bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular and Functional Expression of Voltage-Operated Calcium Channels During Osteogenic Differentiation of Human Mesenchymal Stem Cells
Authors: Zahanich Ihor , Graf Eva M , Heubach Jürgen F , Hempel Ute , Boxberger Sabine , Ravens Ursula
Published: April 2006

We used the patch-clamp technique and RT-PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L-type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L-type Ca2+ channel function.

Introduction: During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs.

Materials and Methods: Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, β-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel α1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique.

Results: mRNA for the pore-forming α1C and α1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the α1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization.

Conclusion: These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.
Skeletal Retention of Bisphosphonate (Pamidronate) and Its Relation to the Rate of Bone Resorption in Patients With Breast Cancer and Bone Metastases
Authors: Cremers Serge CLM , Papapoulos Socrates E , Gelderblom Hans , Seynaeve Caroline , den Hartigh Jan , Vermeij Pieter , van derRijt Carin CD , van Zuylen Lia
Published: April 2006

Bisphosphonate pharmacokinetics may affect individual responses. Skeletal retention of pamidronate infused monthly to patients with bone metastases was highly variable (12-98%) and did not diminish with time, showing the capacity of the skeleton to retain large amounts of bisphosphonate. Relationships between skeletal retention of pamidronate and rate of bone resorption are complex and depend on previous treatment and the total amount of retained bisphosphonate.

Introduction: Bisphosphonates (BPs) given intravenously every 3–4 weeks are effective in the management of metastatic bone disease from breast cancer, but responses among patients vary, and it is not known whether current dose and dose intervals are appropriate for an individual patient. An influence of pharmacokinetics of BPs on antiresorptive action may contribute to this variation in response. To test this hypothesis, we determined the skeletal retention of intravenous pamidronate and its association to the rate of bone resorption in patients with bone metastases from breast cancer.

Materials and Methods: In a cross-sectional study, 24-h urinary excretion of pamidronate and the biochemical marker of bone resorption N-terminal telopeptide of type 1 collagen and serum alkaline phosphatase were measured in 40 patients with bone metastases from breast cancer at the beginning, after 3–6 months, and after 1 year of treatment with intravenous pamidronate 90 mg every 3–4 weeks.

Results and Conclusions: Skeletal retention (dose − amount excreted into urine) 2 days after infusion varied between 12% and 98% (mean, 62%) of the administered dose, but there were no differences in retention between patients receiving pamidronate for the first time or after 3–6 months or after 1 year of treatment. Retention of pamidronate was related to the prevalent rate of bone turnover in previously untreated patients, whereas no such relationship was found in previously treated patients. Rate of bone resorption after treatment seemed to be related to the amount of pamidronate retained. During 1 year of treatment, retention of pamidronate remained constant, indicating no saturation of skeletal binding sites with treatment. The variability in retention among individual patients can be attributed to the number of available binding sites. This is related, however, to bone turnover only before the start of treatment. The apparent relationships between skeletal retention and antiresorptive effect could have implications for the design of optimal therapeutic regimens with BPs in patients with bone metastases from breast cancer.
Exogenous PTH-Related Protein and PTH Improve Mineral and Skeletal Status in 25-Hydroxyvitamin D-1α-Hydroxylase and PTH Double Knockout Mice
Authors: Xue Yingben , Zhang Zengli , Karaplis Andrew C , Hendy Geoffrey N , Goltzman David , Miao Dengshun
Published: April 2006

We examined the effect of NH2-terminal fragments of PTHrP and PTH in young mutant mice deficient in both PTH and 1,25-dihydroxyvitamin D. Both proteins prolonged murine survival by increasing serum calcium, apparently by enhancing renal calcium transporter expression. The dominant effect on the skeleton was an increase in both endochondral bone and appositional formation without increased bone resorption.

Introduction: PTH-related protein (PTHrP) was discovered as a hypercalcemic agent responsible for the syndrome of humeral hypercalcemia of malignancy, and PTH is the major protein hormone regulating calcium homeostasis. Both proteins have skeletal anabolic actions when administered intermittently. We examined effects of exogenous PTHrP(1-86) and PTH(1-34) in double null mutant mice deficient in both PTH and 25-hydroxyvitamin D-1α-hydroxylase {1α(OH)ase} to determine the action of these proteins in the absence of the two major regulators of calcium and skeletal homeostasis.

Materials and Methods: Mice heterozygous for the PTH null allele and for the 1α(OH)ase null allele were mated to generate pups homozygous for both null alleles. PTHrP(1-86) and PTH(1-34) were administered subcutaneously starting 4 days after birth. Serum biochemistry and skeletal radiology, histology, and histomorphometry were performed, and indices of bone formation, resorption, and renal calcium transport were determined by real time RT-PCR, Western blot, and immunohistochemical approaches.

Results: In the double mutant mice, which die within 3 weeks after birth with severe hypocalcemia, tetany, and skeletal defects, exogenous PTHrP and PTH enhanced survival of the animals by improving serum calcium. Both proteins increased renal calcium transporter expression and long bone length and augmented growth plate chondrocyte proliferation, differentiation, and cartilage matrix mineralization. Cortical and trabecular bone mass was increased with augmented osteoblast number and activity; however, bone resorption was not increased.

