Bone

Editorial Board

2012-03-01

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2012-03-01

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2012-03-01

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2012-03-01

Mechanical Stretch Induced Calcium Efflux from Bone Matrix Stimulates Osteoblasts

2012-03-01

Abstract: The mechanisms by which bone cells sense critically loaded regions of bone are still a matter of ongoing debate. Animal models to investigate response to microdamage involve post mortem immunohistological analysis and do not allow real-time monitoring of cellular response during the emergence of the damage in bone. Most in vitro mechanical stimulation studies are conducted on non-bone substrates, neglecting the damage-related alterations in the pericellular niche and their potential effects on bone cells. The current study reports spontaneous efflux of calcium ions (Ca2+) (1.924±0.742 pmol cm−2s−1) from regions of devitalized bone matrix undergoing post-yield strains, induced by a stress concentrator. When these samples are seeded with MC3T3-E1 osteoblasts, the strain-induced Ca2+ efflux from bone elicits cell response at the stress concentration site as manifested by activation of intracellular calcium signaling (increase in fluorescence by 52%±27%). This activity is associated with extracellular calcium because the intracellular calcium signaling in response to mechanical loading subsides when experiments are repeated using demineralized bone substrates (increase in fluorescence by 6%±10%). These results imply a novel perspective where bone matrix acts as an intermediary mechanochemical transducer by converting mechanical strain into a chemical signal (pericellular calcium) to which cells respond. Such a mechanism may be responsible for triggering repair at locations of bone matrix undergoing critical deformation levels.

Molecular characterisation of the Hyp deletion and an improved assay for its detection

Campbell R. Sheen, Genay O.W. Pilarowski, Wei Wang, Jose Luis Millan — 2012-01-24

Abstract: The Hyp mouse is a commonly used model for the study of the phosphate wasting disease X-linked hypophosphataemia. The defect in this mouse line is a deletion that includes exons 16 to 22 of Phex, although the exact extent of this X chromosome deletion remains unknown. This complicates genotyping which increases costs, time and difficulty of working with this important model. We aimed to determine the molecular breakpoints of this deletion in order develop a robust assay for its detection. We designed short mapping PCRs around the Phex locus to refine the putative breakpoint locations, then used gap PCR to amplify a product containing the breakpoint junction. DNA sequencing showed the deleted region was approximately 297kb, significantly larger than previous reports, but did not contain any genes other than Phex. DNA sequence analysis revealed that this deletion may be the result of microhomology-mediated end joining. Finally, we designed a multiplex PCR assay for genotyping Hyp colonies and validated it using a panel of Hyp colony mice. This study provides confirmation of the Hyp phenotype as a single gene defect, a potential mechanism for its formation and an improved method for genotyping that will make working with this strain significantly easier.Highlights: ► The deletion in the Hyp mouse model of X-linked hypophosphataemia was characterised. ► The Hyp deletion was more extensive than previously described but only affects Phex. ► A genotyping method for direct detection of the Hyp deletion was established.

Changes in intracortical microporosities induced by pharmaceutical treatment of osteoporosis as detected by high resolution micro-CT

2012-01-19

Abstract: Bone's microporosities play important biologic and mechanical roles. Here, we quantified 3D changes in cortical osteocyte-lacunae and other small porosities induced by estrogen withdrawal and two different osteoporosis treatments. Unlike 2D measurements, these data collected via synchrotron radiation-based μCT describe the size and 3D spatial distribution of a large number of porous structures. Six-month old female Sprague–Dawley rats were separated into four groups of age-matched controls, untreated OVX, OVX treated with PTH, and OVX treated with Alendronate (ALN). Intracortical microporosity of the medial quadrant of the femoral diaphysis was quantified at endosteal, intracortical, and periosteal regions of the samples, allowing the quantification of osteocyte lacunae that were formed primarily before versus after the start of treatment. Across the overall thickness of the medial cortex, lacunar volume fraction (Lc.V/TV) was significantly lower in ALN treated rats compared to PTH. In the endosteal region, average osteocyte lacunar volume ( ) of untreated OVX rats was significantly lower than in age-matched controls, indicating a decrease in osteocyte lacunar size in bone formed on the endosteal surface after estrogen withdrawal. The effect of treatment (OVX, ALN, PTH) on the number of lacunae per tissue volume (Lc.N/TV) was dependent on the specific location within the cortex (endosteal, intracortical, periosteal). In both the endosteal and intracortical regions, Lc.N/TV was significantly lower in ALN than in untreated OVX, suggesting a site-specific effect in osteocyte lacuna density with ALN treatment. There also were a significantly greater number of small pores (5–100μm3 in volume) in the endosteal region for PTH compared to ALN. The mechanical impact of this altered microporosity structure is unknown, but might serve to enhance, rather than deteriorate bone strength with PTH treatment, as smaller osteocyte lacunae may be better able to absorb shear forces than larger lacunae. Together, these data demonstrate that current treatments of osteoporosis can alter the number, size, and distribution of microporosities in cortical rat lamellar bone.Highlights: ► We quantified the effect of two osteoporosis treatments on 3D microporosity in the rat. ► These data were collected via synchrotron radiation-based micro-CT. ► Estrogen withdrawal resulted in smaller osteocyte lacunae near the endosteal surface. ► ALN resulted in fewer osteocyte lacunae and PTH rats had larger osteocyte lacunae. ► ALN rats had a lower lacunar volume fraction in bone formed during treatment.

Determinants of vitamin D status in a general population of Danish adults

2012-01-24

Abstract: Background and aims: Danish legislation regarding food fortification has been very restrictive and vitamin D deficiency is thought to be common in Denmark due to inadequate dietary intakes and the fact that in Denmark (latitude 56°N) vitamin D is only synthesized in the skin after exposure to solar radiation during summertime (April–September). The purpose of this study was to evaluate the vitamin D status of a general adult population in Denmark and, in addition, associations between vitamin D status and distinct lifestyle factors were studied.Methods: A random sample of 6784 persons from a general population aged 30–60years participated in a health examination in 1999–2001. Serum samples from all participants were stored and levels of 25-hydroxyvitamin D (25(OH)D) were measured by HPLC in 2009. The method was compared to another HPLC method. Information on dietary intake of vitamin D and other lifestyle factors were obtained by questionnaires. A total of 6146 persons defined as ethnic Danes and with successful measurements of 25(OH)D were included in the analyses.Results: The overall prevalence of vitamin D deficiency (25(OH)D<25nmol/l) and insufficiency (25(OH)D<50nmol/l) were 13.8% and 52.2%, respectively. A marked seasonal fluctuation was seen in serum levels of 25(OH)D — median values of 25(OH)D were lowest in February and highest in August. In multiple logistic regression models (n=5506), low vitamin D status was significantly associated with obesity (BMI≥30), daily smoking and a sedentary lifestyle. However, measurements of 25(OH)D were not associated with the estimated dietary intake of vitamin D. Comparison of two HPLC methods demonstrated considerable differences in accuracy.Discussion and conclusions: Our results suggest that poor vitamin D status is common among adults in a Northern European country without food fortification with vitamin D. Methodological issues are, however, of great importance when using cut-off values to define poor vitamin D status. In addition, we demonstrated that low serum levels of 25(OH)D were associated with several lifestyle factors.Highlights: ► Vitamin D insufficiency and deficiency are common among Danish adults. ► Low vitamin D status is associated with obesity, smoking and a sedentary lifestyle. ► Methodological differences limit the use of cut-off values to define deficiency.

