bluuebunny/crossref_metadata_2025 huggingface dataset:
Alendronate Does Not Block the Anabolic Effect of PTH in Postmenopausal Osteoporotic Women
Authors: Cosman Felicia , Nieves Jeri , Woelfert Lillian , Shen Victor , Lindsay Robert
Published: April 2006

In rodent osteoporosis models, anabolic activity of parathyroid hormone (PTH) is preserved in the presence of antiresorptive agents. Anabolic activity is also preserved when PTH is administered to estrogenized postmenopausal women. In contrast, in the ewe treated with tiludronate, PTH-induced stimulation of bone turnover did not occur. To determine whether PTH in combination with alendronate could be a viable treatment for osteoporosis, we performed a short-term study of postmenopausal women with osteoporosis (n = 10) already on alendronate 10 mg/day to determine whether PTH could increase bone formation assessed biochemically. Patients continued alendronate alone (n = 5) or continued alendronate with 400 IU/day subcutaneous human PTH(1–34) added for 6 weeks. Subjects receiving PTH had serum and urine sampling weekly during PTH treatment and for 5 weeks thereafter. Sampling was performed approximately biweekly for subjects who had been on alendronate alone for 11 weeks. Samples were analyzed for osteocalcin (OC), propeptide of type I procollagen (PICP), bone-specific alkaline phosphatase (BSAP), cross-linked urinary N-telopeptide (NTX), and free urinary pyridinoline (PYD). Markers of bone formation increased within 3 weeks in the PTH plus alendronate group, with mean peak levels at 5–7 weeks: OC 49%, p < 0.01; PICP 61%, p < 0.01; and BSAP 24%, p = 0.12. Levels returned to baseline after discontinuing PTH, with PICP declining the most rapidly. There were no significant changes at any time in the alendronate alone group. There were no increments in either urinary NTX or PYD in either treatment group throughout the observation period. The bone turnover marker changes seen with PTH plus alendronate were similar to those seen with PTH plus hormone replacement. These data suggest that: PTH can stimulate bone formation, evidenced by elevations of bone formation markers, even in the presence of a potent bisphosphonate; in the presence of alendronate, PTH-stimulated bone formation precedes stimulation of bone resorption, suggesting that PTH stimulates bone formation de novo; and the combination of PTH and alendronate may be a viable treatment option for postmenopausal women with osteoporosis.
Recombinant Human Basic Fibroblast Growth Factor Accelerates Fracture Healing by Enhancing Callus Remodeling in Experimental Dog Tibial Fracture
Authors: Nakamura Toshiyuki , Hara Yasushi , Tagawa Masahiro , Tamura Makoto , Yuge Takuro , Fukuda Hiroshi , Nigi Hideo
Published: April 2006

Effect of recombinant human basic fibroblast growth factor (bFGF) on fracture healing was investigated using a tibial fracture in beagle dogs. Transverse fractures in the middle of the diaphyses were created in the right tibiae and bFGF was injected into the fracture sites at a single dose of 200 μg. The time course of changes in callus volume and morphology of the fracture sites were evaluated at weeks 2, 4, 8, 16, and 32 after treatment, and the fracture strength was analyzed at weeks 16 and 32. At week 2, a radiogram of the fracture site showed obvious membranous ossification in the group injected with bFGF. Basic FGF extended the callus area at week 4 and increased the bone mineral content (BMC) in the callus at week 8. bFGF also increased the osteoclast number in the periosteal callus at weeks 2 and 4. In the bFGF group, a maximal increase in the osteoclast index was found at week 4, and an identical increase was recognized in the control group at weeks 8 and 16. These findings strongly suggested that bFGF stimulated not only callus formation but osteoclastic callus resorption. BMC in the bFGF group was followed by a rapid decrease from week 8, while that in the control group was identical from week 4. Fracture strength of the bFGF group showed significant recovery by week 16, and recovery was still evident by week 32. We conclude that bFGF promotes the fracture healing in dogs by the stimulation of bone remodeling.
No Difference in Intestinal Strontium Absorption After an Oral or an Intravenous 1,25(OH)2D3 bolus in Normal Subjects
Authors: Bianchi M. L. , Ardissino G. L. , Schmitt C. P. , Daccó V. , Barletta L. , Claris-Appiani A. , Mehls O. ,   FOR THE EUROPEAN STUDY GROUP ON VITAMIN D IN CHILDREN WITH RENAL FAILURE
Published: April 2006

It has been suggested that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) stimulates intestinal calcium absorption less via the intravenous (iv) than the oral route, because the first avoids direct contact of the drug with the enterocytes. However, no study has addressed the issue directly. This investigation was designed to measure the effect of a single oral or iv dose of 1,25(OH)2D3 on calcium absorption, using stable strontium (Sr) as a surrogate for calcium, and measuring the Sr fractional absorbed dose (FAD%) over 240 minutes after Sr administration. In 10 healthy volunteers, five tests were performed in a cross-over design, with a wash-out period between two consecutive tests: Sr absorption without 1,25(OH)2D3 (test A); Sr absorption immediately after either oral (test B) or iv (test C) 1,25(OH)2D3 (1.5 μg/m2 of body surface area [BSA]); Sr absorption (24 hr after either oral (test D) or iv (test E) 1,25(OH)2D3 (1.5 μg/m2 BSA). The concurrent administration of 1,25(OH)2D3 and Sr (tests B and C) did not significantly change the area under the Sr FAD%–time curve with respect to test A (test A: 4090 ± 345; test B: 4510 ± 345; test C: 4210 ± 345), whereas Sr absorption was significantly increased (p < 0.001) when Sr was given 24 hr after either oral or iv 1,25(OH)2D3 (test D: 5710 ± 345; test E: 5510 ± 345). It was concluded that 1,25(OH)2D3 is likely to influence calcium absorption significantly only via its genomic effect, independent of its administration route.
Recurrence of Vertebral Fracture with Cyclical Etidronate Therapy in Osteoporosis: Histomorphometry and X-Ray Microanalysis Evaluation
Authors: Thomas Thierry , Barou Odile , Vico Laurence , Alexandre Christian , Lafage-Proust Marie-Helene
Published: April 2006

