Chondral ossification centers next to dental primordia in the human mandible: A study of the prenatal development ranging between 68 to 270mm CRL.

The human mandible is said to arise from desmal ossification, which, however, is not true for the entire body of the mandible: Meckel's cartilage itself is prone to ossification, at least its anterior part in the canine and incisor region. Also, within the coronoid and in the condylar processes there are cartilaginous cores, which eventually undergo ossification. Furthermore, there are a number of additional single cartilaginous islets arising in fetuses of 95mm CRL and more. They are located predominantly within the bone at the buccal sides of the brims of the dental compartments, mostly in the gussets between the dental primordia. They become wedge-shaped or elongated with a diameter of around 150-500µm and were also found in older stages up to 225mm CRL, which was the oldest specimen used in this study. This report is intended to visualize these single cartilaginous islets histologically and in 3-D reconstructions in stereoscopic images. Although some singular cartilaginous tissue within the mandible may be remains of the decaying Meckel's cartilage, our 3-D reconstructions clearly show that the aforementioned cartilaginous islets are independent thereof, as can be derived from their separate locations within the mandibular bone. The reasons that lead to these cartilaginous formations have remained unknown so far.

The remodeling pattern of human mandibular alveolar bone during prenatal formation from 19 to 270mm CRL.

The underlying mechanisms of human bone morphogenesis leading to a topologically specific shape remain unknown, despite increasing knowledge of the basic molecular aspects of bone formation and its regulation. The formation of the alveolar bone, which houses the dental primordia, and later the dental roots, may serve as a model to approach general questions of bone formation. Twenty-five heads of human embryos and fetuses (Radlanski-Collection, Berlin) ranging from 19mm to 270mm (crown-rump-length) CRL were prepared as histological serial sections. For each stage, virtual 3D-reconstructions were made in order to study the morphogenesis of the mandibular molar primordia with their surrounding bone. Special focus was given to recording the bone-remodeling pattern, as diagnosed from the histological sections. In early stages (19-31mm CRL) developing bone was characterized by appositional only. At 41, in the canine region, mm CRL bony extensions were found forming on the bottom of the trough. Besides general apposition, regions with resting surfaces were also found. At a fetal size of 53mm CRL, septa have developed and led to a compartment for canine development. Furthermore, one shared compartment for the incisor primordia and another shared compartment for the molars also developed. Moreover, the inner surfaces of the dental crypts showed resorption of bone. From this stage on, a general pattern became established such that the compartmentalizing ridges and septa between all of the dental primordia and the brims of the crypts were noted, and were due to appositional growth of bone, while the crypts enlarged on their inner surfaces by resorption. By 160mm CRL, the dental primordia were larger, and all of the bony septa had become reduced in size. The primordia for the permanent teeth became visible at 225mm CRL and shared the crypts of their corresponding deciduous primordia.

Morphogenesis of the compartmentalizing bone around the molar primordia in the mouse mandible during dental developmental stages between lamina, bell-stage, and root formation (E13-P20).

Despite increasing knowledge of the basic molecular aspects of bone formation and its regulation, the mechanisms of bone morphogenesis leading to a topologically specific shape remain unknown. The formation of the alveolar bone, which houses the dental primordia and later, the dental roots, may serve as a model to understand the formation of bone form in general. Thirty-eight heads of mice (C57 Bl/6J) ranging from stages E13-P20 were used to prepare histological serial sections. For each stage, virtual 3D-reconstructions were made in order to study the morphogenesis of the mandibular molar primordia concomitantly with their surrounding bone. Special focus was given to recording the remodeling pattern. It has been shown that, in early stages (E13, E14), bone formation is characterized by apposition only. In stage E15, the bony crypt around the dental primordia is remodeled mostly by resorption of bone. In stage E18, the bone remodeling pattern shows resorption all along the bony gutter, which houses the molar primordia. The medial and lateral margins are characterized by apposition. At birth (stage P0), a bony septum has begun to form between the primordium m1 and of m2, arising from both sides and characterized by pure apposition of bone. In stage P4, the crypts of m1 and m2, and also that of m3, show bone resorption inside, while the medial and lateral bony margins show apposition of bone throughout. Generally, during development, the bone gradually encapsulates the dental primordia, in such a way that the bone reaches over the dental primordia and leaves only a continuous longish opening of about 200µm width. The opening at the occlusal surface of m1, at the time of eruption, starting at stage P14, appears to have increased in size again. The distance between bone and dental primordium undergoes change during development. In erupted molars, it is around 100µm, during early developmental stages, it may be as less as 20µm. These data show the inevitability of bone remodeling.

