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1.  Skeletal Recovery After Weaning Does Not Require PTHrP 
Journal of Bone and Mineral Research  2011;26(6):1242-1251.
Mice lose 20% to 25% of trabecular bone mineral content (BMC) during lactation and restore it after weaning through unknown mechanisms. We found that tibial Pthrp mRNA expression was upregulated fivefold by 7 days after weaning versus end of lactation in wild-type (WT) mice. To determine whether parathyroid hormone–related protein (PTHrP) stimulates bone formation after weaning, we studied a conditional knockout in which PTHrP is deleted from preosteoblasts and osteoblasts by collagen I promoter–driven Cre (CreColI). These mice are osteopenic as adults but have normal serum calcium, calcitriol, and parathyroid hormone (PTH). Pairs of Pthrpflox/flox;CreColI (null) and WT;CreColI (WT) females were mated and studied through pregnancy, lactation, and 3 weeks of postweaning recovery. By end of lactation, both genotypes lost lumbar spine BMC: WT declined by 20.6% ± 3.3%, and null decreased by 22.5% ± 3.5% (p < .0001 versus baseline; p = NS between genotypes). During postweaning recovery, both restored BMC to baseline: WT to –3.6% ± 3.7% and null to 0.3% ± 3.7% (p = NS versus baseline or between genotypes). Similar loss and full recovery of BMC were seen at the whole body and hind limb. Histomorphometry confirmed that nulls had lower bone mass at baseline and that this was equal to the value achieved after weaning. Osteocalcin, propeptide of type 1 collagen (P1NP), and deoxypyridinoline increased equally during recovery in WT and null mice; PTH decreased and calcitriol increased equally; serum calcium was unchanged. Urine calcium increased during recovery but remained no different between genotypes. Although osteoblast-derived PTHrP is required to maintain adult bone mass and Pthrp mRNA upregulates in bone after weaning, it is not required for recovery of bone mass after lactation. The factors that stimulate postweaning bone formation remain unknown. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179289  PMID: 21308774
Pregnancy; Lactation; PTH/PTHRP; Bone Mineralization; Histomorphometry; Knockout; Animal Models/rodent; Growth and Development
2.  Selective Venous Catheterization for the Localization of Phosphaturic Mesenchymal Tumors 
Journal of Bone and Mineral Research  2010;26(6):1295-1302.
Tumor-induced osteomalacia (TIO) is characterized by renal phosphate wasting, hypophosphatemia, and aberrant vitamin D3 metabolism and is caused by fibroblast growth factor 23 (FGF-23)–producing mesenchymal tumors, which are often difficult to locate. We investigated the utility of selective venous sampling in tumor localization. The primary endpoint was identification of the FGF-23 concentration ratio between the venous drainage of the tumor bed and the general circulation that was diagnostic of the location of an FGF-23-secreting tumor. Fourteen subjects underwent 15 sampling procedures after functional and anatomic imaging studies. Subjects fit into three imaging categories: no suspicious site, multiple sites, and single site (positive controls). FGF-23 levels were measured by ELISA. Suspicious tumors were resected for diagnosis, confirmation, and cure. In subjects with a positive venous sampling study and subsequent cure, a minimum ratio of 1.6 was diagnostic. In 7 of 14 subjects there was suggestive imaging, a diagnostic ratio, and an associated TIO tumor (true positive). Four of these required complicated resection procedures. In 4 of 14 subjects with no suspicious site on imaging studies, an FGF-23 diagnostic ratio was not detected (true negative). Biopsy or resection of a single lesion in 2 of 14 subjects with a diagnostic ratio failed to identify a TIO tumor (false positive). A diagnostic FGF-23 ratio was absent in 1 of 14 subjects whose tumor was a single highly suspicious lesion on imaging studies (false negative). These data yield a sensitivity of 0.87 [95% confidence interval (CI) 0.47–0.99] and a specificity of 0.71 (95% CI 0.29–0.96). Selective venous sampling for FGF-23 was particularly useful in subjects with multiple suspicious sites or an anatomically challenging planned resection but not in the absence of a suspicious lesion on imaging studies. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179290  PMID: 21611969
FGF-23; Osteomalacia; Rickets; Tumor-Induced Osteomalacia; Oncogenic Osteomalacia
3.  The Calcium-Sensing Receptor Mediates Bone Turnover Induced by Dietary Calcium and Parathyroid Hormone in Neonates 
Journal of Bone and Mineral Research  2010;26(5):1057-1071.
