bluuebunny/crossref_metadata_2025 huggingface dataset:
Reductions in Bone Mass, Structure, and Strength in Axial and Appendicular Skeletons Associated With Increased Turnover After Ovariectomy in Mature Cynomolgus Monkeys and Preventive Effects of Clodronate
Authors: Itoh Fumiaki , Kojima Masami , Furihata-Komatsu Hanako , Aoyagi Shigemi , Kusama Hiroshi , Komatsu Hidetada , Nakamura Toshitaka
Published: April 2006

Over 16 months, we evaluated the effects of ovariectomy (OVX) and bisphosphonate clodronate (CLO) on bone in 48 cynomolgus monkeys (9-15 years old) fed a normal calcium diet. We established three OVX groups(oral CLO at 0 [OVX control], 12, or 60 mg/kg per day) and one sham-operated (SHAM) group. At 16 months, the bone mineral density (BMD) values (percentage of group baseline; OVX control vs. SHAM) for lumbar bone (L3-L5), proximal femur, midfemur, radius, and tibia were −2.6% versus 11.2%, −3.5% versus 8.9%, −3.0% versus 9.0%, −5.5% versus 15.7%, and −6.7% versus 13.9%, respectively. In OVX control (i) tibia showed significant loss of bone mineral content (BMC; vs. baseline), (ii) urinary deoxypyridinoline (DPD) and serum osteocalcin (OC) levels increased (peak = 182% and 168%, respectively, of SHAM), (iii) in lumbar bone and midfemur, ultimate load (UL) was reduced (vs. SHAM), (iv) in lumbar bone, trabecular bone-formation rates (BFRs) were not changed significantly, but tibial endocortical and intracortical bone formation rates were significantly raised (vs. SHAM), (v) the volumetric BMD (vBMD) and geometry of the tibial cortex (measured by peripheral quantitative computed tomography [pQCT]) were significantly reduced (vs. SHAM). CLO, 60 mg/kg per day but not 12 mg/kg per day, significantly inhibited OVX-induced changes, age-dependent increases in bone mass, and ability to maintain structure. Thus, in OVX mature cynomolgus monkeys (possibly, a unique model of the cortical bone loss secondary to estrogen deficiency), the post-OVX increases in systemic bone markers were slight, but stimulation of local turnover in the cortical envelope was enough to cause bone loss (more so in tibia than in lumbar trabecular bone). High-dose CLO prevented these changes.
Two Different Pathways for the Maintenance of Trabecular Bone in Adult Male Mice
Authors: Lindberg Marie K. , Movérare Sofia , Skrtic Stanko , Alatalo Sari , Halleen Jussi , Mohan Subburaman , Gustafsson J-Å , Ohlsson Claes
Published: April 2006

Androgens may regulate the male skeleton either directly via activation of the androgen receptor (AR) or indirectly via aromatization of androgens into estrogen and, thereafter, via activation of estrogen receptors (ERs). There are two known estrogen receptors, ER-α and ER-β. The aim of this study was to investigate the relative roles of ER-α, ER-β, and AR in the maintenance of trabecular bone in male mice. Seven-month-old male mice, lacking ER-α (ERKO), ER-β (BERKO), or both receptors (DERKO), were orchidectomized (orx) and treated for 3 weeks with 0.7 μg/mouse per day of 17β-estradiol or vehicle. No reduction in trabecular bone mineral density (BMD) was seen in ERKO, BERKO, or DERKO mice before orx, showing that neither ER-α nor ER-β is required for the maintenance of a normal trabecular BMD in male mice. After orx, there was a pronounced decrease in trabecular BMD, similar for all groups, resulting in equal levels of trabecular BMD in all genotypes. This reduction was reversed completely in wild-type (WT) and BERKO mice treated with estrogen, and no significant effect of estrogen was found in ERKO or DERKO mice. In summary, the trabecular bone is preserved both by a testicular factor, presumably testosterone acting via AR and by an estrogen-induced activation of ER-α. These results indicate that AR and ER-α are redundant in the maintenance of the trabecular bone in male mice. In contrast, ER-β is of no importance for the regulation of trabecular bone in male mice.
Estrogen Receptor Isoform-Specific Induction of Progesterone Receptors in Human Osteoblasts
Authors: Rickard David J. , Waters Katrina M. , Ruesink Terry J. , Khosla Sundeep , Katzenellenbogen John A. , Katzenellenbogen Benita S. , Riggs B. Lawrence , Spelsberg Thomas C.
Published: April 2006

