Postnatal development and histofunctional differentiation of the oviduct in the broad-snouted caiman (Caiman latirostris).

Caiman latirostris is a South American crocodilian species characterized as a sentinel of the presence of endocrine-disrupting compounds (EDCs). Evaluating developmental events in hormone-dependent organs, such as the oviduct, is crucial to understand physiological postnatal development, to identify putative periods of exposure sensitive to EDCs, and/or to identify biomarkers useful to evaluate the effects of EDC exposure. In this study, we describe the histomorphological features of C. latirostris oviducts by establishing the ontogeny of changes at cellular, tissue and molecular levels from the neonatal to the pre-pubertal juvenile stages. Since the histological diagnosis of the adenogenic oviduct lies on a group of features, here we defined a histofunctional score system and a cut-off value to distinguish between preadenogenic and adenogenic oviducts. Our results showed that the maturation of the C. latirostris oviduct is completed postnatally and characterized by changes that mimic the pattern of histological modifications described for the mammalian uterus. Ontogenic changes in the oviductal epithelium parallel changes at subepithelial level, and include collagen remodeling and characteristic spatial-temporal patterns of α-actin and desmin. The expression pattern of estrogen receptor alpha and progesterone receptor evidenced that, even at early postnatal developmental stages, the oviduct of C. latirostris is a target organ of endogenous and environmental hormones. Besides, oviductal adenogenesis seems to be an estrogen-dependent process. Results presented here provide not only insights into the histophysiological aspect of caiman female reproductive ducts but also new tools to better characterize caimans as sentinels of endocrine disruption.

The mechanism of transduction of mechanical strains into biological signals at the bone cellular level.

As appears from the literature, the majority of bone researchers consider osteoblasts and osteoclasts the only very important bony cells. In the present report we provide evidence, based on personal morphofunctional investigations, that such a view is incorrect and misleading. Indeed osteoblasts and osteoclasts undoubtedly are the only bone forming and bone reabsorbing cells, but they are transient cells, thus they cannot be the first to be involved in sensing both mechanical and non-mechanical agents which control bone modeling and remodeling processes. Briefly, according to our view, osteoblasts and osteoclasts represent the arms of a worker; the actual operation center is constituted by the cells of the osteogenic lineage in the resting state. Such a resting phase is characterized by osteocytes, bone lining cells and stromal cells, all connected in a functional syncytium by gap junctions, which extends from the bone to the vessels. We named this syncytium the Bone Basic Cellular System (BBCS), because it represents the only permanent cellular background capable first of sensing mechanical strains and biochemical factors and then of triggering and driving both processes of bone formation and bone resorption. As shown by our studies, signalling throughout BBCS can occur by volume transmission (VT) and/or wiring transmission (WT). VT corresponds to the routes followed by soluble substances (hormones, cytokines etc.), whereas WT represents the diffusion of ionic currents along cytoplasmic processes in a neuron-like manner. It is likely that non-mechanical agents first affect stromal cells and diffuse by VT to reach the other cells of BBCS, whereas mechanical agents are first sensed by osteocytes and then issued throughout

Different skeletal regional response to continuous brain infusion of leptin in the rat.

This study was designed to evaluate whether or not continuous intracerebroventricular infusion of leptin (1.5 microg/rat/24 h, for 28 days) produced different regional response on the skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content (BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydinoline and serum osteocalcin remained more elevated in the leptin-treated group as compared to controls. The results demonstrate that long-term central infusion of leptin activates bone remodeling with a negative balance. Leptin induces distinct responses in the different structure of bone and in the axial and appendicular skeleton.

In vivo leptin expression in cartilage and bone cells of growing rats and adult humans.

