Hot off the presses! May 01 Bone

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Latest Articles Include:

• Editorial Board
  - Bone 44(5):i-ii (2009)
  
• IBMS Board of Directors 2007–2009
  - Bone 44(5):731 (2009)
  
• IBMS Corporate Partners
  - Bone 44(5):732 (2009)
  
• IBMS Membership Application
  - Bone 44(5):733 (2009)
  
• FRAX® and its applications to clinical practice
  - Bone 44(5):734-743 (2009)
  The introduction of the WHO FRAX® algorithms has facilitated the assessment of fracture risk on the basis of fracture probability. FRAX® integrates the influence of several well validated risk factors for fracture with or without the use of BMD. Its use in fracture risk prediction poses challenges for patient assessment, the development of practice guidelines, the evaluation of drug efficacy and reimbursement, as well as for health economics which are the topics outlined in this review.
• Efficacy and safety of pharmacological agents in managing osteoporosis in the old old: Review of the evidence
  - Bone 44(5):744-751 (2009)
  Introduction Osteoporosis and fracture risk increase exponentially in postmenopausal females. This places a significant burden in terms of morbidity, mortality and costs that are likely to increase with an ageing population. Despite this there is very limited data on pharmacological management of osteoporosis in this high risk group. Objectives of this review To review the published literature on the clinical efficacy and safety of specific anti osteoporosis treatments in the reduction in fracture risk in females ≥ 75 years of age. The following major endpoints were used in this review:1. Vertebral fracture reduction at 1 year and 3 years. 2. Non-vertebral fracture and hip fracture reduction at 1 year and 3 years. 3. Safety data in this group. Search methods for identification of studies We performed an electronic search of Medline (1970 to June 2007) and the Cochrane Library (1996 to June 2007). Our search strategy included MeSH terms for osteoporosis and treatments. We reviewed the reference list of identified articles for additional relevant published trials. Results Two hundred and fifty-two potentially relevant abstracts were identified. Only six publications were deemed to meet full eligibility criteria and one met most criteria. There is evidence for significant vertebral fracture relative risk reduction(RR) at 1 year for Risedronate (RR 81%; p < 0.001), Teriparatide (RR 65%; p < 0.05) and Strontium Ranelate (RR 59%; p = 0.002) and 3 years for Risedronate (RR 44%; p = 0.003), Alendronate (RR 38%; p < 0.05) and Strontium Ranelate (RR 32%; p = 0.013). There is evidence for significant non-vertebral fracture relative risk reduction at 1 year for Strontium Ranelate (RR 41%; p = 0.027) but not Teriparatide (p = 0.66) and 3 years for Strontium Ranelate (RR 31%; p = 0.011) but not Risedronate (p = 0.66). The only study to report a reduction in hip fracture at 3 years is the TROPOS study with Strontium Ranelate (RR 36%; p = 0.046). Discussion This review reinforces the irony that the least evidence is available for fragility fracture reduction in the group at greatest risk; the old old and those with non vertebral and hip fracture. Although there is good evidence for the benefit of the bisphosphonates (Alendronate and Risedronate), Teriparatide and Strontium Ranelate in vertebral fracture reduction, there are very limited data for non vertebral and hip fracture reduction. Strontium Ranelate is the only agent to date that has demonstrated a reduction in non vertebral and hip fracture events in this high risk elderly female population. Perhaps we need to adopt different strategies in managing older patients with osteoporosis as their fracture risks and treatment strategies may be quite different from younger populations.
• Tracking of bone mass from childhood to adolescence and factors that predict deviation from tracking
  - Bone 44(5):752-757 (2009)
  It has been hypothesized that bone density tracks but long term studies in children are lacking. As such, the aim of this study was to describe tracking of dual X-ray absorptiometry measures from age 8 to age 16 years, whether this was independent of change in body size and whether deviation from tracking could be predicted. 116 males and 67 females had anthropometric (height and weight), questionnaire (medication use, sports, breastfeeding), fitness (PWC170) and DXA measures (bone free lean mass [LM], fat mass [FM] and bone mass) at baseline and follow-up. BMC and aBMD were assessed at the spine and hip and total body and bone mineral apparent density (BMAD) at the spine and hip. We found all DXA measures tracked significantly after adjustment for change in height and change in weight (males: R2: BMC 24–62%; aBMD 41–48%; BMAD 30–37%, females: R2: BMC 45–72%; aBMD 36–56%; BMAD 30–48%). Factors that predicted subjects would deviate positively, that is improv! e in tertile or remain in the highest tertile of spine and hip aBMD included having been breastfed, increase in LM, PWC170 at age 8 and sport participation in males. LM at age 8 was beneficial in males while in females; FM at age 8 predicted subjects would deviate positively. Boys who gained absolute and percent FM and girls who gained percent FM, were more likely to deviate negatively, that is, decrease in tertile or remain in the lowest tertile of spine and hip aBMD. ICS use at age 8 also predicted subjects, particularly males would not improve in bone mass relative to their peers. In conclusion, DXA measures track moderately to strongly from childhood to adolescence. This was independent of linear growth and sex indicating bone development and physical growth are under largely separate mechanistic control. Body composition was the main predictor of altered tracking but environmental factors also appear important.
• Risk of fracture in women treated with monthly oral ibandronate or weekly bisphosphonates: The eValuation of IBandronate Efficacy (VIBE) database fracture study
  - Bone 44(5):758-765 (2009)
  The eValuation of IBandronate Efficacy (VIBE) head-to-head database fracture study compared fracture rates between patients treated with monthly ibandronate and weekly oral bisphosphonates (BPs). This large study included women ≥ 45 years old, newly prescribed monthly oral ibandronate or weekly oral alendronate or risedronate, and without malignancy or Paget's disease of bone. The primary analysis included patients who were adherent to treatment during the first 90 days after the index date. The risks of hip, nonvertebral, vertebral and any clinical fracture were compared using Cox proportional hazards models and adjusted for potential confounding factors. A secondary, "intent-to-treat" analysis included all patients who received at least one BP prescription. Sensitivity analyses based on the primary analysis compared patients receiving ibandronate with patients receiving weekly alendronate or risedronate separately, and explored the effect of excluding patients with potential confounding factors from the analysis. Further sensitivity analyses varied the requirement for adherence during the first 90 days after the index date. The primary analysis population included 7345 monthly ibandronate and 56,837 weekly BP patients. Fracture rates after the 12-month observational period were < 2% and fracture risk was not significantly different between patients receiving monthly ibandronate or weekly BPs for hip, nonvertebral or any clinical fracture (adjusted relative risk: hip = 1.06, p = 0.84; nonvertebral = 0.88, p = 0.255; any clinical fracture = 0.82, p = 0.052). Ibandronate patients had a significantly lower risk of vertebral fracture than weekly BP patients (adjusted relative risk 0.36, 95% confidence interval 0.18–0.75, p = 0.006). In the secondary, "intent-to-treat" analysis, relative risks of fracture were not significantly different between treatment groups for any fracture type. The results of the sensitivity analyses were generally consistent with the primary analysis. This retrospective cohort study found that patients treated with oral monthly ibandronate or weekly BPs (alendronate and risedronate) had similar, low risks of hip fracture, nonvertebral fracture and any clinical fracture. Ibandronate patients had a significantly lower relative risk of vertebral fracture than weekly BP patients; the clinical implications of these findings require further exploration and validation.
• Bisphosphonate dose and incidence of fractures in postmenopausal osteoporosis
  - Bone 44(5):766-771 (2009)
  Introduction The specific pharmacological properties of bisphosphonates have raised concerns about their long-term effects on skeletal fragility that may be related to the total dose of bisphosphonate given. However, the effect of different doses on the incidence of osteoporotic fractures has not been adequately studied. Methods In this retrospective analysis, we investigated the effect of different doses of intravenous pamidronate given at 3-monthly intervals on the incidence of fractures in 92 women with severe postmenopausal osteoporosis. Results The risk of sustaining a new vertebral fracture on treatment was significantly increased by 32% for every prevalent vertebral fracture (OR: 1.32, CI: 1.05, 1.66; p = 0.02). Patients with nonvertebral fractures received a significantly lower dose of pamidronate and their risk for these fractures increased by 25% for every prevalent vertebral fracture at baseline (OR: 1.25, CI: 1.01, 1.53; p = 0.03). Patients who had received oral bisphosphonate before intravenous pamidronate had a significantly higher incidence of nonvertebral fractures which, however, did not hold true after adjustment for baseline BMD and prevalent fractures. Conclusions In patients with established osteoporosis bone fragility during treatment with intravenous pamidronate is mainly determined by the severity of the disease, assessed by the presence and numbers of prevalent fractures, rather than the dose of the bisphosphonate or the rate of bone turnover.