Estrogen induction of progesterone receptor (PR) expression may be important to bone physiology because progesterone has been implicated in the control of bone formation and resorption. Although PR gene expression can be induced in osteoblasts by estrogen signaling through the estrogen receptor (ER) α isoform, it is unknown whether the ER-β isoform is involved in this regulation. The effect of estrogen on PR expression was examined in human fetal osteoblast (hFOB) cell lines stably transfected with either ER-α or ER-β. Estrogen treatment of hFOB/ER-α cells induced PR messenger RNA (mRNA) steady-state levels after 24 h and protein levels after 48 h, as established by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Interestingly, no induction of PR expression was observed in the hFOB/ER-β cells during this period. However, PR mRNA was induced progressively after 48 h of treatment with estrogen with maximum levels achieved at 12 days posttreatment. ER protein also was increased after 12 days of treatment. Both A and B isoforms of PR (PRA and PRB) were induced by estrogen in the hFOB/ER-α cells as well as much later in hFOB/ER-β cells. The pure antiestrogen ICI 182,780 prevented PR induction by estrogen in both cell lines. An ER-β-selective antagonist R, R-tetrahydrochrysene (THC) abolished the induction of PR mRNA in hFOB/ER-β but not in hFOB/ER-α cells, verifying that the response in the former cell line was ER-β-mediated. Transient cotransfection of hFOB cells with ER-α or ER-β together with either a human PRA or PRB promoter linked to a reporter plasmid revealed that although the PRB promoter was stimulated equally by estrogen activation of either ER isoform, PRA was activated preferentially by ER-α. Together, these results show that although estrogen can up-regulate endogenous PR gene expression in osteoblasts via both ER isoforms, ER-α is the predominant inducer.
The Mode of Mechanical Integrin Stressing Controls Intracellular Signaling in Osteoblasts
Authors: Pommerenke Hagen , Schmidt Christian , Dürr Frieda , Nebe Barbara , Lüthen Frank , Müller Petra , Dr. Rychly Joachim
Published: April 2006

Following the idea that integrin receptors function as mechanotransducers, we applied defined physical forces to integrins in osteoblastic cells using a magnetic drag force device to show how cells sense different modes of physical forces. Application of mechanical stress to the β1-integrin subunit revealed that cyclic forces of 1 Hz were more effective to stimulate the cellular calcium response than continuous load. Cyclic forces also induced an enhanced cytoskeletal anchorage of tyrosine-phosphorylated proteins and increased activation of the focal adhesion kinase (FAK) and mitogen activated protein (MAP) kinase. These events were dependent on an intact cytoskeleton and the presence of intracellular calcium. Analyses of the intracellular spatial organization of the calcium responses revealed that calcium signals originate in a restricted region in the vicinity of the stressed receptors, which indicates that cells are able to sense locally applied stress on the cell surface via integrins. The calcium signals can spread throughout the cell including the nucleus, which shows that calcium also is a candidate to transmit mechanically induced information into different cellular compartments.
Cartilage Calcification Studied by Proton Nuclear Magnetic Resonance Microscopy
Authors: Potter K. , Leapman R. D. , Basser P. J. , Landis W. J.
Published: April 2006

A three-dimensional (3D) mineralizing culture system using hollow fiber bioreactors has been developed to study the early stages of endochondral ossification by proton nuclear magnetic resonance (NMR) microscopy. Chondrocytes harvested from the cephalic half of the sterna from 17-day-old chick embryos were terminally differentiated with 33 nM of retinoic acid for 1 week and mineralization was initiated by the addition of 1% β-glycerophosphate to the culture medium. Histological sections taken after 6 weeks of development in culture confirmed calcification of the cartilage matrix formed in bioreactors. Calcium to phosphorus ratios (1.62-1.68) from X-ray microanalysis supported electron diffraction of thin tissue sections showing the presence of a poorly crystalline hydroxyapatite mineral phase in the cultures. After 4 weeks of culture, quantitative proton NMR images showed water proton magnetization transfer rate constants (km) were higher in premineralized cartilage compared with uncalcified cartilage, a result suggesting collagen enrichment of the matrix. Notably after 5 weeks mineral deposits formed in bioreactors principally in the collagen-enriched zones of the cartilage with increased km values. This caused marked reductions in water proton longitudinal (T1) and transverse (T2) relaxation times and water diffusion coefficients (D). These results support the hypothesis that mineralization proceeds in association with a collagen template. After 6 weeks of culture development, the water proton T2 values decreased by 13% and D increased by 7% in uncalcified areas, compared with the same regions of tissue examined 1 week earlier. These changes could be attributed to the formation of small mineral inclusions in the cartilage, possibly mediated by matrix vesicles, which may play an important role in cartilage calcification. In summary, NMR images acquired before and after the onset of mineralization of the same tissue provide unique insights into the matrix events leading to endochondral mineral formation.
Resistance to Unloading-Induced Three-Dimensional Bone Loss in Osteopontin-Deficient Mice
Authors: Ishijima Muneaki , Tsuji Kunikazu , Rittling Susan R. , Yamashita Teruhito , Kurosawa Hisashi , Denhardt David T. , Nifuji Akira , Noda Masaki
Published: April 2006

Recent development in three-dimensional (3D) imaging of cancellous bone has made possible true 3D quantification of trabecular architecture. This provides a significant improvement in the measures available to study and understand the mechanical functions of cancellous bone. We recently reported that the presence of osteopontin (OPN) was required for the effects of mechanical stress on bone as OPN-null (OPN−/−) mice showed neither enhancement of bone resorption nor suppression of bone formation when they were subjected to unloading by tail suspension. However, in this previous study, morphological analyses were limited to two-dimensional (2D) evaluation. Although bone structure is 3D and thus stress effect should be evaluated based on 3D parameters, no such 3D morphological features underlying the phenomenon have been known. To elucidate the role of OPN in mediating mechanical stress effect based on true quantitative examination of bone, we evaluated 3D trabecular structures of hindlimb bones of OPN−/− mice after tail suspension. Tail suspension significantly reduced 3D parameters of bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and anisotropy and increased 3D parameters on trabecular separation (Tb.Sp) in wild-type mice. In contrast, these 3D parameters were not altered after tail suspension in OPN−/− mice. These data provided evidence that OPN is required for unloading-induced 3D bone loss.
Transforming Growth Factor β2 Inhibits Adipocyte Differentiation Induced by Skeletal Unloading in Rat Bone Marrow Stroma
Authors: Ahdjoudj Souhila , Lasmoles Françoise , Holy Xavier , Zerath Erik , Marie Pierre J.
Published: April 2006

