Increased bone mass in a mouse model with low fat mass.

Mice with impaired acute inflammatory responses within adipose tissue display reduced diet-induced fat mass gain associated with glucose intolerance and systemic inflammation. Therefore, acute adipose tissue inflammation is needed for a healthy expansion of adipose tissue. Because inflammatory disorders are associated with bone loss, we hypothesized that impaired acute adipose tissue inflammation leading to increased systemic inflammation results in a lower bone mass. To test this hypothesis, we used mice overexpressing an adenoviral protein complex, the receptor internalization and degradation (RID) complex that inhibits proinflammatory signaling, under the control of the aP2 promotor (RID tg mice), resulting in suppressed inflammatory signaling in adipocytes. As expected, RID tg mice had lower high-fat diet-induced weight and fat mass gain and higher systemic inflammation than littermate wild-type control mice. Contrary to our hypothesis, RID tg mice had increased bone mass in long bones and vertebrae, affecting trabecular and cortical parameters, as well as improved humeral biomechanical properties. We did not find any differences in bone formation or resorption parameters as determined by histology or enzyme immunoassay. However, bone marrow adiposity, often negatively associated with bone mass, was decreased in male RID tg mice as determined by histological analysis of tibia. In conclusion, mice with reduced fat mass due to impaired adipose tissue inflammation have increased bone mass.

Correction to: Compressive loading of the murine tibia reveals site-specific micro-scale differences in adaptation and maturation rates of bone.

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Compressive loading of the murine tibia reveals site-specific micro-scale differences in adaptation and maturation rates of bone.

Loading increases bone mass and strength in a site-specific manner; however, possible effects of loading on bone matrix composition have not been evaluated. Site-specific structural and material properties of mouse bone were analyzed on the macro- and micro/molecular scale in the presence and absence of axial loading. The response of bone to load is heterogeneous, adapting at molecular, micro-, and macro-levels. INTRODUCTION: Osteoporosis is a degenerative disease resulting in reduced bone mineral density, structure, and strength. The overall aim was to explore the hypothesis that changes in loading environment result in site-specific adaptations at molecular/micro- and macro-scale in mouse bone. METHODS: Right tibiae of adult mice were subjected to well-defined cyclic axial loading for 2 weeks; left tibiae were used as physiologically loaded controls. The bones were analyzed with µCT (structure), reference point indentation (material properties), Raman spectroscopy (chemical), and small-angle X-ray scattering (mineral crystallization and structure). RESULTS: The cranial and caudal sites of tibiae are structurally and biochemically different within control bones. In response to loading, cranial and caudal sites increase in cortical thickness with reduced mineralization (-14 and -3%, p < 0.01, respectively) and crystallinity (-1.4 and -0.3%, p < 0.05, respectively). Along the length of the loaded bones, collagen content becomes more heterogeneous on the caudal site and the mineral/collagen increases distally at both sites. CONCLUSION: Bone structure and composition are heterogeneous, finely tuned, adaptive, and site-specifically responsive at the micro-scale to maintain optimal function. Manipulation of this heterogeneity may affect bone strength, relative to specific applied loads.

Effects of developmental exposure to perfluorooctanoic acid (PFOA) on long bone morphology and bone cell differentiation.

