Hypothesis for the lack of a muscular antagonist to the lateral pterygoid.

BACKGROUND: During physiological function of the temporomandibular joint, we have to rely only on elastic structures (in particular the bilaminar zone) for repositioning of the articular disc. A real muscle, however, would be functionally more reasonable. In patients, a decrease of this elasticity is possibly one of the reasons for temporomandibular joint malfunctions, which affect between 16% and 36% of the population. METHOD: This study assesses the morphogenesis of the murine (Mus musculus) temporomandibular joint with particular regard to the masticatory muscles, to throw light on this topic. To that end, a collection of 11 murine heads ranging from prenatal stages E13.25 to E20 was used and early postnatal stages P0 to P4, which were prepared as histological sections (thickness 8-10 µm) and stained conventionally in order to examine them with light microscopy. Next, the temporomandibular joint and selected surrounding structures, along with the masticatory muscles, were threedimensionally reconstructed using analySIS® software. Subsequently, specific morphometric analyses were performed. RESULTS AND CONCLUSIONS: The evaluation of the results led to the following conclusions.

A method to calibrate fetal µCT scans using histological sections from the same specimens.

Craniofacial morphogenesis is an intricate developmental process in 3D, which therefore merits visualization and investigation in 3D. To better understand the process, we utilize µCT imaging, and describe a method to calibrate each cone beam µCT individually. Calibration is necessary, because during development, fetuses undergo tissue differentiation, which affects the acquisition process for radiographic images. Additionally, tissue fixation and conservation agents may influence the physical properties of the specimens and may affect image acquisition. After taking a µCT scan from each specimen, we separated a horizontal slice from each neck (which is inconsequential to our question with relation to the whole head). These neck specimens were prepared as horizontal histological serial sections and stained. With these as a reference, the µCT visualization parameters could be adjusted until they matched the selected virtual section planes, which correspond exactly to the planes of the histological sections with a precision (pixel size) of 0.69µm.

[Cleft lip and palate]. / Lippen-Kiefer-Gaumen-Spalten.

BACKGROUND: Cleft lip and palate (CLP) represents a group of malformations of unknown etiology but similar phenotypes. This implies consequences for the diagnostics, therapy, prevention, prognosis and risk estimation. OBJECTIVE: Definition of CLP subtypes and the embryonic development, clarification of correlations and differences between entities using epidemiological data, overview of the present state of genetic analyses, correlation to syndromes, sequences and associations and resulting consequences for clinical practice. MATERIAL AND METHODS: Update on embryological development of the face, summary of epidemiological and genetic studies and considerations on pedopathological and forensic aspects. RESULTS: Syndromic and non-syndromic CLP exhibit different and highly variable etiologies, therapeutic needs and prognosis. A thorough understanding is mandatory to distinguish between the different subgroups. In addition to specific aspects of CLP for the pediatric (forensic) pathologist this article provides an overall view of the topic which aims to help understand these malformations.

Mouse Incisor Stem Cell Niche and Myb Transcription Factors.

Dental hard tissues are formed particularly by odontoblasts (dentin) and ameloblasts (enamel). Whereas the reparation of dentin is often observed, enamel does not regenerate in most species. However, in mouse incisor, a population of somatic stem cells in the cervical loop is responsible for the incisor regeneration. Understanding of the specificities of these cells is therefore of an interest in basic research as well as regenerative therapies. The Myb transcription factors are involved in essential cellular processes. B-Myb is often linked to the stem cell phenotype, and c-Myb expression marks undifferentiated and proliferating cells such as the stem cells. In the presented study, temporo-spatial expression of B-Myb and c-Myb proteins was correlated with localisation of putative somatic stem cells in the mouse incisor cervical loop by immunohistochemistry. B-Myb expression was localised mostly in the zone of transit-amplifying cells, and c-Myb was found in the inner enamel epithelium, the surrounding mesenchyme and in differentiated cells. Taken together, neither B-Myb nor c-Myb was exclusively present or abundant in the area of the incisor stem cell niche. Their distribution, however, supports recently reported novel functions of c-Myb in differentiation of hard tissue cells.

Microdamage in the alveolar process of rat maxillae after orthodontic tooth movement.

