The mosasaur tooth attachment apparatus as paradigm for the evolution of the gnathostome periodontium

SUMMARY Vertebrate teeth are attached to jaws by a variety of mechanisms, including acrodont, pleurodont, and thecodont modes of attachment. Recent studies have suggested that various modes of attachment exist within each subcategory. Especially squamates feature a broad diversity of modes of attachment. Here we have investigated tooth attachment tissues in the late cretaceous mosasaur Clidastes and compared mosasaur tooth attachment with modes of attachment found in other extant reptiles. Using histologic analysis of ultrathin ground sections, four distinct mineralized tissues that anchor mosasaur teeth to the jaw were identified: (i) an acellular cementum layer at the interface between root and cellular cementum, (ii) a massive cone consisting of trabecular cellular cementum, (iii) the mineralized periodontal ligament containing mineralized Sharpey's fibers, and (iv) the interdental ridges connecting adjacent teeth. The complex, multilayered attachment apparatus in mosasaurs was compared with attachment tissues in extant reptiles, including Iguana and Caiman . Based on our comparative analysis we postulate the presence of a quadruple-layer tissue architecture underlying reptilian tooth attachment, comprised of acellular cementum, cellular cementum, mineralized periodontal ligament, and interdental ridge (alveolar bone). We propose that the mineralization status of the periodontal ligament is a dynamic feature in vertebrate evolution subject to functional adaptation.     

Seasonal development of secondary xylem and phloem in <Emphasis Type="Italic">Schizolobium parahyba</Emphasis> (Vell.) Blake (Leguminosae: Caesalpinioideae)

The cambial activity and periodicity of secondary xylem and phloem formation have been less studied in tropical tree species than in temperate ones. This paper describes the relationship between seasonal cambial activity, xylem and phloem development, and phenology in Schizolobium parahyba, a fast growing semideciduous seasonal forest tree from southeastern Brazil. From 2002 to 2003, wood samples were collected periodically and phenology and climate were recorded monthly in the same period. S. parahyba forms annual growth increments in wood, delimited by narrow initial parenchyma bands. The reduction of the cambial activity to a minimum correlates to the dry season and leaf fall. The higher cambial activity correlates to the wet season and the presence of mature leaves. In phloem, a larger conductive region was observed in the wet season, when the trees were in full foliage. The secondary phloem did not exhibit any incremental zone marker; however, we found that the axial parenchyma tends to form irregular bands.     

The cytology of submandibular glands of the opossum

Tooth attachment in the majority of the bony fish is by ankylosis or fibrous membrane. However, in one group of the osteichthys, the trigger-fish or balistids, tooth attachment is by means of a periodontium composed of a shallow alveolar socket, a periodontal ligament and acellular cementum. Histologically, the balistid periodontal ligament is composed of a dense fibro-cellular connective tissue possessing an abundance of typical fibrocytes, collagen fiber bundles, and oxytalan fibers. The collagen fiber bundles which resemble the principal fiber bundles of the mammalian periodontal ligament are inserted into the bone of the shallow alveolar sockets and are anchored to the teeth by means of a layer of amorphous acellular cementum that covers the radicular dentin. No cementoblasts were found in functional teeth, and epithelial rests are lacking. The mid-central zone of the balistid periodontal ligament is occupied by small blood vessels.     

Growth layers and incremental markings in hard tissues; a review of the literature and some preliminary observations about enamel structure in Paranthropus boisei

The general factors underlying the formation of growth layers and incremental markings in hard tissues are reviewed with particular reference to fossil hominid tooth enamel. The experimental and circumstantial evidence that point to a slowing of enamel matrix secretion in a daily (circadian) and near weekly (circaseptan) mode during tooth formation is also reviewed. Data from previous studies in which the number of daily increments between adjacent striae of Retzius have been recorded in primates are reviewed and new data are presented for this repeat interval in fossil hominids. The factors likely to influence the number of striae of Retzius beneath the cuspal regions of anterior teeth are outlined and the limitations of employing surface incremental features to obtain estimates for age at death of an individual are also discussed. It is concluded that there is good evidence to support the hypothesis that perikymata are near weekly incremental phenomena with a likely periodicity of 7,8 or 9 days in fossil hominids. It can also be concluded that at present, better estimates for the age at death of an individual during early phases of the growth period can be obtained from studies of perikymata than by any other non-destructive technique.     