Further improvements on the factors affecting bone mineral density measured by quantitative micro-computed tomography

2011-12-23

Abstract: The effects of imaging parameters and special configuration of objects within the reconstruction space on the micro computed tomography (μCT) based mineral density have been explored, and a series of density correction curves have been presented. A manufacturer-provided calibration phantom (0, 100, 200, 400, 800mg HA/cm3) was imaged at all possible imaging conditions (n=216) based on energy, resolution, vial diameter, beam hardening correction factor and averaging. For each imaging condition, a linear regression model was fitted to the observed versus expected densities, and the intercepts (β0) and slopes (β1) of the regression lines and each density level were modeled using multiple regression modeling. Additionally, a custom made phantom (0, 50, 150, 500, 800, 1000 and 1500mg HA/cm3) was scanned in order to study the effects of location and orientation of an object within the reconstruction space and presence of surrounding objects on μCT based mineral density. The energy, vial diameter and beam hardening correction factor were significant predictors of cumineral density (P values<0.001), while averaging and resolution did not have a significant effect on the observed density values (P values>0.1) except for 0.0 density (P values<0.04). Varying the location of an object within the reconstruction space from the center to the periphery resulted in a drop in observed mineral density up to 10% (P values<0.005). The presence of surrounding densities resulted in decreased observed mineral density up to 17% at the center and up to 14% at the periphery of the reconstruction space (P values<0.001 for all densities). Changing the orientation of the sample also had a significant effect on the observed mineral density, resulting in up to 16% lower observed mineral density for vertical vs. horizontal orientation at the center of the reconstruction space (P value<0.001). We conclude that energy, resolution and post processing correction factor are significant predictors of the observed mineral density in μCT.Highlights: ► Changing the location of an object within the reconstruction space affects the observed mineral density in micro computed tomography. ► Presence of surrounding densities affects the observed mineral density in micro computed tomography. ► The orientation of the object relative to the X-ray beam affects the observed mineral density in micro computed tomography.

GSK-3 inhibition by an orally active small molecule increases bone mass in rats

2011-12-19

Abstract: Glycogen synthase kinase 3β (GSK-3β) actions are central in the canonical Wnt pathway, important in many biological processes and a potential drug target for treating several diseases. It is appreciated that a balanced Wnt canonical signaling is crucial for the maintenance of normal bone mass. In this study we investigated the effects of a potent orally active GSK-3 inhibitor, AZD2858, on bone mass in rats.Treatment (1μM) of human osteoblast cells with AZD2858 in vitro increased β-catenin levels after a short period of time. In rats, oral AZD2858 treatment caused a dose-dependent increase in trabecular bone mass compared to control after a two-week treatment with a maximum effect at a dose of 20mg/kg once daily (total BMC: 172% of control; p<0.001). A small but significant effect was also seen at cortical sites (total BMC: 111% of control; p<0.001). Biomechanical testing demonstrated an increase in both vertebral compression strength at a dose of 20mg/kg once daily (Load at failure: 370% of control, p<0.001) and diaphyseal strength of femora subjected to a three point bending test (Load at failure: 115% of control; p<0.01). Furthermore, histomorphometry showed a dramatic increase in bone formation indices, and serum markers of both bone formation (Osteocalcin, 146% of control; p<0.001) and resorption (CTX, 189% of control; p<0.001) were elevated.Our conclusion is that a GSK-3 inhibitor drug may prove effective as an anabolic strategy in the treatment of diseases characterized by low bone mass, since AZD2858 has extensive bone building effects at predominantly trabecular sites.Highlights: ► The Wnt-signaling pathway, which includes GSK-3, is important in bone metabolism. ► The small molecule drug AZ2858 inhibits GSK-3. ► Oral administration of AZD2858 increases bone mineral density and content in rats. ► This increase enhances both vertebral compression and diaphyseal bending strength. ► AZD2858 is a potent anabolic drug candidate for future treatment of bone disease.

Bone turnover markers in peripheral blood and marrow plasma reflect trabecular bone loss but not endocortical expansion in aging mice

2011-12-26

Abstract: We examined age-related changes in biochemical markers and regulators of osteoblast and osteoclast activity in C57BL/6 mice to assess their utility in explaining age-related changes in bone. Several recently discovered regulators of osteoclasts and osteoblasts were also measured to assess concordance between their systemic levels versus their levels in marrow plasma, to which bone cells are directly exposed. MicroCT of 6-, 12-, and 24-month-old mice indicated an early age-related loss of trabecular bone volume and surface, followed by endocortical bone loss and periosteal expansion. Trabecular bone loss temporally correlated with reductions in biomarkers of bone formation and resorption in both peripheral blood and bone marrow. Endocortical bone loss and periosteal bone gain were not reflected in these protein biomarkers, but were well correlated with increased expression of osteocalcin, rank, tracp5b, and cathepsinK in RNA extracted from cortical bone. While age-related changes in bone turnover markers remained concordant in blood versus marrow, aging led to divergent changes in blood versus marrow for the bone cell regulators RANKL, OPG, sclerostin, DKK1, and serotonin. Bone expression of runx2 and osterix increased progressively with aging and was associated with an increase in the number of osteoprogenitors and osteoclast precursors. In summary, levels of biochemical markers of bone turnover in blood and bone marrow plasma were predictive of an age-related loss of trabecular surfaces in adult C57BL/6 mice, but did not predict gains in cortical surfaces resulting from cortical expansion. Unlike these turnover markers, a panel of bone cell regulatory proteins exhibited divergent age-related changes in marrow versus peripheral blood, suggesting that their circulating levels may not reflect local levels to which osteoclasts and osteoblasts are directly exposed.Highlights: ► Aging is associated with reductions in biochemical markers of bone turnover. ► Bone cell regulatory proteins change divergently in marrow versus blood with age. ► Marrow biomarkers deserve more research in pathophysiology of age-related bone loss.

Longitudinal effects of fat and lean mass on bone accrual in infants

Ramu G. Sudhagoni, Howard E. Wey, Gemechis D. Djira, Bonny L. Specker — 2011-12-19

Abstract: There are conflicting reports on the influence of lean and fat mass on bone accrual during childhood. No infant's studies have been reported that describe the influence of changes in body composition with changes in bone accrual during the first year of life. The objective of this research was to test the hypothesis that greater gains in lean mass will have a positive effect on bone mineral content (BMC) accrual, while greater gains in fat mass will have a negative effect on BMC accrual in infants. Longitudinal data from 3 previous infant studies were used. Linear mixed models, adjusting for age, sex, dietary calcium, and length were used to investigate longitudinal and cross-sectional associations between total body BMC and lean and fat mass in the individual studies and in a combined analysis. In both individual and combined analyses, we found that lean and fat mass were positively associated with whole body BMC accrual (all, P<0.001). The cross-sectional association of BMC and dietary calcium was negative in one study (P<0.05). No differences in BMC change between sexes were observed in three studies. Our results showed positive cross-sectional and longitudinal associations between total body BMC and lean mass in infants. In contradiction to our hypothesis for fat mass, we found a positive cross-sectional and longitudinal association between total body BMC and fat mass in infants.Highlights: ► No longitudinal studies have investigated the role of lean and fat mass on bone accrual in infants. ► Data on 362 infants from 3 longitudinal studies showed positive relationships between lean and fat mass on bone accrual. ► Results were similar for males and females, and changes in calcium intake had a positive effect on bone accrual.