OBJECTIVE: Playing a decisive role in bone remodeling, microdamage was recently associated with orthodontic tooth movement in pigs. The present study was conducted to evaluate microdamage and its potential association with orthodontic tooth movement in the alveolar process of rat maxillae. MATERIAL AND METHODS: The upper right molars of 24 male Wistar rats (10 weeks old) were splinted and loaded against the (likewise splinted) upper incisors with 25 cN using a Nitinol coil spring. Four groups of 6 animals were treated in this fashion for 1, 2, 4, or 7 days. The upper left quadrants served as controls. The maxillae were halved, gently prepared, and stained en bloc with basic fuchsin. After embedding in resin, 80-µm-thick parasagittal sections were ground parallel to the mesial root of the first molar. These were used to assess microdamage under transmitted and epifluorescent light, also counting and measuring the length of microcracks. Differences between the loaded and unloaded side and between mesial and distal were checked using a Wilcoxon test and were considered significant at ≤ 0.05. RESULTS: Microdamage (in the form of diffuse damage and microcracks) was observed in both the loaded and control jaw halves, as well as on the mesial and distal sides in all four groups. Microcracks averaged 30-100 µm in length and 0.3-1.7/mm(2) in density. While they were more prevalent in the loaded than the control jaw halves, this difference was not statistically significant. CONCLUSION: The alveolar process of rat maxillae is characterized by microdamage (in the form of microcracks and diffuse damage) regardless of whether and for how long orthodontic loading has taken place. Within the limitations of this experimental study, our results do not confirm previous findings of significantly higher prevalence on the pressure side on the first day after initiating orthodontic tooth movement.

Histomorphometry in antigen-induced arthritis of the rabbit temporomandibular joint.

BACKGROUND: Juvenile idiopathic arthritis (JIA) of the temporomandibular joint (TMJ) can cause severe growth disturbances of the craniomandibular system. Antigen-induced arthritis (AIA) of the rabbit TMJ is simulating the inflammatory process of the TMJ in JIA. The aim of this study was to investigate the effect of a systemic administration of the tumor necrosis factor-alpha (TNF-α) antagonist etanercept on AIA in rabbits by means of three different histological staining methods. METHODS: After sensitization, a bilateral arthritis of the TMJ was induced and maintained by repeated intra-articular administrations of ovalbumin in 12 New Zealand white rabbits aged 10 weeks. From the 13th week of age, 6 of the 12 rabbits received weekly subcutaneous injections of etanercept, and the other 6 animals remained without therapy. Another 6 animals served as controls, receiving no treatment or intra-articular injections at all. After euthanasia at the age of 22 weeks, all TMJs were retrieved en bloc. Sagittal sections were cut and stained with hematoxylin-eosin (H-E), Safranin-O for the evaluation of the Mankin score, and tartrate-resistant acid phosphatase (TRAP). RESULTS: In the arthritis group, a chronic inflammation with degeneration of the articular cartilage was visible. In the etanercept group, the signs of cartilage degeneration were significantly reduced but present. In contrast, the joints in the control group were inconspicuous. A strong correlation between the Mankin score and TRAP-positive cells could be found. CONCLUSIONS: Antigen-induced arthritis causes severe damage in the TMJ of young rabbits. An improvement seems to be achievable by a systemic administration of etanercept.

Cephalometric assessment of human fetal head specimens.

BACKGROUND: Past investigations of prenatal craniofacial growth have largely relied on histological sections. Few studies have taken measurements on three-dimensional representations (3D reconstruction, 3D CT, postmortem) or varying depth levels (ultrasound), and we know of no craniofacial growth studies done on cleared-and-stained specimens of whole fetal heads. MATERIALS AND METHODS: This study comprised 14 human fetal head specimens cleared and stained with alizarin red and alcian blue. They had been stored in glycerol and represented weeks 8-12 of gestation, with crown-rump lengths ranging from 23-145 mm. These specimens were cephalometrically analyzed in norma frontalis and norma lateralis, which notably included the opportunity for side-to-side comparison. RESULTS: As the cranial membrane bones progressively approached each other, the orbits, maxilla, and mandible gradually grew wider. Likewise, the sagittal dimensions of the maxilla and mandible increased continuously and synchronically. We noted side-to-side differences ranging from 2-5 mm. Another notable finding concerned the inclination of the maxilla relative to the cranial base, which increased more on the right than on the left side. CONCLUSION: This is the first investigation presenting side-to-side comparative measurements of human fetal head specimens. Such measurements are essential in the quest toward validating the findings of other imaging techniques such as CT or MRI and-most importantly-intrauterine sonography.

Formation of the tooth-bone interface.