Perfluorooctanoic acid (PFOA) is a ubiquitous and persistent environmental chemical, which has been used extensively due to its stability and surface tension-lowering properties. Toxicological effects include induction of neonatal mortality and reproductive toxicity. In this study, pregnant C57BL/6 mice were exposed orally to 0.3mg PFOA/kg/day throughout pregnancy, and female offspring were studied at the age of 13 or 17months. Morphometrical and biomechanical properties of femurs and tibias were analyzed with micro-computed tomography and 3-point bending, and bone PFOA concentrations were determined by mass spectrometry. The effects of PFOA on bone cell differentiation were studied in osteoclasts from C57BL/6 mice and in the MC3T3 pre-osteoblast cell line. PFOA exposed mice showed increased femoral periosteal area as well as decreased mineral density of tibias. Biomechanical properties of these bones were not affected. Bone PFOA concentrations were clearly elevated even at the age of 17months. In osteoblasts, low concentrations of PFOA increased osteocalcin (OCN) expression and calcium secretion, but at PFOA concentrations of 100µM and above osteocalcin (OCN) expression and calcium secretion were decreased. The number of osteoclasts was increased at all PFOA concentrations tested and resorption activity dose-dependently increased from 0.1-1.0µM, but decreased at higher concentrations. The results show that PFOA accumulates in bone and is present in bones until the old age. PFOA has the potential to influence bone turnover over a long period of time. Therefore bone is a target tissue for PFOA, and altered bone geometry and mineral density seem to persist throughout the life of the animal.

Multiple miliary osteoma cutis is a distinct disease entity: four case reports and review of the literature.

BACKGROUND: Multiple miliary osteoma cutis (MMOC) is a rare nodular skin disease characterized by tiny bone nodules which usually form on the facial skin, typically in middle age. The aetiology of this phenomenon is poorly understood. OBJECTIVES: To search for possible bone formation progenitors and to look for a possible association with mutations in the GNAS gene (encoding the G-protein α-stimulatory subunit) and related hormonal parameters in patients with MMOC. We also reviewed the literature and discuss the aetiology and pathogenesis of adult-onset primary osteomas. METHODS: We report four cases of MMOC. Histological samples were analysed for bone morphogenetic protein (BMP)-2, BMP-4 and oestrogen receptor-α known to be involved in bone formation. Endocrinological laboratory investigations and hand X-rays were performed to exclude a systemic disease. The GNAS gene was sequenced from DNA extracted from peripheral blood in all four patients and from a skin sample in one patient to exclude somatic mutations. RESULTS: Histological analyses revealed intramembranous cutaneous bone formation resembling the findings seen in GNAS gene-based osteoma cutis disorders. However, we did not find any germline or somatic GNAS gene mutations in our patients and all laboratory investigations gave normal results. BMP-2 and -4 were expressed normally in MMOC samples, but oestrogen receptor-α was not expressed. Altogether 47 MMOC cases, 41 female and six male, have been published between 1928 and 2009. Of these cases, 55% had a history of pre-existing acne and only 15% had extrafacial osteomas. CONCLUSIONS: MMOC is a rare but distinct disease entity of unknown aetiology. Histologically, the tiny nodular osteomas show intramembranous superficial ossification but the aetiology appears to be different from GNAS-related disorders. The osteomas seem to increase slowly in number after appearing in middle age.

Evidence for the presence of a proton pump of the vacuolar H(+)-ATPase type in the ruffled borders of osteoclasts.

Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H(+)-transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60- and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclasts in chicken and rat bone in immunohistochemistry. Immunoelectronmicroscopy localized these antigens in apical membranes of rat osteoclasts and kidney intercalated cells of inner stripe of outer medulla. Pretreatment of animals with parathyroid hormone enhanced the immunoreaction in the apical membranes of osteoclasts. No immunoreaction was seen in osteoclasts when antibodies against gastric H+,K(+)-ATPase were used. These results suggest that osteoclast resorbs bone by secreting protons through vacuolar H(+)-ATPase.

Influence of intensity and changes of physical activity on bone mineral density of immature equine subchondral bone.