The present investigation was carried out to analyse, immunohistochemically, in vivo leptin expression in cartilage and bone cells, the latter restricted to the elements of the osteogenic system (stromal cells, osteoblasts, osteocytes, bone lining cells). Observations were performed on the first lumbar vertebra, tibia and femur of four rats and on the humerus, femur and acromion of four patients. Histological sections of paraffin-embedded bone samples were immunostained using antibody to leptin. The results showed that, in growing rat bone, leptin is expressed in chondrocytes and stromal cells, but not in osteoblasts; bone lining cells were not found in the microscopic fields examined. In adult human bone, leptin is expressed in chondrocytes, stromal cells and bone lining cells; osteoblasts were not found in the microscopic fields examined. Osteocytes were found to be leptin positive only occasionally and focally in both rat and human bone. The in vivo findings reported show, for the first time, that leptin appears to be expressed only in the cells of the osteogenic lineage (stromal cells, bone lining cells, osteocytes) that, with respect to osteoblasts, are permanent and inactive, i.e. in those cells that according to our terminology constitute the bone basic cellular system (BBCS). Because the BBCS seems to be primarily involved in sensing and integrating mechanical strains and biochemical factors and then in triggering and driving bone formation and/or bone resorption, it appears that leptin seems to be mainly involved in modulating the initial phases of bone modelling and remodelling processes.

Bone as an ion exchange system: evidence for a link between mechanotransduction and metabolic needs.

To detect whether the mutual interaction occurring between the osteocytes-bone lining cells system (OBLCS) and the bone extracellular fluid (BECF) is affected by load through a modification of the BECF-extracellular fluid (ECF; systemic extracellular fluid) gradient, mice metatarsal bones immersed in ECF were subjected ex vivo to a 2-min cyclic axial load of different amplitudes and frequencies. The electric (ionic) currents at the bone surface were measured by a vibrating probe after having exposed BECF to ECF through a transcortical hole. The application of different loads and different frequencies increased the ionic current in a dose-dependent manner. The postload current density subsequently decayed following an exponential pattern. Postload increment's amplitude and decay were dependent on bone viability. Dummy and static loads did not induce current density modifications. Because BECF is perturbed by loading, it is conceivable that OBLCS tends to restore BECF preload conditions by controlling ion fluxes at the bone-plasma interface to fulfill metabolic needs. Because the electric current reflects the integrated activity of OBLCS, its evaluation in transgenic mice engineered to possess genetic lesions in channels or matrix constituents could be helpful in the characterization of the mechanical and metabolic functions of bone.

Osteocyte-osteoclast morphological relationships and the putative role of osteocytes in bone remodeling.

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.

The osteocyte as a wiring transmission system.

The mechanism of transduction of mechanical strains into biological signals remains one of the more baffling problems of skeletal homeostasis. The updated literature ascribes to osteocytes the function of sensing the strains induced into the bone matrix by mechanical stresses. Whether the osteocytes perform such function by themselves or they are helped by other cells is also unknown. Indeed TEM investigations carried out in our laboratory pointed out the existence of a functional syncytium among all the cells of the osteogenic lineage (COL; stromal cells, osteoblasts or bone lining cells, osteocytes). On the basis of this finding, we suggested that COL may reciprocally modulate their function not only by volume transmission (paracrine and autocrine stimulation) but also by wiring transmission, namely in a neuronal like manner. Thanks to their location, osteocytes should theoretically be the first cells of COL functional syncytium to sense mechanical strains, whereas stromal cells should be the first to be activated by hormonal molecules diffusing across the endothelial lining. Since PTH and Estrogen receptors have also been localized on osteocytes, and considering that such hormones have been suggested to modulate the sensitivity to strain of the bone mechanosensor, we suggested that the osteocyte syncytium may constitute the microscopic bone structure that sense both mechanical strain and biochemical factors and, at any moment, after having combined the two types of stimuli, issues the appropriate signals to the other bone cells by volume and/or wiring-transmission. Stromal cells, on the other hand, besides transmitting signals from vascular endothelium to bone cells, may control the differentiation and then direct the course of the osteoblasts around the vascular framework.

Morphometric investigation on osteocytes in human auditory ossicles.