Not only are teeth essential for mastication, but also missing teeth are considered a social handicap due to speech and aesthetic problems, with a resulting high impact on emotional well-being. Several treatment procedures are currently available for tooth replacement with mostly inert prosthetic materials and implants. Natural tooth substitution based on copying the developmental process of tooth formation is particularly challenging and creates a rapidly developing area of molecular dentistry. In any approach, functional interactions among the tooth, the surrounding bone, and the periodontium must be established. Therefore, recent research in craniofacial genetics searches for mechanisms responsible for correct cell and tissue interactions, not only within a specific structure, but also in the context of supporting structures. A tooth crown that is not functionally anchored to roots and bone is useless. This review aims to summarize the developmental and tissue homeostatic aspects of the tooth-bone interface, from the initial patterning toward tooth eruption and lifelong interactions between the tooth and its surrounding alveolar bone.

Lysyl oxidase (lox) gene deficiency affects osteoblastic phenotype.

Lysyl oxidase (LOX) catalyzes cross-linking of elastin and collagen, which is essential for the structural integrity and function of bone tissue. The present study examined the role of Lox gene deficiency for the osteoblast phenotype in primary calvarial osteoblasts from E18.5 Lox knockout (Lox ( -/- )) and wild type (wt) (C57BL/6) mice. Next to Lox gene depletion, mRNA expression of Lox isoforms, LOXL1-4, was significantly downregulated in Lox ( -/- ) bone tissue. A significant decrease of DNA synthesis of Lox ( -/- ) osteoblasts compared to wt was found. Early stages of osteoblastic apoptosis studied by annexin-V binding as well as later stages of DNA fragmentation were not affected. However, mineral nodule formation and osteoblastic differentiation were markedly decreased, as revealed by significant downregulation of osteoblastic markers, type I collagen, bone sialoprotein, and Runx2/Cbfa1.

Anodic cell-protein deposition on inverse inkjet printed micro structured gold surfaces.

The transformation of fibrinogen into fibrin is biologically activated in a complex multi-step process known as the coagulation cascade. This transformation can also be triggered by anodic surfaces. It has been suggested that this mechanism is a result of an electron transfer from the anode to the fibrinogen molecule resulting in the formation of fibrin. In this study we used this pathway to simultaneously deposit vital cells (fibroblasts and keratinocytes) and fibrin on micro structured gold electrodes. The electrodes were produced using a novel inverse inkjet-printing technology in combination with subsequent gold-sputtering, resulting in minimal structure-sizes of 35 microm (+/-6 microm). Cell deposition and fibrin-coagulation were found to occur on the anode only, following exactly the micro structured electrode surface. Successful deposition was limited by the minimal voltage (0.8 V) needed for the formation of fibrin and the maximum voltage (1.85 V) resulting in the deterioration of the Au-electrodes due to electrolysis and possible damaging of the deposited cells due to the formation of molecular chlorine. Furthermore, it was demonstrated that this technique is suitable to co-cultivate different cell types in a layered fashion. Subsequent to the electrically mediated anodic cell-protein deposition, cells were cultivated for up to 4 days and then characterized by vital fluorescence staining, methyl violet-staining and scanning electron microscopy. Cell-vitality was found to be dependent on the experimental setup; in this study non-vital cells were only observed, when sequentially depositing two different cell types. Finally, the coagulation mechanism was studied using HPLC, SDS-gel-chromatography and ATR/FTIR.

Incremental lines in root cementum of human teeth--a reliable age marker?

In root cementum of teeth, alternating dark and light lines become visible in cross-sections under the light microscope. These lines bear an apparent resemblance to the annual rings of trees. Numerous studies have been done to correlate the number of cementum lines with the dental age by examining a great number of teeth of known age. Our study used a different approach. If lines in root cementum develop in an annual rhythm and are thus comparable to annual rings of trees, the same or at least a very similar number of these structures should be found in all areas of the root cementum of the same tooth. We counted cementum lines in the buccal, lingual, distal and mesial region of different sections, all from the middle third of the same root. This was repeated in eight teeth. To our surprise, we had immense difficulty in counting reproducible line numbers in the same cementum area at repeated counts. Nevertheless, the same tooth was found to differ markedly in the number of lines in different sections as well as in different regions of the same sections. These differences cannot be ascribed to variations caused by difficulties with reproducible line counting. Therefore, we are more than skeptical about the reliability of counting lines in root cementum as a method for determining the age of human teeth.

Prenatal morphogenesis of the human incisive canal.

