In Vitro and in Vivo Bone Remodeling Models: Complementary Pieces of the same Puzzle.

The present letter to editor comments on the manuscript entitled "Assembling the Puzzle Pieces. Insights for in Vitro Bone Remodeling" by O. Krasnova & I. Neganova; in this context, we underlie the importance of in vivo models to corroborate in vitro bone remodeling systems.

Morphology and cell biology - two sides of the same coin: Importance of morphology in choosing Cre experimental models for targeting osteoblasts vs osteocytes.

The present letter to editor comments the manuscript entitled "Targeting osteocytes vs osteoblasts" by Y. Kitase and M. Prideaux, where we underlie the importance morphology in choosing Cre experimental models for targeting osteoblasts vs osteocytes.

Unexpected Absence of Skeletal Responses to Dietary Magnesium Depletion: Basis for Future Perspectives?

It's known that a magnesium (Mg)-deficient diet is associated with an increased risk of osteoporosis. The aim of this work is to investigate, by a histological approach, the effects of a Mg-deprived diet on the bone of 8-weeks-old C57BL/6J male mice. Treated and control mice were supplied with a Mg-deprived or normal diet for 8 weeks, respectively. Body weight, serum Mg concentration, expression of kidney magnesiotropic genes, and histomorphometry on L5 vertebrae, femurs, and tibiae were evaluated. Body weight gain and serum Mg concentration were significantly reduced, while a trend toward increase was found in gene expression in mice receiving the Mg-deficient diet, suggesting the onset of an adaptive response to Mg depletion. Histomorphometric parameters on the amount of trabecular and cortical bone, number of osteoclasts, and thickness of the growth plate in femoral distal and tibial proximal metaphyses did not differ between groups; these findings partially differ from most data present in the literature showing that animals fed a Mg-deprived diet develop bone loss and may be only in part explained by differences among the experimental protocols. However, the unexpected findings we recorded on bones could be attributed to genetic differences that may have developed after multiple generations of inbreeding.

The Osteocyte: From "Prisoner" to "Orchestrator".

Osteocytes are the most abundant bone cells, entrapped inside the mineralized bone matrix. They derive from osteoblasts through a complex series of morpho-functional modifications; such modifications not only concern the cell shape (from prismatic to dendritic) and location (along the vascular bone surfaces or enclosed inside the lacuno-canalicular cavities, respectively) but also their role in bone processes (secretion/mineralization of preosseous matrix and/or regulation of bone remodeling). Osteocytes are connected with each other by means of different types of junctions, among which the gap junctions enable osteocytes inside the matrix to act in a neuronal-like manner, as a functional syncytium together with the cells placed on the vascular bone surfaces (osteoblasts or bone lining cells), the stromal cells and the endothelial cells, i.e., the bone basic cellular system (BBCS). Within the BBCS, osteocytes can communicate in two ways: by means of volume transmission and wiring transmission, depending on the type of signals (metabolic or mechanical, respectively) received and/or to be forwarded. The capability of osteocytes in maintaining skeletal and mineral homeostasis is due to the fact that it acts as a mechano-sensor, able to transduce mechanical strains into biological signals and to trigger/modulate the bone remodeling, also because of the relevant role of sclerostin secreted by osteocytes, thus regulating different bone cell signaling pathways. The authors want to emphasize that the present review is centered on the morphological aspects of the osteocytes that clearly explain their functional implications and their role as bone orchestrators.

WISP-2 expression induced by Teriparatide treatment affects in vitro osteoblast differentiation and improves in vivo osteogenesis.

The Osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances occurring during bone remodeling, caused by hormone perturbations or by mechanical loading alterations, can induce bone pathologies such as osteoporosis. Recently, the active fraction of parathormone, PTH (1-34) or Teriparatide (TPTD), was chosen as election treatment for osteoporosis. The effect of such therapy is dependent on the temporal manner of administration. The molecular reasons why the type of administration regimen is so critical for the fate of bone remodeling are numerous and not yet well known. Our study attempts to analyze diverse signaling pathways directly activated in osteocytes upon TPTD treatment. By means of gene array analysis, we found many molecules upregulated or downregulated in osteocytes. Later, we paid attention to Wisp-2, a protein involved in the Wnt pathway, that is secreted by MLO-Y4 cells and increases upon TPTD treatment and that is able to positively influence the early phases of osteogenic differentiation. We also confirmed the pro osteogenic property of Wisp-2 during mesenchymal stem cell differentiation into the preliminary osteoblast phenotype. The same results were confirmed with an in vivo approach confirming a remarkable Wisp-2 expression in metaphyseal trabecular bone. These results highlighted the anabolic roles unrolled by osteocytes in controlling the action of neighboring cells, suggesting that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation.

Interaction among Calcium Diet Content, PTH (1-34) Treatment and Balance of Bone Homeostasis in Rat Model: The Trabecular Bone as Keystone.

The present study is the second step (concerning normal diet restoration) of the our previous study (concerning the calcium-free diet) to determine whether normal diet restoration, with/without concomitant PTH (1-34) administration, can influence amounts and deposition sites of the total bone mass. Histomorphometric evaluations and immunohistochemical analysis for Sclerostin expression were conducted on the vertebral bodies and femurs in the rat model. The final goals are (i) to define timing and manners of bone mass changes when calcium is restored to the diet, (ii) to analyze the different involvement of the two bony architectures having different metabolism (i.e., trabecular versus cortical bone), and (iii) to verify the eventual role of PTH (1-34) administration. Results evidenced the greater involvement of the trabecular bone with respect to the cortical bone, in response to different levels of calcium content in the diet, and the effect of PTH, mostly in the recovery of trabecular bony architecture. The main findings emerged from the present study are (i) the importance of the interplay between mineral homeostasis and skeletal homeostasis in modulating and guiding bone's response to dietary/metabolic alterations and (ii) the evidence that the more involved bony architecture is the trabecular bone, the most susceptible to the dynamical balance of the two homeostases.

Role of Osteocytes in Myeloma Bone Disease: Anti-sclerostin Antibody as New Therapeutic Strategy.

Osteocytes are terminally differentiated cells of the osteoblast lineage. They are involved in the regulation of bone remodeling by increasing osteoclast formation or decreasing bone formation by the secretion of the osteoblast inhibitor sclerostin. Monoclonal antibody anti-sclerostin, Romosozumab, has been developed and tested in clinical trials in patients with osteoporosis. In the last years, the role of osteocytes in the development of osteolytic bone lesions that occurs in multiple myeloma, have been underlined. Myeloma cells increase osteocyte death through the up-regulation of both apoptosis and autophagy that, in turn, triggers osteoclast formation, and activity. When compared to healthy controls, myeloma patients with bone disease have higher osteocyte cell death, but the treatment with proteasome inhibitor bortezomib has been shown to maintain osteocyte viability. In preclinical mouse models of multiple myeloma, treatment with blocking anti-sclerostin antibody increased osteoblast numbers and bone formation rate reducing osteolytic bone lesions. Moreover, the combination of anti-sclerostin antibody and the osteoclast inhibitor zoledronic acid increased bone mass and fracture resistance synergistically. However, anti-sclerostin antibody did not affect tumor burden in vivo or the efficacy of anti-myeloma drugs in vitro. Nevertheless, the combination therapy of anti-sclerostin antibody and the proteasome inhibitor carfilzomib, displayed potent anti-myeloma activity as well as positive effects on bone disease in vivo. In conclusion, all these data suggest that osteocytes are involved in myeloma bone disease and may be considered a novel target for the use of antibody-mediated anti-sclerostin therapy also in multiple myeloma patients.

Structural and ultrastructural analyses of bone regeneration in rabbit cranial osteotomy: Piezosurgery versus traditional osteotomes.

