bluuebunny/crossref_metadata_2025 huggingface dataset:
Risk Factors for Hip Fracture in Asian Men and Women: The Asian Osteoporosis Study
Authors: Lau E. M. C. , Suriwongpaisal P. , Lee J. K. , Das De S. , Festin M. R. , Saw S. M. , Khir A. , Torralba T. , Sham A. , Sambrook P.
Published: April 2006

The objectives of the Asian Osteoporosis Study (AOS) were to determine risk factors for hip fracture in men and women in four Asian countries, that is, Singapore, Malaysia, Thailand, and the Philippines. A total of 451 men and 725 women (aged 50 years and over) with hip fractures were compared with an equal number of community controls. A standardized questionnaire was administered by interview. The following relative risks (RRs) were found in women and men by multiple logistic regression: dietary calcium intake < 498 mg/day, 2.0 for women (95% CI, 1.5–2.8) and 1.5 for men (95% CI, 1.0–2.2); no load bearing activity in the immediate past, 2.0 for women (95% CI, 1.4–2.7) and 3.4 for men (95% CI, 2.3–5.1); no vigorous sport activities in young adulthood, 7.2 for women (95% CI, 4.0–13.0) and 2.4 for men (95% CI, 1.6–3.6); cigarette smoking, 1.5 for men (95% CI, 1.0–2.1); alcohol consumption 7 days a week, 2.9 for women (95% CI, 1.0–8.6) and 1.9 for men (95% CI, 1.1–3.2); fell twice or more in the last 12 months, 3.0 for women (95% CI, 1.8–4.8) and 3.4 for men (95% CI, 1.8–6.6); a history of fractures after 50 years of age, 1.8 for women (95% CI, 1.1–2.9) and 3.0 for men (95% CI, 1.6–5.6); a history of stroke, 3.8 for women (95% CI, 2.0–7.1) and 3.6 for men (95% CI, 1.8–7.1); use of sedatives, 2.5 for women (95% CI, 1.0–6.3) and 3.0 for men (95% CI, 1.0–9.7); and use of thyroid drugs, 7.1 for women (95% CI, 2.0–25.9) and 11.8 for men (95% CI, 1.3–106.0). Women who were 1.56 m or taller had an RR of 2.0 (95% CI, 1.3–3.0) for hip fracture and men who were 1.69 m or taller had an RR of 1.9 (95% CI, 1.2–3.1) for hip fracture. Based on these findings, primary preventive programs for hip fracture could be planned in Asia.
Use of Inhaled Corticosteroids and Risk of Fractures
Authors: Van Staa T. P. , Leufkens H. G. M. , Cooper C.
Published: April 2006

Treatment with systemic corticosteroids is known to increase the risk of fractures but little is known of the fracture risks associated with inhaled corticosteroids. A retrospective cohort study was conducted using a large UK primary care database (the General Practice Research Database [GPRD]). Inhaled corticosteroid users aged 18 years or older were compared with matched control patients and to a group of noncorticosteroid bronchodilator users. Patients with concomitant use of systemic corticosteroids were excluded. The study comprised 170,818 inhaled corticosteroid users, 108,786 bronchodilator users, and 170,818 control patients. The average age was 45.1 years in the inhaled corticosteroid, 49.3 years in the bronchodilator, and 45.2 years in the control groups. In the inhaled corticosteroid cohort, 54.5% were female. The relative rates (RRs) of nonvertebral, hip, and vertebral fractures during inhaled corticosteroid treatment compared with control were 1.15 (95% CI, 1.10–1.20), 1.22 (95% CI, 1.04–1.43), and 1.51 (95% CI, 1.22–1.85), respectively. No differences were found between the inhaled corticosteroid and bronchodilator groups (nonvertebral fracture RR = 1.00; 95% CI, 0.94–1.06). The rates of nonvertebral fractures among users of budesonide (RR = 0.95; 95% CI, 0.85–1.07) and fluticasone propionate (RR = 1.03; 95% CI, 0.71–1.49) were similar to the rate determined for users of beclomethasone dipropionate. We conclude that users of inhaled corticosteroids have an increased risk of fracture, particularly at the hip and spine. However, this excess risk may be related more to the underlying respiratory disease than to inhaled corticosteroid.
Development of a Novel Immunoradiometric Assay Exclusively for Biologically Active Whole Parathyroid Hormone 1–84: Implications for Improvement of Accurate Assessment of Parathyroid Function
Authors: Gao Ping , Scheibel Stephen , D'Amour Pierre , John Markus R. , Rao Sudhaker D. , Schmidt-Gayk Heinrich , Cantor Thomas L.
Published: April 2006

