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J Appl Physiol 95: 672-676, 2003. First published April 11, 2003; doi:10.1152/japplphysiol.00056.2003
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Receptor activator of NF-{kappa}B ligand arrests bone growth and promotes cortical bone resorption in growing rats

Nansie A. McHugh, Haydee M. Vercesi, Robert W. Egan, and John A. Hey

Allergy Department, Schering-Plough Research Institute, Kenilworth, New Jersey 07033

Submitted 21 January 2003 ; accepted in final form 4 April 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptor activator of NF-{kappa}B ligand (RANKL), produced by osteoblastic lineage cells and activated T cells, is an essential factor for osteoclast differentiation, activation, and survival. Therefore, RANKL is a focal point of therapies targeting bone diseases where there is an imbalance of bone metabolism in favor of bone resorption. The present study assesses the effects of exogenous RANKL on growing bone. RANKL (100 µg·kg-1·day-1 for 7 days) administered to Sprague-Dawley weanling rats caused major deficits in growth, appearance, and bone mineral densities (BMD). Urinary deoxypyridinoline crosslinks, a measure of bone turnover, were higher in the RANKL-treated rats (P = 0.031), and the bone mineral content was lower (P < 0.001). The final BMD in the RANKL-treated rats was lower (P = 0.039) than in the control rats (19 ± 7 vs. 38 ± 5 mg/cm3). Moreover, calculated cortical bone density in each bone slice (total BMD - trabecular BMD) indicated there was only 5% cortical bone remaining in RANKL-treated rats. We conclude that therapies targeting RANKL are likely to have effects on cortical as well as trabecular bone density.

receptor activator of nuclear factor-{kappa}B ligand; computed tomography; in vivo; osteopenia; juvenile

THE OSTEOCLAST IS a highly specialized cell with the unique ability to resorb bone. Recent studies (4, 6, 7, 9, 15, 17) have demonstrated that the activity of this cell is regulated by receptor activator of NF-{kappa}B ligand (RANKL), a soluble factor produced by cells of osteoblastic lineage. The osteoblast upregulates RANKL production in response to numerous resorptive stimuli such as parathyroid hormone, vitamin D3, PGE2, and TNF-{alpha} (4, 7–9, 11). Along with granulocyte macrophage colony-stimulating factor, RANKL has been shown to induce osteoclast differentiation and activation in vitro and support their survival in culture (4, 15, 17). Consistent with this, Li et al. (10) showed that administration of RANKL to mice induced a time-dependent rise in serum Ca2+ levels. Furthermore, the continuous infusion of parathyroid hormone to parathyroidectomized weanling rats, fed a calcium-depleted diet, caused an increase in RANKL and a decrease in its soluble decoy receptor, osteoprotegerin (OPG) (11). Furthermore, knockout receptor activator of NF-{kappa}B (RANK) -/- mice lack osteoclasts and develop osteopetrotic bone (6), and OPG -/- mice that lack OPG, the secreted protein that inhibits RANKL-induced osteoclastogenesis, are osteoporotic (2). These findings add to the growing evidence that the RANK/RANKL system plays a pivotal role in regulating the available pool of active osteoclasts and hence promotes normal bone remodeling. Recent studies (8) show that the balance between RANKL and OPG plays a major determining factor as to whether there is bone formation or bone resorption. Furthermore, elevated levels of RANKL have been demonstrated to be causal in the epidemiology of osteoporosis due to estrogen deficiency (12) and senescence (3). We hypothesize that an excess of RANKL also impacts juvenile bone growth and metabolism. In the present study, we evaluated the in vivo effects of RANKL on growing bone in the rat as measured by peripheral quantitative computed tomography (pQCT). To conduct these studies, we used a weanling rat model that displays quantifiable changes in bone remodeling by pQCT within 7 days of onset of treatment (14).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General. This study was conducted under a protocol approved by the Animal Care and Use Committee of Schering-Plough Research Institute. Sprague-Dawley weanling (21 days old) rats weighing 44 ± 2 g were purchased from Charles River Laboratories (Wilmington, MA). All rats were housed in a temperature- and humidity-controlled room on a 12:12-h light-dark cycle. Rats were given free access to standard rat chow (Harlan Teklad Labdiet, Madison, WI) and reverse-osmosis water. Two studies were conducted by using the same protocol. An initial study compared effects of RANKL treatment [10 µg/day sc of 462-TR, a chimeric protein consisting of the CD33 signal peptide plus 6 histidine residues fused to the amino-terminus of the mouse RANKL (amino acids 72–316); R&D Systems, Minneapolis, MN] vs. vehicle-treated rats (control, PBS given subcutaneously). On the basis of the lack of effects observed in the first study, a second study was conducted by using a supraphysiological dose of newly available RANKL [462-TEC; 100 µg/day sc, mouse RANKL (amino acids 158–317 expressed in Escherichia coli); R&D Systems]. Each group included eight rats.