REASONS FOR PERFORMING STUDY: Subchondral bone provides structural support to overlying articular cartilage and plays an important biomechanical role in osteochondral diseases. Mechanical features of bone correlate strongly with bone mineral density, which is directed by the loading conditions to which the tissue is subjected. OBJECTIVE: To investigate the influence of physical activity levels on subchondral bone mineral density (sBMD) in foals during early development. METHODS: Three groups of foals were subjected to different physical activity levels from birth until age 5 months. A proportion of these foals were subjected to euthanasia at 5 months while remaining foals were subjected to similar physical activity levels for 6 months until euthanasia at 11 months. Osteochondral specimens were collected for measurement of sBMD with peripheral quantitative computed tomography at 2 differently loaded anatomical sites of the proximal phalangeal bone at 1, 2, 3, 4 and 5 mm depth from the osteochondral junction. RESULTS: Growth significantly increased sBMD but by a different amount depending on anatomical location and physical activity level. Significantly higher sBMD was found at the habitually loaded central area in comparison to the intermittently peak loaded marginal site. Exercise increased sBMD throughout the whole depth of analysed tissue, but changes were generally more obvious at a depth of 2 mm. Interestingly, foals subjected to additional sprint training preserved the exercise-induced sBMD increase at the habitually loaded central area during the 6 months of the second phase of the study. CONCLUSIONS: Habitual low-intensity loading elicits a greater response in sBMD in quantitative terms than high-intensity low-frequency loading at the sites investigated in this study. Future sBMD may be influenced by means of well-tailored exercise regimens at young age. POTENTIAL RELEVANCE: Specific physical activity levels during early development may potentially reduce the prevalence of osteochondral injury later in life.

Differentiation of osteoblasts on pectin-coated titanium.

The gold standard for implant metals is titanium, and coatings such as collagen-I, RGD-peptide, chondroitin sulfate, and calcium phosphate have been used to modify its biocompatibility. We investigated how titanium coated with pectins, adaptable bioactive plant polysaccharides with anti-inflammatory effects, supports osteoblast differentiation. MC3T3-E1 cells, primary murine osteoblasts, and human mesenchymal cells (hMC) were cultured on titanium coated with rhamnogalacturonan-rich modified hairy regions (MHR-A and MHR-B) of apple pectin. Alkaline phosphatase (ALP) expression and activity, calcium deposition, and cell spreading were investigated. MHR-B, but not MHR-A, supports osteoblast differentiation. The MHR-A surface was not mineralized, but on MHR-B, the average mineralized area was 14.0% with MC3T3-E1 cells and 26.6% with primary osteoblasts. The ALP activity of hMCs on MHR-A was 58.3% at day 7 and 9.3% from that of MHR-B at day 10. These data indicate that modified pectin nanocoatings may enhance the biocompatibility of bone and dental implants.

Prednisolone treatment reduces the osteogenic effects of loading in mice.

Glucocorticoid treatment, a major cause of drug-induced osteoporosis and fractures, is widely used to treat inflammatory conditions and diseases. By contrast, mechanical loading increases bone mass and decreases fracture risk. With these relationships in mind, we investigated whether mechanical loading interacts with GC treatment in bone. Three-month-old female C57BL/6 mice were treated with high-dose prednisolone (15 mg/60 day pellets/mouse) or vehicle for two weeks. During the treatment, right tibiae were subjected to short periods of cyclic compressive loading three times weekly, while left tibiae were used as physiologically loaded controls. The bones were analyzed using peripheral quantitative computed tomography, histomorphometry, real-time PCR, three-point bending and Fourier transform infrared micro-spectroscopy. Loading alone increased trabecular volumetric bone mineral density (vBMD), cortical thickness, cortical area, osteoblast-associated gene expression, osteocyte- and osteoclast number, and bone strength. Prednisolone alone decreased cortical area and thickness and osteoblast-associated gene expression. Importantly, prednisolone treatment decreased the load-induced increase in trabecular vBMD by 57% (p < 0.001) and expression of osteoblast-associated genes, while completely abolishing the load-induced increase in cortical area, cortical thickness, number of osteocytes and osteoclasts, and bone strength. When combined, loading and prednisolone decreased the collagen content. In conclusion, high-dose prednisolone treatment strongly inhibits the loading-induced increase in trabecular BMD, and abolishes the loading-induced increase in cortical bone mass. This phenomenon could be due to prednisolone inhibition of osteoblast differentiation and function.