Quantitative second-harmonic generation microscopy for imaging porcine cortical bone: Comparison to SEM and its potential to investigate age-related changes

Raghu Ambekar, Michael Chittenden, Iwona Jasiuk, Kimani C. Toussaint — 2011-12-22

Abstract: We propose the use of second-harmonic generation (SHG) microscopy for imaging collagen fibers in porcine femoral cortical bone. The technique is compared with scanning electron microscopy (SEM). SHG microscopy is shown to have excellent potential for bone imaging primarily due its intrinsic specificity to collagen fibers, which results in high contrast images without the need for specimen staining. Furthermore, this technique's ability to quantitatively assess collagen fiber organization is evaluated through an exploratory examination of bone structure as a function of age, from very young to mature bone. In particular, four different age groups: 1month, 3.5months, 6months, and 30months, were studied. Specifically, we employ the recently developed Fourier transform-second harmonic generation (FT-SHG) imaging technique for the quantification of the structural changes, and observe that as the bone develops, there is an overall reduction in porosity, the number of osteons increases, and the collagen fibers become comparatively more organized. It is also observed that the variations in structure across the whole cross-section of the bone increase with age. The results of this work show that quantitative SHG microscopy can serve as a valuable tool for evaluating the structural organization of collagen fibers in ex vivo bone studies.

Botulinum toxin in masticatory muscles: Short- and long-term effects on muscle, bone, and craniofacial function in adult rabbits

2011-12-21

Abstract: Paralysis of the masticatory muscles using botulinum toxin (BTX) is a common treatment for cosmetic reduction of the masseters as well as for conditions involving muscle spasm and pain. The effects of this treatment on mastication have not been evaluated, and claims that the treatment unloads the jaw joint and mandible have not been validated. If BTX treatment does decrease mandibular loading, osteopenia might ensue as an adverse result. Rabbits received a single dose of BTX or saline into one randomly chosen masseter muscle and were followed for 4 or 12weeks. Masticatory muscle activity was assessed weekly, and incisor bite force elicited by stimulation of each masseter was measured periodically. At the endpoint, strain gages were installed on the neck of the mandibular condyle and on the molar area of the mandible for in vivo bone strain recording during mastication and muscle stimulation. After termination, muscles were weighed and mandibular segments were scanned with micro CT. BTX paralysis of one masseter did not alter chewing side or rate, in part because of compensation by the medial pterygoid muscle. Masseter-induced bite force was dramatically decreased. Analysis of bone strain data suggested that at 4weeks, the mandibular condyle of the BTX-injected side was underloaded, as were both sides of the molar area. Bone quantity and quality were severely decreased specifically at these underloaded locations, especially the injection-side condylar head. At 12weeks, most functional parameters were near their pre-injection levels, but the injected masseter still exhibited atrophy and percent bone area was still low in the condylar head. In conclusion, although the performance of mastication was only minimally harmed by BTX paralysis of the masseter, the resulting underloading was sufficient to cause notable and persistent bone loss, particularly at the temporomandibular joint.Highlights: ► An injection of botulinum toxin into the rabbit masseter caused persistent atrophy. ► Despite normal chewing, masseter-induced bite force was greatly reduced. ► In vivo bone strain recording indicated underloading of the mandible. ► Substantial bone loss occurred in the underloaded regions, especially the condyle.

TGF-β regulates sclerostin expression via the ECR5 enhancer

2011-12-26

Abstract: Wnt signaling is critical for skeletal development and homeostasis. Sclerostin (Sost) has emerged as a potent inhibitor of Wnt signaling and, thereby, bone formation. Thus, strategies to reduce sclerostin expression may be used to treat osteoporosis or non-union fractures. Transforming growth factor-beta (TGF-β) elicits various effects upon the skeleton both in vitro and in vivo depending on the duration and timing of administration. In vitro and in vivo studies demonstrate that TGF-β increases osteoprogenitor differentiation but decreases matrix mineralization of committed osteoblasts. Because sclerostin decreases matrix mineralization, this study aimed to examine whether TGF-β achieves such inhibitory effects via transcriptional modulation of Sost. Using the UMR106.01 mature osteoblast cell line, we demonstrated that TGF-βTGF-β1-β2-β3 and Activin A increase Sost transcript expression. Pharmacologic inhibition of Alk4/5/7 in vitro and in vivo decreased endogenous Sost expression, and siRNA against Alk4 and Alk5 demonstrated their requirement for endogenous Sost expression. TGF-β1 targeted the Sost bone enhancer ECR5 and did not affect the transcriptional activity of the endogenous Sost promoter. These results indicate that TGF-β1 controls Sost transcription in mature osteoblasts, suggesting that sclerostin may mediate the inhibitory effect of TGF-β upon osteoblast differentiation.Highlights: ► TGF-β isoforms and Activin A increase Sost expression via Smad3. ► Inhibition of Alk4 and Alk5 both in vitro and in vivo reduces Sost expression. ► TGF-β targets ECR5 to enhance Sost.

Ucma is not necessary for normal development of the mouse skeleton

2012-01-03

Abstract: Ucma (Upper zone of growth plate and Cartilage Matrix Associated protein) is a highly conserved tyrosine-sulphated secreted protein of Mw 17kDa, which is expressed by juvenile chondrocytes.To evaluate the physiological function of this novel cartilage protein, we generated a Ucma-deficient mouse strain by introducing a lacZ/neoR-cassette into the first exon of the Ucma gene. This mutation results in the complete loss of Ucma mRNA and protein expression. Surprisingly, however, although previous in vitro studies implied a role for Ucma in calcification and ossification, these processes were not affected in Ucma-deficient mice during normal development. Likewise, cartilage development was normal. While in previous works Ucma was mainly detected in the cartilage of embryonic and young mice, we detected Ucma expression also in the adult cartilage of the ribs using the lacZ cassette under the control of the Ucma promoter. Moreover, Ucma protein was specifically detected in adult growth plate cartilage by immunohistochemistry. Considering that skeletal development in Ucma-deficient mice is not significantly impaired, protein expression in adult cartilage indicates that Ucma might be involved in skeletal homeostasis and in the mechanical properties of the skeleton during challenging conditions such as ageing or disease.Highlights: ► This is the first analysis of a mammalian in vivo loss-of-function model for Ucma. ► Previous data in vitro and in zebrafish indicate a role in skeletal development. ► Earlier studies demonstrate Ucma in young cartilage. ► Surprisingly, Ucma KO mice develop normal. ► We provide the first evidence for Ucma protein deposition in adult mouse cartilage.

Dietary silicon interacts with oestrogen to influence bone health: Evidence from the Aberdeen Prospective Osteoporosis Screening Study

2011-12-26

Abstract: Background: Silicon (Si), as Si(OH)4, is derived mainly from plant-based foods. Dietary Si is associated with bone mineral density (BMD) in premenopausal but not postmenopausal women.Objective: To examine the association between Si intake and markers of bone health in middle-aged women and to test for interaction with oestrogen status.Methods: Femoral neck (FN) and lumbar spine (LS) BMD, urinary markers of bone resorption (free pyridinoline and deoxypyridinoline cross-links relative to creatinine, fPYD/Cr and fDPD/Cr) and serum markers of bone formation (N-terminal propeptide of type 1 collagen, P1NP) were measured in a cohort of 3198 women aged 50–62years (n=1170 current HRT users, n=1018 never used HRT). Dietary Si, bioavailable Si and dietary confounders were estimated by food frequency questionnaire.Results: Mean FN BMD was 2% lower (p<0.005) in the lowest quartile (Q1) compared to the top quartile of energy-adjusted Si intake (Q4) (mean (SD) Q1, 16 (4.0) mg/d; Q4, 31.5 (7.3) mg/d). Energy-adjusted Si intake was associated with FN BMD for oestrogen-replete women only (late premenopausal women (r=+0.21, p=0.03); women on HRT [r=+0.09, p<0.001]). There was an interaction between oestrogen status and quartile of energy-adjusted Si intake on FN BMD, which was significant after adjustment for confounders (F=3.3, p=0.020), and stronger for bioavailable Si (F=5.0. p=0.002). Quartile of energy-adjusted dietary Si intake was negatively associated with fDPD/Cr and fPYD/Cr (p<0.001) and positively with P1NP (p<0.05).Conclusions: This study suggests that oestrogen status is important for Si metabolism in bone health. Further work is required to elucidate the mechanism.Highlights: ► The mechanism for the benefits of fruit and vegetable intakes on bone is uncertain. ► Silicon, a mineral influencing animal bone health may affect human bone health. ► Dietary silicon was associated with postmenopausal bone turnover and BMD. ► Adjusting for confounders, dietary silicon interacted with estrogen status on BMD.

