Resorptive Capacity Research Articles

CSF Flow in the Brain in the Context of Normal Pressure Hydrocephalus.

CSF normally flows back and forth through the aqueduct during the cardiac cycle. During systole, the brain and intracranial vasculature expand and compress the lateral and third ventricles, forcing CSF craniocaudad. During diastole, they contract and flow through the aqueduct reverses. Hyperdynamic CSF flow through the aqueduct is seen when there is ventricular enlargement without cerebral atrophy. Therefore, patients presenting with clinical normal pressure hydrocephalus who have hyperdynamic CSF flow have been found to respond better to ventriculoperitoneal shunting than those with normal or decreased CSF flow. Patients with normal pressure hydrocephalus have also been found to have larger intracranial volumes than sex-matched controls, suggesting that they may have had benign external hydrocephalus as infants. While their arachnoidal granulations clearly have decreased CSF resorptive capacity, it now appears that this is fixed and that the arachnoidal granulations are not merely immature. Such patients appear to develop a parallel pathway for CSF to exit the ventricles through the extracellular space of the brain and the venous side of the glymphatic system. This pathway remains functional until late adulthood when the patient develops deep white matter ischemia, which is characterized histologically by myelin pallor (ie, loss of lipid). The attraction between the bare myelin protein and the CSF increases resistance to the extracellular outflow of CSF, causing it to back up, resulting in hydrocephalus. Thus idiopathic normal pressure hydrocephalus appears to be a "2 hit" disease: benign external hydrocephalus in infancy followed by deep white matter ischemia in late adulthood.

Osteoclastic resorptive capacity is suppressed in patients receiving hyperbaric oxygen therapy

Background and purpose — Hypoxia, necrosis, and bone loss are hallmarks of many skeletal diseases. Hyperbaric oxygen therapy (HBO) is often used as an adjunctive therapy in these cases. However the in vivo effect of HBO on osteoclast formation has not been fully established. We therefore carried out a longitudinal study to examine the effect of HBO on osteoclast formation and bone resorptive capacity in patients who were referred to the Plymouth Hyperbaric Medical Centre.Methods — Osteoclast precursors were isolated from peripheral blood prior to and following 10 and 25 daily hyperbaric treatments (100% O2 at 2.4 atmospheres absolute ATA for 90 min) to determine osteoclast formation and resorptive capacity. The expression of key regulators of osteoclast differentiation RANK, Dc-STAMP, and NFATc1 was also assessed by quantitative real-time PCR.Results — HBO reduced the ability of precursors to form osteoclasts and reduced bone resorption in a treatment-dependent manner. The initial suppressive effect of HBO was more pronounced on mononuclear osteoclast formation than on multinuclear osteoclast formation, and this was accompanied by reduction in the expression of key regulators of osteoclast formation, RANK and Dc-STAMP.Interpretation — This study shows for the first time that in vivo, HBO suppresses the ability of monocytic precursors to form resorptive osteoclasts.

NOD2 Contributes to Porphyromonas gingivalis–induced Bone Resorption

The NOD-like receptors are cytoplasmic proteins that sense microbial by-products released by invasive bacteria. Although NOD1 and NOD2 are functionally expressed in cells from oral tissues and play a role triggering immune responses, the role of NOD2 receptor in the bone resorption and in the modulation of osteoclastogenesis is still unclear. We show that in an experimental model of periodontitis with Porphyromonas gingivalis W83, NOD2-/- mice showed lower bone resorption when compared to wild type. Quantitative polymerase chain reaction analysis revealed that wild-type infected mice showed an elevated RANKL/OPG ratio when compared to NOD2-/- infected mice. Moreover, the expression of 2 osteoclast activity markers—cathepsin K and matrix metalloproteinase 9—was significantly lower in gingival tissue from NOD2-/- infected mice compared to WT infected ones. The in vitro study reported an increase in the expression of the NOD2 receptor 24 hr after stimulation of hematopoietic bone marrow cells with M-CSF and RANKL. We also evaluated the effect of direct activation of NOD2 receptor on osteoclastogenesis, by the activation of this receptor in preosteoclasts culture, with different concentrations of muramyl dipeptide. The results show no difference in the number of TRAP-positive cells. Although it did not alter the osteoclasts differentiation, the activation of NOD2 receptor led to a significant increase of cathepsin K expression. We confirm that this enzyme was active, since the osteoclasts resorption capacity was enhanced by muramyl dipeptide stimulation, evaluated in osteoassay plate. These results show that the lack of NOD2 receptor impairs the bone resorption, suggesting that NOD2 receptor could contribute to the progression of bone resorption in experimental model of periodontitis. The stimulation of NOD2 by its agonist, muramyl dipeptide, did not affect osteoclastogenesis, but it does favor the bone resorption capacity identified by increased osteoclast activity.