Conclusions: PTHrP and PTH reduced hypocalcemia by enhancing renal calcium reabsorption but not by increasing bone resorption. The major skeletal effects of exogenous PTHrP and PTH were to increase bone anabolism.
Multi-Detector Row CT Imaging of Vertebral Microstructure for Evaluation of Fracture Risk
Authors: Ito Masako , Ikeda Kyoji , Nishiguchi Masahiko , Shindo Hiroyuki , Uetani Masataka , Hosoi Takayuki , Orimo Hajime
Published: April 2006

We applied MDCT for in vivo evaluation of the microarchitecture of human vertebrae. Microstructure parameters, such as structure model index, Euler's number, and bone volume fraction, revealed higher relative risk for prevalent vertebral fracture than did BMD obtained by DXA. Thus, microstructure analysis by MDCT, together with simultaneously obtained volumetric BMD values, is useful for clinical assessment of fracture risk.

Introduction: BMD measurement by DXA alone has limitations in predicting fracture, and methods for clinical assessment of bone quality, such as microstructure, are awaited. This study was undertaken to examine the applicability of multidetector row CT (MDCT) for in vivo evaluation of trabecular microstructure.

Materials and Methods: Optimal conditions for MDCT scanning were determined at a spatial resolution of 250 × 250 × 500 μm, using μCT data of excised human vertebra specimens as a reference. We analyzed the trabecular microstructure of the vertebrae of 82 postmenopausal women (55-76 years old), including 39 women with and 43 without a recent vertebral fracture.

Results: Microstructure indices obtained by MDCT scanning revealed higher relative risk for prevalent vertebral fracture (OR: 16.0 for structure model index, 13.6 for bone volume fraction, and 13.1 for Euler's number) than did spinal BMD obtained by DXA (OR: 4.8). MDCT could also provide volumetric BMD data, which had higher diagnostic value (OR: 12.7) than did DXA.

Conclusion: Vertebral microarchitecture can be visualized by MDCT, and microstructure parameters obtained by MDCT, together with volumetric BMD, provided better diagnostic performance for assessing fracture risk than DXA measurement.
Effect of Long-Term Growth Hormone Treatment on Bone Mass and Bone Metabolism in Growth Hormone-Deficient Men
Authors: Bravenboer Nathalie , Holzmann Paulien J , ter Maaten Jan C , Stuurman Lotte M , Roos Jan C , Lips Paul
Published: April 2006

Long-term GH treatment in GH-deficient men resulted in a continuous increase in bone turnover as shown by histomorphometry. BMD continuously increased in all regions of interest, but more in the regions with predominantly cortical bone.

Introduction: Adults with growth hormone (GH) deficiency have reduced rates of bone turnover and subnormal BMD. GH treatment is effective in enhancing bone turnover as shown by biochemical markers and bone histomorphometric studies. However, it is uncertain whether long-term treatment will result in higher bone mass. In this study, we present BMD and histomorphometric data on 5 years of GH treatment in GH-deficient men.

Materials and Methods: Thirty-eight adult men with childhood onset GH deficiency (20-35 years) were included in the study. Twenty-six of these had multiple pituitary hormone deficiencies and were on stable conventional hormone replacement. BMC (total body) and BMD (lumbar spine and hip) were measured before and after 1, 2, 3, 4, and 5 years of treatment. BMD in various regions of the total body was calculated by computer software (head, trunk, arms, and legs). Transiliac bone biopsies were obtained before and after 1 and 5 years of GH treatment.

Results: Total body BMC increased 18% after 5 years of treatment. This increase was observed in all regions of interest: head, 13.7%; trunk, 27.8%; arms, 24.4%; legs, 13.8%. BMD also increased in all separately measured regions: lumbar spine, 9%; femoral neck, 11%; femoral trochanter, 16%. Lumbar spine area significantly increased (p = 0.0002). Histomorphometric data showed increased osteoid surface (p < 0.02), osteoid volume (p < 0.01), and activation frequency (p < 0.006), but trabecular bone volume did not increase significantly. Qualitative assessment of the cortical bone showed endosteal and periosteal bone formation.

Conclusions: In conclusion, GH considerably increases BMC after long-term treatment. The combination of BMD and histomorphometric data suggests that GH has a greater effect on cortical than on trabecular bone.
Application of Histomorphometric Methods to the Study of Bone Repair
Authors: Gerstenfeld Louis C , Wronski Thomas J , Hollinger Jeffrey O , Einhorn Thomas A
Published: April 2006