Skeletal unloading induced by hindlimb suspension in rats reduces bone formation and induces osteopenia, but its effect on adipogenesis is unknown. We assessed the effects of unloading and transforming growth factor (TGF) β2 on bone marrow stromal cell adipocyte differentiation in relation with osteoblast differentiation. Skeletal unloading rapidly (4-7 days) decreased osteoblast transcription factor Runx2, osteocalcin (OC), and type I collagen messenger RNA (mRNA) levels and reduced bone formation in the long bone metaphysis. Conversely, unloading increased expression of the adipocyte transcription factor peroxisome proliferator-activated receptor γ2 (PPARγ2) at 4 days and increased expression of the adipocyte differentiation genes lipoprotein lipase (LPL) and aP2 in the bone marrow stroma at 7 days. Consistently, unloading increased the number and volume of adipocytes in the bone marrow stroma. Continuous (0-7 days) and late (4-7 days) treatments with TGF-β2 corrected the abnormal expression of Cbfa1/Runx2, OC, and type I collagen mRNAs and normalized bone formation in unloaded metaphyseal bone. Moreover, both TGF-β2 treatments decreased PPARγ2 and C/EBPα mRNA levels at 4 days and normalized aP2 and LPL expression and adipocyte number and volume at 7 days. These results show that skeletal unloading increases adipocyte differentiation concomitantly with inhibition of osteoblast differentiation. These abnormalities are prevented and reversed by TGF-β2, suggesting a role for TGF-β in the control of adipogenic differentiation in the bone marrow stroma.
Effect of Crohn's Disease on Bone Metabolism In Vitro: A Role for Interleukin-6
Authors: Sylvester Francisco A. , Wyzga Nancy , Hyams Jeffrey S. , Gronowicz Gloria A.
Published: April 2006

Circulating proinflammatory cytokines may be involved in osteopenia associated with Crohn's disease (CD). Therefore, the effect of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF) α contained in Crohn's serum on bone formation was examined in a bone organ culture system. Initially, serum levels of IL-6, IL-1β, and TNF-α were determined by ELISA in newly diagnosed, untreated children with CD and healthy age-matched controls. Serum IL-6 levels were significantly higher in patients with CD than in controls (23.9 ± 2.8 pg/ml vs. 0.7 pg/ml ± 0.2; p < 0.001), whereas IL-1β and TNF-α serum levels were not. In the organ culture studies, 20-day-old fetal rat parietal bones were incubated for 96 h with CD or control serum, serum preincubated with a neutralizing antibody to each cytokine or a nonimmune immunoglobulin control, and with IL-6. Bone formation measured by assaying calcium content and dry weight was significantly decreased in bones exposed to Crohn's serum. Light microscopy of the bones treated with CD serum revealed a discontinuous, uneven mineralized bone matrix and disorganized osteoblasts with altered morphology. Incubation with an antibody that neutralized IL-6 activity prevented the change in osteoblast and bone morphology. TNF-α and IL-1β antibodies had no apparent effects. Collagen synthesis and DNA content were not affected by CD serum. Also, addition of IL-6 to the culture medium decreased mineralization. These results suggest that IL-6 is a mediator of the effects of Crohn's serum on in vitro mineralization and may be a contributing factor to the osteopenia associated with CD.
Adjusting Bone Mass for Differences in Projected Bone Area and Other Confounding Variables: An Allometric Perspective
Authors: Nevill Alan M. , Holder Roger L. , Maffulli Nicola , Cheng Jack C. Y. , Leung Sophie S. S. F. , Lee Warren T. K. , Lau Joseph T. F.
Published: April 2006

The traditional method of assessing bone mineral density (BMD; given by bone mineral content [BMC] divided by projected bone area [Ap], BMD = BMC/Ap) has come under strong criticism by various authors. Their criticism being that the projected bone “area” (Ap) will systematically underestimate the skeletal bone “volume” of taller subjects. To reduce the confounding effects of bone size, an alternative ratio has been proposed called bone mineral apparent density [BMAD = BMC/(Ap)3/2]. However, bone size is not the only confounding variable associated with BMC. Others include age, sex, body size, and maturation. To assess the dimensional relationship between BMC and projected bone area, independent of other confounding variables, we proposed and fitted a proportional allometric model to the BMC data of the L2-L4 vertebrae from a previously published study. The projected bone area exponents were greater than unity for both boys (1.43) and girls (1.02), but only the boy's fitted exponent was not different from that predicted by geometric similarity (1.5). Based on these exponents, it is not clear whether bone mass acquisition increases in proportion to the projected bone area (Ap) or an estimate of projected bone volume (Ap)3/2. However, by adopting the proposed methods, the analysis will automatically adjust BMC for differences in projected bone size and other confounding variables for the particular population being studied. Hence, the necessity to speculate as to the theoretical value of the exponent of Ap, although interesting, becomes redundant.
Universal Standardization of Forearm Bone Densitometry
Authors: Shepherd John A. , Cheng Xiao Guang , Lu Ying , Njeh Chris , Toschke Jorg , Engelke Klaus , Grigorian Michael , Genant Harry K.
Published: April 2006

