Crown formation times of deciduous teeth and age at death in neolithic newborns / Tiempos de formación de la corona de los dientes deciduos y edad al morir en neonatos neolíticos

SUMMARY: The aim of the present study was to investigate the possibility of estimating crown formation times of immature deciduous teeth and age at death in Neolithic newborns. In the Neolithic-Mesolithic transition, the health of the population deteriorated. Leaving the intrauterine environment for the newborn is the first obstacle in the process of adaptation and survival in the outside world. The fetus is protected by the mother's immune system and receives the necessary nutrients through the umbilical cord, but external factors indirectly affect its development. At birth deciduous teeth are not fully formed and are only partially mineralized. Variations in the rhythmic activity of ameloblasts and the secretion of the enamel matrix lead to the formation of incremental lines in the enamel. The sample consisted of unerupted deciduous teeth removed from the baby jaws from Neolithic archaeological graves, LepenskiVir Serbia. The skeletal age of the babies was from 38 to 40 gestational weeks. The daily enamel apposition rate was obtained for each tooth. The age of individuals was estimated using crown formation time. The average value of daily secretion rates for the primary teeth from the Neolithic age was 3.78 µm. There was no statistically significant difference in age at death determined by skeletal age assessment and crown formation time. Three babies were born preterm. The results of the present study show that the calculation of the time required for the formation of deciduous tooth enamel is applicable to archaeological samples of newborns.The age estimation using crown formation time together with the analysis of other anthropological parameters, can contribute to a more accurate determination of neonatal death in anthropological, archaeological and forensic contexts.

RESUMEN: El objetivo del estudio fue investigar la posibilidad de estimar el tiempo de formación de la corona de los dientes deciduos inmaduros y la edad, al momento de la muerte en neonatos neolíticos. Durante la transición Neolítico-Mesolítico, la salud de la población deterioró significativamente. Para el recién nacido dejar el medio intrauterino es el primer obstáculo en el proceso de adaptación y supervivencia en el mundo exterior. El feto está protegido por el sistema inmunológico de la madre y recibe los nutrientes necesarios a través del cordón umbilical, pero factores externos afectan indirectamente su desarrollo. Al nacer, los dientes deciduos no están completamente formados y solo están parcialmente mineralizados. Las variaciones en la actividad rítmica de los ameloblastos y la secreción de la matriz del esmalte conducen a la formación de líneas incrementales en el esmalte. La muestra consistió en dientes sin erupción extraídos de las mandíbulas de neonatos de tumbas arqueológicas neolíticas, LepenskiVir Serbia. La edad esquelética de los bebés fue de 38 a 40 semanas de gestación. Se obtuvo la tasa diaria de aposición de esmalte para cada diente. La edad de los individuos se estimó utilizando el tiempo de formación de la copa. El valor promedio de las tasas de secreción diaria para los dientes temporales del Neolítico fue de 3,78µm. No hubo diferencia estadísticamente significativa en la edad al momento de la muerte determinada por la evaluación de la edad esquelética y el tiempo de formación de la corona. Tres bebés nacieron prematuros. Los resultados del presente estudio muestran que el cálculo del tiempo requerido para la formación del esmalte dental deciduo es aplicable a muestras arqueológicas de recién nacidos. La estimación de la edad utilizando el tiempo de formación de la corona junto con el análisis de otros parámetros antropológicos, puede contribuir a una mayor determinación precisa de la muerte neonatal en contextos antropológicos, arqueológicos y forenses.

Cellular and molecular mechanisms of tooth root development.

The tooth root is an integral, functionally important part of our dentition. The formation of a functional root depends on epithelial-mesenchymal interactions and integration of the root with the jaw bone, blood supply and nerve innervations. The root development process therefore offers an attractive model for investigating organogenesis. Understanding how roots develop and how they can be bioengineered is also of great interest in the field of regenerative medicine. Here, we discuss recent advances in understanding the cellular and molecular mechanisms underlying tooth root formation. We review the function of cellular structure and components such as Hertwig's epithelial root sheath, cranial neural crest cells and stem cells residing in developing and adult teeth. We also highlight how complex signaling networks together with multiple transcription factors mediate tissue-tissue interactions that guide root development. Finally, we discuss the possible role of stem cells in establishing the crown-to-root transition, and provide an overview of root malformations and diseases in humans.