Clinical advantages of piezosurgery have been already proved. However, few investigations have focused on the dynamics of bone healing. The aim of this study was to evaluate, in adult rabbits, bone regeneration after cranial linear osteotomies with two piezoelectrical devices (Piezosurgery® Medical - PM and Piezosurgery® Plus - PP), comparing them with conventional rotary osteotomes (RO). PP was characterized by an output power three times higher than PM. Fifteen days after surgery, histomorphometric analyses showed that the osteotomy gap produced with PM and PP was about half the size of that produced by RO, and in a more advanced stage of recovery. Values of regenerated bone area with respect to the total osteotomy area were about double in PM and PP samples compared with RO ones, while the number of TRAP-positive (tartrate-resistant acid phosphatase positive) osteoclasts per linear surface showed a significant increase, suggesting greater bone remodelling. Under scanning electron microscopy, regenerated bone displayed higher cell density and less mineralized matrix compared with pre-existent bone for all devices used. Nanoindentation tests showed no changes in elastic modulus. In conclusion, PM/PP osteotomies can be considered equivalent to each other, and result in more rapid healing compared with those using RO.

Expression and functional proteomic analyses of osteocytes from Xenopus laevis tested under mechanical stress conditions: preliminary observations on an appropriate new animal model.

Hitherto, the role of the osteocyte as transducer of mechanical stimuli into biological signals is far from settled. In this study, we used an appropriate model represented by the cortex of Xenopus laevis long bone diaphysis lacking (unlike the mammalian one) of vascular structures and containing only osteocytes inside the bone matrix. These structural features allow any change of protein profile that might be observed upon different experimental conditions, such as bone adaptation to stress/mechanical loading, to be ascribed specifically to osteocytes. The study was conducted by combining ultrastructural observations and two-dimensional electrophoresis for proteomic analysis. The osteocyte population was extracted from long bones of lower limbs of amphibian skeletons after different protocols (free and forced swimming). The experiments were performed on 210 frogs subdivided into five trials, each including free swimming frogs (controls) and frogs submitted to forced swimming (stressed). The stressed groups were obliged to swim (on movable spheres covering the bottom of a pool on a vibrating plate) continuously for 8 h, and killed 24 h later along with the control groups. Long bones free of soft tissues (periosteum, endosteum and bone marrow), as well as muscles of posterior limbs, were processed and analyzed for proteins differentially expressed or phosphorylated between the two sample groups. The comparative analysis showed that protein phosphorylation profiles differ between control and stressed groups. In particular, we found in long bones of stressed samples that both Erk1/2 and Akt are hyperphosphorylated; moreover, the different phosphorylation of putative Akt substrates (recognized by specific Akt phosphosubstrates-antibody) in stressed vs. control samples clearly demonstrated that Akt signaling is boosted by forced swimming (leading to an increase of mechanical stress) of amphibian long bones. In parallel, we found in posterior limb muscles that the expression of heat shock protein HSP27 and HSP70 stress markers increased upon the forced swimming condition. Because the cortexes of frog long bones are characterized by the presence of only osteocytes, all our results establish the suitability of the X. laevis animal model to study the bone response to stress conditions mediated by this cell type and pave the way for further analysis of the signaling pathways involved in these signal transduction mechanisms.

Biocompatibility Analyses of Al2O3-Treated Titanium Plates Tested with Osteocyte and Fibroblast Cell Lines.