In an open prospective study, we evaluated differences between patients with (wRVF group) and without recurrence of vertebral fracture (woRVF group) during cyclical etidronate therapy for osteoporosis. Thirty-two patients (age 64 ± 1.8 years) characterized by at least one osteoporotic VF were treated during 1 year. At baseline, body mass index was significantly lower (23.3 ± 0.6 vs. 26.9 ± 1.0 kg/m2, p< 0.05), the number of previous VFs was higher (4.0 ± 0. 4 vs. 2.4 ± 0.4, NS), and patients were older in the wRVF group as compared with the woRVF group (67.8 ± 3 vs. 62.6± 2.2 year, NS). Trabecular bone volume (11.6 ± 1.2 vs. 15 ± 0.9%, p< 0.05) and trabecular number (1.06 ± 0.08 vs. 1.27 ± 0.05, p < 0.05) were significantly lower in the wRVF group. None of the baseline resorptive variables differed, whereas the bone formation rate (BFR) was 2-fold lower in the wRVF group (p< 0. 05). After 1 year of treatment, osteoclast number, active eroded surfaces, and resorption depth dramatically decreased in both groups (p< 0.01). To a lesser extent, the mineral apposition rate and serum alkaline phosphatase level were significantly reduced (p< 0.05). No impaired mineralization was observed. Using X-ray microanalysis, we found no abnormality in bone mineral but a significant increase of the calcium/phosphorus ratio during treatment in the wRVF group. Our results demonstrate that recurrence of VFs within the first year of cyclical etidronate therapy was related neither to a lack of histologic response to the treatment nor induction of an abnormality of mineralization. VFs were more likely in the presence of a decreased BFR and lower trabecular connectivity, providing support for treating osteoporotic patients with etidronate early in the course of the disease.
Mechanism of Bone Loss After Liver Transplantation: A Histomorphometric Analysis
Authors: Vedi Shobna , Greer Shaun , Skingle Susan J. , Garrahan Nigel J. , Ninkovic Mary , Alexander Graeme A. , Dr. Compston Juliet E.
Published: April 2006

Organ transplantation is associated with increased bone loss and high fracture risk, but the pathophysiological mechanisms responsible have not been established. We have performed a histomorphometric analysis of bone remodeling before and 3 months after liver transplantation in 21 patients (14 male, 7 female) aged 38–68 years with chronic liver disease. Eight-micrometer undecalcified sections of trans-iliac biopsies were assessed using image analysis. Preoperatively, bone turnover was low with a tendency toward reduced wall width and erosion depth. The bone formation rate increased from 0.021 ± 0.016 (mean ± SD) to 0.067 ± 0.055 μm2/μm/day after transplantation (p < 0.0002) and activation frequency from 0.24 ± 0.21/year−1 to 0.81 ± 0.67/year−1 (p < 0.0001). No significant change was observed in wall width, but there was a trend toward an increase in indices of resorption cavity size. There was a small increase in osteoid seam width postoperatively (p< 0.02) and decrease in mineralization lag time (p < 0.001). No significant changes in indices of cancellous bone structure were observed in the postoperative biopsies. These results demonstrate a highly significant and quantitatively large increase in bone turnover in the first 3 months after liver transplantation. Although no significant disruption of cancellous bone structure was demonstrated during the time course of the study, the observed changes in bone remodeling predispose to trabecular penetration and may thus result in long-term adverse effects on bone strength.
Vitamin D Receptor Gene Fok1 Polymorphism Predicts Calcium Absorption and Bone Mineral Density in Children
Authors: Ames Sharla K. , Ellis Kenneth J. , Gunn Sheila K. , Copeland Kenneth C. , Abrams Steven A.
Published: April 2006

The vitamin D receptor (VDR) gene has been implicated as one of the major genetic components of osteoporosis. We evaluated the relationship between markers of mineral status and restriction fragment length polymorphisms of the VDR gene in 72 healthy children age 7–12 years. Using stable isotope techniques and dual-energy X-ray absorptiometry, we measured dietary calcium absorption, bone calcium deposition rates, and total body bone mineral density (BMD). The Fok1 polymorphism at the VDR translation initiation site was significantly associated with BMD (p = 0.02) and calcium absorption (p = 0.04). Children who were FF homozygotes had a mean calcium absorption that was 41.5% greater than those who were ff homozygotes and 17% greater absorption than Ff heterozygotes. BMD was 8.2% greater in the FF genotype than the ff genotype and 4.8% higher than the Ff genotype. These results suggest a substantial relationship between the VDR gene and bone metabolism at one or more levels, including dietary absorption of calcium and BMD in growing children.
Developmental Expression of Creatine Kinase Isoenzymes in Chicken Growth Cartilage
Authors: Hobson Grace M. , Funanage Vicky L. , Elsemore Jennifer , Yagami Machiko , Rajpurohit Ramesh , Perriard Jean-Claude , Hickok Noreen J. , Shapiro Irving M. , Tuan Rocky S.
Published: April 2006