Protocol. On day 1, the rats were anesthetized with isoflurane ("to effect", IsoFlo, Abbott Laboratories, Chicago, IL), and baseline three-dimensional bone mineral density (BMD) measurements were taken by pQCT (Stratec XCT Research SA, Pforzheim, Germany). The rats were given their supplements/vehicle daily on days 1–7. On the day 8, the rats were anesthetized with pentobarbital sodium (50 mg/kg ip, Nembutal, Abbott Laboratories), and the final BMD measurements were taken. The rats were then euthanized by exsanguination, and the scanned tibia was harvested for bone ash content.

pQCT measurement and analysis. The settings for pQCT scanning were research SA collimation at a voxel size of 0.1 mm3; therefore, the slice width was 0.1 mm. The voxel is equivalent to a pixel with volume (3 dimensional). This small voxel size minimizes partial volume effect errors (i.e., including voxels that are not completely filled with bone). Comparable placement of slices was ensured by measuring a slice in the metaphysis 2 mm from the reference line that was placed at the proximal edge of the growth plate. This placement results in the most consistent baseline measurements (14). All bone slices were analyzed with the same parameters by using Stratec Software (Stratec Medizintechnik). An automatic ContourMode 1 was used to define the outer edge of the cortical bone, and PeelMode 20 (an adaptation of PeelMode 2 that determines the threshold to be used by evaluating the BMD at a predefined percentage of total bone) was used to define the inner edge of the cortical bone and the beginning of the trabecular bone. The region of interest for the final scans of the RANKL-treated rats had to be manually drawn due to the lack of cortical bone. For determining trabecular BMD, the percent option was used with trabecular area defined at 30% with a threshold of 280 mg/cm3.

Biochemical markers. In the second study, the rats were placed in metabolic cages on day 7 for a 24-h urine collection. Urine samples were processed [Beckman Synchron Clinical Analyzer (CX3delta), Fullerton, CA] for Ca2+ concentration and creatinine; aliquots of the urine were used to measure deoxypyridinoline (Dpd) crosslinks via ELISA (Quidel, Mountain View, CA). Dpd values are reported as nanomoles of Dpd per millimole of creatinine.

Bone ash content. The scanned tibia was harvested and cleaned of tissue. The bone was vacuum dried overnight, weighed, then placed in a furnace at 950°C, and reduced to its mineral content. Ash data are reported as a percentage of total bone dry weight.

Statistics. Statistical analysis of all data were assessed by using ANOVA with Dunnett's test for treatments with a control (SigmaStat, SPSS Software, Chicago, IL).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Initial study. The baseline measurements of BMD data as measured by pQCT were not significantly different between groups (176 ± 6 mg/cm3 in the control group vs. 172 ± 3 mg/cm3 in the RANKL-treated group). Rats that were treated with the lower dose of RANKL (462-TR, 10 µg/day) showed no differences in their final BMD or bone ash content measurements compared with the vehicle-treated control rats (Fig. 1A). For both RANKL-treated and control rats, final trabecular BMD measurements were significantly higher than their respective baseline, a result expected in growing rats. Bone ash content data also showed no significant differences between the two groups (Fig. 1B).