We have investigated, in neonates, whether the calcium-sensing receptor (CaR) mediates the effects of dietary calcium on bone turnover and/or modulates parathyroid hormone (PTH)–induced bone turnover. Wild-type (WT) pups and pups with targeted deletion of the Pth (Pth–/–) gene or of both Pth and CaR (Pth–/–CaR–/–) genes were nursed by dams on a normal or high-calcium diet. Pups nursed by dams on a normal diet received daily injections of vehicle or of PTH(1–34) (80 µg/kg) for 2 weeks starting from 1 week of age. In pups receiving vehicle and fed by dams on a normal diet, trabecular bone volume, osteoblast number, type 1 collagen–positive area, and mineral apposition rate, as well as the expression of bone-formation-related genes, all were reduced significantly in Pth–/– pups compared with WT pups and were decreased even more dramatically in Pth–/–CaR–/– pups. These parameters were increased in WT and Pth–/– pups but not in Pth–/–CaR–/– pups fed by dams on a high-calcium diet compared with pups fed by dams on a normal diet. These parameters also were increased in WT, Pth–/–, and Pth–/–CaR–/– pups following exogenous PTH treatment; however, the percentage increase was less in Pth–/–CaR–/– pups than in WT and Pth–/– pups. In vehicle-treated pups fed by dams on either the normal or high-calcium diet and in PTH-treated pups fed by dams on a normal diet, the number and surfaces of osteoclasts and the ratio of RANKL/OPG were reduced significantly in Pth–/– pups and less significantly in Pth–/–CaR–/– pups compared with WT pups. These parameters were further reduced significantly in WT and Pth–/– pups from dams fed a high-calcium diet but did not decrease significantly in similarly treated Pth–/–CaR–/– pups, and they increased significantly in PTH-treated pups compared with vehicle-treated, genotype-matched pups fed by dams on the normal diet. These results indicate that in neonates, the CaR mediates alterations in bone turnover in response to changes in dietary calcium and modulates PTH-stimulated bone turnover. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179300  PMID: 21542007
4.  Epidermal Growth Factor Receptor Plays an Anabolic Role in Bone Metabolism In Vivo 
Journal of Bone and Mineral Research  2010;26(5):1022-1034.
While the epidermal growth factor receptor (EGFR)–mediated signaling pathway has been shown to have vital roles in many developmental and pathologic processes, its functions in the development and homeostasis of the skeletal system has been poorly defined. To address its in vivo role, we constructed transgenic and pharmacologic mouse models and used peripheral quantitative computed tomography (pQCT), micro–computed tomography (µCT) and histomorphometry to analyze their trabecular and cortical bone phenotypes. We initially deleted the EGFR in preosteoblasts/osteoblasts using a Cre/loxP system (Col-Cre Egfrf/f), but no bone phenotype was observed because of incomplete deletion of the Egfr genomic locus. To further reduce the remaining osteoblastic EGFR activity, we introduced an EGFR dominant-negative allele, Wa5, and generated Col-Cre EgfrWa5/f mice. At 3 and 7 months of age, both male and female mice exhibited a remarkable decrease in tibial trabecular bone mass with abnormalities in trabecular number and thickness. Histologic analyses revealed decreases in osteoblast number and mineralization activity and an increase in osteoclast number. Significant increases in trabecular pattern factor and structural model index indicate that trabecular microarchitecture was altered. The femurs of these mice were shorter and smaller with reduced cortical area and periosteal perimeter. Moreover, colony-forming unit–fibroblast (CFU-F) assay indicates that these mice had fewer bone marrow mesenchymal stem cells and committed progenitors. Similarly, administration of an EGFR inhibitor into wild-type mice caused a significant reduction in trabecular bone volume. In contrast, EgfrDsk5/+ mice with a constitutively active EGFR allele displayed increases in trabecular and cortical bone content. Taken together, these data demonstrate that the EGFR signaling pathway is an important bone regulator and that it primarily plays an anabolic role in bone metabolism. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179301  PMID: 21542005
5.  Vitamin K Status in Spaceflight and Ground-Based Models of Spaceflight 
Bone loss is a well-documented change during and after long-duration spaceflight. Many types of countermeasures to bone loss have been proposed, including vitamin K supplementation. The objective of this series of studies was to measure change in vitamin K status in response to microgravity under a variety of spaceflight and spaceflight analog (model) conditions, including long-duration spaceflight studies (n = 15), three bed rest studies (n = 15, 49, and 24), and a 14-day saturation dive (n= 6). In crew members who flew 2–6 months on the International Space Station, in-flight and postflight plasma phylloquinone concentrations were unchanged from the preflight mean. Consistent with this finding, urinary γ-carboxyglutamic acid (GLA), a measure of vitamin K-dependent protein turnover, did not change in response to flight. Serum undercarboxylated osteocalcin (%ucOC), a measure of vitamin K function, was generally unchanged in response to flight. Spaceflight findings were corroborated by findings of no changes in phylloquinone, urinary GLA, or %ucOC during or after bed rest in three separate bed rest studies (21–90 days in duration) or after a 14-day saturation dive. The data presented here do not support either a need for vitamin K supplementation during spaceflight or the suggestion of using vitamin K as a bone loss countermeasure in spaceflight. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179302  PMID: 21541997
6.  Effects of 25-Hydroxyvitamin D3 on Proliferation and Osteoblast Differentiation of Human Marrow Stromal Cells Require CYP27B1/1α-Hydroxylase 
Journal of Bone and Mineral Research  2010;26(5):1145-1153.
