Chlorthalidone improves vertebral bone quality in genetic hypercalciuric stone-forming rats

Authors

David A. Bushinsky, Thomas Willett, John R. Asplin, Christopher Culbertson, Sara P.Y. Che, Marc Grynpas Ph.D

Abstract

We have bred a strain of rats to maximize urine (U) calcium (Ca) excretion and model hypercalciuric nephrolithiasis. These genetic hypercalciuric stone-forming (GHS) rats excrete more UCa than control Sprague-Dawley rats, uniformly form kidney stones and, similar to patients, demonstrate lower bone mineral density. Clinically thiazide diuretics reduce UCa and prevent stone formation; however, whether they benefit bone is not clear. We used GHS rats to test the hypothesis that the thiazide diuretic chlorthalidone (CTD) would have a favorable effect on bone density and quality. Twenty GHS rats received a fixed amount of a 1.2% Ca diet and half were also fed CTD (4-5 mg/kg/day). Rats fed CTD had a marked reduction in UCa. The axial and appendicular skeletons were studied. An increase in trabecular mineralization was observed with CTD compared to controls. CTD also improved the architecture of trabecular bone. Using µCT, trabecular bone volume (BV/TV), trabecular thickness and trabecular number were increased with CTD. A significant increase in trabecular thickness with CTD was confirmed by static histomorphometry. CTD also improved the connectivity of trabecular bone. Significant improvements in vertebral strength and stiffness were measured by vertebral compression. Conversely, a slight loss of bending strength was detected in the femoral diaphysis with CTD. Thus, results obtained in hypercalciuric rats suggest that CTD can favorably influence vertebral fracture risk. CTD did not alter formation parameters suggesting that the improved vertebral bone strength was due to decreased bone resorption and retention of bone structure.

Link to Article

http://dx.doi.org/10.1002/jbmr.374

Regenerate Healing Outcomes in Unilateral Mandibular Distraction Osteogenesis Using Quantitative Histomorphometry

Authors

Daniel Schwarz, Krikor Arman, Mehreen Kakwan, Ameen Jamali, Ayman Elmeligy, Steven Buchman

Abstract

The authors' goal was to ascertain regenerate bone-healing metrics using quantitative histomorphometry at a single consolidation period. Rats underwent either mandibular distraction osteogenesis (n = 7) or partially reduced fractures (n = 7); their contralateral mandibles were used as controls (n = 11). External fixators were secured and unilateral osteotomies performed, followed by either mandibular distraction osteogenesis (4 days' latency, then 0.3 mm every 12 hours for 8 days; 5.1 mm) or partially reduced fractures (fixed immediately postoperatively; 2.1 mm); both groups underwent 4 weeks of consolidation. After tissue processing, bone volume/tissue volume ratio, osteoid volume/tissue volume ratio, and osteocyte count per high-power field were analyzed by means of quantitative histomorphometry. Contralateral mandibles had statistically greater bone volume/tissue volume ratio and osteocyte count per high-power field compared with both mandibular distraction osteogenesis and partially reduced fractures by almost 50 percent, whereas osteoid volume/tissue volume ratio was statistically greater in both mandibular distraction osteogenesis specimens and partially reduced fractures compared with contralateral mandibles. No statistical difference in bone volume/tissue volume ratio, osteoid volume/tissue volume ratio, or osteocyte count per high-power field was found between mandibular distraction osteogenesis specimens and partially reduced fractures. The authors' findings demonstrate significantly decreased bone quantity and maturity in mandibular distraction osteogenesis specimens and partially reduced fractures compared with contralateral mandibles using the clinically analogous protocols. If these results are extrapolated clinically, treatment strategies may require modification to ensure reliable, predictable, and improved outcomes.

Link to Article

http://dx.doi.org/10.1097/PRS.0b013e3181e3b351

Bone mineralization defects and vitamin D deficiency: Histomorphometric analysis of iliac crest bone biopsies and circulating 25-hydroxyvitamin D in 675 patients

Authors

Matthias Priemel, Christoph von Domarus, Till Orla Klatte, Steffen Kessler, Julia Schlie, Simon Meier, Nils Proksch, Frederic Pastor, Clemens Netter, Thomas Streichert, Klaus Püschel, Michael Amling

Abstract

Parathyroid hormone (PTH) is only one measurable index of skeletal health, and we reasoned that a histomorphometric analysis of iliac crest biopsies would be another and even more direct approach to assess bone health and address the required minimum 25-Hydroxyvitamin D [25(OH)D] level. A cohort from the northern European population with its known high prevalence of vitamin D deficiency therefore would be ideal to answer the latter question. We examined 675 iliac crest biopsies from male and female individuals, excluding all patients who showed any signs of secondary bone diseases at autopsy. Structural histomorphometric parameters, including osteoid indices, were quantified using the Osteomeasure System according to ASBMR standards, and serum 25(OH)D levels were measured for all patients. Statistical analysis was performed by Student's t test. The histologic results demonstrate an unexpected high prevalence of mineralization defects, that is, a pathologic increase in osteoid. Indeed, 36.15% of the analyzed patients presented with an osteoid surface per bone surface (OS/BS) of more than 20%. Based on the most conservative threshold that defines osteomalacia at the histomorphometric level with a pathologic increase in osteoid volume per bone volume (OV/BV) greater than 2% manifest mineralization defects were present in 25.63% of the patients. The latter were found independent of bone volume per trabecular volume (BV/TV) throughout all ages and affected both sexes equally. While we could not establish a minimum 25(OH)D level that was inevitably associated with mineralization defects, we did not find pathologic accumulation of osteoid in any patient with circulating 25(OH)D above 75 nmol/L. Our data demonstrate that pathologic mineralization defects of bone occur in patients with a serum 25(OH)D below 75 nmol/L and strongly argue that in conjunction with a sufficient calcium intake, the dose of vitamin D supplementation should ensure that circulating levels of 25(OH)D reach this minimum threshold (75 nmol/L or 30 ng/mL) to maintain skeletal health.