Increased proportion of hypermineralized osteocyte lacunae in osteoporotic and osteoarthritic human trabecular bone: Implications for bone remodeling

2011-12-30

Abstract: Hypermineralized osteocyte lacunae (micropetrosis) have received little research attention. While they are a known aspect of the aging human skeleton, no data are available for pathological bone. In this study, intertrochanteric trabecular bone cores were obtained from patients at surgery for osteoporotic (OP) femoral neck fracture (10F, 4M, 65–94years), for hip osteoarthritis (OA; 7F, 8M, 62–87years), and femora at autopsy (CTL; 5F, 11M, 60–84years). Vertebral trabecular bone cores were also obtained from the vertebra of autopsy cases (CVB; 3F, 6M, 53–83years). Specimens were resin-embedded, polished, and carbon coated for quantitative backscattered electron imaging (qBEI), energy dispersive X-ray (EDX) spectrometry, and imaging analysis. Bone mineralization (Wt %Ca) was not different between OP, OA, and CTL; but was greater in femoral CTL than in CVB. The percent of hypermineralized osteocyte lacunae relative to the total number (HL/TL) was greater in OP and OA than in CTL. However, relative to bone mineral area, OP was characterised by increased hypermineralized osteocyte lacunar number density (Hd.Lc.Dn), whereas OA was characterised by decreased osteocyte lacunar number density (Lc.Dn) and total osteocyte lacunar number density (Tt.Lc.Dn). Lc.Dn was higher in CVB than in femoral CTL. The calcium–phosphorus ratio (RCa/P) was not different between hypermineralized osteocyte lacunae and bone matrix in each group. In addition, this study focused on the phenomenon of osteocyte lacunae hypermineralization using qBEI. Seven morphological types of osteocyte lacunae hypermineralization were described according to the presence of one or several hypermineralized spherites, associated or not with a hypermineralized lacunar ring. This study has described, for the first time, the morphology of hypermineralized osteocyte lacunae in OP and OA human bone. Further studies are suggested to investigate the functional influence of hypermineralized osteocyte lacunae on bone remodeling and bone biomechanical properties.Highlights: ► Increased % of hypermineralized osteocyte lacunae in osteoporosis and osteoarthritis. ► Increased hypermineralized osteocyte lacunar number density in osteoporosis. ► Decreased unmineralized and total osteocyte lacunar number density in osteoarthritis. ► Seven morphological types of osteocyte lacunae hypermineralization are described.

Effect of transforming growth factor beta (TGF-β) receptor I kinase inhibitor on prostate cancer bone growth

2011-12-26

Abstract: Transforming growth factor beta 1 (TGF-β1) has been implicated in the pathogenesis of prostate cancer (PCa) bone metastasis. In this study, we tested the antitumor efficacy of a selective TGF-β receptor I kinase inhibitor, LY2109761, in preclinical models. The effect of LY2109761 on the growth of MDA PCa 2b and PC-3 human PCa cells and primary mouse osteoblasts (PMOs) was assessed in vitro by measuring radiolabeled thymidine incorporation into DNA. In vivo, the right femurs of male SCID mice were injected with PCa cells. We monitored the tumor burden in control- and LY2109761-treated mice with MRI analysis and the PCa-induced bone response with X-ray and micro-CT analyses. Histologic changes in bone were studied by performing bone histomorphometric evaluations. PCa cells and PMOs expressed TGF-β receptor I. TGF-β1 induced pathway activation (as assessed by induced expression of p-Smad2) and inhibited cell growth in PC-3 cells and PMOs but not in MDA PCa 2b cells. LY2109761 had no effect on PCa cells but induced PMO proliferation in vitro. As expected, LY2109761 reversed the TGF-β1-induced pathway activation and growth inhibition in PC-3 cells and PMOs. In vivo, LY2109761 treatment for 6weeks resulted in increased volume in normal bone and increased osteoblast and osteoclast parameters. In addition, LY2109761 treatment significantly inhibited the growth of MDA PCa 2b and PC-3 in the bone of SCID mice (p<0.05); moreover, it resulted in significantly less bone loss and change in osteoclast-associated parameters in the PC-3 tumor-bearing bones than in the untreated mice. In summary, we report for the first time that targeting TGF-β receptors with LY2109761 can control PCa bone growth while increasing the mass of normal bone. This increased bone mass in nontumorous bone may be a desirable side effect of LY2109761 treatment for men with osteopenia or osteoporosis secondary to androgen-ablation therapy, reinforcing the benefit of effectively controlling PCa growth in bone. Thus, targeting TGF-β receptor I is a valuable intervention in men with advanced PCa.Highlights: ► TGF-β receptor I inhibitor LY2109761 increased the volume of normal bone. ► TGF-β receptor I inhibitor LY2109761 inhibited prostate cancer growth in bone. ► LY2109761-increased bone volume may help men undergoing androgen-ablation therapy.

Proteinase-activated receptor-2 is required for normal osteoblast and osteoclast differentiation during skeletal growth and repair

2012-01-06

Abstract: Proteinase-activated receptor-2 (PAR2) is a G-protein coupled receptor expressed by osteoblasts and monocytes. PAR2 is activated by a number of proteinases including coagulation factors and proteinases released by inflammatory cells. The aim of the current study was to investigate the role of PAR2 in skeletal growth and repair using wild type (WT) and PAR2 knockout (KO) mice. Micro computed tomography and histomorphometry were used to examine the structure of tibias isolated from uninjured mice at 50 and 90days of age, and from 98-day-old mice in a bone repair model in which a hole had been drilled through the tibias. Bone marrow was cultured and investigated for the presence of osteoblast precursors (alkaline phosphatase-positive fibroblastic colonies), and osteoclasts were counted in cultures treated with M-CSF and RANKL. Polymerase chain reaction (PCR) was used to determine which proteinases that activate PAR2 are expressed in bone marrow. Regulation of PAR2 expression in primary calvarial osteoblasts from WT mice was investigated by quantitative PCR. Cortical and trabecular bone volumes were significantly greater in the tibias of PAR2 KO mice than in those of WT mice at 50days of age. In trabecular bone, osteoclast surface, osteoblast surface and osteoid volume were significantly lower in KO than in WT mice. Bone marrow cultures from KO mice showed significantly fewer alkaline phosphatase-positive colony-forming units and osteoclasts compared to cultures from WT mice. Significantly less new bone and significantly fewer osteoclasts were observed in the drill sites of PAR2 KO mice compared to WT mice 7days post-surgery. A number of activators of PAR2, including matriptase and kallikrein 4, were found to be expressed by normal bone marrow. Parathyroid hormone, 1,25 dihydroxyvitamin D3, or interleukin-6 in combination with its soluble receptor down-regulated PAR2 mRNA expression, and fibroblast growth factor-2 or thrombin stimulated PAR2 expression. These results suggest that PAR2 activation contributes to determination of cells of both osteoblast and osteoclast lineages within bone marrow, and thereby participates in the regulation of skeletal growth and bone repair.Highlights: ► We describe bone structure in mice lacking proteinase-activated receptor-2 (PAR2). ► In the absence of PAR2 bone volume is elevated during growth but repair is delayed. ► Mice lacking PAR2 are deficient in osteoblast and osteoclast precursors. ► Activators of PAR2 are expressed in bone marrow. ► PAR2 antagonists used as therapeutic agents may delay bone repair.