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has many noncalcemic actions that rest on inhibition of proliferation and promotion of differentiation in malignant and normal cell types. 1,25(OH)2D3 stimulates osteoblast differentiation of human marrow stromal cells (hMSCs), but little is known about the effects of 25-hydroxyvitamin D3 [25(OH)D3] on these cells. Recent evidence shows that hMSCs participate in vitamin D metabolism and can activate 25(OH)D3 by CYP27B1/1α-hydroxylase. These studies test the hypothesis that antiproliferative and prodifferentiation effects of 25(OH)D3 in hMSCs depend on CYP27B1. We studied hMSCs that constitutively express high (hMSCshi-1α) or low (hMSCslo-1α) levels of CYP27B1 with equivalent expression of CYP24A1 and vitamin D receptor. In hMSCshi-1α, 25(OH)D3 reduced proliferation, downregulated proliferating cell nuclear antigen (PCNA), upregulated p21Waf1/Cip1, and decreased cyclin D1. Unlike 1,25(OH)2D3, the antiapoptotic effects of 25(OH)D3 on Bax and Bcl-2 were blocked by the P450 inhibitor ketoconazole. The antiproliferative effects of 25(OH)D3 in hMSCshi-1α and of 1,25(OH)2D3 in both samples of hMSCs were explained by cell cycle arrest, not by increased apoptosis. Stimulation of osteoblast differentiation in hMSCshi-1α by 25(OH)D3 was prevented by ketoconazole and upon transfection with CYP27B1 siRNA. These data indicate that CYP27B1 is required for 25(OH)D3's action in hMSCs. Three lines of evidence indicate that CYP27B1 is required for the antiproliferative and prodifferentiation effects of 25(OH)D3 on hMSCs: Those effects were not seen (1) in hMSCs with low constitutive expression of CYP27B1, (2) in hMSCs treated with ketoconazole, and (3) in hMSCs in which CYP27B1 expression was silenced. Osteoblast differentiation and skeletal homeostasis may be regulated by autocrine/paracrine actions of 25(OH)D3 in hMSCs. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179303  PMID: 21542014
7.  Impact of Maturational Status on the Ability of Osteoblasts to Enhance the Hematopoietic Function of Stem and Progenitor Cells 
Journal of Bone and Mineral Research  2010;26(5):1111-1121.
Osteoblasts (OBs) exert a prominent regulatory effect on hematopoietic stem cells (HSCs). We evaluated the difference in hematopoietic expansion and function in response to co-culture with OBs at various stages of development. Murine calvarial OBs were seeded directly (fresh) or cultured for 1, 2, or 3 weeks prior to seeding with 1000 Lin-Sca1 + cKit+ (LSK) cells for 1 week. Significant increases in the following hematopoietic parameters were detected when comparing co-cultures of fresh OBs to co-cultures containing OBs cultured for 1, 2, or 3 weeks: total hematopoietic cell number (up to a 3.4-fold increase), total colony forming unit (CFU) number in LSK progeny (up to an 18.1-fold increase), and percentage of Lin-Sca1+ cells (up to a 31.8-fold increase). Importantly, these studies were corroborated by in vivo reconstitution studies in which LSK cells maintained in fresh OB co-cultures supported a significantly higher level of chimerism than cells maintained in co-cultures containing 3-week OBs. Characterization of OBs cultured for 1, 2, or 3 weeks with real-time PCR and functional mineralization assays showed that OB maturation increased with culture duration but was not affected by the presence of LSK cells in culture. Linear regression analyses of multiple parameters measured in these studies show that fresh, most likely more immature OBs better promote hematopoietic expansion and function than cultured, presumably more mature OBs and suggest that the hematopoiesis-enhancing activity is mediated by cells present in fresh OB cultures de novo. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179304  PMID: 21542011
8.  Unique Roles of Phosphorus in Endochondral Bone Formation and Osteocyte Maturation 
Journal of Bone and Mineral Research  2010;26(5):1047-1056.
The mechanisms by which inorganic phosphate (Pi) homeostasis controls bone biology are poorly understood. Here we used Dmp1 null mice, a hypophosphatemic rickets/osteomalacia model, combined with a metatarsal organ culture and an application of neutralizing fibroblast growth factor 23 (FGF-23) antibodies to gain insight into the roles of Pi in bone biology. We showed (1) that abnormal bone remodeling in Dmp1 null mice is due to reduced osteoclast number, which is secondary to a reduced ratio of RANKL/OPG expressed by osteoclast supporting cells and (2) that osteoblast extracellular matrix mineralization, growth plate maturation, secondary ossification center formation, and osteoblast differentiation are phosphate-dependent. Finally, a working hypothesis is proposed to explain how phosphate and DMP1 control osteocyte maturation. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179305  PMID: 21542006
9.  Mechanical Contributions of the Cortical and Trabecular Compartments Contribute to Differences in Age-Related Changes in Vertebral Body Strength in Men and Women Assessed by QCT-Based Finite Element Analysis 
The biomechanical mechanisms underlying sex-specific differences in age-related vertebral fracture rates are ill defined. To gain insight into this issue, we used finite element analysis of clinical computed tomography (CT) scans of the vertebral bodies of L3 and T10 of young and old men and women to assess age- and sex-related differences in the strength of the whole vertebra, the trabecular compartment, and the peripheral compartment (the outer 2 mm of vertebral bone, including the thin cortical shell). We sought to determine whether structural and geometric changes with age differ in men and women, making women more susceptible to vertebral fractures. As expected, we found that vertebral strength decreased with age 2-fold more in women than in men. The strength of the trabecular compartment declined significantly with age for both sexes, whereas the strength of the peripheral compartment decreased with age in women but was largely maintained in men. The proportion of mechanical strength attributable to the peripheral compartment increased with age in both sexes and at both vertebral levels. Taken together, these results indicate that men and women lose vertebral bone differently with age, particularly in the peripheral (cortical) compartment. This differential bone loss explains, in part, a greater decline in bone strength in women and may contribute to the higher incidence of vertebral fractures among women than men. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179306  PMID: 21542000
10.  PTH Receptor Signaling in Osteocytes Governs Periosteal Bone Formation and Intracortical Remodeling 
Journal of Bone and Mineral Research  2010;26(5):1035-1046.