Membrane estrogen receptor α is essential for estrogen signaling in the male skeleton.

The importance of estrogen receptor α (ERα) for the regulation of bone mass in males is well established. ERα mediates estrogenic effects both via nuclear and membrane-initiated ERα (mERα) signaling. The role of mERα signaling for the effects of estrogen on bone in male mice is unknown. To investigate the role of mERα signaling, we have used mice (Nuclear-Only-ER; NOER) with a point mutation (C451A), which results in inhibited trafficking of ERα to the plasma membrane. Gonadal-intact male NOER mice had a significantly decreased total body areal bone mineral density (aBMD) compared to WT littermates at 3, 6 and 9 months of age as measured by dual-energy X-ray absorptiometry (DEXA). High-resolution microcomputed tomography (µCT) analysis of tibia in 3-month-old males demonstrated a decrease in cortical and trabecular thickness in NOER mice compared to WT littermates. As expected, estradiol (E2) treatment of orchidectomized (ORX) WT mice increased total body aBMD, trabecular BV/TV and cortical thickness in tibia compared to placebo treatment. E2 treatment increased these skeletal parameters also in ORX NOER mice. However, the estrogenic responses were significantly decreased in ORX NOER mice compared with ORX WT mice. In conclusion, mERα is essential for normal estrogen signaling in both trabecular and cortical bone in male mice. Increased knowledge of estrogen signaling mechanisms in the regulation of the male skeleton may aid in the development of new treatment options for male osteoporosis.

Perfluoroalkyl substances in human bone: concentrations in bones and effects on bone cell differentiation.

Perfluoroalkyl substances (PFAS), including two most commonly studied compounds perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), are widely distributed environmental pollutants, used extensively earlier. Due to their toxicological effects the use of PFAS is now regulated. Based on earlier studies on PFOA's distribution in bone and bone marrow in mice, we investigated PFAS levels and their possible link to bone microarchitecture of human femoral bone samples (n = 18). Soft tissue and bone biopsies were also taken from a 49-year old female cadaver for PFAS analyses. We also studied how PFOA exposure affects differentiation of human osteoblasts and osteoclasts. PFAS were detectable from all dry bone and bone marrow samples, PFOS and PFOA being the most prominent. In cadaver biopsies, lungs and liver contained the highest concentrations of PFAS, whereas PFAS were absent in bone marrow. Perfluorononanoic acid (PFNA) was present in the bones, PFOA and PFOS were absent. In vitro results showed no disturbance in osteogenic differentiation after PFOA exposure, but in osteoclasts, lower concentrations led to increased resorption, which eventually dropped to zero after increase in PFOA concentration. In conclusion, PFAS are present in bone and have the potential to affect human bone cells partly at environmentally relevant concentrations.

The role of membrane ERα signaling in bone and other major estrogen responsive tissues.

Estrogen receptor α (ERα) signaling leads to cellular responses in several tissues and in addition to nuclear ERα-mediated effects, membrane ERα (mERα) signaling may be of importance. To elucidate the significance, in vivo, of mERα signaling in multiple estrogen-responsive tissues, we have used female mice lacking the ability to localize ERα to the membrane due to a point mutation in the palmitoylation site (C451A), so called Nuclear-Only-ER (NOER) mice. Interestingly, the role of mERα signaling for the estrogen response was highly tissue-dependent, with trabecular bone in the axial skeleton being strongly dependent (>80% reduction in estrogen response in NOER mice), cortical and trabecular bone in long bones, as well as uterus and thymus being partly dependent (40-70% reduction in estrogen response in NOER mice) and effects on liver weight and total body fat mass being essentially independent of mERα (<35% reduction in estrogen response in NOER mice). In conclusion, mERα signaling is important for the estrogenic response in female mice in a tissue-dependent manner. Increased knowledge regarding membrane initiated ERα actions may provide means to develop new selective estrogen receptor modulators with improved profiles.

