Osteoclast Response to Low Extracellular Sodium and the Mechanism of Hyponatremia-induced Bone Loss

Authors

Julia Barsony, Yoshihisa Sugimura, and Joseph G. Verbalis

Abstract

Our recent animal and human studies revealed that chronic hyponatremia is a previously unrecognized cause of osteoporosis that is associated with increased osteoclast numbers in a rat model of the human disease of the syndrome of inappropriate antidiuretic hormone secretion (SIADH). We used cellular and molecular approaches to demonstrate that sustained low extracellular sodium ion concentrations ([Na+]) directly stimulate osteoclastogenesis and resorptive activity and to explore the mechanisms underlying this effect. Assays on murine preosteoclastic RAW 264.7 cells and on primary bone marrow monocytes both indicated that lowering the medium [Na+] dose-dependently increased osteoclast formation and resorptive activity. Low [Na+], rather than low osmolality, triggered these effects. Chronic reduction of [Na+] dose-dependently decreased intracellular calcium without depleting endoplasmic reticulum calcium stores. Moreover, we found that reduction of [Na+] dose-dependently decreased cellular uptake of radiolabeled ascorbic acid, and reduction of ascorbic acid in the culture medium mimicked the osteoclastogenic effect of low [Na+]. We also detected downstream effects of reduced ascorbic acid uptake, namely evidence of hyponatremia-induced oxidative stress. This was manifested by increased intracellular free oxygen radical accumulation and proportional changes in protein expression and phosphorylation, as indicated by Western blot analysis from cellular extracts and by increased serum 8-hydroxy-2′-deoxyguanosine levels in vivo in rats. Our results therefore reveal novel sodium signaling mechanisms in osteoclasts that may serve to mobilize sodium from bone stores during prolonged hyponatremia, thereby leading to a resorptive osteoporosis in patients with SIADH.

Link to Article

http://dx.doi.org/10.1074/jbc.M110.155002

Contrast-enhanced micro-computed tomography of fatigue microdamage accumulation in human cortical bone

Authors

Matthew D. Landrigan, Jiliang Li, Travis L. Turnbull, David B. Burr, Glen L. Niebur, Ryan K. Roeder

Abstract

Conventional methods used to image and quantify microdamage accumulation in bone are limited to histological sections, which are inherently invasive, destructive, two-dimensional, and tedious. These limitations inhibit investigation of microdamage accumulation with respect to volumetric spatial variation in mechanical loading, bone mineral density, and microarchitecture. Therefore, the objective of this study was to investigate non-destructive, three-dimensional (3-D) detection of microdamage accumulation in human cortical bone using contrast-enhanced micro-computed tomography (micro-CT), and to validate micro-CT measurements against conventional histological methods. Unloaded controls and specimens loaded in cyclic uniaxial tension to a 5% and 10% reduction in secant modulus were labeled with a precipitated BaSO4 stain for micro-CT and basic fuchsin for histomorphometry. Linear microcracks were similarly labeled by BaSO4 and basic fuchsin as shown by backscattered electron microscopy and light microscopy, respectively. The higher X-ray attenuation of BaSO4 relative to the bone extracellular matrix provided enhanced contrast for the detection of damage that was otherwise not able to be detected by micro-CT prior to staining. Therefore, contrast-enhanced micro-CT was able to nondestructively detect the presence, 3-D spatial location, and accumulation of fatigue microdamage in human cortical bone specimens in vitro. Microdamage accumulation was quantified on segmented micro-CT reconstructions as the ratio of BaSO4 stain volume (SV) to total bone volume (BV). The amount of microdamage measured by both micro-CT (SV/BV) and histomorphometry (Cr.N, Cr.Dn, Cr.S.Dn) progressively increased from unloaded controls to specimens loaded to a 5% and 10% reduction in secant modulus (p<0.001). Group means for micro-CT measurements of damage accumulation were strongly correlated to those using histomorphometry (p<0.05), validating the new methods. Limitations of the new methods in the present study included that the precipitated BaSO4 stain was non-specific and non-biocompatible, and that micro-CT measurements exhibited greater variability compared to conventional histology. Nonetheless, contrast-enhanced micro-CT enabled non-destructive imaging and 3-D spatial information, which are not possible using conventional histological methods.

