Bone - Articles in Press

Increased Dickkopf-1 expression accelerates bone cell apoptosis in femoral head osteonecrosis - Corrected Proof

2009-11-20

Abstract: Intensive bone cell apoptosis contributes to osteonecrosis of femoral head (ONFH). Dickkopf-1 (DKK1) reportedly mediates various types of skeletal disorders. This study investigated whether DKK1 was linked to the occurrence of ONFH. Thirty-nine patients with various stages of ONFH were recruited. Bone specimens were harvested from 34 ONFH patients underwent hip arthroplasty, and from 10 femoral neck fracture patients. Bad, Bcl2 TNFα, DKK1, Wnt3a, LRP5, and Axin1 expressions were analyzed by quantitative RT-PCR and ELISA. Apoptotic cells were assayed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL). Primary bone-marrow mesenchymal cells were treated with DKK1 RNA interference and recombinant DKK1 protein. ONFH patients with the histories of being administrated corticosteroids and excessive alcohol consumption had significantly higher Bad and DKK1 mRNA expressions in bone tissue and DKK1 abundances in serum than femoral neck fracture patients. Bone cells adjacent to osteonecrotic bone displayed strong DKK1 immunoreactivity and TUNEL staining. Increased DKK1 expression in bone tissue and serum correlated with Bad expression and TUNEL staining. Serum DKK1 abundance correlated with the severity of ONFH. The DKK1 RNA interference and recombinant DKK1 protein regulated Bad expression and apoptosis of primary bone-marrow mesenchymal cells. Knock down of DKK1 reduced dexamethasone-induced apoptosis of mesenchymal cells. Taken together, promoted DKK1 expression was associated with bone cell apoptosis in the occurrence of ONFH patients with the histories of corticosteroid and alcohol intake and progression of ONFH. DKK1 expression in injured tissue provides new insight into ONFH pathogenesis.

Distraction osteogenesis induces endothelial progenitor cell mobilization without inflammatory response in man - Corrected Proof

2009-11-19

Abstract: Introduction: Distraction osteogenesis (DO) is a unique postnatal bone formation process, which is characterized by a profuse increase in vascularization. Recently endothelial progenitor cells (EPCs) have been reported to circulate in substantial numbers under physiologic conditions and to contribute to bone regeneration. The authors investigated EPC mobilization in patients undergoing limb lengthening.Materials and methods: Thirteen of 24 consecutive patients who underwent limb-lengthening procedures on weight-bearing long bones (femur and tibia) were included in this study. Peripheral blood samples were taken at four different time points from each patient, that is, before operation (pre-Op), 2 or 3 days after osteotomy (early-PO), before the start of distraction (pre-Dist), and at 7 to 14 days after the start of distraction (during-Dist). Numbers of leukocytes and levels of plasma C-reactive protein (CRP) were determined. After isolating mononuclear cells (MNCs) by centrifugation, we performed FACS analysis on freshly isolated MNCs using antibodies to the cell surface markers; CD34, CD133, vascular endothelial growth factor receptor 2 (VEGFR2), and alkaline phosphatase. MNCs were also cultured in endothelial cell growth medium and numbers of EPC colony-forming units were counted. Plasma levels of EPC-mobilizing cytokines, such as, VEGF, SDF-1, and MCP-1, were determined by ELISA.Results: Numbers of leukocytes and CRP plasma levels increased significantly during the early-PO period (p<0.01) but were maintained within normal range in the during-Dist period. FACS analysis of freshly isolated MNCs showed that EPC-enriched cell fractions increased after distraction, but that alkaline phosphatase-positive cell numbers were unchanged. Numbers of EPC colony-forming units significantly increased in the during-Dist period (p<0.01). Plasma levels of VEGF and SDF-1 significantly increased in the during-Dist period (p<0.05). In femoral lengthening patients whose healing index was less than 30 days/cm, the number of CFUs was 46.8 in the during-Dist period, whereas it was 12.7 in patients whose healing index was more than 30 days/cm (p=0.088).Conclusions: This study demonstrates a mobilization of EPC population during distraction osteogenesis in human limb-lengthening patients. Distraction strain provoked increases in the plasma levels of EPC-mobilizing cytokines, such as, VEGF and SDF-1. These findings suggest a possibility that therapeutical approaches which modulate EPC mobilization may speed bone healing by angiogenesis–osteogenesis coupling during distraction osteogenesis.

Serum TSH values and risk of vertebral fractures in euthyroid post-menopausal women with low bone mineral density - Corrected Proof

2009-11-19

Abstract: Introduction: There is evidence that variations of thyrotropin (TSH) even in its reference range may influence bone mineral density (BMD). In fact, low-normal TSH values have been associated with high prevalence of osteoporosis in post-menopausal women. However, data associating TSH and risk of fractures are scanty and limited to subjects with subclinical thyrotoxicosis.Materials and methods: In this observational study, we investigated the correlation between serum TSH and prevalence of radiological vertebral fractures in a cohort of 130 post-menopausal women with normal thyroid function.Results: Osteoporosis was observed in 80 women (61.5%), whereas 49 women (37.7%) had osteopenia. Vertebral fractures were found in 49 women (37.7%), who were significantly older, with higher prevalence of osteoporosis and with lower serum TSH values as compared with women who did not fracture. Stratifying the patients according to serum TSH values, vertebral fractures were found to be significantly (p=0.004) more prevalent in first tertile (56.8%) of TSH values as compared with the second (23.3%) and third tertiles (32.6%). Multivariate logistic regression analysis demonstrated that low serum TSH maintained a significant correlation with vertebral fractures (odds ratio 2.8, C.I. 95% 1.20–6.79) even after correction for age, BMD, BMI and serum free-thyroxine values.Discussion: Low-normal TSH values are associated with high prevalence of vertebral fractures in women with post-menopausal osteoporosis or osteopenia, independently of thyroid hormones, age and BMD.

Associations between leisure physical activity participation and cortical bone mass and geometry at the radius and tibia in a Canadian cohort of postmenopausal women - Corrected Proof

Celeste J. Hamilton, Scott G. Thomas, Sophie A. Jamal — 2009-11-19

Abstract: Few studies have examined the effects of leisure physical activity (PA) participation on bone mass and geometry in postmenopausal women using peripheral quantitative computed tomography (pQCT). The purpose of this study was to determine associations between leisure PA participation and bone mass and geometry at the radius and tibia in a Canadian cohort of healthy postmenopausal women (n=234, mean age 62 years). Leisure PA participation was assessed using the Minnesota Leisure Time Physical Activity Questionnaire and by generating a total activity score (TAS, mean=105, range=0–840). Bone mass and geometry at the distal and midshaft sites of the non-dominant radius and tibia were measured using pQCT. Associations between TAS and bone mass and geometry variables were determined using linear regression models adjusted for clinically relevant confounding variables. TAS was positively and significantly associated with total content, total area, cortical content and cortical area at the midshaft sites of the radius and tibia (p<0.05 for all associations). TAS was also positively and significantly associated with bone bending and torsional strength parameters at the midshaft radius and tibia (p<0.05 for all associations). No associations were observed between TAS and trabecular bone parameters. Leisure PA participation is positively associated with cortical bone mass and geometry as well as bending and torsional strength at weight-bearing and non-weight-bearing bone sites and may have the potential to modify bone strength and influence bone fragility in postmenopausal women.

The impact of zoledronic acid on regenerate and native bone after consolidation and removal of the external fixator: An animal model study - Corrected Proof

2009-11-18

Abstract: We investigated the role of zoledronic acid on the regenerate and native bone after consolidation and removal of the external fixator in a rabbit model of distraction osteogenesis using 28 New Zealand white rabbits.The rabbits were randomly distributed into two groups. The first group received three doses of zoledronic acid (ZA) 0.1 mg/kg subcutaneously at weekly intervals while the second group received injections of sterile saline. Distraction started on day 7 at a rate of 0.8 mm/day for 12 days.At week 3 the average lengthening, regenerate density, and regenerate continuity were comparable between the two groups.At week 11 the regenerate in the treated group had a significant increase in Bone Mineral Density (BMD) and Bone Mineral Content (BMC) compared to the placebo group. On axial compression, the regenerate showed an increase in the peak load and a higher modulus of elasticity in the treated group. At 6 months, radiographs demonstrated signs of osteopenia of the proximal metaphysis in the control group, and failure of new bone formation around the pin sites in the treated group. BMC and BMD value differences between the two groups were not statistically significant. Histologically, there was persistence of more bone trabeculae in the medullary canal of the regenerate with the persistence of the pin-holes in the treated group. Mechanically, the regenerates in the treated group remain stronger in resisting the axial compression. The proximal fragment in the treated group exhibited a statistically significant decrease in the peak load, toughness and efail %. In conclusion, bisphosphonate-treated rabbits have a stronger regenerate during distraction, and directly after removal of the fixator. They do not develop disuse osteopenia in their lengthened tibia. This treatment may shorten the time in the external fixator and prevent fragility fractures in the treated extremity. However, its long-term safety has not yet been established.

CRTAP deficiency leads to abnormally high bone matrix mineralization in a murine model and in children with osteogenesis imperfecta type VII - Corrected Proof

2009-11-18

Abstract: Cartilage-associated protein (CRTAP) is an essential cofactor for the proper post-translational chain modification and collagen folding. CRTAP mutations lead mice (Crtap−/− mice) and humans (OI type VII) to a severe/lethal osteochondrodystrophy; patients have fractures at birth, deformities of the lower extremities and impaired growth. The consequences of CRTAP deficiency on intrinsic bone material properties are still unknown. In the present study we evaluated bone quality based on quantitative backscattered electron imaging (qBEI) to assess bone mineralization density distribution (BMDD) in femurs from 12 weeks old Crtap−/− mice and transiliac bone biopsies from 4 children with hypomorphic mutations and having residual CRTAP expression.The analyses revealed in the bone matrix of Crtap−/− animals and OI type VII patients a significant increase in mean (CaMean) and most frequent mineral concentration (CaPeak) compared to wild-type littermates and control children, respectively. The heterogeneity of mineralization (CaWidth) was reduced in Crtap−/− mice but normal in OI type VII patients. The fraction of highly mineralized bone matrix (CaHigh) was remarkably increased in the patients: cancellous bone from 2.1 to 3.7 times and cortical bone from 7.6 to 25.5 times, associated with an increased persistence of primary bone. In conclusion, the BMDD data show that CRTAP deficiency results in a shift towards higher mineral content of the bone matrix similar to classical OI with collagen gene mutations. Our data further suggest altered mineralization kinetics resulting ultimately in an overall elevated tissue mineralization density. Finally, in OI type VII patients the increased portion of primary bone is most likely reflecting a disturbed bone development.