An osteocyte lacunae differential count (1-lacunae with live osteocytes, 2-lacunae with degenerating osteocytes, 3-empty lacunae) was carried out on ear ossicles and clavicles from cadavers as well as on stapes removed by stapedotomy. The distance of the three types of lacunae from the vascular source was also determined by a computer-assisted light microscope. Results showed that the delayed fixation of bone from cadavers does not significantly interfere with osteocyte preservation, at least with the scope of this investigation. The results of osteocyte differential count show that the number of empty lacunae and lacunae with degenerating osteocytes: (a) is significantly higher in ear ossicles than in clavicles, (b) increases with age, (c) is higher in stapes than in incuses and mallei, (d) increases with the distance from the vascular sources in both ear ossicles and clavicles. Additionally it appeared that the process of osteocyte degeneration in ear ossicles is very rapid and widespread, over 40% of the cells being dead within the 2nd year of age. In the light of the recent literature and personal findings, which ascribe to osteocytes the function of mechanical detectors, and considering that bone remodeling occasionally occurs in ear ossicles, it is postulated that osteocyte death in these bones could be a programmed phenomenon (apoptosis?), due to which they lose the ability to react to strains and stresses and achieve the structural stability they need to perform their peculiar stereotyped function.

Osteocyte-bone lining cell system at the origin of steady ionic current in damaged amphibian bone.

A wound-generated steady electric current was measured by a two-dimensional vibrating probe system in the metatarsal bones of 22 adult frogs (Xenopus laevis) placed in amphibian Ringer. Inward currents were recorded entering a micrometric hole drilled through the cortex at middiaphyseal level. These steady state currents (mean +/- SD 8.50 +/- 2.77 microA/cm2) last approximately 2 hours, were dependent on the presence of sodium in the incubation medium, were no more detectable after fixation, and were reduced to background level when the cell membranes were solubilized. These results agree with previous recordings of metatarsal bones of weanling mice, under identical conditions. Both results suggest that the measured ionic currents have a cellular origin. Metatarsal bones of adult amphibian were purposely selected for this study because, unlike mammalian bones, their shafts are avascular and only contain an osteocyte-bone lining cell system, as documented by scanning and transmission electron observations. Thus, unlike the data from previous investigations on mammals, the results succeeded in giving the first convincing evidence that the osteocyte-bone lining cell system is the origin of damage-generated ionic currents. As damage exposes bone ionic compartment to plasma, damage-generated ionic currents are representative of ion fluxes at bone plasma interface, and cells at the origin of the current generate the driving force of such fluxes. By demonstrating that osteocytes and bone lining cells are at the origin of the current, this study suggests that the osteocyte-bone lining cell system, though operating as a cellular membrane partition, regulates ionic flow between bone and plasma. Since strain-related adaptive remodeling could also depend on ionic characteristics and flow of the bone fluid through the osteocyte lacuno-canalicular network, the results reported here support the view that osteocyte and bone lining cells may constitute a functional syncytium involved in mineral homeostasis as well as in bone adaptation to mechanical loading.

Stromal cell structure and relationships in perimedullary spaces of chick embryo shaft bones.

Structure and relationships of stromal cells were studied by light (LM) and transmission electron microscopy (TEM) in the perimedullary spaces that form the growing cortex of the chick embryo tibia. Observation under LM showed that in all perimedullary spaces the interstices between the cells carpeting the bone surface and the endothelial lining contain stromal cells surrounded by an amorphous matrix. Two types of stromal cells were distinguished: stellate and spindle-shaped. All stromal cells are alkaline phosphatase-positive. TEM showed that both types of stromal cells have cytoplasmic processes of various length and calibre, coming into contact with each other as well as with endothelial cells and osteoblasts or bone lining cells. Capillaries were found to have a continuous endothelial lining; occasionally endothelial cells radiate cytoplasmic processes towards stromal cells. Along all the above-mentioned cellular contacts adherens and/or gap junctions were often observed. The results of the present study, together with our previous findings on osteoblast-osteocyte relationships, show that the cells of the osteogenic lineage form a continuous protoplasmic network that extends from the osteocytes to the vascular endothelium, passing through osteoblasts (or bone lining cells) and stromal cells. The occurrence of gap junctions among this cytoplasmic network, namely of junctions enabling metabolic and electric coupling, indicates that it forms a functional syncytium, suggesting the hypothesis that the activity of the cells pertaining to the osteogenic lineage might be regulated not only by diffusion (volume transmission) through the intercellular fluids of systemic (hormones) and local factors (cytokines, etc.) but also by signals issued through the cytoplasmic network of the osteogenic cells (wiring transmission).