The literature describing the formation of the incisive canal is very bizarre. The fusion of the primary and secondary palatal processes leads to formation of a triangular seam, which erroneously has been taken for the future incisive canal. If so, the nasopalatine (incisive) nerve and its accompanying vessels were to run through the primary oronasal cavity, which is not compatible with our biological experience. This study was undertaken to shed light on this region of fusion. We focus on the formation of the incisive canal; the neighboring nasopalatine ducts, which are a transient formation, are mentioned where present. A series of seven horizontal cross-sections of human embryos and fetuses from the 7th to the 24th week of pregnancy (between 25 and 225 mm CRL, crown-rump-length) were examined histologically and partly reconstructed in 3D applying the software analySIS (Soft Imaging Systems, Münster, Germany). The incisive canal did not develop at the junction of the primary and the secondary palate, but within the primary palate rostral to that location. The nasopalatine nerve and the nasopalatine artery are structures that exist before ossification starts in the area of the future incisive canal. The neighboring nasopalatine ducts were found in regions laterally and anterolaterally of the nasopalatine nerve, and it was mostly separated from it by bone. In advanced stages of development, the nasopalatine duct only existed as single epithelial remnants and was prone to obliteration.

A possible interdependency between the wavy path of enamel rods, distances of Retzius lines, and mitotic activity at the cervical loop in human teeth: a hypothesis.

In human enamel, the enamel rods do not run straight in most regions. Instead, they obtain an undulated path. The diameter of the enamel rods remains constant all the way, and a wavy pattern is necessary to produce the volume of the enamel mantle. It is not understood, how this undulated migration of ameloblasts is created. Considerations are presented to explain causal interdependencies between the wavy path of the enamel rods, unequal growth of the cervical loop cells and unequal distances of the striae of Retzius. To test these considerations, further research must record mitotic activity at the cervical loop during the stages of dental development. The distances of Retzius lines must be measured at different spots all over the whole enamel mantle, preferably in 3D. As a result, knowledge of the interdependency between the inner structure and the outer form of each tooth crown will lead to an understanding of tooth form and occlusion.

Development of the dentition: four-dimensional visualization and open questions concerning the morphogenesis of tooth form and occlusion.

OBJECTIVES: The formation of the dental primordial should be visualized with special reference to characteristic differences for each single primordium. Until today, it has not been clear how traffic of the ameloblasts is controlled, how the folding pattern of the occlusal relief is generated or how the enamel production is terminated at the enamel surface. DESIGN: Using computer-aided reconstructions from histological serial sections, the dental primordial were visualized and, using fractured enamel specimens, the traces of each single ameloblast were followed by means of scanning electron microscopy. In this way, the developmental movements of the inner enamel epithelium can be reconstructed. RESULTS: Gathering morphological knowledge, three-dimensional polygon sets of shape data were input into a computer workstation and animated by means of the software Soft Image (Microsoft). CONCLUSIONS: The development of the human primary and permanent dentition was animated to simulate growth.

Prenatal craniofacial morphogenesis: four-dimensional visualization of morphogenetic processes.

OBJECTIVES: Basic research concerning craniofacial development presently runs along two pathways, namely the molecular and the morphometric. This gap needs to be bridged. DESIGN: Using histological serial sections of human fetuses computer-aided three-dimensional reconstructions were made (Software Analysis, SIS) with special focus given to all anatomical structures of the orofacial region of the growing head. RESULTS: All reconstructions can be viewed from any rotation and they are available for virtual dissection according to anatomical rules. As an example, the prenatal development of the human mandible with the formation of the mental foramen therein is described. Furthermore, the spatial arrangement of bone, cartilage and nerves is presented in three dimensions in different developmental stages. The interaction of tissues with possible morphogenetic interaction is discussed. CONCLUSIONS: This work serves as a reference system for prenatal development in comparison with pathological development.

In vitro investigation of titanium and hydroxyapatite dental implant surfaces using a rat bone marrow stromal cell culture system.

In this study, rat bone marrow cells (RBM) were used to evaluate different titanium and hydroxyapatite dental implant surfaces. The implant surfaces investigated were: a titanium surface having a porous titanium plasma-sprayed coating (sample code Ti-TPS), a titanium surface with a deep profile structure (sample code Ti-DPS), an uncoated titanium substrate with a machined surface (sample code Ti-ma) and a machined titanium substrate with a porous hydroxyapatite plasma-sprayed coating (sample code Ti-HA). RBM cells were cultured on the disc-shaped test substrates for 14 days. The culture medium was changed daily and examined for calcium and phosphate concentrations. After 14 days specimens were examined by light microscopy, scanning electron microscopy, energy dispersive X-ray analysis and morphometry of the cell-covered substrate surface. All test substrates facilitated RBM growth of extracellular matrix formation. Ti-DPS and Ti-TPS to the highest degree, followed by Ti-ma and Ti-HA. Ti-DPS and Ti-TPS displayed the highest cell density and thus seem to be well suited for the endosseous portion of dental implants. RBM cells cultured on Ti-HA showed a delayed growth pattern. This may be related to its high phosphate ion release.