Osseointegration of a titanium implant is still an issue in dental/orthopedic implants durable over time. The good integration of these implants is mainly due to their surface and topography. We obtained an innovative titanium surface by shooting different-in-size particles of Al2O3 against the titanium scaffolds which seems to be ideal for bone integration. To corroborate that, we used two different cell lines: MLO-Y4 (murine osteocytes) and 293 (human fibroblasts) and tested the titanium scaffolds untreated and treated (i.e., Al2O3 shot-peened titanium surfaces). Distribution, density, and expression of adhesion molecules (fibronectin and vitronectin) were evaluated under scanning electron microscope (SEM) and confocal microscope (CM). DAPI and fluorochrome-conjugated antibodies were used to highlight nuclei, fibronectin, and vitronectin, under CM; cell distribution was analyzed after gold-palladium sputtering of samples by SEM. The engineered biomaterial surfaces showed under SEM irregular morphology displaying variously-shaped spicules. Both SEM and CM observations showed better outcome in terms of cell adhesion and distribution in treated titanium surfaces with respect to the untreated ones. The results obtained clearly showed that this kind of surface-treated titanium, used to manufacture devices for dental implantology: (i) is very suitable for cell colonization, essential prerequisite for the best osseointegration, and (ii) represents an excellent solution for the development of further engineered implants with the target to obtain recovery of stable dental function over time.

Morphological Study: Ultrastructural Aspects of Articular Cartilage and Subchondral Bone in Patients Affected by Post-Traumatic Shoulder Instability.

Post-traumatic shoulder instability is a frequent condition in active population, representing one of most disabling pathologies, due to altered balance involving joints. No data are so far available on early ultrastructural osteo-chondral damages, associated with the onset of invalidating pathologies, like osteoarthritis-OA. Biopsies of glenoid articular cartilage and sub-chondral bone were taken from 10 adult patients underwent arthroscopic stabilization. Observations were performed under Transmission Electron Microscopy-TEM in tangential, arcuate and radial layers of the articular cartilage and in the sub-chondral bone. In tangential and arcuate layers chondrocytes display normal and very well preserved ultrastructure, probably due to the synovial liquid supply; otherwise, throughout the radial layer (un-calcified and calcified) chondrocytes show various degrees of degeneration; occasionally, in the radial layer evidences of apoptosis/autophagy were also observed. Concerning sub-chondral bone, osteocytes next to the calcified cartilage also show signs of degeneration, while osteocytes farther from the osteo-chondral border display normal ultrastructure, probably due to the bone vascular supply. The ultrastructural features of the osteo-chondral complex are not age-dependent. This study represents the first complete ultrastructural investigation of the articular osteo-chondral complex in shoulder instability, evaluating the state of preservation/viability of both chondrocytes and osteocytes throughout the successive layers of articular cartilage and sub-chondral bone. Preliminary observations here collected represent the morphological basis for further deepening of pathogenesis related to shoulder instability, enhancing the relationship between cell shape and microenvironment; in particular, they could be useful in understanding if the early surgical treatment in shoulder instability could avoid the onset of OA. Anat Rec, 300:1208-1218, 2017. © 2016 Wiley Periodicals, Inc.

PTH(1-34) effects on repairing experimentally drilled holes in rat femur: novel aspects - qualitative vs. quantitative improvement of osteogenesis.

The timetable of effects on bone repair of the active fraction-parathyroid hormone, PTH(1-34), was analytically investigated from the morphometric viewpoint in 3-month-old male Sprague-Dawley rats, whose femurs were drilled at mid-diaphyseal level (transcortical holes). The animals were divided into groups with/without PTH(1-34) administration, and sacrificed at different times (10, 28, 45 days after surgery). The observations reported here need to be framed in the context of our previous investigations regarding bone histogenesis (Ferretti et al. Anat Embryol. 2002; 206: 21-29) in which we demonstrated the occurrence of two successive bone-forming processes during both skeletal organogenesis and bone repair, i.e. static and dynamic osteogenesis: the former (due to stationary osteoblasts, haphazardly grouped in cords) producing preliminary bad quality trabecular bone, the latter (due to typical polarized osteoblasts organized in ordered movable laminae) producing mechanically valid bone tissue. The primary function of static osteogenesis is to provide a rigid scaffold containing osteocytes (i.e. mechano-sensors) for osteoblast laminae acting in dynamic osteogenesis. In the present work, histomorphometric analysis revealed that, already 10 days after drilling, despite the holes being temporarily filled by the same amount of newly formed trabecular bone by static osteogenesis independently of the treatment, the extent of the surface of movable osteoblast-laminae (covering the trabecular surface) was statistically higher in animals submitted to PTH(1-34) administration than in control ones; this datum strongly suggests the effect of PTH(1-34) alone in anticipating the occurrence of dynamic osteogenesis involved in the production of good quality bone (with more ordered collagen texture) more suitable for loading. This study could be crucial in further translational clinical research in humans for defining the best therapeutic strategies to be applied in recovering severe skeletal lesions, particularly as regards the time of PTH(1-34) administration.