Dynamics of incisor growth and daily increments on the incisor surface in three species of small rodents

The lower and upper incisors has been studied in striped field mice (Apodemus agrarius), yellow-necked mice (A. flavicollis), and migratory hamsters (Cricetulus migratorius) trapped in the Volgograd Region from April to March. The incisors of all animals have been found to have a striated surface, with series of slight ridges and grooves. As shown by analyzing the incisors of animals labeled with tetracycline, these are daily increments of dentin. The total numbers of increments (the period of complete tooth renewal) in the upper and lower incisors of the same animal are similar, but the degrees of their distinctness can be different. The number of daily increments increases but their width (daily growth rate) decreases with age even in adult animals and, in addition, varies by seasons. These two trends—the decrease in tooth increment with age and its seasonal variation—have proved to mask each other when an uneven-aged sample of animals trapped over several months is studied. Therefore, the season of death of an animal cannot be reliably estimated from the width of daily tooth increment unless the age of this animal is taken into account. The increment width in young animals in autumn may be the same as in older animals in spring and summer. In attempts to use the incisor surface sculpture for determining the season of animal death, it is necessary to separate the young from adults (at least by the criterion of incisor length). The decrease of increment width with age and its seasonal variation in the absence of obvious changes in the diet and pattern of incisor attrition indicate that the incisor growth rate is subject to age-dependent and seasonal changes, as is the growth rate of the organism as a whole. The distinctness and internal structure of daily increments also vary depending on animal age and species, which is due apparently to differences in the circadian rhythms of incisor growth. The presence of one or, less frequently, several grooves within a daily increment, which has occurred in all species studied, is evidence for not only circadian but also ultradian rhythms in the growth of incisors.     

A new method for the automated age-at-death evaluation by tooth-cementum annulation (TCA)

A valid age at death estimation is required in historical and also forensic anthropology. Tooth cementum annulation (TCA) is a method for age at death estimation of adult individuals. The method is based on light microscope images taken from tooth-root cross sections. The age is then estimated by manually counting the cementum incremental lines and adding this to the chronological age at the assumed point of tooth eruption. Manual line counting, however, is time consuming, potentially subjective and the number of individual counts is insufficient for statistical evaluations. Software developed for the automated evaluation of TCA images, that uses Fourier analysis and algorithms for image analysis and pattern recognition is presented here. It involves "line-by-line" scanning and the counting of gray scale peaks within a selected region-of-interest (ROI). Each scanning process of a particular ROI yields up to 400 counts that are subsequently statistically evaluated. This simple and time saving program seeks to substitute manual counting and supply consistent and reproducible results as well as reduce the demand of human error by eliminating unavoidable factors such as subjectivity and fatigue.     

Growth layers in dental cementum ofSaguinus monkeys in South America

Dark stained layers were observed in the cementum of the teeth ofSaguinus monkeys living in South America. Since the number of layers correlated with the degree of attrition in the incisor, the dark layers probably showed the growth lines. The teeth of the monkeys captured at the beginning of the wet season showed the incremental dark line around most of the exterior of the cementum.     

Human and mouse cementum proteins immunologically related to enamel proteins

SDS-polyacrylamide gel electrophoresis, immunoblot and amino acid composition analyses were applied to human and mouse acellular cementum proteins immunologically related to enamelins and amelogenins. In this analysis, anti-mouse amelogenin, anti-human enamelin and synthetic peptide (e.g., -LPPHPGHPGYIC-) antibodies were shown to cross-react with tooth crown-derived enamelin with a molecular mass of 72,000 Da (72 kDa), amelogenins (26 kDa), and also to four human cementum proteins (72, 58, 50 and 26 kDa) and two mouse cementum proteins (72 and 26 kDa). Each of the antibodies recognized tooth root-derived cementum polypeptides which share one or more epitopes with tooth crown-derived enamel proteins. The molecular mass and isoelectric points for crown-derived and root-derived enamel-related proteins were similar. Analysis of human and mouse cementum proteins revealed a characteristic amino acid composition enriched in glutamyl, serine, glycine, alanine, proline, valine and leucine residues; compared to the major enamel protein amelogenin, cementum proteins were low in proline, histidine and methionine. The human and mouse putative intermediate cementum proteins appear to represent a distinct class of enamel-related proteins. Moreover, these results support the hypothesis that epithelial root sheath epithelia express several cementum proteins immunologically related to canonical enamel proteins.     