The Phosphoinositide 3‐Kinase Isoform PI3Kβ Regulates Osteoclast‐Mediated Bone Resorption in Humans and Mice

ObjectiveWhile phosphoinositide 3-kinases (PI3Ks) are involved in various intracellular signal transduction processes, the specific functions of the different PI3K isoforms are poorly understood. We have previously shown that the PI3Kβ isoform is required for arthritis development in the K/BxN serum–transfer model. Since osteoclasts play a critical role in pathologic bone loss during inflammatory arthritis and other diseases, we undertook this study to test the role of PI3Kβ in osteoclast development and function using a combined genetic and pharmacologic approach.MethodsThe role of PI3Kβ in primary human and murine osteoclast cultures was tested with the PI3Kβ-selective inhibitor TGX221 and by using PI3Kβ−/− mice. The trabecular bone architecture of PI3Kβ−/− mice was evaluated using micro–computed tomography and histomorphometric analyses.ResultsThe expression of PI3Kβ was strongly and specifically up-regulated during in vitro osteoclast differentiation. In vitro development of large multinucleated osteoclasts from human or murine progenitors and their resorption capacity were strongly reduced by the PI3Kβ inhibitor TGX221 or by the genetic deficiency of PI3Kβ. This was likely due to defective cytoskeletal reorganization and vesicular trafficking, since PI3Kβ−/− mouse multinucleated cells failed to form actin rings and retained intracellular acidic vesicles and cathepsin K. In contrast, osteoclast-specific gene expression and the survival and apoptosis of osteoclasts were not affected. PI3Kβ−/− mice had significantly increased trabecular bone volume and showed abnormal osteoclast morphology with defective resorption pit formation.ConclusionPI3Kβ plays an important role in osteoclast development and function and is required for in vivo bone homeostasis.

Dual impact of live Staphylococcus aureus on the osteoclast lineage, leading to increased bone resorption.

Bone and joint infection, mainly caused by Staphylococcus aureus, is associated with significant morbidity and mortality, characterized by severe inflammation and progressive bone destruction. Studies mostly focused on the interaction between S. aureus and osteoblasts, the bone matrix-forming cells, while interactions between S. aureus and osteoclasts, the only cells known to be able to degrade bone, have been poorly explored. We developed an in vitro infection model of primary murine osteoclasts to study the direct impact of live S. aureus on osteoclastogenesis and osteoclast resorption activity. Staphylococcal infection of bone marrow-derived osteoclast precursors induced their differentiation into activated macrophages that actively secreted proinflammatory cytokines. These cytokines enhanced the bone resorption capacity of uninfected mature osteoclasts and promoted osteoclastogenesis of the uninfected precursors at the site of infection. Moreover, infection of mature osteoclasts by live S. aureus directly enhanced their ability to resorb bone by promoting cellular fusion. Our results highlighted two complementary mechanisms involved in bone loss during bone and joint infection, suggesting that osteoclasts could be a pivotal target for limiting bone destruction.

Nutrient resorption in tropical savanna forests and woodlands of central Brazil

Nutrient limitation in Brazilian savanna (known as cerrado) presumably causes trees to maximize nutrient resorption from senesced leaves to reduce their dependence on nutrient availability. To assess patterns between nutrient resorption and soil fertility, we measured community-level nitrogen (N), phosphorus (P), and potassium (K) concentra- tions in mature and senesced leaves and soil fertility in the upper 50 cm soil layer in structurally diverse cerrado ecosystems in the Cuiaba Basin (CB) and Pantanal (PAN) of Mato Grosso, Brazil. Foliar nutrient concentration data were used to estimate resorption efficiency and proficiency, and correlation was used to determine whether resorption efficiency and proficiency varied across soil fertility gradients. We found that N and P resorption proficiency (NRP and PRP, respectively) and P resorption efficiency (PRE) increased significantly as total soil N (NRP) and extractable P (PRP and PRE) declined. In contrast, K resorption efficiency (KRE) declined as soil sand content and bulk density increased, which was likely due to a reduction in soil water-holding capacity. Leaf N/P ratios indicate potential N limitation and/or N ? P co-limitation for ecosys- tems in the PAN and P limitation and/or N ? P co- limitation for ecosystems in the CB, while trends in leaf N/K ratios indicate possible K or K ? P co- limitation for the CB only. Our results illustrate that cerrado forests and woodlands have highly variable nutrient resorption capacities that vary predictably across soil fertility or textural gradients and indicate that cerrado communities have flexible nutrient resorption that can reduce their dependence on soil