ABSTRACT: Standardized methods for the histomorphometric assessment of bone are essential features of most studies of metabolic bone diseases and their treatments. These methodologies were developed to assess coupled remodeling, focusing primarily on osteoblasts and osteoclasts, the anabolic and catabolic rates of these cells, and structural features of mature bone. Research studies on bone healing and the development of new therapeutic approaches for the enhancement of bone repair also require a comprehensive understanding of the basic cellular and tissue level mechanisms that underlie these processes. However, the histological methods developed for metabolic bone disease studies are not completely suitable for studies of bone repair because they are based on assumptions that there is little variation in tissue composition within a sample of bone and not generally designed to quantify other types of tissues, such as cartilage, that contribute to bone healing. These techniques also do not provide tissue-based structural measurements that are relatable to the specific types of biomechanical and radiographic structural assessments that are used to determine rates of bone healing. These deficiencies in current histological approaches therefore point to the need to establish standardized criteria for the histomorphometric assessments that are specifically adapted for the study of bone repair in models of fracture healing and bone regeneration. In this Perspective, we outline what we believe to be the specific structural, tissue. and cellular aspects that need to be addressed to establish these standardized criteria for the histomorphometric assessment of bone repair. We present the specific technical considerations that need to be addressed to appropriately sample repair tissues to obtain statistically meaningful results and suggest specific procedures and definitions of nomenclatures for the application of this technology to bone repair. Finally, we present how aspects of histomorphometric measurements of bone repair can be related to biomechanical and radiographic imaging properties that functionally define rates of bone healing, and thus, how these tools can be used to provide corroborating data.
Rate of Forearm Bone Loss Is Associated With an Increased Risk of Fracture Independently of Bone Mass in Postmenopausal Women: The OFELY Study
Authors: Sornay-Rendu Elisabeth , Munoz Françoise , Duboeuf François , Delmas Pierre D
Published: April 2006

BMD is a major determinant of the risk of fragility fractures, but the role of the rate of postmenopausal bone loss is still unclear. In 671 postmenopausal women from the OFELY cohort, we found that the rate of bone loss was significantly associated with fracture risk independently of other well-known predictors including BMD and previous fractures.

Introduction: The level of BMD is a major determinant of the risk of fragility fractures, but the role of the rate of postmenopausal bone loss is still unclear.

Materials and Methods: In the OFELY study, we analyzed the risk of fracture in 671 postmenopausal women (mean age, 62.2 ± 9 years), according to the rate of bone loss. BMD was measured annually by DXA at the forearm, with a mean number of measurements of 10.3 ± 2.6. Peripheral fractures, all confirmed by radiographs, were prospectively registered, and vertebral fractures were evaluated with spine radiographs every 4 years.

Results: During a median (interquartile range [IQ]) of 11.2 years (11–12.3 years) of follow-up, 183 incident fragility fractures including 53 vertebral and 130 nonvertebral fractures were recorded in 134 women. The annual median ± IQ rate of bone loss, calculated from the slope, was −0.30 ± 0.76% at the mid-radius, −0.55 ± 0.79% at the distal radius, and −0.40 ± 0.96% at the ultradistal radius. Women with incident fracture had a rate of bone loss (before fracture) higher by 38–53% than those without fracture (p = 0.0003–0.016). Using multivariate Cox regression models, we found that bone loss in the highest tertile at the mid-radius, distal radius, and ultradistal radius was associated with a significant increased risk of all fractures with an hazard ratio from 1.45 to 1.70 (p = 0.02 to p = 0.009 after adjusting for age, previous fractures, maternal history of fracture, physical activity, grip strength, falls, and baseline BMD).

Conclusions: The rate of bone loss in postmenopausal women is significantly associated with fracture risk independently of other well-known predictors such as BMD and history of fractures.
Missense Mutations in LRP5 Are Not a Common Cause of Idiopathic Osteoporosis in Adult Men
Authors: Crabbe Patricia , Balemans Wendy , Willaert Andy , Van Pottelbergh Inge , Cleiren Erna , Coucke Paul J , Ai Minrong , Goemaere Stefan , Van Hul Wim , De Paepe Anne , Kaufman Jean-Marc
Published: April 2006

We studied whether the LRP5 gene contributes to the clinical phenotype of IO in men. Mutation analysis in 66 IO men revealed a range of sequence variants, of which two missense variants were shown to be of functional relevance.

Introduction: Mutations in the LDL receptor-related protein 5 (LRP5) gene have been associated with extreme bone phenotypes, which makes LRP5 a plausible candidate gene for idiopathic osteoporosis (IO).

Materials and Methods: In 66 men with IO, all 23 exons and exon-intron boundaries of the LRP5 gene were screened for mutations, and functional analyses were performed for those that were putatively involved in the phenotype.

Results: Mutation analysis in the IO probands revealed five missense mutations, of which 1067C>T (S356L), 1364C>T (S455L), and 4609G>A (A1537T) were of potential functional significance because they were located in highly conserved regions of LRP5 and not found in a control panel. Segregation analysis in the respective families could not exclude their possible causality for IO. Furthermore, functional analyses clearly showed an inhibitory effect of mutations 1067C>T and 1364C>T on Wnt signal transduction. These effects are most likely caused by impaired LRP5 synthesis in the case of 1067C>T and failure of protein trafficking to the cell surface for 1364C>T.

Conclusions: For 2 of 66 IO probands, a mutation in the LRP5 gene with proven functionality was found. The findings indicate that carrying an LRP5 mutation is a risk factor for IO, but that overall, IO in men is infrequently underlied by such a mutation.
Self-Assembled RANK Induces Osteoclastogenesis Ligand-Independently
Authors: Kanazawa Kiyoshi , Kudo Akira
Published: April 2006

By immunoprecipitation assay, we showed that mouse RANK was self-assembled through its cytoplasmic domain located at position 534–539, whose domain was different form TRAF binding domains. Moreover, overexpression experiments showed that oligomeric RANK, which was self-associated, induced osteoclastogenesis ligand-independently.