Independent predictors of all osteoporosis-related fractures among healthy Saudi postmenopausal women: The CEOR Study

2012-01-03

Abstract: This study was designed to identify independent predictors of all osteoporosis-related fractures (ORFs) among healthy Saudi postmenopausal women. We prospectively followed a cohort of 707 healthy postmenopausal women (mean age, 61.3±7.2years) for 5.2±1.3years. Data were collected on demographic characteristics, medical history, personal and family history of fractures, lifestyle factors, daily calcium intake, vitamin D supplementation, and physical activity score. Anthropometric parameters, total fractures (30.01 per 1000women/year), special physical performance tests, bone turnover markers, hormone levels, and bone mineral density (BMD) measurements were performed. The final model consisted of seven independent predictors of ORFs: [lowest quartile (Q1) vs highest quartile (Q4)] physical activity score (Q1 vs Q4: ≤12.61 vs ≥15.38); relative risk estimate [RR], 2.87; (95% confidence interval [CI]: 1.88–4.38); age≥60years vs age<60years (RR=2.43; 95% CI: 1.49–3.95); hand grip strength (Q1 vs Q4: ≤13.88 vs ≥17.28kg) (RR=1.88; 95% CI: 1.15–3.05); BMD total hip (Q1 vs Q4: ≤0.784 vs 0.973g/cm2) (RR=1.86; 95% CI: 1.26–2.75); dietary calcium intake (Q1 vs Q4: ≤391 vs ≥648mg/day) (RR=1.66; 95% CI: 1.08–2.53); serum 25(OH)D (Q1 vs Q4: ≤17.9 vs ≥45.1nmol/L) (RR=1.63; 95% CI: 1.06–2.51); and past year history of falls (RR=1.61; 95% CI: 1.06–2.48). Compared with having none (41.9% of women), having three or more clinical risk factors (4.8% of women) increased fracture risk by more than 4-fold, independent of BMD. Having three or more risk factors and being in the lowest tertile of T-score of [total hip/lumbar spine (L1–L4)] was associated with a 14.2-fold greater risk than having no risk factors and being in the highest T-score tertile. Several clinical risk factors were independently associated with all ORFs in healthy Saudi postmenopausal women. The combination of multiple clinical risk factors and low BMD is a very powerful indicator of fracture risk.Highlights: ► The clinical risk factors for fractures in postmenopausal women were studied. ► Risk factors include: lowest quartiles of physical activity score, hand grip strength, and BMD of total hip.

Krüppel-like factors KLF2 and 6 and Ki-67 are direct targets of zoledronic acid in MCF-7 cells

2012-01-03

Abstract: Bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases due to breast and prostate cancer. Recent clinical studies indicated a benefit in survival and tumor relapse with the supportive treatment of breast cancer using zoledronic acid (ZA), thus stimulating the debate about its putative anti-tumor activity in vivo. MCF-7 breast cancer cells were treated for 3h (pulse treatment) and 72h (permanent treatment) with ZA, and apoptosis rates and cell viability, defined as ATP content, were determined after 72h. Permanent and pulse stimulation with ZA inhibited the viability of MCF-7 cells, which could partly be rescued by atorvastatin (Ator) pre-treatment but not by geranylgeranyl pyrophosphate (GGPP) co-treatment. Microarray analysis of ZA treated MCF-7 cells identified genes of the mevalonate pathway as significantly upregulated, which was verified by qPCR. Additionally the putative tumor suppressors krüppel-like factor 2 and 6 (KLF2 and KLF6) were markedly upregulated, while the classical proliferation marker Ki-67 was clearly downregulated. The expression of all three genes was confirmed by qPCR on mRNA level and by immunocytochemistry or Western blot staining. Expression of target genes were also analyzed in other breast (MDA-MB-231, BT-20, ZR75-1, T47D) and prostate (LNCaP, PC3) cancer cell lines by qPCR. ZA responsiveness of KLF2, KLF6 and Ki-67 could be verified in PC3 and T47D cells, KLF6 responsiveness in LNCaP and KLF2 responsiveness in MDA-MB-231 and BT-20 cells. Here we demonstrate in the apoptosis insensitive MCF-7 cell line a remarkable impact of ZA exposure on cell viability and on the regulation of putative tumor suppressors of the KLF family. The molecular mechanism involved might be the accumulation of isopentenyl pyrophosphate (IPP) and ApppI, since we could partly rescue the ZA effect by Ator pre-treatment and GGPP co-treatment. These data should stimulate further research into both the role of the mevalonate pathway and the accumulation of pyrophosphate compounds like ApppI in tumorigenesis and differentiation and their potential apart from the inhibition of mitochondrial ADP/ATP translocase and apoptosis, since such effects might well be responsible for the adjuvant ZA treatment benefit of patients suffering from breast cancer.Highlights: ► Zoledronic acid induces tumor suppressor genes in MCF-7 breast cancer cells. ► The proliferation marker Ki-67 is downregulated in MCF-7 cells by zoledronic acid. ► Zoledronic acid effects on gene regulation are mediated by the mevalonate pathway.

The different contributions of cortical and trabecular bone to implant anchorage in a human vertebra

2011-12-30

Abstract: The quality of the peri-implant bone and the strength of the bone–implant interface are important factors for implant anchorage. With regard to peri-implant bone, cortical and trabecular compartments both contribute to the load transfer from the implant to the surrounding bone but their relative roles have yet to be investigated in detail. However, this knowledge is crucial for the better understanding of implant failure and for the development of new implants. This is especially true for osteoporotic bone, which is characterized by a deterioration of the trabecular architecture and a thinning of the cortical shell, leading to a higher probability of implant loosening. The aim of this study was to investigate the relative biomechanical roles of cortical and trabecular bone on implant pull-out stiffness in human vertebrae. The starting point of our investigation was a micro-computed tomography scan of an adult human vertebra. The cortical shell was identified and an implant was digitally inserted into the vertebral body. Pull-out tests were simulated with micro-finite element analysis and the apparent stiffness of the system with various degrees of shell thickness and bone volume fraction was computed. Our computational models demonstrated that cortical bone, although being very thin, plays a major role in the mechanical competence of the bone–implant construct.Graphical abstract: Highlights: ► Osteoporotic bone is characterized by a deterioration of the trabecular network and a thinning of the cortical shell. ► The interplay between trabecular and cortical bone is a key factor for implant fixation. ► Implant pull-out stiffness was investigated by micro-finite element analysis based on a micro-computed tomography scan of an adult human vertebra. ► Cortical bone, although being very thin, contributed as much as 60% to the total stiffness of the bone-implant system. ► Highly porous or damaged cortical bone can have a significant negative impact on implant fixation.

