report refinements to the procedures used to isolate porcine enamel and dentin proteins, recent advances in the characterization of enamel matrix protein posttranslational modifications, and summarize the results of human genetic studies that associate specific mutations in the genes encoding matrix proteins with a range of dental phenotypes.     

Development of circadian periodicity in base and stress levels of corticosterone.

Base and stress levels of corticosterone were assessed at 4-h intervals over a 24-h period in male and female rats at age 18, 22, and 26 days. A significant periodicity in base levels of corticosterone is present at 22 days of age; however, a rhythm in stress values does not appear until age 26 days. At age 26 days the pattern of the circadian periodicity in both base and stress concentrations of corticosterone resembles that of the mature rhythm.     

An ultrastructural study of the effect of streptozotocin on the secretory ameloblasts of the rat incisor

The acute effect of a single injection of streptozotocin (75 mg/kg) on the secretory ameloblasts of rat incisor was found to be reversible after 4 h. This effect was manifested ultrastructurally by an accumulation of secretion granules within the Golgi apparatus and a diminished accumulation of secretion granules within Tomes' process, thus suggesting a temporary inhibition of secretion. The long term effect of streptozotocin was characterized mainly by large accumulations of secretion granules within the Golgi apparatus, the presence of many lysosomal structures, an abnormal redistribution of secretion granules, and the presence of large amounts of electron-dense material within the intercellular spaces. Because the electron-dense material resembled the enamel matrix, this, and the above changes, suggested that there was a pronounced inhibition in the movement of secretion granules into Tomes' process, as well as an ectopic secretion of the enamel matrix protein.     

Distribution of Amelotin in Mouse Tooth Development

Amelotin is expressed and secreted by ameloblasts in tooth development, but amelotin distribution during enamel development is not clear. In this report, we first investigated amelotin expression in developing teeth by immunohistochemistry. Amelotin was detected in the enamel matrix at the secretion and maturation stages of enamel development. Amelotin was also observed at Tomes' processes on the apical ends of secretory ameloblasts. We then compared amelotin gene expression with those of amelogenin, enamelin, and ameloblastin in the mandibles of postnatal mice by RT‐PCR. The expression of amelotin was detected as early as in postnatal day 0 mandibles and amelotin was coexpressed with amelogenin, ameloblastin, and enamelin during tooth development. These data strongly suggest that amelotin is an enamel matrix protein expressed at the secretion and maturation stages of enamel development. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Splitting of the locomotor activity rhythm in rats by exposure to continuous light

Female rats exposed to low intensities (0.1-1.5 lx) of continuous light (LL), displayed regular estrous cycles and free-running circadian rhythms of locomotor activity. In most rats, as the intensity of LL was increased to greater than 2.0 lx, components within the active portion (alpha) of the locomotor rhythm remained synchronized as the periodicity of the rhythm lengthened. However, in a few rats alpha split into two components; one of which free-ran with a period shorter than 24 h, while the other free-ran with a period longer than 24 h. As soon as the two components became maximally separated they spontaneously rejoined. In most rats, estrous cycles ceased shortly after the intensity of LL was increased to greater than 2.0 lx even though the locomotor activity rhythm retained its unsplit free-running nature. These observations suggest that the multiple oscillators that control the rhythms of locomotor activity and the estrous cycle are normally coupled to one another. In certain intensities of LL, these oscillators uncouple and free-run with different periodicities, a condition which causes estrous cycles to cease and sometimes produces a split locomotor activity rhythm.