Primary tooth size asymmetry in twins and singletons.

OBJECTIVES: To explore asymmetry values of antimeric deciduous tooth crown dimensions in three types of twins: monozygotic (MZ), dizygotic same-sex (DZ) and opposite-sex (OS) vs. single-born controls. SETTING AND SAMPLE POPULATION: Mesiodistal and labio-lingual crown dimensions of second deciduous molars and mesiodistal canine and first molar crown dimensions of 2159 children at 6-12 years of age were evaluated, originating from the US cross-sectional Collaborative Perinatal Study from the 1970s, including altogether MZ (n = 28), DZ same-sex (n = 33) and OS (n = 39) pairs. Single born (n = 1959) were used as controls. MATERIAL AND METHODS: Dental casts were measured for comparison of variance relationships calculated from antimeric teeth, exhibiting fluctuating (FA), and directional (DA) asymmetry using anova. RESULTS: Significant differences appeared in MZ and OS girls in DA of deciduous canines, which gain size in the first and second trimester, and deciduous second molars, which finally stop crown growth during the early post-natal period. Significantly, increased FA values appeared for lower deciduous canines and second molars, indicating greatest environmental stress in OS girls, MZ girls and DZ boys. Twin girls had more fluctuating and directional crown asymmetry than twin boys, but in some dimensions, the twins were more symmetric than controls. CONCLUSIONS: Transmembrane hormonal influence between opposite-sex twins, and late gestational stress factors, caused by placental malfunction and/or monochorionicity, may be involved in asymmetric growth of antimers, during critical periods of crown size gain.

Lobodontia: Genetic entity with specific pattern of dental dysmorphology.

A characteristic pattern of dental anomalies including cone-shaped premolars, multitubercular molar crowns, pyramidal molar roots with single root canals, shovel-shaped incisors with palatal invaginations and hypodontia usually described as lobodontia was recognised as a separate entity. Only a few family reports on this condition have been published until now. The prevalence of the condition is estimated to be less than 1:1000,000. In the present paper we tried to delineate and clarify some additional aspects of this rare genetic entity in three families with 17 affected members. This represents the largest number of cases recorded since now. The analyses of dental morphology, crown-size profile patterns, pedigree analyses, and analyses of digitopalmar dermatoglyphics were performed in 7 examined patients. Crown-size profile pattern was calculated for seven patients and compared with standards for the Croatian population. The most striking features of the condition are conical premolars, tritubercular canines, single pyramidal molar roots, multitubercular molar crowns and invaginated upper incisors. A considerable reduction of crown-size was observed for all premolars, particularly in mandible. The alveolar process in the premolar region was hypoplastic and thin in all patients studied. Gender ratio of affected individuals was approximately M1:F1. Our data suggest that the prevalence of this condition is less than 1:300,000 in the Croatian population, which is considerably higher than previously reported in the literature. The analysis of the anomaly in all the families showed a slight variability in the clinical picture and autosomal dominant (AD) mode of inheritance. It could be concluded that this rare condition described as lobodontia represents a true genetic entity which follows AD mode of inheritance and displays variability in its expression.

[Biological dental implant: myth or reality?]. / L'implant dentaire biologique : mythe ou réalité ?

The currently available options for tooth-loss are prostheses, implants, or surgery (auto-transplantation). They all have their limitations. The emergence of tissue engineering, 15 years ago, was made possible by a better knowledge of the various stages of dental development, and the mastery of stem cell differentiation. It opened a new alternative approach for tooth regeneration. Even if animal experiments have demonstrated that it was possible to obtain a biological tooth from stem cells, two major issues remain to be discussed. Is it possible to use induced pluripotent stem cells instead of embryonic stem cells, which raise an ethical problem? Is it possible to reproduce a dental crown with an adapted shape and colour? Or should we consider the simpler creation of a biological root secondarily covered by a ceramic prosthesis? Our study mentions the main landmarks and the key cells involved in the embryological development of the tooth, establishes a mapping and a list of the various types of stem cells. It details the various methods used to create a biological implant.

Cell differentiation and matrix organization in engineered teeth.