We developed a novel immunoradiometric assay (IRMA; whole parathyroid hormone [PTH] IRMA) for PTH, which specifically measures biologically active whole PTH(1–84). The assay is based on a solid phase coated with anti-PTH(39–84) antibody, a tracer of125I-labeled antibody with a unique specificity to the first N-terminal amino acid of PTH(1–84), and calibrators of diluted synthetic PTH(1–84). In contrast to the Nichols intact PTH IRMA, this new assay does not detect PTH(7–84) fragments and only detects one immunoreactive peak in chromatographically fractionated patient samples. The assay was shown to have an analytical sensitivity of 1.0 pg/ml with a linear measurement range up to 2300 pg/ml. With this assay, we further identified that the previously described non-(1–84)PTH fragments are aminoterminally truncated with similar hydrophobicity as PTH(7–84), and these PTH fragments are present not only in patients with secondary hyperparathyroidism (2°-HPT) of uremia, but also in patients with primary hyperparathyroidism (1°-HPT) and normal persons. The plasma normal range of the whole PTH(1–84) was 7–36 pg/ml (mean ± SD: 22.7 ± 7.2 pg/ml, n = 135), whereas over 93.9% (155/165) of patients with 1°-HPT had whole PTH(1–84) values above the normal cut-off. The percentage of biologically active whole PTH(1–84) (pB%) in the pool of total immunoreactive “intact” PTH is higher in the normal population (median: 67.3%; SD: 15.8%; n = 56) than in uremic patients (median:53.8%; SD: 15.5%; n = 318; p < 0.001), although the whole PTH(1–84) values from uremic patients displayed a more significant heterogeneous distribution when compared with that of 1°-HPT patients and normals. Moreover, the pB% displayed a nearly Gaussian distribution pattern from 20% to over 90% in patients with either 1°-HPT or uremia. The specificity of this newly developed whole PTH(1–84) IRMA is the assurance, for the first time, of being able to measure only the biologically active whole PTH(1–84) without cross-reaction to the high concentrations of the aminoterminally truncated PTH fragments found in both normal subjects and patients. Because of the significant variations of pB% in patients, it is necessary to use the whole PTH assay to determine biologically active PTH levels clinically and, thus, to avoid overestimating the concentration of the true biologically active hormone. This new assay could provide a more meaningful standardization of future PTH measurements with improved accuracy in the clinical assessment of parathyroid function.
Increased Vitamin D Receptor Level Enhances 1,25-Dihydroxyvitamin D3-Mediated Gene Expression and Calcium Transport in Caco-2 Cells
Authors: Shao A. , Wood R. J. , Fleet J. C.
Published: April 2006

Altered vitamin D receptor (VDR) level has been proposed to explain differences in intestinal responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We tested whether the enterocyte VDR level influences 1,25(OH)2D3-mediated gene expression and transepithelial calcium (Ca) transport in the human intestinal cell line Caco-2. Cells were stably transfected with a human metallothionein (hMT) IIA promoter-human VDR (hVDR) complementary DNA (cDNA) transgene that overexpressed hVDR in response to heavy metals. In MTVDR clones, induction of 25-hyroxyvitamin D3-24-hydroxylase (24-OHase) messenger RNA (mRNA) expression by 1,25(OH)2D3 (10−9 M, 4 h) was correlated to metal-induced changes in nuclear VDR level (r2 = 0.99). In MTVDR clones, basal VDR level was 2-fold greater and 1,25(OH)2D3-mediated Ca transport (10−7 M, 24 h) was 43% higher than in parental Caco-2 cells. Treatment of MTVDR clones with Cd (1 μM, 28 h) increased VDR level by 68%, significantly enhanced 1,25(OH)2D3-mediated Ca transport by 24%, and increased accumulation of calbindin D9K mRNA by 76% relative to 1,25(OH)2D3 alone. These observations support the hypothesis that the enterocyte VDR level is an important modulator of intestinal responsiveness to 1,25(OH)2D3.
Interaction of Two Novel 14-Epivitamin D3 Analogs with Vitamin D3 Receptor-Retinoid X Receptor Heterodimers on Vitamin D3 Responsive Elements
Authors: Verlinden Lieve , Verstuyf Annemieke , Quack Marcus , Van Camp Mark , Van Etten Evelyne , De Clercq Pierre , Vandewalle Maurits , Carlberg Carsten , Bouillon Roger
Published: April 2006

This study provides a detailed and exact evaluation of the interactions between vitamin D3 receptor (VDR), retinoid X receptor (RXR), and vitamin D3 responsive elements (VDREs) mediated by two novel 14-epianalogs of 1,25-dihydroxyvitamin D [1,25(OH)2D3], 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527). Both analogs were more potent (14- and 75-fold, respectively) than 1,25(OH)2D3 in inhibiting cell proliferation and inducing cell differentiation. However, DNA-independent experiments indicated that both analogs had a lower affinity to VDR and that the stability of the induced VDR conformation, as measured by limited protease digestion assays, was similar (TX 527) or even weaker (TX 522) than that induced by the parent compound. However, DNA-dependent assays such as gel shift experiments revealed that those analogs were slightly more potent (3–7 times) than 1,25(OH)2D3 in enhancing binding of VDR-RXR heterodimers to a direct repeat spaced by three nucleotides (DR3) type VDRE. The functional consequences of the ligand-VDR-RXR-VDRE interactions observed in vitro were subsequently evaluated in transfection experiments. Both 14-epianalogs enhanced transcription of VDRE containing reporter constructs more efficiently than 1,25(OH)2D3 in COS-1 and MCF-7 cells regardless of the presence of ketoconazole. Transactivation activity is suggested to be a cell-specific process because maximal transcriptional induction and the half-maximal transactivation concentration for each reporter construct were different in both cell lines. The superagonistic transactivation activity closely resembled the biological potency of these analogs on the inhibition of MCF-7 cell proliferation. These data clearly indicate that superagonistic activity starts beyond the binding of the ligand-heterodimer (VDR-RXR) complex to VDRE and thus probably involves coactivator/corepressor molecules.
Evidence for a Direct Role of Cyclo-Oxygenase 2 in Implant Wear Debris-Induced Osteolysis
Authors: Zhang Xinping , Morham Scott G. , Langenbach Robert , Young Donald A. , Xing Lianping , Boyce Brendan F. , Puzas Edward J. , Rosier Randy N. , O'Keefe Regis J. , Schwarz Edward M.
Published: April 2006