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  		Fig. 1. Lower dose study that shows the baseline and change from baseline (growth) bone mineral density (BMD) in receptor activator of NF-{kappa}B ligand (RANKL)-treated (10 µg/day) and control rats (A) and the bone ash weight (bone mineral content; B). Both BMD and bone ash weight showed the typical growth pattern of the weanling rat, and there were no significant differences between treated and untreated groups. Values are means ± SE.

 

Second study. In the high-dose study, the control group's final trabecular BMD measurements were also significantly higher than their respective baseline; however, RANKL-treated (462-TEC, 100 µg/day) rats exhibited major differences in growth, appearance, and BMD values. The final weights of the RANKL-treated rats were 58 ± 5 vs. 96 ± 2 g in control rats. Two RANKL-treated rats were euthanized early on days 6 and 7 because of adverse effects on their health that were likely due to the treatment with RANKL. The effects observed included shivering and lethargy compared with the other members of their group and control rats. Although the RANKL-treated group showed a small, nonsignificant increase in final trabecular BMD values compared with their baseline measurements, this was likely due to the fact that all the bone was now the consistency of trabecular bone. The change from baseline BMD measurements between the two groups was significantly different in trabecular bone measurements (Fig. 2A; P = 0.039). Furthermore, when the difference between the total bone density less the trabecular bone density was examined, the RANKL-treated group was devoid of cortical bone (Fig. 2B; P < 0.001). Bone ash content data confirm these findings showing a decrement in percentage of bone ash to total bone content (Fig. 3; P < 0.001). The pQCT scan images were dramatically different after 1 wk of treatment. We observed no distinct cortical rim in the pQCT image of the RANKL-treated rats and the entire slice appeared to be trabecular bone (Fig. 4). Moreover, we had to hand-draw the region of interest for these slices because the automated computerized tomography software uses the cortical edge density as a reference point for determining the region of interest. Furthermore, after the tibia for bone ash content was removed, most of the RANKL-treated bones could not be removed without fracture. The RANKL-treated group had significantly higher urinary Dpd values than the untreated controls (Fig. 5; P = 0.031); however, their urinary and serum Ca2+ levels were not significantly different. There was a trend toward a lower urinary Ca2+ levels in the RANKL-treated group suggesting a compensatory mechanism to conserve calcium.



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  		Fig. 2. Changes in the trabecular BMD in RANKL-treated (100 µg/day) 21-day-old growing rats. A: values of baseline and change from baseline (growth) trabecular BMD after 7 days of treatment with RANKL. *Significant difference compared with control, P = 0.039. B: amount of cortical bone as defined by total BMD less trabecular BMD after RANKL treatment. #Significant difference compared with control, P < 0.001. Data are means ± SE.

 


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  		Fig. 3. RANKL-treated rats (100 µg·kg-1·day-1) exhibited significantly less bone mineral content than control rats. Bone mineral content is reported as bone ash weight normalized to total bone weight. Data are means ± SE. *Significant difference compared with control, P < 0.001.

 


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  		Fig. 4. Representative peripheral quantitative computed tomography scan images of a control rat (A) and a RANKL-treated rat (B). The normal cortical bone is absent in the bone scans from rats treated with RANKL (100 µg·kg-1·day-1).