• Prevalence and risk factors of osteoporosis and vertebral fractures in patients with ankylosing spondylitis
  - Bone 44(5):772-776 (2009)
  Ankylosing spondylitis (AS) is characterized by inflammation of the entheses and paravertebral structures, leading in time to bone formation at those sites. As well, vertebral bone loss is also a recognized feature of AS Objective: To calculate the prevalence and risk factors of osteoporosis and vertebral fractures in patients with AS. Methods: Eighty patients with AS were enrolled in the study. Clinical, biological and radiological status was assessed by the Bath AS Disease Activity Index (BASDAI), Bath AS Functional Index (BASFI), ESR and C-reactive protein (CRP), Bath AS Radiology Index (BASRI) and modified stoke AS spine score (mSASSS). BMD of the hip and spine was measured and vertebral fractures were defined using a combination of Genant semiquantitative (SQ) approach and morphometry by VFA (fracture vertebral assessment). Results: The years ± 11.8. The mean BMI was 22.8 kg/m2 ± 4.1 and the mean disease duration was 10.8 years ± 6.6. Prevalence of osteoporosis was 25%. 18.8% of patients had a vertebral fracture (grades 2 and 3). Factors associated with osteoporosis were low weight and BMI and longer disease duration, higher ESR, CRP, BASFI and BASDAI. Vertebral fractures were associated with advanced age, longer disease duration, higher BASFI, BASRI and mSASSS and reduced BMD and T-score at the hip site, presence of osteoporosis at any site. Multiple logistic regression analysis (Table 4) revealed that parameters significantly associated with osteoporosis were BASDAI (OR = 1.05, 95% confidence interval [CI]: 1.03–1.09); disease duration (OR = 1.13, 95%CI: 1.03–1.25); and BMI (OR = 0.82, 95%CI: 0.69–0.93). The presence of VFs (grades 2 and 3) were independently associated with disease duration (OR = 1.50, 95%CI: 1.07–2.10); and mSASSS (OR = 1.17, 95%CI: 1.05–1.30). Conclusion: Osteoporosis is common in patients with AS and seems to be related to disease activity while vertebral fractures appear to be related to the duration and structural severity of the disease rather than BMD.
• Novel and unexpected functions of zebrafish CCAAT box binding transcription factor (NF-Y) B subunit during cartilages development
  - Bone 44(5):777-784 (2009)
  We used zebrafish as a model to study the biological functions of NF-YB during early development. Both RT-PCR and whole-mount in situ hybridization experiments revealed that nf-yb was a maternally inherited gene. Later, its expression was restricted in the future head cartilages as well as in the developing notochord. Embryos after injection with nf-yb-morpholino displayed reduced-head phenotypes, including smaller head (WT, length of head, L: 0.515 ± 0.019 mm, width of head, W: 0.323 ± 0.077 mm; nf-yb-morphant, L: 0.347 ± 0.037 mm; W: 0.266 ± 0.018 mm), sharpen Meckel's cartilage, loss of ceratobranchial, and enlarged angles of ceratohyal (WT: 72.6 ± 9.4°; nf-yb-morphant: 110.0 ± 32.5°). Subsequently, those abnormalities can be rescued after injection with capped nf-yb mRNA. TUNEL assay suggested that large amounts of cell apoptosis appeared in the head region of nf-yb-morphants. Staining with digoxigenin-labeled dlx2a, sox9a, runx2b and col2a1 riboprobes show! ed that nf-yb-morphants displayed reduced amounts of cranial neural crest cells which are required for mandibular and branchial arches formation. These observations clearly indicate that knockdown of nf-yb translation induced parts of cranial neural crest cells apoptosis, affected cartilages formation and consequently caused reduced-head phenotypes. These findings uncover a novel and unexpected role for NF-YB as a critical modulator of neural crest cell's gene expression governing embryonic cartilage growth.
• Osteocyte-derived HB-GAM (pleiotrophin) is associated with bone formation and mechanical loading
  - Bone 44(5):785-794 (2009)
  HB-GAM (also known as pleiotrophin) is a cell matrix-associated protein that is highly expressed in bone. It affects osteoblast function, and might therefore play a role in bone development and remodeling. We aimed to investigate the role of HB-GAM in bone in vivo and in vitro. The bones of HB-GAM deficient mice with an inbred mouse background were studied by histological, histomorphometrical, radiological, biomechanical and μ-CT analyses and the effect of immobilization was evaluated. HB-GAM localization in vivo was studied. MLO-Y4 osteocytes were subjected to fluid shear stress in vitro, and gene and protein expression were studied by subtractive hybridization, quantitative PCR and Western blot. Human osteoclasts were cultured in the presence of rhHB-GAM and their formation and resorption activities were assayed. In agreement with previous reports, the skeletal structure of the HB-GAM knockout mice developed normally. However, a growth retardation of the weight-bearing bones was observed by 2 months of age, suggesting a link to physical activity. Adult HB-GAM deficient mice were characterized by low bone formation and osteopenia, as well as resistance to immobilization-dependent bone remodeling. HB-GAM was localized around osteocytes and their processes in vivo and furthermore, osteocytic HB-GAM expression was upregulated by mechanical loading in vitro. HB-GAM did not affect on human osteoclast formation or resorption in vitro. Taken together, our results suggest that HB-GAM is an osteocyte-derived factor that could participate in mediating the osteogenic effects of mechanical loading on bone.
• Glutaredoxin 5 regulates osteoblast apoptosis by protecting against oxidative stress
  - Bone 44(5):795-804 (2009)
  There is now increasing evidence which suggests an important role for reactive oxygen species (ROS) in the pathogenesis of osteoporosis. However, little is known on the molecular components of the oxidative stress pathway or their functions in bone. In this study, we evaluated the role and mechanism of action of glutaredoxin (Grx) 5, a protein that is highly expressed in bone. Osteoblasts were transfected with Grx5 siRNA and treated with hydrogen peroxide (H2O2). Grx5 siRNA treatment increased apoptosis while Grx5 overexpression protected MC3T3-E1 cells against H2O2 induced apoptosis and ROS formation. Grx5 deficiency results in impaired biogenesis of Fe–S cluster in yeast. Accordingly, activity of mitochondrial aconitase, whose activity is dependent on Fe–S cluster, decreased in Grx5 siRNA treated cells. Since reduced formation of Fe–S cluster would lead to increased level of free iron, a competitive inhibitor of manganese superoxide dismutase (MnSOD), we measur! ed MnSOD activity in Grx5 deficient osteoblasts and found MnSOD activity was significantly reduced. The consequence of long term inhibition of Grx5 on osteoblast apoptosis was evaluated using lentiviral shRNA technology. Grx5 shRNA cells exhibited higher caspase activity and cardiolipin oxidation in the presence of H2O2. MnSOD activity was rescued by the addition of MnCl2 to Grx5 shRNA osteoblasts in the presence of H2O2. Our findings are consistent with the hypothesis that Grx5 is an important determinant of osteoblast apoptosis and acts via a molecular pathway that involves regulation of ROS production, cardiolipin oxidation, caspase activity, Fe–S cluster formation, and MnSOD activity.
• Wnt signaling inhibits cementoblast differentiation and promotes proliferation
  - Bone 44(5):805-812 (2009)
  Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. In this study, we have identified a consistent expression profile of Wnt signaling molecules in cementoblasts, in vitro by RT-PCR. Exposure of cells to LiCl, which promotes canonical Wnt signaling by inhibiting GSK-3β, increased β-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive promoters, suggesting that an endogenous canonical Wnt pathway functions in cementoblasts. Activation of endogenous canonical Wnt signaling with LiCl suppressed alkaline phosphatase (ALP) acti! vity and expression of genes associated with cementum function; ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Exposure to Wnt3a, as a representative canonical Wnt member, also inhibited the expression of ALP, BSP, and OCN gene. This effect was accompanied by decreased gene expression of Runx2 and Osterix and by increased gene expression of lymphoid enhancer factor-1. Pretreatment with Dickkopf (Dkk)-1, a potent canonical Wnt antagonist, which binds to a low-density lipoprotein-receptor-related protein (LRP)-5/6 co-receptor, attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of selective transcription factors. Wnt3a also increased the expression of cyclin D1, known as a cell cycle regulator, as well as cell proliferation. In conclusion, these observations suggest that Wnt signaling inhibits cementoblast d! ifferentiation and promotes cell proliferation. Elucidating th! e role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.