We have shown previously that creatine kinase (CK) activity is required for normal development and mineralization of chicken growth cartilage and that expression of the cytosolic isoforms of CK is related to the biosynthetic and energy status of the chondrocyte. In this study, we have characterized changes in isoenzyme activity and mRNA levels of CK (muscle-specific CK, M-CK; brain-type CK, B-CK; and mitochondrial CK subunits, MiaCK and MibCK) in the growth plate in situ and in chondrocyte culture systems that model the development/maturation program of the cartilage. The in vitro culture systems analyzed were as follows: tibial chondrocytes, which undergo hypertrophy; embryonic cephalic and caudal sternal chondrocytes, which differ from each other in their mineralization response to retinoic acid; and long-term micromass cultures of embryonic limb mesenchymal cells, which recapitulate the chondrocyte differentiation program. In all systems analyzed, B-CK was found to be the predominant isoform. In the growth plate, B-CK expression was highest in the most calcified regions, and M-CK was less abundant than B-CK in all regions of the growth plate. In tibial chondrocytes, an increase in B-CK expression was seen when the cells became hypertrophic. Expression of B-CK increased slightly over 15 days in mineralizing, retinoic acid–treated cephalic chondrocytes, but it decreased in nonmineralizing caudal chondrocytes, while there was little expression of M-CK. Interestingly, in limb mesenchyme cultures, significant M-CK expression was detected during chondrogenesis (days 2–7), whereas hypertrophic cells expressed only B-CK. Finally, expression of MiaCK and MibCK was low both in situ and in vitro. These observations suggest that the CK genes are differentially regulated during cartilage development and maturation and that an increase in CK expression is important in initiating chondrocyte maturation.
Parathyroid Hormone Regulates the Expression of Fibroblast Growth Factor-2 mRNA and Fibroblast Growth Factor Receptor mRNA in Osteoblastic Cells
Authors: Hurley Marja M. , Tetradis Sotirios , Huang Yu-Feng , Hock Janet , Kream Barbara E. , Raisz Lawrence G. , Sabbieti Maria Giovanna
Published: April 2006

We examined the effect of parathyroid hormone (PTH) on basic fibroblast growth factor-2 (FGF-2) and FGF receptor (FGFR) expression in osteoblastic MC3T3-E1 cells and in neonatal mouse calvariae. Treatment of MC3T3-E1 cells with PTH(1–34) (10–8M) or forskolin (FSK; 10–5M) transiently increased a 7 kb FGF-2 transcript with a peak at 2 h. The PTH increase in FGF-2 mRNA was maintained in the presence of cycloheximide. PTH also increased FGFR-1 mRNA at 2 h and transiently increased FGFR-2 mRNA at 1 h. FGFR-3 and FGFR-4 mRNA transcripts were not detected in MC3T3-E1 cells. In cells transiently transfected with an 1800-bp FGF-2 promoter-luciferase reporter, PTH and FSK increased luciferase activity at 2 h and 4 h. Immunohistochemistry showed that PTH and FSK increased FGF-2 protein labeling in the nuclei of MC3T3-E1 cells. PTH also increased FGF-2 mRNA, and FGFR-1 and FGFR-2 mRNA levels within 30 minutes in neonatal mouse calvarial organ cultures. We conclude that PTH and cAMP stimulate FGF-2 mRNA abundance in part through a transcriptional mechanism. PTH also regulated FGFR gene expression. We hypothesize that some effects of PTH on bone remodeling may be mediated by regulation of FGF-2 and FGFR expression in osteoblastic cells.
Prevalent Vertebral Deformities Predict Hip Fractures and New Vertebral Deformities but Not Wrist Fractures
Authors: Black Dennis M. , Arden Nigel K. , Palermo Lisa , Pearson Jim , Cummings Steven R. ,   for the Study of Osteoporotic Fractures Research Group
Published: April 2006

Although vertebral deformities are known to predict future vertebral deformities, little is known about their ability to predict other osteoporotic fractures. We examined the association between prevalent vertebral deformities and incident osteoporotic fractures in the Study of Osteoporotic Fractures, a prospective study of 9704 women aged 65 years and older. Prevalent vertebral deformities were determined morphometrically from spinal radiographs at baseline and incident deformities from repeat spinal radiographs after a mean of 3.7 years. Appendicular fractures were collected by postcard every 4 months for a mean of 8.3 years. During follow-up, 389 women with new vertebral deformities, 464 with hip fractures, and 574 with wrist fractures were identified. Prevalent vertebral deformities were associated with a 5-fold increased risk (relative risk 5.4, 95% confidence interval [CI] 4.4, 6.6) of sustaining a further vertebral deformity; the risk increased dramatically with both the number and severity of the prevalent deformities. Similarly, the risks of hip and any nonvertebral fractures were increased with baseline prevalent deformity, with relative risks of 2.8 (95% CI 2.3, 3.4) and 1.9 (95% CI 1.7, 2.1), respectively. Risk increased with number and severity of deformities. These associations remained significant after adjustment for age and calcaneal bone mineral density (BMD). Although there was a small increased risk of wrist fracture, this was not significant after adjusting for age and BMD. In conclusion, the presence of prevalent morphometrically defined vertebral deformities predicts future vertebral and nonvertebral fractures, including hip but not wrist fractures. Spinal radiographs identifying prevalent vertebral deformities may be a useful additional measurement to classify further a woman's risk of future fracture.
Failure to Detect the Extracellular Calcium-Sensing Receptor (CasR) in Human Osteoblast Cell Lines
Authors: Pi Min , Hinson Todd K. , Quarles L. Darryl
Published: April 2006