Aseptic loosening is a major complication of prosthetic joint surgery and is manifested as chronic inflammation, pain, and osteolysis at the bone implant interface. The osteolysis is believed to be driven by a host inflammatory response to wear debris generated from the implant. In our current study, we use a selective inhibitor (celecoxib) of cyclo-oxygenase 2 (COX-2) and mice that lack either COX-1 (COX-1−/−) or COX-2 (COX-2−/−) to show that COX-2, but not COX-1, plays an important role in wear debris-induced osteolysis. Titanium (Ti) wear debris was implanted surgically onto the calvaria of the mice. An intense inflammatory reaction and extensive bone resorption, which closely resembles that observed in patients with aseptic loosening, developed within 10 days of implantation in wild-type and COX-1−/− mice. COX-2 and prostaglandin E2 (PGE2) production increased in the calvaria and inflammatory tissue overlying it after Ti implantation. Celecoxib (25 mg/kg per day) significantly reduced the inflammation, the local PGE2 production, and osteolysis. In comparison with wild-type and COX-1−/− mice, COX-2−/− mice implanted with Ti had a significantly reduced calvarial bone resorption response, independent of the inflammatory response, and significantly fewer osteoclasts were formed from cultures of their bone marrow cells. These results provide direct evidence that COX-2 is an important mediator of wear debris-induced osteolysis and suggests that COX-2 inhibitors are potential therapeutic agents for the prevention of wear debris-induced osteolysis.
Intervertebral Disc Disorganization Is Related to Trabecular Bone Architecture in the Lumbar Spine
Authors: Simpson E. K. , Parkinson I. H. , Manthey B. , Fazzalari N. L.
Published: April 2006

Cancellous bone morphometry was investigated in the sagittal plane of lumbar vertebrae using histoquantitation. The aim of this study was to identify variations in cancellous bone architecture at increasing states of intervertebral disc (IVD) disorganization after age adjustment and to investigate regional variations within the whole vertebral body. Measurements were taken of the ratio of bone volume (BV) to total volume (TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N). Lumbar spines (T12-L5) of 19 men and 8 women were removed at autopsy from an adult sample with no clinical history of bone-related disease or histologically identifiable bone disease. It was found that degeneration of the IVD becomes more common with increasing age. After age-adjustment, significant increases in the proportion of BV/TV were observed in the presence of advancing IVD disorganization. Significant architectural changes were observed in the anterior regions of the vertebral body with increases in Tb.Th and Tb.N and decreases in Tb.Sp. Minimal alterations were found at posterior regions. Bone loss was observed in central regions (most distant from the cortex) as IVD disorganization increased through reduction in both Tb.N and Tb.Th. The BV/TV increase in anterior areas of the centrum may be a response to a redistribution of load to the vertebral body periphery as a result of IVD disorganization. It appears that trabecular morphology is related to the condition of the associated IVD, rather than being the sole consequence of a loss of BV/TV with age. This relationship could influence the occurrence of vertebral body crush fracture.
A Three-Dimensional Simulation of Age-Related Remodeling in Trabecular Bone
Authors: Van Der Linden J. C. , Verhaar J. A. N. , Weinans H.
Published: April 2006

After peak bone mass has been reached, the bone remodeling process results in a decrease in bone mass and strength. The formation deficit, the deficit of bone formation compared with previous resorption, results in bone loss. Moreover, trabeculae disconnected by resorption cavities probably are not repaired. The contributions of these mechanisms to the total bone loss are unclear. To investigate these contributions and the concomitant changes in trabecular architecture and mechanical properties, we made a computer simulation model of bone remodeling using microcomputed tomography (micro-CT) scans of human vertebral trabecular bone specimens. Up to 50 years of physiological remodeling were simulated. Resorption cavities were created and refilled 3 months later. These cavities were not refilled completely, to simulate the formation deficit. Disconnected trabeculae were not repaired; loose fragments generated during the simulation were removed. Resorption depth, formation deficit, and remodeling space were based on biological data. The rate of bone loss varied between 0.3% and 1.1% per year. Stiffness anisotropy increased, and morphological anisotropy (mean intercept length [MIL]) was almost unaffected. Connectivity density increased or decreased, depending on the remodeling parameters. The formation deficit accounted for 69–95%, disconnected trabeculae for 1–21%, and loose fragments for 1–17% of the bone loss. Increasing formation deficit from 1.8% to 5.4% tripled bone loss but only doubled the decrease in stiffness. Increasing resorption depth from 28 to 56 μm slightly increased bone loss but drastically decreased stiffness. Decreasing the formation deficit helps to prevent bone loss, but reducing resorption depth is more effective in preventing loss of mechanical stiffness.
Endogenous Prostaglandin E2 and Insulin-like Growth Factor 1 Can Modulate the Levels of Parathyroid Hormone Receptor in Human Osteoarthritic Osteoblasts
Authors: Hilal George , Massicotte Frédéric , Martel-Pelletier Johanne , Fernandes Julio C. , Pelletier Jean-Pierre , Lajeunesse Daniel
Published: April 2006

Subchondral bone sclerosis may be important for the onset and/or progression of cartilage loss/damage in human osteoarthritis (OA). OA osteoblasts are resistant to parathyroid hormone (PTH) stimulation, which could explain bone sclerosis via the inhibition of PTH-dependent catabolism. Here, we investigated the molecular mechanism(s) responsible for reduced PTH-dependent cyclic adenosine monophosphate (cAMP) synthesis in OA subchondral osteoblasts. Although cholera toxin (CTX) increased basal cAMP formation in these cells, it failed to stimulate PTH-dependent cAMP synthesis, whereas pertussis toxin (PTX) did not inhibit basal cAMP, yet diminished PTH-dependent cAMP production. Binding of125I-PTH indicated lower PTH receptor levels in OA than in normal osteoblasts (−50.5 ± 9.5%). This could be attributed to either reduced expression of the PTH receptor (PTH-R) or altered recycling of existing pools of receptors. Reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated decreased PTH-R messenger RNA (mRNA) levels in OA cells that were highly variable (ranging from −10% to −60%), a situation that reflects disease severity. Interestingly, OA osteoblasts produced more prostaglandin E2 (PGE2) than normal osteoblasts, and using naproxen, a cyclo-oxygenase inhibitor, increased PTH-dependent cAMP formation to a level similar to normal osteoblasts. Because heterologous desensitization can explain a decrease in PTH binding but cannot account for reduced PTH-R expression, we looked at the possible effect of insulin-like growth factor 1 (IGF-1) on this parameter. Blocking IGF-1 signaling with a neutralizing receptor antibody increased125I-PTH binding in both normal and OA osteoblasts. Conversely, treatments with IGF-1 receptor (IGF-1R) antibody only slightly increased the levels of PTH-R mRNA whereas the addition of IGF-1 significantly reduced PTH-R mRNA levels (−24.1 ± 7.1%), yet neither PGE2 nor naproxen modified PTH-R levels. These results suggest that both IGF-1 signaling and PGE2 formation repress PTH-dependent response in OA osteoblasts, a situation that can contribute to abnormal bone remodeling and bone sclerosis in OA.
Mild Heat Shock Induces Proliferation, Alkaline Phosphatase Activity, and Mineralization in Human Bone Marrow Stromal Cells and Mg-63 Cells In Vitro
Authors: Shui Chaoxiang , Scutt Andrew
Published: April 2006