The RhoGAP Activity of Myosin IXB Is Critical for Osteoclast Podosome Patterning, Motility, and Resorptive Capacity

Osteoclasts are large, multinucleated cells of the monocyte-macrophage lineage that generate specialized substrate adhesion complexes to facilitate their function as bone-degrading cells. The patterning and function of these actin-based complexes, podosomes and sealing zones, are regulated by the small GTPase Rho. Myosin IXB (Myo9b) is a unique actin-based motor protein that contains a RhoGAP domain, which, like other RhoGAPs, is inhibitory to Rho signaling. In this study, Myo9b is shown to be expressed in osteoclasts and act as a critical regulator of podosome patterning and osteoclast function. SiRNA-mediated knockdown of Myo9b results in increased activity of Rho but not Rac in osteoclasts. Knockdown in osteoclasts on glass results in altered podosome patterning and decreased motility, and this effect is reversed by addition of a Rho inhibitor. SiRNA-mediated suppression of Myo9b expression in osteoclasts on bone results in a dramatic loss of resorptive capacity even though sealing zones appear normal. This loss of resorption is also reversible with addition of a Rho inhibitor. Cells with diminished Myo9b levels display mislocalization and suppressed activation of Src, a tyrosine kinase with critical effects on osteoclast actin cytoskeletal rearrangement and function. In addition, siRNA-treated cells display poorly formed microtubule networks and a lack of tubulin acetylation, a marker of microtubule stability. However, short-term addition of TNFα to cells with suppressed Myo9b levels overcomes or circumvents these defects and causes increased sealing zone size and resorptive capacity. These results indicate that the RhoGAP activity of Myo9b plays a key role in regulating the actin-based structures necessary for osteoclast motility and resorption, and confirms that Myo9b can act as a motorized signaling molecule that links Rho signaling to the dynamic actin cytoskeleton.

Impairment of rat tooth eruption in pups born to mothers exposed to chronic stress during pregnancy

ObjectiveTooth eruption is a multifactorial process in which bone tissue plays a prevailing role. In this study we evaluated the bone overlying the developing tooth germ and the degree of tooth eruption of the first mandibular molar in pups born to mothers subjected to constant light during pregnancy. DesignPregnant rats were divided into two groups: mothers chronically exposed to a 12:12 light/light cycle (LL) from day 10 to 20 of pregnancy and controls (C) maintained on a 12:12h light/dark cycle. Pups from each group were euthanized at the age 3 or 15 days.Buccolingually oriented sections of mandibles were stained with haematoxylin–eosin or for histochemical detection of tartrate resistant acid phosphatase (TRAP). The histomorphometric parameters evaluated were bone volume, number of osteoclasts, TRAP+ bone surface, number of TRAP+ and TRAP− osteoclasts per mm2 and degree of tooth eruption (mm). ResultsIt was found an increase in bone volume (LL: 58.14±4.24 vs. C: 32.31±2.16; p<0.01) and a decrease in the number of osteoclasts (LL: 3.5±0.65 vs. C: 8.03±1.31; p<0.01) and TRAP+ cells (LL: 0.84±0.53 vs. C: 8.59±1.26; p<0.01) in 3-day-old pups born to LL-exposed mothers. These observations are consistent with the decrease in the degree of tooth eruption observed in 15-day-old experimental pups (LL: −0.605±0.05 vs. C: −0.342±0.02; p<0.0001). ConclusionOur results suggest that chronic constant light applied as a pre-natal stressor impairs the resorptive capacity of osteoclasts involved in the formation of the eruption pathway and consequently the degree of tooth eruption.