Whether the known calcium-sensing receptor (CasR) is present in osteoblasts is a source of considerable controversy. Prior studies failed to detect CasR in osteoblasts, but more recent investigations purport the detection of CasR in several osteoblast cell lines by immunoblot analysis with polyclonal anti-CasR antisera (4637) and low stringency reverse transcriptase-polymerase chain reaction (RT-PCR). To explain these disparate findings, we performed immunoblot analysis with the 4637 anti-CasR antisera and a highly specific monoclonal antibody to CasR (ADD), and we compared the ability of low and high stringency RT-PCR to amplify CasR transcripts. We found that the ADD antibody detected the anticipated CasR immunoreactive bands, including a ∼165 kDa and ∼140 kDa glycosylated doublet and a >250 kDa dimerized receptor, in positive control mouse kidney, human parathyroid, and human embryonic kidney (HEK) 293 cells transfected with rat CasR, but we did not detect these bands in either wild-type HEK 293 cells or Saos2, MG-63, or U-2 OS osteoblast-like cell lines. Standard two-step RT-PCR using CasR-specific primers confirmed these results by detecting CasR transcripts in positive controls but not in negative control HEK 293 cells or osteoblast cell lines. In contrast, the 4637 antisera did not recognize CasR by immunoblot analysis under the conditions studied and our low stringency RT-PCR procedure amplified nonspecific products in wild-type HEK 293 cells and osteoblasts. Since we failed to detect CasR in human osteoblast cell lines using either the highly specific ADD antibody or RT-PCR under standard conditions, it is possible that the cation response in osteoblasts is mediated by a functionally similar but molecularly distinct calcium sensing receptor.
Polarity of Osteoblasts and Osteoblast-like UMR-108 Cells
Authors: Ilvesaro Joanna , Metsikkö Kalervo , Väänänen Kalervo , Tuukkanen Juha
Published: April 2006

Enveloped viruses, such as vesicular stomatitis virus (VSV) and Influenza virus, have been widely used in studying epithelial cell polarity. Viral particles of VSV-infected epithelial cells bud from the basolateral membrane, which is in contact with the internal milieu and the blood supply. Influenza-infected cells bud viral particles from the apical surface facing the external milieu. This feature can be utilized in labeling polarized membrane domains. We studied the polarity of mesenchymal osteoblasts using osteosarcoma cell line UMR-108 and endosteal osteoblasts in situ in bone tissue cultures. Immunofluorescence confocal microscopy revealed that the VSV glycoprotein (VSV G) was targeted to the culture medium–facing surface. In endosteal osteoblasts, VSV G protein was found in the surface facing bone marrow and circulation. On the contrary, Influenza virus hemagglutinin (HA) was localized to the bone substrate-facing surface of the UMR-108 cells. Electron microscopy showed that in the cases where the cells were growing as a single layer, VSV particles were budding from the culture medium–facing surface, whereas Influenza viruses budded from the bone substrate–facing surface. When the cells overlapped, this polarity was lost. Cell surface biotinylation revealed that 55% of VSV G protein was biotinylated, whereas Influenza virus HA was only 22% biotinylated. These findings suggest that osteoblasts are polarized at some point of their life cycle. The bone-attaching plasma membrane of osteoblasts is apical, and the circulation or bone marrow–facing plasma membrane is basolateral in nature.
Urinary Galactosyl-Hydroxylysine in Postmenopausal Osteoporotic Women: A Potential Marker of Bone Fragility
Authors: Cascio Vincenzo Lo , Bertoldo Francesco , Gambaro Greta , Gasperi Elisabetta , Furlan Federico , Colapietro Francesca , Cascio Claudia Lo , Campagnola Mario
Published: April 2006

Alterations of the collagen matrix, e.g., increased hydroxylation and glycosylation of lysyl residues in collagen I, were found in human osteoporotic bone, and it was suggested that they could alter the mechanical properties of skeleton. To test this hypothesis, we evaluated the content of galactosyl-hydroxylysine (GHYL) in bone collagen, as assessed by its urinary excretion, and related it to the occurrence of fracture. Two hundred and fifteen unselected postmenopausal women with osteoporosis were divided in two subgroups (comparable for age, age of menopause, bone mineral density, and biochemical parameters of bone turnover) on the basis of the history of fragility fracture; 115 patients had suffered no fracture and 100 patients had suffered one or more fractures 3 or more years before. Four urinary markers of bone turnover (hydroxyproline, cross-linked N-telopeptide, free deoxypyridoline, and GHYL) were evaluated in all patients. There was no difference between the two groups with regard to all the parameters studied except for GHYL, which was significantly higher in the group with a history of fracture (1.35 ± 0.82 mmol/mol of creatinine [Cr] versus 1.03 ± <0.48 mmol/mol Cr, p < 0.001); this marker did not correlate with other markers of bone remodeling in the fracture group, indicating a possible defect in bone collagen. In conclusion, provided that increased levels of urinary GHYL do reflect overglycosylation of hydroxylysine in bone collagen, the GHYL may be considered a marker of bone collagen quality. Our results, showing higher urinary GHYL in osteoporosis patients with fracture, seem to confirm this suggestion.
Biological Properties of Salmon Calcitonin IV
Authors: Schecroun Nadia , Pidoux Elisabeth , Horne William C. , Hernandez-Lagunas Laura , Baron Roland , Dr. Cressent Michele
Published: April 2006