A new carbide finishing bur for bracket debonding.

BACKGROUND: The removal of residual bonding resin with rotary instruments after bracket debonding may damage the enamel surface. Conventional carbide burs may scratch the enamel due to the shape and sharpness of their blades. CARBIDE FINISHING BUR: A new carbide finishing bur with a slightly tapered shape, rounded tip and eight twisted blades has been developed. As a special feature the wedge angle of the blades has been enlarged to ca. 130-135 degrees by means of an oblique ground chamfer (relief angle ca. 0-5 degrees). The transition from head to shaft has been smoothed off with a safety chamfer. Overall, the cutting capacity has been reduced in the enamel, while the bur has remained sufficiently sharp within the adhesive resin. PROTOTYPE TESTING: For prototype testing, incisor brackets were bonded in vitro to 70 human incisors according to the standard clinical technique and removed after 7 days. Residual bonding resin was removed with conventional carbide burs and with gradually modified prototypes of the new finishing instrument respectively. The resulting enamel surfaces were evaluated by scanning electron microscopy. The specimens were then treated with a polishing paste and evaluated again. RESULTS: Conventional carbide burs remove not only residual bonding resin but also some enamel; scoring may occur. The newly developed finishing bur has been proven by morphological evaluation to be less aggressive in removing residual bonding resin after bracket debonding.

Bone remodeling of the human mandible during prenatal development.

MATERIAL AND METHOD: Based on histological serial sections of human fetuses (22-117 mm crown-rump length) the remodeling of the bone surfaces of the fetal mandible was characterized and visualized in 3D reconstructions. RESULTS: In the embryo of 22 mm crown-rump length the mandible was completely covered by cells producing bone apposition, but beginning with the embryo of 25 mm crown-rump length, morphological differentiations of these cells leading to bone modeling were visible. These modeling processes were found to be much more complicated than described in the literature so far. CONCLUSION: Bone growth of the fetal mandible is a complex process comprising not only lingual resorption and buccal apposition. Instead, we found well differentiated remodeling processes, represented by variable portions of apposition, resorption and resting areas seamed by lining cells.

Outline and arrangement of enamel rods in human deciduous and permanent enamel. 3D-reconstructions obtained from CLSM and SEM images based on serial ground sections.

Human enamel rods were made visible continuously from the dentino-enamel junction (DEJ) up to the enamel surface. From 12 teeth (1st and 2nd dentition) enamel blocks from the cervical third were prepared with perpendicular planes, embedded in resin, and ground down in steps of 15 microm parallel to the enamel surface. Enamel rods were made visible by acid etching (35% H3PO4, 45 s), sputtered and examined in the scanning electron microscope (SEM). Prior to this, the enamel blocks were viewed under the CLSM and optical sections at distances of 2 microm were obtained, starting in the same plane as the grinding surface. The outlines of the rods were digitized and reconstructed three dimensionally. For the first time, the path of single and grouped enamel rods on their way through the entire enamel layer was depicted. 3D images obtained from confocal laser scanning microscopy (CLSM) data were similar to those gained from SEM images. Single rods did not maintain their same outline throughout their path; arcade outlines predominated close to the DEJ, while keyhole outlines prevailed at the enamel surface. Within a group of rods, neighborhood relations changed, and neighbor rods influenced their outlines mutually, including the variable extent of the tail. The interdependence between the plasticity of the rods and the ameloblasts' forms should be topics of further research.

Prenatal development of the muscles in the floor of the mouth in human embryos and fetuses from 6.9 to 76 mm CRL.

The development of the muscles in the floor of the mouth is described in 10 human embryos and fetuses ranging from 6.9 to 76 mm CRL by means of computer-aided graphical 3D-reconstructions. All primordia of the muscles in the floor of the mouth could be identified from the 15.6 mm CRL stage on. The proportions and insertion lines of the early muscles were found to be different from adult anatomy. Each muscle first inserted in the medial surface of Meckels cartilage, but during the developmental period between 19 and 68 mm CRL the insertion lines were gradually transposed to the bony ridges of the mandible which progrediently embraced Meckels cartilage. The fibers of the mylohyoid muscles left the anterior region near the symphysis mentalis free during all stages of this study. The digastric muscle revealed only one belly with a constriction of its continuous fibers where it passed the hyoid bone primordium. There was no attachment of digastric muscle fibers to the hyoid; only geniohyoid and mylohyoid fibers. Geniohyoid and genioglossus muscles basically correspond to their definite arrangement, but they underwent proportional changes. Individual specimens embodied irregularities such as accessory geniohyoid and hyoid portions and muscle fibers separate from the mylohyoide muscle.