Link to Article

http://dx.doi.org/10.1016/j.bone.2010.10.160

Temporal and spatial expression of osteoactivin during fracture repair

Authors

Samir M. Abdelmagid, Mary F. Barbe, Michael Hadjiargyrou, Thomas A. Owen, Roshanak Razmpour, Saqib Rehman, Steven N. Popoff, Fayez F. Safadi

Abstract

We previously identified osteoactivin (OA) as a novel secreted osteogenic factor with high expression in developing long bones and calvaria, and that stimulates osteoblast differentiation and matrix mineralization in vitro. In this study, we report on OA mRNA and protein expression in intact long bone and growth plate, and in fracture calluses collected at several time points up to 21 days post-fracture (PF). OA mRNA and protein were highly expressed in osteoblasts localized in the metaphysis of intact tibia, and in hypertrophic chondrocytes localized in growth plate, findings assessed by in situ hybridization and immunohistochemistry, respectively. Using a rat fracture model, Northern blot analysis showed that expression of OA mRNA was significantly higher in day-3 and day-10 PF calluses than in intact rat femurs. Using in situ hybridization, we examined OA mRNA expression during fracture healing and found that OA was temporally regulated, with positive signals seen as early as day-3 PF, reaching a maximal intensity at day-10 PF, and finally declining at day-21 PF. At day-5 PF, which correlates with chondrogenesis, OA mRNA levels were significantly higher in the soft callus than in intact femurs. Similarly, we detected high OA protein immunoexpression throughout the reparative phase of the hard callus compared to intact femurs. Interestingly, the secreted OA protein was also detected within the newly made cartilage matrix and osteoid tissue. Taken together, these results suggest the possibility that OA plays an important role in bone formation and serves as a positive regulator of fracture healing.

Link to Article

http://dx.doi.org/10.1002/jcb.22702

Dose response of bone-targeted enzyme replacement for murine hypophosphatasia

Authors

Manisha C. Yadav, Isabelle Lemire, Pierre Leonard, Guy Boileau, Laurent Blond, Martin Beliveau, Esther Cory, Robert L. Sah, Michael P. Whyte, Philippe Crine and José Luis Millán

Abstract

Hypophosphatasia (HPP) features rickets or osteomalacia from tissue-nonspecific alkaline phosphatase (TNSALP) deficiency due to deactivating mutations within the ALPL gene. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP (sALP-FcD10, renamed ENB-0040) prevents manifestations of HPP when initiated at birth in TNSALP knockout (Akp2−/−) mice. Here, we evaluated the dose–response relationship of ENB-0040 to various phenotypic traits of Akp2−/− mice receiving daily subcutaneous (SC) injections of ENB-0040 from birth at 0.5, 2.0, or 8.2 mg/kg for 43 days. Radiographs, μCT, and histomorphometric analyses documented better bone mineralization with increasing doses of ENB-0040. We found a clear, positive correlation between ENB-0040 dose and prevention of mineralization defects of the feet, rib cage, lower limbs, and jaw bones. According to a dose–response model, the ED80 (the dose that prevents bone defects in 80% of mice) was 3.2, 2.8 and 2.9 mg/kg/day for these sites, respectively. Long bones seemed to respond to lower daily doses of ENB-0040. There was also a positive relationship between ENB-0040 dose and survival. Median survival, body weight, and bone length all improved with increasing doses of ENB-0040. Urinary PPi concentrations remained elevated in all treatment groups, indicating that while this parameter is a good biochemical marker for diagnosing HPP, it may not be a good follow up marker for evaluating response to treatment when administering bone-targeted TNSALP. These dose–response relationships strongly support the pharmacological efficacy of ENB-0040 for HPP, and provide the experimental basis for the therapeutic range of ENB-0040 chosen for clinical trials. We find a positive correlation between ENB-0040 dose and prevention of mineralization defects in a murine model of hypophosphatasia. We found a positive relationship between ENB-0040 dose and survival. Urinary pyrophosphate is not a good follow up marker for evaluating response when using bone-targeted TNSALP treatment. These studies help set the therapeutic range of ENB-0040 to be chosen for clinical trials.