In this study we characterized the biological activity of the recently identified salmon calcitonin (sCT) IV, in order to evaluate its potential therapeutic value. In the rat bioassay, sCT IV exhibited a 30% higher hypocalcemic activity than sCT I. The capacity of the molecule to inhibit bone resorption was assessed in vitro by the bone resorbing assay and the pit assay. An inhibitory effect, similar to that of sCT I, was observed in both assays. The interaction of sCT IV with the rabbit CT receptor was also studied. The affinity of sCT IV for the receptor was similar to that of sCT I, as was the potency for stimulating cAMP production. The antigenicity of the two molecules was not identical. Thus, this new CT could represent a useful novel therapeutic agent for the treatment of bone disorders.
Cardiac L-Type Calcium Channel α1-Subunit Is Increased by Cyclic Adenosine Monophosphate: Messenger RNA and Protein Expression in Intact Bone
Authors: Wang Xi-Tao , Nagaba Shizuka , Nagaba Yasushi , Leung Steven W. , Wang Jinsong , Qiu Weiping , Zhao Pei-Lin , Guggino Sandra E.
Published: April 2006

L-type calcium channels have been identified previously in both osteoblast-like osteosarcoma cell lines and primary cultures of osteoblasts using numerous techniques such as patch clamp recording, drug inhibited45Ca2+ uptake, and Fura-2 measurements, but intact bone has not been investigated. Using reverse-transcription polymerase chain reaction (RT-PCR) we found that the three major isoforms of the α1-subunit of L-type calcium channels, (α1C, α1D, and α1S) are present in RNA extracted from ROS 17/2.8 osteosarcoma cells, rat femur, and rat skull. Sequencing of most of the α1C-subunit from rat femur and ROS cells revealed that the splice variants in osteosarcoma cells and intact bone differ, but there are no unique sequence variations compared with those found in other tissues. Northern blot analysis of ROS cell RNA indicated that cyclic adenosine monophosphate (cAMP), but not 1α,25-dihydroxyvitamin D3, increased the messenger RNA (mRNA) of the α1C-subunit. Western blot of ROS cell lysates revealed a band of more then 220 kDa, the amount of which increased in cells treated with cAMP. Using confocal microscopy combined with immunohistochemistry in ROS cells, intact bone, and cartilage, we found that the α1C-subunit of this channel is expressed in osteoblasts and chondrocytes suggesting this channel may be a pathway for signal transduction in intact tissue, because it is in osteosarcoma cell lines and primary osteoblasts grown in tissue culture.
Structure, Composition, and Maturation of Newly Deposited Calcium-Phosphate Crystals in Chicken Osteoblast Cell Cultures
Authors: Kuhn Liisa T. , Wu Yaotang , Rey Christian , Gerstenfeld Louis C. , Grynpas Marc D. , Ackerman Jerome L. , Kim Hyun-Man , Dr. Glimcher Melvin J.
Published: April 2006

Characterization of the very early calcium phosphate (CaP) crystals deposited in bone or in osteoblast cell cultures has been hampered by the overwhelming presence of organic matrix components and cells that obscure spectral analyses. We have overcome this problem using isolated protein-free crystals and have obtained new data including31P nuclear magnetic resonance (NMR) spectra for the first time from mineral crystals deposited during osteoblast calcification in culture. Crystals were isolated from cultures at two time points: (a) at first calcium accumulation (day 8–10) and (b) after 60 days of culture, to assess maturational changes. The analyses show that the chemical composition overall and short range order of the early and mature crystals are characteristic of the apatite crystals found in young embryonic chick bone in vivo. No mineral phase other than apatite was detected by any of the methods used.31P NMR spectroscopy identified the HPO4 groups as those present in bone apatite. Similar to bone apatites, no OH groups were detected by Fourier transform infrared (FTIR) spectroscopy. The temporal maturational changes in composition and structure of the mineral phase were difficult to assess because of the continuous deposition of crystals throughout culturing. The pathway of the maturational changes observed were similar to those occurring in chick bone in vivo and synthetic apatite crystals in vitro although to a much smaller extent.
Spaceflight Inhibits Bone Formation Independent of Corticosteroid Status in Growing Rats
Authors: Zerath Erik , Holy Xavier , Roberts Sydney G. , Andre Catherine , Renault Sylvie , Hott Monique , Marie Pierre J.
Published: April 2006