• Thrombin-stimulated growth factor and cytokine expression in osteoblasts is mediated by protease-activated receptor-1 and prostanoids
  - Bone 44(5):813-821 (2009)
  Thrombin exerts multiple effects upon osteoblasts including stimulating proliferation, and inhibiting osteoblast differentiation and apoptosis. Some of these effects are believed to be mediated by the synthesis and secretion of autocrine factors such as growth factors and cytokines. Many but not all cellular responses to thrombin are mediated by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. The current study was undertaken to investigate the nature of thrombin's induction of autocrine factors by analysing the expression of twelve candidate genes in thrombin-stimulated primary mouse osteoblasts. Analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that thrombin induced transforming growth factor β, cyclooxygenase-2, tenascin C, fibroblast growth factor-1 and -2, connective tissue growth factor and interleukin-6 expression in wild type osteoblasts, but not PAR-1 null mouse osteoblasts. ! Induction of all the thrombin-responsive genes was blocked by the presence of the non-selective cyclooxygenase inhibitor indomethacin. Further studies were conducted on interleukin-6, which was the gene that showed the greatest increase in expression following stimulation of osteoblast-like cells with thrombin. A PAR-1-specific activating peptide, but neither a PAR-4-activating peptide nor catalytically inactive thrombin induced release of interleukin-6 by osteoblasts. Furthermore, in the presence of the selective cyclooxygenase-1 and -2 inhibitors SC-560 and NS-398 thrombin-induced interleukin-6 release was prevented. Levels of both prostaglandin E2 and interleukin-6 in medium conditioned by thrombin-stimulated osteoblast-like cells were found to be significantly increased compared to medium conditioned by non-stimulated cells, however release of prostaglandin E2 was found to precede release of interleukin-6. Treatment of isolated osteoblast-like cells with a number of syn! thetic prostanoids stimulated secretion of interleukin-6 with ! differing potencies. These studies suggest that activation of PAR-1 on osteoblasts by thrombin induces cyclooxygenase activity, which in turn results in the increased expression of multiple secreted factors. The induction of these secreted factors may act in an autocrine fashion to alter osteoblast function, allowing these cells to participate in the earliest stages of bone healing by both autocrine and paracrine mechanisms.
• Use of rapidly mineralising osteoblasts and short periods of mechanical loading to accelerate matrix maturation in 3D scaffolds
  - Bone 44(5):822-829 (2009)
  MLO-A5 cells are a fully differentiated osteoblastic cell line with the ability to rapidly synthesise mineralised extracellular matrix (ECM). We used MLO-A5 cells to develop a system for studying the mechanical modulation of bone matrix formation in 3D using a cyclic compressive loading stimulus. Polyurethane (PU) open cell foam scaffolds were seeded with MLO-A5 cells under static conditions and loaded in compression at 1 Hz, 5% strain in a sterile fluid-filled chamber. Loading was applied for only 2 h per day on days 5, 10 and 15 of culture and cell-seeded scaffolds were assayed on days 10, 15 and 20 of culture. Collagen content as assayed by Sirius red was significantly (2 fold) higher at days 15 and 20 in loaded samples compared with static controls. Calcium content as assayed by alizarin red was significantly (4 fold) higher by day 20. The number of viable cells as assayed by MTS was higher in loaded samples at day 10 but there was no difference by days 15 and 20. ! Loaded samples also had higher stiffness in compression by the end of the experiment. The mRNA expression of type I collagen, osteopontin and osteocalcin was higher, after a single bout of loading, in loaded than in non-loaded samples as assayed by RT-PCR. In conclusion, mineralisation by fully differentiated osteoblasts, MLO-A5s, was shown to be highly sensitive to mechanical loading, with short bouts of mechanical loading having a strong effect on mineralised matrix production. The 3D system developed will be useful for systematic investigation of the modulators of in vitro matrix mineralisation by osteoblasts in mechanobiology and tissue engineering studies.
• Long term oncostatin M treatment induces an osteocyte-like differentiation on osteosarcoma and calvaria cells
  - Bone 44(5):830-839 (2009)
  Previous in vitro studies on primary osteoblastic and osteosarcoma cells (normal and transformed osteoblasts) have shown that oncostatin M (OSM), a member of the interleukin-6 family, possesses cytostatic and pro-apoptotic effects in association with complex and poorly understood activities on osteoblast differentiation. In this study, we use rat osteosarcoma cells transduced with lentiviral particles encoding OSM (lvOSM) to stably produce this cytokine. We show that after several weeks of culture, transduced OSRGA and ROS 17/2.8 cells are growth inhibited and sensitized to apoptosis induced by the kinase inhibitor Staurosporine (Sts). Moreover, this long term OSM treatment induces (i) a decrease in osteoblastic markers, (ii) morphological changes leading to an elongated and/or stellate shape and (iii) an increase in osteocytic markers (sclerostin and/or E11), suggesting an osteocyte-like differentiation. We also show that non transformed rat calvaria cells transduced ! with lvOSM differentiate into stellate shaped cells expressing sclerostin, E11, Phex and functional hemichannels. Together, these results indicate that osteosarcoma cells stably producing OSM do not develop resistance to this cytokine and thus could be a valuable new tool to study the anti-cancer effect of OSM in vivo. Moreover, OSM-over-expressing osteoblastic cells differentiate into osteocyte-like cells, the major cellular contingent in bone, providing new culture conditions for this cell type which is difficult to obtain in vitro.
• A new tool to assess the mechanical properties of bone due to collagen degradation
  - Bone 44(5):840-848 (2009)
  Current clinical tools for evaluating fracture risk focus only on the mineral phase of bone. However, changes in the collagen matrix may affect bone mechanical properties, increasing fracture risk while remaining undetected by conventional screening methods such as dual energy x-ray absorptiometry (DXA) and quantitative ultrasound (QUS). The mechanical response tissue analyzer (MRTA) is a non-invasive, radiation-free potential clinical tool for evaluating fracture risk. The objectives of this study were two-fold: to investigate the ability of the MRTA to detect changes in mechanical properties of bone as a result of treatment with 1 M potassium hydroxide (KOH) and to evaluate the differences between male and female bone in an emu model. DXA, QUS, MRTA and three-point bending measurements were performed on ex vivo emu tibiae before and after KOH treatment. Male and female emu tibiae were endocortically treated with 1 M KOH solution for 1–14 days, resulting in negligib! le collagen loss (0.05%; by hydroxyproline assay) and overall mass loss (0.5%). Three-point bending and MRTA detected significant changes in modulus between days 1 and 14 of KOH treatment (− 18%) while all values measured by DXA and QUS varied by less than 2%. This close correlation between MRTA and three-point bending results support the utility of the MRTA as a clinical tool to predict fracture risk. In addition, the significant reduction in modulus contrasted with the negligible amount of collagen removal from the bone after KOH exposure. As such, the significant changes in bone mechanical properties may be due to partial debonding between the mineral and organic matrix or in situ collagen degradation rather than collagen removal. In terms of sex differences, male emu tibiae had significantly decreased failure stress and increased failure strain and toughness compared to female tibiae with increasing KOH treatment time.
• Galectin-9 accelerates transforming growth factor β3-induced differentiation of human mesenchymal stem cells to chondrocytes
  - Bone 44(5):849-857 (2009)
  Galectin-9 (Gal-9), a β-galactoside binding lectin, plays a crucial role in innate and adaptive immunity. In the rat collagen-induced arthritis model, administration of Gal-9 induced repair of existing cartilage injury even when joints were already swollen with cartilage destruction. We thus attempted to explore the role of Gal-9 in chondrocyte differentiation utilizing human mesenchymal stem cell (MSC) pellet cultures. During chondrogenesis induced by transforming growth factor β3 (TGFβ3), MSCs strongly expressed endogenous Gal-9. Expression of Gal-9 peaked on day 14 and the neutralization of endogenous Gal-9 resulted in the reduced chondrogenesis, indicating possible involvement of Gal-9 in TGFβ-mediated chondrogenesis. In pellets, addition of Gal-9 significantly enhanced TGFβ3-induced chondrogenesis, as evidenced by increasing proteoglycan content, but not cell proliferation. In the absence of Gal-9, collagen expression by MSCs switched from type I to type II o! n 28 days after stimulation with TGFβ3. When MSCs were co-stimulated with Gal-9, the class switch occurred on day 21. In addition, Gal-9 synergistically enhanced TGFβ3-induced phosphorylation of Smad2, though Gal-9 did not itself induce detectable Smad2 phosphorylation. These results suggest that Gal-9 has a beneficial effect on cartilage repair in injured joints by induction of differentiation of MSCs into chondrocytes.
