Immunohistochemical PGP 9.5 positivity in human osteoblasts may indicate that compensatory and dysplastic craniofacial growth are under control by peripheral nerves.

OBJECTIVES: The purpose was to examine human osteoblasts immunohistochemically in order to clarify the significance of the innervation for alveolar bone growth. SETTING AND SAMPLE POPULATION: Unstained sections available from 21 normal human mandibles (foetal gestational ages: 14-22 weeks). MATERIAL AND METHODS: Before sectioning in 1980-1990, the mandibular tissue had been fixed in 4% neutral-buffered formaldehyde for 5 days. Tissue blocks were then decalcified in equal parts of 2% citric acid and 20% sodium citrate for 7-15 days, dehydrated, embedded in paraffin, and sagittally cut into 4-mum-thick serial sections and mounted on Superfrost Plus microscope slides. Sections were dried overnight at 40 degrees C. In the present study, paraffin sections were deparaffinized and treated with Tris-EDTA (Merck, Germany), pH 9.0, and immunohistochemically tested with polyclonal rabbit anti-PGP 9.5, and the EnVision +/HRP dual link (K4065; DAKO Denmark A/S, Denmark) method. RESULTS: A pronounced protein gene product (PGP) 9.5 activity was registered in osteoblasts from alveolar bone in all specimens. In all cases, the activity was intense at the top of and labially to the alveolar bone, while less or no activity was observed on the inner lingual aspects of the alveolar processes. Osteoclasts and osteocytes reacted vaguely or negatively. CONCLUSION: As the present study has demonstrated that human osteoblast activity in the alveolar bone seemingly responds to innervation, it is suggested that the peripheral nervous system via the trigeminal ganglion regulates compensatory and dysplastic alveolar bone formation.

Unexpected early apical resorption of primary molars and canines.

AIM: This was to examine the resorption pattern of primary molars and canines in dentitions with advanced apical resorption even though the permanent successor had barely begun root formation and to verify the resorption process histologically. STUDY DESIGN: Panoramic or bite-wing radiographs were selected from a dentition archive of radiographs from 142 children with deviant resorption patterns. METHODS: There were 14 patients (10 boys, 4 girls) aged 6 years 2 months to 8 years 1 month selected. In 11 patients abnormal resorption occurred only in the roots (group I). In 3 patients resorption occurred in the roots as well as in the crown (group II). The degree of resorption was evaluated using Haavikko's five resorption stages, and the degree of development of the permanent successor was evaluated using Haavikko's ten formation stages [Haavikko, 1973]. For histologic study 7 teeth were decalcified, paraffin-embedded, sectioned and stained with hematoxylin and eosin and examined microscopically. RESULTS: Group I: in 5 patients severe root resorption occurred before crown formation of the permanent successor was completed. In 6 children the abnormal resorption pattern was less severe. Group II: in 3 subjects full resorption of the root complex was observed as well as partial resorption of the crown. A histological analysis confirmed the resorption process. CONCLUSION: This is the first study focussing on unexpected early resorption of primary molars and canines in 14 patients without agenesis. The study showed an abnormal resorption pattern of roots and crown of primary teeth before the permanent successor had barely begun root formation. This indicates that resorption of primary molars and canines may occur independently from eruptional processes in the succeeding permanent tooth.

The pre-natal development and osseous growth of the human cerebellar field.

OBJECTIVES: To describe the pre-natal development of the bones that enclose the cerebellum and part of the brain stem (the neuro-osteological cerebellar field) in the mid-sagittal plane. DESIGN: Radiographic, cephalometric and histologic examination of normal pre-natal human fetuses; 50 normal fetuses, with crown-rump length of 18-227 mm and approximate gestational age from 6 to 26 weeks. RESULTS: The cerebellar field expressed extensive growth during development both sagittally and vertically. Because of changes in shape, the field was displaced in an anterio-caudal direction. CONCLUSION: In the present study we recorded normal measurements of size, shape and position of the cerebellar field. These standards can be used as references in skeletal analysis of cases with cranial abnormalities and cerebellar malformations.

A histological and radiological investigation of the nasal bone in fetuses with Down syndrome.