Female Mice Lacking Estrogen Receptor-α in Hypothalamic Proopiomelanocortin (POMC) Neurons Display Enhanced Estrogenic Response on Cortical Bone Mass.

Estrogens are important regulators of bone mass and their effects are mainly mediated via estrogen receptor (ER)α. Central ERα exerts an inhibitory role on bone mass. ERα is highly expressed in the arcuate (ARC) and the ventromedial (VMN) nuclei in the hypothalamus. To test whether ERα in proopiomelanocortin (POMC) neurons, located in ARC, is involved in the regulation of bone mass, we used mice lacking ERα expression specifically in POMC neurons (POMC-ERα(-/-)). Female POMC-ERα(-/-) and control mice were ovariectomized (OVX) and treated with vehicle or estradiol (0.5 µg/d) for 6 weeks. As expected, estradiol treatment increased the cortical bone thickness in femur, the cortical bone mechanical strength in tibia and the trabecular bone volume fraction in both femur and vertebrae in OVX control mice. Importantly, the estrogenic responses were substantially increased in OVX POMC-ERα(-/-) mice compared with the estrogenic responses in OVX control mice for cortical bone thickness (+126 ± 34%, P < .01) and mechanical strength (+193 ± 38%, P < .01). To test whether ERα in VMN is involved in the regulation of bone mass, ERα was silenced using an adeno-associated viral vector. Silencing of ERα in hypothalamic VMN resulted in unchanged bone mass. In conclusion, mice lacking ERα in POMC neurons display enhanced estrogenic response on cortical bone mass and mechanical strength. We propose that the balance between inhibitory effects of central ERα activity in hypothalamic POMC neurons in ARC and stimulatory peripheral ERα-mediated effects in bone determines cortical bone mass in female mice.

The phase state of NiTi implant material affects osteoclastic attachment.

In the present work, the responses of mature osteoclasts cultured on austenite and martensite phases of NiTi shape memory implant material were studied. We used the sensitivity of osteoclasts to the underlying substrate and actin ring formation as an indicator of the adequacy of the implant surface. The results showed osteoclasts with actin ring on both NiTi phases. However, significantly more osteoclasts were present on the austenitic NiTi than on the martensitic NiTi. We also analyzed the surface free energy of the samples but found no significant difference between austenite and martensite phases. The results revealed that osteoclasts tolerated well the austenite phase of NiTi. The chemically identical martensitic NiTi was not as well tolerated by osteoclasts (e.g., indicated by diminished actin ring formation). This leads to the conclusion that certain physical properties specific to the martensitic NiTi have an adverse effect to the surviving of osteoclasts on this NiTi phase. These results confirm that mature, authentic osteoclasts can act as cell probes in experiments concerning aspects of biocompatibility of bone implant materials.

Bone-resorbing osteoclasts contain gap-junctional connexin-43.

Intercellular gap junctions have been previously described at contact sites between surface osteoblasts, between osteoblasts and underlying osteocytes, and between osteocyte cell processes in the canaliculi. The subunits of gap junction channels are assembled from a family of proteins called connexins. In the present work, we show that rat osteoclasts cultured on bovine bone slices show connexin-43 (Cx43) staining localizing in the plasma membrane of the cells in cell-cell contacts and over the basolateral membrane of osteoclasts. The effect of heptanol, a known gap-junctional inhibitor, was studied using the well-characterized pit formation assay. Heptanol decreased the number and activity of osteoclasts. The proportion of mononuclear tartrate-resistant acid phosphatase (TRAP)-positive cells out of all TRAP-positive cells increased on heptanol treatment, suggesting a defect in the fusion of mononuclear osteoclast precursors to multinucleated mature osteoclasts. Furthermore, the total resorbed area and the number of resorption pits also decreased in the heptanol-treated cultures. These results suggest that gap-junctional Cx43 plays a functional role in osteoclasts and that the blocking of gap junctions decreases both the number and the activity of osteoclasts. This can indicate both a direct communication between multinucleated osteoclasts and mononuclear cells through gap junctions or an indirect effect through gap junctions between osteoblasts.