Bisphosphonate treatment of postmenopausal osteoporosis is associated with a dose dependent increase in serum sclerostin

2012-01-09

Abstract: The benefits coming from long-term treatment of postmenopausal osteoporosis with bisphophonates are limited by a coupled decrease in bone formation. The objective of this study is to determine whether this decrease in bone formation is associated with changes in serum levels of the WNT signaling antagonist sclerostin or Dickkopf-1 (DKK1).This is an ancillary observation from patients participating in a 12months, phase 2, randomized clinical trial. We analyzed 107 patients given either monthly intramuscular neridronate (12.5, 25 or 50mg) or placebo.Serum C-terminal telopeptide of type I collagen (sCTX, a bone-resorption marker) decreased by 61%, 75% and 73% in the 12.5, 25 and 50mg dose groups, respectively.Mean changes in bone alkaline phosphatase (bAP) at 12months were −47%, −60.0% and −52.6% in the groups receiving 12.5, 25 or 50mg neridronate, respectively. Serum DKK1 remained unchanged at all time points in the 3 groups. Serum sclerostin increased versus placebo group gradually and significantly only in patients treated with 25 or 50mg neridronate monthly, reaching 138–148% of baseline values (P<0.001). Changes in serum sclerostin at 12months were negatively correlated with changes in bAP (P<0.001) even when data were adjusted for sCTX changes and only treated patients were included.In conclusions, decreased bone formation after several months of bisphosphonate therapy is associated with increased serum levels of sclerostin. This might suggest that Wnt signaling may play a role in the coupling between resorption and formation.Highlights: ► Neridronate treatment is associated with significant increases in serum sclerostin. ► This is associated with decreases in serum bone alkaline phosphatase. ► Formation- resorption coupling after bisphosphonates may involve Wnt signaling.

Age-related loss of proximal femoral strength in elderly men and women: The Age Gene/Environment Susceptibility Study — Reykjavik

2011-12-26

Abstract: The risk of hip fracture rises rapidly with age, and is particularly high in women. This increase in fracture risk reflects both the age-related change in the risk of falling and decrements in the strength of the proximal femur. To better understand the extent to which proximal femoral density, structure and strength change with age as a function of gender, we have carried out a longitudinal analysis of proximal femoral volumetric quantitative computed tomographic (vQCT) images in men and women, analyzing changes in trabecular and cortical bone properties, and using subject-specific finite element modeling (FEM) to estimate changes in bone strength. In the AGES-Reykjavik Study vQCT scans of the hip were performed at a baseline visit in 2002–2006 and at a second visit 5.05±0.25years later. From these, 223 subjects (111 men, 112 women, aged 68–87years) were randomly selected. The subjects were evaluated for longitudinal changes in three bone variables assessed in a region similar to the total femur region quantified by DXA: areal bone mineral density (aBMD), trabecular volumetric bone mineral density (tBMD) and the ratio of cortical to total tissue volume (cvol/ivol). They were also evaluated for changes in bone strength using FEM models of the left proximal femur. Models were analyzed under single-limb stance loading (FStance), which approximates normal physiologic loading of the hip, as well as a load approximating a fall onto the posterolateral aspect of the greater trochanter (FFall). We computed five-year absolute and percentage changes in aBMD, tBMD, cvol/ivol, FFall and FStance. The Mann–Whitney Test was employed to compare changes in bone variables between genders and the Wilcoxon Signed Rank Test was used to compare changes in bone strength between loading conditions. Multiple (linear) regression was employed to determine the association of changes in FFall and FStance with baseline age and five-year weight loss. Both men and women showed declines in indices of proximal femoral density and structure (aBMD: men −3.9±6.0%, women −6.1±6.2%; tBMD: men −14.8±20.3%, women −23.9±26.8%; cvol/ivol: men −2.6±4.6%, women −4.7±4.8%, gender difference: p<0.001). Both men and women lost bone strength in each loading condition (FStance: men −4.2±9.9%, women −8.3±8.5%; FFall: men −7.0±15.7%, women −12.8±13.2%; all changes from baseline p<0.0001). The gender difference in bone strength loss was statistically significant in both loading conditions (p<0.001 for FStance and P<0.01 for FFall) and FFall was lost at a higher rate than FStance in men (p<0.01) and women (p<0.0001). The gender difference in strength loss was statistically significant after adjustment for baseline age and weight loss in both loading conditions (p<0.01). In these multi-linear models, men showed increasing rates of bone loss with increasing age (FFall: p=0.002; FStance: p=0.03), and women showed increasing bone strength loss with higher degrees of weight loss (FStance: p=0.003). The higher loss of FFall compared to FStance supports previous findings in animal and human studies that the sub-volumes of bone stressed under normal physiologic loading are relatively better protected in aging. The gender difference in hip bone strength loss is consistent with the higher incidence of hip fracture among elderly women.Highlights: ► We determined age-related changes in hip strength in elderly men and women. ► Finite element modeling estimated strength changes under stance and fall loading. ► Women lost twice as much hip strength as men. ► Women and men had different risk factors for loss of bone strength. ► Hip strength in fall loading was lost at a 50% greater rate than in stance loading.

Ultrashort echo time (UTE) imaging with bi-component analysis: Bound and free water evaluation of bovine cortical bone subject to sequential drying

2012-01-09

Abstract: Recent proton magnetic resonance (MR) spectroscopy studies have shown that cortical bone exists as different components which have distinct transverse relaxation times (T2s). However, cortical bone shows zero or near zero signal with all conventional MR sequences on clinical scanners and the different water components cannot be assessed with this approach. In order to detect signal in this situation a two-dimensional (2D) non-slice selective ultrashort echo time (UTE) pulse sequence with a nominal TE of 8μs was used together with bi-component analysis to quantify bound and free water in bovine cortical bone at 3T. Total water concentration was quantified using a 3D UTE sequence together with a reference water phantom. 2D and 3D UTE imaging were performed on 14 bovine bone samples which were subjected to sequential air drying to evaluate free water loss, followed by oven drying to evaluate bound water loss. Sequential bone weight loss was measured concurrently using a precision balance. Bone porosity was measured with micro computed tomography (μCT) imaging. UTE measured free water loss was higher than the volume of cortical pores measured with μCT, but lower than the gravimetric bone water loss measured during air drying. UTE assessed bound water loss was about 82% of gravimetric bone water loss during oven drying. On average bovine cortical bone showed about 13% free water and 87% bound water. There was a high correlation (R=0.91; P<0.0001) between UTE MR measured free water loss and gravimetric bone weight loss during sequential air drying, and a significant correlation (R=0.69; P<0.01) between UTE bound water loss and gravimetric bone weight loss during oven drying. These results show that UTE bi-component analysis can reliably quantify bound and free water in cortical bone. The technique has potential applications for the in vivo evaluation of bone porosity and organic matrix.Highlights: ► UTE MR techniques can measure bound and free water in cortical bone. ► UTE assessed free water loss correlates with gravimetric water loss in air-drying. ► UTE assessed bound water loss correlates with gravimetric water loss in oven-drying. ► The techniques have potential evaluating bone porosity and organic matrix in vivo.