Ultrastructural immunolocalization of dentin matrix protein 1 on Sharpey's fibers in monkey tooth cementum

Despite the importance of dentin matrix protein 1 (DMP1) in the formation of mineralized tissue, including dentinogenesis and osteogenesis, its precise role in cementogenesis remains to be clarified fully. The purpose of our study was to demonstrate the ultrastructural immunolocalization of DMP1 in monkey molar tooth cementum. Japanese Macaca fuscata monkeys were fixed by perfusion. The upper molar teeth and accompanying periodontium then were dissected and demineralized with EDTA. Cryosections were obtained, incubated in anti-DMP1 polyclonal antibody, and processed by immunoperoxidase and immunogold labeling. Intense immunoperoxidase staining for DMP1 was observed in acellular extrinsic fiber cementum, particularly in Sharpey's fibers. Cementocyte lacunae with canaliculi showed DMP1 staining in the apical region of the tooth root. Electron immunomicroscopy revealed the close proximity of DMP1 to collagen fibrils in Sharpey's fibers at the mineralization front. Intense immunogold labeling was localized on the walls of the cementocyte lacunae in cellular cementum. These results should contribute to better understanding of the role of DMP1, not only in Sharpey's fiber biomineralization, but also in the maintenance of the cementocyte lacunar space in cementum.     

Reconstruction of individual life histories of rodents from their teeth and bone

Age changes of bone tissue and molar cementum, and formation of daily layers in dentin of incisors enable us to estimate age and maximum lifespan, season of birth, season of death, sexual maturation, sex in adults, and in combination with tetracycline prebaiting, movements and space distribution of rodents. In hibernating rodents, the hibernation zone formed in incisor dentin can be used to assess inter- and intrapopulation variations in seasonal activity and growth. This approach is illustrated by examples from the study of rodents of the generaApodemus andSicista.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

Structural characteristics of dental cementum of skeletal remains of the first Catholic cemetery in Berlin (St. Hedwig's Cemetery,Central Berlin; 1777-1834)

Analysis of the incremental lines of human tooth cementum permits the chronological age-at-death diagnosis in skeletal finds. In addition, certain life history parameters, especially times of higher physiological calcium demand, should also manifest themselves in the cementum in the form of hypomineralized incremental lines. It has been shown previously that counting the incremental lines leads to a chronological age-at-death diagnosis more precise than the morphological age-at-death assessment, particularly in less well preserved skeletal finds. Both aspects were subject of this investigation of an underprivileged early modern human population.     

Ligamental increments of the mid-Holocene Pacific oyster Crassostrea gigas are reliable independent proxies for seasonality in the western Bohai Sea,China

Modern living and mid-Holocene shells of Crassostrea gigas from the western Bohai Sea in China were subjected to sclerochronological analysis with the aim of determining whether their concave bottoms and growth breaks on resilifer surfaces, as well as corresponding translucent growth bands visible in cross-section, have seasonal significance. An additional aim was to obtain ontogenetic information from mid-Holocene fossil shells. We compared δ¹⁸O and δ¹³C profiles compiled with high and low sampling resolution for the same shell in order to test the consistency of the results and identify a suitable sampling strategy for future sclerochronological studies. The results show that concave bottoms on the resilifer surface and corresponding translucent growth bands in cross-section formed during the cold season. These features are appropriate indicators of annual growth increments in fossil C. gigas shells. Moreover, growth breaks located near convex tops indicate annual growth increments for that part of the shell which grew when the organism was sexually mature. Therefore, spawning-related growth breaks and alternating winter freeze shocks (concave bottoms) are excellent morphological features in determining annual growth increments. Consequently, the life span, growth rate, and timing of spawning and death can be determined from the increments of ligament growth of these fossil oyster shells. For future sclerochronological studies, an adaptive micro-sampling strategy could be used for different increments of ligament growth that represent different seasons. Such a strategy would be more efficient in providing reliable insights into growth history of shells with variable growth rates of annual increments.     

The effects of day length, hibernation, and ambient temperature on incisor dentin in the Turkish hamster (Mesocricetus brandti)

Dentin is deposited on a circadian basis, and daily layers manifest as bands on the medial surfaces of rodent incisors. Hibernation alters dentin deposition, and a distinct hibernation mark has been described on incisor surfaces of several rodent species; the factors that influence the morphology of this mark are poorly understood. We tested the effects of day length, torpor expression, and ambient temperature on incisor surface morphology in Turkish hamsters housed in one of four conditions: long days (LDs) at 22 °C, short days (SDs) at 22 °C, SDs at 5 °C, and SDs at 13 °C. Body temperature was monitored continuously with implanted radio transmitters, and teeth examined postmortem. Teeth of SD hamsters had narrower, less distinct circadian increments than those of LD hamsters, but the width of ultradian increments was similar in both photoperiods. Hibernation at both 5 and 13 °C was associated in most specimens with very narrow, sharply defined dentin increments and increased tooth heterogeneity. Hamsters in SDs at 5 °C that did not hibernate lacked characteristic hibernation increments. At 5 °C, but not 13 °C, the number and cumulative width of hibernation increments were related to number and cumulative duration of periodic arousals. Our results suggest that incremental deposition of dentin in rodent incisors may be a useful trait for characterizing hibernation behavior in both evolutionary and historical contexts.     