Bone formation has been shown to be stimulated by local diathermy in vivo; however, the mechanisms involved in this heat-induced osteogenesis are unclear. In this study, we investigated the direct effect of temperature on human bone marrow-derived stromal cells (BMSCs) and the human osteoblast-like, osteosarcoma-derived MG-63 cells in culture conditions. Both cell types were shown to tolerate the transient exposure to mild heat shock conditions (1 h at 39–41°C), and long-term (96 h) exposure at 39°C stimulated DNA synthesis in BMSC but caused growth arrest in MG-63 cells. Furthermore, 1-h exposure to higher temperatures (42.5–45°C) or continuous 96-h exposure to 40°C or 41°C inhibited the proliferation of both BMSCs and MG63 cells. The level of alkaline phosphatase (ALP) in these cells linearly correlated with the increase in temperature, and the ALP expression, either at the basal level or in response to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], was enhanced after a single 1-h exposure to 42.5°C. In addition, continuous incubation at 39°C or repeated transient exposure to 39/41°C greatly enhanced the ability of BMSCs to form mineralizing nodules. The heat shock protein HSP70, which was expressed constitutively by BMSCs, was found to be up-regulated by hyperthermia (39°C) and down-regulated at 33°C. The expression of HSP70 could be induced in MG-63 cells by both low- and high-temperature conditions. These data suggest that treatment with a mild heat shock induces the proliferation and differentiation of osteoprogenitor cells, and the direct effects of temperature on bone-forming cells might be one of the mechanisms involved in heat-induced bone formation in vivo.
Glutamate Does Not Play a Major Role in Controlling Bone Growth
Authors: Gray Colin , Marie Hélène , Arora Maureen , Tanaka Kohichi , Boyde Alan , Jones Sheila , Attwell David
Published: April 2006

Bone cells express glutamate-gated Ca2+-permeable N-methyl-D-aspartate (NMDA) receptors and GLAST glutamate transporters. Blocking NMDA receptors has been reported to reduce the number of bone resorption pits produced by osteoclasts, and mechanical loading alters GLAST transporter expression, which should change the extracellular glutamate concentration and NMDA receptor activation. Thus, by analogy with the brain, glutamate is postulated to be an important intercellular messenger in bone, controlling bone formation and resorption. We found that activating or blocking NMDA receptors had no effect on bone formation by rat osteoblasts in culture. The number of resorption pits produced by osteoclasts was reduced by the NMDA receptor blocker MK-801 but not by another blocker AP-5, implying that this effect of MK-801 is unrelated to its glutamate-blocking action. By contrast, MK-801, AP-5, and NMDA had no consistent effect on the volume of pits. In mice with GLAST glutamate transporters knocked out, no differences were detected in mandible and long bone size, morphology, trabeculation, regions of muscle attachment, resorption lacunae, or areas of formation versus resorption of bone, compared with wild-type siblings. These data suggest that glutamate does not play a major role in controlling bone growth.
Genetic Control of Bone Density and Turnover: Role of the Collagen 1α1, Estrogen Receptor, and Vitamin D Receptor Genes
Authors: Brown Matthew A. , Haughton Margaret A. , Grant Struan F. A. , Gunnell Anthony S. , Henderson N. Kathryn , Eisman John A.
Published: April 2006

Genetic factors are known to influence both the peak bone mass and probably the rate of change in bone density. A range of regulatory and structural genes has been proposed to be involved including collagen 1α1 (COL1A1), the estrogen receptor (ER), and the vitamin D receptor (VDR), but the actual genes involved are uncertain. We therefore studied the role of the COL1A1 and VDR loci in control of bone density by linkage in 45 dizygotic twin pairs and 29 nuclear families comprising 120 individuals. The influences on bone density of polymorphisms of COL1A1, VDR, and ER were studied by association both cross-sectionally and longitudinally in 193 elderly postmenopausal women (average age, 69 years) over a mean follow-up time of 6.3 years. Weak linkage of the COL1A1 locus with bone density was observed in both twins and families (p = 0.02 in both data sets), confirming previous observations of linkage of this locus with bone density. Association between the MscI polymorphism of COL1A1 and rate of lumbar spine bone loss was observed with significant gene-environment interaction related to dietary calcium intake (p = 0.0006). In the lowest tertile of dietary calcium intake, carriers of “s” alleles lost more bone than “SS” homozygotes (p = 0.01), whereas the opposite was observed in the highest dietary calcium intake (p = 0.003). Association also was observed between rate of bone loss at both the femoral neck and the lumbar spine and the TaqI VDR polymorphism (p = 0.03). This association was strongest in those in the lowest tertile of calcium intake, also suggesting the presence of gene-environment interaction involving dietary calcium and VDR, influencing bone turnover. No significant association was observed between the PvuII ER polymorphism alone or in combination with VDR or COL1A1 genotypes, with either bone density or its rate of change. These data support the involvement of COL1A1 in determination of bone density and the interaction of both COL1A1 and VDR with calcium intake in regulation of change of bone density over time.
Lasofoxifene (CP-336,156) Protects Against the Age-Related Changes in Bone Mass, Bone Strength, and Total Serum Cholesterol in Intact Aged Male Rats
Authors: Ke Hua Zhu , Qi Hong , Chidsey-Frink Kristin L. , Crawford D. Todd , Thompson David D.
Published: April 2006