Embryonic dental cells were used to check a series of criteria to be achieved for tooth engineering. Implantation of cultured cell-cell re-associations led to crown morphogenesis, epithelial histogenesis, organ vascularization, and root and periodontium development. The present work aimed to investigate the organization of predentin/dentin, enamel, and cementum which formed and mineralized after implantation. These implants were processed for histology, transmission electron microscopy, x-ray microanalysis, and electron diffraction. After two weeks of implantation, the re-associations showed gradients of differentiating odontoblasts. There were ciliated, polarized, and extended cell processes in predentin/dentin. Ameloblasts became functional. Enamel crystals showed a typical oriented arrangement in the inner and outer enamel. In the developing root, odontoblasts differentiated, cementogenesis occurred, and periodontal ligament fibroblasts interacted with the root surface and newly formed bone. The implantation of cultured dental cell re-associations allows for reproduction of complete functional differentiation at the cell, matrix, and mineral levels.

The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions.

Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the number and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth.

Correlation between dental maturity and cervical vertebral maturity.

OBJECTIVE: The aim of this study was to investigate the association between dental and skeletal maturity. STUDY DESIGN: Digital panoramic radiographs and lateral skull cephalograms of 302 patients (134 boys and 168 girls, ranging from 8 to 16 years of age) were examined. Dental maturity was assessed by calcification stages of the mandibular canines, first and second premolars, and second molars, whereas skeletal maturity was estimated by the cervical vertebral maturation (CVM) stages. The Spearman rank-order correlation coefficient was used to measure the association between CVM stage and dental calcification stage of individual teeth. RESULTS: The mean chronologic age of girls was significantly lower than that of boys in each CVM stage. The Spearman rank-order correlation coefficients between dental maturity and cervical vertebral maturity ranged from 0.391 to 0.582 for girls and from 0.464 to 0.496 for boys (P < 0.05). In girls, the mandibular second molar had the highest and the canine the lowest correlation. In boys, the canine had the highest and the first premolar the lowest correlation. CONCLUSIONS: Tooth calcification stage was significantly correlated with cervical vertebral maturation stage. The development of the mandibular second molar in females and that of the mandibular canine in males had the strongest correlations with cervical vertebral maturity. Therefore, it is practical to consider the relationship between dental and skeletal maturity when planning orthodontic treatment.

PDGFs regulate tooth germ proliferation and ameloblast differentiation.

OBJECTIVE: The purpose of this study was to elucidate the effects of platelet-derived growth factors (PDGFs) during tooth development, as well as the mechanisms underlying the interactions of growth factors with PDGF signalling during odontogenesis. DESIGN: We used an ex vivo tooth germ organ culture system and two dental cell lines, SF2 cells and mDP cells, as models of odontogenesis. AG17, a tyrosine kinase inhibitor, was utilised for blocking PDGF receptor signalling. To analyse the expressions of PDGFs, reverse transcriptase (RT)-PCR and immunohistochemistry were performed. Proliferation was examined using a BrdU incorporation assay for the organ cultures and a cell counting kit for the cell lines. The expressions of Fgf2 and ameloblastin were analysed by real-time RT-PCR. RESULTS: The PDGF ligands PDGF-A and PDGF-B, and their receptors, PDGFRalpha and PDGFRbeta, were expressed throughout the initial stages of tooth development. In the tooth germ organ cultures, PDGF-AA, but not PDGF-BB, accelerated cusp formation. Conversely, AG17 suppressed both growth and cusp formation of tooth germs. Exogenous PDGF-BB promoted mDP cell proliferation. Furthermore, PDGF-AA decreased Fgf2 expression and increased that of ameloblastin, a marker of differentiated ameloblasts. CONCLUSION: Our results indicate that PDGFs are involved in initial tooth development and regulate tooth size and shape, as well as ameloblast differentiation.

Accelerated development of the first and second mandibular molars after distraction osteogenesis of the mandible in patients with hemifacial microsomia.

