Immunohistochemical studies of the periodontal membrane in primary teeth.

OBJECTIVES: To describe the periodontal membrane of human primary teeth immunohistochemically, while focusing on the epithelial layer of Malassez, fibers, and peripheral nerves, and to compare the findings with those of a previous study of human permanent teeth. MATERIAL AND METHODS: Nineteen human primary teeth extracted in late childhood in connection with treatment were fixed, decalcified, dehydrated, and embedded in paraffin. Paraffin sections were stained with wide spectrum screening (WSS), Vimentin, and NeuN in order to mark the epithelial layer of Malassez, fibers, and peripheral nerves. RESULTS: For root surfaces without resorption, the epithelial rests of Malassez appeared as small scattered islands. The fibers varied from tightly packed close to the root surface to a messy and loose organization. Innervation could be seen in close proximity to the root surface. The epithelial cells of Malassez were not usually seen along root surfaces with resorption. The fibers were sparse or not present. Innervation was seen in close proximity to the root. In regions with repair of resorption lacunae, the immunohistochemical reactions for epithelial cells of Malassez, fibers, and innervation pattern could be identical to those in regions with no resorption. CONCLUSION: In regions without resorption, spatial organization of the periodontal membrane of primary teeth was similar to that of permanent teeth, although the number and distribution of epithelial cells and fibers differed. In regions with repair of root resorption, the epithelial cells of Malassez, fibers, and innervation appeared as root surfaces without resorption.

The human periodontal membrane: focusing on the spatial interrelation between the epithelial layer of Malassez, fibers, and innervation.

OBJECTIVE: The purpose of the present study was to map the spatial interrelation of fibers, peripheral nerves, and epithelial layer of Malassez in human periodontal membrane in areas close to the root surfaces. MATERIAL AND METHODS: Four healthy permanent teeth extracted from four patients during puberty due to orthodontic treatment planning were analyzed. The extracted teeth, fixed in 4% neutral buffered formaldehyde for 5 days, were decalcified in 0.5 M EDTA. Paraffin blocks were sagittally cut in 5 microm thick serial sections and mounted on Superfrost Plus microscope slides. For survey, every fifth slide was stained with Alcian Blue/Van Gieson. Immunohistochemical reactions: Cytokeratin (wide spectrum screening) for epithelium, anti-vimentin for fibers, and anti-neuronal nuclei (NeuN) for innervation. RESULTS: The study indicates that the epithelial layer of Malassez is a border between different fiber morphologies and innervation patterns. Innervation is identified predominantly in the periodontal layer with tightly packed fibers close to the root surface. CONCLUSION: It is suggested that the genetic composition of the epithelial layer of Malassez in the periodontal membrane may be the key to understanding the different functions of the periodontal membrane and also the individual differences of these functions.

Cervical vertebrae, cranial base, and mandibular retrognathia in human triploid fetuses.

On profile radiographs of adults, an association between fusions of cervical vertebrae, deviations in the cranial base and mandibular retrognathia has been documented radiographically. An elaboration of this association on a histological level is needed. In human triploid fetuses severe mandibular retrognathia and deviations in the cranial base have previously been described radiographically (without cephalometry) and cervical column fusions radiographically as well as histologically. Therefore, triploid fetuses were chosen to elucidate the cranial base cephalomterically and histologically. In the present study, eight triploid fetuses were analyzed radiographically and histologically focusing especially on the cranial base, which borders to the spine and to which the jaws are attached. A histological analysis of the cranial base has not previously been performed in triploid cases. An enlarged cranial base angle and a retrognathic position of the mandible were documented cephalometrically on radiographs of all cases. Histologically, malformations were observed in the cranial base as well as in the spine. These are new findings indicating the association between the occipital bone and the uppermost vertebra in the body axis. As the notochord connects the cervical column and the cranial base in early prenatal life, molecular signaling from the notochord may in future studies support the notochord as the developmental link between abnormal development in the spine and the cranial base.

Association between the development of the body axis and the craniofacial skeleton studied by immunohistochemical analyses using collagen II, Pax9, Pax1, and Noggin antibodies.