As part of an effort to quantify device-dependent differences in forearm bone density, 101 women, aged 20-80 years (∼16 women in each age decade), were scanned on six forearm bone densitometers: the Aloka DCS-600EX, the Hologic QDR-4500A, the Lunar PIXI, the Norland pDEXA, the Osteometer DTX-200, and the Pronosco X-posure System. Regression statistics are reported for all similar regions of interest (ROIs). However, comparisons were confounded because of large differences in the ROI size and placement. The number of ROIs reported for a single scan by each device varied from 1 to 12. The correlation coefficients ranged from 0.7 < r < 0.97, with the highest correlation coefficients and lowest SEs for comparisons between the most similar ROIs. Standardized units of bone mineral density are derived for distal (sdBMD), mid- (smBMD), and proximal (spBMD) ROIs that allow for comparable mean bone densities to be derived for patient populations. Five phantoms were scanned and characterized on five of the devices and the precision and mean values were reported. These phantom values will aid in the in vitro cross-calibration between manufacturers to recreate the presented in vivo relationships. Care should be exercised when using these equations for cross-calibrating patient databases or pooling clinical data from different devices because the least significant differences detectable from measurements taken on two different machines can be increased substantially.
Synchrotron Radiation Microtomography Allows the Analysis of Three-Dimensional Microarchitecture and Degree of Mineralization of Human Iliac Crest Biopsy Specimens: Effects of Etidronate Treatment
Authors: Nuzzo S. , Lafage-Proust M. H. , Martin-Badosa E. , Boivin G. , Thomas T. , Alexandre C. , Peyrin F.
Published: April 2006

Quantitative microcomputed tomography using synchrotron radiation (SR μCT) was used to assess the effects of a sequential etidronate therapy on both three-dimensional (3D) microarchitecture and degree of mineralization of bone (DMB) in postmenopausal osteoporosis. Thirty-two iliac crest biopsy specimens were taken from 14 patients with osteoporosis (aged 64 ± 1.8 years) before (baseline) and after 1 year of etidronate treatment, and after 2 years of treatment for four of the patients. The samples were imaged at high spatial resolution (voxel size = 10 μm) using the microtomography system developed at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. Three-dimensional microarchitecture parameters were calculated and compared with those obtained from conventional histomorphometry. In addition, the DMB was evaluated also in 3D. No significant statistical changes regarding bone mass and structural parameters were observed in histomorphometry or 3D analyses. The distribution of the DMB in cortical and trabecular bone showed a trend to a shift toward highest mineralization values after 1 year of etidronate treatment (3.88% and 1.24% in cortical and trabecular bone, respectively). This trend was more evident after 2 years. The study also showed that SR μCT is an accurate technique and the only one for quantifying both the mineralization and the microarchitecture of bone samples at the same time in 3D.
Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In Vivo
Authors: Chikazu Daichi , Li Xiaodong , Kawaguchi Hiroshi , Sakuma Yoko , Voznesensky Olga S. , Adams Douglas J. , Xu Manshan , Hoshi Kazuto , Katavic Vedran , Herschman Harvey R. , Raisz Lawrence G. , Pilbeam Carol C.
Published: April 2006

We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the −300/−213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfa1) consensus sequence (5′-AACCACA-3′) at −267/−261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX-2 knockout (−/−) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2+/+ mice but not in cells from COX-2−/− mice. In vivo, BMP-2 (10 μg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (μCT), was decreased by 78% in COX-2−/− mice compared with COX-2+/+ mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfa1 binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo.
Localization of Nitric Oxide Synthases During Fracture Healing
Authors: Zhu Wei , Murrell George A. C. , Lin Jianhao , Gardiner Edith M. , Diwan Ashish D.
Published: April 2006

Previously, we have reported that nitric oxide synthases (NOSs), which generate NO, modulate fracture healing. However, the cellular sources of the NOS isoforms during the course of fracture healing have not been studied systematically. The purpose of this study was to localize the cellular distribution of NOS isoforms (inducible NOS [iNOS], endothelial NOS [eNOS], and neuronal NOS [bNOS]) by in situ hybridization and immunohistology after femoral fractures in rats. The iNOS signal was detected during the initial stages (on day 4 and day 7) of fracture healing in 52 ± 2% (mean ± SE, n = 7) of cells within the intramembranous region, along the edge of the periosteal callus. The iNOS signal in callus cells declined to an undetectable level on day 14. eNOS was detected during the middle stages (on day 7 and day 14) of fracture healing in cells lining the blood vessels and also in 49 ± 3% of cells in the chondral region. The bNOS signal was found to be increased at the later stages (day 14 and day 21) of fracture healing in 51 ± 3% of cells at the junction between fibrous tissue and cartilage within the fibrochondral region. In summary, the expression of NOS isoforms during fracture healing was time dependent and cellular distinctive.
The Comparative Efficacy of Drug Therapies Used for the Management of Corticosteroid-Induced Osteoporosis: A Meta-Regression
Authors: Amin Shreyasee , Lavalley Michael P. , Simms Robert W. , Felson David T.
Published: April 2006