Bone formation and structure have been shown repeatedly to be altered after spaceflight. However, it is not known whether these changes are related to a stress-related altered status of the corticosteroid axis. We investigated the role of corticosteroids on spaceflight-induced effects in rat pelvis and thoracic vertebrae. Thirty-six male Sprague-Dawley rats were assigned to a flight, flight control, or vivarium group (n = 12/group). Bilateral adrenalectomy was performed in six rats per group, the additional six rats undergoing sham surgery. Adrenalectomized (ADX) rats were implanted with corticosteroid pellets. On recovery from spaceflight, thoracic vertebrae and the whole pelvis were removed and processed for biochemistry, histomorphometry, or bone cell culture studies. The 17-day spaceflight resulted in decreased bone volume (BV) in the cotyle area of pelvic bones (−12%; p < 0.05) associated with ∼50% inhibition of bone formation in the cancellous area of pelvic metaphyses and in thoracic vertebral bodies. The latter effect was associated with a decreased number of endosteal bone cells isolated from the bone surface (BS) in these samples (−42%; p < 0.05). This also was associated with a decreased number of alkaline phosphatase positive (ALP+) endosteal bone cells at 2 days and 4 days of culture, indicating decreased osteoblast precursor cell recruitment. Maintaining basal serum corticosterone levels in flight-ADX rats did not counteract the impaired bone formation in vertebral or pelvic bones. Moreover, the decreased ex vivo number of total and ALP+ endosteal bone cells induced by spaceflight occurred independent of endogenous corticosteroid hormone levels. These results indicate that the microgravity-induced inhibition of bone formation and resulting decreased trabecular bone mass in specific areas of weight-bearing skeleton in growing rats occur independently of endogenous glucocorticoid secretion.
Parathyroid Hormone Added to Established Hormone Therapy: Effects on Vertebral Fracture and Maintenance of Bone Mass After Parathyroid Hormone Withdrawal
Authors: Cosman F. , Nieves J. , Woelfert L. , Formica C. , Gordon S. , Shen V. , Lindsay R.
Published: April 2006

Our best pharmacologic agents for osteoporosis treatment prevent no more than 40-60% of osteoporotic fractures in patients at highest risk. Thus, there is a need for agents that can further augment bone mass and reduce fracture risk more substantially. To this end, we investigated the utility of parathyroid hormone (PTH) in combination with established hormone-replacement therapy (HRT) in women with osteoporosis. Fifty-two women who had been on HRT for at least 2 years were enrolled in this trial in which 25 were assigned randomly to remain on HRT alone and 27 were assigned to remain on HRT and also receive daily subcutaneous PTH(1-34) 400 U (25 μg) per day for 3 years. Bone mineral density (BMD) measurements at the spine, hip, and total body as well as biochemical determinations of bone turnover and calcium homeostasis were obtained every 6 months. Lateral thoracic and lumbar spine X-rays were obtained at baseline and annually. Subjects also had measurements of bone density and biochemical indices of bone turnover 1 year after discontinuation of PTH, while HRT was continued. In the group receiving HRT alone, bone density and biochemical variables of bone turnover remained stable throughout the 3-year treatment trial and 1-year follow-up. In the PTH + HRT group, biochemical variables of bone formation and resorption peaked at 6 months and subsequently remained elevated until 30 months at which time levels were indistinguishable from baseline. Subjects in the PTH + HRT group increased bone mass by 13.4 ± 1.4% in the spine, 4.4 ± 1.0% in the total hip, and 3.7 ± 1.4% in the total body. Bone density measurements remained stable 1 year after discontinuation of PTH without any significant loss while women continued HRT. Biochemical variables did not change significantly after cessation of PTH through the 1-year follow-up period. PTH + HRT reduced the percent of women who had vertebral fractures from 37.5% to 8.3% (using a 15% height reduction criterion) and from 25% to 0% (using a 20% height reduction criterion) compared with women receiving HRT alone (p < 0.02 for both). We conclude that ongoing HRT maintains almost all of the PTH-induced bone mass increment for 1 year after discontinuation of PTH. Furthermore, PTH in combination with hormone therapy is an effective means of increasing bone mass throughout the skeleton and specifically reducing vertebral fracture occurrence by 75-100%, compared with HRT alone.
Eating Attitudes and Habitual Calcium Intake in Peripubertal Girls Are Associated with Initial Bone Mineral Content and Its Change over 2 Years
Authors: Barr Susan I. , Petit Moira A. , Vigna Yvette M. , Prior Jerilynn C.
Published: April 2006

This 2-year prospective study examined associations among bone mineral acquisition and physical, maturational, and lifestyle variables during the pubertal transition in healthy girls. Forty-five girls, initially 10.5 ± 0.6 years, participated. Body composition and bone mineral content (BMC) at the spine and total body (TB) were assessed at baseline and annually thereafter using dual-energy X-ray absorptiometry (DXA). Nutrient intakes were assessed using 3-day diet records and a calcium food frequency questionnaire (FFQ), physical activity by questionnaire, sexual maturation using Tanner's stages of breast and pubic hair maturation, growth by height and weight, and eating attitudes using the children's Eating Attitudes Test (Children's EAT). Mean children's EAT subscale scores (dieting, oral control [OC], and bulimia) were stable over time. Median split of OC subscale scores was used to form high and low OC groups. Groups had similar body composition, dietary intake, activity, and Tanner stage at baseline and 2 years. Using height, weight, and Tanner breast stage as covariates, girls with low OC scores had greater TB BMC at baseline (1452 ± 221 g vs. 1387 ± 197 g; p = 0.030) and 2 years (2003 ± 323 g vs. 1909 ± 299 g; p = 0.049) and greater lumbar spine (LS) BMC at 2 years (45.2 ± 8.8 g vs. 41.2 ± 9.6 g; p = 0.042). In multiple regression analysis, OC score predicted baseline, 2 years, and 2-year change in TB and spinal BMC, contributing 0.9-7.6% to explained variance. Calcium intake predicted baseline, 2 years, and 2-year change in TB BMC, explaining 1.6-5.3% of variance. We conclude that both OC and habitual calcium intake may influence bone mineral acquisition.
COL1A1 Sp1 Polymorphism Predicts Perimenopausal and Early Postmenopausal Spinal Bone Loss
Authors: Macdonald Helen M. , McGuigan Fiona A. , New Susan A. , Campbell Marion K. , Golden Michael H. N. , Ralston Stuart H. , Reid David M.
Published: April 2006