• Pulse treatment with zoledronic acid causes sustained commitment of bone marrow derived mesenchymal stem cells for osteogenic differentiation
  - Bone 44(5):858-864 (2009)
  The aminobisphosphonate zoledronic acid (ZA) is a bone seeking specific inhibitor of protein farnesylation and geranylgeranylation, which causes inhibition of osteoclast function and apoptosis. It is widely used as an osteoclast targeted antiresorptive treatment of metastatic bone disease, Paget's disease and osteoporosis. Mesenchymal stem cells (MSC) and osteoblast precursors can also be targets of bisphosphonates, but the clinical relevance of these effects is under debate. We show here that ZA in vitro causes inhibition of proliferation and induction of apoptosis in hMSC, when applied in concentrations of 20 and 50 μM for more than 24 h which can be rescued by treatment with 10 μM geranylgeranyl pyrophosphate (GGPP). However, pulse stimulation for 3 and 6 h with these concentrations and subsequent culture for up to 2 weeks under osteogenic conditions exerts sustained regulation of osteogenic marker genes in hMSC. The effect on gene regulation translates into marke! d enhancement of mineralization, as shown by alizarin red and alkaline phosphatase staining after 4 weeks of osteogenic culture. ZA, when applied as a pulse stimulus, might therefore also stimulate osteogenic differentiation in vivo, since μM plasma concentrations can be achieved by intravenous application of 5 mg in patients. These data set the stage for the future dissection of the effects of ZA and other aminobisphosphonates on cells beyond osteoclasts, with respect to cell differentiation in benign metabolic and to antitumor efficacy in metastatic bone diseases, as well as adverse events due to putative substance accumulation in bone during long-term treatment.
• Collagenase expression and activity in the stromal cells from giant cell tumour of bone
  - Bone 44(5):865-871 (2009)
  The characteristic bone destruction in giant cell tumour of bone (GCT) is largely attributed to the osteoclast-like giant cells. However, experimental analyses of bone resorption by cells from GCT often fail to exclude the neoplastic spindle-like stromal cells, and several studies have demonstrated that bone resorption by GCT cells is increased in the presence of stromal cells. The spindle-like stromal cells from GCT may therefore actively contribute to the bone resorption observed in the tumour. Type I collagen, a major organic constituent of bone, is effectively degraded by three matrix metalloproteinases (MMPs) known as the collagenases: MMP-1, MMP-8 and MMP-13. We established primary cell cultures from nine patients with GCT and the stromal cell populations were isolated in culture. The production of collagenases by primary cultures of GCT stromal cells was determined through real-time PCR, western blot analysis and a multiplex assay system. Results show that the c! ells produce MMP-1 and MMP-13 but not MMP-8. Immunohistochemistry confirmed the presence of MMP-1 and MMP-13 in paraffin-embedded GCT tissue samples. Medium conditioned by the stromal cell cultures was capable of proteolytic activity as determined by MMP-1 and MMP-13-specific standardized enzyme activity assays. The spindle-like stromal cells from GCT may therefore actively participate in the bone destruction that is characteristic of the tumour.
• Parathyroid hormone enhances bone morphogenetic protein activity by increasing intracellular 3′, 5′-cyclic adenosine monophosphate accumulation in osteoblastic MC3T3-E1 cells
  - Bone 44(5):872-877 (2009)
  Intermittent subcutaneous injections of parathyroid hormone (PTH) increase bone mass in a variety of animal models and humans. The anabolic actions of PTH on osteogenic cells are mainly mediated through the protein kinase A (PKA) signaling pathway via PTH receptor 1 (PTHR1). We have already reported 3′, 5′-cyclic adenosine monophosphate (cAMP)/PKA-mediated enhancement of bone morphogenetic protein (BMP) signaling. Herein, we focused on the involvement of PTH in BMP signaling pathways in the MC3T3-E1 mouse osteoblastic cell line, to elucidate a potential mechanism of the anabolic actions of PTH on bone formation. Elevation of intracellular cAMP level in MC3T3-E1 cells by addition of PTH (10− 7 M) to culture media was transient without significant effect on biological actions of BMP. Cyclic addition of PTH (10 cyclic additions of 10− 8 M PTH at 3-min intervals) maintained a high intracellular cAMP level for about 2 h and mRNA expression and enzymatic activity of ! alkaline phosphatase (ALP) by BMP was enhanced by this addition. Relative luciferase expression assay in MC3T3-E1 cells using the Id1 promoter, an early response gene to BMPs, enhanced elevation of transcriptional activity in response to recombinant human BMP-2 by concomitant addition of PTH and BMP. Furthermore, cyclic PTH treatment significantly further suppressed BMP-induced inhibitory Smad6 expression. H89 (PKA inhibitor) almost completely abolished PTH actions on BMP signaling. IBMX (phosphodiesterase inhibitor) enhanced PTH actions. These results suggest that PTH enhances BMP signaling when PTH-induced intracellular cAMP level is maintained for a few hours, accelerating BMP actions to promote osteoblastic function and anabolic actions of new bone formation.
• Potential roles of growth factor PDGF-BB in the bony repair of injured growth plate
  - Bone 44(5):878-885 (2009)
  Injured growth plate cartilage is often repaired by bony tissue resulting in impaired bone growth in children. Using a rat injury model, our previous studies show that following the injury-induced initial inflammatory response, an influx of mesenchymal-like cells occurs within the growth plate injury site prior to formation of bony tissue. As platelet-derived growth factor (PDGF-BB) is a potent chemotactic factor of mesenchymal cells during skeletal tissue repair, we examined its role during the early fibrogenic response and the subsequent bony repair of injured growth plate. Following growth plate injury, rats received daily injection of the PDGF receptor (PDGFR) inhibitor, Imatinib, for 7 days. Immunohistochemical analysis of injured growth plate at day 1 showed the presence of PDGF-BB expression in some inflammatory cells, while at day 4 PDGFR was expressed by a proportion of the infiltrating mesenchymal cells at the injury site. By day 4, PDGFR inhibition reduced m! esenchymal infiltrate (P < 0.05); by day 14, Imatinib-treated rats exhibited less bony trabeculae and cartilaginous repair tissues, fewer osteoclasts and less bone marrow (BM) at the injury site, compared to vehicle controls (P < 0.01). In vitro "scratch" migration assays with rat BM mesenchymal cells revealed that recombinant PDGF-BB increased cell migration into the "wound" (P < 0.05), while Imatinib inhibited this chemotactic response. Quantitative RT-PCR analysis showed that Imatinib treatment decreased expression of the cartilage and bone related genes, Col2a1 and osteocalcin, respectively. These results suggest that PDGF-BB contributes to growth plate injury repair by promoting mesenchymal progenitor cell infiltration, the chondrogenic and osteogenic responses, and remodelling of the repair tissues.
• COMP-Angiopoietin-1 ameliorates surgery-induced ischemic necrosis of the femoral head in rats
  - Bone 44(5):886-892 (2009)
  Introduction Ischemic necrosis of the femoral head (INFH) can lead to loss of femoral head architecture and deformity. Moreover, the process of bone healing is intimately associated with angiogenesis. We considered that COMP-Ang1 (an angiogenic factor) might preserve femoral head structure and facilitate bone repair. Methods INFH was induced in the femoral head of rats by dissecting the cervical periosteum and placing a ligature tightly around the femoral neck. Two weeks later, COMP-Ang1 was injected directly into infarcted areas. Rats were divided into the following groups; 1) the sham-operated group (the sham group), 2) the bovine serum albumin-injected group (the BSA group), and 3) the COMP-Ang1-injected group (the COMP-Ang1 group) (n = 20/group). At 8 weeks post-surgery animals were sacrificed and radiologic and histomorphometric assessments were performed. Results Radiographs obtained at 8 weeks post-surgery showed better preservation of femoral head architecture in the COMP-Ang1 group than in the BSA group. Histological findings and immunostainings of endothelial cells for factor VIII revealed that COMP-Ang1 group animals showed higher levels of vascularity in the secondary ossification center of infarcted femoral heads. Conclusions When INFH was surgically induced in rats, an intraosseous injection of COMP-Ang1 preserved the trabecular framework of the osseous epiphysis and prevented femoral head deformities by promoting angiogenesis and bone remodeling.