OBJECTIVES: Previous studies of nasal bone development in Down syndrome have used radiographs or ultrasound for the detection of nasal bone length or nasal bone absence. The aim of this study was to investigate the presence and size of the nasal bones in postmortem Down syndrome fetuses by means of radiographs and histological examination. METHODS: Thirty-three aborted human fetuses (gestational age 14-25 weeks) with Down syndrome were included. A mid-sagittal tissue block was excised from the skull base to the foramen magnum and along the lateral aspect of the spine. Radiographs of the tissue block were taken in lateral, frontal and axial projections. The length of the nasal bone was measured. The tissue blocks were cut in serial sections and stained. The crown-rump length (CRL), foot length (FL) and number of ossified bones in the hand and foot (CNO) were recorded. RESULTS: A total of 8/33 fetuses had bilateral nasal bone absence and two had unilateral absence. In fetuses with radiographically diagnosed nasal bone absence, no nasal bone could be found histologically. The majority of the Down syndrome fetuses had CRL, FL and CNO values within the range of those for normal age-matched fetuses. Nasal bone length was normal or reduced. CONCLUSIONS: Absence of the nasal bone was registered by postmortem examination in one-third of fetuses with Down syndrome. In some fetuses this could be a result of delayed maturation associated with Down syndrome. The phenotypic differences in nasal bone appearance may reflect genotypic differences in the Down syndrome group.

A molecular oyster: a neutral anion receptor containing two cyclopeptide subunits with a remarkable sulfate affinity in aqueous solution.

An artificial anion receptor is presented, in which two cyclohexapeptide subunits containing l-proline and 6-aminopicolinic acid subunits in an alternating sequence are connected via an adipinic acid spacer. This compound was devised to stabilize the 2:1 sandwich-type anion complexes that are observed when the two cyclopeptide moieties are not covalently connected and to obtain a 1:1 stoichiometry for these aggregates. Electrospray ionization mass spectrometry and NMR spectroscopic investigations showed that the bridged bis(cyclopeptide) does indeed form defined 1:1 complexes with halides, sulfate, and nitrate. ROESY NMR spectroscopy and molecular modeling allowed a structural assignment of the sulfate complex in solution. The stabilities of various anion complexes were determined by means of NMR titrations and isothermal titration microcalorimetry in 50% water/methanol. Both methods gave essentially the same quantitative results, namely stability constants that varied in the range 105-102 M-1 and decreased in the order SO42- > I- > Br- > Cl- > NO3-. This order was rationalized in terms of the size of the anions with the larger anions forming the more stable complexes because they better fit into the cavity of the host. The ability of sulfate to form stronger hydrogen bonds to the NH groups of the receptor, in addition to its slightly larger ionic radius with respect to iodide, causes the higher stability of the sulfate complex. No significant effect of the countercation on complex stability was observed. Furthermore, complex stability is enthalpically as well as entropically favored. A comparison of the iodide and sulfate complex stabilities of the ditopic receptor with those of a cyclopeptide that forms 1:1 anion complexes in solution showed that the presence of a second binding site increases complex stability by a factor of 100-350.

Spatiotemporal expression of NGFR during pre-natal human tooth development.

OBJECTIVES: The relation between nerve growth factor receptor (NGFR) in the human pre-natal tooth buds and the dental follicle was investigated. In particular, we sought to determine if there is a specific pattern of p75NGFR expression in developing human tooth buds and their surrounding tissue. SETTING AND SAMPLE POPULATION: The Department of Orthodontics at Copenhagen University, Denmark. Histological sections from 11 fetuses, aged 11-21 gestational weeks. METHOD: The sections were studied by conventional immunohistochemistry. RESULTS: Specific spatiotemporal patterns of p75NGFR reactions were observed in the tooth buds and dental follicle: Before matrix production by the ameloblasts, the entire inner enamel epithelium and the entire dental follicle display p75NGFR immunoreactivity; after matrix production is initiated, the immunoreactivity of the matrix producing cells is lost, as is that of the dental follicle adjacent to these matrix-producing cells. CONCLUSION: A unique spatiotemporal distribution of NGFR in the pre-eruptive human tooth bud was demonstrated.

p75NGFR immunoreactivity in normal prenatal human dorsal root ganglia.

The purpose of the present study was to examine immunohistochemically the expression of the low-affinity p75 nerve growth factor receptor in the dorsal root ganglia from 12 human fetuses (gestational ages, 10-24 weeks) located in three different spinal segments (cervical, thoracic, and lumbosacral), using a monoclonal mouse-antihuman low-affinity p75 nerve growth factor receptor antibody. The low-affinity p75 nerve growth factor receptor immunoreactivity was present within the dorsal root ganglia and the surrounding nerve fibers in all spinal segments at the different gestational ages examined. From 10 weeks of gestation, three different types of neuronal staining were observed: dorsal root ganglia neurons without low-affinity p75 nerve growth factor receptor immunoreactivity (classified as type I neurons), neurons displaying weak low-affinity p75 nerve growth factor receptor immunoreactivity (classified as type II neurons), and neurons manifesting intense low-affinity p75 nerve growth factor receptor immunoreactivity (classified as type III neurons). The distribution of the three types of neurons in the dorsal root ganglia was identical in the three spinal segments and did not change between 10 and 24 weeks of gestation. This study provides the first demonstration of the low-affinity p75 nerve growth factor receptor immunoreactivity in the dorsal root ganglia from human fetuses at different gestational ages.