Quantitative evaluation on osteocyte canalicular density in human secondary osteons.

Osteocyte canalicular density (OCD) was evaluated at different levels of the wall of human secondary osteons, in subjects of different ages, to find out whether any correlation exists between the extension of the canalicular network and the exponential decrement of the appositional growth rate (AGR), which has been shown to occur during osteon formation. Scanning electron microscopy (SEM) was used to count the number of canalicular openings per unit surface on large Haversian canals of forming osteons as well as on small canals of completed osteons. Reflected polarized light microscopy (RPL) enabled the number per unit length of canaliculi to be counted at different concentric levels of the osteons. The results of both techniques agree in showing that, in the subjects examined, OCD does not change significantly throughout the osteon wall. Since no correlation exists between OCD and AGR, it follows that osteoblast flattening which was shown to occur in parallel to the decrement of the rate of concentric bone deposition, does not seem to depend primarily on the number of osteoblast-osteocyte contacts, but on other factors.

Collagen texture and osteocyte distribution in lamellar bone.

A comparative scanning and transmission electron microscopy study was carried out on collagen fiber texture and osteocyte lacunae distribution in human lamellar bone. The results show that bony lamellae are not made up of parallel-arranged collagen fibers, as classically maintained. They are instead made up of highly interlaced fibers, and the lamellation appears to be due to the alternation of collagen-rich and collagen-poor layers, namely of dense and loose lamellae. The present study additionally shows that osteocyte lacunae are only located inside loose lamellae. Such structural organization of lamellar bone is briefly discussed in terms of bone biomechanics and osteogenesis.

Structure and function of lamellar bone.

A comparative scanning and transmission electron microscopy study was carried out on human compact lamellar bone. The results obtained fully confirm our previous findings which show that bony lamellae are not made up of parallel-arranged collagen fibers, as classically maintained. They are instead made up of highly interlaced fibers, and the lamellation appears to be due to the alternation of collagen-rich and collagen-poor layers, namely of dense and loose lamellae. In the light of these findings, lamellar bone must be considered a variety of woven- instead of parallel-fibered bone, as given in almost all the textbook of histology as an established datum. Such collagen organization of lamellar bone is briefly discussed in terms of bone biomechanics and osteogenesis.

Histomorphological analysis of the effect of rigid fixation on growing sutures in the rabbit.

The effect of internal rigid fixation (IRF) on bone growth was studied in an experimental model set up in the rabbit. The frontonasal suture of the right side was surgically bridged by a microplate. As reference for bone growth, four screws were placed symmetrically in the four bony segments including the frontonasal suture on both sides. The suture development was followed dynamically for 40 days on the basis of the position of the screws established radiographically. The rate of bone formation along the sutural bones was evaluated by means of the tetracycline labelling technique. The structure of the newly formed bone and its degree of mineralization were respectively analysed under polarized light and with microradiography. It was demonstrated that IRF prevents growth of the sutural membrane but not of the osteogenic process; as a consequence the constrained sutures soon undergo synostosis. This fact must be taken into consideration when IRF is employed in children in order to avoid delayed removal of the plate irreversibly stopping the growth of the constrained suture.

A new theory of bone lamellation.