Signaling pathways implicated in androgen regulation of endocortical bone - Corrected Proof

Kristine M. Wiren, Anthony A. Semirale, Joel G. Hashimoto, Xiao-Wei Zhang — 2009-11-18

Abstract: Periosteal expansion is a recognized response to androgen exposure during bone development and in profoundly hypogonadal adults. However, androgen also suppresses endocortical bone formation, indicating that its effects on bone are dichotomous and envelope-specific. In fact, enhanced androgen signaling has been shown to have dramatic detrimental effects on whole bone biomechanical properties in two different transgenic models with skeletally targeted androgen receptor (AR) overexpression. As the mechanisms underlying this response are uncharacterized, we compared patterns of gene expression in periosteum-free cortical bone samples derived from AR-overexpressing transgenic male mice and their wild-type counterparts. We then assessed direct androgen effects in both wild-type and AR-overexpressing osteoblasts in primary culture. Among major signaling pathways associated with bone formation, focused quantitative RT-PCR (qPCR) array-based analysis of endocortical bone gene expression from wild-type vs. transgenic males identified the transforming growth factor-beta (TGF-β) superfamily and bone morphogenetic protein (BMP) signaling as significantly altered by androgen in vivo. Bioinformatic analyses indicated proliferation, osteoblast differentiation and mineralization as major biological processes affected. Consistent with the in vivo array data and bioinformatic analyses, inhibition of differentiation observed with androgen exposure was reduced by exogenous BMP2 treatment of AR-overexpressing cultures to stimulate BMP signaling, confirming array pathway analysis. In addition, nonaromatizable dihydrotestosterone (DHT) inhibited osteoblast proliferation, differentiation and several indices of mineralization, including mineral accumulation and mineralized nodule formation in primary cultures from both wild-type and AR-transgenic mice. These findings identify a molecular mechanism based on altered BMP signaling that contributes to androgen inhibition of osteoblast differentiation and mineralization. Such detrimental effects of androgen on osteoblast function may underlie the generally disappointing results of androgen therapy.

Increased matrix mineralization in the immature femoral head following ischemic osteonecrosis - Corrected Proof

Jochen G. Hofstaetter, Paul Roschger, Klaus Klaushofer, Harry K.W. Kim — 2009-11-16

Abstract: Traditionally, it is believed that structural failure of the ischemic epiphysis as well as changes in radiodensity seen in the early stage of Legg–Calvé–Perthes disease is due to the repair process. However, little is known if matrix properties are altered following ischemic injury of the juvenile femoral head. The purpose of this study was to determine the matrix mineralization density, an important determinant of material quality and strength, of the proximal femoral epiphysis in an experimental animal model of Perthes disease.Ten piglets were surgically induced with femoral head ischemia and euthanized at 4 and 8 weeks following surgery. Contralateral, unoperated femoral heads were used as controls. Bone and calcified cartilage mineralization density distribution parameters were determined using quantitative backscattered electron imaging (qBEI) in the epiphyseal calcified articular cartilage, subchondral bone and central trabecular bone region. Histological as well as radiographic assessment was also performed.In the necrotic calcified epiphyseal cartilage matrix, a significant increase in the mean degree of mineralization (CaMean: +24%, p<0.0001) as well as the homogeneity of mineralization (CaWidth: −21%, p<0.05) and a significantly reduced amount of low mineralized matrix (CaLow: −49%, p<0.0001) were already present at 4 weeks post-ischemia induction. Similar changes, but more moderate, were also seen in the subchondral bone region. In contrast, in the necrotic central trabecular region, significant changes in matrix mineralization were found at 8 weeks (CaMean: +4%, p<0.05; CaWidth: −22%, p<0.05; CaLow: −8%, p<0.05) but not at 4 weeks post-ischemia induction.Our findings indicate that the process of matrix mineralization continues in necrotic calcified articular cartilage and bone following femoral head ischemia, which leads to a higher and more homogenous mineralized tissue matrix altering its intrinsic material properties.This may also explain the increased radiodensity seen in the early stage of Perthes disease prior to the initiation of the repair process.

Loading-related regulation of gene expression in bone in the contexts of estrogen deficiency, lack of estrogen receptor α and disuse - Corrected Proof

2009-11-16

Abstract: Loading-related changes in gene expression in resident cells in the tibia of female mice in the contexts of normality (WT), estrogen deficiency (WT-OVX), absence of estrogen receptor α (ERα−/−) and disuse due to sciatic neurectomy (WT-SN) were established by microarray. Total RNA was extracted from loaded and contra-lateral non-loaded tibiae at selected time points after a single, short period of dynamic loading sufficient to engender an osteogenic response. There were marked changes in the expression of many genes according to context as well as in response to loading within those contexts. In WT mice at 3, 8, 12 and 24 h after loading the expression of 642, 341, 171 and 24 genes, respectively, were differentially regulated compared with contra-lateral bones which were not loaded. Only a few of the genes differentially regulated by loading in the tibiae of WT mice have recognized roles in bone metabolism or have been linked previously to osteogenesis (Opn, Sost, Esr1, Tgfb1, Lrp1, Ostn, Timp, Mmp, Ctgf, Postn and Irs1, BMP and DLX5). The canonical pathways showing the greatest loading-related regulation were those involving pyruvate metabolism, mitochondrial dysfunction, calcium-induced apoptosis, glycolysis/gluconeogenesis, aryl hydrocarbon receptor and oxidative phosphorylation. In the tibiae from WT-OVX, ERα−/− and WT-SN mice, 440, 439 and 987 genes respectively were differentially regulated by context alone compared to WT. The early response to loading in tibiae of WT-OVX mice involved differential regulation compared to their contra-lateral non-loaded pair of fewer genes than in WT, more down-regulation than up-regulation and a later response. This was shared by WT-SN. In tibiae of ERα−/− mice, the number of genes differentially regulated by loading was markedly reduced at all time points.These data indicate that in resident bone cells, both basal and loading-related gene expression is substantially modified by context. Many of the genes differentially regulated by the earliest loading-related response were primarily involved in energy metabolism and were not specific to bone.

Transient muscle paralysis disrupts bone homeostasis by rapid degradation of bone morphology - Corrected Proof

2009-11-16

Abstract: We have previously shown that transient paralysis of murine hindlimb muscles causes profound degradation of both trabecular and cortical bone in the adjacent skeleton within 3 weeks. Morphologically, the acute loss of bone tissue appeared to arise primarily due to osteoclastic bone resorption. Given that the loss of muscle function in this model is transient, we speculated that the stimulus for osteoclastic activation would be rapid and morphologic evidence of bone resorption would appear before 21 days. We therefore utilized high-resolution in vivo serial micro-CT to assess longitudinal alterations in lower hindlimb muscle volume, proximal tibia trabecular, and tibia mid-diaphysis cortical bone morphology in 16-week-old female C57 mice following transient calf paralysis from a single injection of botulinum toxin A (BtA; 2U/100 g body weight). In an acute study, we evaluated muscle and bone alterations at days 0, 3, 5, and 12 following transient calf paralysis. In a chronic study, following day 0 imaging, we assessed the recovery of these tissues following the maximum observed trabecular degradation (day 12) through day 84 post-paralysis. The time course and degree of recovery of muscle, trabecular, and cortical bone varied substantially. Significant atrophy of lower limb muscle was evident by day 5 of paralysis, maximal at day 28 (−34.1±0.9%) and partially recovered by day 84. Trabecular degradation within the proximal tibia metaphysis occurred more rapidly, with significant reduction in BV/TV by day 3, maximal loss at day 12 (−76.8±2.9%) with only limited recovery by day 84 (−51.7±5.1% vs. day 0). Significant cortical bone volume degradation at the tibia mid-diaphysis was first identified at day 12, was maximal at day 28 (−9.6±1.2%), but completely recovered by day 84. The timing, magnitude, and morphology of the observed bone erosion induced by transient muscle paralysis were consistent with a rapid recruitment and prolific activation of osteoclastic resorption. In a broader context, understanding how brief paralysis of a single muscle group can precipitate such rapid and profound bone resorption in an adjacent bone is likely to provide new insight into how normal muscle function modulates bone homeostasis.

The daily habitual in vivo strain history of a non-weight-bearing bone - Corrected Proof

W.C. de Jong, J.H. Koolstra, J.A.M. Korfage, L.J. van Ruijven, G.E.J. Langenbach — 2009-11-16

Abstract: Daily mechanical loading strongly influences the architecture and composition of bone tissue. Throughout the day, the amplitudes, rates, frequencies, and the dispersion over time of these loads vary. Nevertheless, most experimental and descriptive studies on the aforementioned relationship consider only cyclic loading and, in addition, focus on weight-bearing bones. A more complete assessment of the daily loading of bone might lead to a better understanding of the natural everyday stimulus for bone maintenance or adaptive responses. In the present study, we measured the daily habitual strain history of the non-weight-bearing mandible bone in the rabbit. Long-term continuous strain recordings were made using an implantable telemetry device able to read out bone-bonded strain gauges. The lateral surface of the rabbit mandibular corpus was chosen as the bone surface of interest. During the recordings, which lasted up to 33 h, the rabbits (N=7) were able to move unrestrictedly in their cages, performing their habitual behaviours. Analysis of the recordings revealed that the measured bone surface was subjected to 2.9 (±1.4)×103 strain events per hour of which 1.8 (±1.0)×103 had amplitudes ≤10 microstrains (μɛ). Larger strain amplitudes occurred less often and principal strains fell within the range of −517 (±118) μɛ to 298 (±81) μɛ. Strain rates never exceeded 10,000 μɛ/s and only 8.9% (±7.2%) of the habitual strain rates were higher than 1000 μɛ/s. Strain frequency spectra displayed clear peaks at 4–5 and 9 Hz. The wirelessly recorded daily strain history of the rabbit mandible featured peak strain amplitudes resembling those of other mammalian mandibles, but much smaller than those found in many long-bone strain measurements.

Effects of alcohol on skeletal response to growth hormone in hypophysectomized rats - Corrected Proof

Russell T. Turner, Clifford J. Rosen, Urszula T. Iwaniec — 2009-11-16

Abstract: Chronic alcohol abuse is an established risk factor for osteoporosis. However, the precise mechanisms for the bone loss are largely unknown. Alcohol decreases skeletal expression of insulin-like growth factor-I (IGF-I), an important growth hormone (GH)-regulated skeletal growth factor. Therefore, we investigated the effects of alcohol on the skeletal response to GH in male Sprague–Dawley rats made GH-deficient by hypophysectomy (HYPOX). Four groups of sexually mature (3-month-old) rats were studied: pituitary-intact (control), HYPOX, HYPOX + GH, and HYPOX + alcohol + GH. All animals were transferred to a liquid diet 6 days following surgery. The alcohol-fed group was adapted to a graded increase in alcohol beginning 11 days following surgery. GH or vehicle was administered during the final 8 days of study and all animals were sacrificed 25 days following surgery. HYPOX resulted in cessation of body weight gain and tibial growth. Compared to controls, longitudinal bone growth and cancellous bone formation were lower following HYPOX. The latter was associated with lower mineralizing perimeter/bone perimeter. Bone marrow adiposity was higher following HYPOX. Compared to HYPOX, GH treatment increased body weight gain and bone formation rate, and decreased bone marrow adiposity. In contrast to the effects of GH treatment without alcohol, bone marrow adiposity did not differ between HYPOX and alcohol-fed GH-treated HYPOX rats. Alcohol did not alter GH-induced weight gain or increases in serum IGF-I levels, but significantly impaired the effects of GH on tibial growth and cancellous bone formation. We conclude that the detrimental skeletal effects of alcohol abuse observed in this experiment are mediated, at least in part, by skeletal resistance to GH.

HO-1 expression increases mesenchymal stem cell-derived osteoblasts but decreases adipocyte lineage - Corrected Proof

2009-11-12

Abstract: Human bone marrow mesenchymal stem cells (MSC) are pleiotropic cells that differentiate to either adipocytes or osteoblasts as a result of cross-talk by specific signaling pathways including heme oxygenase (HO)-1/-2 expression. We examined the effect of inducers of HO-1 expression and inhibitors of HO activity on MSC differentiation to the osteoblast and adipocyte lineage. HO-1 expression is increased during osteoblast stem cell development but remains elevated at 25 days. The increase in HO-1 levels precedes an increase in alkaline phosphatase (AP) activity and an increase in BMP, osteonectin and RUNX-2 mRNA. Induction of HO-1 by osteogenic growth peptide (OGP) was associated with an increase in BMP-2 and osteonectin. Exposure of MSC to high glucose levels decreased osteocalcin and osteogenic protein expression, which was reversed by upregulation of the OGP-mediated increase in HO-1 expression. The glucose-mediated decrease in HO-1 resulted in decreased levels of pAMPK, pAKT and the eNOS signaling pathway and was reversed by OGP. In contrast, MSC-derived adipocytes were increased by glucose. HO-1 siRNA decreased HO-1 expression but increased adipocyte stem cell differentiation and the adipogenesis marker, PPARγ. Thus, upregulation of HO-1 expression shifts the balance of MSC differentiation in favor of the osteoblast lineage. In contrast, a decrease in HO-1 or exposure to glucose drives the MSC towards adipogenesis. Thus, targeting HO-1 expression is a portal to increased osteoblast stem cell differentiation and to the attenuation of osteoporosis by the promotion of bone formation.

Bisphosphonates do not inhibit periosteal bone formation in estrogen deficient animals and allow enhanced bone modeling in response to mechanical loading - Corrected Proof

2009-11-11

Abstract: The suppressive effects of bisphosphonates (BPs) on bone remodeling are clear yet there is conflicting data concerning the effects of BPs on modeling (specifically formation modeling on the periosteal surface). The normal periosteal expansion that occurs during aging has significant benefits to maintaining/improving the bones' mechanical properties and thus it is important to understand whether BPs affect this bone surface. Therefore, the purpose of this study was to determine the effects of BPs on periosteal bone formation modeling induced by ovariectomy (OVX) and mechanical loading. Six-month-old Sprague–Dawley OVX rats (n=60; 12/group) were administered vehicle, risedronate, alendronate, or zoledronate at doses used clinically for treatment of post-menopausal osteoporosis. Three weeks after initiating BP treatment, all animals underwent in vivo ulnar loading of the right limb every other day for 1 week (3 total sessions). Periosteal surface mineral apposition rate, mineralizing surface, and bone formation rate were determined at the mid-diaphysis of both loaded (right) and non-loaded (left) ulnae. There was no significant effect of any of the BPs on periosteal bone formation parameters compared to VEH-treated animals in the non-loaded limb, suggesting that BP treatment does not compromise the normal periosteal expansion associated with estrogen loss. Mechanical loading significantly increased BFR in the loaded limb compared to the non-loaded limb in all BP-treated groups, with no difference in the magnitude of this effect among the various BPs. Collectively, these data show that BP treatment, at doses comparable to those used for treatment of post-menopausal osteoporosis, (1) does not alter the periosteal formation activity that occurs in the absence of estrogen and (2) allows normal stimulation of periosteal bone formation in response to the anabolic stimulation of mechanical loading.

Rehydration of vertebral trabecular bone: Influences on its anisotropy, its stiffness and the indentation work with a view to age, gender and vertebral level - Corrected Proof

Uwe Wolfram, Hans-Joachim Wilke, Philippe K. Zysset — 2009-11-09

Abstract: For understanding the fracture risk of vertebral bodies the macroscopic mechanical properties of the cancellous core are of major interest. Due to the hierarchical nature of bone, these depend in turn on the micromechanical properties of bone extracellular matrix which is at least linear elastic transverse isotropic. The experimental determination of local elastic properties of bone ex vivo necessitates a high spatial resolution which can be provided by depth-sensing indentation techniques. Using microindentation, this study investigated the effects of rehydration on the transverse isotropic elastic properties of vertebral trabecular bone matrix obtained from two orthogonal directions with a view to microanatomical location, age, gender, vertebral level and anatomic direction in a conjoint statistics.Biopsies were gained from 104 human vertebrae (T1–L3) with a median age of 65 years (21–94). Wet elastic moduli were 29% lower (p<0.05) than dry elastic moduli. For wet indentation the ratio of mean elastic moduli tested in axial to those tested in transverse indentation direction were 1.13 to 1.23 times higher than for dry indentation. The ratio of elastic moduli tested in the core to those tested in the periphery of trabeculae was 1.05 to 1.16 times higher when testing wet. Age and gender did not show any influence on the elastic moduli for wet and dry measurements. The correlation between vertebral level and elastic moduli became weaker after rehydration (pwet<0.09, rwet2=0.14) and (pdry<0.01, rwet2=0.38). Elastic and dissipated energies were similarly affected by rehydration compared to the elastic modulus. No significant difference in the energies could be found for gender (p>0.05). Significant differences in the energies were found for age (p<0.05) after rehydration.Qualitative and quantitative insights into the transverse isotropic elastic properties of trabecular bone matrix under two testing conditions over a broad spectrum of vertebrae could be given. This study could help to further improve understanding of the mechanical properties of vertebral trabecular bone.

Maximizing bone mineral mass gain during growth for the prevention of fractures in the adolescents and the elderly - Corrected Proof

2009-11-09

Abstract: Bone mass is a key determinant of fracture risk. Maximizing bone mineral mass during childhood and adolescence may contribute to fracture risk reduction during adolescence and possibly in the elderly. Although more than 60% of the variance of peak bone mass (PBM), the amount of bone present in the skeleton at the end of its maturation process, is genetically determined, the remainder is likely influenced by factors amenable to positive intervention, such as adequate dietary intake of dairy products as a natural source of calcium and proteins, vitamin D, and regular weight-bearing physical activity. Low calcium and vitamin D intakes are associated with negative effects on bone, including suboptimal PBM acquisition. As suggested by intervention studies, regular intake of dairy products may have positive and possibly sustained effects on bone mineral mass gain, contributing thereby to fracture risk reduction. Further evidence from intervention studies suggests that weight-bearing physical activities, such as jumping, may contribute to bone mineral mass gain in children. Optimizing PBM acquisition through dietary and physical exercise measures may represent a valuable primary method for the prevention of fractures.

Relaxin stimulates osteoclast differentiation and activation - Corrected Proof

2009-11-09

Abstract: Relaxin is a pleiotropic hormone with actions in reproductive and non-reproductive tissues, and has a role in tumor biology. It can promote growth, differentiation and invasiveness of different tumors, especially those that give bone metastases, and relaxin serum concentrations are increased in patients with bone metastasis. In osteolytic metastasis the destruction of bone is mediated by osteoclasts that are multinucleated cells derived from hematopoietic progenitors. We found that human hematopoietic precursors and mature osteoclasts express the relaxin receptor RXFP1. Then, we investigated the effects of relaxin on the differentiation, activation and gene expression of osteoclasts during in vitro osteoclastogenesis from human hematopoietic progenitor cells. Relaxin alone was able to induce the multistep differentiation process of human osteoclastogenesis with timing similar to that obtained with the classical stimulators of osteoclastogenesis RANKL, M-CSF and PTH. The expression profile of several osteoclast genes was studied with quantitative RT-PCR during the entire process of osteoclastogenesis. This analysis showed that relaxin induced genes that are implicated in the differentiation, survival and activation of osteoclasts. Relaxin-induced osteoclasts were fully differentiated, positive for tartrate resistant acid phosphatase and vitronectin receptor, expressing a typical F-actin ring and able to resorb the bone. Furthermore, relaxin induced the expression of its specific receptor RXFP1 in osteoclasts. This study demonstrates for the first time that relaxin is a potent stimulator of osteoclastogenesis from hematopoietic precursors and regulates the activity of mature osteoclasts, opening new perspectives on the role of this hormone in bone physiology, diseases and metastasis.

Osteoarthritic cartilage chondrocytes alter subchondral bone osteoblast differentiation via MAPK signalling pathway involving ERK1/2 - Corrected Proof

Indira Prasadam, Thor Friis, Wei Shi, Stijn van Gennip, Ross Crawford, Yin Xiao — 2009-11-09

Abstract: Osteoarthritic subchondral bone is characterized by abnormal bone density and enhanced production of bone turnover markers, an indication of osteoblast dysfunction. Several studies have proposed that pathological changes in articular cartilage influence the subchondral bone changes, which are typical of the progression of osteoarthritis; however, direct evidence of this has yet to be reported. The aim of the present study was to investigate what effects articular cartilage cells, isolated from normal and osteoarthritic joints, may have on the subchondral bone osteoblast phenotype, and also the potential involvement of the mitogen activated protein kinase (MAPK) signalling pathway during this process. Our results suggest that chondrocytes isolated from a normal joint inhibited osteoblast differentiation, whereas chondrocytes isolated from an osteoarthritic joint enhanced osteoblast differentiation, both via a direct and indirect cell interaction mechanisms. Furthermore, the interaction of subchondral bone osteoblasts with osteoarthritic chondrocyte conditioned media appeared to significantly activate ERK1/2 phosphorylation. On the other hand, conditioned media from normal articular chondrocytes did not affect ERK1/2 phosphorylation. Inhibition of the MAPK–ERK1/2 pathways reversed the phenotype changes of subchondral bone osteoblast, which would otherwise be induced by the conditioned media from osteoarthritic chondrocytes. In conclusion, our findings provide evidence that osteoarthritic chondrocytes affect subchondral bone osteoblast metabolism via an ERK1/2 dependent pathway.

Reduced size-independent mechanical properties of cortical bone in high-fat diet-induced obesity - Corrected Proof

2009-11-09

Abstract: Overweight and obesity are rapidly expanding health problems in children and adolescents. Obesity is associated with greater bone mineral content that might be expected to protect against fracture, which has been observed in adults. Paradoxically, however, the incidence of bone fractures has been found to increase in overweight and obese children and adolescents. Prior studies have shown some reduced mechanical properties as a result of high-fat diet (HFD) but do not fully address size-independent measures of mechanical properties, which are important to understand material behavior. To clarify the effects of HFD on the mechanical properties and microstructure of bone, femora from C57BL/6 mice fed either a HFD or standard laboratory chow (Chow) were evaluated for structural changes and tested for bending strength, bending stiffness and fracture toughness. Here, we find that in young, obese, high-fat fed mice, all geometric parameters of the femoral bone, except length, are increased, but strength, bending stiffness, and fracture toughness are all reduced. This increased bone size and reduced size-independent mechanical properties suggests that obesity leads to a general reduction in bone quality despite an increase in bone quantity; yield and maximum loads, however, remained unchanged, suggesting compensatory mechanisms. We conclude that diet-induced obesity increases bone size and reduces size-independent mechanical properties of cortical bone in mice. This study indicates that bone quantity and bone quality play important compensatory roles in determining fracture risk.

Muscle and bone follow similar temporal patterns of recovery from muscle-induced disuse due to botulinum toxin injection - Corrected Proof

Sarah L. Manske, Steven K. Boyd, Ronald F. Zernicke — 2009-11-09

Abstract: If muscle force is a primary source for triggering bone adaptation, with disuse and reloading, bone changes should follow muscle changes. We examined the timing and magnitude of changes in muscle cross-sectional area (MCSA) and bone architecture in response to muscle inactivity following botulinum toxin (BTX) injection. We hypothesized that MCSA would return to baseline levels sooner than bone properties following BTX injection.Female BALB mice (15 weeks old) were injected with 20 μL of BTX (1 U/100 g body mass, n=18) or saline (SAL, n=18) into the posterior calf musculature of one limb. The contralateral limb (CON) served as an internal control. MCSA and bone properties were assessed at baseline, 2, 4, 8, 12, and 16 weeks post-injection using in vivo micro-CT at the tibia proximal metaphysis (bone only) and diaphysis. Muscles were dissected and weighed after sacrifice.Significant Group×Leg×Time interactions indicated that the maximal decrease in MCSA (56%), proximal metaphyseal BV/TV (38%) and proximal diaphyseal Ct.Ar (7%) occurred 4 weeks after injection. There was no delay prior to bone recovery as both muscle and bone properties began to recover after this time, but MCSA and BV/TV remained 15% and 20% lower, respectively, in the BTX-injected leg than the BTX-CON leg 16 weeks post-injection. Gastrocnemius mass (primarily fast-twitch) was 14% lower in the BTX-injected leg than the SAL-injected leg, while soleus mass (primarily slow-twitch) was 15% greater in the BTX group than the SAL group.Our finding that muscle size and bone began to recover at similar times after BTX injection was unexpected. This suggested that partial weight-bearing and/or return of slow-twitch muscle activity in the BTX leg may have been sufficient to stimulate bone recovery. Alternatively, muscle function may have recovered sooner than MCSA. Our results indicated that muscle cross-sectional area, while important, may not be the primary factor associated with bone loss and recovery when muscle atrophy is induced through BTX injection. To understand the nature of the interaction between muscle and bone, future work should focus on the functional recovery of individual muscles in relation to bone.

International longitudinal pediatric reference standards for bone mineral content - Corrected Proof

2009-11-09

Abstract: To render a diagnosis pediatricians rely upon reference standards for bone mineral density or bone mineral content, which are based on cross-sectional data from a relatively small sample of children. These standards are unable to adequately represent growth in a diverse pediatric population. Thus, the goal of this study was to develop sex and site-specific standards for BMC using longitudinal data collected from four international sites in Canada and the United States. Data from four studies were combined; Saskatchewan Paediatric Bone Mineral Accrual Study (n=251), UBC Healthy Bones Study (n=382); Penn State Young Women's Health Study (n=112) and Stanford's Bone Mineral Accretion study (n=423). Males and females (8 to 25 years) were measured for whole body (WB), total proximal femur (PF), femoral neck (FN) and lumbar spine (LS) BMC (g). Data were analyzed using random effects models. Bland–Altman was used to investigate agreement between predicted and actual data. Age, height, weight and ethnicity independently predicted BMC accrual across sites (P<0.05). Compared to White males, Asian males had 31.8 (6.8) g less WB BMC accrual; Hispanic 75.4 (28.2) g less BMC accrual; Blacks 82.8 (26.3) g more BMC accrual with confounders of age, height and weight controlled. We report similar findings for the PF and FN. Models for females for all sites were similar with age, height and weight as independent significant predictors of BMC accrual (P<0.05). We provide a tool to calculate a child's BMC Z-score, accounting for age, size, sex and ethnicity. In conclusion, when interpreting BMC in pediatrics we recommend standards that are sex, age, size and ethnic specific.

Association between collagen cross-links and trabecular microarchitecture properties of human vertebral bone - Corrected Proof

2009-11-06

Abstract: It has been suggested that age-related deterioration in trabecular microarchitecture and changes in collagen cross-link concentrations may contribute to skeletal fragility. To further explore this hypothesis, we determined the relationships among trabecular bone volume fraction (BV/TV), microarchitecture, collagen cross-link content, and bone turnover in human vertebral trabecular bone. Trabecular bone specimens from L2 vertebrae were collected from 51 recently deceased donors (54–95 years of age; 20 men and 30 women). Trabecular bone volume and microarchitecture was assessed by microCT and bone formation, reflected by osteoid surface (OS/BS, %), was measured by 2D histomorphometry. Pyridinoline (PYD), deoxypyridinoline (DPD), pentosidine (PEN) and collagen content in the cancellous bone were analysed by high-performance liquid chromatography. Associations between variables were investigated by Pearson correlations and multiple regression models, which were constructed with BV/TV and collagen cross-links as explanatory variables and microarchitecture parameters as the dependent variables. Results: Microarchitecture parameters were modestly to strongly correlated with BV/TV (r2=0.10–0.71). The amount of mature enzymatic PYD and DPD cross-links were not associated with the microarchitecture, either before or after adjustment for BV/TV. However, there was a positive correlation between PEN content and trabecular number (r=0.45, p=0.001) and connectivity density (r=0.40, p=0.004), and a negative correlation between PEN content and trabecular separation (r=−0.29, p=0.04). In the multiple regression models including BV/TV, age and PEN content was still significantly associated with several of the microarchitecture variables. In summary, this study suggests a link between trabecular microarchitecture and the collagen cross-link profile. As PEN reflects non-enzymatic glycation of collagen and generally increases with bone age, the association between PEN and trabecular architecture suggests that the preserved trabeculae may contain mainly old bone and have undergone little remodeling. Thus, vertebral fragility may not only be due to alterations in bone architecture but also to modification of collagen cross-link patterns thereby influencing bone's mechanical behavior.

Evidence for anti-osteoporosis therapy in acute fracture situations—Recommendations of a multidisciplinary workshop of the International Society for Fracture Repair - Corrected Proof

2009-11-05

Abstract: The International Society for Fracture Repair convened a multidisciplinary workshop to assess the current evidence around the interaction between anti-osteoporosis drugs and the healing of incident fractures, with a view to making recommendations for clinical practice. The consensus was that there is no evidence-based reason to withhold anti-resorptive therapy while a fracture heals, whether or not the patient was taking such therapy when the fracture occurred. The workshop also considered existing models of service provision for secondary prevention and concluded that the essential ingredient for reliable delivery is the inclusion of a dedicated coordinator role. Several unresolved issues were defined as subjects for further research, including the question of whether continuous long-term administration of anti-resorptives may impair bone quality. The rapidly changing area requires re-assessment of drugs and their interaction with fracture healing in the near future.

Insulin-like growth factor-1 is associated with bone formation markers, PTH and bone mineral density in healthy premenopausal women - Corrected Proof

2009-11-05

Abstract: Bone turnover markers (BTM) progressively decrease in young adult women. This might be linked to changes in insulin-like growth factor-1 (IGF-I).Four serum BTMs [serum C-telopeptide of type 1 collagen (CTX), osteocalcin (OC), N-terminal propeptide of type 1 procollagen (P1NP), and bone alkaline phosphatase (bone AP)], serum calcium (sCa), phosphate (sPO4), magnesium, 25-hydroxyvitamin D [25(OH)D], intact parathyroid hormone (PTH) and IGF-I were measured in 531 young healthy premenopausal women aged 20–50 years participating in the BONTURNO study. In all subjects bone mineral density (BMD) was measured at the spine and at the hip by dual-energy X-ray densitometry.Hip BMD, IGF-I, the four BTMs, sCa and sPO4 progressively decreased with advancing age and this was associated with proportional increases in PTH.IGF-I levels were significantly and positively correlated with sCa, sPO4, CTX, OC, P1NP, bone AP, spine BMD, femoral neck BMD and total hip BMD and negatively with age, BMI and serum PTH. When the IGF-I levels were adjusted for age and BMI, the only correlations maintaining a statistical significance were those with serum PTH, P1NP and bone AP. These associations were weak and IGF-I accounted for a only a small proportion of the BTM variance.The mean, age-adjusted IGF-I values were significantly higher in women practicing physical exercises for more then 60 min per week than in sedentary women.In conclusion, in this study we provide evidence of an association between the age-related decline in IGF-I with the progressive decrease in bone formation markers in premenopausal women.

Peak bone mineral density, lean body mass and fractures - Corrected Proof

2009-11-02

Abstract: Background: During childhood and adolescence, bone mass and lean body mass (LBM) increase till a plateau is reached. In this longitudinal and cross-sectional study, the age of reaching the plateau was evaluated for lumbar spine and total body bone mass measurements and lean body mass. The association between fractures and bone mineral density (BMD) was studied.Patients and methods: We included 501 healthy participants, 141 males and 360 females, aged 13–29 years. Of these 90 had participated in a previous longitudinal study of 444 participants, aged 4–20 years (for the first measurement) and 198 participants, aged 8–25 years (for a second measurement). BMD and body composition were measured with dual energy X-ray absorptiometry (DXA). Volumetric BMD (bone mineral apparent density, BMAD) was calculated. All the data were used to determine the age of reaching the plateau.Results: The plateau for lumbar spine BMD, BMAD, total body BMD, bone mineral content and LBM was reached between 18 and 20 years of age in females and between 18 and 23 years in males. The prevalence of fractures was 37% in males and 28% in females. Total body BMD Z-score was significantly lower in all participants who had had a fracture (p<0.05), whereas lumbar spine BMD and BMAD was only significantly lower in females who had had fractures (p=0.007 and p<0.001, respectively). Mean lumbar spine BMAD Z-score at the previous measurement was significantly lower in the participants who had a first fracture between the last two measurements (p=0.04).Conclusion: Peak BMD and peak LBM were attained between 18 and 20 years in females and between 18 and 23 years in males in this study using longitudinal and cross sectional data in the age range of 4 to 30 years. A significantly lower total body BMD was seen in participants who had had a fracture and a lower lumbar spine BMD and BMAD in females who had had a fracture. Lumbar spine BMAD Z-score seems to be a good predictor for future fractures.

Hip fractures and femoral bone mineral density in male former elite athletes - Corrected Proof

2009-11-02

Abstract: Introduction: We studied whether vigorous physical activity in young adulthood is associated with higher femoral bone density and lower risk of hip fracture at older age in men.Materials: A cohort of former male elite athletes (n=2147) and matched control subjects (n=1467) were studied for their leisure physical activity, and for fragility fractures at the hip (proximal femur) by Cox regression. Areal bone mineral densities (aBMD) at femoral neck and trochanter region were measured using dual-energy X-ray absorptiometry in a subgroup of the former athletes (n=87; median age 59 years) and in a population-based control group (n=194) and compared by general linear models.Results: After their active sporting careers, the former athletes participated in leisure physical activity more than the matched control subjects (p<0.0001). The hazard ratio (HR) of osteoporotic hip fracture adjusted for the occupational group was 0.77 (95% CI 0.45 to 1.32, p=0.34) in the athletes compared with the control subjects. The mean age at the time of the fracture event was 76.9 years (95% CI 73.2 to 78.8) for the athletes and 70.6 years (95% CI 67.1 to 72.9) for the matched control subjects (p=0.005). Adjusted for age and body mass index, aBMD at the proximal femur was significantly higher in the former athletes compared with the population-based control group (p<0.0001 for both measurement sites).Conclusions: Osteoporotic hip fractures were sustained at a significantly older age among former athletes compared with control subjects. Clear skeletal benefits of long-term physical loading were also observed in comparative DXA measurements of aBMD.

Temporal pattern of gene expression and histology of stress fracture healing - Corrected Proof

L.J. Kidd, A.S. Stephens, J.S. Kuliwaba, N.L. Fazzalari, A.C.K. Wu, M.R. Forwood — 2009-11-02

Abstract: Loading of the rat ulna is an ideal model to examine stress fracture healing. The aim of this study was to undertake a detailed examination of the histology, histomorphometry and gene expression of the healing and remodelling process initiated by fatigue loading of the rat ulna. Ulnae were harvested 1, 2, 4, 6, 8, and 10 weeks following creation of a stress fracture. Stress fracture healing involved direct remodelling that progressed along the fracture line as well as woven bone proliferation at the site of the fracture. Histomorphometry demonstrated rapid progression of basic multicellular units from 1 to 4 weeks with significant slowing down of healing by 10 weeks after loading. Quantitative PCR was performed at 4 hours, 24 hours, 4 days, 7 days, and 14 days after loading. Gene expression was compared to an unloaded control group. At 4 hours after fracture, there was a marked 220-fold increase (P<0.0001) in expression of IL-6. There were also prominent peak increases in mRNA expression for OPG, COX-2, and VEGF (all P<0.0001). At 24 hours, there was a peak increase in mRNA expression for IL-11 (73-fold increase, P<0.0001). At 4 days, there was a significant increase in mRNA expression for Bcl-2, COX-1, IGF-1, OPN, and SDF-1. At 7 days, there was significantly increased mRNA expression of RANKL and OPN. Prominent, upregulation of COX-2, VEGF, OPG, SDF-1, BMP-2, and SOST prior to peak expression of RANKL indicates the importance of these factors in mediating directed remodelling of the fracture line. Dramatic, early upregulation of IL-6 and IL-11 demonstrate their central role in initiating signalling events for remodelling and stress fracture healing.

Regeneration of bone marrow after tibial ablation in immunocompromised rats is age dependent - Corrected Proof

Maya Fisher, Sharon Hyzy, Robert E. Guldberg, Zvi Schwartz, Barbara D. Boyan — 2009-10-30

Abstract: Injuries to the marrow cavity result in rapid endosteal bone formation followed by remodeling and regeneration of the marrow. It is not known whether this process is affected by age, although marrow quality is markedly different in young and old animals. To test if marrow regeneration differs with age, we used a bone marrow ablation model that has been used to examine calcification, osteointegration of metal implants, and remodeling of bone graft substitutes. Marrow was ablated in the left tibia of seven immunocompromised rats (rNu/rNu) per time point. At 0, 7, 14, 21, 28, 35 and 42 days post-surgery, treated and contralateral tibias were harvested and fixed in buffered formalin. Both tibias were scanned using microCT and trabecular and cortical BVF calculated. Mid-sagittal histological sections of the treated limbs were stained with haematoxylin and eosin and BV/TV calculated. MicroCT and histomorphometry showed the greatest increase in bone formation was in young animals and was seen on day 7. Remodeling also occurred at an earlier time point in young rats. Bone formation peaked on day 7 in adult rats, but remodeling was slower than in young rats. Aged animals showed a delay in bone formation. Moreover, aged rats produced less primary bone than younger animals and remodeling was initiated later. These results show that response to injury in immunocompromised rats is reduced in aging and restoration of normal tissue quality is age-dependent.

Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism - Corrected Proof

2009-10-28

Abstract: S-adenosylmethionine (SAM)-dependent methylation of biological molecules including DNA and proteins is rapidly being uncovered as a critical mechanism for regulation of cellular processes. We investigated the effects of reduced SAM-dependent methylation on osteoblast differentiation by using periodate oxidized adenosine (ADOX), an inhibitor of SAM-dependent methyltransferases. The capacity of this agent to modulate osteoblast differentiation was analyzed under non-osteogenic control conditions and during growth factor-induced differentiation and compared with the effect of inhibition of DNA methylation by 5-Aza-2′-deoxycytidine (5-Aza-CdR). Without applying specific osteogenic triggers, both ADOX and 5-Aza-CdR induced mRNA expression of the osteoblast markers Alp, Osx, and Ocn in murine C2C12 cells. Under osteogenic conditions, ADOX inhibited differentiation of both human mesenchymal stem cells and C2C12 cells. Gene expression analysis of early (Msx2, Dlx5, Runx2) and late (Alp, Osx, Ocn) osteoblast markers during bone morphogenetic protein 2-induced C2C12 osteoblast differentiation revealed that ADOX only reduced expression of the late phase Runx2 target genes. By using a Runx2-responsive luciferase reporter (6xOSE), we showed that ADOX reduced the activity of Runx2, while 5-Aza-CdR had no effect. Taken together, our data suggest that decreased SAM-dependent methyltransferase activity leads to impaired osteoblast differentiation via non-DNA-dependent methylation mechanisms and that methylation is a regulator of Runx2-controlled gene expression.

A microarray based identification of osteoporosis-related genes in primary culture of human osteoblasts - Corrected Proof

2009-10-26

Abstract: Genetic factors influencing the pathogenesis of osteoporosis are still largely unknown. We employed genome-wide gene expression approach in order to discover novel genes involved in the pathogenesis of osteoporosis. To this end, primary cultures of osteoblasts isolated from osteoporotic and non-osteoporotic human bone tissue samples were prepared. One thousand six hundred six genes were found to be differentially expressed, indicating increased demand for protein synthesis and decreased cell proliferation rate in osteoblasts from osteoporotic tissue as compared to osteoblasts from non-osteoporotic tissue. At first, top four genes, based on the microarray data and potential role in bone metabolism, were further studied in bone tissue samples of 55 patients. PTN and COL15A1 were both downregulated in osteoporotic bone tissue (6.2- and 3.4-fold, respectively, both p<0.05), while IBSP and CXCL2 were both upregulated (5.7-fold, p<0.05, and 2.1-fold, p>0.05). Further biostatistical analysis of the microarray data by gene set enrichment analysis suggested oxidative stress may have an important part in the pathogenesis of osteoporosis. Thus, secondly, we tested it by an in vitro assay on human osteosarcoma cell line cells treated with hydrogen peroxide. After 72 h of treatment with 500 μM hydrogen peroxide, the upregulation of the same genes involved in the response to oxidative stress as on the microarrays was observed: MT1G (metallothionein 1G, 22.1-fold, p<0.05), TXNRD1 (thioredoxin reductase 1, 3.7-fold, p<0.05), AOX1 (aldehyde oxidase 1, 24.5-fold, p<0.05) and GSR (glutathione reductase, 4.7-fold, p<0.05). Our results present a novel list of genes and metabolic pathways that may be associated with the pathogenesis of osteoporosis. PTN, CXCL2, COL15A1, IBSP, AOX1, MT1G, GSR and TXNRD1 are candidate genes for further studies in the assessment of the genetic susceptibility to osteoporosis. In addition, differences in protein synthesis, cell proliferation rate and response to oxidative stress may also be involved in the pathogenesis of osteoporosis.

A biodegradable porous composite scaffold of PGA/β-TCP for bone tissue engineering - Corrected Proof

Hong Cao, Noboru Kuboyama — 2009-10-26

Abstract: Polyglycolic acid (PGA) and beta-tricalcium phosphate (β-TCP) each have many applications as tissue repair materials. In this study, three-dimensional (3D) porous composite scaffolds of PGA/β-TCP (in 1:1 and 1:3 weight ratios) were fabricated using the solvent casting and particulate leaching method. PGA/β-TCP scaffolds with high porosity, interconnected 3D pores and rough surfaces were obtained and were observed using scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). The PGA/β-TCP scaffolds were investigated during the repair of critical bone defects (3 mm diameter, 2 mm depth) in rat femoral medial-epicondyles, compared with hydroxylapatite (HAP) and no implant as controls. Quantitative imageology analysis (volume and density of new bone) and qualitative histological evaluations (hematoxylin and eosin staining; tartrate-resistant acid phosphatase-hematoxylin counterstaining) were characterized using in vivo micro-CT images and histological sections at 0, 14, 30 and 90 days after surgery. Significant differences of all variables were tested by multivariate analysis (p<0.05). The results showed that the bone reformation by using the PGA/β-TCP scaffolds began within 14 days of surgery, and were healing well at 30 days after surgery. By 90 days after surgery, the bone replacement was almost completed and presented a healthy bone appearance. The new bone mineral densities (mg/cm3) with HAP, PGA/β-TCP (1:1) and PGA/β-TCP (1:3) at 90 days after surgery were: 390.4±18.1, 563.8±26.9 and 606.3±26.9, respectively. The new bone mineral density with the PGA/β-TCP scaffold was higher than with HAP (p<0.001), and with the PGA/β-TCP (1:3) scaffold was higher than with the PGA/β-TCP (1:1) scaffold at each time examined (p<0.05). The biodegradation percents (%) of HAP, PGA/β-TCP (1:1) and PGA/β-TCP (1:3) at 90 days after surgery were: 35.1±5.5, 99.0±1.0 and 96.2±3.3, respectively. The biodegradation percents of the PGA/β-TCP scaffolds were higher than HAP at each time examined (p<0.01), and matched the osteogenesis rates. The PGA/β-TCP scaffolds were almost replaced by new growing bone within 90 days after surgery. Thus the PGA/β-TCP composite scaffold, especially weight ratio 1:3, exhibited a strong ability for osteogenesis, mineralization and biodegradation for bone replacement.