Exercise can provide protection against bone loss and prevent the decrease in mechanical strength of femoral neck in ovariectomized rats.

The effect of treadmill exercise on bone loss in ovariectomized (OVX) rats was studied in two different sets of experiments. In the first experiment rats were either ovariectomized (n = 38) or sham operated (n = 18) at the age of 12 weeks. Half the OVX rats were trained twice a day for 30 minutes by running at 10 m/minute for 7 or 17 weeks. In the second experiment 40 female rats, aged 12 weeks, were divided into five groups (n = 8). One group of rats was sacrificed on day 0 for the baseline data. Other rats were sham operated or ovariectomized for 9 weeks. Half of both groups were trained using the same training program as in the first experiment. OVX reduced trabecular bone volume (TBV) in the distal femur to 42.7 and 48.3% in 8 and 18 weeks, respectively. Exercise opposed this effect significantly but could not prevent it totally. Exercise did not have any significant effect on sham-operated animals. OVX induced a 17.7 and 30.7% decrease in maximal failure load of femoral neck in 8 and 18 weeks, respectively. A corresponding decrease was also observed in the torque capacity of tibia. Exercise was able to prevent almost totally the decrease in bone strength of femoral neck, tibia, and humerus. In conclusion, our results suggest that the measurement of bone strength in aging female rat femoral neck can be used as a useful indicator of the deleterious effect of OVX in bone. These results further indicate that exercise can overcome a significant part of the decrease in trabecular bone volume and maintain the mechanical strength of femoral neck and tibial shaft in the OVX rats.

Polarity of osteoblasts and osteoblast-like UMR-108 cells.

Enveloped viruses, such as vesicular stomatitis virus (VSV) and Influenza virus, have been widely used in studying epithelial cell polarity. Viral particles of VSV-infected epithelial cells bud from the basolateral membrane, which is in contact with the internal milieu and the blood supply. Influenza-infected cells bud viral particles from the apical surface facing the external milieu. This feature can be utilized in labeling polarized membrane domains. We studied the polarity of mesenchymal osteoblasts using osteosarcoma cell line UMR-108 and endosteal osteoblasts in situ in bone tissue cultures. Immunofluorescence confocal microscopy revealed that the VSV glycoprotein (VSV G) was targeted to the culture medium-facing surface. In endosteal osteoblasts, VSV G protein was found in the surface facing bone marrow and circulation. On the contrary, Influenza virus hemagglutinin (HA) was localized to the bone substrate-facing surface of the UMR-108 cells. Electron microscopy showed that in the cases where the cells were growing as a single layer, VSV particles were budding from the culture medium-facing surface, whereas Influenza viruses budded from the bone substrate-facing surface. When the cells overlapped, this polarity was lost. Cell surface biotinylation revealed that 55% of VSV G protein was biotinylated, whereas Influenza virus HA was only 22% biotinylated. These findings suggest that osteoblasts are polarized at some point of their life cycle. The bone-attaching plasma membrane of osteoblasts is apical, and the circulation or bone marrow-facing plasma membrane is basolateral in nature.

Femoral neck is a sensitive indicator of bone loss in immobilized hind limb of mouse.