 


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  		Fig. 5. Deoxypyridinoline (Dpd) cross-links, a urinary marker of bone resorption, were significantly increased in the RANKL-treated rats (100 µg·kg-1·day-1) compared with control rats. Data are means ± SE. *Significant difference compared with control rats, P = 0.031.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies show that various cytokines and humoral factors such as parathyroid hormone, PGE2, vitamin D3, and IL-6 that regulate osteoclast function in normal bone remodeling and pathophysiological bone diseases do so by modifying the expression of RANKL (8, 11, 17). We presently sought to investigate the role of RANKL in the developing mammal at a time when basal bone mass is being established, and it is assumed that the balance is tipped in favor of bone formation. This model was first developed by Schenk et al. (16) to test antiresorptive compounds such as bisphosphonates. We have previously employed this model to evaluate antiresorptive compounds by pQCT quantification within 1 wk (14).

In the present study, we found that a 10 µg/day dose of RANKL for 7 days elicited no observable changes in bone mineral content or BMD. We had attributed the lack of effect to the dose of RANKL that was used; however, recently Takayanagi et al. (18) reported that RANKL can limit its own osteoclastogenic effect by stimulating production of IFN-{beta} by osteoclast precursors. Consequently, the lower dose may not have generated serum levels high enough to elicit pathological effects. In a second attempt to show proof of principle for the role of RANKL in the growing rat, we used a newly developed protein that was assured to be effective. We chose a dose that was 10 times that used in the first study, a supraphysiological dose to define the maximal effect on the growing rat. We found that a dose of 100 µg·kg-1·day-1 exerted a significant resorptive action on cortical bone by pQCT scanning techniques, suggesting that RANKL may play a large role in the developing bone.

Clinical studies reporting the effects of osteoporosis on bone primarily show the effects on trabecular bone, which, due to its porous composition and architecture, is commonly first to manifest the effects of increased or decreased osteoclastic activity. Meanwhile, cortical bone comprises 85% of the total body bone content. Remodeling of the cortical bone occurs by endosteal resorption, and the decline in cortical bone mass normally occurs later than trabecular bone loss (23). For example, the vertebra is composed largely of trabecular bone, and, therefore, the incidence of vertebral fracture occurs earlier in life (~60 yr of age) (23). Conversely, long bones such as the femur have a large cortical component, and, therefore, the incidence of fracture in the femur and femoral neck occur more at an age of >=70 yr (23). Osteoporosis studies are conducted on adult humans that have achieved peak bone mass, and the diagnosis of osteoporosis is determined by using dual-energy X-ray absorptiometry, a two-dimensional scan (22). In humans, the most common form of adult-onset osteoporosis is found in the postmenopausal woman and the early progression of the disease can be observed in the trabecular compartment with the loss of some trabeculae entirely leaving the remaining trabeculae with wide separations and not connected (12). Cortical bone loss occurs through enlargement of the subendocortical spaces and deeper erosion due to increased osteoclastic activity (12). Present therapies addressing this form of osteoporosis have been relatively successful in maintaining bone by inhibition of resorption, and emerging therapies are aimed at replacing bone loss.

In this study, we found that, in the developing bone, RANKL not only had effects on trabecular bone but also elicited profound effects on cortical bone. Cortical bone was virtually remodeled to the density and architecture of trabecular bone or never formed. These data have serious implications for osteopenic diseases or conditions of the juvenile bone. Osteoporotic diseases that compromise bone growth through the RANK/RANKL pathway may also compromise peak cortical bone mass. These include early nutritional deficiencies (Ca2+, vitamin K, vitamin D, and anorexia nervosa) or may be secondary to therapeutic steroid use. Peak bone mass has been determined to be an important factor in osteoporotic fracture risk assessment (1). It has been well established that women develop osteoporosis earlier and more severely than men who have higher initial BMD (12). Moreover, perimenopausal women who are small in stature and slight of build are predisposed to develop osteoporosis because their initial bone mass is less than their sturdier peers. This risk factor is not specific to the female gender because men who do not achieve bone masses comparable to their peers are also predisposed to the early onset of osteoporosis. For example, Van Pottelbergh et al. (21) found that men with idiopathic osteoporosis showed no indications of accelerated bone loss compared with age-matched controls. However, by measuring the BMD of three generations within the families of these men with idiopathic osteoporosis, the authors found that there was a deficit in bone acquisition. Therefore, they suggest that these men were genetically predisposed to lower peak bone masses. Therefore, if peak bone mass is not achieved during early development, initial bone mass values will be lower despite gender or hormonal issues, and it can, therefore, be predicted that the onset of osteoporosis will also be earlier.