We determined the comparative efficacy of vitamin D, calcitonin, fluoride, and bisphosphonates for the management of corticosteroid-induced osteoporosis using meta-regression models. A systematic search for trials was conducted using MEDLINE, bibliographic references, abstracts from national meetings, and contact with pharmaceutical companies and content experts. We included all randomized controlled trials, lasting at least 6 months, of adult patients on oral corticosteroids that evaluated treatment comparisons between vitamin D, calcitonin, bisphosphonates, or fluoride either with no therapy/calcium or with each other and that reported extractable results. The outcome measure of interest was change in lumbar spine bone mineral density (BMD). We identified 45 eligible trials, which provided 49 eligible treatment comparisons (some trials had three arms or more). Our results indicated that bisphosphonates were the most effective class (effect size 1.03; 95% CI: 0.85, 1.17); results were similar even when newer generations of nitrogen-containing bisphosphonates were excluded from analysis. We found the efficacy of bisphosphonates was enhanced further when used in combination with vitamin D (effect size, 1.31; 95% CI: 1.07, 1.50). Vitamin D and calcitonin were more effective than no therapy/calcium (effect size, 0.46; 95% CI: 0.27, 0.62; and effect size, 0.51; 95% CI: 0.33, 0.67, respectively) and were of similar efficacy, but both were significantly less effective than bisphosphonates. Fluoride appeared effective, but there were too few studies (n = 5) to draw robust conclusions regarding its efficacy compared with the other three therapies. In summary, bisphosphonates are the most effective of evaluated agents for managing corticosteroid-induced osteoporosis. The efficacy of bisphosphonates is enhanced further with concomitant use of vitamin D.
Insulin‐Like Growth Factor I Effect on the Number of Osteoblast Progenitors Is Impaired in Ovariectomized Mice
Authors: Kasukawa Yuji , Stabnov Lisa , Miyakoshi Naohisa , Baylink David J. , Mohan Subburaman
Published: April 2006

Because insulin‐like growth factor (IGF) I is an important regulator of bone formation, we proposed the hypothesis that IGF‐I could contribute in regulating the number of osteoblast progenitors (colony‐forming unit fibroblast with ALP activity [CFU‐F/ALP+]). To test ex vivo and in vivo effects of IGF‐I on the number of CFU‐F/ALP+, bone marrow cells (BMCs) derived from normal mice, growth hormone (GH)‐deficient lit/lit mice, or ovariectomized (OVX) mice were cultured and the CFU‐F/ALP+ number was counted. Ex vivo treatment of IGF‐I increased the CFU‐F/ALP+ number in a dose‐dependent manner compared with vehicle‐treated control cultures. The CFU‐F/ALP+ number was decreased by 20% (p < 0.01; n = 7−9) in GH‐deficient lit/lit mice compared with age‐matched control mice. Four weeks after OVX or sham operation, IGF‐I (2 μg/g body wt) or vehicle was administered twice on day 1, and 5 days later, BMCs were removed from the femur and cultured for 10 days (n = 9−10 per group). IGF‐I administration increased the CFU‐F/ALP+ number by 63% (p < 0.01) and 19% (NS), respectively, in sham‐operated (sham) and OVX mice compared with the vehicle‐treated control group. The serum IGF‐I level was similar in OVX mice compared with sham mice; this finding is different from that found in rats in which OVX increases the serum IGF‐I level. This study showed that IGF‐I is an important regulator of osteoblast‐progenitor number in the BMCs of mice both ex vivo and in vivo and that the IGF‐I response to increase the number of osteoblast progenitors was impaired in OVX mice.
Long Bones From the Senescence Accelerated Mouse SAMP6 Have Increased Size But Reduced Whole‐bone Strength and Resistance to Fracture
Authors: Silva Matthew J. , Brodt Michael D. , Ettner Sara L.
Published: April 2006

The senescence accelerated mouse strain P6 (SAMP6) has emerged as a useful model of senile osteoporosis because it has many features of the disease, including low trabecular bone formation and low areal bone density. We further characterized the SAMP6 model of senile osteoporosis by comparing morphological, mechanical, and densitometric properties of femurs and tibias from SAMP6 mice to those of the control strain (SAMR1) at 4 months and 12 months of age. SAMP6 long bones had increased periosteal width and endosteal area (p < 0.05), resulting in an average increase of 30% in moments of inertia (p < 0.05), but no difference in bone area (p > 0.05) compared with control. Despite their increased moments of inertia, long bones from SAMP6 mice were relatively weak and brittle. Ultimate bending moment was reduced by 25%, and both postyield displacement and energy‐to‐fracture were reduced by 60% compared with SAMR1 controls (p < 0.001). Average cortical ash fraction was increased slightly from 0.74 in SAMR1 to 0.76 in SAMP6 bones (p < 0.05), indicating that increased mineralization may have contributed to the brittleness of SAMP6 bones. The relative differences we observed—increased endosteal and periosteal dimensions, reduced bending strength, increased brittleness, and increased mineralization—are analogous to changes that occur in the aging human skeleton. Moreover, these features were consistently observed in young (4‐month) and old (12‐month) animals. These findings extend the previous descriptions of the SAMP6 mouse and identify key mechanical features that further validate its relevance as a unique and functionally relevant model of senile osteoporosis.
Low‐Magnitude Mechanical Loading Becomes Osteogenic When Rest Is Inserted Between Each Load Cycle
Authors: Srinivasan Sundar , Weimer David A. , Agans Steven C. , Bain Steven D. , Gross Ted S.
Published: April 2006