The Proteasome Inhibitor Bortezomib Maintains Osteocyte Viability in Multiple Myeloma Patients by Reducing Both Apoptosis and Autophagy: A New Function for Proteasome Inhibitors.

Multiple myeloma (MM) is characterized by severely imbalanced bone remodeling. In this study, we investigated the potential effect of proteasome inhibitors (PIs), a class of drugs known to stimulate bone formation, on the mechanisms involved in osteocyte death induced by MM cells. First, we performed a histological analysis of osteocyte viability on bone biopsies on a cohort of 37 MM patients with symptomatic disease. A significantly higher number of viable osteocytes was detected in patients treated with a bortezomib (BOR)-based regimen compared with those treated without BOR. Interestingly, both osteocyte autophagy and apoptosis were affected in vivo by BOR treatment. Thereafter, we checked the in vitro effect of BOR to understand the mechanisms whereby BOR maintains osteocyte viability in bone from MM patients. We found that osteocyte and preosteocyte autophagic death was triggered during coculturing with MM cells. Our evaluation was conducted by analyzing either autophagy markers microtubule-associated protein light chain 3 beta (LC3B) and SQSTM1/sequestome 1 (p62) levels, or the cell ultrastructure by transmission electron microscopy. PIs were found to increase the basal levels of LC3 expression in the osteocytes while blunting the myeloma-induced osteocyte death. PIs also reduced the autophagic death of osteocytes induced by high-dose dexamethasone (DEX) and potentiated the anabolic effect of PTH(1-34). Our data identify osteocyte autophagy as a new potential target in MM bone disease and support the use of PIs to maintain osteocyte viability and improve bone integrity in MM patients.

Mineral and Skeletal Homeostasis Influence the Manner of Bone Loss in Metabolic Osteoporosis due to Calcium-Deprived Diet in Different Sites of Rat Vertebra and Femur.

Rats fed calcium-deprived diet develop osteoporosis due to enhanced bone resorption, secondary to parathyroid overactivity resulting from nutritional hypocalcemia. Therefore, rats provide a good experimental animal model for studying bone modelling alterations during biochemical osteoporosis. Three-month-old Sprague-Dawley male rats were divided into 4 groups: (1) baseline, (2) normal diet for 4 weeks, (3) calcium-deprived diet for 4 weeks, and (4) calcium-deprived diet for 4 weeks and concomitant administration of PTH (1-34) 40 µg/Kg/day. Histomorphometrical analyses were made on cortical and trabecular bone of lumbar vertebral body as well as of mid-diaphysis and distal metaphysis of femur. In all rats fed calcium-deprived diet, despite the reduction of trabecular number (due to the maintenance of mineral homeostasis), an intense activity of bone deposition occurs on the surface of the few remaining trabeculae (in answering to mechanical stresses and, consequently, to maintain the skeletal homeostasis). Different responses were detected in different sites of cortical bone, depending on their main function in answering mineral or skeletal homeostasis. This study represents the starting point for work-in-progress researches, with the aim of defining in detail timing and manners of evolution and recovery of biochemical osteoporosis.

Ferutinin dose-dependent effects on uterus and mammary gland in ovariectomized rats.