The biomechanical properties and strength of bones are determined by the composition of the bone, BMD, and the cross-sectional architecture. The size and shape of the cortical bone, the composition and architecture of trabecular bone, as well as the ratio of cortical to trabecular bone all play a role in determining the strength or fragility of bone (19, 20). Simply stated, the biomechanical strength of the bone is determined by the ability of its structure to withstand the stresses demanded of it. As people age, bones generally become more brittle at a time when they are more prone to falls (24). Normal young bone is more flexible and will deform due to stresses before breaking; however, the juvenile bone is subject to additional stresses due to sports-related falls and injuries (13). The decrease in cortical bone that we observed would affect the cortical-to-trabecular bone ratios and would, therefore, potentially have serious life-long consequences. Much attention has been given to the changes in trabecular bone with osteoporosis; however, cortical bone is an important factor in determining bone strength as shown by Crabtree et al. (5) in their intracapsular hip fracture studies. The fragility of the bone is dependent as much on the amount of bone tissue as on the material properties and architecture.

In conclusion, this study has shown that the RANK/RANKL pathway potentially plays a major role in the development of peak cortical bone mass as well as its role in normal bone turnover. Further studies are warranted to determine whether lower doses affect the bone strength of juvenile bone by decreasing cortical wall thickness. Furthermore, the mouse RANKL was able to activate the rat RANK receptor to affect bone resorption and will be a useful tool in evaluating this relationship.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. A. McHugh, Schering-Plough Research Institute, Allergy Dept., 2015 Galloping Hill Rd., K15-1-1600, Kenilworth, NJ 07033 (E-mail: nansie.mchugh{at}spcorp.com).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bonjour JP, Theintz G, Law F, Slosman D, and Rizzoli R. Peak bone mass. Osteoporosis Int 4, Suppl 1: 7-13, 1994.
  2. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, and Simonet WS. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 12: 1260-1268, 1998.[Abstract/Free Full Text]
  3. Cao J, Venton L, Sakata T, and Halloran BP. Expression of RANKL and OPG correlates with age-related bone loss in male C57BL/6 mice. J Bone Miner Res 18: 270-277, 2003.[Web of Science][Medline]
  4. Chambers TJ. Regulation of the differentiation and function of osteoclasts. J Pathol 192: 4-13, 2000.[Web of Science][Medline]
  5. Crabtree N, Loveridge N, Parker M, Rushton N, Power J, Bell KL, Beck TJ, and Reeve J. Intracapsular hip fracture and the region-specific loss of cortical bone: analysis by peripheral quantitative computed tomography. J Bone Miner Res 16: 1318-1328, 2001.[Web of Science][Medline]
  6. Dougall WC, Glaccum M, Charrier K, Rohrbach K, Brasel K, De Smedt T, Daro E, Smith J, Tometsko ME, Maliszewski CR, Armstrong A, Shen V, Bain S, Cosman D, Anderson D, Morrissey PJ, Peschon JJ, and Schuh J. RANK is essential for osteoclast and lymph node development. Genes Dev 13: 2412-2424, 1999.[Abstract/Free Full Text]
  7. Fuller K, Wong B, Choi Y, and Chambers TJ. TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 188: 997-1001, 1998.[Abstract/Free Full Text]
  8. Kanematsu M, Sato T, Takai H, Watanabe K, Ikeda K, and Yamada Y. Prostaglandin E2 induced expression of receptor activator of nuclear factor-{kappa}B ligand/osteoprotegerin ligand on pre-B cells: implications for accelerated osteoclastogenesis in estrogen deficiency. J Bone Miner Res 15: 1321-1329, 2000.[Web of Science][Medline]