Sexual dimorphism in teeth? Clinical relevance.

Many morphometric studies show a sexual dimorphism in human teeth. We wanted to know whether it is possible to determine the sex of an individual if only the anterior teeth are visible. Fifty intraoral photographs showing the front tooth region of female and male individuals (age: from 7 to 75 years) were randomly arranged in actual size on a questionnaire. The lip region was covered in each case. Besides "female" and "male", one was also able to check "?" if undecided. The questionnaires were distributed to 50 expert test persons (dentists, dental technicians, dental assistants, and students of dental medicine) and to 50 laymen and were all returned for evaluation. Although the correct sex was recognized on single photographs to a maximum of 76%, it was incorrect in 69% on other photographs. Altogether, the statistical evaluation showed that in most cases, the sex was only recognized correctly by one half, and incorrect by the other half. It can be concluded that a sexual dimorphism of human teeth-although measurable morphometrically-could not be recognized visually on the basis of photographs of the front tooth region. Neither experts in the field of dentistry nor laymen were able to properly distinguish between male and female teeth.

Tooth-bone morphogenesis during postnatal stages of mouse first molar development.

The first mouse molar (M1) is the most common model for odontogenesis, with research particularly focused on prenatal development. However, the functional dentition forms postnatally, when the histogenesis and morphogenesis of the tooth is completed, the roots form and the tooth physically anchors into the jaw. In this work, M1 was studied from birth to eruption, assessing morphogenesis, proliferation and apoptosis, and correlating these with remodeling of the surrounding bony tissue. The M1 completed crown formation between postnatal (P) days 0-2, and the development of the tooth root was initiated at P4. From P2 until P12, cell proliferation in the dental epithelium reduced and shifted downward to the apical region of the forming root. In contrast, proliferation was maintained or increased in the mesenchymal cells of the dental follicle. At later stages, before tooth eruption (P20), cell proliferation suddenly ceased. This withdrawal from the cell cycle correlated with tooth mineralization and mesenchymal differentiation. Apoptosis was observed during all stages of M1 postnatal morphogenesis, playing a role in the removal of cells such as osteoblasts in the mandibular region and working together with osteoclasts to remodel the bone around the developing tooth. At more advanced developmental stages, apoptotic cells and bodies accumulated in the cell layers above the tooth cusps, in the path of eruption. Three-dimensional reconstruction of the developing postnatal tooth and bone indicates that the alveolar crypts form by resorption underneath the primordia, whereas the ridges form by active bone growth between the teeth and roots to form a functional complex.

An atlas of prenatal development of the human orofacial region.

There are several atlases available showing prenatal human development. However, none is focused on prenatal orofacial development during maxillary and mandibular bone formation. These events, together with dental development and formation of the temporomandibular joint, take place during several fetal stages. While photographic atlases are limited to depicting the outer shape, and atlases based on histological sections only show a series of single sections, an atlas based on three-dimensional reconstructions from serial sections can show both the outer skin and the structures underneath, which can be electronically dissected layer by layer. In this Focus article, we present our atlas on prenatal human orofacial development, which is accessible online at the Journal's website.

Validation of two dual fluorescence techniques for confocal microscopic visualization of resin penetration into enamel caries lesions.