The periosteal and endocortical surfaces of cortical bone dictate the geometry and overall mechanical properties of bone. Yet the cellular and molecular mechanisms that regulate activity on these surfaces are far from being understood. Parathyroid hormone (PTH) has profound effects in cortical bone, stimulating periosteal expansion and at the same time accelerating intracortical bone remodeling. We report herein that transgenic mice expressing a constitutive active PTH receptor in osteocytes (DMP1-caPTHR1 mice) exhibit increased cortical bone area and an elevated rate of periosteal and endocortical bone formation. In addition, DMP1-caPTHR1 mice display a marked increase in intracortical remodeling and cortical porosity. Crossing DMP1-caPTHR1 mice with mice lacking the Wnt coreceptor, LDL-related receptor 5 (LRP5), or with mice overexpressing the Wnt antagonist Sost in osteocytes (DMP1-Sost mice) reduced or abolished, respectively, the increased cortical bone area, periosteal bone formation rate, and expression of osteoblast markers and Wnt target genes exhibited by the DMP1-caPTHR1 mice. In addition, DMP1-caPTHR1 lacking LRP5 or double transgenic DMP1-caPTHR1;DMP1-Sost mice exhibit exacerbated intracortical remodeling and increased osteoclast numbers, and markedly decreased expression of the RANK decoy receptor osteoprotegerin. Thus, whereas Sost downregulation and the consequent Wnt activation is required for the stimulatory effect of PTH receptor signaling on periosteal bone formation, the Wnt-independent increase in osteoclastogenesis induced by PTH receptor activation in osteocytes overrides the effect on Sost. These findings demonstrate that PTH receptor signaling influences cortical bone through actions on osteocytes and defines the role of Wnt signaling in PTH receptor action. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179307  PMID: 21140374
11.  Camurati-Engelmann Disease: Unique Variant Featuring a Novel Mutation in TGFβ1 Encoding Transforming Growth Factor Beta 1 and a Missense Change in TNFSF11 Encoding RANK Ligand 
We report a 32-year-old man and his 59-year-old mother with a unique and extensive variant of Camurati-Engelmann disease (CED) featuring histopathological changes of osteomalacia and alterations within TGFβ1 and TNFSF11 encoding TGFβ1 and RANKL, respectively. He suffered leg pain and weakness since childhood and reportedly grew until his late 20s, reaching 7 feet in height. He had deafness, perforated nasal septum, torus palatinus, disproportionately long limbs with knock-knees, low muscle mass, and pseudoclubbing. Radiographs revealed generalized skeletal abnormalities, including wide bones and cortical and trabecular bone thickening in keeping with CED, except that long bone ends were also affected. Lumbar spine and hip BMD Z-scores were + 7.7 and + 4.4, respectively. Biochemical markers of bone turnover were elevated. Hypocalciuria accompanied low serum 25-hydroxyvitamin D (25[OH]D) levels. Pituitary hypogonadism and low serum insulin-like growth factor (IGF)-1 were present. Karyotype was normal. Despite vitamin D repletion, iliac crest histology revealed severe osteomalacia. Exon 1 of TNFRSF11A (RANK), exons 2, 3, and 4 of LRP5, and all coding exons and adjacent mRNA splice junctions of TNFRSF11B (OPG), SQSTM1 (sequestosome 1), and TNSALP (tissue nonspecific alkaline phosphatase) were intact. His asymptomatic and less dysmorphic 5′11″ mother, also with low serum 25(OH)D, had milder clinical, radiological, biochemical, and histopathological findings. Both individuals were heterozygous for a novel 12-bp duplication (c.27_38dup, p.L10_L13dup) in exon 1 of TGFβ1, predicting four additional leucine residues in the latency-associated-peptide segment of TGFβ1, consistent with CED. The son was also homozygous for a single base transversion in TNFSF11, predicting a nonconservative amino acid change (c.107C > G, p.Pro36Arg) in the intracellular domain of RANKL that was heterozygous in his nonconsanguineous parents. This TNFSF11 variant was not found in the SNP Database, nor in published TNFSF11 association studies, but it occurred in four of the 134 TNFSF11 alleles (3.0%) we tested randomly among individuals without CED. Perhaps the unique phenotype of this CED family is conditioned by altered RANKL activity. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179308  PMID: 21541994
12.  Association of Menopausal Vasomotor Symptoms With Increased Bone Turnover During the Menopausal Transition 
The purpose of this study was to determine the longitudinal association between menopausal vasomotor symptoms (VMS) and urinary N-telopeptide level (NTX) according to menopausal stage. We analyzed data from 2283 participants of the Study of Women's Health Across the Nation, a longitudinal community-based cohort study of women aged 42 to 52 years at baseline. At baseline and annually through follow-up visit 8, participants provided questionnaire data, urine samples, serum samples, and anthropometric measurements. Using multivariable repeated-measures mixed models, we examined associations between annually assessed VMS frequency and annual NTX measurements. Our results show that mean adjusted NTX was 1.94 nM of bone collagen equivalents (BCE)/mM of creatinine higher among early perimenopausal women with any VMS than among early perimenopausal women with no VMS (p < .0001). Mean adjusted NTX was 2.44 nM BCE/mM of creatinine higher among late perimenopausal women with any VMS than among late perimenopausal women with no VMS (p = .03). Among premenopausal women, VMS frequency was not significantly associated with NTX level. When NTX values among women with frequent VMS (≥6 days in past 2 weeks) were expressed as percentages of NTX values among women without frequent VMS, the differences were 3% for premenopausal women, 9% for early perimenopausal women, 7% for late perimenopausal women, and 4% for postmenopausal women. Adjustment for serum follicle-stimulating hormone (FSH) level greatly reduced the magnitudes of associations between VMS and NTX level. We conclude that among early perimenopausal and late perimenopausal women, those with VMS had higher bone turnover than those without VMS. Prior to the final menstrual period, VMS may be a marker for risk of adverse bone health. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179323  PMID: 20878774
13.  Nell-1, a key Functional Mediator of Runx2, Partially Rescues Calvarial Defects in Runx2+/− Mice 
Mesenchymal stem cell commitment to an osteoprogenitor lineage requires the activity of Runx2, a molecule implicated in the etiopathology of multiple congenital craniofacial anomalies. Through promoter analyses, we have recently identified a new direct transcriptional target of Runx2, Nell-1, a craniosynostosis (CS)–associated molecule with potent osteogenic properties. This study investigated the mechanistic and functional relationship between Nell-1 and Runx2 in regulating osteoblast differentiation. The results showed that spatiotemporal distribution and expression levels of Nell-1 correlated closely with those of endogenous Runx2 during craniofacial development. Phenotypically, cross-mating Nell-1 overexpression transgenic (CMV-Nell-1) mice with Runx2 haploinsufficient (Runx2+/−) mice partially rescued the calvarial defects in the cleidocranial dysplasia (CCD)–like phenotype of Runx2+/− mice, whereas Nell-1 protein induced mineralization and bone formation in Runx2+/− but not Runx2−/− calvarial explants. Runx2-mediated osteoblastic gene expression and/or mineralization was severely reduced by Nell-1 siRNA oligos transfection into Runx2+/+ newborn mouse calvarial cells (NMCCs) or in N-ethyl-N-nitrosourea (ENU)–induced Nell-1−/− NMCCs. Meanwhile, Nell-1 overexpression partially rescued osteoblastic gene expression but not mineralization in Runx2 null (Runx2−/−) NMCCs. Mechanistically, irrespective of Runx2 genotype, Nell-1 signaling activates ERK1/2 and JNK1 mitogen-activated protein kinase (MAPK) pathways in NMCCs and enhances Runx2 phosphorylation and activity when Runx2 is present. Collectively, these data demonstrate that Nell-1 is a critical downstream Runx2 functional mediator insofar as Runx2-regulated Nell-1 promotes osteoblastic differentiation through, in part, activation of MAPK and enhanced phosphorylation of Runx2, and Runx2 activity is significantly reduced when Nell-1 is blocked or absent. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179324  PMID: 20939017
14.  Determinants of the Mechanical Behavior of Human Lumbar Vertebrae After Simulated Mild Fracture 
The ability of a vertebra to carry load after an initial deformation and the determinants of this postfracture load-bearing capacity are critical but poorly understood. This study aimed to determine the mechanical behavior of vertebrae after simulated mild fracture and to identify the determinants of this postfracture behavior. Twenty-one human L3 vertebrae were analyzed for bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA) and for microarchitecture by micro–computed tomography (µCT). Mechanical testing was performed in two phases: initial compression of vertebra to 25% deformity, followed, after 30 minutes of relaxation, by a similar test to failure to determine postfracture behavior. We assessed (1) initial and postfracture mechanical parameters, (2) changes in mechanical parameters, (3) postfracture elastic behavior by recovery of vertebral height after relaxation, and (4) postfracture plastic behavior by residual strength and stiffness. Postfracture failure load and stiffness were 11% ± 19% and 53% ± 18% lower than initial values (p = .021 and p < .0001, respectively), with 29% to 69% of the variation in the postfracture mechanical behavior explained by the initial values. Both initial and postfracture mechanical behaviors were significantly correlated with bone mass and microarchitecture. Vertebral deformation recovery averaged 31% ± 7% and was associated with trabecular and cortical thickness (r = 0.47 and r = 0.64; p = .03 and p = .002, respectively). Residual strength and stiffness were independent of bone mass and initial mechanical behavior but were related to trabecular and cortical microarchitecture (|r| = 0.50 to 0.58; p = .02 to .006). In summary, we found marked variation in the postfracture load-bearing capacity following simulated mild vertebral fractures. Bone microarchitecture, but not bone mass, was associated with postfracture mechanical behavior of vertebrae. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179325  PMID: 20928886
15.  Blockade of Receptor-Activated Gi Signaling in Osteoblasts In Vivo Leads to Site-Specific Increases in Cortical and Cancellous Bone Formation 
Osteoblasts play a critical role in the maintenance of bone mass through bone formation and regulation of bone resorption. Targeted expression of a constitutively active engineered Gi-coupled G protein–coupled receptor (GPCR) to osteoblasts in vivo leads to severe osteopenia. However, little is known about the role of endogenous receptor-mediated Gi signaling in regulating osteoblast function. In this study, we investigated the skeletal effects of blocking Gi-coupled signaling in osteoblasts in vivo. This was accomplished by transgenic expression of the catalytic subunit of pertussis toxin (PTX) under control of the collagen Iα 2.3-kb promoter. These mice, designated Col1(2.3)+/PTX+, showed increased cortical thickness at the femoral midshaft at 12 weeks of age. This correlated with increased periosteal bone formation associated with expanded mineralizing surface observed in 8-week-old mice of both genders. The cancellous bone phenotype of the Col1(2.3)+/PTX+ mice was sexually dimorphic, with increases in fractional bone volume at the distal femur seen only in females. Similarly, while cancellous bone-formation rates were unchanged in males, they could not be quantified for female Col1(2.3)+/PTX+ mice owing to the disorganized nature of the labeling pattern, which was consistent with rapid formation of woven bone. Alterations in osteoclast activity did not appear to participate in the phenotype. These data demonstrate that Gi-coupled signaling by GPCRs endogenous to osteoblasts plays a complex role in the regulation of bone formation in a manner that is dependent on both gender and the anatomic site within bone. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179326  PMID: 20939063
16.  Systems Genetics Analysis of Mouse Chondrocyte Differentiation 
One of the goals of systems genetics is the reconstruction of gene networks that underlie key processes in development and disease. To identify cartilage gene networks that play an important role in bone development, we used a systems genetics approach that integrated microarray gene expression profiles from cartilage and bone phenotypic data from two sets of recombinant inbred strains. Microarray profiles generated from isolated chondrocytes were used to generate weighted gene coexpression networks. This analysis resulted in the identification of subnetworks (modules) of coexpressed genes that then were examined for relationships with bone geometry and density. One module exhibited significant correlation with femur length (r = 0.416), anteroposterior diameter (r = 0.418), mediolateral diameter (r = 0.576), and bone mineral density (r = 0.475). Highly connected genes (n = 28) from this and other modules were tested in vitro using prechondrocyte ATDC5 cells and RNA interference. Five of the 28 genes were found to play a role in chondrocyte differentiation. Two of these, Hspd1 and Cdkn1a, were known previously to function in chondrocyte development, whereas the other three, Bhlhb9, Cugbp1, and Spcs3, are novel genes. Our integrative analysis provided a systems-level view of cartilage development and identified genes that may be involved in bone development. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179327  PMID: 20954177
17.  EP1−/− Mice Have Enhanced Osteoblast Differentiation and Accelerated Fracture Repair 
As a downstream product of cyclooxygenase 2 (COX-2), prostaglandin E2 (PGE2) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro–computed tomographic (µCT), and biomechanical measures. EP1−/− mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1−/− mice also had an earlier appearance of tartrate-resistant acid phosphatase (TRAcP)–positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1−/− mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE2 receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179328  PMID: 20939055
18.  The Relationship Between Bisphosphonate Adherence and Fracture: Is It the Behavior or the Medication? Results From the Placebo Arm of the Fracture Intervention Trial 
Medication compliance may be a surrogate for factors that improve health outcomes such as fractures. Little is known about the size of this potential “healthy adherer” effect. We evaluated the hypothesis that compliance with placebo is associated inversely with bone loss and fractures among women participating in the Fracture Intervention Trial (FIT). Compliance with placebo and alendronate was evaluated using daily medication diaries. Women were defined as having high compliance if they took 80% or more of dispensed study medication. Change in bone mineral density (BMD) was assessed using mixed models comparing women with high versus lower compliance with placebo. Cox proportional-hazards models analyzed the association between placebo compliance and various types of fractures. Among 3169 women randomized to placebo, 82% had high compliance. Compared with women with lower placebo compliance, bone loss at the total hip was lower in compliant placebo-treated women (−0.43%/year versus −0.58%/year, p = .04). Among placebo-treated women, there were 46 hip, 110 wrist, 77 clinical vertebral, and 492 total clinical fractures. Compared with women with lower placebo compliance, women with high placebo compliance had a nonsignificant reduced risk for hip fracture [adjusted hazard ratio (HR) = 0.67, 95% confidence interval (CI) 0.30–1.45]. This trend was not observed for other fractures. Medication compliance may be a proxy for factors that confers benefit on reducing hip fracture (but not other types of fractures) independent of the effect of the medication itself. Nonrandomized studies of interventions designed to maintain or improve bone density and/or hip fracture may need to consider medication compliance as a confounder to better estimate true intervention effects. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179329  PMID: 20939064
19.  Growth Hormone Mediates Pubertal Skeletal Development Independent of Hepatic IGF-1 Production 
Deficiencies in either growth hormone (GH) or insulin-like growth factor 1 (IGF-1) are associated with reductions in bone size during growth in humans and animal models. Liver-specific IGF-1-deficient (LID) mice, which have 75% reductions in serum IGF-1, were created previously to separate the effects of endocrine (serum) IGF-1 from autocrine/paracrine IGF-1. However, LID mice also have two- to threefold increases in GH, and this may contribute to the observed pubertal skeletal phenotype. To clarify the role of GH in skeletal development under conditions of significantly reduced serum IGF-1 levels (but normal tissue IGF-1 levels), we studied the skeletal response of male LID and control mice to GH inhibition by pegvisomant from 4 to 8 weeks of age. Treatment of LID mice with pegvisomant resulted in significant reductions in body weight, femur length (Le), and femur total area (Tt.Ar), as well as further reductions in serum IGF-1 levels by 8 weeks of age, compared with the mean values of vehicle-treated LID mice. Reductions in both Tt.Ar and Le were proportional after treatment with pegvisomant. On the other hand, the relative amount of cortical tissue formed (RCA) in LID mice treated with pegvisomant was significantly less than that in both vehicle-treated LID and control mice, indicating that antagonizing GH action, either directly (through GH receptor signaling inhibition) or indirectly (through further reductions in serum/tissue IGF-1 levels), results in disproportionate reductions in the amount of cortical bone formed. This resulted in bones with significantly reduced mechanical properties (femoral whole-bone stiffness and work to failure were markedly decreased), suggesting that compensatory increases of GH in states of IGF-1 deficiency (LID mice) act to protect against a severe inhibition of bone modeling during growth, which otherwise would result in bones that are too weak for normal and/or extreme loading conditions. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179330  PMID: 20928887