Link to Article

http://dx.doi.org/10.1359/jbmr.090728

Infrared spectroscopy indicates altered bone turnover and remodeling activity in renal osteodystrophy

Authors

Hanna Isaksson, Mikael J Turunen, Lassi Rieppo, Simo Saarakkala, Inari S Tamminen, Jarno Rieppo, Heikki Kröger, Jukka S Jurvelin

Abstract

Renal osteodystrophy alters metabolic activity and remodeling rate of bone and also may lead to different bone composition. The objective of this study was to characterize the composition of bone in high-turnover renal osteodystrophy patients by means of Fourier transform infrared spectroscopic imaging (FTIRI). Iliac crest biopsies from healthy bone (n = 11) and patients with renal osteodystrophy (ROD, n = 11) were used in this study. The ROD samples were from patients with hyperparathyroid disease. By using FTIRI, phosphate-to-amide I ratio (mineral-to-matrix ratio), carbonate-to-phosphate ratio, and carbonate-to-amide I ratio (turnover rate/remodeling activity), as well as the collagen cross-link ratio (collagen maturity), were quantified. Histomorphometric analyses were conducted for comparison. The ROD samples showed significantly lower carbonate-to-phosphate (p < .01) and carbonate-to-amide I (p < .001) ratios. The spatial variation across the trabeculae highlighted a significantly lower degree of mineralization (p < .05) at the edges of the trabeculae in the ROD samples than in normal bone. Statistically significant linear correlations were found between histomorphometric parameters related to bone-remodeling activity and number of bone cells and FTIRI-calculated parameters based on carbonate-to-phosphate and carbonate-to-amide I ratios. Hence the results suggested that FTIRI parameters related to carbonate may be indicative of turnover and remodeling rate of bone.

Link to Article

http://dx.doi.org/10.1002/jbmr.10

VAMP3 regulates podosome organisation in macrophages and together with Stx4/SNAP23 mediates adhesion, cell spreading and persistent migration

Authors

Kelly J. Veale, Carolin Offenhäuser, Nazi Lei, Amanda C. Stanley, Jennifer L. Stow, and Rachael Z. Murray

Abstract

The ability of cells to adhere, spread and migrate is essential to many physiological processes, particularly in the immune system where cells must traffic to sites of inflammation and injury. By altering the levels of individual components of the VAMP3/Stx4/SNAP23 complex we show here that this SNARE complex regulates efficient macrophage adhesion, spreading and migration on fibronectin. During cell spreading this complex mediates the polarised exocytosis of VAMP3-positive recycling endosome membrane into areas of membrane expansion, where VAMP3's surface partner Q-SNARE complex Stx4/SNAP23 was found to accumulate. Lowering the levels of VAMP3 in spreading cells resulted in a more rounded cell morphology and most cells were found to be devoid of the typical ring-like podosome superstructures seen normally in spreading cells. In migrating cells lowering VAMP3 levels disrupted the polarised localisation of podosome clusters. The reduced trafficking of recycling endosome membrane to sites of cell spreading and the disorganised podosome localisation in migrating macrophages greatly reduced their ability to persistently migrate on fibronectin. Thus, this important SNARE complex facilitates macrophage adhesion, spreading, and persistent macrophage migration on fibronectin through the delivery of VAMP3-positive membrane with its cargo to expand the plasma membrane and to participate in organising adhesive podosome structures.

Link to Article

http://dx.doi.org/10.1016/j.yexcr.2011.04.016

Adenosine A1 receptors regulate bone resorption in mice...

Title

Adenosine A1 receptors regulate bone resorption in mice: Adenosine A1 receptor blockade or deletion increases bone density and prevents ovariectomy-induced bone loss in adenosine A1 receptor–knockout mice

Authors

Firas M. Kara, Stephen B. Doty, Adele Boskey, Steven Goldring, Mone Zaidi, Bertil B. Fredholm, Bruce N. Cronstein

Abstract

Accelerated osteoclastic bone resorption plays a central role in the pathogenesis of osteoporosis and other bone diseases. Because identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and developing new treatments, we studied the effect of adenosine A1 receptor blockade or deletion on bone density. The bone mineral density (BMD) in adenosine A1 receptor–knockout (A1R-knockout) mice was analyzed by dual x-ray absorptiometry (DXA) scanning, and the trabecular and cortical bone volume was determined by microfocal computed tomography (micro-CT). The mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several parameters were analyzed by DXA scanning and micro-CT. A histologic examination of bones obtained from A1R-knockout and wild-type mice was carried out. Visualization of osteoblast function (bone formation) after tetracycline double-labeling was performed by fluorescence microscopy. Micro-CT analysis of bones from A1R-knockout mice showed significantly increased bone volume. Electron microscopy of bones from A1R-knockout mice showed the absence of ruffled borders of osteoclasts and osteoclast bone resorption. Immunohistologic analysis demonstrated that although osteoclasts were present in the A1R-knockout mice, they were smaller and often not associated with bone. No morphologic changes in osteoblasts were observed, and bone-labeling studies revealed no change in the bone formation rates in A1R-knockout mice. These results suggest that the adenosine A1 receptor may be a useful target in treating diseases characterized by excessive bone turnover, such as osteoporosis and prosthetic joint loosening.

Link to Article

http://dx.doi.org/10.1002/art.27219