Although distraction osteogenesis of the mandible in patients with hemifacial microsomia (HFM) before the mixed dentition period is widely performed, long-term follow-up information on dental problems is limited. We evaluated the long-term effect of distraction osteogenesis on dental development by comparing Nolla's developmental stages of the first and second molars between the affected and unaffected sides of the mandible. Forty-five patients with unilateral HFM who received distraction osteogenesis of the mandible between the ages of 5 and 8 years and whose dental status was followed for more than 2 years were selected for this study. The patients were divided into 2 groups: group I (n = 28) consisted of patients with Pruzansky types I and IIA and group II (n = 17) consisted of patients with types IIB and III. Panoramic radiographs at T0 (1 month before distraction), T1 (6 months to 1 year after distraction), T2 (2-3 years after distraction) were reviewed. Nolla's stages for the first and second molars were compared at each of the abovementioned times between the affected and unaffected sides using Wilcoxon signed-rank test. In group I, there was no differences observed in Nolla's stages of the first and second molars in comparisons between the affected and unaffected sides of the mandible. However, in group II, at T0, there was a significant difference in Nolla's stage of the first and second molars in comparisons between the affected and unaffected sides. Over time, tooth development was accelerated, and eventually, there was no difference in Nolla's stage between the affected and unaffected sides at T1 and T2. Patients with severe HFM can benefit from distraction osteogenesis of the mandible. The results of this study showed that this procedure could normalize the delayed development and eruption of the first and second molars.

The location and characteristics of two populations of dental pulp cells affect tooth development.

This study investigated the characteristics of two dental pulp cell populations during the early stages of crown formation in porcine teeth. A transplantation method was developed to reproduce epithelial cell-mesenchymal cell interactions during odontogenesis (tooth development). The technique allowed two types of cells/tissue to be combined in vivo. Populations of cells localized in the cervical loop epithelium region, dental pulp horn, and dental pulp core chambers were isolated and dissociated into single cells. Each population was examined for its gene-expression pattern using both semiquantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses, and for its tissue-formation capability by combining the cervical loop epithelial cells with either pulp horn cells or pulp core cells on biodegradable collagen scaffolds that were subsequently examined using histology and immunohistology. Gene-expression patterns showed that pulp horn cells were more mature than pulp core cells. Cervical loop epithelial cells combined with pulp horn cells mainly reconstituted dentin-cementum structures. By contrast, cervical loop epithelial cells combined with pulp core cells reconstituted enamel-dentin structures. These results suggest that mesenchymal cells residing in a specific location of the pulp possess a specific tissue-formation potential when combined with epithelial cells.

Crown formation during tooth development and tissue engineering.

Considering tooth crown engineering, three main parameters have to be taken into account: (1) the relationship between crown morphology and tooth functionality, (2) the growth of the organ, which is hardly compatible with the use of preformed scaffolds, and (3) the need for easily available nondental competent cell sources. In vitro reassociation experiments using either dental tissues or bone marrow-derived cells (BMDC) have been designed to get information about the mechanisms to be preserved in order to allow crown engineering. As the primary enamel knot (PEK) is involved in signaling crown morphogenesis, the formation and fate of this structure was investigated (1) in heterotopic reassociations between embryonic day 14 (ED14) incisor and molar enamel organs and mesenchymes, and (2) in reassociations between ED14 molar enamel organs and BMDC. A PEK formed in cultured heterotopic dental tissue reassociations. The mesenchyme controls the fate of the EK cells, incisor or molar-specific using apoptosis as criterion, and functionality to drive single/multiple cusps tooth development. Although previous investigations showed that they might differentiate as odontoblast- or ameloblast-like cells, BMDC reassociated to an enamel organ could not support the development of multicusp teeth. These cells apparently could neither maintain nor stimulate the formation of a PEK.

Temporal and spatial expression of TGF-beta2 in tooth crown development in mouse first lower molar.