The emerging role of lysophosphatidic acid (LPA) in skeletal biology

Julia Blackburn, Jason P. Mansell — 2011-12-30

Abstract: Lysophosphatidic acid (LPA) is the simplest signalling lipid eliciting pleiotropic actions upon most mammalian cell types. Although LPA has an established role in many biological processes, particularly wound healing and cancer, the participation of LPA in skeletal biology is just beginning to emerge. Early studies, identified in this review, gave a solid indication that LPA, via binding to one of several cell surface receptors, activated multiple intracellular systems culminating in altered cell morphology, growth, motility and survival. More recently the ablation of murine LPA1 and 4 receptors implies that this lipid has a role in skeletal development and post natal bone accrual. Greater understanding of the ability of LPA to influence, for example, osteoblast growth, maturation and survival could be advantageous in developing novel strategies aimed at improving skeletal tissue repair and regeneration. Herein this review provides an insight into the diversity of studies exploring the actions of a small lipid on those major cell types key to skeletal tissue health and homeostasis.Highlights: ► Cell surface receptors for LPA have been identified for most skeletal cell types. ► LPA influences the growth, migration, morphology and maturation of these cells. ► LPA appears to have a role in skeletogenesis via LPA receptors 1 and 4. ► LPA co-operates with calcitriol to enhance human osteoblastogenesis and maturation. ► Selected LPA receptor ligands may find a role in bone repair and regeneration.

Mice with increased angiogenesis and osteogenesis due to conditional activation of HIF pathway in osteoblasts are protected from ovariectomy induced bone loss

Qiang Zhao, Xing Shen, Wei Zhang, Guochun Zhu, Jin Qi, Lianfu Deng — 2012-01-12

Abstract: Postmenopausal osteoporosis is characterized by a reduction in the numbers of sinusoidal and arterial capillaries in the bone marrow and reduced bone perfusion suggesting a role of vascular component in the pathogenesis of osteoporosis. Previous studies have shown that bone formation and angiogenesis are positively coupled through activation of the hypoxia inducible factor (HIF1α) signaling pathway. Therefore, we hypothesized that mice with increased angiogenesis and osteogenesis due to activation of the HIF signaling pathway in osteoblasts, via osteoblast specific disruption of HIF degrading protein von Hippel–Lindau (VHL) (ΔVhl), are protected from ovariectomy induced bone loss. ΔVhl mice and control littermates were ovariectomized or sham operated and four weeks later bone quality was evaluated along with blood vessel formation. Trabecular and cortical bone volume was strikingly increased in ΔVhl mice along with blood vessel formation as compared to control littermates. In control mice, ovariectomy significantly decreased bone mineral density, deteriorated bone microarchitecture, and decreased mechanical strength compared to the sham operated control mice. This was accompanied with a significant decrease in blood vessel volume and expressions of HIF1α, HIF2α, and VEGF proteins at the distal femur in ovariectomized control mice. In contrast, ovariectomy in ΔVhl mice had absolutely no effect on either the blood vessel formation or the bone structural and mechanical quality parameters. These data indicate that activation of HIF signaling pathway in osteoblasts may prevent estrogen deficiency-induced bone loss and decrease in blood vessels in bone marrow.Highlights: ► Osteoporotic bones display reduced perfusion and numbers of capillaries. ► Bone formation and angiogenesis are coupled through HIF signaling in osteoblasts. ► Active HIF signaling pathway in osteoblasts prevents ovariectomy induced bone loss.

Is the spinal motion segment a diarthrodial polyaxial joint: What a nice nucleus like you doing in a joint like this?

Irving M. Shapiro, Edward J. Vresilovic, Makarand V. Risbud — 2012-01-06

Abstract: This review challenges an earlier view that the intervertebral joint could not be classified as a diarthrodial joint and should remain as an amphiarthrosis. However, a careful analysis of the relevant literature and in light of more recent studies, it is clear that while some differences exist between the spinal articulation and the generic synovial joint, there are clear structural, functional and developmental similarities between the joints that in sum outweigh the differences. Further, since the intervertebral motion segment displays movement in three dimensions and the whole spine itself provides integrated rotatory movements, it is proposed that it should be classified not as an amphiarthrose, “a slightly moveable joint” but as a complex polyaxial joint. Hopefully, reclassification will encourage further analysis of the structure and function of the two types of overlapping joints and provide common new insights into diseases that afflict the many joints of the human skeleton.Highlights: ► We critique the assumption that the intervertebral disk is an amphiarthosis. ► We compare the development, structure and function of the disk with a synovial joint. ► The intervertebral disk has many similarities with synovial joints. ► The intervertebral disk should be re-classified as a polyaxial diarthrodial joint.

Variations in vertebral body dimensions in women measured by 3D-XA: A longitudinal in vivo study

S. Kolta, S. Kerkeni, C. Travert, W. Skalli, R. Eastell, C.C. Glüer, C. Roux — 2012-01-12

Abstract: Bone size and shape play an important role in bone strength, as shown by biomechanical testing and clinical studies. Vertebral body dimensions determine vertebral body strength even after adjustment for bone mineral density. We have recently proposed an in vivo method for 3D reconstruction of vertebral bodies using the whole spine imaging on a standard DXA device (3D-XA). The aim of our study was to measure in vivo vertebral body dimension changes by 3D-XA in women over a 6year period. A total of 174 women were included in this study. They were divided into 3 groups: premenopausal (20–40years; N=53), postmenopausal women (55–60years; N=65) and elderly women (70–80years; N=56). Thoracic and lumbar spine (T4–L4) were reconstructed using the 3D-XA method at baseline and 6years later. Biochemical markers of bone remodeling were measured at baseline. In premenopausal women, there was an increase in minimal cross-sectional area (minCSA), vertebral body volume as well as end plate width of the lumbar vertebrae, without statistically significant change of these parameters at the thoracic spine; there was no change in anterior heights. In postmenopausal women, there was a decrease in vertebral body anterior height and depth, driven by results in the elderly group at both the thoracic and lumbar spine. Vertebral body width decreased at the thoracic spine but increased at the lumbar spine. MinCSA and volume decreased at the thoracic spine, in contrast with an increase of these 2 parameters at the lumbar spine in early postmenopausal women (55–60years). In elderly women (70–80years), the change in minCSA and volume of the lumbar spine was not statistically significant over 6years. In postmenopausal women, there was no correlation between changes in vertebral dimensions and baseline biochemical markers of bone remodeling except for NTX/Cr and anterior height decrease. Our study confirms that an increase in geometric dimensions of lumbar vertebrae occurs through adult life. This could be related to a compensation for bone loss, aiming to maintain bone strength through increase in size. However, this phenomenon is not observed at all levels in the spine; since we do not confirm this increase at the thoracic spine. This might be one of the determinants of the higher risk of fractures in this part of the spine.Highlights: ► We measured in vivo vertebral dimension changes by 3D-XA in 174 women over 6 years. ► In pre and postmenopausal women, minCSA and volume increased at the lumbar spine. ► In post menopausal women, these parameters decreased at the thoracic spine. ► Vertebral body dimension changes are different at the thoracic and lumbar levels. ► This might explain part of the higher risk of fractures at the thoracic spine.