• Effect of 1,25-dihydroxy vitamin D3 on fracture healing and bone remodeling in ovariectomized rat femora
  - Bone 44(5):893-898 (2009)
  Osteoporosis is a major health problem characterized by compromised bone strength that predisposes patients to an increased risk of fracture, more and more investigations are focusing on the treatment of osteoporotic fracture healing. However, there are few studies elucidating the efficacy of vitamin D, 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), on osteoporotic fracture healing. In the present study we have established an osteoporotic fracture rat model to evaluate the effects of 1,25(OH)2D3 on fracture healing. Female SD rats of six-month-old (n = 40) allocated randomly into two groups were given ovariectomy. Bilateral midshaft femoral osteotomy was performed 12 weeks post-ovariectomy. Then treatment was begun at the second day after osteotomy and continued until sacrifice at 6 and 16 weeks post-fracture with middle chain triglyceride (MCT) vehicle and 1,25(OH)2D3 at 0.1 μg/kg/day by oral gavage. Fracture callus was evaluated by soft X-ray radiography, micro-computed t! omography (micro-CT), biomechanical testing and histology. Soft X-ray radiography, at 6 weeks post-fracture, showed a less distinct fracture line in the 1,25(OH)2D3 group compared with the MCT-vehicle group, however, the fracture line was invisible in both groups at 16 weeks post-fracture. Micro-CT based histomorphometric data, at 6 weeks post-fracture, showed that the total volume of callus (TV) was approximately 23% higher in the 1,25(OH)2D3 group than that in the MCT-vehicle group (P < 0.001), and the new bone volume (BV), BV/TV, the trabecular number (Tb.N), and density of TV also showed the same trend. At 16 weeks post-fracture, the increment still existed as shown by Tb.Th and density of TV (P < 0.001, vs control). Biomechanical testing data, at 6 weeks post-fracture, showed that the ultimate load at failure and energy absorption of the 1,25(OH)2D3 group were nearly one fold higher than that of the MCT-vehicle group (P < 0.001). At 16 weeks post-fracture, the ultimate! load and energy absorption were also higher with the treatmen! t of 1,25(OH)2D3 (P < 0.01 vs control). Histology showed that the fracture callus in the 1,25(OH)2D3 group was remodeled better compared to the control group. In conclusion, 1,25(OH)2D3 could promote fracture healing by improving the histomorphometric parameters, mechanical strength and tendency to increase transformation of woven bone into lamellar bone in an ovariectomized rat model.
• A comparison of cortical and trabecular bone from C57 Black 6 mice using Raman spectroscopy
  - Bone 44(5):899-907 (2009)
  Cortical and trabecular bone are both produced and maintained by the same cell types. At the microscopic scale they have a similar lamellar structure but at a macroscopic scale they are very different. Raman microscopy has been used to investigate compositional differences in the two bone types using bone from standard laboratory mice in physiological conditions. Clear differences were observed when complete spectra were compared by principal component analysis (PCA). Analysis of individual bands showed cortical bone to have compositional characteristics of older bone when compared with trabecular material, possibly due to the higher bone turnover traditionally reported in the trabecular compartment.
• The bisphosphonate zoledronic acid decreases tumor growth in bone in mice with defective osteoclasts
  - Bone 44(5):908-916 (2009)
  Bisphosphonates (BPs), bone targeted drugs that disrupt osteoclast function, are routinely used to treat complications of bone metastasis. Studies in preclinical models of cancer have shown that BPs reduce skeletal tumor burden and increase survival. Similarly, we observed in the present study that administration of the Nitrogen-containing BP (N-BP), zoledronic acid (ZA) to osteolytic tumor-bearing Tax+ mice beginning at 6 months of age led to resolution of radiographic skeletal lesions. N-BPs inhibit farnesyl diphosphate (FPP) synthase, thereby inhibiting protein prenylation and causing cellular toxicity. We found that ZA decreased Tax+ tumor and B16 melanoma viability and caused the accumulation of unprenylated Rap1a proteins in vitro. However, it is presently unclear whether N-BPs exert anti-tumor effects in bone independent of inhibition of osteoclast (OC) function in vivo. Therefore, we evaluated the impact of treatment with ZA on B16 melanoma bone tumor burden in! irradiated mice transplanted with splenic cells from src-/- mice, which have non-functioning OCs. OC-defective mice treated with ZA demonstrated a significant 88% decrease in tumor growth in bone compared to vehicle-treated OC-defective mice. These data support an osteoclast-independent role for N-BP therapy in bone metastasis.
• Optimal increase in bone mass by continuous local infusion of alendronate during distraction osteogenesis in rabbits
  - Bone 44(5):917-923 (2009)
  Several methods have been used to increase bone mass in distraction osteogenesis. Since bone resorption as well as regeneration is stimulated in the distracted segment, bisphosphonate can be a beneficial agent for distraction osteogenesis. Here, we examined the effects of bisphosphonate injected continuously into the regenerate on bone volume, and architectural and mechanical properties of distraction osteogenesis. The left tibia of Japanese White rabbits (n = 66) was subjected to slow distraction using an external fixator. At the beginning of the consolidation phase, alendronate (7 μg/kg/day) was infused directly into the lengthened segment for 14 days using an osmotic pump. Control rabbits were infused with phosphate buffered saline (PBS). The tibiae were monitored weekly by soft X-ray and dual-energy X-ray absorptiometry (DXA). The animals were sacrificed at 4, 6, and 8 weeks after operation to examine bone mineral density (BMD) and cortical bone thickness (CBT) by! peripheral quantitative computerized tomography (pQCT), while the mechanical property of the lengthened tibia was measured by three-point bending test. In PBS-infused control animals, bone mineral content around the lengthened segment began to decrease after the first week of consolidation phase, forming a tubular bone structure with thin cortex. Infusion of alendronate increased peak bone mineral content around the lengthened segment. At the end of the experiment, volumetric BMD, CBT and mechanical strength of the lengthened segment of the treatment group were approximately twice those of the control animals. Alendronate infused in this manner significantly prevented the osteopenia that critically began early in the consolidation phase, though the dose used in this study was relatively low and no adverse events were noted.
• Density and architecture have greater effects on the toughness of trabecular bone than damage
  - Bone 44(5):924-929 (2009)
  Bone damage has been cited as an important aspect of bone quality. As such, understanding the effects of damage on the toughness of trabecular bone should provide insight into trabecular bone behavior during energy-limiting cases, such as falls. The effects of damage on the toughness of 35 bovine trabecular bone specimens were studied. Damage was induced by compressing the on-axis specimens to either 1.5% or 2.5% strain, followed by compression to 7.5% strain. The overloads resulted in significant decreases in both modulus and elastic toughness, with significantly greater decreases for the high-damage group than the low-damage group. Following damage, the elastic toughness of the high-damage group was also lower than the undamaged elastic toughness of the control group. In contrast, there was no detectable effect of damage level on toughness measured to 7.5% strain. Toughness increased linearly with BMD (R2 = 0.50) and by a power law relationship with volume fraction (! BV/TV) (R2 = 0.65). Microarchitectural parameters also predicted the toughness in the absence of BV/TV or BMD. Toughness decreased with increasing slenderness ratio (Tb.Sp/Tb.Th) and structure model index (SMI) (R2 = 0.68, multiple regression), again independent of damage level, suggesting that failure is influenced by trabecular buckling. Taken together, the results show that normal variations in toughness due to density and architecture dominate the changes due to damage at the levels induced in this study. Moreover, measuring toughness is sensitive to the final strain, as differences found in the elastic and initial plastic regions were undetectable at higher strains. The self-limiting nature of microcracks in trabecular bone, or the trabecular architecture itself, may inhibit microcracks from propagating to macroscopic trabecular fractures, thereby limiting the effect of damage on toughness and making it difficult to detect in comparison to normal population variability.