Strategies to counteract bone loss with exercise have had fairly limited success, particularly those regimens subjecting the skeleton to mild activity such as walking. In contrast, here we show that it is possible to induce substantial bone formation with low‐magnitude loading. In two distinct in vivo models of bone adaptation, we found that insertion of a 10‐s rest interval between each load cycle transformed a locomotion‐like loading regime that minimally influenced osteoblast activity into a potent anabolic stimulus. In the avian ulna model, the minimal mean (+SE) periosteal labeled surface (Ps.LS) observed in the intact contralateral bones (1.6 ± 1.5%) was doubled after 3 consecutive days of low‐magnitude loading (3.8 ± 1.5%; p = 0.03). However, modifying the regimen by inserting 10 s of rest between each load cycle significantly enhanced the periosteal response (21.9 + 4.5%; p = 0.03). In the murine tibia model, 5 consecutive days of 100 low‐magnitude loading cycles did not significantly alter mean periosteal bone formation rate (BFR) compared with contralateral bones (0.011 ± 0.005 μm3/μm2 per day vs. 0.021 ± 0.013 μm3/μm2 per day). In contrast, separating each of 10 of the same loading cycles with 10 s of rest significantly elevated periosteal BFR (0.167 ± 0.049 μm3/μm2 per day; p = 0.01). Endocortical bone formation parameters were not altered by any loading regimen in either model. We conclude that 10 s of rest between each load cycle of a low‐magnitude loading protocol greatly enhances the osteogenic potential of the regimen.
Long‐Term Predictions of the Therapeutic Equivalence of Daily and Less Than Daily Alendronate Dosing
Authors: Hernandez C. J. , Beaupré G. S. , Marcus R. , Carter D. R.
Published: April 2006

Less than daily alendronate dosing has been identified as an attractive alternative to daily dosing for patients and physicians. A recent 2‐year study found bone mineral density (BMD) changes caused by weekly alendronate dosing therapeutically equivalent to that caused by daily dosing. There are no methods that can be used to predict how long therapeutic equivalence will be maintained after the first 2 years of treatment. In addition, it is unclear if dosing less frequently than weekly also might be therapeutically equivalent to daily dosing. In this study we use a computer simulation to develop predictions of the therapeutic equivalence of daily and less than daily dosing over time periods as long as a decade. The computer simulation uses a cell‐based computer model of bone remodeling and a quantitative description of alendronate pharmacokinetics/pharmacodynamics (PK/PD). The analyses suggest that less than daily dosing regimens do not increase BMD as much as daily dosing. However, model predictions suggest that dosing as frequent as weekly still may be therapeutically equivalent to daily dosing over periods as long as 10 years. In addition, the simulations predict dosing less frequently than weekly may be therapeutically equivalent to daily dosing within the first year of treatment but may not be therapeutically equivalent after 10 years. Hypotheses based on these simulations may be useful for determining which dosing regimen may be most attractive for clinical trials.
Cyclic Adenosine Monophosphate/Protein Kinase A Mediates Parathyroid Hormone/Parathyroid Hormone‐Related Protein Receptor Regulation of Osteoclastogenesis and Expression of RANKL and Osteoprotegerin mRNAs by Marrow Stromal Cells
Authors: Kondo Hisatomo , Guo Jun , Bringhurst F. Richard
Published: April 2006

Parathyroid hormone (PTH) is a major regulator of osteoclast formation and activation, effects that are associated with reciprocal up‐ and down‐regulation of RANKL and osteoprotegerin (OPG), respectively. The roles of specific downstream signals generated by the activated PTH/PTH‐related protein (PTHrP) receptor (PTH1R), such as cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and phospholipase C/protein kinase C (PLC/PKC), in controlling RANKL and OPG expression and osteoclastogenesis remain uncertain. In MS1 conditionally transformed clonal murine marrow stromal cells, which support PTH‐induced osteoclast formation from cocultured normal spleen cells, PTH(1–34) increased RANKL and macrophage colony‐stimulating factor (M‐CSF) mRNA expression and decreased that of OPG when present continuously for 7–20 days at 37°C in the presence of dexamethasone (Dex). In cells precultured for 7 days and then treated with PTH(1–34), similar reciprocal regulation of RANKL and OPG occurred, maximally at 6–24 h, that was of greater amplitude than the changes induced by chronic (7–10 days) PTH exposure. These acute effects of PTH(1–34) were mimicked by PKA stimulators (8‐bromoadenosine [8Br]‐cAMP or forskolin [FSK]), blocked by the PKA inhibitor Rp‐cAMPs but unaffected by the PKC inhibitor GF109203X. Amino‐truncated PTH(1–34) analogs PTH(5–34) and PTH(7–34) neither increased cAMP production in MS1 cells nor regulated RANKL or OPG mRNA. Reciprocal RANKL/OPG mRNA regulation was induced in MS1 cells by PTH(3–34) but only at high concentrations that also increased cAMP. The highly PKA‐selective PTH analog [Gly1,Arg19]human PTH(1–28) exerted effects similar to PTH(1–34) on RANKL and OPG mRNAs and on osteoclast formation, both in MS1/spleen cell cocultures and in normal murine bone marrow cultures. The direct PKC stimulator 12‐O‐tetradecanoylphorbol‐13‐acetate (PMA) did not induce RANKL mRNA in MS1 cells, but it did up‐regulate OPG mRNA and also antagonized osteoclast formation induced by PTH(1–34) in both MS1/spleen cocultures and normal bone marrow cultures. Thus, cAMP/PKA signaling via the PTH1R is the primary mechanism for controlling RANKL‐dependent osteoclastogenesis, although direct PKC activation may negatively regulate this effect of PTH by inducing expression of OPG.
Direct Effect of Acute Metabolic and Respiratory Acidosis on Parathyroid Hormone Secretion in the Dog
Authors: Lopez Ignacio , Aguilera‐Tejero Escolastico , Felsenfeld Arnold J. , Estepa Jose Carlos , Rodriguez Mariano
Published: April 2006