Link to Article

http://dx.doi.org/10.1016/j.bone.2011.03.770

Vitamin D deficiency promotes growth of MCF-7 human breast cancer in a rodent model of osteosclerotic bone metastasis

Authors

Li Laine Ooi, Yu Zheng, Hong Zhou, Trupti Trivedi, Arthur D. Conigraveb, Markus J. Seibel, Colin R. Dunstan

Abstract

Breast cancer metastases to bone are common in advanced stage disease. We have recently demonstrated that vitamin D deficiency enhances breast cancer growth in an osteolytic mouse model of breast cancer metastasis. In this study, we examined the effects of vitamin D deficiency on tumor growth in an osteosclerotic model of intra-skeletal breast cancer in mice. The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on proliferation and apoptosis of MCF-7 breast cancer cells, and changes in the expression of genes within the vitamin D metabolic pathway (VDR, 1α- and 24-hydroxylase) were examined in vitro. MCF-7 breast cancer cells were injected intra-tibially into vitamin D deficient and vitamin D sufficient mice co-treated with and without osteoprotegerin (OPG). The development of tumor-related lesions was monitored via serial X-ray analysis. Tumor burden and indices of proliferation and apoptosis were determined by histology along with markers of bone turnover and serum intact PTH levels. In vitro, MCF-7 cells expressed critical genes for vitamin D signalling and metabolism. Treatment with 1,25(OH)2D3 inhibited cell growth and proliferation, and increased apoptosis. In vivo, osteosclerotic lesions developed faster and were larger at endpoint in the tibiae of vitamin D deficient mice compared to vitamin D sufficient mice (1.49±0.08mm2 versus 1.68±0.15mm2, P<0.05). Tumor area was increased by 55.8% in vitamin D deficient mice (0.81±0.13mm2 versus 0.52±0.11mm2 in vitamin D sufficient mice). OPG treatment inhibited bone turnover and caused an increase in PTH levels, while tumor burden was reduced by 90.4% in vitamin D sufficient mice and by 92.6% in vitamin D deficient mice. Tumor mitotic activity was increased in the tibiae of vitamin D deficient mice and apoptosis was decreased, consistent with faster growth. Vitamin D deficiency enhances both the growth of tumors and the tumor-induced osteosclerotic changes in the tibiae of mice following intratibial implantation of MCF-7 cells. Enhancement of tumor growth appears dependent on increased bone resorption rather than increased bone formation induced by these tumors.

Link to Article

http://dx.doi.org/10.1016/j.bone.2010.07.012

Quantitative Histomorphometric Assessment of Regenerate Cellularity and Bone Quality in Mandibular Distraction Osteogenesis After Radiation Therapy

Authors

Alero F. Inyang, Daniel Schwarz, Ameen M. Jamali, Steven R. Buchman

Abstract

Background: The use of mandibular distraction osteogenesis (MDO) for tissue replacement after oncologic resection in head and neck cancer could have immense therapeutic ramifications. We have previously demonstrated significantly decreased mechanical and microdensitomeric metrics of our MDO regenerate after 36-Gy radiation. Quantitative histomorphometry, a third metric, would permit objective investigation of the effects of radiation on tissue and cellular composition. Our hypothesis is that radiation-induced cellular depletion and diminution in function impair optimal bone regeneration. Five rats received radiation to the left mandible; 5 received none. All animals underwent surgical placement of external fixators, creation of mandibular osteotomies, distraction to a 5.1-mm gap width, and consolidation. Point counting and color thresholding were performed. There was a significant increase in empty lacunae and a corresponding diminution in osteocytes after radiation. Whereas the volume fraction of mineralized, mature bone was not different, that of nonmineralized, immature osteoid was significantly increased in the radiated group compared with that in the nonradiated group. Our findings confirm our prior 2 metrics. Actually, all 3 diverse metrics-microdensitometry, biomechanical analysis, and histomorphometry-corroborate our hypothesis of cellular depletion and diminution of function as the potential mechanism of radiation-induced attenuation in the distracted regenerate. Furthermore, our findings of tissue and cellular changes in the irradiated regenerate elucidate the pathophysiology of decreased bone quality when amalgamated with our previous results. Therapeutic agents may now be introduced, and their effects on the irradiated regenerate critically measured, so that MDO may be used as a viable reconstructive option in patients with head and neck cancer.

Link to Article

http://dx.doi.org/10.1097/SCS.0b013e3181ec693f