The purpose of this study was to evaluate if long-term (6 months) treatment with lasofoxifene (LAS), a new selective estrogen receptor modulator (SERM), can protect against age-related changes in bone mass and bone strength in intact aged male rats. Sprague-Dawley male rats at 15 months of age were treated (daily oral gavage) with either vehicle (n = 12) or LAS at 0.01 mg/kg per day (n = 12) or 0.1 mg/kg per day (n = 11) for 6 months. A group of 15 rats was necropsied at 15 months of age and served as basal controls. No significant change was found in body weight between basal and vehicle controls. However, an age-related increase in fat body mass (+42%) and decrease in lean body mass (−8.5%) was observed in controls. Compared with vehicle controls, LAS at both doses significantly decreased body weight and fat body mass but did not affect lean body mass. No significant difference was found in prostate wet weight among all groups. Total serum cholesterol was significantly decreased in all LAS-treated rats compared with both the basal and the vehicle controls. Both doses of LAS treatment completely prevented the age-related increase in serum osteocalcin. Peripheral quantitative computerized tomography (pQCT) analysis at the distal femoral metaphysis indicated that the age-related decrease in total density, trabecular density, and cortical thickness was completely prevented by treatment with LAS at 0.01 mg/kg per day or 0.1 mg/kg per day. Histomorphometric analysis of proximal tibial cancellous bone showed an age-related decrease in trabecular bone volume (TBV; −46%), trabecular number (Tb.N), wall thickness (W.Th), mineral apposition rate, and bone formation rate-tissue area referent. Moreover, an age-related increase in trabecular separation (Tb.Sp) and eroded surface was observed. LAS at 0.01 mg/kg per day or 0.1 mg/kg per day completely prevented these age-related changes in bone mass, bone structure, and bone turnover. Similarly, the age-related decrease in TBV and trabecular thickness (Tb.Th) and the age-related increase in osteoclast number (Oc.N) and osteoclast surface (Oc.S) in the third lumbar vertebral cancellous bone were completely prevented by treatment with LAS at both doses. Further, LAS at both doses completely prevented the age-related decrease in ultimate strength (−47%) and stiffness (−37%) of the fifth lumbar vertebral body. These results show that treatment with LAS for 6 months in male rats completely prevents the age-related decreases in bone mass and bone strength by inhibiting the increased bone resorption and bone turnover associated with aging. Further, LAS reduced total serum cholesterol and did not affect the prostate weight in these rats. Our data support the potential use of a SERM for protecting against the age-related changes in bone and serum cholesterol in elderly men.
The Src Signaling Pathway Regulates Osteoclast Lysosomal Enzyme Secretion and Is Rapidly Modulated by Estrogen
Authors: Pascoe David , Oursler Merry Jo
Published: April 2006

To investigate the role of the pp60src signaling pathway in osteoclast activity, we have used dominant negative pp60src, c-ras, and c-raf expression vectors to individually disrupt their functions in osteoclasts. Osteoclasts were transiently transfected and secretions of cathepsin B/K and tartrate-resistant acid phosphatase (TRAP) were monitored. Expression of these constructs increased secretion of lysosomal enzymes. In contrast, constitutively active pp60src expression caused decreased lysosomal enzyme secretion. Osteoclasts respond to 17-β estradiol (17βE2) treatment with decreased lysosomal enzyme secretion. Therefore, we investigated the effects of E2 on pp60src kinase activity and observed an E2 time- and dose-dependent decrease in cytoskeletal membrane-associated pp60src tyrosine kinase activity. We have shown that estrogen decreases lysosomal enzyme gene expression and secretion; so we have examined the effects of the expression constructs on estrogen regulation of enzyme secretion. Constitutively active pp60src blocked E2 effects on secretion whereas expression of dominant negative pp60src, c-Ras, or c-Raf enhanced E2 effects. These data support that the kinase domain of cytoskeletal-associated pp60src is likely to be involved in the regulation of lysosomal enzyme secretion.
Modulation of 11β-Hydroxysteroid Dehydrogenase Isozymes by Proinflammatory Cytokines in Osteoblasts: An Autocrine Switch from Glucocorticoid Inactivation to Activation
Authors: Cooper Mark S. , Bujalska Iwona , Rabbitt Elizabeth , Walker Elizabeth A. , Bland Rosemary , Sheppard Michael C. , Hewison Martin , Stewart Paul M.
Published: April 2006

