Ossification centers of human femur.

The purpose of the present work was to investigate the appearance of ossification centers in human femur by Dual-energy X-ray Absorptiometry (DXA), comparing densitometric data with morphogenetic events. Posteroanterior scans were performed on 31 dried femora (from 11.5 weeks of conceptual age to 1 year of postnatal life) by a Hologic QDR 1000 X-ray densitometer with Ultra-Hi-Resolution software. The results were expressed as bone mineral content (BMC, g) and bone mineral density (BMD, g/cm2). The analyses were performed on a rectangular area corresponding to the minimum width and total length of the shaft. The rectangular area was divided into five equal sections along its longitudinal axis, and BMC was calculated on each section. To distinguish the ossification area of the lesser trochanter with respect to the area of the greater trochanter, the proximal femoral end was divided into two portions, medial and lateral, respectively; BMC and BMD were calculated on each portion. Our data show that the ossification center of the femoral shaft extends prevalently in the proximal direction. A denser area was recognizable on the densitometric images at the level of the lesser trochanter from the 19th week of prenatal life and at the level of the greater trochanter at term. During development, the trends of BMC and BMD are similar in both trochanteric areas, but these parameters are fairly constantly higher at the level of the lesser trochanter. Our findings disagree with the data of the current literature on the postnatal appearance of the trochanteric ossification centers and suggest a different biomechanical interpretation of the secondary ossification of the femur.

Bone mineral density in the growing human mandible.

The objective of this study was to investigate bone mineral density (BMD) in the growing human mandible by dual-energy X-ray absorptiometry, comparing densitometric data with the nature of the dentition and hence, indirectly, with variation of mechanical stresses. Postero-anterior scans were performed on 58 dried mandibles (between 15 months and 14 years) by a Hologic QDR 1000 X-ray densitometer with "Ultra-Hi-Resolution" software (Hologic Inc., Waltham, MA). The analyses were performed on a polygonal area corresponding to the infero-median region of the mandibular body, and divided into three sections. The sample was divided into two groups according to the appearance of the first permanent molar. A high correlation is detectable between BMD values and age in the group with decidual dentition, while BMD values are significantly less correlated with age in the group with mixed dentition. There are no significant differences between the BMD values of males and females.

Densitometric study of developing femur.

The purpose of this work was to extend to long bones the study on the ossification of human fetal skeleton in relation to conceptual age by a quantitative methodological approach. Postero-anterior scans were performed on 29 dried fetal femora (from 11.5 weeks of conceptual age to term) by a Hologic QDR 1000 X-ray densitometer with Ultra-Hi-Resolution software. The results were expressed as bone mineral content (BMC, g) and bone mineral density (BMD, g/cm2). BMD was calculated on a rectangular area corresponding to the total length and minimum width of the shaft. This area was divided into five equal sections along its longitudinal axis in order to estimate the rate of ossification from a spatio-temporal point of view. Our data show that BMC has a high correlation with conceptual age during the whole prenatal life, increasing in the third trimester (r >/= 0. 96). During development, BMD is progressively less correlated with conceptual age (r = 0.95 in the first half of development, r = 0.68 in the second half), particularly according to a bidirectional gradient from the middle to the proximal and distal ends of the shaft. Our findings confirm the data obtained in our previous studies on the ossification of fetal human spines, and suggest an individual variability in bone density at term of development and particularly at the level of spongiosa, viz. in the areas mostly involved in architectural changes during the morphogenesis of the long bones.

Notochord degeneration in chick embryo: a particular case of developmental cell death?

To define the type of cell death occurring in notochordal tissue, the cytological features of degenerating notochord were studied by transmission electron-microscope in thirty chick embryos from the 20th hour to the 15th incubation day. During the first two days the notochordal cells show nuclei with large nucleoli and cytoplasm with yolk granules, lipid droplets, phagolysosomes and deposits of glycogen. From the 3rd to the 5th incubation day, besides the peculiar vacuolization, disaggregation of the endoplasmic reticulum, transformation of the mitochondrial morphology, and disintegration of the cell membrane are detectable. Nuclei are normal up to advanced stages of cytoplasmic degeneration. On the 6th day a large number of cells are dying and, later on, the tissue disintegrates at the level of the vertebral bodies. Cell death in the notochord does not seem to be classifiable as one of the types of developmental cell death described in literature: the comparison with similar cytological features referred by pathologists as a consequence of metabolic damage, suggests that the degeneration of the notochord may be related to its morphological isolation and thus to trophic deprivation.

Densitometric study of developing vertebral bodies.

To define the quantitative aspects of ossification of human fetal spine, we performed a high resolution densitometric study by lateral and postero-anterior scanning of five fetal spines (18-36 weeks of conceptual age) and one spine of a 4-month-old infant. The data were plotted against developmental age for each spine and vertebra. Bone mineral content increased with developmental age, with a peak at the upper lumbar level, in agreement with the ossification pattern of the spine, reported in embryological literature. Bone mineral density (BMD) was unrelated to developmental age, and showed similar trends for each vertebra in all the vertebral columns examined. The changes of BMD seems to be a phenomenon related to individual variability. This study also demonstrates that densitometric techniques may provide useful and interesting information in studies on skeletal development.

Ontogeny of calbindin-D28K and calretinin in developing chick kidney.

The ontogeny of two calcium-binding proteins (calbindin-D28k and calretinin) was studied by immunohistochemical techniques in developing chick kidney. This study showed the presence of calbindin on the 5th incubation day and calretinin on the 7th incubation day in mesonephric distal and connecting tubules, and in the medial wall of the Wolffian duct. At later stages, immunostaining for these two proteins, in particular for calretinin, was also demonstrated in some metanephric proximal tubules. Glomeruli and Bowman's capsules were negative both in the mesonephros and metanephros. The presence of calretinin in the developing kidney has thus been demonstrated for the first time. The early expression of calbindin and calretinin in mesonephric distal tubules suggests their role in regulating the final excretion of calcium. The different patterns of immunoreactivity of the walls of the Wolffian duct can be correlated with their different histogenetic and histological features.

X-ray densitometry applied to the study of prenatal ossification.

To evaluate bone mass (BMC) and bone density (BMD) it is possible to utilize x-ray absorptiometry. This method is usually employed in clinical practice for the evaluation of metabolic osteopathies. The authors describe an application of this diagnostic technique to the study of the ossification pattern of the human vertebral column in pre- and post-natal life. On this regard, an ultra high resolution software allowed to analyze quantitative aspects of the ossification of small size vertebral bodies by lateral scans directly performed on bone specimens.

Cytodifferentiation of the notochord in early chick embryo related to the development of the axial and paraxial structures.

The cytology of the notochord was investigated by means of L.M. and T.E.M. in fifty chick embryos from the 7th HH stage to hatching, at different levels of the longitudinal axis and at different levels in each somite. The cytodifferentiation of the notochord was compared with the one of the surrounding axial and paraxial structures (neuro-epithelium and somites). Close segmental relations among notochord, neural groove and sclerotome, i.e. reciprocal contacts of cell processes and filopodia, where observed in 7-9 HH stages embryos. During the following stages in the notochordal cells signs of secretory activity are detectable, but at the same time degenerative changes are evident and they will increase up to the total regression of the notochord. These morphological observations seem to suggest the presence of reciprocal morphogenetic influences among notochord, neuro-epithelium and sclerotome, only before the 9th stage, rather than a determinant role of the notochord in the surrounding structures at later developmental stages.

"Chordoid tissue": biological significance and relevance of the term.

The significance and relevance of the term "chordoid tissue" are discussed on the ground of some ultrastructural features of the notochordal cells, observed in 15 chick embryos from stage 5HH to hatching. The characteristics of the cytoplasmic organelles and of the nucleus, the membrane alterations and in particular the vacuolization show the notochordal cells undergo a process of necrobiosis rather than of apoptosis. The degeneration of the notochord, that not casually starts in the notochordal core, seems to be due to a progressive metabolic isolation, related to the lack of blood vessels and to the formation of a thick perichordal sheath. These findings suggest that the notochordal tissue shows the same cytologic features of any tissue degenerating owing to a metabolic damage. The term "chordoid tissue", in the meaning of a tissue with peculiar structure and function, does not seem hence to be relevant, also on the ground of the fact that in the species in which a nucleous pulposus arises from notochord remnants, the notochordal cells show the same ultrastructural characteristics detectable in chick embryos.

Expression of calcium binding proteins in the developing spine: preliminary report.

Previous ultrastructural studies on the developing spine induced the Authors to analyse the presence of Ca-binding proteins in the vertebral anlagen of the chick embryo, by immunostaining for calretinin. At 7 incubation days, intense positivity for calretinin at level of the intervertebral region is detectable. From the 7th day the positivity is localized in particular to the intervertebral menisci and it appears also in the degenerating chondrocytes of the chondrifying vertebrae and at level of the peripheral zones of vascular erosion in the ossifying vertebrae. The intervertebral positivity for calretinin can be related to the presence at this level of fibroblast-like cells, that for such immunoreactivity are distinguishable from true fibroblasts. Since other Authors showed presence of S-100 protein in fibroblast-like cells during histogenesis of white adipose tissue, the significance of the expression of some Ca-binding proteins in the differentiation of these cell types can be supposed. The meaning of the immunoreactivity in the vertebral bodies is still uncertain and needs further immunohistochemical studies.

Relations between subcellular events in the degenerating notochord and histopathological features of the spinal chordomas.