Fluorescence confocal microscopy is a useful tool to analyze the infiltration of enamel caries lesions with low-viscosity resins (infiltrants) in vitro. The conventionally used staining technique, which comprises dye labeling of the resin, has been shown to be limited by chromatographic separation of the resin-dye-mixture during penetration. The aim of this study was to develop an improved dual staining technique and to compare validity and reproducibility of both methods. Human molars with proximal white spots were cut across the demineralizations. After varnishing the cut surfaces, paired lesion halves were infiltrated with an infiltrant using either one of two different staining techniques. For the conventional direct technique (A) the infiltrant was labeled with rhodamine isothiocyanate (RITC) prior to application. Using the new indirect technique (B) lesions were stained with RITC solution and subsequently infiltrated with pure infiltrant. After light curing, unbound dye was bleached by immersion in hydrogen peroxide. Remaining lesion pores were stained with sodium fluorescein solution. Penetration depths (PD) and lesion depths (LD) were evaluated by five examiners using confocal microscopy and compared with the results of scanning electron microscopic (SEM; PD) and microradiographic (TMR; LD) analysis. The indirect technique showed better correlation (intraclass coefficients) with SEM (0.990) and TMR (0.982) compared with the direct technique (SEM: 0.513; TMR: 0.702). Inter- and intrarater reliability was higher for technique B compared with technique A. The new indirect technique yields to more valid and reliable results to visualize infiltrant penetration into natural enamel caries lesions compared with the conventional method.

[Genes, forces and forms: mechanical aspects of prenatal craniofacial development]. / Gènes, forces et formes: aspects mécaniques du développement cranio-facial prénatal.

Current knowledge of molecular signaling during craniofacial development is advancing rapidly. We know that cells can respond to mechanical stimuli by biochemical signaling. Thus, the link between mechanical stimuli and gene expression has become a new and important area of the morphological sciences. This field of research seems to be a revival of the old approach of developmental mechanics, which goes back to the embryologists His [36], Carey [13, 14], and Blechschmidt [5]. These researchers argued that forces play a fundamental role in tissue differentiation and morphogenesis. They understood morphogenesis as a closed system with living cells as the active part and biological, chemical, and physical laws as the rules. This review reports on linking mechanical aspects of developmental biology with the contemporary knowledge of tissue differentiation. We focus on the formation of cartilage (in relation to pressure), bone (in relation to shearing forces), and muscles (in relation to dilation forces). The cascade of molecules may be triggered by forces, which arise during physical cell and tissue interaction. Detailed morphological knowledge is mandatory to elucidate the exact location and timing of the regions where forces are exerted. Because this finding also holds true for the exact timing and location of signals, more 3D images of the developmental processes are required. Further research is also required to create methods for measuring forces within a tissue. The molecules whose presence and indispensability we are investigating appear to be mediators rather than creators of form.

Insular dentin formation pattern in human odontogenesis in relation to the scalloped dentino-enamel junction.

This study is a first report on the modality of early dentin formation in respect to the scalloped pattern of the dentino-enamel junction (DEJ). We applied scanning electron microscopy (SEM), transmission electron microscopy (TEM), histological serial sections, and three-dimensional (3D) reconstructions. TEM and SEM showed scallops and secondary scallops on the DEJ of deciduous dental primordia and on deciduous teeth with the enamel cap removed. This peculiar outline of the DEJ requires a specific dentin formation pattern; histological sections showed that dentin formation began at the brims of the scallops, seen as triangular spikes in serial sections. The dentin formation front was not uniform; instead, it was characterized by multiple, insular forming centers, as revealed by our 3D reconstructions. As thicker dentin layers formed, the islands became confluent. Factors are discussed, which may lead to crimpling of the inner enamel epithelium, and maintained as the scalloped pattern of the DEJ develops. Signaling patterns in accordance with the insular dentin formation are unknown so far.

Developmental movements of the inner enamel epithelium as derived from micromorphological features.

It was the purpose of this article to analyze the (micro) morphological structure of enamel at different stages of development in order to deduce movement patterns of ameloblasts during formation of the human dental primordium. Developing enamel and overlying ameloblasts were dried and fractured for scanning electron microscopy (SEM) and sectioned for transmission electron microscopy (TEM). Specimens of human permanent enamel were either fractured and/or ground and etched to visualize the enamel rods. All specimens were viewed by SEM. Moreover, three-dimensional reconstructions were made from serial ground sections of enamel blocks to follow the enamel rods for a longer distance. In addition, the outline of the dentino-enamel junction was analyzed under the SEM after removal (using nitric acid) of the enamel cap, and in serial histological sections. Two basic movements of the inner enamel epithelium can be derived from the micromorphological features: (i) the scalloped dentino-enamel junction may be a consequence of a bulged inner enamel epithelium owing to initial spatial impediment; and (ii) the undulating path of the enamel rods may be a consequence of unequal growth of the cells in the cervical loop.