Tissue damage by proinflammatory cytokines is attenuated at both systemic and cellular levels by counter anti-inflammatory factors such as corticosteroids. Target cell responses to corticosteroids are dependent on several factors including prereceptor regulation via local steroidogenic enzymes. In particular, two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD), by interconverting hormonally active cortisol (F) to inactive cortisone (E), regulate the peripheral action of corticosteroids 11β-HSD1 by converting E to F and 11β-HSD2 by inactivating F to E. In different in vitro and in vivo systems both 11β-HSD isozymes have been shown to be expressed in osteoblasts (OBs). Using the MG-63 human osteosarcoma cell-line and primary cultures of human OBs, we have studied the regulation of osteoblastic 11β-HSD isozyme expression and activity by cytokines and hormones with established roles in bone physiology. In MG-63 cells, interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) potently inhibited 11β-HSD2 activity (cortisol-cortisone conversion) and messenger RNA (mRNA) levels in a dose-dependent manner while stimulating reciprocal expression of 11β-HSD1 mRNA and activity (cortisone-cortisol conversion). A similar rise in 11β-HSD1 reductase activity also was observed in primary cultures of OBs treated with 10 ng/ml TNF-α. Pretreatment of MG-63 cells with 0.1 ng/ml IL-1β resulted in increased cellular sensitivity to physiological glucocorticoids as shown by induction of serum and glucocorticoid-inducible kinase (SGK; relative increase with 50 nM F but no IL-1β pretreatment 1.12 ± 0.34; with pretreatment 2.63 ± 0.50; p < 0.01). These results highlight a novel mechanism within bone cells whereby inflammatory cytokines cause an autocrine switch in intracellular corticosteroid metabolism by disabling glucocorticoid inactivation (11β-HSD2) while inducing glucocorticoid activation (11β-HSD1). Therefore, it can be postulated that some of the effects of proinflammatory cytokines within bone (e.g., periarticular erosions in inflammatory arthritis) are mediated by this mechanism.
Monitoring of Periprosthetic BMD After Uncemented Total Hip Arthroplasty with Dual-Energy X-Ray Absorptiometry—a 3-Year Follow-Up Study
Authors: Venesmaa Petri K. , Kröger Heikki P. J. , Miettinen Hannu J. A. , Jurvelin Jukka S. , Suomalainen Olavi T. , Alhava Esko M.
Published: April 2006

Insertion of a metallic implant into the femur changes bone loading conditions and results in remodeling of femoral bone. To quantify changes in bone mass after uncemented total hip arthroplasty (THA), we monitored femoral bone with dual-energy X-ray absorptiometry (DXA). The periprosthetic bone mineral density (BMD) was measured with Lunar DPX densitometry in seven Gruen zones and the total periprosthetic area at scheduled time intervals in 22 patients during a 3-year follow-up. BMD decreased significantly almost in all Gruen zones during the first 3 months, ranging from 3.4% to 14.4% (p < 0.05 to p < 0.001). At the end of the first year, the most remarkable decrease in BMD was found in the calcar (zone 7; −22.9%). During the second postoperative year, a slight restoration of periprosthetic bone mass was recorded. During the third year, no significant changes in BMD were found. The preoperative BMD was the only factor that was significantly related to the periprosthetic bone loss. Clearly, the early periprosthetic bone loss noticed during the 3 months after THA is caused by mainly limited weight bearing to the operated hip and stress shielding. We suggest that the restoration of bone mass is a sign of successful osteointegration between bone and metallic implant. DXA is a suitable tool to follow the bone response to prosthetization and will increase our knowledge on the behavior of bone after THA.
Number and Proliferative Capacity of Osteogenic Stem Cells Are Maintained During Aging and in Patients with Osteoporosis
Authors: Stenderup Karin , Justesen Jeannette , Eriksen Erik F. , Rattan Suresh I. S. , Kassem Moustapha
Published: April 2006

Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units-fibroblastic [CFU-Fs]) when cultured in vitro. To examine the effect of aging and osteoporosis on the MSC population, we quantified the number of MSCs and their proliferative capacity in vitro. Fifty-one individuals were studied: 38 normal volunteers (23 young individuals [age, 22-44 years] and 15 old individuals [age, 66-74 years]) and 13 patients with osteoporosis (age, 58-83 years). Bone marrow was aspirated from iliac crest; mononuclear cells were enriched in MSCs by magnetic activated cell sorting (MACS) using STRO-1 antibody. Total CFU-F number, size distribution, cell density per CFU-F, number of alkaline phosphatase positive (ALP+) CFU-Fs, and the total ALP+ cells were determined. In addition, matrix mineralization as estimated by alizarin red S (AR-S) staining was quantified. No significant difference in colony-forming efficiency between young individuals (mean ± SEM; 87 ± 12 CFU-Fs/culture), old individuals (99 ± 19 CFU-Fs/culture), and patients with osteoporosis (129 ± 13 CFU-Fs/culture; p = 0.20) was found. Average CFU-F size and cell density per colony were similar in the three groups. Neither the percentage of ALP+ CFU-Fs (66 ± 6%, 65 ± 7%, and 72 ± 4% for young individuals, old individuals, and patients with osteoporosis, respectively) nor the percentage of ALP+ cells per culture (34 ± 5%, 40 ± 6%, and 41 ± 4%) differed between groups. Finally, mineralized matrix formation was similar in young individuals, old individuals, and patients with osteoporosis. Our study shows that the number and proliferative capacity of osteoprogenitor cells are maintained during aging and in patients with osteoporosis and that other mechanisms must be responsible for the defective osteoblast (OB) functions observed in these conditions.
Genome Screen for Quantitative Trait Loci Underlying Normal Variation in Femoral Structure
Authors: Koller Daniel L. , Liu Guangda , Econs Michael J. , Hui Siu L. , Morin Phillip A. , Joslyn Geoff , Rodriguez Lawrence A. , Conneally P. Michael , Christian Joe C. , Johnston C. Conrad , Foroud Tatiana , Peacock Munro
Published: April 2006

Femoral structure contributes to bone strength at the proximal femur and predicts hip fracture risk independently of bone mass. Quantitative components of femoral structure are highly heritable traits. To identify genetic loci underlying variation in these structural phenotypes, we conducted an autosomal genome screen in 309 white sister pairs. Seven structural variables were measured from femoral radiographs and used in multipoint sib-pair linkage analyses. Three chromosomal regions were identified with significant evidence of linkage (log10 of the odds ratio [LOD] > 3.6) to at least one femoral structure phenotype. The maximum LOD score of 4.3 was obtained for femur neck axis length on chromosome 5q. Evidence of linkage to chromosome 4q was found with both femur neck axis length (LOD = 3.9) and midfemur width (LOD = 3.5). Significant evidence of linkage also was found to chromosome 17q, with a LOD score of 3.6 for femur head width. Two additional chromosomal regions 3q and 19p gave suggestive (LOD > 2.2) evidence of linkage with at least two of the structure phenotypes. Chromosome 3 showed evidence of linkage with pelvic axis length (LOD = 3.1), midfemur width (LOD = 2.8), and femur head width (LOD = 2.3), spanning a broad (60 cm) region of chromosome 3q. Linkage to chromosome 19 was supported by two phenotypes, femur neck axis length (LOD = 2.8) and femur head width (LOD = 2.8). This study is the first genome screen for loci underlying variation in femoral structure and represents an important step toward identifying genes contributing to the risk of osteoporotic hip fracture in the general population.
Transforming Growth Factor-β Induces Osteoclast Ruffling and Chemotaxis: Potential Role in Osteoclast Recruitment
Authors: Pilkington Mary F. , Sims Stephen M. , Dixon S. Jeffrey
Published: April 2006

Transforming growth factor-β (TGF-β) is released from the matrix during bone resorption and has been implicated in the pathogenesis of giant cell tumors of bone and the expansion of breast cancer metastases in bone. Because osteoclasts mediate tumor-induced osteolysis, we investigated whether TGF-β stimulates osteoclast recruitment. Osteoclasts were isolated from rat long bones and time-lapse video microscopy was used to monitor their morphology and motility. Within 5 minutes, TGF-β (0.1 nM) induced dynamic ruffling, with 65% of osteoclasts displaying membrane ruffles compared with 35% in untreated controls. Over a 2-h period, osteoclasts exhibited significant directed migration toward a source of TGF-β, indicating chemotaxis. echistatin, an αvβ3 integrin blocker that inhibits macrophage colony-stimulating factor (M-CSF)-induced osteoclast migration, did not prevent the migration of osteoclasts toward TGF-β. In contrast, a β1 integrin blocking antibody inhibited osteoclast chemotaxis toward TGF-β but not M-CSF. These data indicate the selective use of integrins by osteoclasts migrating in response to different chemotaxins. In addition, wortmannin and U0126 inhibited TGF-β-induced chemotaxis, suggesting involvement of the phosphatidylinositol 3 (PI 3) kinase and mitogen-activated protein (MAP) kinase signaling pathways. Physiologically, TGF-β may coordinate osteoclast activity by recruiting osteoclasts to existing sites of resorption. Pathologically, TGF-β-induced osteoclast recruitment may be critical for expansion of primary and metastatic tumors in bone.
Organization of Cytoskeletal F-Actin, G-Actin, and Gelsolin in the Adhesion Structures in Cultured Osteoclast
Authors: Akisaka Toshitaka , Yoshida Hisaho , Inoue Satoshi , Shimizu Kouichi
Published: April 2006

Immunofluorescence using Gc protein (group-specific component or vitamin D binding protein [DBP]) as a marker of G-actin showed that nonfilamentous, monomeric G-actin is a component of the podosomes of osteoclasts cultured on glass plates or bone slices. Typical individual podosomes of the well-spread cells on glass plates were rosette in form. When viewed from the basolateral surface, the core portion of the dotlike podosomes was associated with packed F-actin filaments surrounded by G-actin organized in a ringlike structure. The podosomes, when viewed perpendicular to the substrate, showed a conical shape as a bundle of short F-actin core and a ring of G-actin. With cell spreading on glass plates, the clustering of the podosomes formed a continuous belt of tightly packed podosomes as an adhesion structure at the paramarginal area. In addition, these structures were seen on the ventral cell surface. Similar changes in cell shape were seen in the osteoclasts when they were plated on bone slices. With the loss of dotlike podosomes, a continuous band of F-actin was formed around the resorption lacunae. It became evident then that F- and G-actin dissociated from each other in the podosomes. The staining patterns of G-actin varied from a discrete dot to a diffuse one. Toward the nonresorption phase, the osteoclasts lost their continuous F-actin band but dotlike podosomes appeared in the leading and the trailing edges. In such a cell undergoing translational movements, G-actin was located diffusely in the cytoplasm behind the lamellipodia and along some segments of the leading edge. Cytochalasin B treatment caused cells to disorganize the actin cytoskeletal architecture, which indicated the disassembling of F-actin into G-actin in podosomes and disappearance of actin-ring of cultured osteoclasts. Staining with polyclonal actin antibody or monoclonal β-actin was overlapped with the distribution pattern of G- and F-actin. Gelsolin was detected in the region of the adhesion area corresponding to the podosome. The observation that F-actin, G-actin, and gelsolin were detected in the osteoclastic adhesion structures suggests that the podosomes may represent sites where a rapid polymerization/depolymerization of actin occurs. These dynamic changes in cytoskeletal organization and reorganization of G-actin may reflect changes in the functional polarization of the osteoclast during the bone resorption cycle and suggest the important role of G-actin in the regulation of osteoclast adhesion.
Targeted Disruption of Cadherin-11 Leads to a Reduction in Bone Density in Calvaria and Long Bone Metaphyses
Authors: Kawaguchi Jitsutaro , Azuma Yoshiaki , Hoshi Kazuto , Kii Isao , Takeshita Sunao , Ohta Tomohiro , Ozawa Hidehiro , Takeichi Masatoshi , Chisaka Osamu , Kudo Akira
Published: April 2006