The present study was carried out to evaluate a unilateral hind limb immobilization model in the mouse. The right legs of male mice (age 10-12 weeks) were immobilized for 3 weeks against the abdomen by an elastic bandage. Body weight decreased significantly during the immobilization. Peripheral quantitative computed tomography (pQCT) analysis showed that the cross-sectional cortical area (CSA), the bone mineral content (BMC), and the bone mineral density (BMD) of the tibial diaphysis were lower in both legs of the immobilized animals than in age-matched controls, but the difference was mainly due to weight reduction. At the tibial metaphysis, CSA, BMC, and BMD were reduced in both legs of the immobilized animals, even after weight adjustment. At the femoral neck, CSA, BMC, and BMD were significantly lower in both legs of the immobilized animals, and the difference between the hind legs of the immobilized animals was also highly significant. The findings of the pQCT study were in good agreement with the changes in mechanical strength. The tibia was a more sensitive indicator of diaphyseal bone weakening than the femur when measuring the bending breaking force of the diaphysis. The femoral neck showed significantly decreased strength, and the difference between the immobilized leg and the contralateral leg was most clearly seen in lateral loading. We conclude that 3 weeks of hind limb immobilization weakened the tibia and femur significantly compared with their contralateral counterparts. The reduction was more significantly seen in the mechanical bending strength than in the pQCT evaluation, and the femoral neck was the most sensitive indicator of bone weakening.

Alteration in the mechanical competence and structural properties in the femoral neck and vertebrae of ovariectomized rats.

The structural and mechanical properties of bone in the femoral neck and various other sites were investigated in intact (INT), sham-operated (Sham), and ovariectomized (OVX) rats. Six weeks after operation, the maximal load and energy absorption of the femoral neck were significantly lower in the OVX than in the INT or Sham groups, being 73.2 +/- 1.4 (SE) N, 86.3 +/- 4.1 N, and 87.1 +/- 3.2 N, respectively (p < 0.01) for load. The total cross-sectional area of the femoral neck did not change after OVX, but the marrow cavity area was enlarged, leading to a reduced bone area (including both cortical and trabecular bone) (p < 0.01). Histomorphometric analysis showed that new bone formation could not be detected at the periosteum of the femoral neck below the femoral head, but at the endocortical surfaces the double tetracycline labeling revealed an increased mineral apposition rate (MAR) and bone formation rate (BFR) in OVX animals (p < 0.001). In contrast, MAR and BFR were significantly increased in both periosteal and endocortical surfaces of the tibia, humerus, and femoral shaft, thus preventing a decrease in cortical bone area. The maximal bending loads of the tibia and humerus were not different in the various groups of animals. The correlation coefficient between maximal load and bone area revealed positive relationships in the femoral neck (r = 0.54, p < 0.01), tibia (r = 0.46, p < 0.01), and humerus (r = 0.51, p < 0.01). Ovariectomy resulted in a decreased trabecular bone volume of lumbar vertebra VI (L6) decreased compressive loads of lumbar vertebrae I, III, and IV. These lumbar bone loads were positively related to their L6 bone area (L4/L6: r = 0.66, p < 0.001). Element analyses (energy dispersion spectrometer) from trabecular and cortical areas of bone showed some changes related to aging but not to OVX. These results indicate that ovariectomy influences the biomechanical properties of rat bone by changing structural properties rather than material ones.

Organization of osteoclast microfilaments during the attachment to bone surface in vitro.

Rat and chicken osteoclasts were cultured on bone slices, where they showed active resorption with resorption lacunae, which cold be seen by toluidine blue staining or with a scanning electron microscope. Osteoclast microfilaments, F-actin, vinculin, and talin were studied by immunofluorescence. In attached osteoclasts, vinculin appeared as a double circle in the periphery of the cell, and the most intense F-actin staining was located between these vinculin zones. Some chicken osteoclasts showed also intense F-actin staining throughout the center of the cell. Talin appeared in a similar double circle to vinculin. This kind of distribution of microfilaments was always associated with resorption lacunae, and F-actin, vinculin, and talin zones correspond roughly to the edge of lacunae. Osteoclasts showing a diffuse staining pattern were not associated with a resorption pit. The results suggest that this specific microfilament arrangement is located at the attachment zone of the osteoclast and is obligatory for the attachment and resorption. However, this arrangement of microfilaments is quite different from the one that has been previously described in osteoclasts cultured on glass.