Introduction: TNF receptor I or II (TNF-RI or TNF-RII) is thought to induce its own trimerization by ligand binding; however, recently TNF-RI or TNF-RII was shown to form a trimer through its extracellular domain without ligand binding. RANK, which plays an important role in osteoclast differentiation, is a member of the TNF receptor family. Here, we studied the self-assembly of mouse RANK.

Materials and Methods: Self-assembly of mouse RANK was examined by immunoprecipitation assay using 293T cells that had been transfected with the full-length RANK (Full) fused to FLAG tag (Full-FLAG) and Full fused to HA tag (Full-HA) without soluble RANKL (sRANKL). To explore the binding site for self-assembly, FLAG-tagged RANK C-terminal deletion mutants, 461-, 511-, 533-, 539-, and 544-FLAG, were constructed, and immunoprecipitation was performed. To examine whether RANK overexpression induced osteoclastogenesis, osteoclast progenitors that were derived from wildtype bone marrow cells, in which RANK was overexpressed, were cultured with monocyte-macrophage colony-stimulating factor (M-CSF), and TRACP staining was performed. We examined whether overexpression of each five individual C-terminal mutants induced osteoclastogenesis in osteoclast progenitors. To study the involvement of TRAF6 in RANK-induced osteoclastogenesis, osteoclast progenitors, in which RANK was overexpressed, were cultured with M-CSF and TNF receptor-associated factor (TRAF)6 decoy peptides (T6DP) that inhibit the interaction of RANK with TRAF6.

Results and Conclusions: Immunoprecipitation experiments showed that RANK was self-assembled without sRANKL. Among the five individual mutants, only 539- and 544-FLAG mutants were associated with Full-HA ligand-independently, suggesting that self-association of RANK was regulated by its cytoplasmic domain located at position 534–539. Overexpression of full-length RANK induced osteoclast differentiation, and this differentiation was suppressed by treatment with T6DP. Overexpression of RANK deletion mutants revealed that only 539- and 544-FLAG induced osteoclastogenesis. The five C-terminal mutants had the TRAF6 binding domain in their cytoplasmic regions, suggesting that ligand-independent osteoclastogenesis requires the receptor oligomerization of RANK.
Association of Amount of Physical Activity With Cortical Bone Size and Trabecular Volumetric BMD in Young Adult Men: The GOOD Study
Authors: Lorentzon Mattias , Mellström Dan , Ohlsson Claes
Published: April 2006

In this population-based study, amount of PA was associated with cortical bone size (increased thickness and periosteal circumference) and trabecular vBMD, but not with cortical vBMD or length of the long bones in young men. The lowest effective amount of PA was ≥4 h/week.

Introduction: Physical activity (PA) is believed to have positive effects on the skeleton and possibly help in preventing the occurrence of osteoporosis. Neither the lowest effective amount of PA needed to induce an osteogenic response nor its effect on the BMD and size of the different bone compartments (i.e., trabecular and cortical bone) has yet been clarified.

Materials and Methods: In this population-based study, we investigated the amount of all types of PA in relation to areal BMD (aBMD), trabecular and cortical volumetric BMD (vBMD), and cortical bone size in 1068 men (age, 18.9 ± 0.02 years), included in the Gothenburg Osteoporosis and Obesity Determinants (GOOD) study. aBMD was measured by DXA, whereas cortical and trabecular vBMD and bone size were measured by pQCT.

Results and Conclusions: The amount of PA was associated with aBMD of the total body, radius, femoral neck, and lumbar spine, as well as with cortical bone size (increased thickness and periosteal circumference) and trabecular vBMD, but not with cortical vBMD or length of the long bones. The lowest effective amount of PA was ≥4 h/week. aBMD, cortical bone size, and trabecular vBMD were higher in subjects who started their training before age 13 than in subjects who started their training later in life. Our data indicate that ≥4 h/week of PA is required to increase bone mass in young men and that exercise before and during the pubertal growth is of importance. These findings suggest that PA is imperative for the augmentation of cortical bone size and trabecular vBMD but does not affect the cortical vBMD in young men.
Novel Assessment of Bone Using Time-Resolved Transcutaneous Raman Spectroscopy
Authors: Draper Edward RC , Morris Michael D , Camacho Nancy P , Matousek Pavel , Towrie Mike , Parker Anthony W , Goodship Allen E
Published: April 2006

With fragility fractures increasing as the population ages, there is a need for improved means to estimate risk of fracture. We recorded Raman spectra of both the mineral and organic phases of bone transcutaneously, a technology with potential to enhance bone quality and fracture risk assessment.

Introduction: The current “gold standard” assessment of bone quality is BMD determined by DXA. However, this accounts for only 60–70% of bone strength. X-rays are absorbed by the mineral phase of bone, whereas the organic phase remains essentially invisible; however, bone strength is critically dependent on both phases. We report, for the first time, a Raman spectroscopic technique that analyses both phases of bone beneath unbroken skin by eliminating spectral components of overlying tissues.