Biomechanical effects of teriparatide in women with osteoporosis treated previously with alendronate and risedronate: Results from quantitative computed tomography-based finite element analysis of the vertebral body - Corrected Proof

2009-10-26

Abstract: Previous antiresorptive treatment may influence the anabolic response to teriparatide. The OPTAMISE (Open-label Study to Determine How Prior Therapy with Alendronate or Risedronate in Postmenopausal Women with Osteoporosis Influences the Clinical Effectiveness of Teriparatide) study reported greater increases in biochemical markers of bone turnover and volumetric bone mineral density (BMD) when 12 months of teriparatide treatment was preceded by 2 years or more of risedronate versus alendronate treatment. The objective of this study was to use quantitative computed tomography (CT)-based nonlinear finite element modeling to evaluate how prior therapy with alendronate or risedronate in postmenopausal women with osteoporosis influences the biomechanical effectiveness of teriparatide. Finite element models of the L1 vertebra were created from quantitative CT scans, acquired before and after 12 months of therapy with teriparatide, from 171 patients from the OPTAMISE study. These models were subjected to uniaxial compression. Total BMD-derived bone volume fraction (BV/TVd, i.e., bone volume [BV]/total volume [TV]), estimated from quantitative CT-based volumetric BMD, vertebral stiffness, and failure load (strength) were calculated for each time measurement point. The results of this study demonstrated that 12 months of treatment with teriparatide following prior treatment with either risedronate or alendronate increased BMD-derived BV/TVd, the predicted vertebral stiffness, and failure load. However, the effects of teriparatide were more pronounced in patients treated previously with risedronate, which is consistent with the findings of the OPTAMISE study. The mean (±standard error) increase in stiffness was greater in the prior risedronate group than the prior alendronate group (24.6±3.2% versus 14.4±2.8%, respectively; p=0.0073). Similarly, vertebral failure load increased by 27.2±3.5% in the prior risedronate group versus 15.3±3.1% in the prior alendronate group (p=0.0042). The mechanical variables increased in greater proportion than BV/TVd, which increased by 6.9±0.9% versus 4.6±0.8% in the prior-risedronate and prior-alendronate groups, respectively (p=0.0290). Our finding indicated that while teriparatide can be used with success on patients who have previously undergone treatment with risedronate and alendronate, it demonstrated greater anabolic effect on biomechanical properties in prior-risedronate patients in the first year of teriparatide treatment.

Characterisation of human bone marrow stromal cell heterogeneity for skeletal regeneration strategies using a two-stage colony assay and computational modelling - Corrected Proof

Bram G. Sengers, Jonathan I. Dawson, Richard O.C. Oreffo — 2009-10-26

Abstract: Skeletal regeneration and tissue engineering strategies rely critically on the efficient expansion of progenitor cell populations whilst simultaneously preserving multipotentiality and the ability to induce differentiation towards bone and cartilage. Cell population heterogeneity has a significant impact on this process, but is currently poorly quantified, hampering the interpretation of experimental results and the design of optimised expansion protocols.The objective of this study was to characterise individual human bone marrow stromal cell heterogeneity in terms of colony expansion potential. For this purpose, a novel two-stage CFU-F assay was developed in which cells from primary single cell-derived colonies were detached and reseeded again at clonal density as single cells to form new secondary colonies. This clearly demonstrated how secondary colony growth potential varies markedly both between and within primary colonies. Depending on the primary colony, cells either generated small secondary colonies only, or else a wide range of colony sizes. Using computational modelling it was shown how such colony heterogeneity could arise from hierarchical progenitor cell populations and what the limits of such a population structure were in explaining the experimental data. In addition the model demonstrated the significant potential impact of cell mobility on expansion potential and its implications for inducing population heterogeneity.This combined experimental-computational approach will ascertain the impact of cell culture protocols on the expansion potential and functional composition of heterogeneous progenitor populations. Such insights are likely to be of crucial importance for the success of skeletal regeneration strategies.

Sex-specific effect of Pirin gene on bone mineral density in a cohort of 4000 Chinese - Corrected Proof

2009-10-23

Abstract: Background: Osteoporosis is a common condition among elderly. Genetic mapping studies repeatedly located the distal short arms of X-chromosome as the quantitative trait loci (QTL) for BMD in mice. Fine mapping of a syntenic segment on Xp22 in a Caucasian female population suggested a moderate association between lumbar spine (LS) BMD and 2 intronic SNPs in the Pirin (PIR) gene, which encodes an iron-binding nuclear protein. This study aimed to examine genetic variations in the PIR gene by a comprehensive tagging method and its sex-specific effects on BMD and osteoporotic risk.Methods: Two thousand men and 2000 women aged 65 or above were recruited from the community. BMDs at the LS, femoral neck, total hip and whole body were measured and followed up at 4-year. Genotyping was performed for tagSNPs of PIR gene including adjacent regions, and the PIR haplotypes were inferred using PHASE program.Results: Analysis by linear regression showed a significant association between SNP rs5935970 and LS-BMD, while haplotype T-T-A was significantly associated with BMD of all measured sites. However, none of such associations were found in men. Linear Mixed Model also confirmed the same sex-specific and site-specific effect for longitudinal BMD changes.Conclusion: In addition to confirming the association between BMDs and the PIR gene, we also revealed that this finding is sex-specific, possibly due to an X-linked effect. This study demonstrated the importance of considering sex and genetic interactions in studies of disease predisposition and complex traits.

Bone involvement in clusters of autoimmune diseases: Just a complication? - Corrected Proof

2009-10-23

Abstract: Bone loss, described in individual groups of patients with Type 1 diabetes (T1D), autoimmune thyroid disease (ATD) or celiac disease (CD) is usually viewed as a complication of these diseases. There is increasing evidence that alterations in the immune system may directly affect bone mass. Clustering of autoimmune diseases in the same individual might predispose to higher risk of osteopenia due to imbalance in immune regulation. The aim of this study was to evaluate bone involvement in clusters of the most common autoimmune diseases (T1D, ATD and CD) in children.The study was performed at a tertiary care center for the care of pediatric diabetes. One-hundred-two patients with T1D alone or associated with ATD and/or CD were studied; 13 patients had cluster of three autoimmune diseases. Amplitude-dependent speed of sound (AD-SoS) was measured by phalangeal quantitative ultrasound and expressed as standard deviation score (SDS). AD-SoS SDS < −2 was considered indicative of osteopenia.Osteopenia was equally distributed among children with T1D alone (8.1%), T1D associated with ATD (7.7%) or CD (10.3%), while it was 53.8% in patients presenting with three autoimmune diseases. Poor compliance to gluten-free diet increased osteopenia to 18.8% in patients with T1D and CD and 80% in patients with three autoimmune disorders. No difference among groups was found with regard to gluco-metabolic control, calcium metabolism, thyroid function.In conclusion bone impairment in multiple autoimmune diseases might be considered not only a complication due to endocrine or nutritional mechanisms, but also a consequence of an immunoregulatory imbalance. Alterations of homeostatic mechanisms might explain an imbalance of osteoclast activity leading to osteopenia.

Nmp4/CIZ: Road block at the intersection of PTH and load - Corrected Proof

Paul Childress, Alexander G. Robling, Joseph P. Bidwell — 2009-10-19

Abstract: Teriparatide (parathyroid hormone, [PTH]) is the only FDA-approved drug that replaces bone lost to osteoporosis. Enhancing PTH efficacy will improve cost-effectiveness and ameliorate contraindications. Combining this hormone with load-bearing exercise may enhance therapeutic potential consistent with a growing body of evidence that these agonists are synergistic and share common signaling pathways. Additionally, neutralizing molecules that naturally suppress the anabolic response to PTH may also improve the efficacy of treatment with this hormone. Nmp4/CIZ (nuclear matrix protein 4/cas interacting zinc finger)-null mice have enhanced responses to intermittent PTH with respect to increasing trabecular bone mass and are also immune to disuse-induced bone loss likely by the removal of Nmp4/CIZ suppressive action on osteoblast function. Nmp4/CIZ activity may be sensitive to changes in the mechanical environment of the bone cell brought about by hormone- or mechanical load-induced changes in cell shape and adhesion. Nmp4 was identified in a screen for PTH-responsive nuclear matrix architectural transcription factors (ATFs) that we proposed translate hormone-induced changes in cell shape and adhesion into changes in target gene DNA conformation. CIZ was independently identified as a nucleocytoplasmic shuttling transcription factor associating with the mechano-sensitive focal adhesion proteins p130Cas and zxyin. The p130Cas/zyxin/Nmp4/CIZ pathway resembles the β-catenin/TCF/LEF1 mechanotransduction response limb and both share features with the HMGB1 (high mobility group box 1)/RAGE (receptor for advanced glycation end products) signaling axis. Here we describe Nmp4/CIZ within the context of the PTH-induced anabolic response and consider the place of this molecule in the hierarchy of the PTH-load response network.