A comparative polarized light (PLM), scanning (SEM), and transmission (TEM) electron microscopy study was carried out on cross- and longitudinal sections of human lamellar bone in the tibiae of four male subjects aged 9, 23, 45, and 70 years. SEM analysis was also performed on rectangular-prismatic samples in order to observe each lamella sectioned both transversely and longitudinally. The results obtained do not confirm the model hitherto suggested to explain the lamellar appearance of bone. In particular, the classic description by Gebhardt (still accepted by the majority of bone researchers), which suggests that collagen fibers alternate between longitudinal and transversal in successive lamellae, or that they have spiral paths of different pitches, appears to be no longer acceptable in the light of our findings. In fact, SEM and TEM observations here reported agree in demonstrating that lamellar bone is made up of alternating collagen-rich (dense lamellae) and collagen-poor (loose lamellae) layers, all having an interwoven arrangement of fibers. No interlamellar cementing substance was observed between the lamellae, and collagen bundles form a continuum throughout lamellar bone. Preliminary measurements of lamellar thickness indicate that dense lamellae are significantly (P < 0.001) thinner than loose lamellae. Compared with the classic model of Gebhardt, the dense lamellae correspond to the transverse lamellae and are birifringent under PLM, whereas the loose lamellae correspond to the longitudinal lamellae and are extinguished. Collagen-fiber organization in dense and loose lamellae is discussed in terms of bone biomechanics and osteogenesis.

A quantitative evaluation of osteoblast-osteocyte relationships on growing endosteal surface of rabbit tibiae.

Scanning electron microscopy (SEM) was used to quantify the intercellular relationships between osteoblasts and osteocytes on the growing endosteal surfaces of the medullary canal of the tibia in four rabbits of different ages. The area of each osteoblast was measured on the SEM micrographs by means of an Image Analyzer. The number of osteocyte cytoplasmic processes was indirectly evaluated by counting the canalicular openings present on the same microscopic fields after the removal of the osteoblasts. The metabolic activity of the osteoblasts was indirectly evaluated from their shape, and the structure was analyzed by transmission electron microscope (TEM) in sections taken from the samples studied by SEM. In all four animals, the surface area of the osteoblasts (OA) was found to vary a great deal, whereas the density of canalicular openings was fairly uniform. Moreover, although the OA mean value increases significantly with the age of the animals, the density of canalicular openings does not; it would therefore appear that the older the animal and the more flattened the osteoblasts, the greater the number of canaliculi beneath them. Since osteoblast activity has previously been shown to be inversely proportional to the area of the protoplasm in contact with the bone surface, it appears that the less active osteoblasts should contact a greater number of osteocyte cytoplasmic processes. These findings suggest that osteocytes might play an important role in modulating osteoblast activity and in recruiting osteoblasts that differentiate into osteocytes, possibly by means of inhibitory signals transmitted via gap junctions.

Osteocyte differentiation in the tibia of newborn rabbit: an ultrastructural study of the formation of cytoplasmic processes.

The morphological changes undergone by the osteoblast at the ultrastructural level, during its differentiation into osteocyte, were studied in the primary parallel-fibred bone of the newborn rabbit by means of incomplete three-dimensional reconstruction from partially serial-sectioned preosteocytes. The findings obtained suggest that the formation of osteocyte cytoplasmic processes is an asynchronous and asymmetrical phenomenon that seems to precede the mineralization of the organic matrix and to give rise to an asymmetrical mature osteocyte. The functions of cytoplasmic processes as regards bone formation, cell nutrition and osteoblast modulation are discussed. The mechanism by which the osteoblast 'enters' the bone matrix is hypothesized.

Morphological study of intercellular junctions during osteocyte differentiation.

Ultrastructural studies were carried out on intercellular relationships during osteocyte differentiation in primary parallel-fibred bone of newborn rabbit. It was found that throughout the whole differentiative process preosteocytes are in close relationship with the neighboring cells (osteoblasts, osteocytes) by means of variously-shaped intercellular contacts (invaginated finger-like, side-to-side, and end-to-end) and two types of specialized junctions: gap and adherens. The pivotal role played by these contacts and junctions in osteocyte differentiation and activity is considered in the context of their particular functional significance.

A light and scanning electron microscopic study of osteocyte activity in calcium-deficient rats.

The material herein is an extension of an earlier study of osteocyte lacunae in calcium-deficient rats, utilizing morphometric measurements in undecalcified bone sections and scanning electron microscopy. The results confirm our earlier finding that bone resorption resulting from a low-calcium diet is not accompanied by osteocytic resorption.