• The mouse fibula as a suitable bone for the study of functional adaptation to mechanical loading
  - Bone 44(5):930-935 (2009)
  Bones' functionally adaptive responses to mechanical loading can usefully be studied in the tibia by the application of loads between the knee and ankle in normal and genetically modified mice. Such loading also deforms the fibula. Our present study was designed to ascertain whether the fibula adapts to loading in a similar way to the tibia and could thus provide an additional bone in which to study functional adaptation. The right tibiae/fibulae in C57BL/6 mice were subjected to a single period of axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle; approximately 7 min/day, 3 alternate days/week, 2 weeks). The left tibiae/fibulae were used as non-loaded, internal controls. Both left and right fibulae and tibiae were analyzed by micro-computed tomography at the levels of the mid-shaft of the fibula and 25% from its proximal and distal ends. We also investigated the effects of intermittent parathyroid hormone (iPTH) on the (re)modelling respons! e to 2-weeks of loading and the effect of 2-consecutive days of loading on osteocytes' sclerostin expression. These in vivo experiments confirmed that the fibula showed similar loading-related (re)modelling responses to those previously documented in the tibia and similar synergistic increases in osteogenesis between loading and iPTH. The numbers of sclerostin-positive osteocytes at the proximal and middle fibulae were markedly decreased by loading. Collectively, these data suggest that the mouse fibula, as well as the tibia and ulna, is a useful bone in which to assess bone cells' early responses to mechanical loading and the adaptive (re)modelling that this engenders.
• Thioredoxin-1 overexpression in transgenic mice attenuates streptozotocin-induced diabetic osteopenia: A novel role of oxidative stress and therapeutic implications
  - Bone 44(5):936-941 (2009)
  Diabetes mellitus is associated with increased risk of osteopenia and bone fracture. However, the mechanisms accounting for diabetic bone disorder are unclear. We have previously reported that streptozotocin-induced diabetic mice develop low turnover osteopenia associated with increased oxidative stress in the diabetic condition. To determine the role of oxidative stress in the development of diabetic osteopenia, we presently investigated the effect of overexpression of thioredoxin-1 (TRX), a major intracellular antioxidant, on the development of diabetic osteopenia, using TRX transgenic mice (TRX-Tg). TRX-Tg are C57BL/6 mice that carry the human TRX transgene under the control of β-actin promoter. Eight-week-old male TRX-Tg mice and wild type (WT) littermates were intraperitoneally injected with either streptozotocin or vehicle. Mice were grouped as 1) non-diabetic WT, 2) non-diabetic TRX-Tg, 3) diabetic WT, and 4) diabetic TRX-Tg. After 12 weeks of streptozotocin treatment, oxidative stress on the whole body and bone was evaluated, and the physical properties of the femora, and histomorphometry parameters of the tibiae were assessed. TRX overexpression did not affect either body weight or hemoglobin A1c levels. There were no significant differences in renal function and in serum levels of calcium, phosphate, and intact parathyroid hormone among the four groups. On the other hand, urinary excretion of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, was significantly elevated in diabetic WT and attenuated in diabetic TRX-Tg. Immunohistochemical staining for 8-OHdG revealed marked intensity in the bone tissue of diabetic WT compared with non-diabetic WT, while staining was attenuated in diabetic TRX-Tg. TRX overexpression partially restored reduced bone mineral density and prevented the suppression of bone formation observed in diabetic WT. Increased oxidative stress in diabetic condition contributes to the development of diabetic osteopenia. Suppression of increased oxidative stress by TRX induction could be a potential therapeutic approach for diabetic osteopenia.
• Effects of cod bone gelatin on bone metabolism and bone microarchitecture in ovariectomized rats
  - Bone 44(5):942-947 (2009)
  Several animal studies have showed that gelatin may be effective for minimizing bone loss in OVX rats with established osteopenia. To gain insight into how cod bone gelatin administration affects bone loss after ovariectomy, studies were carried out focusing on bone quality and the molecular mechanisms. Eighty-four female rats were ovariectomized, 12 sham-operated, divided into six groups of 12 each and treated one week after ovariectomy either with vehicle or cod bone gelatin (0.375, 0.75, 1.5, 3, 6 mg/kg body weight) for 90 days. Bone densitometry, μCT analysis, real-time PCR analysis and biochemical analysis were used at the end of the study. After 90 days, BMD of proximal tibia and femoral neck decreased in OVX rats, whereas the loss of BMD in those regions was prevented at 3 g/kg (P < 0.05). However, the BMD of midshaft femurs showed no significant differences. BV/TV, Tb.N. and Tb.Th. in the 3 g/kg group were, respectively, 30.4% (P < 0.05), 145.5% (P < 0.05) and! 81.5% (P < 0.05) higher than in the OVX group. A significant decrease was detected in urine CTX, NTX and DPD, suggesting decreased bone resorption. Treatment with 3 g/kg and 6 g/kg cod bone gelatin attenuated the increase in serum IL-1β, IL-6 and TNF-α observed in the OVX group. Real-time PCR showed significantly decreased levels of mRNA expression for RANKL at the dosage of 6 g/kg and the RANKL/OPG mRNA ratio in the 3 g/kg and 6 g/kg group significantly decreased compared to the OVX group (P < 0.05). In conclusion, our data confirmed that the cod bone gelatin treatment at 3 g/kg is effective in the prevention of estrogen deficient bone loss by modulating the expression of RANKL and OPG and suppressing the release of proinflammatory cytokines.
• Effect of long-term intervention of soy isoflavones on bone mineral density in women: A meta-analysis of randomized controlled trials
  - Bone 44(5):948-953 (2009)
  Introduction A number of RCTs have examined the role of soy isoflavones on bone mineral density (BMD) and yielded inconsistent results. This meta-analyses aims to assess the overall effect of soy isoflavones on BMD. Methods We searched for all articles published in English from January 1990 to March, 2008. We included RCTs of soy isoflavone supplementation in women of at least one year duration. The main outcomes were BMD changes from baseline at the lumbar spine, total hip and femoral neck. Results We identified 10 eligible RCTs containing 896 women. A mean dose of 87 mg soy isoflavones for at least one year did not significantly affect BMD changes. The mean (95%CI) differences in BMD changes (in mg/cm2/year) were 4.1 (− 1.6, 9.8) (0.4%) at the lumbar spine, − 1.5 (− 7.2, 4.3) (− 0.3%) at the femoral neck under random-effects model, and 2.5 (− 0.5, 5.4) (0.2%) at the total hip by fix-effects model, respectively. Similar results were obtained in subgroup analyses by isoflavone sources (soy protein vs. isoflavone extract), ethnic differences (Asian vs. Western). Larger dose (≥ 80 mg/d), but not lower dose (< 80 mg/d), of isoflavone intervention tended to have a weak beneficial effect on spine BMD (p = 0.08 vs. p = 0.94). Conclusions Soy isoflavone supplementation is unlikely to have significant favorable on BMD at the lumbar spine and hip in women.
• Cysteine, homocysteine and bone mineral density: A role for body composition?
  - Bone 44(5):954-958 (2009)
  Background Plasma total cysteine (tCys) and homocysteine (tHcy) are associated with body composition, which in turn affects bone mineral density (BMD). Objectives To investigate whether associations of tCys and tHcy with BMD are mediated through body composition (fat mass and/or lean mass). Design Using data from 5238 Hordaland Homocysteine Study participants, we fit multiple linear regression models and concentration–response curves to explore the relationships between tCys, tHcy, and BMD, with and without adjustment for body mass index (BMI), lean mass and/or fat mass. Results All associations were stronger in women. tCys was positively associated with BMD (women, partial r = 0.11; men, partial r = 0.07, p ≤ 0.001 for both), but this association was markedly attenuated after adjustment for fat mass. tHcy showed an inverse association with BMD in women (partial r = − 0.09, p < 0.001), which remained significant after adjustment for lean mass and fat mass. In men and women, changes in tCys or tHcy during 6 years were not associated with BMD at follow-up. Weight gain during 6 years predicted higher BMD at follow-up (p ≤ 0.009) independent of nutrient intakes, physical activity and baseline BMI. Baseline tHcy inversely predicted BMD measured 6 years later (partial r = − 0.11, p < 0.001 in women; partial r = − 0.07, p = 0.002 in men) independent of baseline BMI, while a positive association of baseline tCys with BMD at follow-up (partial r = 0.10 in women, 0.09 in men, p ≤ 0.001) disappeared after adjustment for baseline BMI. Conclusion tHcy is inversely associated with BMD independent of body composition, while the positive association of tCys with BMD appears to be mainly mediated through fat mass.