Identification of a novel dentin matrix protein-1 (DMP-1) mutation and dental anomalies in a kindred with autosomal recessive hypophosphatemia - Corrected Proof

2009-10-19

Abstract: An autosomal recessive form of hypophosphatemia (ARHP) was recently shown to be caused by homozygous mutations in DMP1, the gene encoding dentin matrix protein-1 (DMP-1), a non-collagenous bone matrix protein with an important role in the development and mineralization of bone and teeth. Here, we describe a previously not reported consanguineous ARHP kindred in which the three affected individuals carry a novel homozygous DMP-1 mutation. The index case presented at the age of 3 years with bowing of his legs and showed hypophosphatemia due to insufficient renal phosphate retention. Serum alkaline phosphatase activity was elevated, with initially normal PTH. FGF23 was inappropriately normal at an older age while being treated with oral phosphate and 1,25(OH)2D. Similar clinical and biochemical findings, except for elevated FGF23 levels, were present in his 16-month-old brother and his 12.5-year-old female cousin; the parents of the three affected children are first-degree cousins. Nucleotide sequence analysis was performed on PCR-amplified exons encoding DMP-1 and flanking intronic regions. A novel homozygous frame-shift mutation (c.485Tdel; p.Glu163ArgfsX53) in exon 6 resulting in a premature stop codon was identified in all effected individuals. The parents and available unaffected siblings were heterozygous for c.485Tdel. Tooth growth and shape were normal for the index case, his affected brother and cousin, but their permanent and deciduous teeth displayed enlarged pulp chambers. The identified genetic mutation underscores the importance of DMP-1 mutations in the pathogenesis of ARHP. Furthermore, DMP-1 mutations appear to contribute, through yet unknown mechanisms, to tooth development.

Fatigue in bone: A novel phenomenon attributable to bisphosphonate use - Corrected Proof

Athanassios Kyrgidis, Eugenia Verrou — 2009-10-19

We read with interest and appreciation the article by Lesclous et al. attempting to illustrate the role of inflammation in the aetiopathogenesis of bisphosphonate (BP) associated osteonecrosis of the jaw (ONJ).

Reply to “Fatigue in bone: A novel phenomenon attributable to bisphosphonate use” - Corrected Proof

2009-10-19

We thank Kirgidis and Verrou for their interest in our paper “Bisphosphonate-associated osteonecrosis of the jaw: a key role of inflammation?” and we found their comments an interesting contribution.

An approach to quantifying bone overloading and hypertrophy with applications to multiple experimental studies - Corrected Proof

J.C. Chen, G.S. Beaupré, D.R. Carter — 2009-10-19

Abstract: Many studies have investigated mechanically induced bone formation in mice and rats by applying loads to the long bones, and measuring changes in periosteal cortical bone apposition rates. However, the results are difficult to compare among each other because the loading schemes are generally different. The purpose of the present study was to develop a theoretical framework for evaluating the mechanical stimulus based on the bone daily strain stimulus, which is a function of loading cycles and bone strains. The daily strain stimulus would act as a single unifying parameter for directly comparing data from existing in vivo experiments, and is applied here to twenty previous rat and mouse studies. To calculate the daily strain stimulus, we determined the periosteal daily strain stimulus necessary for bone maintenance (ξperi,0) and the strain-cycle weighting exponent (m). In the first approach, we applied data from Rubin and Lanyon's bone maintenance studies. We calculated ξperi,0 to be 2793 microstrain/day, and m to be 4.5. In the second approach, we used Fritton et al. 's strain gage recordings to calculate ξperi,0 to be 1496 microstrain/day, and used an m value of 11.88, equal to human bone compressive fatigue properties. Fatigue data provided physiological relevance, and was useful for applying an established remodeling theory to in vivo studies. For both approaches, ξperi,0 was below the fracture level. We then analyzed the applied strains, cycles, and periosteal bone apposition rates from the previous studies. The range of daily strain stimuli calculated using the first approach was much larger than the range using the second approach (2793–17312 microstrain/day compared to 1496–7681 microstrain/day). None of the studies applied a daily strain stimulus above the complete fatigue failure level, but some studies applied loading that could result in major fatigue microdamage. Bone apposition rates generally increased with increasing daily strain stimulus, which was consistent with previous theoretical models. The results suggest that the daily strain stimulus may be a reasonable first approximation for predicting bone apposition rates in a consistent manner. The use of the daily strain stimulus may be helpful for improving the design of future bone loading studies.

Pathway-based genome-wide association analysis identified the importance of EphrinA–EphR pathway for femoral neck bone geometry - Corrected Proof

2009-10-16

Abstract: Femoral neck (FN) bone geometry is an important predictor of bone strength with high heritability. Previous studies have revealed certain candidate genes for FN bone geometry. However, the majority of the underlying genetic factors remain to be discovered. In this study, pathway-based genome-wide association analysis was performed to explore the joint effects of genes within biological pathways on FN bone geometry variations in a cohort of 1000 unrelated US whites. Nominal significant associations (nominal p value<0.05) were observed between 76 pathways and a key FN bone geometry variable—section modulus (Z), biomechanically indicative of bone strength subject to bending. Among them, EphrinA–EphR pathway was most significantly associated with FN Z even after multiple testing adjustments (pFWER value=0.035). The association of EphrinA–EphR pathway with FN Z was also observed in an independent sample from Framingham Osteoporosis Study. Overall, these results suggest the significant genetic contribution of EphrinA–EphR pathway to femoral neck bone geometry.

A multicentre, retrospective case–control study assessing the role of trabecular bone score (TBS) in menopausal Caucasian women with low areal bone mineral density (BMDa): Analysing the odds of vertebral fracture - Corrected Proof

2009-10-15

Abstract: Introduction: The trabecular bone score (TBS) is a new parameter that is determined from grey level analysis of DXA images. It relies on the mean thickness and volume fraction of trabecular bone microarchitecture. This was a preliminary case–control study to evaluate the potential diagnostic value of TBS, both alone and combined with bone mineral density (BMDa), in the assessment of vertebral fracture.Methods: Out of a subject pool of 441 Caucasian, postmenopausal women between the ages of 50 and 80 years, we identified 42 women with osteoporosis-related vertebral fractures, and compared them with 126 age-matched women without any fractures (1 case: 3 controls). Primary outcomes were BMDa and TBS. Inter-group comparisons were undertaken using Student's t-tests and Wilcoxon signed ranks tests for parametric and non-parametric data, respectively. Odds ratios for vertebral fracture were calculated for each incremental one standard deviation decrease in BMDa and TBS, and areas under the receiver operating curve (AUC) calculated and sensitivity analysis were conducted to compare BMDa alone, TBS alone, and the combination of BMDa and TBS. Subgroup analyses were performed specifically for women with osteopenia, and for women with T-score-defined osteoporosis.Results: Across all subjects (n=42, 126) weight and body mass index were greater and BMDa and TBS both less in women with fractures. The odds of vertebral fracture were 3.20 (95% CI, 2.01–5.08) for each incremental decrease in TBS, 1.95 (1.34–2.84) for BMDa, and 3.62 (2.32–5.65) for BMDa + TBS combined. The AUC was greater for TBS than for BMDa (0.746 vs. 0.662, p=0.011). At iso-specificity (61.9%) or iso-sensitivity (61.9%) for both BMDa and TBS, TBS + BMDa sensitivity or specificity was 19.1% or 16.7% greater than for either BMDa or TBS alone. Among subjects with osteoporosis (n=11, 40) both BMDa (p=0.0008) and TBS (p=0.0001) were lower in subjects with fractures, and both OR and AUC (p=0.013) for BMDa + TBS were greater than for BMDa alone (OR=4.04 [2.35–6.92] vs. 2.43 [1.49–3.95]; AUC=0.835 [0.755–0.897] vs. 0.718 [0.627–0.797], p=0.013). Among subjects with osteopenia, TBS was lower in women with fractures (p=0.0296), but BMDa was not (p=0.75). Similarly, the OR for TBS was statistically greater than 1.00 (2.82, 1.27–6.26), but not for BMDa (1.12, 0.56–2.22), as was the AUC (p=0.035), but there was no statistical difference in specificity (p=0.357) or sensitivity (p=0.678).Conclusions: The trabecular bone score warrants further study as to whether it has any clinical application in osteoporosis detection and the evaluation of fracture risk.

A phenotypically restricted set of primary afferent nerve fibers innervate the bone versus skin: Therapeutic opportunity for treating skeletal pain - Corrected Proof

2009-10-15

Abstract: Although musculoskeletal pain is one of the most common causes of chronic pain and physical disability in both developing and developed countries, relatively little is known about the nerve fibers and mechanisms that drive skeletal pain. Small diameter sensory nerve fibers, most of which are C-fiber nociceptors, can be separated into two broad populations: the peptide-rich and peptide-poor nerve fibers. Peptide-rich nerve fibers express substance P (SP) and calcitonin gene-related peptide (CGRP). In contrast, the peptide-poor nerve fibers bind to isolectin B4 (IB4) and express the purinergic receptor P2X3 and Mas-related G protein-coupled receptor member d (Mrgprd). In the present report, we used mice in which the Mrgprd+ nerve fibers express genetically encoded axonal tracers to determine the peptide-rich and peptide-poor sensory nerve fibers that innervate the glabrous skin of the hindpaw as compared to the bone marrow, mineralized bone and periosteum of the femur. Whereas the skin is richly innervated by CGRP+, SP+, P2X3+ and Mrgprd+ sensory nerve fibers, the bone marrow, mineralized bone and periosteum receive a significant innervation by SP+ and CGRP+, but not Mrgprd+ and P2X3+ nerve fibers. This lack of redundancy in the populations of C-fibers that innervate the bone may present a unique therapeutic opportunity for targeting skeletal pain as the peptide-rich and peptide-poor sensory nerve fibers generally express a different repertoire of receptors and channels to detect noxious stimuli. Thus, therapies that target the specific types of C-nerve fibers that innervate the bone may be uniquely effective in attenuating skeletal pain as compared to skin pain.

Low-intensity pulsed ultrasound-dependent osteoblast proliferation occurs by via activation of the P2Y receptor: Role of the P2Y1 receptor - Corrected Proof

2009-10-15

Abstract: Low-intensity pulsed ultrasound (LIPUS) is commonly used in the treatment of fractures and nonunion-promoting acceleration of healing fractures. In this report, we investigated the implication of the P2 receptors in osteoblast proliferation induced with LIPUS treatment. We observed that ADP, ATP, UTP, and UDP promote osteoblast increase and an increase of intracellular Ca2+, through activation of P2Y receptors. Osteoblasts' expression of the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13 receptors was confirmed. In addition, the participation of the P2Y1 receptor in osteoblast increase and the ADP-dependent increase of Ca2+ concentration were shown. Furthermore, release of ATP/purines was induced by LIPUS treatment. Finally, LIPUS-dependent osteoblast increase was abolished in the presence of the Ca2+ chelator (BAPTA), the inositol 1,4,5-trisphosphate receptor antagonist (2-APB), and the selective P2Y1 receptor antagonist (MRS2179). In conclusion, LIPUS treatment induces osteoblastogenesis via the release of purines, such as ATP, activating P2Y receptors, mainly the P2Y1 receptor.