Materials and Methods: We used an 800-nm laser (1-kHz, 1-ps pulses) with a synchronized 4-ps Kerr gate with variable picosecond delay that effectively shuttered out photons from overlying tissues. We measured bone Raman spectra at a point 2 mm above the carpus from two mouse genotypes with extreme differences in bone matrix quality: wildtype and oim/oim (matched for age, sex, and weight). Typical depth was 1.1 mm. We repeated the measurements with overlying tissues removed down to bone. Oim/oim mice produce only homotrimeric collagen, which results in poorly mineralized bone tissue.

Results: The main spectral features were present from both bone phases. The spectral bands were in similar ratios when measured through the skin or directly from bone (in both genotypes). The band of the mineral phase (phosphate ν1) was smaller in oim/oim mice when measured directly from bone and through skin. The band associated with a particular vibrational mode of organic phase collagen (CH2 wag) showed a frequency shift between the genotypes.

Conclusions: This novel technique allowed us, for the first time, to make objective transcutaneous spectral measurements of both the mineral and the organic phases of bones and distinguish between normal and unhealthy bone tissue. After further optimization, this technology may help improve fracture risk assessments and open opportunities for screening in anticipation of the predicted increase in fragility fractures.
Relationship Between Osteoporosis and Cardiovascular Disease in Postmenopausal Women
Authors: Tankó László B , Christiansen Claus , Cox David A , Geiger Mary Jane , McNabb Michelle A , Cummings Steven R
Published: April 2006

In the placebo group of the MORE study, including 2576 postmenopausal women (mean age, 66.5 years), the authors describe a strong linear association between the severity grade of osteoporosis (from low BMD to presence of severe vertebral fractures) and the future risk of cardiovascular events. Accordingly, treatment of postmenopausal osteoporosis should include consideration of measures to prevent adverse cardiovascular outcomes.

Introduction: Observations indicate an inverse association between BMD and the severity of peripheral atherosclerosis in postmenopausal women. The predictive value of osteoporosis and its different severity stages for the risk of acute cardiovascular events remains unknown.

Materials and Methods: Participants were 2576 women (mean age, 66.5 years) assigned to placebo and followed for 4 years in an osteoporosis treatment trial. Those with at least one vertebral fracture or total hip BMD T score ≤ −2.5 at baseline were defined as having osteoporosis, whereas those without vertebral fracture and total hip BMD T score between −2.5 and −1 were defined as having low bone mass. The primary outcome for these posthoc analyses was the incidence of adjudicated fatal or nonfatal cardiovascular events.

Results: After adjustment for potential confounders, women with osteoporosis had a 3.9-fold (95% CI, 2.0–7.7; p < 0.001) increased risk for cardiovascular events compared with women with low bone mass. Under the same boundaries, a total hip BMD T score ≤ −2.5 versus a T score between −2.5 and −1 was associated with a 2.1-fold (95% CI, 1.2–3.6; p < 0.01) increase in risk, whereas presence of at least one vertebral fracture versus no vertebral fracture at baseline was associated with a 3.0-fold (95% CI, 1.8–5.1; p < 0.001) increase in risk. The risk of cardiovascular events increased incrementally with the number and increasing severity of baseline vertebral fractures (both p < 0.001).

Conclusions: Postmenopausal women with osteoporosis are at an increased risk for cardiovascular events that is proportional to the severity of osteoporosis at the time of the diagnosis. Treatment of postmenopausal osteoporosis should include consideration of measures to prevent cardiovascular outcomes.
Skeletal Effects of Estrogen Are Mediated by Opposing Actions of Classical and Nonclassical Estrogen Receptor Pathways
Authors: Syed Farhan A , Mödder Ulrike IL , Fraser Daniel G , Spelsberg Thomas C , Rosen Clifford J , Krust Andree , Chambon Pierre , Jameson J Larry , Khosla Sundeep
Published: April 2006

ERα acts either through classical (ERE-mediated) or nonclassical (non-ERE) pathways. The generation of mice carrying a mutation that eliminates classical ERα signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice.

Introduction: Estrogen receptor α (ERα) can act either through classical estrogen response elements (EREs) or through non-ERE (nonclassical) pathways. To unravel these in bone, we crossed mice heterozygous for a knock-in mutation abolishing ERE binding (nonclassical ERα knock-in [NERKI]) with heterozygote ERα knockout mice and studied the resulting female ERα+/+, ERα+/NERKI, and ERα−/NERKI mice. The only ERα present in ERα−/NERKI mice is incapable of activating EREs but can signal through nonclassical pathways, whereas ERα+/NERKI mice may have a less drastic alteration in the balance between classical and nonclassical estrogen signaling pathways.

Materials and Methods: BMD was measured using DXA and pQCT at 3 months of age (n = 46–48/genotype). The mice were randomly assigned to sham surgery, ovariectomy, ovariectomy + estradiol (0.25 μg/day), or ovariectomy + estradiol (1.0 μg/day; n = 10–12/group) and restudied 60 days later.