The authors focused their attention on the cytology of the degenerating notochord at the level of the vertebral anlagen and compared it with data of the literature on the histopathology of the spinal chordomas. The purpose of the research was to investigate the notochordal histological features justifying the fact that the remnants of a structure destined to atrophy during prenatal life, maintain a proliferative potential in postnatal life. Therefore, from the earliest stages (24th incubation hour) to the terms of development, the notochord was studied by electron microscope in chick embryo. The data obtained show that, in most notochordal cells, signs of metabolic damage and progressive degeneration coexist with signs of secretory and mitotic activity, from which the proliferative potential of these cells seems to derive. Vacuolar degeneration is also likely due to the progressive metabolic segregation of the notochord, owing to the absence of blood-vessels and the appearance of perichordal sheath.

Notochordal histogenesis: preliminary report of ultrastructural observations in chick embryo.

Transverse, frontal and sagittal notochord sections of chick embryos at different developmental stages were ultrastructurally examined. The notochord tissue organization appears to be more likely to build a stiff but flexible body axis than to have an inducting role on the surrounding embryonic structures.

Chondrogenic gradients in the developing vertebral body.

Since in literature the question of the spatio-temporal sequence of the cartilage maturation in the developing vertebra is still controversial, the authors studied by light and electron microscope, the chondrification of the vertebral body in chick embryo from the 6th to the 13th incubation days, in order to define the correlations between morphology and distribution of the cartilage cells in this phase of vertebral development. The results show that the chondrogenesis follows spatio-temporal gradients, starting at about the 8th incubation day from a zone located between notochord and neural tube, slightly cranially to the midvertebral level. From this starting point the chondrification proceeds with dorso-lateral and radial progression, and at the same time extends towards the cranial and caudal plates of the developing vertebra. These data are compared to the findings obtained by other authors on the ultrastructural and biochemical aspects of the vertebral development.

Xenobiotic-metabolizing enzymes in human respiratory nasal mucosa.

Study of oxidative and non-oxidative xenobiotic-metabolizing enzymes was undertaken in microsomal and cytosolic fractions of two human livers, 10 individual and several pooled samples of human respiratory nasal mucosa obtained by surgical operation of male and female patients affected by hypertrophy of the inferior turbinates. The purity of nasal microsomes was checked by electron microscopy and marker enzyme assay. The pooled samples of respiratory nasal epithelium contained, relative to liver, a low amount of cytochrome P450 (about 25 pmol/mg protein) and associated biotransformation activities, and a low level of other components of the mixed-function oxidase system such as cytochrome b5, NADH and NADPH-cytochrome c reductase however the NADH-cytochrome b5 reductase activity was comparable to that of liver. The P450-dependent monooxygenase activities such as ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase and the dimethylnitrosamine N-demethylase were found in nearly all nasal microsomal specimens. The aniline hydroxylase and the aminopyrine or hexamethylphosphoramide N-demethylases were detected only in the pooled nasal samples. With regard to the non-oxidative enzymes, the activities of glutathione S-transferase, DT-diaphorase, epoxide hydrolase, UDP-glucuronyl-transferase, carbonyl reductase, benzaldehyde and propionaldehyde dehydrogenases, were investigated both in the individual and pooled nasal tissues and livers. These activities were similar in nasal and liver tissue, except for UDP-glucuronyltransferase which was not detected in nasal mucosa. The present findings demonstrate that the respiratory section of human nose contains a wide array of oxidative and non-oxidative enzymes, which could play a crucial role in the bioactivation or detoxication in situ of inhaled xenobiotics.

Volumetric growth of the different regions of the fetal spine.

The Author analysed the volumetric growth of the spine ("centra" + intervertebral discs) from the 13th week to term of prenatal life. The data were obtained from the osteological collection of the Department of Human Anatomy and Physiology (University of Turin), by means of a geometric procedure. The growth rate was compared with the observations of other Authors on the growth of the linear measures and with the results of previous works on the anteroposterior curvatures of the developing spine. The analysis of the growth in volume of the various spinal regions, seems to admit temporo-spatial relationships only between the growth velocity of the lumbar region and the appearance of the lumbar lordosis.

Fetal motor activity and spine development.

The Authors, on the basis of previous data obtained by ultrasonography, analysed more closely the quantitative and qualitative correlations between movements and spine development in the human fetus. The bending angles of cervical lordosis and thoracic kyphosis showed a trend directly and inversely proportional, respectively, to the trend of the overall motility of the fetus, measured as the percentage of time employed by the fetus in motor activity. The trend of the lumbo-sacral angle, that appears later, seemed to be less related to the motor behaviour of the fetus, while the bending angle of lumbar lordosis increases very rapidly when also the fetal motility shows an increase at 25-30 weeks. The significance that the developmental stages and the actions of the muscles involved in early fetal motility, and particularly in spine movements, have in the ossification and in the postural attitude of the axial skeleton, was finally discussed.