Transforming Growth Factor beta2 (TGF-beta2) is involved in the regulation of many important cellular processes during tooth development. In this study we systematically characterized the expression pattern of TGF-beta2 in vivo and further analyzed its possible roles during different developmental stages of mouse first lower molar using immunofluorescence histochemical method with confocal microscopy. TGF-beta2 signaling was detected in different developing stages in both dental epithelium and surrounding dental mesenchyme. For the first time, we found that the basement membrane and epithelial cells in the basal layer showed no immunostaining from embryonic day 11 to 13; the primary enamel knot and secondary enamel knot exhibited pronounced immunostaining with different expression patterns at embryonic day 14 and 16. In addition, the mature ameloblast lost immunoreactivity, but the secretory ameloblast still exhibited positive immunoreaction at day 2 of postnatal development. Collectively, the temporospatial distribution patterns of TGF- beta2, especially in the basement membrane, epithelial cells in the basal layer, enamel knot, mature odontoblast and ameloblast, suggested a close association between TGF-beta2 signaling and tooth crown development, and indicated that TGF-beta2 might participate in tooth initiation, epithelial morphogenesis, formation of dentine matrix, and ameloblast differentiation.

Regulating the role of bone morphogenetic protein 4 in tooth bioengineering.

PURPOSE: Culture of the whole organ and regulation of its development using biologic and engineering principles can be used to produce structures and organs for reconstructing defects. The application of these bioengineering approaches in artificial tooth development may be the alternative way to replace missing dentition. MATERIALS AND METHODS: For the artificial bioengineering of a mouse tooth, tooth buds were dissected and transplanted into the diastema of the developing mandible. The mandiblular primordia containing transplanted tooth buds were culture in vitro and in vivo using a bioengineering method. In addition, to regulate the development of tooth germs, bone morphogenetic protein 4 (BMP4) or its antagonist, Noggin was administered. RESULTS: After the period of in vitro and in vivo culture, the transplanted tooth germ in the diastema showed tooth development with supportive structure formation. In the BMP-treated group, the bioengineered tooth was observed with increased maturation of cusp and enamel matrix. However, in the Noggin-treated tooth germs, the developing molar had a crater-like appearance with the immature development of the cusp and suppressed formation of the enamel matrix. CONCLUSIONS: This study confirmed that tooth germ transplantation in the diastema and culture with administration of BMP4 could lead to the mature development of the dental structures. In addition, these results suggest the possibility of bioengineering the tooth in morphogenesis and differentiation even in the toothless area.

Inhibition of apoptosis in early tooth development alters tooth shape and size.

Apoptosis plays important roles in various stages of organogenesis. In this study, we hypothesized that apoptosis would play an important role in tooth morphogenesis. We examined the role of apoptosis in early tooth development by using a caspase inhibitor, z-VAD-fmk, concomitant with in vitro organ culture and tooth germ transplantation into the kidney capsule. Inhibition of apoptosis at the early cap stage did not disrupt the cell proliferation level when compared with controls. However, the macroscopic morphology of mice molar teeth exhibited dramatic alterations after the inhibition of apoptosis. Crown height was reduced, and mesiodistal diameter was increased in a concentration-dependent manner with z-VAD-fmk treatment. Overall, apoptosis in the enamel knot would be necessary for the proper formation of molar teeth, including appropriate shape and size.

Cessation of Fgf10 signaling, resulting in a defective dental epithelial stem cell compartment, leads to the transition from crown to root formation.

Mouse, rat and human molars begin to form root after the completion of crown formation. In these teeth, fibroblast growth factor (Fgf) 10 disappears in the transitional stage from crown formation to root. By contrast, rodent incisors and vole molars demonstrate continuous growth, owing to the formation and maintenance of a stem cell compartment by the constant expression of Fgf10. To clarify the relationship between root formation and disappearance of Fgf10, we carried out two experiments for the loss and gain of Fgf10 function. First, we examined postnatal growth in the incisors of Fgf10-deficient mice, which have the defect of a dental epithelial stem cell compartment referred to as ;apical bud', after implantation under the kidney capsule. The growth at the labial side in the mutant mice mimics the development of limited-growth teeth. 5'-Bromo-2'-deoxyuridine (BrdU) labeling and cytokeratin (CK) 14 and Notch2 immunostaining suggested that the inhibition of inner enamel epithelium growth and the more-active proliferation of the outer enamel epithelium and/or stellate reticulum result in Hertwig's epithelial root sheath formation. Second, we examined the effects of Fgf10 overexpression in the transitional stage of molar germs, which led to the formation of apical bud involving in the inhibition of HERS formation. Taken together, these results suggest that the disappearance of Fgf10 signaling leads to the transition from crown to root formation, owing to the loss of a dental epithelial stem cell compartment.