Because both metabolic (Met Acid) and respiratory acidosis (Resp Acid) have diverse effects on mineral metabolism, it has been difficult to establish whether acidosis directly affects parathyroid hormone (PTH) secretion. Our goal was to determine whether acute Met Acid and Resp Acid directly affected PTH secretion. Three groups of dogs were studied: control, acute Met Acid induced by HCl infusion, and acute Resp Acid induced by hypoventilation. EDTA was infused to prevent acidosis‐induced increases in ionized calcium, but more EDTA was needed in Met Acid than in Resp Acid. The PTH response to EDTA‐induced hypocalcemia was evaluated also. Magnesium needed to be infused in groups receiving EDTA to prevent hypomagnesemia. The half‐life of intact PTH (iPTH) was determined during hypocalcemia when PTH was measured after parathyroidectomy. During normocalcemia, PTH values were greater (p < 0.05) in Met Acid (92 ± 19 pg/ml) and Resp Acid (77 ± 22 pg/ml) than in controls (27 ± 5 pg/ml); the respective pH values were 7.23 ± 0.01, 7.24 ± 0.01, and 7.39 ± 0.02. The maximal PTH response to hypocalcemia was greater (p < 0.05) in Met Acid (443 ± 54 pg/ml) than in Resp Acid (267 ± 37 pg/ml) and controls (262 ± 48 pg/ml). The half‐life of PTH was greater (p < 0.05) in Met Acid than in controls, but the PTH secretion rate also was greater (p < 0.05) in Met Acid than in the other two groups. In conclusion, (1) both acute Met Acid and Resp Acid increase PTH secretion when the ionized calcium concentration is normal; (2) acute Met Acid may increase the bone efflux of calcium more than Resp Acid; (3) acute Met Acid acts as a secretogogue for PTH secretion because it enhances the maximal PTH response to hypocalcemia.
Tryptophan Missense Mutation in the Ligand‐Binding Domain of the Vitamin D Receptor Causes Severe Resistance to 1,25‐Dihydroxyvitamin D
Authors: Nguyen T. M. , Adiceam P. , Kottler M. L. , Guillozo H. , Rizk‐Rabin M. , Brouillard F. , Lagier P. , Palix C. , Garnier J. M. , Garabedian M.
Published: April 2006

In this study, two related young children, brother and sister, exhibited severe vitamin D‐resistant rickets without alopecia. Sequence analysis of the total vitamin D receptor (VDR) cDNA from skin fibroblasts revealed a substitution of the unique tryptophan of the VDR by arginine at amino acid 286 (W286R). Cultured skin fibroblasts of the two patients expressed normal‐size VDR protein (immunocytochemistry and Western blotting) and normal length VDR mRNA (Northern blotting). But, these fibroblasts, as well as COS‐7 cells transfected with the W286R mutant, failed to bind 3H 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3]. The tryptophan substitution did not affect VDR trafficking toward the nucleus but abolished the 24‐hydroxylase gene response to 1,25(OH)2D3, even at 10−6 M concentrations. In conclusion, this case report of a new family with hereditary vitamin D‐ resistant rickets (HVDRR) emphasizes the crucial role of the VDR tryptophan for ligand binding and for transactivation of 1,25(OH)2D3 target genes. It clearly shows the clinical significance of this VDR amino acid for calcium homeostasis and bone mineralization. This observation suggests further that the presence of a stable VDR‐bound ligand may not be obligatory for normal hair follicle development.
Biglycan Deficiency Interferes With Ovariectomy-Induced Bone Loss
Authors: Nielsen Karina L , Allen Matthew R , Bloomfield Susan A , Andersen Thomas L , Chen Xiao-Dong , Poulsen Hans S , Young Marian F , Heegaard Anne-Marie
Published: April 2006

Biglycan is a matrix proteoglycan with a possible role in bone turnover. In a 4-week study with sham-operated or OVX biglycan-deficient or wildtype mice, we show that biglycan-deficient mice are resistant to OVX-induced trabecular bone loss and that there is a gender difference in the response to biglycan deficiency.

Introduction: Biglycan (bgn) is a small extracellular matrix proteoglycan enriched in skeletal tissues, and biglycan-deficient male mice have decreased trabecular bone mass and bone strength. The purpose of this study was to investigate the bone phenotype of the biglycan-deficient female mice and to investigate the effect of estrogen depletion by ovariectomy (OVX).

Materials and Methods: OVX or sham operations were performed on 21-week-old mice that were divided into four groups: wt sham (n = 7), wt OVX (n = 9), bgn-deficient sham (n = 10) and bgn-deficient OVX (n = 10). The mice were killed 4 weeks after surgery. Bone mass and bone turnover were analyzed by peripheral quantitative computed tomography (pQCT), biochemical markers, and histomorphometry.