Histology of tooth attachment tissues and plicidentine in Varanus (Reptilia: Squamata), and a discussion of the evolution of amniote tooth attachment

Few recent studies have examined the histological basis for tooth attachment in squamates. In the past few years, a surge of interest in this topic has led to the intriguing suggestion that the major tissues derived from the tooth germ (enamel, dentine, cementum and alveolar bone), are conservative and are present in all amniotes. In this study, we describe the histology and development of the tooth attachment complex in Varanus rudicollis, the rough‐neck monitor. We provide the first published evidence for the role of cementum and alveolar bone in tooth attachment in varanoid lizards. In Varanus, cementum is deposited on the external surface of the tooth root as well as at the base of the tooth, where it plays a role in the attachment of the tooth to the jawbone. Alveolar bone is also involved in tooth ankylosis. Our results support the hypothesis that the major tooth germ tissues are found in all amniotes. We provide insights into the structure and development of plicidentine, defined as infolding of the dentine around the tooth base. This feature is unique to varanoids among extant tetrapods and is the third tissue implicated in tooth attachment in Varanus. Plicidentine develops asymmetrically along the labial‐lingual axis of a tooth. Varanus is characterized by the presence of both primary and higher‐order lamellae, which anastomose to form a honeycomb‐like surface that then interacts with the more basal attachment tissues. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

Wnt3a promotes differentiation of human bone marrow-derived mesenchymal stem cells into cementoblast-like cells

Cementum is a calcified, avascular connective tissue that laminates the root of a tooth and plays a pivotal role in the development, homeostasis, and regeneration of a periodontal tissue. As a potential treatment for periodontal tissue defects in the patient with chronic periodontitis, much attention has been paid to tissue engineering combined with mesenchymal stem cells for regenerating periodontal tissues including cementum. However, limited information is available for the molecular factors that have impacts on the differentiation of mesenchymal stem cells into cementoblasts. Here, we focus on the effect of Wnt3a as a potential inducer and tested the effect of this protein in vitro using human bone marrow-derived mesenchymal stem cells. It was found that, when cells were cultured in an osteogenic medium containing Wnt3a, cementoblast-specific genes, such as cementum protein 1 and cementum attachment protein, as well as bone-related genes were significantly upregulated. These results suggest that Wnt3a promotes differentiation of the cells into cementoblast-like cells. Further experiments were carried out using inhibitors to gain deeper insights into molecular mechanisms underlying the observed differentiation. As a result, we conclude that Wnt3a-triggered differentiation into cementoblast-like cells is the consequence of the activation of the canonical Wnt signaling pathway with possible involvement of the non-canonical pathway.     

Immunolocalization of proteoglycans and bone-related noncollagenous glycoproteins in developing acellular cementum of rat molars

To elucidate the roles of proteoglycans (PGs), bone sialoprotein (BSP), and osteopontin (OPN) in cementogenesis, their distribution was investigated in developing and established acellular cementum of rat molars by an immunoperoxidase method. To characterize PGs, antibodies against five species of glycosaminoglycans (GAGs), chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), unsulfated chondroitin (C0S), dermatan sulfate (DS), and keratan sulfate (KS) were used. Routine histological staining was also applied. With onset of dentin mineralization, the initial cementum appeared on the dentin surface as a hematoxylin-stained fibril-poor layer. Subsequently, primitive principal fibers attached to the initial cementum. As the acellular cementum containing extrinsic fibers covered the initial cementum, the initial cementum formed the cemento-dentinal junction. Following immunohistochemistry at the earliest time of cementogenesis, the initial cementum was intensely immunoreactive for C4S, C6S, C0S, BSP, and OPN. After the initial cementum was embedded, neither the cemento-dentinal junction nor the cementum was immunoreactive for any GAG species. However, the cementum and cemento-dentinal junction were consistently immunoreactive for BSP. Although the cemento-dentinal junction was consistently immunoreactive for OPN, the remaining cementum showed no significant immunoreactivity. Thus, initial acellular cementogenesis requires a dense accumulation of PGs, BSP, and OPN, which may be associated with the mineralization process independently of collagen fibrils and initial principal fiber attachment.