• Bone matrix quality and plasma homocysteine levels
  - Bone 44(5):959-964 (2009)
  It has recently been reported in the clinical literature that blood homocysteine levels correlate well with fracture risk, although a couple of reports exist to the opposite. Bone strength depends on both bone quantity and quality. The purpose of the present study was to investigate possible correlations between plasma homocysteine levels and bone material properties (Bone Mineral Density Distribution; BMDD, and collagen cross-link ratio). In the present study, femoral heads from subjects (N = 19, females, age range 70–95 years old) with known homocysteine plasma levels were investigated. The bone material was collected during hemiarthroplasty surgery. We have determined collagen cross-link ratio and bone mineralization density distribution (BMDD) in bone tissue from patients with acute femoral neck fractures, by Fourier Transform Infrared Imaging (FTIRI) and quantitative Backscattered Electron Imaging (qBEI), respectively. The collagen cross-link ratio that was spectroscopically determined was pyridinoline/divalent cross-links (pyr/divalent). The BMDD variables quantified were: CaMean: the weighted mean calcium concentration; CaPeak: the most frequent Ca concentration; CaWidth: the width of the distribution, a measure of the mineralization homogeneity; CaLow: the percentage of bone area that is mineralized below the 5th percentile in the reference range; CaHigh: the percentage of bone area that is mineraliz! ed above the 95th percentile in the reference range. There was a significant correlation between plasma homocysteine levels and collagen cross-link ratio in areas of primary mineralized bone (p < 0.0001), unlike the case of trabecular bone surfaces undergoing resorption (p > 0.05). On the other hand there was no correlation in any of the BMDD parameters and plasma homocysteine levels (p > 0.05). The results are consistent with the known effect of homocysteine on collagen post-translational modifications. These changes were independent of bone mineral characteristics. The results of the present study offer a mechanism by which homocysteine affects bone quality, but caution should be exercised since all patients examined had sustained fracture.
• Bone mineral density of the spine and femur in a group of healthy Moroccan men
  - Bone 44(5):965-969 (2009)
  Background Bone mineral density (BMD) measurements using dual-energy X-rays absorptiometry (DXA) are widely used to diagnose osteoporosis and to assess its severity. Previous studies show the necessity to establish reference data for bone mass measurements for each particular population. Such data are lacking for the male Moroccan population. Aim To establish reference values for the healthy Moroccan male population and to compare them with those for Caucasian and Arab males, and to study the impact of different curves implemented in the DXA system on the diagnosis of osteoporosis. Methods A cross-sectional study of 592 Moroccan men, recruited from the area of Rabat, the capital of Morocco, aged between 20 and 79 years was carried to establish reference values of bone mineral density. Measurements were taken at the lumbar spine and proximal femurs using DXA (Lunar Prodigy Vision, GE). The data were compared with published normative taken by US, European, Iranian, Lebanese, and Saudi men over six decades of age. Impact on osteoporosis diagnosis according to the WHO criteria using the personalized curve and US (NHANES), European and Middle-East reference curves (as implemented in the Lunar densitometers) was studied. Results Our results showed that the Moroccan men showed the expected decline in BMD at both sites with age after peaking at 20–29 years age group. Every anatomical region has a different rate of bone loss: lumbar spine (0.3% per year) femoral neck (0.6%), trochanter (0.3%), and total hip (0.4%). The lumbar spine and femoral subregions BMD exhibited increases from 0.3 to 0.5% per kilogram of body weight. In the spine, the US/European Lunar reference values classified a larger proportion of men as osteoporotic (18.1% vs. 7.4%) while using the Arabic Lunar reference values, only 7.8% were classified as osteoporotic. However, using Arabic curve for the femurs resulted in underdiagnosis of osteoporosis (1.8% vs. 6.0%), whereas the US/European Lunar reference values classified men as osteoporotic in 3.9% and 5.3% respectively. Discussion In comparison with the other Countries, the spine BMD of Moroccan men were slightly lower than Iranian's, Europeans and Brazilians but higher than the Saudi and Lebanese males. We found BMD values taken at the lumbar spine to be around 4% lower than European values between ages 50 and 59 years, and 10% lower for older subjects. These values were 4–6% higher than Saudis/Lebanese values between ages 20–39. For older subjects, Moroccan values were more than 10% higher than Saudis and almost similar to Lebanese. Femoral neck BMD values were 8% higher in young adults (age 20–39 years) to US/Saudis/Lebanese values, but about 10% lower in ages over 60 to US values whereas it was similar to Saudis and Lebanese values. Conclusion Our study emphasises the importance of using population-specific reference values for BMD measurements to avoid over or underdiagnosis of osteoporosis.
• Ethnic differences in bone geometry and strength are apparent in childhood
  - Bone 44(5):970-975 (2009)
  Purpose Ethnic differences in bone strength and structure likely contribute to the disparity in fracture rates, however few studies have assessed bone structure in multiethnic cohorts of children. The purpose of this study was to investigate ethnic differences in bone strength in childhood and to characterize the structural bases for these differences. Methods Peripheral quantitative computed tomography (pQCT 3000, Orthometrix) was used to assess bone parameters at the radius and tibia in Caucasian (CA, n = 21), African American (AA, n = 23), and Hispanic (HI, n = 29) children (10.9 ± 0.1 yrs). At the distal site (8%), we measured compressive bone strength (BSI), trabecular and total bone density, and total bone area. Polar strength-strain index, total and cortical bone area, and cortical density were assessed at the midshaft (50%). Muscle cross-sectional area (CSA) and fat CSA were measured at the tibia (66%) and the radius (50%). Physical activity and calcium intake were assessed by questionnaire. Analysis of covariance was used to compare bone outcomes among ethnic groups adjusting for age, sex, limb length and muscle CSA. Results Age, BMI, and body composition were similar among the 3 groups, however AA children were taller and had longer bone length. At all sites, AA and HI children had higher bone strength (SSIp and BSI + 10–37%) than CA children due mainly to greater bone tissue density (2–18% > CA) at the distal sites of the radius and tibia. The greater bone strength at the midshaft was due to both a higher bone density (2–5%) and greater bone area than CA (7–18%). Conclusion AA and HI children have significantly higher bone strength than CA children, due to greater bone volumetric density and greater cortical area. AA and HI children also have higher bone strength relative to load. These observations suggest that ethnic differences in bone strength manifest in childhood.
• Assessment of trabecular bone structure of the calcaneus using multi-detector CT: Correlation with microCT and biomechanical testing
  - Bone 44(5):976-983 (2009)
  The prediction of bone strength can be improved when determining bone mineral density (BMD) in combination with measures of trabecular microarchitecture. The goal of this study was to assess parameters of trabecular bone structure and texture of the calcaneus by clinical multi-detector row computed tomography (MDCT) in an experimental in situ setup and to correlate these parameters with microCT (μCT) and biomechanical testing. Thirty calcanei in 15 intact cadavers were scanned using three different protocols on a 64-slice MDCT scanner with an in-plane pixel size of 208 μm and 500 μm slice thickness. Bone cores were harvested from each specimen and μCT images with a voxel size of 16 μm were obtained. After image coregistration, trabecular bone structure and texture were evaluated in identical regions on the MDCT images. After data acquisition, uniaxial compression testing was performed. Significant correlations between MDCT- and μCT-derived measures of bone volume! fraction (BV/TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) were found (range, R2 = 0.19–0.65, p < 0.01 or 0.05). The MDCT-derived parameters of volumetric BMD, app. BV/TV, app. Tb.Th and app. Tb.Sp were capable of predicting 60%, 63%, 53% and 25% of the variation in bone strength (p < 0.01). When combining those measures with one additional texture index (either GLCM, TOGLCM or MF.euler), prediction of mechanical competence was significantly improved to 86%, 85%, 71% and 63% (p < 0.01). In conclusion, this study showed the feasibility of trabecular microarchitecture assessment using MDCT in an experimental setup simulating the clinical situation. Multivariate models of BMD or structural parameters combined with texture indices improved prediction of bone strength significantly and might provide more reliable estimates of fracture risk in patients.