  9. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, and Boyle WJ. Osteoprotegrin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93: 165-176, 1998.[Web of Science][Medline]
  10. Li J, Sarosi I, Yan XQ, Morony S, Capparelli C, Tan HL, McCabe S, Elliott R, Scully S, Van G, Kaufman S, Juan SC, Sun Y, Tarpley J, Martin L, Christensen K, McCabe J, Kostenuik P, Hsu H, Fletcher F, Dunstan CR, Lacey DL, and Boyle WJ. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci USA 97: 1566-1571, 2000.[Abstract/Free Full Text]
  11. Ma YL, Cain RL, Halladay DL, Yang X, Zeng Q, Miles RR, Chandrasedhar S, Martin TJ, and Onyia JE. Catabolic effects of continuous human PTH (1–38) in vivo is associated with sustained stimulation of RANKL and inhibition of osteoprotegerin and gene-associated bone formation. Endocrinology 142: 4047-4054, 2001.[Abstract/Free Full Text]
  12. Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev 21: 115-137, 2000.[Abstract/Free Full Text]
  13. McCalden R, McGeough J, Barker M, and Court-Brown C. Age-related changes in the tensile properties of cortical bone: the relative importance of changes in porosity, mineralization and microstructure. J Bone Joint Surg Am 75: 1193-1205, 1993.[Abstract/Free Full Text]
  14. McHugh NA, Vercesi HM, Egan RW, and Hey JA. In vivo rat assay: bone remodeling and steroid effects on juvenile bone by pQCT quantification in 7 days. Am J Physiol Endocrinol Metab 284: E70-E75, 2003. First published September 3, 2002, 10.1152/ajpendo.00102.2002.[Abstract/Free Full Text]
  15. Quinn JMW, Elliot J, Gillespie MT, and Martin TJ. A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro. Endocrinology 139: 4424-4427, 1998.[Abstract/Free Full Text]
  16. Schenk R, Eggli P, Fleisch H, and Rosini S. Quantitative morphometric evaluation of the inhibitory activity of new aminobisphosphonates on bone resorption in the rat. Calcif Tissue Int 38: 342-349, 1986.[Web of Science][Medline]
  17. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, and Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20: 345-357, 1999.[Abstract/Free Full Text]
  18. Takayanagi H, Kim S, Matsuo K, Suzuki H, Suzuki T, Sato K, Yokochi T, Oda H, Nakamura K, Ida N, Wagner EF, and Taniguchi T. RANKL maintains bone homeostasis through c-Fos-dependent induction of interferon-beta. Nature 416: 744-749, 2002.[Medline]
  19. Turner CH and Burr DB. Basic biomechanical measurements of bone: a tutorial. Bone 14: 595-608, 1993.[Medline]
  20. Turner RT, Bolander ME, Sarkar G, An KN, Maran A, and Ritman EL. An integrated approach to assess structure-to-function relationships in the skeleton. J Musculoskel Neuron Interact 2: 3-8, 2001.
  21. Van Pottelbergh I, Goemaere S, Zmierczak H, De Bacquer D, and Kaufman JM. Deficient acquisition of bone during maturation underlies idiopathic osteoporosis in men: evidence from a three-generation family study. J Bone Miner Res 18: 303-311, 2003.[Web of Science][Medline]
  22. Warming L, Hassager C, and Christiansen C. Changes in bone mineral density with age in men and women: a longitudinal study. Osteoporos Int 13: 105-112, 2002.[Web of Science][Medline]
  23. Wasnich RD. Epidemiology of osteroporosis. In: Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, edited by Kleerekoper M and Khosla S. Philadelphia, PA: Lippincott, Williams & Wilkins, 1999, p. 30-31, 257.
  24. Winner S, Morgan C, and Grimley-Evans J. Perimenopausal risk of falling and incidence of distal forearm fracture. BMJ 298: 1486-1488, 1989.




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