[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.

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.

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.

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.

Morphogenesis of human tooth primordia: the importance of 3D computer-assisted reconstruction.

Three-dimensional reconstruction of embryological structures from histological serial sections is necessary when researching questions concerning formal development and mutual influences due to neighboring relationships. Graphical reconstructions can be performed by personal computers. In the present paper the need for 3D-reconstructions is explained, and examples showing the development of human dental primordia and their surrounding structures of specimens ranging between 18 and 64 mm CRL are presented. The contour-line plots that are provided by the software HISTOL are graphically reworked to anatomical drawings. Spatial impediment as one of the factors responsible for development of tooth form is discussed on the basis of the 3D-reconstructions.

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.

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.

Morphological evaluation of osteoblasts cultured on different calcium phosphate ceramics.

The objective of these investigations was to develop an in vitro test system for evaluating novel rapidly resorbable calcium phosphate ceramics of varying composition. Rat bone marrow cells were cultured on the disc-shaped test substrates for 14 days. Five calcium phosphates were examined: R1 CaNaPO4; R1/M2, composed of CaNaPO4 and MgNaPO4; R1/2, composed of CaNaPO4 and Mg2SiO4; R1 + 9% SiO2 consisting of CaNaPO4 and 9% SiO2 (wt%) and R17, Ca2KNa(PO4)2. Two studies were performed. In study I cultures were re-fed every two to three days. In study II the medium was changed daily, and calcium and phosphate concentrations of the medium were determined daily. Specimens were prepared for light microscopy and morphometric evaluation of the cell-covered substrate area, scanning electron microscopy and energy-dispersive X-ray analysis. With all materials tested except for R1/2, an increase of cellular growth was observed after changing the medium daily. Of the different calcium phosphate ceramics tested, R17 and R1/M2 facilitated osteoblast growth and elaboration of extracellular matrix to the highest degree. The inhibition of cell growth encountered with R1 in study I and R1/2 in both studies seemed to be related to a high phosphate-ion release from these materials.

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 scanning electron-microscopic study of developing human deciduous enamel on the dependence of the outline of surface pits on the angle of observation.

On the developing enamel surfaces of fetal human deciduous teeth, many of the surface pits were arcade-shaped with the arcade preferentially pointing in a cervical direction. The configuration of the interprism ridges between the pits contributed to this appearance. Surface cracks allowed verification of an incisal inclination of the subsurface prisms. This apparent paradox was solved when the specimens were tilted so that the pits were viewed in the directions of the prisms, giving the pits a compressed arcade-shape with the arcades pointing incisally. It is recommended that care should be exercised and due attention paid to the angle of observation when determining the orientation of pit arcades. Pit entry direction seems to be a more reliable feature for inferring the direction of tangential ameloblast movement.

In vitro abrasion using an air-powder polishing device and its quantification by radiotracer measurement.

OBJECTIVES: This study was conducted to examine the assessment of abrasion of two different materials after neutron activation. METHODS: A fissure sealant material (Fissurit F/VOCO with a compressive strength of 235 MPa) and a glass ionomer cement (Aqua Ionofil/VOCO with a compressive strength of 170 MPa) were activated by irradiation with neutrons. Subsequent measurements of the full-energy-peak (FEP) (1368.55 keV of 24Na) were made of the sample materials before and after abrasion via exposure to the air-powder polishing to accurately describe substance loss. RESULTS: Abrasion varied more than three-fold between the two materials. SIGNIFICANCE: Neutron activation and radiotracer measurement allows the quantification of abrasion effects in different materials. In comparison with other current methods its use may allow a superior measurement accuracy and precision in determining the abraded mass.

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.

The origin of tooth number of the human deciduous dentition: a hypothesis.

The constant number of 5 teeth per quadrant in the human deciduous dentition is seen to be a result of the mitotic activity within the dental lamina which leads to a constant number of centers in which cells are compressed together. These centers in which, due to the compression, the cells become narrow and cylindrical and, due to subsequent bulging, wedge-shaped, are the prerequisite for the formation of the constant number of five tooth buds per quadrant.

The origin of tooth cusps--a hypothesis.

An embryological concept is offered to explain the formation of tooth cusps. The epithelium is considered to play the active part in morphogenesis. The folding of the inner sheath of the enamel organ must be seen to be the origin of tooth cusps. This folding can be considered to be a consequence of the ability to span only a certain area without folding. The regularity of the folding--and thus the regularity of the molar occlusal pattern--might be a result of the unique material properties of the inner enamel epithelium.

Development of the parotid gland and its closer neighboring structures in human embryos and fetuses of 19-67 mm CRL.

The fetal development of the human parotid gland was studied by means of serial sections of human embryos and fetuses ranging from 19 mm to 67 mm CRL. Analysis of computer assisted 3-dimensional reconstructions and anatomical drawings leads to the following observations: 1. The parotid gland anlage is found at the most lateral and cranial point of the sulcus buccalis. 2. The location of the orifice of the parotid duct is dependent upon the developmental processes of the fetal skeleton. 3. The anlagen of anatomical structures dominating the parotid bed in the adult are found prior to the enlargement of the parotid gland. 4. The surface of the parotid gland shows impressions of the surrounding structures. 5. There is no evidence that the parotid gland is subdivided into two lobes by the facial nerve.

Development of the submandibular gland and its closer neighboring structures in human embryos and fetuses of 19-67 mm CRL.

The fetal development and arrangement of the human submandibular gland was studied by means of serial sections of human embryos and fetuses ranging from 19 mm to 67 mm CRL. Computer assisted 3-dimensional reconstruction leads to the following observations: 1. The orifice of the submandibular duct is located in the medial paralingual sulcus. 2. There is evidence that the extension and location of WHARTON's duct is influenced by the surrounding structures. 3. The surface of the submandibular gland primordium shows impressions of the neighboring structures. 4. The glandular tissue is encapsulated in condensed mesenchyme.

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.

4D-computerized visualisation of human craniofacial skeletal growth and of the development of the dentition.

The understanding of growth and developmental changes can be improved when shapes and changes in size, proportion, and relationships are visualized in 3 dimensions and at different stages. This applies particularly to craniofacial skeletal growth and the development of the dentition. For that purpose 3D-data were collected from prenatal human heads ranging from 18 up to 275 mm CRL and from a collection of macerated fetal and postnatal skulls. Computer-aided graphical reconstructions were obtained from histological serial sections of embryonic and early fetal specimens. Proportional changes in the growing skull were recorded by means of radiological and cephalometric evaluation. In addition, computed tomography was applied to fetal and postnatal skulls. Furthermore, the prenatal and postnatal development of the dentition was digitized. To that end 3D-polygone sets of these data were read into a workstation computer and animated by means of the software Soft Image (Microsoft). This comprehensive 4D insight into growth facilitates the understanding and teaching of normal and abnormal development.