Treatment with a soluble receptor for activin improves bone mass and structure in the axial and appendicular skeleton of female cynomolgus macaques (Macaca fascicularis) - Corrected Proof

2009-10-15

Abstract: A recent study suggests that activin inhibits bone matrix mineralization, whereas treatment of mice with a soluble form of the activin type IIA receptor markedly increases bone mass and strength. To further extend these observations, we determined the skeletal effects of inhibiting activin signaling through the ActRIIA receptor in a large animal model with a hormonal profile and bone metabolism similar to humans. Ten female cynomolgus monkeys (Macaca fascicularis) were divided into two weight-matched groups and treated biweekly, for 3 months, with either a subcutaneous injection 10 mg/kg of a soluble form of the ActRIIA receptor fused with the Fc portion of human IgG1 (ACE-011) or vehicle (VEH). Bone mineral density (BMD), micro-architecture, compressive mechanical properties, and ash fraction were assessed at the end of the treatment period. BMD was significantly higher in ACE-011 treated individuals compared to VEH: +13% (p=0.003) in the 5th lumbar vertebral body and +15% (p=0.05) in the distal femur. In addition, trabecular volumetric bone density at the distal femur was 72% (p=0.0004) higher than the VEH-treated group. Monkeys treated with ACE-011 also had a significantly higher L5 vertebral body trabecular bone volume (p=0.002) and compressive mechanical properties. Ash fraction of L4 trabecular bone cores did not differ between groups. These results demonstrate that treatment with a soluble form of ActRIIA (ACE-011) enhances bone mass and bone strength in cynomolgus monkeys, and provide strong rationale for exploring the use of ACE-011 to prevent and/or treat skeletal fragility.

COMP-Ang1, a chimeric form of Angiopoietin 1, enhances BMP2-induced osteoblast differentiation and bone formation - Corrected Proof

2009-10-15

Abstract: Introduction: Angiogenesis is closely associated with bone formation, especially endochondral ossification. Angiopoietin 1 (Ang1) is a specific growth factor functioning to generate a stable and matured vasculature through the Tie2 receptor/PI3K/AKT pathway. Recently cartilage oligomeric matrix protein (COMP)-Ang1, an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor and AKT, was developed. This study was designed to examine the effects of angiogenic COMP-Ang1 on BMP2-induced osteoblast differentiation and bone formation.Methods: Expression of endogenous Ang-1 and its binding receptor Tie 2 mRNA was examined in osteoblast-like cells and primary mouse calvarial cells by RT-PCR analysis, and was also monitored during osteoblast differentiation induced by BMP-2 and/or ascorbic acid and β-glycerophosphate. Effects of COMP-Ang-1 on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and osteocalcin (OC) production, and Alizarin red stain. For a molecular mechanism, Western blot and OG2 and 6xOSE promoter assays were done. For in vivo evaluation, adenoviral (Ad) vectors containing COMP-Ang-1 or BMP-2 gene were administered into thigh muscle of mice, and after 2 weeks bone formation was analyzed by micro-computed tomography and histology. Angiogenic event of COMP-Ang1 was confirmed by immunofluorescence analysis with anti-CD31 antibody.Results: Expression of Tie2 receptor was significantly increased in the course of osteoblast differentiation. Treatment or overexpression of COMP-Ang1 enhanced BMP2-induced ALP activity, OC production, and mineral deposition in a dose-dependent manner. In addition, COMP-Ang1 synergistically increased OG2 and 6xOSE promoter activities of BMP2, and sustained p38, Smad and AKT phosphorylation of BMP2. Notably, in vivo intramuscular injection of COMP-Ang1 dose-dependently enhanced BMP2-induced ectopic bone formation with increases in CD31 reactivity.Conclusions: These results suggest that COMP-Ang1 synergistically enhanced osteoblast differentiation and bone formation through potentiating BMP2 signaling pathways and angiogenesis. Combination of BMP2 and COMP-Ang1 should be clinically useful for therapeutic application to fracture and destructive bone diseases.

Circadian rhythm affects the preventive role of pulsed electromagnetic fields on ovariectomy-induced osteoporosis in rats - Corrected Proof

2009-10-15

Abstract: Pulsed electromagnetic fields (PEMF) have been proved effective in the prevention of osteoporosis both experimentally and clinically. Chronotherapy studies have shown that circadian rhythm (CR) played an important role in the occurrence, development and treatment of several diseases. CR has also been recognized as an essential feature of bone metabolism. Therefore, it is of therapeutic significance to investigate the impact of CR on the efficacy of PEMF in the prevention of osteoporosis. However, this issue has never been discussed previously. The objective of this study was to systematically evaluate the impact of CR on the preventive effect of PEMF on osteoporosis in rats. Thirty-two 3 month old female Sprague–Dawley rats were randomly divided into four different groups: sham-operated control (Sham), ovariectomy (OVX), OVX with PEMF stimulation in daytime (OVX+DPEMF) and OVX with PEMF stimulation in nighttime (OVX+NPEMF) groups. The OVX+DPEMF and OVX+NPEMF groups were subjected to daily PEMF exposure on the 2nd post-operative day, from 9:00 to 15:00, and 0:00 to 6:00, respectively. After 12 weeks, the OVX+DPEMF group presented better efficacy in prevention against OVX-induced bone loss and deterioration of trabecular bone architecture compared with the OVX+NPEMF group. This was evidenced by the increased levels of femoral bone mineral density, trabecular area percentage, trabecular thickness, trabecular number and decreased trabecular separation. Furthermore, the bone turnover biomarkers (serum alkaline phosphatase, serum bone Gla protein and urinary deoxypyridinoline) and the dynamic histomorphometric parameters reflecting the trabecular osteoblast and osteoclast activity (bone formation rate with bone volume as referent, osteoclast number, etc.) in the OVX+DPEMF group decreased to a larger extent compared with the OVX+NPEMF group. In conclusion, the results indicated that CR was an important factor determining the preventive effect of PEMF on osteoporosis and PEMF exposure in the daytime presented better stimulus efficacy in rats. The findings might be helpful for the efficacious use of PEMF mediations, evaluation of PEMF action and experimental design in the future studies of biological effect of electromagnetic fields.

Factors affecting calcium balance in Chinese adolescents - Corrected Proof

2009-10-15

Abstract: Chinese dietary reference intakes (DRIs) for calcium were developed mainly from studies conducted amongst Caucasians, yet a recent review showed that reference calcium intakes for Asians are likely to be different from those of Caucasians (Lee and Jiang, 2008). In order to develop calcium DRIs for Chinese adolescents, it is necessary to explore the characteristics and potential influencing factors of calcium metabolic balance in Chinese adolescents. A total of 80 students (15.1±0.8 years) were recruited stratified by gender from a 1-year calcium supplementation study. Subjects were randomly designed to four groups and supplemented with calcium carbonate tablets providing elemental calcium at 63, 354, 660, and 966 mg/day, respectively. Subjects consumed food from a 3-day cycle menu prepared by staff for 10 days. Elemental calcium in samples of foods, feces, and urine was determined in duplicates by inductively coupled plasma atomic emission spectrometry. The total calcium intake ranged from 352 to 1323 mg/day. The calcium apparent absorption efficiency and retention in boys were significantly higher than that in girls (68.7% vs. 46.4%, 480 mg/day vs. 204 mg/day, P<0.05). Calcium retention increased with calcium intakes, but did not reach a plateau. Calcium absorption efficiency in boys increased with calcium intake up to 665 mg/day, and decreased after that. In girls, calcium absorption efficiency decreased with calcium intake. Calcium absorption efficiency increased within 1 year after first spermatorrhea in boys, but decreased with pubertal development in girls. Sex, calcium intake, age, and pubertal development were the most important determinants of calcium absorption (R2=0.508, P<0.01) and retention (R2=0.745, P<0.05). This study indicates that sex, calcium intake, age, and pubertal development are important factors for calcium retention and absorption during growth, which should be considered for the development of calcium DRIs for Chinese adolescents.

Reproducibility of bone micro-architecture measurements in rodents by in vivo micro-computed tomography is maximized with three-dimensional image registration - Corrected Proof

Kyle K. Nishiyama, Graeme M. Campbell, Robert J. Klinck, Steven K. Boyd — 2009-10-15

Abstract: In vivo micro-computed tomography (μCT) is a new method to monitor longitudinal changes of bone micro-architecture. Common animal models of bone diseases are mice and rats, and it is important to know the reproducibility of the bone measurements in order to correctly interpret results. When performing baseline and follow-up acquisitions, variation in the scan region will influence the parameters, and it has yet to be investigated if three-dimensional (3D) registration can improve the reproducibility.Two typical breeds of mice and one typical breed of rats were scanned four times each using μCT and standard bone morphological and density measurements were calculated. Image registration was used to find the overlapping regions between the scans within each series of animal data and only overlapping regions were analyzed for the bone parameters.Reproducibility was determined for each animal both pre- and post-registration. For the rats, results included a bone volume ratio (BV/TV) precision error of 5.46%, cortical thickness (Ct.Th) error of 1.97%, and tissue mineral density (TMD) of 2.00%. For the BL6 mice, precision errors were 3.00% (BV/TV), 0.95% (Ct.Th), and 0.94% (TMD), and for the C3H mice 2.68% (BV/TV), 1.52% (Ct.Th), and 1.72% (TMD). After image registration there was a significant improvement in reproducibility in most parameters for the rats.In general, metric parameters such as bone volume ratio had better reproducibility than the non-metric parameters connectivity density and structure model index. With 3D registration, reproducibility improved the results obtained by the experienced operators in this study. Registration may serve to equalize reproducibility of operators with different skill levels and across laboratories. It also improves efficiency by reducing the amount of hand-contouring required. This reproducibility data will be important for the interpretation of current and future longitudinal μCT studies.

An orally active calcium-sensing receptor antagonist that transiently increases plasma concentrations of PTH and stimulates bone formation - Corrected Proof

2009-10-15

Abstract: Daily subcutaneous administration of exogenous parathyroid hormone (PTH) promotes bone formation in patients with osteoporosis. Here we describe two novel, short-acting calcium-sensing receptor antagonists (SB-423562 and its orally bioavailable precursor, SB-423557) that elicit transient PTH release from the parathyroid gland in several preclinical species and in humans. In an ovariectomized rat model of bone loss, daily oral administration of SB-423557 promoted bone formation and improved parameters of bone strength at lumbar spine, proximal tibia and midshaft femur. Chronic administration of SB-423557 did not increase parathyroid cell proliferation in rats. In healthy human volunteers, single doses of intravenous SB-423562 and oral SB-423557 elicited transient elevations of endogenous PTH concentrations in a profile similar to that observed with subcutaneously administered PTH. Both agents were well tolerated in humans. Transient increases in serum calcium, an expected effect of increased parathyroid hormone concentrations, were observed post-dose at the higher doses of SB-423557 studied. These data constitute an early proof of principle in humans and provide the basis for further development of this class of compound as a novel, orally administered bone-forming treatment for osteoporosis.