20.  Calpain-6, a Target Molecule of Glucocorticoids, Regulates Osteoclastic Bone Resorption via Cytoskeletal Organization and Microtubule Acetylation 
Glucocorticoids (GCs) inhibit the resorptive capacity of the osteoclast by disrupting its cytoskeleton. We find that calpain-6 (Capn6), a unique, nonproteolytic member of its family, is suppressed 12-fold by dexamethasone (DEX) in the bone-degrading cell. While Capn6 abundance parallels commitment of naive bone marrow macrophages (BMMs) to the osteoclast phenotype, its excess or deletion does not affect the cell's differentiation. On the other hand, Capn6 localizes to the sealing zone, and its overexpression promotes osteoclast spreading and large actin ring formation, eventuating in stimulated bone degradation. Conversely, Capn6 knockdown impairs cytoskeletal organization and the cell's resorptive capacity. Capn6 complexes with tubulin, and its absence inhibits microtubule acetylation and stability in the osteoclast. Knockdown of Capn6 also reduces β3-integrin subunit protein, another essential regulator of osteoclast cytoskeletal function. Reflecting Capn6 as a target molecule of GCs, microtubule stability and acetylation, as well as the expression of β3-integrin protein, are similarly suppressed in DEX-treated osteoclasts. Moreover, overexpression of Capn6 rescues GC-mediated disruption of osteoclast cytoskeleton. Thus Capn6 promotes cytoskeletal organization and microtubule stability in osteoclasts, and its inhibition may mediate the resorption-arresting properties of GCs. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179291  PMID: 20814968
21.  Differential Maintenance of Cortical and Cancellous Bone Strength Following Discontinuation of Bone-Active Agents 
Osteoporotic patients treated with antiresorptive or anabolic agents experience an increase in bone mass and a reduction in incident fractures. However, the effects of these medications on bone quality and strength after a prolonged discontinuation of treatment are not known. We evaluated these effects in an osteoporotic rat model. Six-month-old ovariectomized (OVX) rats were treated with placebo, alendronate (ALN, 2 µg/kg), parathyroid hormone [PTH(1–34); 20 µg/kg], or raloxifene (RAL, 2 mg/kg) three times a week for 4 months and withdrawn from the treatments for 8 months. Treatment with ALN, PTH, and RAL increased the vertebral trabecular bone volume (BV/TV) by 47%, 53%, and 31%, with corresponding increases in vertebral compression load by 27%, 51%, and 31%, respectively (p < .001). The resulting bone strength was similar to that of the sham-OVX control group with ALN and RAL and higher (p < .001) with PTH treatment. After 4 months of withdrawal, bone turnover (BFR/BS) remained suppressed in the ALN group versus the OVX controls (p < .001). The vertebral strength was higher than in the OVX group only in ALN-treated group (p < .05), whereas only the PTH-treated animals showed a higher maximum load in tibial bending versus the OVX controls (p < .05). The vertebral BV/TV returned to the OVX group level in both the PTH and RAL groups 4 months after withdrawal but remained 25% higher than the OVX controls up to 8 months after withdrawal of ALN (p < .05). Interestingly, cortical bone mineral density increased only with PTH treatment (p < .05) but was not different among the experimental groups after withdrawal. At 8 months after treatment withdrawal, none of the treatment groups was different from the OVX control group for cortical or cancellous bone strength. In summary, both ALN and PTH maintained bone strength (maximum load) 4 months after discontinuation of treatment despite changes in bone mass and bone turnover; however, PTH maintained cortical bone strength, whereas ALN maintained cancellous bone strength. Additional studies on the long-term effects on bone strength after discontinuation and with combination of osteoporosis medications are needed to improve our treatment of osteoporosis. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179292  PMID: 20839286
22.  Mutations in FKBP10 Cause Recessive Osteogenesis Imperfecta and Bruck Syndrome 
Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by bone fragility and alteration in synthesis and posttranslational modification of type I collagen. Autosomal dominant OI is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Bruck syndrome is a recessive disorder featuring congenital contractures in addition to bone fragility; Bruck syndrome type 2 is caused by mutations in PLOD2 encoding collagen lysyl hydroxylase, whereas Bruck syndrome type 1 has been mapped to chromosome 17, with evidence suggesting region 17p12, but the gene has remained elusive so far. Recently, the molecular spectrum of OI has been expanded with the description of the basis of a unique posttranslational modification of type I procollagen, that is, 3-prolyl-hydroxylation. Three proteins, cartilage-associated protein (CRTAP), prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene), and the prolyl cis-trans isomerase cyclophilin-B (PPIB), form a complex that is required for fibrillar collagen 3-prolyl-hydroxylation, and mutations in each gene have been shown to cause recessive forms of OI. Since then, an additional putative collagen chaperone complex, composed of FKBP10 (also known as FKBP65) and SERPINH1 (also known as HSP47), also has been shown to be mutated in recessive OI. Here we describe five families with OI-like bone fragility in association with congenital contractures who all had FKBP10 mutations. Therefore, we conclude that FKBP10 mutations are a cause of recessive osteogenesis imperfecta and Bruck syndrome, possibly Bruck syndrome Type 1 since the location on chromosome 17 has not been definitely localized. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179293  PMID: 20839288