Craniofacial tissues including tooth buds in fetal hypohidrotic ectodermal dysplasia.

OBJECTIVE: Hypohidrotic ectodermal dysplasia (HED) comprises defects in hair, teeth, and sweat glands. Disturbances in other ectodermal tissues have been associated with the condition. Our objective was to examine ectodermal craniofacial structures histologically in a fetus with HED and to compare the findings to similar structures in normal control fetuses. MATERIALS AND METHODS: A male fetus diagnosed with HED was therapeutically aborted in the 15th week of gestation. One male and two female healthy fetuses were used as normal controls. All fetuses were examined with parental consent, and had comparable sizes. Their bone maturation stage in the hand was identical. Tissue blocks from the craniofacial region were excised from all fetuses and prepared for histological analysis (formalin fixed, stained with toluidine blue or Alcian blue). The tissues examined were: tooth buds, skin and skin appendages, oral mucosa including minor salivary glands, major salivary glands, lacrimal glands, and adenohypophysis. RESULTS: Fewer tooth buds, minor salivary glands, and hair follicles were observed in the HED fetus as compared to controls. The structures of the epidermal components in the developing HED organs were loose and disorganised. The adhesion between the ectodermal and mesenchymal organ components in the HED fetus seemed to be disturbed.

Prenatal localization of the dorsal root ganglion in different segments of the normal human vertebral column.

STUDY DESIGN: Vertebral columns from 11 normal human fetuses (10-24 weeks of gestation) derived from spontaneous abortions were examined as part of the legal autopsy procedure including spinal cord analysis. OBJECTIVES: To study the localization of the dorsal root ganglion in the normal fetal spine and to relate the dorsal root ganglion location to the ossification of the vertebral bodies and vertebral arches. SUMMARY OF BACKGROUND DATA: The normal and pathologic ossification pattern of the fetal human spine has been studied. There has been no study addressing the localization of the dorsal root ganglion in normal and pathologic axial development. METHODS: The dorsal root ganglion were studied by using histology (horizontal sections) and morphometric measurement. RESULTS: The study showed: 1) The dorsal root ganglion appeared before ossification of the spine; 2) The dorsal root ganglion had an oval shape in all cases; 3) The longitudinal axis of dorsal root ganglion was directed anterolaterally in the cervical and lumbosacral segments and mainly laterally in the thoracic segment; 4) During development, the dorsal root ganglion changed position according to the body axis; and 5) The para-axial ossification protected the dorsal root ganglion differently in the different axial segments. CONCLUSIONS: The dorsal root ganglion appeared before ossification. The distance from the dorsal root ganglion to the body axis increased during development. In the different segments of the spine, different orientations and different locations of the dorsal root ganglion were observed in relation to osseous spine components. The results can be used as reference data for future studies on the dorsal root ganglion in pathologic spines.

Aspects of skeletal development in fragile X syndrome fetuses.

The purpose of the present investigation was to describe the skeletal development in prenatal fragile X syndrome. We studied fetuses (4 males, 2 females), with gestational ages (GA) 12-14 weeks, from 5 unrelated, different, known carrier mothers. Because of trauma to the fetus during abortion, different parts of the 6 fetuses were available for investigation. The vertebral column and the facial skeleton of all the fetuses were examined, the feet and hands of 5 fetuses, and the cranial base of 3 fetuses. The tissue remnants were examined radiographically and histochemically, and the results compared with previously published normal findings. Radiographic findings included normal ossification sequence, except for 1 fetus where there was an abnormal sequence in the first finger; normal morphology of ossification centres; and nasal bones were absent in the 5 fetuses and present in 1 (14 weeks of gestation). The histological study suggests presence of an acid mucopolysaccharide malfunction in the supporting tissue, because the normal cartilage resorption and orthochromatic cartilage reactions do not appear during the initial enchondral ossification. In addition, the apoptosis of ectodermally derived cells (notochord and palatal epithelial layers) appears delayed or abnormal. The sella turcica was malformed in the 2 fetuses investigated for sella turcica morphology.

Prenatal malformed lumbar vertebral corpora in trisomies 21, 18, and 13, evaluated radiographically and histologically.

The aim of the present study was to compare, both radiographically and histologically, malformed vertebral lumbar corpora in trisomies 21, 18 and 13 with earlier reported normal corporal development in the axial lumbar region. Axial skeletons of human fetuses (GA 15-22 wk) derived from therapeutically induced abortion were investigated in connection with requested autopsy. The number of lumbar vertebral corpora examined for each genotype was as follows: 20 from trisomy 21, 10 from trisomy 18, and 10 from trisomy 13. After radiography in frontal, lateral and axial projections, the individual vertebral corpora were decalcified and horizontally embedded in paraffin. The blocks were serially sectioned and stained with toluidine blue and alcian blue/van Gieson. The radiographic characteristics of the vertebral corpora varied from an almost normal appearance of the corporal bone to complete clefting of the bony corpora. Histological examination showed accumulations of cartilage centrally, in some cases associated with amorphous material. Pronounced metachromatic differences were observed in the cartilaginous ground substance. The study showed identical phenotypic characteristics in the corpora from trisomy 21, trisomy 18, and trisomy 13. It is characteristic of all three genotypes that there are central anomalies, corresponding to the location of the notochord in normal corpora, and marked regional differences in metachromasia in the ground substance of the cartilage.

Malformations of cranial base structures and pituitary gland in prenatal Meckel syndrome.

The sella turcica region, including the clivus and the pituitary gland, was studied histologically in five human fetuses with Meckel syndrome (MS). All cases had malformed sella turcica and malformed clivus with irregularly shaped notochordal remnants. We consider that these three characteristics are constant phenotypic traits in MS. The adenohypophysis was present in three cases. In one of these, ectopia of the gland occurred with adenopituitary tissue overlying the dorsum sella, and in another remnants were found in the pharyngeal submucosa. In two fetuses the neurohypophysis was not found. The findings in the region were compared to normal findings and to findings in trisomy 18, where cranial base structures radiographically appeared similar to those in MS. We conclude that in MS specific characteristics are found in the cranial base region and that radiographic analysis needs to be supplemented by histological analysis when studying this specific region.

Prenatal development of the normal human vertebral corpora in different segments of the spine.

STUDY DESIGN: Vertebral columns from 13 normal human fetuses (10-24 weeks of gestation) that had aborted spontaneously were investigated as part of the legal autopsy procedure. The investigation included spinal cord analysis. OBJECTIVES: To analyze the formation of the normal human vertebral corpora along the spine, including the early location and disappearance of the notochord. SUMMARY OF BACKGROUND DATA: Reference material on the development of the normal human vertebral corpora is needed for interpretation of published observations on prenatal malformations in the spine, which include observations of various types of malformation (anencephaly, spina bifida) and various genotypes (trisomy 18, 21 and 13, as well as triploidy). METHODS: The vertebral columns were studied by using radiography (Faxitron X-ray apparatus, Faxitron Model 43,855, Hewlett Packard) in lateral, frontal, and axial views and histology (decalcification, followed by toluidine blue and alcian blue staining) in and axial view. Immunohistochemical marking with Keratin Wide Spectrum also was done. RESULTS: Notochordal tissue (positive on marking with Keratin Wide Spectrum [DAKO, Denmark]) was located anterior to the cartilaginous body center in the youngest fetuses. The process of disintegration of the notochord and the morphology of the osseous vertebral corpora in the lumbosacral, thoracic, and cervical segments are described. Marked differences appeared in axial views, which were verified on horizontal histologic sections. Also, the increase in size was different in the different segments, being most pronounced in the thoracic and upper lumbar bodies. The lower thoracic bodies were the first to ossify. The morphologic changes observed by radiography were verified histologically. CONCLUSIONS: In this study, normal prenatal standards were established for the early development of the vertebral column. These standards can be used in the future--for evaluation of pathologic deviations in the human vertebral column in the second trimester.

Pituitary gland and sella turcica in human trisomy 18 fetuses.

The purpose of this study was to elucidate the phenotypic conditions in the sella turcica/pituitary gland complex in human trisomy 18 fetuses. Fourteen human fetuses with gestational ages from 12 to 39 weeks were included in the study. Normal fetuses at corresponding ages were used as controls. Whole body and special radiographic examination was undertaken before the midsagittal cranial base block, including the pituitary gland, was excised and analyzed histologically and immunohistochemically (keratin wide spectrum [KWS], thyroid-stimulating hormone [TSH], and neurophysin [Nph]). In all trisomy 18 fetuses, TSH-positive adenopituitary tissue was present in the sella and in greater or lesser amounts pharyngeally. The neurohypophysis was Nph-positive and located normally in the sella turcica. The adenohypophyseal tissue reacted either KWS-faint or KWS-negative, whereas KWS-positive reaction occurs in normal fetuses. This circumstance might suggest an altered cytoskeletal structure of the surface ectoderm in the pituitary placode in trisomy 18. The sella turcica was malformed in all the fetuses. Very broad craniopharyngeal canals were observed in some of the fetuses. Because endocrine disorders occur in many congenital malformations, it is essential in future studies to chart the sella turcica/pituitary gland region systematically in different genotypes.

Pituitary gland and sella turcica in human trisomy 21 fetuses related to axial skeletal development.

The purpose of the present investigation was to study the sella turcica/pituitary gland region in trisomy 21 fetuses and to relate the findings in the region to the ossification pattern in the axial skeleton formed by the cranial base and spine. Material from 22 human fetuses with trisomy 21, CRL 80 mm to CRL 190 mm, corresponding to gestational ages from 14 to 21 weeks, was examined and compared with material from gestation-matched normal controls. After radiography, tissue blocks from the cranial base, including the pituitary gland, were examined and compared with those of normal fetuses. Four different types of sella turcica/ pituitary gland morphology were observed. Thirteen fetuses (Type I) were morphologically normal. Minor abnormalities occurred in the sella turcica and pituitary gland (adenopituitary gland tissue pharyngeally) in six fetuses (Types II and III). There was agreement between the histologically recorded deviations in the sella turcica and the radiographic observations of the basisphenoid bone. In three cases (Type IV) out of 22, more pronounced structural abnormalities occurred in the sella turcica, and radiographically the basisphenoid bone appeared cleft. All sella turcica changes observed in trisomy 21 were situated anteriorly in the base of the sella. In all cases the basilar part of the occipital bone was normal. Minor changes in the sella turcica region were mainly accompanied by cervical vertebral abnormalities, while the most severe abnormalities occurred in association with malformations in the lumbar vertebrae. There was no association between sella turcica malformations and the absence or presence of the nasal bone.

The association between prenatal sella turcica morphology and notochordal remnants in the dorsum sellae.

The purpose of this study was to describe the location and morphology of notochordal remnants in the cranial base in normal and pathological conditions and to relate these findings to the morphological appearance of the sella turcica. Serially cut sagittal sections of paraffin-embedded sella turcica tissue blocks from 88 normal and pathological fetuses, 13 to 24 weeks of gestation, were examined. Twenty-seven specimens out of 88 had visible notochordal remnants in the cranial base, and these constituted the material available for this study. A straight notochordal course is always seen in normal sella turcica morphology, and a non-straight notochordal course is always seen in malformed sella turcica. Among the fetuses diagnosed at autopsy as "normal fetuses," both normal and pathological findings in the sella turcica regions were observed. The pathological findings were always found in the spontaneously aborted fetuses (five cases). Among the fetuses diagnosed at autopsy as "pathological fetuses," both normal and pathological findings were also observed in the sella region. Our conclusion is that the morphological appearance of the notochordal remnants in the dorsum sellae is associated with the morphology of the sella turcica. These structures ought to be analyzed on larger materials of both normal and pathological fetuses. One of the more obvious perspectives opened up by the present study is the probable disclosure of malformations in spontaneously aborted fetuses without external malformations.

A new craniofacial disorder involving hypertelorism and malformations of external nose, palate and pituitary gland.

The aim of the present study was to describe and pathologically evaluate an apparently unreported craniofacial malformation, based on comparison of the cranial midsagittal components with similar components under normal developmental conditions. A severely malformed fetus with a gestational age of about 17 weeks underwent whole body and special craniofacial radiography. Following autopsy dissection, the midsagittal segment of the cranial base, including the eyes, was radiographed in different projections. Midsagittal tissue blocks were serially sectioned for microscopy. Routine stains and immunohistochemical stains were applied. The face was characterized by hypertelorism, absence of external nose but with open shell-like cavities medio-cranially to the eyes, and by a palate fused in the midline and with extensive bony ridges laterally. There was absence of normal nasal cavities, presence of nasal septum and vomer, normal eyes, and nasal ducts covered with nasal mucosa ending blindly in the cartilage. No olfactory bulbs were found. The palatal ridges consisted of bony tissue. The pituitary gland was severely malformed and consisted solely of adenopituitary gland tissue, located in its full extent in the pharyngeal mucosa. There was no sella turcica. From a pathogenetic point of view, it is suggested that the neural crest cells in the frontonasal region of the crest were reduced in amount or late in migration to the midfacial region compared to the neural crest cells to the maxillary region. Therefore, we believe that the malformations observed in the nasal placodes and in the pituitary placode, combined with abnormal migration or abnormal timing of neural crest cells during the craniofacial development, are important factors behind this disorder.