STUDY DESIGN: Immunohistochemical analyses on the axial skeleton from wild type mice. OBJECTIVE: In the clinic, we have previously observed cervical spine defects associated with deviations in the posterior part of the occipital bone and with morphologic and functional variations in the craniofacial skeleton. As examples, cervical spine fusions occurred frequently in patients with mandibular overjet and even more frequently and more caudally in the cervical spine in patients with sleep apnoea. The aims of the present study were to elucidate this association between the spine and the cranium by comparing gene expression domains of important developmental genes known to be involved in vertebral column formation with gene expression in the craniofacial region. SUMMARY OF BACKGROUND DATA: This is the first study looking specifically on gene expression in the basilar part of the occipital bone that is formed around the cranial part of the notochord, thus connecting the spine and the craniofacial skeleton. METHODS: The material consisted of 4 mouse embryos p.c. day 13.5, NMRI wild-type mice, from the same litter. The body axis, the cranial base, and the craniofacial area were studied by immunohistochemical analyses using Collagen II, Pax9, Pax1, and Noggin antibodies. RESULTS: Pax1 expression was highly similar in the posterior part of the occipital bone and in the vertebral column, indicating that the basilar part of the occipital bone from a developmental standpoint can be considered the uppermost vertebra. Pax9 and Noggin expression domains were in accordance with those described previously. CONCLUSION: The present study supports that the basilar part of the occipital bone may be regulated by similar developmental mechanisms as the vertebral column and may thus be regarded the uppermost vertebra. Thus, the clinically observed association between the cervical column and the craniofacial area has been proved by immunohistochemical methods.

Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification.

The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.

Immunohistochemical localization of epithelial rests of Malassez in human periodontal membrane.

The aim of the present study was to describe the localization and extension of epithelial rests of Malassez (ERM) in the periodontal membrane (PDM) in normal human third molars. The material consisted of 24 normally developed human third molars surgically removed from patients with an age range from 15 to 27 years (six females and six males). The root lengths were developed from close to half-length to complete apex closure. The extracted teeth were fixed in 10 per cent neutral-buffered formalin, decalcified in ethylene diamine tetra acetic acid EDTA, paraffin embedded and cut sagittaly in 5 microm serial sections. Immunohistochemistry was performed using polyclonal rabbit anti-bovine cytokeratin (wide-spectrum screening, WSS) and the EnVision+ dual link system. The results were based on the visual comparison of WSS in the tissue sections using a light microscope. It was demonstrated that the ERM cells were distributed in the PDM in a network-shaped manner along the root surface and in the furcation region. The distribution of ERM was more prominent in teeth with incomplete root formation. The ectodermal tissue layer might influence not only the morphology of the tooth but also tooth eruption. The reaction of this epithelial layer in connection with ankylosis and orthodontic tooth movement may be of future interest.

Human prenatal nasal bone lengths: normal standards and length values in fetuses with cleft lip and cleft palate.

OBJECTIVE: The aim of this study was to present prenatal autopsy standards for nasal bone length in normal fetuses, as well as to compare nasal bone lengths in human fetuses with cleft lip and palate with those standards. MATERIAL AND METHODS: The material consisted of human fetuses investigated in connection with diagnosed and legally approved abortions and spontaneous abortions; 40 were normal and 26 had cleft lip and palate (7 isolated cleft lip, 12 isolated cleft palate, and 7 combined cleft lip and palate). Menstrual ages (MA) ranged from 12 to 25 weeks, crown-rump length (CRL) from 55 to 210 mm and foot length (FL) from 7 to 44 mm. All fetuses were radiographed in lateral projection, and, using these images, the nasal bone lengths were measured with a digital caliper. Regression analyses were performed. RESULTS: Standards for normal nasal bone length at different ages are expressed as menstrual age, crown-rump length, or foot length. Comparisons with normal standards showed that the isolated cleft lip group had a significantly smaller nasal bone length than the normal fetuses had. Fetuses with isolated cleft palate and fetuses with combined cleft lip and palate showed no significant deviation from normal standards. CONCLUSION: Normal nasal bone standards can contribute to information on deviations in nasal bone length for aborted fetuses with known and unknown genotypes. The differences in nasal bone lengths in fetuses with different lip and palate clefting can contribute to understanding the pathogenesis of cleft lip and palate.

Immunohistochemical expression of p63 in human prenatal tooth primordia.

The aim of this study was to investigate the expression of the p63 gene in normal human tooth buds at different gestational stages. This is the first detailed study of p63 expression in normal human prenatal tooth primordia. The material consisted of sections of the midaxial tissue block from the cranial base of three human fetuses of gestational ages (GA) 11, 15, and 21 weeks. The sections included tooth primordia representing cap stages and bell stages of human tooth morphogenesis. In the present study, immunostaining was carried out using the primary antibody, monoclonal mouse anti human p63 protein, clone 4A4. The sections were counterstained with hematoxylin Mayer. p63 immunoreactivity was identified by microscopy. The study showed a positive reaction of p63 in both the cap stage and the bell stage. In both stages, positivity was observed in the cells of the oral mucosa, the inner and outer enamel epithelium, and in the primary and secondary dental lamina. In the early cap stage, there is a strong positive reaction to p63 in the enamel knot, but not in the late cap stage. We suggest that p63 may have an important regulatory function in the enamel knot.

Abnormal vomer development in human fetuses with isolated cleft palate.

OBJECTIVE: The aim of this study was to elucidate the prenatal human development of the vomer with emphasis on the vomeral footplate and to assess vomeral morphology in fetuses with isolated cleft palate. MATERIAL AND METHODS: Nine human fetuses of which four were normal (menstrual age [MA] 13 to 21 weeks) and five with isolated cleft palate (14 to 19 weeks MA) were studied. Midaxial cranial tissue blocks from the fetuses were cut frontally in 4microm serial sections. Sections were stained with toluidine blue in 30% ethanol. RESULTS: From 16 weeks MA, the vomeral footplate of normal fetuses was formed from bilateral ossifications located below a U-shaped vomeral body. Later in development, an osseous connection was found between the footplate and the vomeral body. Neither bilateral areas of ossification below the vomer nor a vomeral footplate was observed in isolated cleft palate fetuses. CONCLUSIONS: In normal fetuses, the base or footplate of the vomeral bone appears from 16 weeks MA in frontal sections. In fetuses with isolated cleft palates, with no connection between the nasal septum and the maxillary processes, this vomeral footplate does not develop in the period observed (14 to 19 weeks MA).

p75-NGFR expression in the human prenatal pituitary gland.

The aim of this study was to elucidate the distribution of nerve growth factor receptor (NGFR) in the prenatal human pituitary gland until 21 weeks of gestation. Eight fetuses, with gestational ages from 13 to 21 weeks, were examined. The midaxial tissue block from the cranial base, including the pituitary gland and the sella turcica, was excised from the fetuses. The tissue was decalcified, embedded in paraffin, and cut in serial sections in 4-micro thicknesses. Immunohistochemistry was performed with monoclonal antibody p75-NGFR. p75-NGFR immunoreactivity was observed in fetuses from 15 weeks of gestation in the borderline between pars intermedia and the neurohypophysis.

Nerve growth factor receptor immunolocalization during human palate and tongue development.

OBJECTIVE: To investigate the temporospatial pattern of nerve growth factor receptor (NGFR) immunolocalization during human palatal closure. MATERIALS: Human palate and tongue tissues from 33 embryos/fetuses, 9 to 22 weeks of fertilization age. METHODS: Tissues were divided according to developmental stage and palatal development (before, during, and after closure) and then subjected to decalcification, paraffin embedding, serial sectioning, survey staining, and p75NGFR immunohistochemical staining. RESULTS: Specific temporospatial patterns of p75NGFR reactivity were observed; reactivity was intense in the soft tissue palatal shelves before and during palatal closure and was weaker in the palate after palatal closure. In the tongue, intense reactivity was seen throughout 9 to 22 weeks. CONCLUSION: The observed patterns suggest that p75NGFR may enable the visualization of physiological events in palatal closure during normal human development.

Histological examinations of bone and cartilage in the axial skeleton of human triploidy fetuses.

The aim of this study was to examine histologically bone and cartilage in vertebral corpora of axial skeletons of eight human triploid fetuses, gestational ages 14-25 weeks, CRL 100-200 mm. The results were compared to earlier studies on vertebral development in trisomies 21, 18, 13, and to normal corpora development. After radiography in frontal and lateral projections, the vertebral column was sectioned into cervical, thoracic and lumbar segments, decalcified, dehydrated, and embedded in paraffin. The blocks were serially sectioned in the vertical plane and stained with Toluidine blue and Alcian blue/van Gieson. The radiographic characteristics of the vertebral corpora observed in frontal and lateral projection varied from small cleft vertebral corpora to fusions between the individual corpora. Histological examination of the vertebral corpora confirmed the abnormal pattern of ossification seen radiographically. As a new finding abnormal metachromasia of the ground substance was observed in the cartilage. Marked borderlines were registered in the cartilage between regions with differences in metachromasia. These borderlines were similar but more extensive than borderlines observed previously in trisomies 21, 18 and 13.