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.

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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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.

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.

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.

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.

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.

Polarity of mature human odontoblasts.

Odontoblast polarization is based on histological appearance as columnar cells with asymmetric disposition of organelles and plasma membrane domains. However, little is known about the odontoblast plasma membrane organization. We investigated odontoblast membrane polarity using influenza virus hemagglutinin and vesicular stomatitis virus glycoprotein as model proteins in mature human odontoblast organ culture. We also examined the distribution patterns of aquaporin 4 and 5, which are basolateral and apical proteins in epithelial cells, respectively. Confocal microscopy immunofluorescence and electron microscopy demonstrated that the apical markers located at the surface toward pulp and basolateral markers located at the plasma membrane of odontoblast processes. Therefore, odontoblast plasma membrane polarity was different from that in epithelial cells. Also, certain lectins stained odontoblast processes while others stained the soma, reflecting the different natures of their membrane domains. Strong ZO-1 and weaker claudin expression suggest weak tight junctions in the odontoblasts. TGF-ß1 showed a tendency to reinstate the expression of selected TJ genes, indicating that TGF-ß1 may control odontoblast cell layer integrity by controlling tight junction protein expression.

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.

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.

Biocompatibility of sol-gel-derived titania-silica coated intramedullary NiTi nails.

We investigated bone response to sol-gel-derived titania-silica coated functional intramedullary NiTi nails that applied a continuous bending force. Nails 26 mm in length, either straight or with a radius of curvature of 28 or 15 mm, were implanted in the cooled martensite form from a proximal to distal direction into the medullary cavity of the right femur in 40 Sprague-Dawley rats. Body temperature restored the austenite form, causing the curved implants to generate a bending force on the bone. The femurs were examined after 24 weeks. Bone length measurements did not reveal any bowing or shortening of the bone in the experimental groups. The results from histomorphometry demonstrated that the stronger bending force, together with sol-gel surface treatment, resulted in more bone deposition around the implant and the formation of significantly less fibrous tissue. Straight intramedullary nails, even those with a titania-silica coating, were poorly attached when compared to the implants with a curved austenite structure.

Fibronectin modulates osteoblast behavior on Nitinol.

We have previously demonstrated that primary rat osteoclasts behave differently when cultured on austenite and martensite Nitinol. In this study, we coated the two phases of Nitinol with plasma fibronectin and studied if this modifies the proliferation and cell cycle of MC3T3-E1 osteoblasts. The influence of the crystalline structure of Nitinol on the remodeling and conformation of fibronectin was also studied. The results on austenite demonstrated that fibronectin was more strongly remodeled and the cells spread better compared with the martensite phase. Interestingly, the conformation of the protein showed no differences between austenite and martensite. In addition, fibronectin improved cell proliferation in both phases, but the effect of fibronectin coating was stronger on the austenite surface. In addition, in both Nitinol phases, the proportion of cells in the G(1) phase was observed to grow in the presence of fibronectin. This could indicate cell differentiation on Nitinol.

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.

Endostatin inhibits endochondral ossification.

BACKGROUND: Angiogenesis is essential for the replacement of cartilage by bone during skeletal growth and regeneration. Vascular endothelial growth factor-A (VEGF-A) is a key regulator of angiogenesis whereas endostatin, a potent inhibitor of endothelial cell proliferation and migration, is a natural antagonist of VEGF-A. The regulatory role of these peptides in angiogenesis and bone formation was investigated using adenoviral gene delivery of VEGF-A and endostatin in a mouse ectopic ossification model. METHODS: Bone formation was induced in the hamstring muscles of adult mice with native bone morphogenetic protein (BMP) extract implemented in gelatine gel together with VEGF-A and endostatin recombinant adenoviral vectors. The mice were sacrificed 1, 2, and 3 weeks after the operation and ectopic bone formation was followed radiographically and histologically. RESULTS: Significant bone formation was induced by BMP extract in all treatment groups. VEGF-A stimulated and endostatin prevented the formation of FVIII-related antigen-positive vessels as well as the number of cartilage-resorbing chondroclasts/osteoclasts. Endostatin alone or in conjugation with VEGF-A reduced bone formation. Excess of VEGF-A stimulated and endostatin reduced bone formation, respectively, at the 3-week time point. CONCLUSIONS: Our findings indicate that endostatin retards the cartilage phase in endochondral ossification which subsequently reduces bone formation in our experimental model. We conclude that bone growth and healing, which share features with ectopic bone formation, may be regulated by endostatin.

The effect of oxide thickness on osteoblast attachment and survival on NiTi alloy.

NiTi alloy is used in various medical applications and the surface titanium oxide layer produced naturally or enhanced artificially has been thought to offer a protecting film against allergic and toxic effects of nickel (Ni). In this study, we investigated the effect of different oxide layer thicknesses on the survival and attachment of osteoblastic cells (ROS-17/2.8). AFM, X-ray diffraction and electrical resistance measurements were used to analyze the surface properties of oxidized NiTi samples and the effect of oxidation on material properties. The results clearly showed that straight correlation between oxide thickness and cellular well-being cannot be maid. However, the different thicknesses of oxide layer on NiTi had surprising impacts on cellular responses and also to the properties of the metal alloy.

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.

Keratinocytes cultured from patients with Hailey-Hailey disease and Darier disease display distinct patterns of calcium regulation.

BACKGROUND: Hailey-Hailey disease (HHD) (OMIM 16960) and Darier disease (DD) (OMIM 124200) are dominantly inherited acantholytic skin diseases, respectively, caused by mutations in the genes encoding the Golgi secretory pathway Ca2+-ATPase (SPCA1, ATP2C1) and the sarco/endoplasmic reticulum Ca2+-ATPase type 2 (SERCA2, ATP2A2) genes. OBJECTIVES: To investigate calcium regulation in keratinocytes cultured from patients with HHD and DD by measuring intracellular calcium resting levels and the cellular responses to ATP and thapsigargin. METHODS: The study was carried out using keratinocyte cultures established from four patients with HHD and four with DD. Calcium concentrations were measured with fluorescence ratio imaging using fura-2 loading. RESULTS: Control and HHD keratinocytes displayed approximately the same Ca2+ levels in resting phase, while DD keratinocytes showed elevated Ca2+ levels. Application of ATP caused less pronounced elevation of intracellular calcium concentration ([Ca2+]i) in both HHD and DD keratinocytes than in control cells. HHD keratinocytes did not lower their [Ca2+]i as efficiently as control keratinocytes after treatment with thapsigargin. In addition, DD keratinocytes were practically incapable of lowering their [Ca2+]i after treatment with thapsigargin. CONCLUSIONS: The results demonstrate that the defects in SPCA1 and SERCA2 calcium ATPases result in distinct patterns of calcium metabolism. This is also supported by the different clinical features of the diseases.