The migration and adhesion of osteoblasts requires several classical cadherins. Cadherin-11, one of the classical cadherins, was expressed in mouse osteoblasts in skull bone and femur, revealed by immunohistochemistry. To elucidate the function of cadherin-11 in osteoblastogenesis, cadherin-11 null mutant mice were investigated. Although apparently normal at birth, Alizarin red staining of null mutant mice showed a reduced calcified area at the frontal suture that caused a round-shaped calvaria with increasing animal age to 3 months. Consequently, there was a reduction in bone density at the femoral metaphyses and the diploë of calvaria in null mutant mice. In the in vitro culture of newborn calvarial cells, the calcified area of mutant cells was smaller than those derived from wild-type littermates. These results show that absence of cadherin-11 leads to reduced bone density in some parts of skeletons including calvaria and long bone metaphyses, and thus suggest that cadherin-11 plays roles in the regulation of osteoblast differentiation and in the mineralization of the osteoid matrix.
Tumor Necrosis Factor α Induces Expression of Genes for Matrix Degradation in Human Chondrocyte-like HCS-2/8 Cells Through Activation of NF-κB: Abrogation of the Tumor Necrosis Factor α Effect by Proteasome Inhibitors
Authors: Sakai Tadahiro , Kambe Fukushi , Mitsuyama Hirohito , Ishiguro Naoki , Kurokouchi Kazutoshi , Takigawa Masaharu , Iwata Hisashi , Seo Hisao
Published: April 2006

Tumor necrosis factor α (TNF-α) has been suggested to induce chondrocytic chondrolysis in both inflammatory and degenerative joint diseases. However, its intracellular signaling pathway leading to the chondrolysis has not been studied in detail. Thus, we investigated whether TNF-α activates a transcription factor nuclear factor κB (NF-κB) in human chondrocyte-like cells (HCS-2/8) and induces the expression of genes involved in the degradation of cartilage matrix. Treatment of the cells with TNF-α markedly increased the levels of matrix metalloproteinase 1 (MMP-1), MMP-3, intercellular adhesion molecule 1 (ICAM-1), and cyclo-oxygenase 2 (COX-2) messenger RNAs (mRNAs). The increase in the mRNAs was associated with the activation of p65/p50 heterodimer NF-κB. IκB-α and IκB-β, cytoplasmic molecules preventing the nuclear translocation of NF-κB, were degraded rapidly by TNF-α followed by their synthesis to the basal level. Treatment with proteasome inhibitors inhibited the degradation of both IκB-α and IκB-β and prevented the TNF-α-dependent nuclear translocation of p65. Furthermore, the inhibitors completely prevented the TNF-α-dependent induction of MMP-1, MMP-3, ICAM-1, and COX-2 mRNAs. Thus, it is shown that the activation of p65/p50 NF-κB by TNF-α plays a cardinal role in inducing the expression of MMP-1, MMP-3, ICAM-1, and COX-2 genes, which are involved in matrix degradation and inflammatory reaction in chondrocytes, leading to chondrocytic chondrolysis.
Does Body Size Account for Gender Differences in Femur Bone Density and Geometry?
Authors: Looker Anne C. , Beck Thomas J. , Orwoll Eric S.
Published: April 2006

The extent to which greater bone strength in men is caused by proportionately greater bone mass versus bigger bone size is not clear, primarily because the larger overall body size of men has made direct comparisons of skeletal measures difficult. We examined gender differences in femur neck (FN) areal bone mineral density (BMD) values collected from 5623 non‐Hispanic whites aged 20+ years in the third National Health and Nutrition Examination Survey (NHANES III, 1988‐1994) before and after correction for measured height and weight. We supplemented the conventional areal BMD data (Hologic QDR 1000) with measurements of areal BMD and geometric properties (subperiosteal width, section modulus, and cortical thickness) made at narrow “cross‐sectional” regions traversing the FN and the proximal shaft using a structural analysis program. Before body size adjustment, men had significantly higher values than women for all variables at the three measurement sites (p< 0.0001). Adjustment for body size reduced the differences between the sexes for all variables but had a greater effect on BMD (1‐8% higher in men) than on geometry (5‐17% higher in men). When examined by age, the sex discrepancy was significantly greater in the older group for all variables except subperiosteal widths. We conclude that although body size difference may account for most of the areal BMD difference between men and women, male bones are still bigger in ways that suggest greater bone strength. These differences may contribute importantly to lower fracture risk in men.
Increased Body Weight and Decreased Radial Cross-Sectional Dimensions in Girls with Forearm Fractures
Authors: Skaggs David L. , Loro M. Luiza , Pitukcheewanont Pisit , Tolo Vernon , Gilsanz Vicente
Published: April 2006

A large number of children sustain fractures after relatively minor trauma and several investigators have associated these fractures to a deficient accumulation of bone during growth. This study was conducted to better characterize the skeletal phenotype associated with low-energy impact fractures of the forearm in girls. The densities of cancellous, cortical, and integral bone and the cross-sectional area were measured in the radius of 100 healthy white girls (aged 4-15 years) using computed tomography (CT); 50 girls had never fractured and 50 girls had sustained a forearm fracture within the previous month. Fractured and nonfractured groups were matched for age, height, weight, and Tanner stage of sexual development. Compared with controls, girls with fractures had, on average, 8% smaller cross-sectional area at the distal radius (1.82 ± 0.50 cm2 vs. 1.97 ± 0.42 cm2; p < 0.0001) but similar cancellous, integral, and cortical bone densities. Neither radial length nor the amount of fat or muscle at the midshaft of the radius differed between girls with and without fractures. Both study subjects and matched controls were overweight. Although mean height was at the 50th percentile, mean weight was at the 90th percentile for age-adjusted normal values. Girls who sustain forearm fractures after minor trauma have small cross-sectional dimensions of the radius and tend to be overweight. The smaller cross-sectional area confers a biomechanical disadvantage that, coupled with the greater body weight, increases the vulnerability to fracture after a fall.