Genes, forces, and forms: mechanical aspects of prenatal craniofacial development.

Current knowledge of molecular signaling during craniofacial development is advancing rapidly. We know that cells can respond to mechanical stimuli by biochemical signaling. Thus, the link between mechanical stimuli and gene expression has become a new and important area of the morphological sciences. This field of research seems to be a revival of the old approach of developmental mechanics, which goes back to the embryologists His (1874), Carey (1920), and Blechschmidt (1948). These researchers argued that forces play a fundamental role in tissue differentiation and morphogenesis. They understood morphogenesis as a closed system with living cells as the active part and biological, chemical, and physical laws as the rules. This review reports on linking mechanical aspects of developmental biology with the contemporary knowledge of tissue differentiation. We focus on the formation of cartilage (in relation to pressure), bone (in relation to shearing forces), and muscles (in relation to dilation forces). The cascade of molecules may be triggered by forces, which arise during physical cell and tissue interaction. Detailed morphological knowledge is mandatory to elucidate the exact location and timing of the regions where forces are exerted. Because this finding also holds true for the exact timing and location of signals, more 3D images of the developmental processes are required. Further research is also required to create methods for measuring forces within a tissue. The molecules whose presence and indispensability we are investigating appear to be mediators rather than creators of form.

The effect of surface chemistry modification of titanium alloy on signalling pathways in human osteoblasts.

Establishing and maintaining mature bone at the bone-device interface is critical to the long-term success of prosthesis. Poor cell adhesion to orthopaedic and dental implants results in implant failure. Considerable effort has been devoted to alter the surface characteristics of these biomaterials in order to improve the initial interlocking of the device and skeleton. We investigated the effect of surface chemistry modification of titanium alloy (Ti-6Al-4V) with zinc, magnesium or alkoxide-derived hydroxy carbonate apatite (CHAP) on the regulation of key intracellular signalling proteins in human bone-derived cells (HBDC) cultured on these modified Ti-6Al-4V surfaces. Western blotting demonstrated that modifying Ti-6Al-4V with CHAP or Mg results in modulation of key intracellular signalling proteins. We showed an enhanced activation of Shc, a common point of integration between integrins and the Ras/Mapkinase pathway. Mapkinase pathway was also upregulated, suggesting its role in mediating osteoblastic cell interactions with biomaterials. The signalling pathway involving c-fos (member of the activated protein-1) was also shown to be upregulated in osteoblasts cultured on the Mg and CHAP modified Ti-6Al-4V. Thus surface modification with CHAP or Mg may contribute to successful osteoblast function and differentiation at the skeletal tissue-device interface.

Explainable and critical periods during human dental morphogenesis and their control.

OBJECTIVE: To what extent is the current knowledge about regulatory and patterning processes gained from research on animal models (predominantly mouse) applicable to describe certain aspect of human prenatal dental development? METHODS: 3D-reconstructions were produced from serial sections of human dental primordia (Radlanski collection, Berlin) and scanning electron microscopic visualisation techniques were applied. RESULTS AND CONCLUSION: There are several examples, where present knowledge of regulatory processes allows the understanding of changes in outline and form. However, many other examples show that much more complex regulatory mechanisms should be expected to explain the details of human prenatal dental development.

Bone remodeling during prenatal morphogenesis of the human mental foramen.