Increased susceptibility to microdamage in Brtl/+ mouse model for osteogenesis imperfecta

2012-01-16

Abstract: Osteogenesis imperfecta (OI) is a genetic disease of collagen or collagen-related proteins that adversely impacts bone mass and fracture resistance. Little is known regarding the role that microdamage plays in OI and whether or not OI bone is more prone to damage accumulation than bone with unaffected collagen. The Brtl/+ mouse is a heterozygous model for OI which contains a Gly349Cys substitution in one COL1A1 allele, and demonstrates a low ductility phenotype. At 8weeks of age, Brtl/+ demonstrates an increase in osteoclast number, which mimics the upregulated bone turnover often found in OI patients. We hypothesize that upregulated osteoclast activity in Brtl/+ is due, in part, to increased remodeling associated with microdamage repair. In the present study, we used Brtl/+ to investigate the susceptibility of OI bone to microdamage. The mouse ulnar loading model was used to induce microdamage and to test the hypothesis that Brtl/+ is more susceptible to damage accumulation than age-matched wild type (WT) counterparts. Linear elastic fracture mechanics (LEFM) was used to investigate the fracture toughness properties of both Brtl/+ and WT bones to determine if there is any correlation with toughness and the degree of microdamage.Results show that Brtl/+ ulnae subject to normal cage activity demonstrate an inherently larger amount of microdamage than WT controls. Following axial compressive loading, Brtl/+ ulnae are more prone to damage than WT counterparts despite demonstrating a greater resistance to whole-bone deformation. Fracture toughness results demonstrate that Brtl/+ specimens, despite not exhibiting a significant difference, display a trend toward lower fracture toughness values than their WT counterparts. Correlations show that microdamage levels tend to increase as fracture toughness decreases. Together, these findings may have strong clinical implications for explaining increased fragility and remodeling activity in OI patients.Highlights: ► Brtl/+ mice, modeling osteogenesis imperfecta, have increased bone microdamage. ► Increased bone microdamage is correlated to reduced bone fracture toughness. ► Fragility and remodeling activity in OI patients may be related to bone microdamage.

Modeling of cortical bone adaptation in a rat ulna: Effect of frequency

N. Chennimalai Kumar, J.A. Dantzig, I.M. Jasiuk — 2012-01-13

Abstract: We employ a recently developed model for the adaptation of cortical bone in response to mechanical loading to study the effect of loading frequency on the computed response, and we compare our results to previous experimental measurements on rat ulnae. We represent the cortical bone as a poroelastic material with orthotropic permeability. Bone adaptation in the model is related to a mechanical stimulus derived from the dissipation energy of the poroelastic flow induced by deformation. We account for a non-locality in the mechanotransduction of osteocytes present in the lacunae by using a “zone of influence.” Calculations are done using the finite element method applied to a rat ulna whose geometry is obtained from micro-computed tomography images. We show that the change in the second moment of inertia of the cross-section increases non-linearly and saturates at higher frequency range. The numerical results are then compared quantitatively to experimental data from the literature. Finally, we examine the role of local narrowing of intramedullary canal in our specific ulna in the development of local irregularities in growth.Highlights: ► We model bone adaptation in a rat ulna using a finite element method. ► We represent bone as a poroelastic material with orthotropic permeability. ► In adaptation law we use the mechanical stimulus derived from the dissipation energy of the poroelastic flow. ► We account for nonlocality of mechanotransduction by osteocytes using a zone of influence. ► We show that the change in the second moment of inertia of the cross-section increases non-linearly.

Osteoclasts derived from patients with neurofibromatosis 1 (NF1) display insensitivity to bisphosphonates in vitro

2012-01-16

Abstract: A total of 20 patients with neurofibromatosis 1 (NF1) were screened for NF1-related osteoporosis, and blood samples were collected for isolation of peripheral blood osteoclast progenitors. Patients with NF1 had higher levels of serum bone turnover markers (CTX and PINP) compared to controls. In addition, persons with high bone resorption in vitro on average had high levels of serum CTX. Of the 20 patients with NF1, 15 had low bone mineral density (osteopenia/osteoporosis), but these 15 patients did not have marked risk factors for low bone mineral density. Thus, we recommend screening for osteoporosis to all adult patients with NF1. Our aim was also to characterize the effects of bisphosphonates on NF1 osteoclasts in vitro. NF1 osteoclasts and osteoclasts from healthy controls in vitro were treated with zoledronic acid, alendronate and clodronate. These bisphosphonates caused a marked reduction in the number of normal control osteoclasts in vitro, while only a slight change was observed in the number of NF1 osteoclasts. Ras-inhibitor FTS counteracted this NF1-related insensitivity to zoledronic acid, suggesting that Ras may play a role in this phenomenon.Highlights: ► Peripheral-blood derived NF1 osteoclasts displayed in vitro insensitivity to zoledronic acid. ► Similar in vitro results were obtained with alendronate and clodronate. ► Ras inhibitor FTS counteracted the in vitro insensitivity to zoledronic acid in NF1 osteoclasts. ► Patients with NF1 have higher levels of serum CTX and PINP compared to controls. ► Persons with high bone resorption in vitro on average have high levels of serum CTX.

Characterization of mesenchymal progenitor cells isolated from human bone marrow by negative selection

2012-01-16

Abstract: Studies on the pathogenesis of osteoporosis and other metabolic bone diseases would be greatly facilitated by the development of approaches to assess changes in gene expression in osteoblast/osteoprogenitor populations in vivo without the potentially confounding effects of in vitro culture and expansion of the cells. While positive selection to identify a progenitor population in human marrow can be used to select for cells capable of osteoblast differentiation, each of the markers that have been used to identify marrow mesenchymal populations (alkaline phosphatase [AP], Stro-1, CD29, CD49a, CD73, CD90, CD105, CD166, CD44, CD146 and CD271) may be expressed on distinct subsets of marrow mesenchymal cells. Thus, positive selection with one or more of these markers could exclude a possibly relevant cell population that may undergo important changes in various clinical conditions. In the present report, we describe the isolation and characterization of human osteoprogenitor cells obtained by depletion of bone marrow cells of all hematopoietic lineage/hematopoietic stem cells and endothelial/endothelial precursor cells (lin−/CD34/CD31−). The yield of lin−/CD34/CD31− cells from ~10mL of bone marrow (~80million mononuclear cells) was ~80,000 cells (0.1% of mononuclear cells). While not selected on the basis of expression for the mesenchymal marker, Stro-1, 68% of these cells were Stro-1+. Using linear whole transcriptome amplification followed by quantitative polymerase chain reaction (QPCR) analysis, we also demonstrated that, compared to lin− cells (which are already depleted of hematopoietic cells), lin−/CD34/31− cells expressed markedly lower mRNA levels for the endothelial/hematopoietic markers, CD34, CD31, CD45, and CD133. Lin−/CD34/31− cells were also enriched for the expression of mesenchymal/osteoblastic markers, with a further increase in runx2, osterix, and AP mRNA expression following in vitro culture under osteogenic conditions. Importantly, lin−/CD34/31− cells contained virtually all of the mineralizing cells in human marrow: while these cells displayed robust calcium deposition in vitro, lin−/CD34/31+ cells demonstrated little or no mineralization when cultured under identical osteogenic conditions. Lin−/CD34/31− cells thus represent a human bone marrow population highly enriched for mesenchymal/osteoblast progenitor cells that can be analyzed without in vitro culture in various metabolic bone disorders, including osteoporosis and aging.Highlights: ► We describe the isolation of human osteoprogenitor cells using negative selection. ► Lin-/CD34/CD31- cells were enriched for expression of osteoblast markers. ► These cells can be analyzed without in vitro culture in metabolic bone disorders.

Corrigendum to “Bisphosphonates and glucocorticoid osteoporosis in men: results of a randomized controlled trial comparing zoledronic acid with risedronate” [Bone 50 (2012) 289-295]

2012-01-09

, cited in the second to last sentence in the first column of page 292, was erroneously omitted from the manuscript. The table appears below:

Corrigendum to “Effects of 50Hz sinusoidal electromagnetic fields of different intensities on proliferation, differentiation and mineralization potentials of rat osteoblasts” [Bone 49 (2011) 753–761]

2012-01-09

In the author line, affiliation “a” and "b" was incomplete. The correct affiliation “a” and "b" appears above. In the reference list, references 4, 10, 29, and 35 were cited incorrectly. The correct references appear below: