bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Liver-Derived Insulin-Like Growth Factor I on Bone Metabolism in Mice
Authors: Sjögren Klara , Sheng Matilda , Movérare Sofia , Liu Jun-Li , Wallenius Kristina , Törnell Jan , Isaksson Olle , Jansson John-Olov , Mohan Subburaman , Ohlsson Claes
Published: April 2006

Insulin-like growth factor (IGF) I is an important regulator of both skeletal growth and adult bone metabolism. To better understand the relative importance of systemic IGF-I versus locally expressed IGF-I we have developed a transgenic mouse model with inducible specific IGF-I gene inactivation in the liver (LI-IGF-I−/−). These mice are growing normally up to 12 weeks of age but have a disturbed carbohydrate and lipid metabolism. In this study, the long-term effects of liver-specific IGF-I inactivation on skeletal growth and adult bone metabolism were investigated. The adult (week 8–55) axial skeletal growth was decreased by 24% in the LI-IGF-I−/− mice whereas no major reduction of the adult appendicular skeletal growth was seen. The cortical cross-sectional bone area, as measured in the middiaphyseal region of the long bones, was decreased in old LI-IGF-I−/− mice. This reduction in the amount of cortical bone was caused mainly by decreased periosteal circumference and was associated with a weaker bone determined by a decrease in ultimate load. In contrast, the amount of trabecular bone was not decreased in the LI-IGF-I−/− mice. DNA microarray analysis of 30-week-old LI-IGF-I−/− and control mice indicated that only four genes were regulated in bone whereas ∼40 genes were regulated in the liver, supporting the hypothesis that liver-derived IGF-I is of minor importance for adult bone metabolism. In summary, liver-derived IGF-I exerts a small but significant effect on cortical periosteal bone growth and on adult axial skeletal growth while it is not required for the maintenance of the trabecular bone in adult mice.
Role of the Cholesterol Biosynthetic Pathway in Osteoblastic Differentiation of Marrow Stromal Cells
Authors: Parhami Farhad , Mody Nilam , Gharavi Nima , Ballard Alex J. , Tintut Yin , Demer Linda L.
Published: April 2006

Cholesterol is an important molecule that plays a key role in regulating cellular differentiation and function. Although the possible role of lipids has been implicated in regulating osteoblastic cells, the role of cholesterol in that process is not well defined. In this study we have examined the role of the cellular cholesterol biosynthetic pathway on osteoblastic differentiation of marrow stromal cells (MSCs). Treatment of pluripotent mouse MSCs M2–10B4 with inhibitors of the cholesterol biosynthetic pathway mevastatin or mevinolin inhibited the maturation of these cells into functional osteoblastic cells. This was determined by the inhibition of the activity and expression of alkaline phosphatase (ALP), a key enzyme involved in differentiation and mineralization of osteoblastic cell cultures, as well as inhibition of mineralization. Mevastatin treatment did not affect expression of the osteoblast-specific gene osteocalcin (OCN). Furthermore, promoter-reporter studies in MSCs showed that mevastatin inhibited activity of the ALP gene promoter, suggesting regulation by derivatives of the cholesterol biosynthetic pathway. The effects of mevastatin and mevinolin were reversed by mevalonate but not by geranylgeraniol or farnesol, intermediates in the cholesterol biosynthetic pathway. Altogether, these results suggest that products of the cholesterol biosynthetic pathway are important for proper development of MSCs into functional osteoblastic cells capable of forming a mineralized matrix. Identification of those molecules may provide new therapeutic approaches to prevent the decline in osteoblastic activity in osteoporosis and aging.
Simvastatin Improves Fracture Healing in Mice
Authors: Skoglund Björn , Forslund Carina , Aspenberg Per
Published: April 2006

Recently, several articles have been published dealing with the anabolic effects on bone by statins. Mundy and associates discovered that several statins were able to activate the promotor of bone morphogenetic protein (BMP) 2. Additionally, oral simvastatin and lovastatin increased the cancellous bone volume in rats, presumably an effect of the increase of BMP-2. Other studies have followed, with conflicting results; some have found a positive bone metabolic effect of statins and others have not. Studies published so far have focused on osteoporosis. In this study, femur fractures were produced in 81 mature male BALB/c mice and stabilized with marrow-nailing. Forty-one mice were given a diet prepared with simvastatin, so that each mouse received an approximate dose of 120 mg/kg of body weight per day. The remaining mice received the same diet with the exception of the simvastatin. Bilateral femurs were harvested at 8, 14, and 21 days postoperatively (po), the marrow-nail was extracted, and diameters were measured. Biomechanical tests were performed on 42 mice, by way of three-point bending. Histological specimens were prepared using standard techniques. For statistical analysis, ANOVA with Scheffé's post hoc test was used. At 8 days, the fracture callus was too soft for meaningful biomechanical testing. At 14 days, the callus of the simvastatin-treated mice had a 53% larger transverse area than controls (p = 0.001), the force required to break the bone was 63% greater (p = 0.001), and the energy uptake was increased by 150% (p = 0.0008). Stiffness and modulus of elasticity were not significantly affected. At 21 days, the fractures were histologically healed and the mechanical differences had disappeared. The contralateral unbroken bone showed a slight increase in transverse area because of the simvastatin treatment, but there was no significant effect on the force required to break the bone or on energy uptake. These results point to a new possibility in the treatment of fractures.
Mechanisms for the Enhancement of Fracture Healing in Rats Treated With Intermittent Low-Dose Human Parathyroid Hormone (1–34)
Authors: Nakajima Arata , Shimoji Naoshi , Shiomi Koji , Shimizu Sumito , Moriya Hideshige , Einhorn Thomas A. , Yamazaki Masashi
Published: April 2006

Recent reports have demonstrated that intermittent treatment with parathyroid hormone (1–34) [PTH(1–34)] increases callus formation and mechanical strength in experimental fracture healing. However, little is known about the optimal dose required for enhancement of fracture repair or the molecular mechanisms by which PTH regulates the healing process. In this study, we analyzed the underlying molecular mechanisms by which PTH affects fracture healing and tested the hypothesis that intermittent low-dose treatment with human PTH(1–34) can increase callus formation and mechanical strength. Unilateral femoral fractures were produced and a daily subcutaneous injection of 10 μg/kg of PTH(1–34) was administered during the entire healing period. Control animals were injected with vehicle solution alone. The results showed that on day 28 and day 42 after fracture, bone mineral content (BMC), bone mineral density (BMD), and ultimate load to failure of the calluses were significantly increased in the PTH-treated group compared with controls (day 28, 61, 46, and 32%; day 42, 119, 74, and 55%, respectively). The number of proliferating cell nuclear antigen (PCNA)-positive subperiosteal osteoprogenitor cells was significantly increased in the calluses of the PTH-treated group on day 2, and TRAP+ multinucleated cells were significantly increased in areas of callus cancellous bone on day 7. The levels of expression of type I collagen (COL1A1), osteonectin (ON), ALP, and osteocalcin (OC) mRNA were increased markedly in the PTH-treated group and accompanied by enhanced expression of insulin-like growth factor (IGF)-I mRNA during the early stages of healing (days 4–7). The increased expression of COL1A1, ON, ALP, and OC mRNA continued during the later stages of healing (days 14–21) despite a lack of up-regulation of IGF-I mRNA. These results suggest that treatment of fractures with intermittent low dose PTH(1–34) enhances callus formation by the early stimulation of proliferation and differentiation of osteoprogenitor cells, increases production of bone matrix proteins, and enhances osteoclastogenesis during the phase of callus remodeling. The resultant effect to increase callus mechanical strength supports the concept that clinical investigations on the ability of injectable low-dose PTH(1–34) to enhance fracture healing are indicated.
Stimulation of Smad1 Transcriptional Activity by Ras-Extracellular Signal-Regulated Kinase Pathway: A Possible Mechanism for Collagen-Dependent Osteoblastic Differentiation
Authors: Suzawa Miyuki , Tamura Yasuhiro , Fukumoto Seiji , Miyazono Kohei , Fujita Toshiro , Kato Shigeaki , Takeuchi YASUHIRO
Published: April 2006

Signals from bone morphogenetic protein receptors (BMPRs) and cell adhesion to type I collagen are both important for osteoblastic differentiation and functions. BMP signals are mediated mostly by Smad and collagen signals are transduced by integrins to activate focal adhesion kinase (FAK) and its downstream molecules. This study was undertaken to clarify how extracellular matrix collagen signals converge with BMP actions. We show that integrin activation by collagen was involved in BMP signals because disruption of either collagen synthesis or collagen-α2β1-integrin binding inhibited the stimulatory effect of BMP-2 on osteoblastic MC3T3-E1 cells. Downstream signals of collagen-integrin might be FAK-Ras-extracellular signal-regulated kinase (ERK) in osteoblastic cells. We further show that Ras-ERK signals enhanced the transcriptional activity of Smad1 in response to BMP in these cells transiently transfected with expression plasmids for a constitutively active mutant RasV12, a dominant negative mutant RasN17, and an ERK phosphatase CL100. Ras-ERK signals did not augment the transcriptional activity of Smad3 in response to transforming growth factor β (TGF-β) receptor activation but that of Smad1 in response to BMPR activation as examined in COS-1 cells. These observations suggest that the Ras-ERK pathway downstream of integrin-FAK is involved in Smad1 signals activated by BMP and provide a possible mechanism for cooperation between intracellular signals activated by integrin and BMPRs in osteoblastic cells.
Platelet-Derived Growth Factor BB Secreted From Osteoclasts Acts as an Osteoblastogenesis Inhibitory Factor
Authors: Kubota Kazuishi , Sakikawa Chisa , Katsumata Mutsumi , Nakamura Takemichi , Wakabayashi Kenji
Published: April 2006

Osteoclasts and osteoblasts are responsible for strict bone maintenance with a balance between bone formation and resorption by interacting with each other. Recently, it has been revealed that osteoblasts/stromal cells regulate differentiation of osteoclasts/hematopoietic cells by two factors, the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) on the plasma membrane, and secreted osteoprotegerin (OPG). However, no factors have yet been reported by which osteoclasts/hematopoietic cells regulate osteoblasts/stromal cells. To elucidate the possibility of signal transduction from osteoclasts to osteoblasts, we studied the conditioned medium of mouse osteoclast-like myeloma cell line RAW264.7 treated with RANKL. We found that this medium contains a factor that inhibits differentiation of mouse osteoblast precursor-like cell line MC3T3-E1 to osteoblasts induced by bone morphogenetic protein 4 (BMP-4) and named this factor osteoblastogenesis inhibitory factor (OBIF). OBIF was purified by successive three-step chromatography by heparin affinity, anion exchange, and reversed-phase columns. Osteoblastogenesis inhibitory activity made one peak in each chromatography step, showing the factor is a single entity. Active fractions were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bands of proteins were excised, digested by trypsin, and analyzed by liquid chromatography equipped with tandem mass spectrometry (LC/MS/MS). Consequently, we have identified this factor to be platelet-derived growth factor BB (PDGF BB) homodimer. Furthermore, this identification of PDGF BB as OBIF was confirmed by neutralization of the inhibitory activity of the medium with anti-PDGF antibody. These results show, for the first time, that osteoclasts regulate osteoblasts directly and suggest that PDGF BB is a key factor in bone remodeling.
The Assembly and Remodeling of the Extracellular Matrix in the Growth Plate in Relationship to Mineral Deposition and Cellular Hypertrophy: An In Situ Study of Collagens II and IX and Proteoglycan
Authors: Mwale Fackson , Tchetina Elena , Wu C. William , Poole A. Robin
Published: April 2006

The recent development of new specific immunoassays has provided an opportunity to study the assembly and resorption of type II and IX collagens of the extracellular matrix in relationship to endochondral calcification in situ. Here, we describe how in the bovine fetal physis prehypertrophic chondrocytes deposit an extensive extracellular matrix that, initially, is rich in both type II and type IX collagens and proteoglycan (PG; principally, aggrecan). The majority of the α1(IX)-chains lack the NC4 domain consistent with our previous studies with cultured chondrocytes. During assembly, the molar ratio of type II/COL2 domain of the α1(IX)-chain varied from 8:1 to 25:1. An increase in the content of Ca2+ and inorganic phosphate (Pi) was initiated in the prehypertrophic zone when the NC4 domain was removed selectively from the α1(IX)-chain. This was followed by the progressive loss of the α1(IX) COL2 domain and type II collagen. In the hypertrophic zone, the Ca2+/Pi molar ratio ranged from 1.56 to a maximum of 1.74, closely corresponding to that of mature hydroxyapatite (1.67). The prehypertrophic zone had an average ratio Ca2+/Pi ranging from 0.25 to 1, suggesting a phase transformation. At hypertrophy, when mineral content was maximal, type II collagen was reduced maximally in content coincident with a peak of cleavage of this molecule by collagenase when matrix metalloproteinase 13 (MMP-13) expression was maximal. In contrast, PG (principally aggrecan) was retained when hydroxyapatite was formed consistent with the view that this PG does not inhibit and might promote calcification in vivo. Taken together with earlier studies, these findings show that matrix remodeling after assembly is linked closely to initial changes in Ca2+ and Pi to subsequent cellular hypertrophy and mineralization. These changes involve a progressive and selective removal of types II and IX collagens with the retention of the PG aggrecan.
The Effects of Dynamic Axial Loading on the Rat Growth Plate
Authors: Ohashi Naoko , Robling Alexander G. , Burr David B. , Turner Charles H.
Published: April 2006

Longitudinal bone growth can be suppressed by compressive loading. In this study, we applied three different magnitudes (17, 8.5, and 4N) of compressive force to growing rat ulnas 10 minutes/day for 8 days and investigated the effects on the distal growth plate biology. Further, to investigate growth rate recovery after cessation of loading, we examined rats 7 days after the loading period. Longitudinal growth of the ulna was suppressed in a dose-dependent manner by applied compressive force. In the 17N group, the longitudinal mineralization rate (LMR) at the distal growth plate was suppressed completely by loading and did not recover. However, in the 8.5N and 4N groups, LMR suppression recovered in 1 week. In the 17N group, growth plate height and hypertrophic zone height were significantly greater than control; the number of hypertrophic chondrocytes was increased; and some traumatic changes such as cracks in the growth plate were found. In addition, 17N loading suppressed cartilage mineralization and capillary invasion beneath the growth plate, although the number of chondrocytes synthesizing vascular endothelial growth factor (VEGF) was increased. Our study shows longitudinal growth suppression caused by axial loading of the ulna, which is proportional to the magnitude of load. Only the largest load (17N) caused morphological changes in the distal growth plate cartilage. There was no association found between mineralization and type X collagen localization or capillary invasion and VEGF expression.
Discriminatory Ability of Quantitative Ultrasound Parameters and Bone Mineral Density in a Population-Based Sample of Postmenopausal Women With Vertebral Fractures: Results of the Basel Osteoporosis Study
Authors: Hartl F. , Tyndall A. , Kraenzlin M. , Bachmeier C. , Gückel C. , Senn U. , Hans D. , Theiler R.
Published: April 2006

The discriminatory potential to classify subjects with or without vertebral fractures was tested cross-sectionally with different methods for the measurement of bone status in a population-based sample of postmenopausal women. Quantitative ultrasound (QUS) measurement at the calcaneus (Lunar Achilles, Hologic Sahara), the proximal phalanges (Igea Bone Profiler), and measurement of bone mineral density (BMD) with dual-energy X-ray absorptiometry (DXA; Lunar Expert) at several anatomic sites was performed in 500 postmenopausal women (aged 65-75 years) randomly selected from the population. In addition, 50 young female subjects (20-40 years old) had QUS measurements and served as controls to express QUS results as T-score values. Radiographs of the lumbar and thoracic spine were performed in the elderly women. Two independent radiologists reviewed the X-rays for the presence of vertebral fractures. Of 486 eligible study participants, no fracture was seen in 396 participants. Single vertebral fractures were observed in 71 subjects; 19 individuals presented multiple fractures. The overall prevalence of vertebral fractures was 18.5%. Participants without vertebral fractures were compared with subjects with vertebral fractures. Normal statistical distributions were found for all bone measurement results. Risk of vertebral fracture in subjects with no and multiple vertebral fracture was estimated using age adjusted odds ratios (ORs) for QUS and dual-energy X-ray absorptiometry (DXA) values. Each SD decrease in bone measurement increased the risk of multiple vertebral fracture by 3.0 (95% CI, 1.6-5.6) for the Achilles stiffness, by 3.8 (95% CI, 1.8-8.2) for the Sahara QUI, 2.1 (95% CI, 1.3-3.4) for the Bone Profiler amplitude-dependent speed of sound (AD-SOS), and 2.1 (95% CI, 1.2-3.9) and 2.4 (95% CI, 1.3-4.3) for DXA lumbar spine and for DXA total hip, respectively. Results of a discriminant analysis showed sensitivities between 84% and 58% and specificities between 72% and 58% for the respective DXA and QUS parameters. Optimum fracture thresholds for QUS measurements derived from this analysis were calculated also. Optimum T-score threshold values for QUS measurements tended to be higher than those for DXA measurements. However, the performance of QUS measurements is at least comparable with DXA measurements in identifying subjects with multiple vertebral fractures randomly selected from the population.
A Randomized School-Based Jumping Intervention Confers Site and Maturity-Specific Benefits on Bone Structural Properties in Girls: A Hip Structural Analysis Study
Authors: Petit M. A. , Mckay H. A. , Mackelvie K. J. , Heinonen A. , Khan K. M. , Beck T. J.
Published: April 2006

We compared 7-month changes in bone structural properties in pre- and early-pubertal girls randomized to exercise intervention (10-minute, 3 times per week, jumping program) or control groups. Girls were classified as prepubertal (PRE; Tanner breast stage 1; n = 43 for intervention [I] and n = 25 for control [C]) or early-pubertal (EARLY; Tanner stages 2 and 3; n = 43 for I and n = 63 for C). Mean ± SD age was 10.0 ± 0.6 and 10.5 ± 0.6 for the PRE and EARLY groups, respectively. Proximal femur scans were analyzed using a hip structural analysis (HSA) program to assess bone mineral density (BMD), subperiosteal width, and cross-sectional area and to estimate cortical thickness, endosteal diameter, and section modulus at the femoral neck (FN), intertrochanter (IT), and femoral shaft (FS) regions. There were no differences between intervention and control groups for baseline height, weight, calcium intake, or physical activity or for change over 7 months (p > 0.05). We used analysis of covariance (ANCOVA) to examine group differences in changes of bone structure, adjusting for baseline weight, height change, Tanner breast stage, and physical activity. There were no differences in change for bone structure in the PRE girls. The more mature girls (EARLY) in the intervention group showed significantly greater gains in FN (+2.6%, p = 0.03) and IT (+1.7%, p = 0.02) BMD. Underpinning these changes were increased bone cross-sectional area and reduced endosteal expansion. Changes in subperiosteal dimensions did not differ. Structural changes improved section modulus (bending strength) at the FN (+4.0%, p = 0.04), but not at the IT region. There were no differences at the primarily cortical FS. These data provide insight into geometric changes that underpin exercise-associated gain in bone strength in early-pubertal girls.
The Secreted Protein Thrombospondin 2 Is an Autocrine Inhibitor of Marrow Stromal Cell Proliferation
Authors: Hankenson Kurt D. , Bornstein Paul
Published: April 2006

Marrow stromal cells (MSCs) are obtained in increased number from mice in which the thrombospondin 2 (TSP2) gene is disrupted, and these cells show increased DNA synthesis in vitro. To examine more closely the role of TSP2 in the physiology and osteogenic differentiation of MSCs, an in-depth characterization of TSP2-null MSCs was conducted. Determination of TSP2 protein content by Western analysis and RNA levels by reverse-transcription polymerase chain reaction (RT-PCR) indicated that MSCs are the primary source of TSP2 in the marrow and secrete abundant TSP2 into culture medium. Morphologically, the TSP2-null and wild-type (WT) cell populations were similar and by flow cytometry contained equivalent numbers of CD44+, Mac1+, intercellular adhesion molecule-1 (ICAM-1+), and ScaI+ cells. TSP2-null cells showed delayed mineralization associated with an increased rate of proliferation. Consistent with this finding, there was a decrease in expression of collagen and osteocalcin RNA by TSP2-null MSCs on day 7 and increased osteopontin expression on day 7 and day 14. In add-back experiments, recombinant TSP2 produced a dose-dependent decrease in proliferation. This reduction was associated with an accumulation of TSP2-treated cells in the G1 phase of the cell cycle and did not result from an increase in apoptosis. When TSP2 treatment was terminated, the cell population reentered the S phase. We conclude that the increased endosteal bone formation observed in TSP2-null mice results primarily from the failure of TSP2 to regulate locally MSC cell cycle progression.
Thyroid Hormones Promote Chondrocyte Differentiation in Mouse ATDC5 Cells and Stimulate Endochondral Ossification in Fetal Mouse Tibias Through Iodothyronine Deiodinases in the Growth Plate
Authors: Miura Masako , Tanaka Kiyoshi , Komatsu Yasato , Suda Michio , Yasoda Akihiro , Sakuma Yoko , Ozasa Ami , Nakao Kazuwa
Published: April 2006

Thyroid hormones (THs), 3,3′,5-triiodo-L-thyronine (T3) and L-thyroxine (T4), are important for the normal development of the growth plate (GP); congenital TH deficiency leads to severe dwarfism. In mouse chondrogenic cell line, ATDC5, T3 enhanced differentiation and increased Alizarin red staining, but did not affect Alcian blue staining. In organ-cultured mouse tibias, THs stimulated the cartilage growth, especially in the hypertrophic zone. Interestingly, T4 was as equally potent as T3 in organ-cultured tibias, which suggests that T4 is metabolized locally to T3, because T4 is a prohormone and must be converted to T3 for its activity. Two enzymes catalyze the conversion; type I deiodinase (D1) and type II deiodinase (D2). D1 has a ubiquitous distribution and D2, with a high affinity for T4, is present where the maintenance of intracellular T3 concentration is critical. Messenger RNAs (mRNAs) for D1 and D2 were detected in neonatal mouse tibias and ATDC5 cells. The enzyme activity was unaffected by the D1 inhibitor 6-propyl-2-thiouracil, suggesting that D2 mainly catalyzes the reaction. D2 mRNA was detected in differentiated ATDC5 cells. In organ-cultured mouse tibias, D2 activity was greater at later stages. In contrast, thyroid hormone receptors (TRs) were expressed in neonatal mouse tibias and ATDC5 cells, but their expression levels in ATDC5 cells were stable throughout the culture periods. Therefore, increased T3 production at later stages by D2 is likely to contribute to the preferential effects of THs in the terminal differentiation of GP. This article is the first to show that T4 is activated locally in GP and enhances the understanding of TH effects in GP.
Regulation of Fibroblast Growth Factor 2 and Fibroblast Growth Factor Receptors by Transforming Growth Factor β in Human Osteoblastic MG-63 Cells
Authors: Sobue T. , Gravely T. , Hand A. , Min Y. K. , Pilbeam C. , Raisz L. G. , Zhang X. , Larocca D. , Florkiewicz R. , Hurley M. M.
Published: April 2006

Fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) are important regulators of bone cell function. Although FGF-2 is a major modulator of bone cell function, its expression and regulation in human osteoblasts have not been investigated. We examined FGF-2 messenger RNA (mRNA) expression and regulation in the human osteosarcoma MG-63 cells. Northern analysis revealed that MG-63 cells expressed FGF-2 mRNA transcripts of 7, 4, 2.2, and 1.3 kilobases (kb). In the absence of serum, treatment with transforming growth factor beta (TGF-β; 0.1-10 ng/ml) increased all FGF-2 mRNA transcripts. Maximal increase was seen with 1 ng/ml of TGF-β. TGF-β increased FGF-2 mRNA expression within 2 h and this was sustained for 24 h. Phorbal myristate acetate (PMA; 1 μM) also increased FGF-2 mRNA at 6 h. Time course studies showed that TGF-β did not significantly alter FGFR1 or FGFR2 mRNA expression in MG-63 cells. Western blotting with anti-human FGF-2 revealed that MG-63 cells synthesize three isoforms of FGF-2 protein of ∼18, 22/23, and 24 kDa, which were increased after either 6 h or 24 h of treatment with TGF-β. Increased FGF-2 mRNA and protein expression in response to TGF-β was markedly reduced by the protein kinase A (PKA) inhibitor H-89. Immunogold labeling of MG-63 cells treated with TGF-β showed increased labeling for FGF-2 and FGFR2 in the nuclei. In contrast, TGF-β treatment significantly decreased FGFR1 labeling in the nuclei. These data show that TGF-β regulates FGF-2 gene expression in human osteosarcoma cells. Furthermore, TGF-β modulates the cellular localization of FGF-2 and its receptors.
A Randomized Trial of Nasal Spray Salmon Calcitonin in Men With Idiopathic Osteoporosis: Effects on Bone Mineral Density and Bone Markers
Authors: Dr. Trovas G. P. , Lyritis G. P. , Galanos A. , Raptou P. , Constantelou E.
Published: April 2006

In a 12-month randomized, double-blind, placebo-controlled trial, we have studied the effects of intranasal salmon calcitonin (SCT) on bone mineral density (BMD) and biochemical markers of bone turnover. Twenty-eight men with idiopathic osteoporosis aged 27-74 years (mean, 52.4 years) were randomized to receive either nasal SCT (200 IU) or a nasal placebo daily for a period of 1 year. All the men received a daily supplement of 0.5 g of calcium. The men who received SCT had a mean (±SEM) increase in BMD of 7.1 ± 1.7% at the lumbar spine. In contrast, the men who received the placebo had an increase of 2.4 ± 1.5% (p > 0.05) for the comparison with baseline. The increase in lumbar BMD in the calcitonin group was significantly greater than that in the placebo group (p < 0.05). There were no significant changes in the femoral neck, trochanter, or Ward's triangle relative to both baseline and placebo after 12 months. Treatment with nasal SCT resulted in a significantly pronounced suppression of bone resorption markers (urinary deoxypyridinoline [DPD], type I cross-linked N-telopeptide [NTX], and type I cross-linked C-telopeptide [CTX]) and to a lesser extent in bone formation markers (serum bone-specific alkaline phosphatase [BALP], osteocalcin [OC], serum C-terminal procollagen type I extension peptides [PICP], and serum N-terminal procollagen type I extension peptides [PINP]), whereas the placebo did not. Therapy was tolerated well and there were no treatment-related adverse events. We conclude that intranasal SCT (200 IU daily) is safe and effective in increasing lumbar BMD and reducing bone turnover in men with idiopathic osteoporosis.
Does Hormone-Replacement Therapy Prevent Fractures in Early Postmenopausal Women?
Authors: Randell Kaisa M. , Honkanen Risto J. , Kröger Heikki , Saarikoski Seppo
Published: April 2006

The purpose of this population-based prospective cohort study was to examine the effect of hormone-replacement therapy (HRT) on the risk of fractures. The study population consisted of 7217 postmenopausal women aged 47-56 years (mean, 53.3 years) at baseline from data taken from the Kuopio Osteoporosis Risk Factor and Prevention Study (OSTPRE) in Finland. We compared fracture incidences between HRT users and nonusers. A total of 679 (9.4%) women recorded validated fractures during the 5-year follow-up. Of these, 268 (39%) women had a distal forearm fracture. Two thousand six hundred seventy women (37%) had used HRT >6 months during the follow-up—one-half of them continuously. The relative risk, estimated as hazard ratio with Cox regression, was 0.69 (95% CI, 0.58-0.82) for any fracture and 0.49 (0.36-0.66) for distal forearm fracture among HRT users as compared with never-users. After adjusting for age, body mass index (BMI), number of chronic health disorders, fracture history, and time since menopause (independent risk factors) the corresponding risks were 0.67 (0.55-0.81) and 0.53 (0.37-0.74), respectively. The respective adjusted risks for continuous HRT users were 0.62 (0.48-0.79) and 0.41 (0.26-0.67). The adjusted risk of other than distal forearm fracture was 0.74 (0.55-0.98). The results suggest that HRT has a beneficial effect on prevention of fractures in general and on that of distal forearm fracture in particular in early postmenopausal women.
Associations of Calcium Intake and Physical Activity With Bone Density and Size in Premenopausal and Postmenopausal Women: A Peripheral Quantitative Computed Tomography Study
Authors: Uusi-Rasi Kirsti , Sievänen Harri , Pasanen Matti , Oja Pekka , Vuori Ilkka
Published: April 2006

The purpose of this cross-sectional study was to examine the impact of long-term physical activity (PA) and calcium intake on non-weight-bearing radius and weight-bearing tibia. Altogether, 218 healthy, nonsmoking women, [92 premenopausal women, mean age, 32.6 years (SD, 2.2 years), and 126 postmenopausal women, mean age, 67.3 years (SD, 2.0 years)] participated. The subjects were divided according to their habitual levels of physical activity (PA+ or PA−) and calcium intake (Ca+ or Ca−). The distal end and shaft regions of the radius and tibia were evaluated with peripheral quantitative tomography (pQCT). For the shaft regions, bone mineral content (BMC), cortical cross-sectional area (CoA), cortical density (CoD), and bone strength index, that is, 1-11.9% of the density-weighted section modulus (BSI) were determined. For the distal ends, BMC, total cross-sectional area (ToA), trabecular density (TrD), and BSI were determined. The BMC at the distal radius in the young PA+ group was 6.6% (95% CI, 1-to 11.9%) lower than that of the PA− group. A similar nonsignificant trend was found for the radial shaft. The radial shaft showed a mechanically more competent structure among the older subjects with a BSI 8.5% (95% CI, 1.8-15.6%) higher in the older PA+ group than in the older PA− group. The associations between calcium intake and the radial bone characteristics were systematically positive in both age groups. PA seemed to benefit the distal tibia. In the younger age group the TrD was 6.9% (95% CI, 1.8-12.4%) higher in the PA+ group, and in the elderly the BMC was 5% (95% CI, 0.3-9.9%) higher in the PA+ group than in the PA− group. Note that in the younger age group the ToA was 5.1% (95% CI, 0-9.1%) smaller in the PA+ group than in the PA− group, and in the older age group the ToA was 4.2% (95% CI, −0.3-8.9%) greater in the PA+ group than in the PA− group. The association of PA and bone characteristics at the tibial shaft was positive in both age groups (statistically significant for the older subjects). The tibial shaft BSI of the older PA+ group was 8.6% (95% CI, 2.6-14.9%) better than that of the old PA− group. There was no association between calcium intake and the tibial bone characteristics in either age group. In conclusion, high calcium intake was positively associated with a mechanically competent structure in the radius among both younger and older women, whereas the influence of PA did not become apparent until older ages. PA seemed to benefit particularly the weight-bearing tibia, whereas calcium intake was not associated with the tibia.
Human Osteoblasts' Proliferative Responses to Strain and 17β-Estradiol Are Mediated by the Estrogen Receptor and the Receptor for Insulin-Like Growth Factor I
Authors: Cheng Ming Zhao , Rawlinson Simon C. F. , Pitsillides Andrew A. , Zaman Gul , Mohan Subburaman , Baylink David J. , Lanyon Lance E.
Published: April 2006

The mechanism by which mechanical strain and estrogen stimulate bone cell proliferation was investigated using monolayer cultures of human osteoblastic TE85 cells and female human primary (first-passage) osteoblasts (fHOBs). Both cell types showed small but statistically significant dose-dependent increases in [3H]thymidine incorporation in response to 17β-estradiol and to a single 10-minute period of uniaxial cyclic strain (1 Hz). In both cell types, the peak response to 17β-estradiol occurred at 10−8-10−7 M and the peak response to strain occurred at 3500 microstrain (μϵ). Both strain-related and 17β-estradiol-related increases in [3H]thymidine incorporation were abolished by the estrogen receptor (ER) modulator ICI 182,780 (10−8 M). Tamoxifen (10−9-10−8 M) increased [3H]thymidine incorporation in both cell types but had no effect on their response to strain. In TE85 cells, tamoxifen reduced the increase in [3H]thymidine incorporation associated with 17β-estradiol to that of tamoxifen alone but had no such effect in fHOBs. In TE85 cells, strain increased medium concentrations of insulin-like growth factor (IGF) II but not IGF-I, whereas 17β-estradiol increased medium concentrations of IGF-I but not IGF-II. Neutralizing monoclonal antibody (MNAb) to IGF-I (3 μg/ml) blocked the effects of 17β-estradiol and exogenous truncated IGF-I (tIGF-I; 50 ng/ml) but not those of strain or tIGF-II (50 ng/ml). Neutralizing antibody to IGF-II (3 μg/ml) blocked the effects of strain and tIGF-II but not those of 17β-estradiol or tIGF-I. MAb αIR-3 (100 ng/ml) to the IGF-I receptor blocked the effects on [3H]thymidine incorporation of strain, tIGF-II, 17β-estradiol, and tIGF-I. HOBs and TE85 cells, act similarly to rat primary osteoblasts and ROS 17/2.8 cells in their dose-related proliferative responses to strain and 17β-estradiol, both of which can be blocked by the ER modulator ICI 182,780. In TE85 cells (as in rat primaries and ROS 17/2.8 cells), the response to 17β-estradiol is mediated by IGF-I, and the response to strain is mediated by IGF-II. Human cells differ from rat cells in that tamoxifen does not block their response to strain and reduces the response to 17β-estradiol in TE85s but not primaries. In both human cell types (unlike rat cells) the effects of strain and IGF-II as well as estradiol and IGF-I can be blocked at the IGF-I receptor.
Possible Involvement of IκB Kinase 2 and MKK7 in Osteoclastogenesis Induced by Receptor Activator of Nuclear Factor κB Ligand
Authors: Yamamoto Aiichiro , Miyazaki Tsuyoshi , Kadono Yuho , Takayanagi Hiroshi , Miura Toshiki , Nishina Hiroshi , Katada Toshiaki , Wakabayashi Kenji , Oda Hiromi , Nakamura Kozo , Tanaka Sakae
Published: April 2006

Recent studies have revealed the essential role of the receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) in osteoclast differentiation and activation. Adenovirus vector could efficiently transduce genes into RAW264.7 cells, which differentiate into osteoclast-like multinucleated cells in the presence of RANKL. The role of NF-κB and c-jun N-terminal kinase (JNK) activation in RANKL-induced osteoclast differentiation was investigated using an adenovirus vector carrying the dominant negative IκB kinase 2 gene (AxIKK2DN) or dominant negative MKK7 gene (AxMKK7DN). IKK2DN and MKK7DN overexpression in RAW cells specifically suppressed the NF-κB activation and JNK activation in response to RANKL, respectively, without affecting other signaling pathways. Either inhibition of NF-κB or JNK pathways dose-dependently inhibited osteoclast formation induced by RANKL. These results suggest that both NF-κB and JNK activation are independently required for osteoclast differentiation.
Vitamin D Hormone Inhibits Osteoclastogenesis In Vivo by Decreasing the Pool of Osteoclast Precursors in Bone Marrow
Authors: Shibata Takeshi , Shira-Ishi Ayako , Sato Takuya , Masaki Toshimi , Sasaki Aya , Masuda Yoshiko , Hishiya Akinori , Ishikura Nobuyuki , Higashi Sayumi , Uchida Yasuhiro , Saito Moto-O , Ito Masako , Ogata Etsuro , Watanabe Ken , Ikeda Kyoji
Published: April 2006

Previous observations that vitamin D hormone induces the expression of the receptor activator of nuclear factor κB (NF-κB) ligand (RANKL), thereby stimulating osteoclastogenesis in vitro, led to the widespread belief that 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is a bone-resorbing hormone. Here, we show that alfacalcidol, a prodrug metabolized to 1α,25(OH)2D3, suppresses bone resorption at pharmacologic doses that maintain normocalcemia in an ovariectomized (OVX) mouse model of osteoporosis. Treatment of OVX mice with pharmacologic doses of alfacalcidol does not increase RANKL expression, whereas toxic doses that cause hypercalcemia markedly reduce the expression of RANKL. When bone marrow (BM) cells from OVX mice were cultured with sufficient amounts of macrophage colony-stimulating factor (M-CSF) and RANKL, osteoclastogenic activity was higher than in sham mice. Marrow cultures from alfacalcidol- or estrogen-treated OVX mice showed significantly less osteoclastogenic potential compared with those from vehicle-treated OVX mice, suggesting that the pool of osteoclast progenitors in the marrow of vitamin D-treated mice as well as estrogen-treated mice was decreased. Frequency analysis showed that the number of osteoclast progenitors in bone marrow was increased by OVX and decreased by in vivo treatment with alfacalcidol or estrogen. We conclude that the pharmacologic action of active vitamin D in vivo is to decrease the pool of osteoclast progenitors in BM, thereby inhibiting bone resorption. Because of its unusual activity of maintaining bone formation while suppressing bone resorption, in contrast to estrogens that depress both processes, vitamin D hormone and its bone-selective analogs may be useful for the management of osteoporosis.
Proteolysis Involving Matrix Metalloproteinase 13 (Collagenase-3) Is Required for Chondrocyte Differentiation That Is Associated with Matrix Mineralization
Authors: Wu C. William , Tchetina Elena V. , Mwale Fackson , Hasty Karen , Pidoux Isabelle , Reiner Agnes , Chen Jeffrey , Van Wart Harold E. , Poole A. Robin
Published: April 2006

Collagenases are involved in cartilage matrix resorption. Using bovine fetal chondrocytes isolated from physeal cartilages and separated into a distinct prehypertrophic subpopulation, we show that in serum-free culture they elaborate an extracellular matrix and differentiate into hypertrophic chondrocytes. This is characterized by expression of type X collagen and the transcription factor Cbfa1 and increased incorporation of45Ca2+ in the extracellular matrix, which is associated with matrix calcification. Collagenase activity, attributable only to matrix metalloproteinase (MMP) 13 (collagenase-3), is up-regulated on differentiation. A nontoxic carboxylate inhibitor of MMP-13 prevents this differentiation; it suppresses expression of type X collagen, Cbfa1, and MMP-13 and inhibits increased calcium incorporation in addition to inhibiting degradation of type II collagen in the extracellular matrix. General synthesis of matrix proteins is unaffected. These results suggest that proteolysis involving MMP-13 is required for chondrocyte differentiation that occurs as part of growth plate development and which is associated with matrix mineralization.
Tests of Linkage and/or Association of Genes for Vitamin D Receptor, Osteocalcin, and Parathyroid Hormone With Bone Mineral Density
Authors: Deng Hong-Wen , Shen Hui , Xu Fu-Hua , Deng Hong-Yi , Conway Theresa , Zhang Hai-Tao , Recker Robert R.
Published: April 2006