Results and Conclusions: In contrast to the male mice, there were only few effects of bgn deficiency on bone metabolism in female mice, showing a clear gender difference. However, when stressed by OVX, the female bgn knockout (KO) mice were resistant to the OVX-induced trabecular bone loss. The wt mice showed a decrease in trabecular bone mineral density by pQCT measurements, a decrease in trabecular bone volume (BV/TV), and an increase in mineral apposition rate. In contrast, no significant changes were detected in bgn KO mice after OVX. In addition, analysis of the bone resorption marker deoxypyridinoline showed no significant increase in the bgn KO OVX mice compared with bgn KO sham mice. Measurements of serum osteoprotegerin (OPG) and RANKL revealed increased levels of OPG and decreased levels of RANKL in the bgn KO mice compared with wt mice. In conclusion, the bgn deficiency protects against increased trabecular bone turnover and bone loss in response to estrogen depletion, supporting the concept that bgn has dual roles in bone, where it may modulate both formation and resorption ultimately influencing the bone turnover process.
Shock Wave Application Enhances Pertussis Toxin Protein-Sensitive Bone Formation of Segmental Femoral Defect in Rats
Authors: Chen Yeung-Jen , Kuo Yur-Ren , Yang Kuender D , Wang Ching-Jen , Huang Hue-Chen , Wang Feng-Sheng
Published: April 2006

Extracorporeal shock waves (ESWs) elicit a dose-dependent effect on the healing of segmental femoral defects in rats. After ESW treatment, the segmental defect underwent progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Along with the intensive bone formation, there was a persistent increase in TGF-β1 and BMP-2 expression. Pretreatment with pertussis toxin reduced ESW-promoted callus formation and gap healing, which presumably suggests that Gi proteins mediate osteogenic signaling.

Introduction: Extracorporeal shock waves (ESWs) have previously been used to promote bone repair. In our previous report, we found that ESWs promoted osteogenic differentiation of mesenchymal cells through membrane perturbation and activation of Ras protein. In this report, we show that ESWs elicit a dose-dependent effect on the healing of segmental defects and that Gi proteins play an important role in mediating ESW stimulation.

Materials and Methods: Rats with segmental femoral defects were subjected to ESW treatment at different energy flux densities (EFD) and impulses. Bone mass (mineral density and calcium content), osteogenic activities (bone alkaline phosphatase activity and osteocalcin content), and immunohistochemistry were assessed.

Results: An optimal ESW energy (500 impulses at 0.16 mJ/mm2 EFD) stimulated complete bone healing without complications. ESW-augmented healing was characterized by significant increases (p < 0.01) in callus size, bone mineral density, and bone tissue formation. With exposure to ESW, alkaline phosphatase activity and osteocalcin production in calluses were found to be significantly enhanced (p < 0.05). After ESW treatment, the histological changes we noted included progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Intensive bone formation was associated with a persistent increase in transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) expression, suggesting both growth factors were active in ESW-promoted bone formation. We also found that pertussis toxin, an inhibitor of membrane-bound Gi proteins, significantly reduced (p < 0.01) ESW promotion of callus formation and fracture healing.

Conclusion: ESW treatments enhanced bone formation and the healing of segmental femoral defects in rats. It also seems likely that TGF-β1 and BMP-2 are important osteogenic factors for ESW promotion of fracture healing, presumably through Gi protein-mediated osteogenic signaling.
Bone Histomorphometric Changes After Liver Transplantation for Chronic Cholestatic Liver Disease
Authors: Guichelaar Maureen MJ , Malinchoc Michael , Sibonga Jean D , Clarke Bart L , Hay J Eileen
Published: April 2006

Thirty-three patients with cholestatic liver disease underwent histomorphometric assessment of paired bone biopsy specimens at time of orthotopic liver transplantation (OLT) and 4 months thereafter. At 4 months after OLT, bone metabolism improved, with bone formation increasing to normal and no change in bone resorption. Early post-transplant bone loss may be attributed to an additional insult to bone formation early after transplantation.

Introduction: Patients with advanced liver disease, especially chronic cholestasis, often have osteopenia, which worsens early after orthotopic liver transplantation (OLT) before starting to recover. The changes in bone metabolism leading to this rapid loss of bone after OLT, and to its recovery, are poorly defined.

Materials and Methods: In thirty-three patients with advanced chronic cholestatic liver disease, tetracycline-labeled bone biopsy specimens were analyzed prospectively at time of OLT and at 4 months after OLT, as part of a randomized trial to study the efficacy of calcitonin on post-transplant bone loss. Hierarchical cluster analysis of histomorphometric parameters was performed in an attempt to establish the functional grouping of individual histomorphometric parameters before and after OLT.

Results and Conclusions: Results showed that from the time of OLT to 4 months after OLT, bone mineral density of the lumbar spine and histomorphometric parameters of bone volume decreased, consistent with early post-transplant bone loss. Histomorphometric resorption parameters were increased before OLT, with no change after OLT. Histomorphometric formation parameters increased from low values before OLT to normal values at 4 months after OLT, with the exception of mean wall thickness values, which further decreased after OLT, suggesting an additional insult to bone formation during the study period. Histomorphometric changes after OLT were similar in female and male patients, pre- and postmenopausal women, and in patients treated and not treated with calcitonin. Hierarchical cluster analysis suggested that before OLT, bone resorption was functioning independently of bone formation, but that by 4 months after OLT, their coupled relationship had improved. Therefore, despite post-transplant bone loss, by 4 months after OLT, bone metabolism had improved, with increased bone formation and more coupled bone balance, as suggested by hierarchical cluster analysis.