Expression of metastasin S100A4 is essential for bone resorption and regulates osteoclast function

ObjectiveS100A4 is a Ca-binding protein that regulates cell growth, survival, and motility. The abundant expression of S100A4 in rheumatiod arthritis contributes to the invasive growth of joint tissue and to bone damage. In the present study, we analysed the role of S100A4 in bone homeostasis. MethodsPeripheral quantitative computed tomography and histomorphometric analysis were performed in mice lacking the entire S100A4 protein (S100A4KO) and in wild-type (WT) counterparts treated with shRNA-lentiviral constructs targeting S100A4 (S100A4-shRNA). Control groups consisted of sex-matched WT counterparts and WT mice treated with a non-targeting RNA construct. ResultsS100A4 deficiency was associated with higher trabecular and cortical bone mass, increased number and thickness of trabeculi combined with larger periosteal circumference and higher predicted bone strength. S100A4 inhibition by shRNA led to an increase in cortical bone in WT mice. S100A4-deficieny was associated with a reduced number of functional osteoclasts. S100A4KO and S100A4-shRNA-treated bone marrow progenitors gave rise to a large number of small TRAP+ cells with few nuclei and few pseudopodial processes. Poor osteoclastogenesis and the low resorptive capacity in S100A4Ko mice may be linked to low levels of surface integrins, impaired adhesion capacity, and poor multinucleation in S100A4-deficient osteoclasts, as well as a low content of proteolytic enzymes cathepsin K and MMP3 and MMP9 to break down the organic matrix. ConclusionS100A4 emerges as a negative regulator of bone metabolism potentially responsible for the excessive bone turnover in conditions marked by high levels of S100A4 protein, such as inflammation and rheumatoid arthritis.

Zap70 inhibits Syk‐mediated osteoclast function

The αvβ3 integrin stimulates the resorptive capacity of the differentiated osteoclast (OC) by organizing its cytoskeleton via the tyrosine kinase, Syk. Thus, Syk-deficient OCs fails to spread or form actin rings, in vitro and in vivo. The Syk family of tyrosine kinases consists of Syk itself and Zap70 which are expressed by different cell types. Because of their structural similarity, and its compensatory properties in other cells, we asked if Zap70 can substitute for absence of Syk in OCs. While expression of Syk, as expected, normalizes the cytoskeletal abnormalities of Syk(-/-) OCs, Zap70 fails do so. In keeping with this observation, Syk, but not Zap70, rescues αvβ3 integrin-induced SLP76 phosphorylation in Syk(-/-) OCs. Furthermore the kinase sequence of Syk partially rescues the Syk(-/-) phenotype but full normalization also requires its SH2 domains. Surprisingly, expression of Zap70 inhibits WT OC spreading, actin ring formation and bone resorptive activity, but not differentiation. In keeping with arrested cytoskeletal organization, Zap70 blocks integrin-activated endogenous Syk and Vav3, SLP76 phosphorylation. Such inhibition requires Zap70 kinase activity, as it is abolished by mutation of the Zap70 kinase domain. Thus, while the kinase domain of Syk is uniquely required for OC function that of Zap70 inhibits it.

Genetics in Endocrinology: Autosomal dominant osteopetrosis revisited: lessons from recent studies

Systematic studies of autosomal dominant osteopetrosis (ADO) were followed by the identification of underlying mutations giving unique possibilities to perform translational studies. What was previously designated ADO1 turned out to be a high bone mass phenotype caused by a missense mutation in the first propeller of LRP5, a region of importance for binding inhibitory proteins. Thereby, ADO1 cannot be regarded as a classical form of osteopetrosis but must now be considered a disease of LRP5 activation. ADO (Albers-Schönberg disease, or previously ADO2) is characterized by increased number of osteoclasts and a defect in the chloride transport system (ClC-7) of importance for acidification of the resorption lacuna (a form of Chloride Channel 7 Deficiency Osteopetrosis). Ex vivo studies of osteoclasts from ADO have shown that cells do form normally but have reduced resorption capacity and an expanded life span. Bone formation seems normal despite decreased osteoclast function. Uncoupling of formation from resorption makes ADO of interest for new strategies for treatment of osteoporosis. Recent studies have integrated bone metabolism in whole-body energy homeostasis. Patients with ADO may have decreased insulin levels indicating importance beyond bone metabolism. There seems to be a paradigm shift in the treatment of osteoporosis. Targeting ClC-7 might introduce a new principle of dual action. Drugs affecting ClC-7 could be antiresorptive, still allowing ongoing bone formation. Inversely, drugs affecting the inhibitory site of LRP5 might stimulate bone formation and inhibit resorption. Thereby, these studies have highlighted several intriguing treatment possibilities, employing novel modes of action, which could provide benefits to the treatment of osteoporosis.