Genetic factors play an important role in the pathogenesis of osteoporosis but the genes that determine susceptibility to poor bone health are defined incompletely. Previous work has shown that a polymorphism that affects an Sp1 binding site in the COL1A1 gene is associated with reduced bone mineral density (BMD) and an increased risk of osteoporotic fracture in several populations. Data from cross-sectional studies have indicated that COL1A1 Sp1 alleles also may be associated with increased rates of bone loss with age, but longitudinal studies, which have examined bone loss in relation to COL1A1> genotype, have yielded conflicting results. In this study, we examined the relationship between COL1A1 Sp1 alleles and early postmenopausal bone loss measured by dual-energy X-ray absorptiometry (DXA) in a population-based cohort of 734 Scottish women who were followed up over a 5- to 7-year period. The distribution of genotypes was as expected in a white population with 484 “SS” homozygotes (65.9%); 225 “Ss” heterozygotes (30.7%), and 25 “ss” homozygotes (3.4%). Women taking hormone-replacement therapy (HRT; n = 239) had considerably reduced rates of bone loss at the spine (−0.40 ± 0.06%/year) and hip (−0.56 ± 0.06%/year) when compared with non-HRT users (n = 352; spine, −1.36 ± 0.06%/year; hip, −1.21 ± 0.05%/year; p < 0.001 for both sites). There was no significant difference in baseline BMD values at the lumbar spine (LS) or femoral neck (FN) between genotypes or in the rates of bone loss between genotypes in HRT users. However, in non-HRT users (n = 352), we found that ss homozygotes (n = 12) lost significantly more bone at the lumbar site than the other genotype groups in which ss = −2.26 ± 0.31%/year compared with SS = −1.38 ± 0.07%/year and Ss = −1.22 ± 0.10%/year (p = 0.004; analysis of variance [ANOVA]) and a similar trend was observed at the FN in which ss = −1.78 ± 0.19%/year compared with SS = −1.21 ± 0.06%/year and Ss = −1.16 ± 0.08%/year (p = 0.06; ANOVA). The differences in spine BMD loss remained significant after correcting for confounding factors. Stepwise multiple regression analysis showed that COL1A1 genotype independently accounted for a further 3.0% of the variation in spine BMD change after age (4.0%), weight (5.0%), and baseline BMD (2.8%). We conclude that women homozygous for the Sp1 polymorphism are at significantly increased risk of excess rates of bone loss at the spine, but this effect may be nullified by the use of HRT.
Tibolone Exerts Its Protective Effect on Trabecular Bone Loss Through the Estrogen Receptor
Authors: Ederveen A. G. H. , Kloosterboer H. J.
Published: April 2006

Tibolone (Org OD14) has estrogenic, progestogenic, and/or androgenic activity depending on the tissue. In postmenopausal women, tibolone prevents bone loss without stimulating the endometrium. Tibolone is effective in preventing trabecular bone loss from the peripheral and axial skeleton of young and old ovariectomized (OVX) rats by reducing bone turnover, that is, bone resorption, like estrogens. We evaluated the contribution of the various hormonal activities to tibolone's bone-conserving effect. Three-month-old OVX rats received tibolone (125 μg/rat or 500 μg/rat, twice daily), alone or combined with an antiestrogen, antiandrogen, or antiprogestogen, and the effects on trabecular bone mass and bone turnover were evaluated. Sham-operated and control OVX groups were treated with vehicle. The remaining OVX groups received oral doses of tibolone twice daily, alone or with twice daily (a) antiestrogen ICI 164.384, (b) antiandrogen flutamide, or (c) antiprogestogen Org 31710. For comparison, the effects of 17β-estradiol and testosterone were examined also. After 4 weeks, trabecular bone mineral density (BMD) in the distal femur, plasma osteocalcin, and urinary deoxypyridinoline/creatinine ratio (Dpyr/Cr) were measured. Tibolone or 17β-estradiol significantly blocked ovariectomy-induced loss of trabecular BMD and inhibited bone resorption and bone turnover as judged by reduced Dpyr/Cr ratio and osteocalcin, respectively. These effects of both compounds were counteracted by the antiestrogen. This suggests a major involvement of the estrogen receptor in the action of tibolone on bone metabolism. However, the antiandrogen and the antiprogestogen did not counteract the effects of tibolone, excluding a major role of the androgenic and progestogenic activities of tibolone in its action against trabecular bone loss. The results indicate that tibolone acts on bone almost entirely through activation of the estrogen receptor.
Z Score Prediction Model for Assessment of Bone Mineral Content in Pediatric Diseases
Authors: Ellis Kenneth J. , Shypailo Roman J. , Hardin Dana S. , Perez Maria D. , Motil Kathleen J. , Wong William W. , Abrams Steven A.
Published: April 2006