Results and Conclusions: At 3 months of age, both the ERα+/NERKI and ERα−/NERKI mice had deficits in cortical, but not in trabecular, bone. Remarkably, changes in cortical bone after ovariectomy and estrogen replacement in ERα−/NERKI mice were the opposite of those in ERα+/+ mice. Relative to sham mice, ovariectomized ERα−/NERKI mice gained more bone (not less, as in ERα+/+ mice), and estrogen suppressed this increase (whereas augmenting it in ERα+/+ mice). Estrogen also had opposite effects on bone formation and resorption parameters on endocortical surfaces in ERα−/NERKI versus ERα+/+ mice. Collectively, these data show that alteration of the balance between classical and nonclassical ERα signaling pathways leads to deficits in cortical bone and also represent the first demonstration, in any tissue, that complete loss of classical ERE signaling can lead to paradoxical responses to estrogen. Our findings strongly support the hypothesis that there exists a balance between classical and nonclassical ERα signaling pathways, which, when altered, can result in a markedly aberrant response to estrogen.
Combination Teriparatide and Raloxifene Therapy for Postmenopausal Osteoporosis: Results From a 6-Month Double-Blind Placebo-Controlled Trial
Authors: Deal Chad , Omizo Molly , Schwartz Elliott N , Eriksen Erik F , Cantor Per , Wang Jingyuan , Glass Emmett V , Myers Stephen L , Krege John H
Published: April 2006

We compared combination treatment with teriparatide plus raloxifene with teriparatide alone in women with postmenopausal osteoporosis in a 6-month double-blind, placebo-controlled trial that measured biochemical markers of bone turnover and BMD. Markers of bone formation and spine BMD increased similarly with teriparatide alone and combination therapy. However, combination therapy induced a significantly smaller increase in bone resorption versus teriparatide alone and significantly increased total hip BMD versus baseline.

Introduction: The effects of combining two approved treatments for osteoporosis with different modes of action were examined by comparing teriparatide [rhPTH(1–34)] monotherapy with combination teriparatide and raloxifene therapy.

Materials and Methods: A 6-month randomized, double-blind trial comparing teriparatide plus raloxifene (n = 69) versus teriparatide plus placebo (n = 68) was conducted in postmenopausal women with osteoporosis.

Results: Bone formation (N-terminal propeptide of type 1 collagen [PINP]) increased similarly in both treatment groups. However, the increase in bone resorption (serum C-terminal telopeptide of type I collagen [CTx]) in the combination group was significantly smaller than in the teriparatide-alone group (p = 0.015). Lumbar spine BMD significantly increased 5.19 ± 0.67% from baseline in the teriparatide-alone group. In the combination group, lumbar spine (6.19 ± 0.65%), femoral neck (2.23 ± 0.64%), and total hip (2.31 ± 0.56%) BMD significantly increased from baseline to study endpoint, and the increase in total hip BMD was significantly greater than in the teriparatide-alone group (p = 0.04). In the teriparatide-alone group, mean serum calcium levels increased from baseline to endpoint (0.30 ± 0.06 mg/dl, p < 0.001), whereas mean serum phosphate remained unchanged. In the combination group, mean serum calcium was unchanged, and mean serum phosphate decreased (−0.20 ± 0.06 mg/dl, p < 0.001) from baseline to endpoint. Changes in serum calcium (p < 0.001) and phosphate (p < 0.004) were significantly different between treatment groups. The safety profile of combination therapy was similar to teriparatide alone.

Conclusions: Combination therapy increased bone formation to a similar degree as teriparatide alone. However, the increase in bone resorption was significantly less and total hip BMD significantly increased for combination therapy compared with teriparatide alone. Combination treatment with raloxifene may thus enhance the bone forming effects of teriparatide. Further studies over longer treatment duration that include fracture endpoints are necessary to fully ascertain the clinical significance of combination raloxifene plus teriparatide therapy in postmenopausal osteoporosis.
Polymorphisms of the CLCN7 Gene Are Associated With BMD in Women
Authors: Pettersson Ulrika , Albagha Omar ME , Mirolo Max , Taranta Anna , Frattini Annalisa , McGuigan Fiona EA , Vezzoni Paolo , Teti Anna , Van Hul Wim , Reid David M , Villa Anna , Ralston Stuart H
Published: April 2006

Here we show that a common polymorphism causing a valine to methionine amino acid substitution at codon 418 (V418M) in the CLCN7 gene is associated with femoral neck BMD in women. Our study adds to accumulating evidence that shows that common allelic variants in monogenic bone disease genes often contribute to BMD regulation in normal subjects.

Introduction: The CLCN7 gene is a strong candidate for regulation of BMD, because mutations in CLCN7 cause some forms of osteopetrosis, a disease characterized by impaired osteoclast function and increased BMD. In this study, we sought to determine whether common allelic variation within CLCN7 was associated with BMD in the normal population.

Materials and Methods: We conducted mutation screening of the exons and intron-exon boundaries in CLCN7 by DNA sequencing in 50 normal subjects. We conducted an association study between common polymorphisms in CLCN7 and haplotypes defined by these polymorphisms and BMD values at the lumbar spine and femoral neck in a population-based cohort study of 1077 Scottish women 45–55 years of age.

Results: We identified 24 polymorphisms, but most were rare and only 4 had allele frequencies of >5%. These were a conservative single nucleotide polymorphism (SNP) in exon 1 (rs3751884), a 50-bp tandem repeat polymorphism within intron 8, and two SNPs within exon 15 (rs12926089 and rs12926669), of which one (rs12926669) predicts an amino acid change from valine to methionine at codon 418 (V418M). The exon 15 SNPs were in strong linkage disequilibrium and were both associated with femoral neck BMD (p = 0.001–0.003). None of the other polymorphisms were associated with BMD, and long-range haplotypes showed a much weaker association with BMD than the exon 15 SNPs. The V418M polymorphism was an independent predictor of femoral neck BMD on multiple regression analysis accounting for 1% of the variance in BMD at this site.