The present paper completes our recent study on the effects of phytoestrogen ferutinin in preventing osteoporosis and demonstrating the superior osteoprotective effect of a 2 mg/kg/day dose in ovariectomized (OVX) rats, compared to both estrogens and lower (0.5, 1 mg/kg/day) ferutinin doses. Morphological and morphometrical analyses were performed on the effects of different doses of ferutinin administrated for one month on uterus and on mammary gland of Sprague-Dawley OVX rats, evaluated in comparison with the results for estradiol benzoate. To verify whether ferutinin provides protection against uterine and breast cancer, estimations were made of both the amount of cell proliferation (by Ki-67), and the occurrence of apoptosis (by TUNEL), two processes that in unbalanced ratio form the basis for cancer onset. The results suggest that the effects of ferutinin are dose dependent and that a 2 mg/kg/day dose might offer a better protective action against the onset of both breast and uterine carcinoma compared to ferutinin in lower doses or estradiol benzoate, increasing cellular apoptosis in glandular epithelia.

The problem of bone lamellation: an attempt to explain different proposed models.

Collagen texture and osteocyte distribution were analyzed in human woven- and lamellar-bone using scanning and transmission electron microscopy. We provide data substantiating the concept that lamellar bone is made up of an alternation of dense-acellular lamellae and loose-cellular lamellae, all exhibiting an interwoven texture of collagen fibers. An attempt is also made to explain how the present findings might conform to those of authors whose models propose orderly, geometric arrangements of collagen fibers inside bony lamellae. Such a comparison is possible because the present investigation analyzes split loose lamellae and tangentially-sectioned dense lamellae. It emerged that only loose lamellae can be dissected, revealing a loose interwoven collagen texture and halved osteocyte lacunae. Dense lamellae cannot be split because of their compactness. The analysis of tangentially sectioned dense lamellae demonstrates that they consist of a network of interwoven collagen fiber bundles. Inside each bundle, collagen fibers run parallel to each other but change direction where they enter adjacent bundles, at angles as described by other authors whose TEM investigations were performed at a much higher magnification than those of the present study. Consequently, what these authors consider to be a lamella are, instead, bundles of collagen fibers inside a lamella. There is discussion of the role played by the manner of osteocyte-recruitment in the deposition of lamellar- and woven-bone and how the presence of these cells is crucial for collagen spatial arrangement in bone tissues.

Immunocytochemical and structural comparative study of committed versus multipotent stem cells cultured with different biomaterials.

The aim of this work was the comparison of the behavior of committed (human osteoblast cells - hOB - from bone biopsies) versus multipotent (human dental pulp stem cells - hDPSC - from extracted teeth) cells, cultured on shot-peened titanium surfaces, since the kind of cell model considered has been shown to be relevant in techniques widely used in studies on composition/morphology of biomaterial surfaces. The titanium surface morphology, with different roughness, and the behavior of cells were analyzed by confocal microscope (CM), scanning electron microscope (SEM) and X-ray microanalysis. The best results, in terms of hOB adhesion/distribution, were highlighted by both CM and SEM in cultured plates having 20-µm-depth cavities. On the contrary, CM and SEM results highlighted the hDPSC growth regardless the different surface morphology, arranged in overlapped layers due to their high proliferation rate, showing their unfitness in biomaterial surface test. Nevertheless, hDPSC cultured inside 3D-matrices reproduced an osteocyte-like three-dimensional network, potentially useful in the repair of critical size bone defects. The behavior of the two cell models suggests a different use in biomaterial cell cultures: committed osteoblast cells could be appropriate in selecting the best surfaces to improve osseointegration, while multipotent cells could be suitable to obtain in vitro osteocyte-like network for regenerative medicine. The originality of the present work consists in studying for the first time two different cell models (committed versus multipotent) compared in parallel different biomaterial cultures, thus suggesting distinct targets for each cellular model.

Striated muscle fiber apoptosis after experimental tendon lesion in a rat model.