Bone mineral density (BMD) is a major determinant of osteoporotic fractures (OFs). The heritability of BMD ranges from 50% to 90% in human populations. Extensive molecular genetic analyses have been performed through traditional linkage or association approaches to test and identify genes or genomic regions underlying BMD variation. The results, particularly those concerning the vitamin D receptor (VDR) gene, have been inconsistent and controversial. In this study, we simultaneously test linkage and/or association of the genes for VDR, osteocalcin (also known as bone Gla protein [BGP]), and parathyroid hormone (PTH) with BMD in 630 subjects from 53 human pedigrees. Each of these pedigrees was ascertained through a proband with an extreme BMD value at the hip or spine (Z score ≤ −1.28). For the raw BMD values, adjusting for significant covariate effects of age, sex, and weight, we performed tests for linkage alone, association alone, and then both linkage and association. For the spine BMD, at the two markers (ApaI and FokI) inside the VDR gene we found evidence for linkage (p < 0.05) and for both linkage and association by the transmission disequilibrium test (TDT; p < 0.05); association was detected (p < 0.07) with regular statistical testing by analyses of variance (ANOVA). In addition, significant results were found for association alone (p < 0.05), linkage alone (p = 0.0005), and for linkage and association (p = 0.0019) for the intragenic marker HindIII of the BGP gene for the hip BMD. Through testing for association, linkage, and linkage and association simultaneously, our data support the VDR gene as a quantitative trait locus (QTL) underlying spine BMD variation and the BGP gene as a QTL underlying hip BMD variation. However, our data do not support the PTH gene as a QTL underlying hip or spine BMD variation. This is the first study in the broad field of bone genetics that tests candidate genes as QTLs for BMD by testing simultaneously for association alone, for linkage alone, and for association and linkage (via the TDT).
Mechanical and Architectural Bone Adaptation in Early Stage Experimental Osteoarthritis
Authors: Boyd Steven K. , Müller Ralph , Dr. Zernicke Ronald F.
Published: April 2006

The purpose of this study was to quantify mechanical and architectural changes to knee joint periarticular subchondral cancellous bone in early stage experimental osteoarthritis (OA). Unilateral anterior cruciate ligament transection (ACLX) was performed on 10 dogs that were assigned randomly to two groups: 3 weeks or 12 weeks post-ACLX. Cylindrical bone cores excised from the medial condyle of the distal femur after death were scanned using high-resolution microcomputed tomography (μCT) and subsequently failed under unconstrained uniaxial compression. The apparent-level elastic modulus was less in the ACLX femur compared with the contralateral control, and the decrease was significant (−45%; p < 0.05) by 12-weeks post-ACLX. A finite element (FE) analysis based on μCT data simulated the uniaxial compression tests on a specimen-by-specimen basis to determine tissue modulus. No change in tissue modulus was detected, and a single tissue modulus of 5100 MPa (95% CI, ±600 MPa) explained the apparent-level modulus changes observed in the disease-related bone adaptation. The three-dimensional (3D) connectivity was evaluated from the original μCT data to quantify architectural alterations in contrast to tissue alterations. Significantly increased connectivity (through plate perforations) occurred as early as 3 weeks post-ACLX and was as high as 127% by 12 weeks post-ACLX in the distal femur. These measured changes indicated that architectural adaptation predominated over tissue modulus changes affecting apparent-level elastic modulus in the early stage of experimental OA and suggests that to maintain normal cancellous bone after a traumatic injury, early intervention should focus on preventing the substantial architectural alterations.
Can Vitamin D Supplementation Reduce the Risk of Fracture in the Elderly? A Randomized Controlled Trial
Authors: Meyer Haakon E. , Smedshaug Guro B. , Kvaavik Elisabeth , Falch Jan A. , Tverdal Aage , Pedersen Jan I.
Published: April 2006

Randomized controlled trials have shown that a combination of vitamin D and calcium can prevent fragility fractures in the elderly. Whether this effect is attributed to the combination of vitamin D and calcium or to one of these nutrients alone is not known. We studied if an intervention with 10 μg of vitamin D3 per day could prevent hip fracture and other osteoporotic fractures in a double-blinded randomized controlled trial. Residents from 51 nursing homes were allocated randomly to receive 5 ml of ordinary cod liver oil (n = 569) or 5 ml of cod liver oil where vitamin D was removed (n = 575). During the study period of 2 years, fractures and deaths were registered, and the principal analysis was performed on the intention-to-treat basis. Biochemical markers were measured at baseline and after 1 year in a subsample. Forty-seven persons in the control group and 50 persons in the vitamin D group suffered a hip fracture. The corresponding figures for all nonvertebral fractures were 76 persons (control group) and 69 persons (vitamin D group). There was no difference in the incidence of hip fracture (p = 0.66, log-rank test), or in the incidence of all nonvertebral fractures (p = 0.60, log-rank test) in the vitamin D group compared with the control group. Compared with the control group, persons in the vitamin D group increased their serum 25-hydroxyvitamin D concentration with 22 nmol/liter (p = 0.001). In conclusion, we found that an intervention with 10 μg of vitamin D3 alone produced no fracture-preventing effect in a nursing home population of frail elderly people.