Conclusions: Our study indicates that the V418M polymorphism of CLCN7 contributes to the genetic regulation of femoral neck BMD in women and adds to accumulating evidence that indicates that subtle polymorphic variation in genes that cause monogenic bone diseases also contribute to regulation of BMD in normal subjects.
Fibroblast Growth Factor-23 Is Regulated by 1α,25-Dihydroxyvitamin D
Authors: Collins Michael T , Lindsay John R , Jain Alka , Kelly Marilyn H , Cutler Carolee M , Weinstein Lee S , Liu Jie , Fedarko Neal S , Winer Karen K
Published: April 2006

Serum FGF-23 regulation was studied in patients with hypoparathyroidism or pseudohypoparathyroidism treated with calcitriol. Serum FGF-23 levels changed in parallel in response to changes in serum 1,25-D, suggesting that FGF-23 may be regulated by 1,25-D. In addition, the phosphaturic effect of FGF-23 may be diminished in the absence of PTH action on the kidney.

Introduction: Fibroblast growth factor (FGF)-23 is a recently described hormone that has been shown to be involved in the regulation of phosphate and vitamin D metabolism. The physiologic role of FGF-23 in mineral metabolism and how serum FGF-23 levels are regulated have yet to be elucidated. Three patients with mineral metabolism defects that allowed for the investigation of the regulation of FGF-23 were studied.

Materials and Methods: Patient 1 had postsurgical hypoparathyroidism and Munchausen's syndrome and consumed a pharmacologic dose of calcitriol. Patient 2 had postsurgical hypoparathyroidism and fibrous dysplasia of bone. She was treated with increasing doses of calcitriol followed by synthetic PTH(1–34). Patient 3 had pseudohypoparathyroidism type 1B and tertiary hyperparathyroidism. She underwent parathyroidectomy, which was followed by the development of hungry bone syndrome and hypocalcemia, requiring treatment with calcitriol. Serum FGF-23 and serum and urine levels of mineral metabolites were measured in all three patients.

Results: Patient 1 had an acute and marked increase in serum FGF-23 (70 to 670 RU/ml; normal range, 18–108 RU/ml) within 24 h in response to high-dose calcitriol administration. Patient 2 showed stepwise increases in serum FGF-23 from 117 to 824 RU/ml in response to increasing serum levels of 1α,25-dihydroxyvitamin D (1,25-D). Finally, before parathyroidectomy, while hypercalcemic, euphosphatemic, with low levels of 1,25-D (10 pg/ml; normal range, 22–67 pg/ml), and with very high serum PTH (863.7 pg/ml; normal range, 6.0–40.0 pg/ml), patient 3 had high serum FGF-23 levels (217 RU/ml). After surgery, while hypocalcemic, euphosphatemic, and with high serum levels of serum 1,25-D (140 pg/ml), FGF-23 levels were higher than preoperative levels (305 RU/ml). It seemed that the phosphaturic effect of FGF-23 was diminished in the absence of PTH or a PTH effect.

Conclusions: Serum FGF-23 may be regulated by serum 1,25-D, and its phosphaturic effect may be less in the absence of PTH.
A New Selective Estrogen Receptor Modulator, CHF 4227.01, Preserves Bone Mass and Microarchitecture in Ovariectomized Rats
Authors: Armamento-Villareal Reina , Sheikh Sharmin , Nawaz Abroo , Napoli Nicola , Mueller Cheryl , Halstead Linda R , Brodt Michael D , Silva Matthew J , Galbiati Elisabetta , Caruso Paola Lorenza , Civelli Maurizio , Civitelli Roberto
Published: April 2006

A new SERM, CHF 4227.01, given to 6-month-old female rats immediately after ovariectomy, preserved bone mass and bone microarchitecture without affecting uterus weight. It also decreased serum cholesterol and fat mass in estrogen-deficient rats.

Introduction: We tested the effect of a new benzopyran derivative, CHF 4227.01, with selective estrogen receptor modulator (SERM) activity on bone mass and biomechanics in ovariectomized (OVX) female rats in comparison with 17α-ethinylestradiol (EST), raloxifene (RLX), and lasofoxifene (LFX).

Materials and Methods: Four doses of CHF 4227.01 (0.001, 0.01, 0.1, and 1 mg/kg body weight [bw]/day) were administered in OVX animals daily by gavage 5 days/week for 4 months. EST was administered at a dose of 0.1 mg/kg bw/day, whereas RLX and LSX were administered at doses of 1 and 0.1 mg/kg bw/day, respectively, by gavage. In one group (Sham), rats were operated but the ovaries not removed; another OVX group was treated only with placebo.