THU0117 Loss of PTEN in Myeloid Cells Controls Inflammatory Bone Destruction by Regulating the Osteoclastogenic Potential of Myeloid Cells

BackgroundLocal bone destruction in rheumatoid arthritis, psoriasisarthritis or ankylosing spondylitis is a serious health burden and the major cause of disability and severely reduced quality of life in these diseases....

THU0037 Immune complex-induced inhibition of osteoclastogenesis is mediated via activating, but not inhibitory FCGAMMA receptors on myeloid precursor cells

BackgroundRheumatoid arthritis is characterized by osteoclast-mediated bone loss. Co-stimulatory signalling via ITAM- and ITIM-coupled receptors is essential for osteoclast formation and function. The ITAM- and ITIM-coupled Fcγ receptors (FcγR) are...

OP0087 Autophagy contributes to TNFα-mediated joint destruction

BackgroundAutophagy is an essential, homeostatic process by which cells digest unnecessary cellular organelles. Beclin1 and Atg7 are markers of autophagy that are required for the initiation of the autophagasomeand fusion...

Lysophosphatidic acid stimulates osteoclast fusion through OC-STAMP and P2X7 receptor signaling

Bone is continuously remodeled by bone formation and resorption, and cooperative bone metabolism is precisely regulated to maintain homeostasis. Osteoclasts, which are responsible for bone resorption, are differentiated through multiple steps that include cell fusion at the last step of differentiation, yielding multinuclear cells. However, the factors involved in and the precise mechanism of cell fusion are still unknown. To determine the molecules involved in osteoclast fusion, we examined the effect of lysophosphatidic acid (LPA), which has been reported to participate in the progression of cancer bone metastasis. LPA had no effect on osteoclast formation and bone resorption under receptor activator of nuclear factor kappa B ligand (RANKL) conditions, whereas LPA stimulated osteoclast fusion, thereby causing increased osteoclast diameter and bone resorptive capacity under a RANKL-limited condition. This result encouraged us to assess what molecules are needed for LPA-stimulated osteoclast fusion. Interestingly, LPA stimulated osteoclast stimulatory transmembrane protein (OC-STAMP) and P2X7 receptor mRNA expression during osteoclast fusion under a RANKL limiting condition. siRNA-induced OC-STAMP or P2X7 receptor knockdown significantly suppressed the LPA-stimulated increase in osteoclast diameter and bone resorptive capacity in differentiating cultures. Using cyclosporin A as an inhibitor, we revealed that NF-ATc1 directly regulates OC-STAMP and P2X7 receptor expression during LPA-stimulated osteoclast fusion. These results suggest that LPA is a critical regulator of osteoclast fusion by inducing the OC-STAMP and P2X7 receptor. Therefore, LPA signaling might be useful to help understand their effects on osteoclast formation and as a therapeutic target for patients with pathologically increased osteoclast formation.

The surface microporosity of ceramic biomaterials influences the resorption capacity of osteoclasts

This study investigated the influence of the surface microporosity of beta-tri-calcium phosphate (β-TCP) ceramics on the resorption capacity of osteoclasts. This was achieved by first compacting commercially available β-TCP powder into disks that were sintered at various temperatures, thereby yielding different surface microporosities. Scanning electron microscopy (SEM) and subsequent image processing verified different degrees of surface microporosity on the disks. Rabbit osteoclasts in a bone marrow derived cell suspension were then seeded onto these disks and incubated for 48 h. Tartrate resistant acid phosphatase (TRAP) staining confirmed the presence of osteoclasts on all disks. Actin ring staining that detected actively resorbing OCs showed an inverse linear correlation between the number of actively resorbing osteoclasts (percentage of total OCs on the surfaces) with surface microporosity. These findings should be taken into consideration for the design and/or production of new β-TCP bone graft substitutes.