23.  Perlecan/Hspg2 Deficiency Alters the Pericellular Space of the Lacunocanalicular System Surrounding Osteocytic Processes in Cortical Bone 
Osteocytes project long, slender processes throughout the mineralized matrix of bone, where they connect and communicate with effector cells. The interconnected cellular projections form the functional lacunocanalicular system, allowing fluid to pass for cell-to-cell communication and nutrient and waste exchange. Prevention of mineralization in the pericellular space of the lacunocanalicular pericellular space is crucial for uninhibited interstitial fluid movement. Factors contributing to the ability of the pericellular space of the lacunocanalicular system to remain open and unmineralized are unclear. Immunofluorescence and immunogold localization by transmission electron microscopy demonstrated perlecan/Hspg2 signal localized to the osteocyte lacunocanalicular system of cortical bone, and this proteoglycan was found in the pericellular space of the lacunocanalicular system. In this study we examined osteocyte lacunocanalicular morphology in mice deficient in the large heparan sulfate proteoglycan perlecan/Hspg2 in this tissue. Ultrastructural measurements with electron microscopy of perlecan/Hspg2-deficient mice demonstrated diminished osteocyte canalicular pericellular area, resulting from a reduction in the total canalicular area. Additionally, perlecan/Hspg2-deficient mice showed decreased canalicular density and a reduced number of transverse tethering elements per canaliculus. These data indicated that perlecan/Hspg2 contributed to the integrity of the osteocyte lacunocanalicular system by maintaining the size of the pericellular space, an essential task to promote uninhibited interstitial fluid movement in this mechanosensitive environment. This work thus identified a new barrier function for perlecan/Hspg2 in murine cortical bone. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179294  PMID: 20814969
24.  Active Shape Modeling of the Hip in the Prediction of Incident Hip Fracture 
The objective of this study was to evaluate right proximal femur shape as a risk factor for incident hip fracture using active shape modeling (ASM). A nested case-control study of white women 65 years of age and older enrolled in the Study of Osteoporotic Fractures (SOF) was performed. Subjects (n = 168) were randomly selected from study participants who experienced hip fracture during the follow-up period (mean 8.3 years). Controls (n = 231) had no fracture during follow-up. Subjects with baseline radiographic hip osteoarthritis were excluded. ASM of digitized right hip radiographs generated 10 independent modes of variation in proximal femur shape that together accounted for 95% of the variance in proximal femur shape. The association of ASM modes with incident hip fracture was analyzed by logistic regression. Together, the 10 ASM modes demonstrated good discrimination of incident hip fracture. In models controlling for age and body mass index (BMI), the area under receiver operating characteristic (AUROC) curve for hip shape was 0.813, 95% confidence interval (CI) 0.771–0.854 compared with models containing femoral neck bone mineral density (AUROC = 0.675, 95% CI 0.620–0.730), intertrochanteric bone mineral density (AUROC = 0.645, 95% CI 0.589–0.701), femoral neck length (AUROC = 0.631, 95% CI 0.573–0.690), or femoral neck width (AUROC = 0.633, 95% CI 0.574–0.691). The accuracy of fracture discrimination was improved by combining ASM modes with femoral neck bone mineral density (AUROC = 0.835, 95% CI 0.795–0.875) or with intertrochanteric bone mineral density (AUROC = 0.834, 95% CI 0.794–0.875). Hips with positive standard deviations of ASM mode 4 had the highest risk of incident hip fracture (odds ratio = 2.48, 95% CI 1.68–3.31, p < .001). We conclude that variations in the relative size of the femoral head and neck are important determinants of incident hip fracture. The addition of hip shape to fracture-prediction tools may improve the risk assessment for osteoporotic hip fractures. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179295  PMID: 20878772
25.  BMI and Fracture Risk in Older Men: The Osteoporotic Fractures in Men Study (MrOS) 
Low body mass index (BMI) is a risk factor for fracture, but little is known about the association between high BMI and fracture risk. We evaluated the association between BMI and fracture in the Osteoporotic Fractures in Men Study (MrOS), a cohort of 5995 US men 65 years of age and older. Standardized measures included weight, height, and hip bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA); medical history; lifestyle; and physical performance. Only 6 men (0.1%) were underweight (<18.5 kg/m2); therefore, men in this category were excluded. Also, 27% of men had normal BMI (18.5 to 24.9 kg/m2), 52% were overweight (25 to 29.9 kg/m2), 18% were obese I (30 to 34.9 kg/m2), and 3% were obese II (35 to 39.9 kg/m2). Overall, nonspine fracture incidence was 16.1 per 1000 person-years, and hip fracture incidence was 3.1 per 1000 person-years. In age-, race-, and BMD-adjusted models, compared with normal weight, the hazard ratio (HR) for nonspine fracture was 1.04 [95% confidence interval (CI) 0.87–1.25] for overweight, 1.29 (95% CI 1.00–1.67) for obese I, and 1.94 (95% CI 1.25–3.02) for obese II. Associations were weaker and not statistically significant after adjustment for mobility limitations and walking pace (HR = 1.02, 95% CI 0.84–1.23, for overweight; HR = 1.12, 95% CI 0.86–1.46, for obese I, and HR = 1.44, 95% CI 0.90–2.28, for obese II). Obesity is common among older men, and when BMD is held constant, it is associated with an increased risk of fracture. This association is at least partially explained by worse physical function in obese men. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179296  PMID: 20814955

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