Results and Conclusions: Treatment with CHF 4227.01 (1.0 and 0.1 mg/kg bw), EST (0.1 mg/kg bw), LFX (0.1 mg/kg bw), or RLX (1.0 mg/kg bw) prevented bone loss on the lumbar spine and the proximal femur assessed in vivo by DXA. Volumetric BMD obtained by pQCT ex vivo confirmed protection from bone loss in the spine and proximal femur among rats treated with CHF 4227.01. This effect was associated with strong inhibition of bone resorption both histologically and biochemically. Furthermore, CHF 4227.01 preserved trabecular microarchitecture, analyzed by μCT, and maintained biomechanical indices of bone strength in the spine and proximal femur, effects also observed for RLX, whereas LSX was less protective of microarchitecture. CHF 4227.01 treatment did not affect uterine weight, prevented the increase in body weight and fat mass seen in OVX animals, and decreased serum cholesterol to below the average of intact animals. In conclusion, CHF 4227.01 exhibits a promising therapeutic and safety profile as a new SERM on both skeletal and extraskeletal outcomes.
Influence of BMPs on the Formation of Osteoblastic Lesions in Metastatic Prostate Cancer
Authors: Feeley Brian T , Gamradt Seth C , Hsu Wellington K , Liu Nancy , Krenek Lucie , Robbins Paul , Huard Johnny , Lieberman Jay R
Published: April 2006

The purpose of this study was to evaluate the role of BMPs on the formation of metastatic prostate cancer lesions to bone. Our results show that BMPs influence the development and progression of osteoblastic lesions and suggest that therapies that inhibit BMP activity may reduce the formation and progression of osteoblastic lesions.

Introduction: Prostate adenocarcinoma is the leading cause of cancer in North American men. The formation of skeletal metastases affects ∼70% of patients with advanced disease, and a majority of these patients have osteoblastic lesions. Although BMPs have been found to be expressed in multiple oncogenic cell lines, their role in the formation of metastatic osteoblastic lesions remains uncharacterized. We hypothesized that BMPs influence the development of metastatic osteoblastic lesions associated with prostate cancer.

Materials and Methods: Western blot analysis and RT-PCR was used to determine BMP receptor expression on osteoblastic prostate cancer cell lines LAPC-4 and LAPC-9. Migration, invasion, and cellular proliferation assays were used to quantify the effects of BMP-2, −4, and −7 on LAPC-4 cells in vitro. LAPC-9 cells alone or transfected with a retrovirus overexpressing noggin were injected into the tibias of SCID mice, and the animals were followed for 8 weeks. Tumor size was determined by radiographs and direct measurement. Histology was performed at the time of death.

Results: We determined that BMP receptor mRNA and protein was expressed on osteoblastic prostate cancer cell lines LAPC-4 and LAPC-9. In vitro studies showed that BMP-2 and −7 stimulated cellular migration and invasion of prostate cancer cells in a dose-dependent fashion, although BMP-4 had no effect. Noggin inhibited cellular migration and invasion of BMP-2- and −7-stimulated LAPC-4 cells. LAPC-9 cells implanted into immunodeficient mouse tibias formed an osteoblastic lesion with sclerotic bone at 8 weeks. Formation of osteoblastic lesions was inhibited by overexpression of noggin by prostate cancer cells transduced with a retrovirus containing the cDNA for noggin.

Conclusions: BMPs are critical in the formation of the osteoblastic lesions associated with prostate cancer metastases, and future treatment strategies that inhibit local BMP activity may reduce the formation and progression of osteoblastic lesions.
Periosteal Progenitor Cell Fate in Segmental Cortical Bone Graft Transplantations: Implications for Functional Tissue Engineering
Authors: Zhang Xinping , Xie Chao , Lin Angela SP , Ito Hiromu , Awad Hani , Lieberman Jay R , Rubery Paul T , Schwarz Edward M , O'Keefe Regis J , Guldberg Robert E
Published: April 2006

A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that ∼70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP-2-producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene-enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair.

Introduction: The loss of cellular activity in a structural bone allograft markedly reduces its healing potential compared with a live autograft. To further understand the cellular mechanisms for structural bone graft healing and repair and to devise a therapeutic strategy aimed at enhancing the performance of allograft, we established a segmental femoral structural bone graft model in mice that permits qualitative and quantitative analyses of graft healing and neovascularization.

Materials and Methods: Using this segmental femoral bone graft model, we transplanted live isografts harvested from Rosa 26A mice that constitutively express β-galactosidase into their wildtype control mice. In an attempt to emulate the osteogenic and angiogenic properties of periosteum, we applied a cell-based, adenovirus-mediated gene therapy approach to engraft BMP-2-producing bone marrow stromal cells onto devitalized allografts.

Results: X-gal staining for donor cells allowed monitoring the progression of periosteal progenitor cell fate and showed that 70% of osteogenesis was attributed to cellular proliferation and differentiation of donor progenitor cells on the surface of the live bone graft. Quantitative μCT analyses showed a 3-fold increase in new bone callus formation and a 6.8-fold increase in neovascularization for BMP-2/stromal cell-treated allograft compared with control acellular allografts. Histologic analyses showed the key features of autograft healing in the BMP-2/stromal cell-treated allografts, including the formation of a mineralized bone callus completely bridging the segmental defects, abundant neovascularization, and extensive resorption of bone graft.

Conclusions: The marked improvement of healing in these cellularized allografts suggests a clinical strategy for engineering a functional periosteum to improve the osteogenic and angiogenic properties of processed allografts.