• Multiple osteoporosis susceptibility genes on chromosome 1p36 in Chinese
  - Bone 44(5):984-988 (2009)
  Introduction Chromosome 1p36 is a region that has previously shown good evidence of linkage to bone mineral density (BMD) in multiple studies, but the genes that are responsible for the linkage signals are unknown. Materials and methods We performed a gene-wide and tag SNP-based association study of four positional and functional candidate genes (TNFRSF1B, PLOD, CNR2, and MTHFR) at 1p36 in 1, 243 case–control Chinese subjects. Twenty-three tag SNPs were selected and genotyped using the high-throughput Sequenom genotyping platform. Binary logistic regression analyses were performed to test for genotype associations between each SNP and BMD. Allelic and haplotype association analyses were conducted by Haploview. Gene–gene interactions were investigated using multifactor dimensionality reduction method. Results The PLOD rs7529452 (C385T; F98F) and MTHFR rs1801133 (C677T; A429E) showed significant genotypic/allelic associations with BMDs at all sites measured (P = 0.08–0.001), and a promising two-locus gene–gene interaction for femoral neck BMD. The CNR2 rs2501431 (A592G; G155G) showed nominally significant allelic associations with trochanter and hip BMD. The TNFRSF1B rs976881 showed genotypic associations with BMDs (P = 0.08–0.04). Conclusions Our results suggest that multiple genes at 1p36, individually or in different combinations, contribute to osteoporosis susceptibility in Chinese.
• Joint loading-driven bone formation and signaling pathways predicted from genome-wide expression profiles
  - Bone 44(5):989-998 (2009)
  Joint loading is a recently developed loading modality that induces anabolic responses by lateral loads applied to a synovial joint such as an elbow and a knee. The present study extended this loading modality to an ankle and addressed a question: does ankle loading promote bone formation in the tibia? If so, what signaling pathways are involved in the anabolic responses? Using C57BL/6 female mice as a model system, lateral loads of 0.5 N were applied to the ankle at 5 Hz for 3 min/day for 3 consecutive days and load-driven bone formation was evaluated at three tibial cross-sections (the proximal, middle, and distal diaphysis). Furthermore, total RNA was isolated for 3 pairs of microarray experiments as well as quantitative real-time PCR analyses. The histomorphometric results revealed that in all cross-sections ankle loading elevated the cortical area and thickness as well as the calcein-labeled surface. Signaling pathway analysis from microarray-derived whole-genome ! mRNA expression profiles and quantitative real-time PCR predicted that molecules in phosphoinositide 3-kinase (PI3K), ECM-receptor interactions, TGFβ signaling, and Wnt signaling were involved in the joint-loading driven responses. Since ankle loading stimulates bone formation throughout the tibia both in the endosteum and the periosteum, it may provide a non-pharmacological approach to effectively activate molecular signaling necessary for preventing bone loss.
• Identification and association analysis of single nucleotide polymorphisms in the human noggin (NOG) gene and osteoporosis phenotypes
  - Bone 44(5):999-1002 (2009)
  Noggin, an extracellular bone morphogenic protein (BMP) antagonist, blocks BMP signaling and decreases osteoblastogenesis. The purpose of this study was to identify novel sequence variations in the human noggin gene and to perform association analyses of these variations with phenotypes related to osteoporosis. Novel single nucleotide polymorphisms (SNPs) were identified by resequencing 7 kb of the noggin gene region in 24 randomly selected Afro-Caribbean men without regard to their bone mineral density (BMD) level. We identified 22 SNPs in the 7 kb noggin gene region, only 2 of which were previously described in dbSNP (build 126). There were also 11 unvalidated SNPs from dbSNP that could not be verified in our sequence analysis. Ten of the 22 identified SNPs showed a minor allele frequency greater than 0.05. Seven of these common SNPs were genotyped in 2060 Afro-Caribbean men age 40 and older. None of the 7 SNPs were associated with BMD at the proximal femur or lumbar! spine. Our analysis suggests that a common variation in the noggin gene is unlikely to have a major impact on BMD among older men of African ancestry.
• Associations of genetic lactase non-persistence and sex with bone loss in young adulthood
  - Bone 44(5):1003-1009 (2009)
  Some studies have reported that after attainment of peak bone mass (PBM), slow bone loss may occur in both men and women; however, findings are inconsistent. Genetic factors play a significant role in bone loss, but the available evidence is conflicting. Genetic lactase non-persistence (lactase C/C−13910 genotype) is suggested to increase risk for inadequate calcium intake predisposing to poorer bone health. We investigated whether this genotype is associated with PBM and bone loss in young Finnish adults. Subjects belong to the Cardiovascular Risk in Young Finns Study that is an ongoing multi-centre follow-up of atherosclerosis risk factors. From the original cohort, randomly selected subjects aged 20–29 participated in baseline bone mineral density (BMD) measurements (n = 358), and in follow-up measurements 12 years later (n = 157). Bone mineral content (BMC) and BMD at lumbar spine (LS) and femoral neck (FN) were measured at baseline and follow-up with dual ener! gy X-ray absorptiometry (DXA). Lactase C/T−13910 polymorphism was determined by PCR and allele-specific fluorogenic probes. Information on lifestyle was elicited with questionnaires. During the follow-up, bone loss at both bone sites was greater in males (LS BMD: − 1.1%, FN BMD: − 5.2%) than in females (LS BMD: + 2.1%, FN BMD: − 0.7%) (both bone sites p = 0.001). Younger age predicted greater loss of FN BMC and BMD in females (p = 0.013 and p = 0.001, respectively). Increased calcium intake predicted FN BMD gain in both sexes (in females B = 0.007 g/cm2/mg, p = 0.002; in males B = 0.006, p = 0.045), and increased physical activity LS BMD gain in females (B = 0.091 g/cm2/physical activity point, p = 0.023). PBM did not differ between the lactase genotypes, but males with the CC−13910 genotype seemed to be prone to greater bone loss during the follow-up (LS BMD: C/C vs. T/T p = 0.081). In conclusion, bone loss in young adulthood was more common in males than in fema! les and seemed to occur mainly at the femoral neck. Young male! s with the lactase CC−13910 genotype may be more susceptible to bone loss; however, calcium intake predicts changes in bone mass more than the lactase genotype.
• An in vivo genome wide gene expression study of circulating monocytes suggested GBP1, STAT1 and CXCL10 as novel risk genes for the differentiation of peak bone mass
  - Bone 44(5):1010-1014 (2009)
  Peak bone mass (PBM) is an important determinant of osteoporosis. Circulating monocytes serve as early progenitors of osteoclasts and produce important molecules for bone metabolism. To search for genes functionally important for PBM variation, we performed a whole genome gene differential expression study of circulating monocytes in human premenopausal subjects with extremely low (N = 12) vs. high (N = 14) PBM. We used Affymetrix HG-U133 plus2.0 GeneChip® arrays. We identified 70 differential expression probe sets (p < 0.01) corresponding to 49 unique genes. After false discovery rate adjustment, three genes [STAT1, signal transducer and activator of transcription 1; GBP1, guanylate binding protein 1; CXCL10, Chemokine (C-X-C motif) ligand 10] expressed significantly differentially (p < 0.05). The RT-PCR results independently confirmed the significantly differential expression of GBP1 gene, and the differential expression trend of STAT1. Functional analyses suggested! that the three genes are associated with the osteoclastogenic processes of proliferation, migration, differentiation, migration, chemotaxis, adhesion. Therefore, we may tentatively hypothesize that the three genes may potentially contribute to differential osteoclastogenesis, which may in the end lead to differential PBM. Our results indicate that the GBP1, STAT1 and CXCL10 may be novel risk genes for the differentiation of PBM at the monocyte stage.
• An effective histological staining process to visualize bone interstitial fluid space using confocal microscopy
  - Bone 44(5):1015-1017 (2009)
  Bone is a composite porous material with two functional levels of porosity: the vascular porosity that surrounds blood vessels and the lacunar–canalicular porosity that surrounds the osteocytes. Both the vascular porosity and lacunar–canalicular porosity are directly involved in interstitial fluid flow, thought to play an important role in bone's maintenance. Because of the small dimensions of the lacunar–canalicular porosity, interstitial fluid space has been difficult to visualize and quantify. We report a new staining protocol that is reliable and easily reproducible, using fluorescein isothiocyanate (FITC) as a probe visualized by confocal microscopy. Reconstructed FITC-stained cross sections enable effective visualization of bone microstructure and microporosities. This new staining process can be used to analyze interstitial fluid space, providing high-resolution quantification of the vascular pores and the lacunar–canalicular network of cortical and canc! ellous bone.
• Silicon and bone
  - Bone 44(5):1018 (2009)
  
• Reply to Dr Exley comments on: Jugdaohsingh R et al., Increased longitudinal growth in rats on a silicon depleted diet. Bone 2008;43:596–606
  - Bone 44(5):1019-1020 (2009)
  
• Announcements
  - Bone 44(5):1021 (2009)