Type I Phosphotidylinosotol 4-Phosphate 5-Kinase γ Regulates Osteoclasts in a Bifunctional Manner*

Type 1 phosphotidylinosotol-4 phosphate 5 kinase γ (PIP5KIγ) is central to generation of phosphotidylinosotol (4,5)P(2) (PI(4,5)P(2)). PIP5KIγ also participates in cytoskeletal organization by delivering talin to integrins, thereby enhancing their ligand binding capacity. As the cytoskeleton is pivotal to osteoclast function, we hypothesized that absence of PIP5KIγ would compromise their resorptive capacity. Absence of the kinase diminishes PI(4,5) abundance and desensitizes precursors to RANK ligand-stimulated differentiation. Thus, PIP5KIγ(-/-) osteoclasts are reduced in number in vitro and confirm physiological relevance in vivo. Despite reduced numbers, PIP5KIγ(-/-) osteoclasts surprisingly have normal cytoskeletons and effectively resorb bone. PIP5KIγ overexpression, which increases PI(4,5)P(2), also delays osteoclast differentiation and reduces cell number but in contrast to cells lacking the kinase, its excess disrupts the cytoskeleton. The cytoskeleton-disruptive effects of excess PIP5KIγ reflect its kinase activity and are independent of talin recognition. The combined arrested differentiation and disorganized cytoskeleton of PIP5KIγ-transduced osteoclasts compromises bone resorption. Thus, optimal PIP5KIγ and PI(4,5)P(2) expression, by osteoclasts, are essential for skeletal homeostasis.

Deterioration of Glaucoma After Carotid Endarterectomy

A 64-year-old man with a long history of untreated diabetes mellitus had suffered from visual disturbance in his right eye. Neovascular glaucoma in the right eye and diabetic retinopathy in both eyes were found, and ischemic ocular syndrome was suspected for the right eye. Neuroimaging revealed severe stenosis of the right internal carotid artery. He was first treated for diabetes and glaucoma, and then, after these conditions were stabilized, right carotid endarterectomy (CEA) was carried out. Although the operation was uneventful, he suffered from headache and his right sight was blurred on the day after surgery. Right intraocular pressure was markedly increased, and corneal edema and increased iris neovascularization were also recognized. Intensive ophthalmologic care was carried out, but his right vision worsened and was eventually lost. Ocular ischemia causes not only neovascularization of the iris, which leads to insufficient resorption of the aqueous humor, but also insufficient production of the aqueous humor. After CEA, production of the humor is immediately activated, but the resorption capacity does not change, which results in an extraordinary increase in intraocular pressure. Neurosurgeons should be aware that CEA not only improves or avoids worsening of vision in patients with ischemic oculopathy, but can also rarely cause paradoxical devastating visual deterioration.

Magnesium

Magnesium

C-Fms Signaling Mediates Neurofibromatosis Type-1 Osteoclast Gain-In-Functions

Skeletal abnormalities including osteoporosis and osteopenia occur frequently in both pediatric and adult neurofibromatosis type 1 (NF1) patients. NF1 (Nf1) haploinsufficient osteoclasts and osteoclast progenitors derived from both NF1 patients and Nf1+/− mice exhibit increased differentiation, migration, and bone resorptive capacity in vitro, mediated by hyperactivation of p21Ras in response to limiting concentrations of macrophage-colony stimulating factor (M-CSF). Here, we show that M-CSF binding to its receptor, c-Fms, results in increased c-Fms activation in Nf1+/ − osteoclast progenitors, mediating multiple gain-in-functions through the downstream effectors Erk1/2 and p90RSK. PLX3397, a potent and selective c-Fms inhibitor, attenuated M-CSF mediated Nf1+/− osteoclast migration by 50%, adhesion by 70%, and pit formation by 60%. In vivo, we administered PLX3397 to Nf1 +/− osteoporotic mice induced by ovariectomy (OVX) and evaluated changes in bone mass and skeletal architecture. We found that PLX3397 prevented bone loss in Nf1+/−-OVX mice by reducing osteoclast differentiation and bone resorptive activity in vivo. Collectively, these results implicate the M-CSF/c-Fms signaling axis as a critical pathway underlying the aberrant functioning of Nf1 haploinsufficient osteoclasts and may provide a potential therapeutic target for treating NF1 associated osteoporosis and osteopenia.