From a morphogenetic point of view, the mental foramen of the mandible is a highly suitable model to study the interactions of different tissues such as nerves, vessels, mesenchymal cells, cartilage, and bone. In previous work, we provided a three-dimensional description of the mental foramen at different developmental stages, and now we complement those studies with a three-dimensional visualization of different bone remodeling activities around the mental foramen. Histological serial sections of human embryos and fetuses, ranging in size from 25 to 117 mm crown-rump-length (CRL), were used to characterize the bone remodeling activity (apposition, inactivity, and resorption). We quantified and reconstructed this activity in three dimensions, and included information on the spatial relationship of the nerves, vessels, and dental primordia. In general, the mandible showed strong apposition at its outer surfaces. The brim of the mental foramen, however, displayed changing remodeling activity at different stages. In the depth of the bony gutter, which provides space for the nerve and the blood vessels, we found bone resorption beneath the inferior alveolar vein. Bone was also resorbed in proximity to the dental primordia. In future studies, we will relate gene expression data to these morphological findings in order to identify molecular mechanisms that regulate this complex system.

Prenatal development of the human mandible. 3D reconstructions, morphometry and bone remodelling pattern, sizes 12-117 mm CRL.

Human embryos and fetuses ( n=25) ranging from 12 to 117 mm CRL (crown-rump-length) were serially sectioned and the mandibles were reconstructed in 3D. In addition, characteristic areas of apposition, resorption and resting zones were projected onto the surface of the mandibular reconstructions after histological evaluation of the remodeling processes. Furthermore, morphometric data were taken to describe growth processes in horizontal views. In this way the changing outlines as seen in 3D could be correlated with the remodeling patterns and with the changes in growth. In these stages the mandible showed a general appositional growth, but resorption areas were found at the posterior margins of the mental foramen and at the lateral and medial posterior bony planes at concave surfaces. The bulging of bone underneath and over Meckel's cartilage could be recognized as active appositional growth areas. Meckel's cartilage itself lay in a trough which could be characterized by less apposition and even resorption. Questions were raised in how much the gap between our present knowledge of genetic expression of signaling molecules and the precise morphologic description of the mandibles can be bridged.

Distribution of the cement film beneath the orthodontic band: a morphometric in vitro study.

BACKGROUND: During orthodontic treatment with a multiband appliance, enamel decalcifications and periodontal irritation may occur due to inevitable plaque retention. Besides the band itself, non-cemented gaps between tooth and band constitute a problem that has not yet been investigated from quantitative aspects. MATERIALS AND METHODS: In this in vitro study, the cement distribution beneath the orthodontic band was investigated on 48 identical transparent resin replicas of upper molars and lower molars, respectively. The replicas with the cemented orthodontic bands were divided into buccal, distal, oral, and mesial segments so that the inner surfaces of the bands could undergo morphometric analysis for areas not covered with cement. Two different molar bands (Dentaform Snap by Dentaurum, Ispringen, Germany, and "Washbon" by Ormco, Orange, CA, USA), and two glass-ionomer cements (OptiBand by Ormco and Ultra Band-Lok Blue by GAC, Gräfelfing, Germany) were used. In this way, 8 test series with twelve specimens each were performed. RESULTS: Not one cement-band combination was without defects in the cement film, with poorer cement flow properties being observed at the upper than at the lower molars. In general, fewer defects were recorded in the occlusal than in the cervical areas. Overall, the buccal surfaces yielded the best results, and the mesial surfaces the poorest. CONCLUSIONS: Since defects in the cement film have so far been unavoidable, the indication for orthodontic treatment with a multiband appliance must continue to be strict. Unless accompanying professional prophylactic care coupled with optimal oral hygiene is ensured, multiband appliances should be used with great caution.

Prenatal morphogenesis of the human mental foramen.

The mental foramen, at first glance, merely looks like a hole where the mental nerve and the vascular bundle runs through. From a morphogenetic point of view, however, the mental foramen is a suitable model to study the development of a structure where different components are involved. To understand this developmental process, a three-dimensional description at different developmental stages first has to be given. From histological serial sections of human embryos and fetuses, ranging in size from 19 to 117 mm crown rump length (CRL), three-dimensional reconstructions of the foraminar regions were made. Outline and form of the developing foramen, size, course of the mental nerve and the adjacent blood vessels could be shown in detail. In this way, the formation of these structures became concrete in three dimensions. In the future, to understand the mechanisms regulating this complex system, where a nerve and blood vessels became successively surrounded by bone, molecular biological data have to be correlated with morphological findings.