Tendon lesions induce muscular atrophy, the nature of which has not yet been clearly related to lesion etiology and entity. In the present study, tendon and muscle alterations were assessed after experimental tendon lesion of the Infraspinatus muscle in young rats. The consequences of lesions differed on the basis of both extension and injured tissue vascularization, that is apoptosis and/or degeneration, differing mainly by energy demands: apoptosis requires high energy levels (proportional to vascular supply), but degeneration does not. It is well known that tendons are poorly supplied with blood compared with muscular masses, which are abundantly vascularized. Five weeks after tendon surgical section, tendon/muscle samples were taken for TUNEL and transmission electron microscopy. The structural results reported here identified different tendon/muscle alterations: degeneration of tendon without signs of apoptosis, and atrophy of muscle fibers due only to apoptosis. This led to the formulation of the following hypothetical sequence of events: a tendon lesion, not recovering quickly due to the poor tendon blood supply, results in degeneration of the injured tendon, which, in turn, induces a partial disuse of the muscle mass, which consequently atrophies (proportionally to the severity of tendon lesion) by striated muscular fiber apoptosis. The authors suggest that the different behavior of the two tissues depends on the marked difference in their vascularization.

Effects of different doses of ferutinin on bone formation/resorption in ovariectomized rats.

This study analyzes the effects of different doses of ferutinin on bone loss caused by estrogen deficiency in ovariectomized rats, in comparison with estradiol benzoate. Thirty female Sprague-Dawley rats were ovariectomized and treated for 30 days from the day after ovariectomy. Static/dynamic histomorphometric analyses were performed on trabecular and cortical bone of lumbar vertebrae and femurs. Very low weight increments were recorded only in all F-OVX groups, with respect to the others. Although the great differences in weight, that could imply a decrease of bone mass in F-OVX groups compared to the control ovariectomized group (C-OVX), trabecular bone in lumbar vertebrae did not show significant differences, suggesting that ferutinin, opposing estrogen deficiency, inhibits bone resorption. Newly formed cortical bone was always low in all F-OVX groups and high in C-OVX, suggesting that it is mainly devoted in answering mechanical demands. In contrast, in distal femoral metaphyses, trabecular bone was reduced and the number of osteoclasts was increased in C-OVX with respect to all other groups, suggesting that it is mainly devoted in answering metabolic demands; moreover, ferutinin dose of 2 mg/kg seemed to be more effective than the lower doses used and estrogens, particularly in those skeletal regions with higher metabolic activity. Our results suggest that the role of ferutinin in preventing osteoporosis caused by estrogen deficiency is expressed in decreasing bone erosion; moreover, in all F-OVX groups bone turnover is very low and seems correlated to the trivial body weight increase, which, in turn, depends on ferutinin treatment.

Osteocyte apoptosis and absence of bone remodeling in human auditory ossicles and scleral ossicles of lower vertebrates: a mere coincidence or linked processes?

Considering the pivotal role as bone mechanosensors ascribed to osteocytes in bone adaptation to mechanical strains, the present study analyzed whether a correlation exists between osteocyte apoptosis and bone remodeling in peculiar bones, such as human auditory ossicles and scleral ossicles of lower vertebrates, which have been shown to undergo substantial osteocyte death and trivial or no bone turnover after cessation of growth. The investigation was performed with a morphological approach under LM (by means of an in situ end-labeling technique) and TEM. The results show that a large amount of osteocyte apoptosis takes place in both auditory and scleral ossicles after they reach their final size. Additionally, no morphological signs of bone remodeling were observed. These facts suggest that (1) bone remodeling is not necessarily triggered by osteocyte death, at least in these ossicles, and (2) bone remodeling does not need to mechanically adapt auditory and scleral ossicles since they appear to be continuously submitted to stereotyped stresses and strains; on the contrary, during the resorption phase, bone remodeling might severely impair the mechanical resistance of extremely small bony segments. Thus, osteocyte apoptosis could represent a programmed process devoted to make stable, when needed, bone structure and mechanical resistance.