The objective of this study was to develop an anthropometry-based prediction model for the assessment of bone mineral content (BMC) in children. Dual-energy X-ray absorptiometry (DXA) was used to measure whole-body BMC in a heterogeneous cohort of 982 healthy children, aged 5–18 years, from three ethnic groups (407 European- American [EA], 285 black, and 290 Mexican-American [MA]). The best model was based on log transformations of BMC and height, adjusted for age, gender, and ethnicity. The mean ± SD for the measured/predicted ln ratio was 1.000 ± 0.017 for the calibration population. The model was verified in a second independent group of 588 healthy children (measured/predicted ln ratio = 1.000 ± 0.018). For clinical use, the ratio values were converted to a standardized Z score scale. The whole-body BMC status of 106 children with various diseases (42 cystic fibrosis [CF], 29 juvenile dermatomyositis [JDM], 15 liver disease [LD], 6 Rett syndrome [RS], and 14 human immunodeficiency virus [HIV]) was evaluated. Thirty-nine patients had Z scores less than −1.5, which suggest low bone mineral mass. Furthermore, 22 of these patients had severe abnormalities as indicated by Z scores less than −2.5. These preliminary findings indicate that the prediction model should prove useful in determining potential bone mineral deficits in individual pediatric patients.
Effect of High-Impact and Low-Repetition Training on Bones in Ovariectomized Rats
Authors: Honda Akiko , Umemura Yoshihisa , Nagasawa Seigo
Published: April 2006

This study was designed to investigate the effect of high-impact and low-repetition jump training on bones in ovariectomized (OVX) rats. Forty female Wistar rats were sham-operated (sham) or OVX at the age of 11 weeks. The rats were divided randomly into the following four groups: sham-sedentary (SS; n = 10), sham-exercised (SE; n = 10), OVX-sedentary (OS; n = 10), and OVX-exercised (OE; n = 10). The rats started the jump training at the age of 12 weeks. The jump-training protocol was 10 times/day, 5 days/week and the jumping-height was 40 cm. After 8 weeks of training, the mass and breaking force in the tibia and ulna, cross-sectional areas of diaphysis in the tibia, and serum bone turnover markers were measured. The jump training significantly increased the fat-free dry weight, ash weight, and ultimate breaking force in the tibia. The rate of increase in these parameters was similar in both the sham and the OVX groups. On the other hand, in the ulna, there were no significant changes in the ultimate breaking force. The jump training significantly increased the periosteal perimeter and cortical area, although the increase in these parameters in OE compared with OS was lower than that in SE compared with SS. The jump training significantly increased serum osteocalcin in the OVX groups, as well as in the sham groups. These results suggest that high-impact and low-repetition training had beneficial effects on bone formation and bone biomechanical properties in OVX rats, as well as in sham rats.
Comprehensive Microarray Analysis of Bone Morphogenetic Protein 2-Induced Osteoblast Differentiation Resulting in the Identification of Novel Markers for Bone Development
Authors: Vaes Bart L. T. , Dechering Koen J. , Feijen Alie , Hendriks José M. A. , Lefèvre Christophe , Mummery Christine L. , Olijve Wiebe , Van Zoelen Everardus J. J. , Steegenga Wilma T.
Published: April 2006

Osteoblasts are cells responsible for matrix deposition during bone development and although temporal expression of many genes has been related to osteoblast differentiation, a complete description of osteoblast-specific gene regulation will lead to a better understanding of osteoblast function. In this study, microarray technology was used to analyze gene expression on a broad scale during osteoblast differentiation. Expression analysis of 9596 sequences revealed 342 genes and expressed sequence tags (ESTs) to be modulated differentially during a time course experiment in which murine C2C12 mesenchymal progenitor cells were induced to differentiate into mature osteoblasts by treatment with bone morphogenetic protein 2 (BMP-2). By means of hierarchical clustering, these genes were grouped by similarities in their expression profiles, resulting in subsets of early, intermediate, and late response genes, which are representative of the distinct stages of osteoblast differentiation. To identify new bone markers, the bone specificity of the late response genes was determined by comparing BMP-induced expression in C2C12 and MC3T3 osteoblasts with that in NIH3T3 fibroblasts. This resulted in the identification of nine novel genes and ESTs that were induced specifically in osteoblasts, in addition to the well-known markers ALP and osteocalcin. For at least one of these novel genes, Wnt inhibitory factor 1, and two of the ESTs, expression in developing bone was verified in vivo by in situ hybridization of E16.5 mouse embryos. In conclusion, by a combination of in vitro and in vivo screening approaches, a set of new genes related to osteoblast differentiation and skeletal development has been identified.
The Zinc Finger Transcription Factor Zfp60 Is a Negative Regulator of Cartilage Differentiation
Authors: Ganss Bernhard , Kobayashi Hiroaki
Published: April 2006

The differentiation of many mesenchyme-derived cells, including cells that form bone and cartilage, is regulated at the level of gene transcription, but many of the factors involved in this regulation remain to be identified. In this study, a modified RNA fingerprinting technique was used to identify the KRAB domain zinc finger transcription factor Zfp60 as a candidate regulator of cell differentiation in mouse calvaria primary cultures. The highest expression of Zfp60 mRNA in vivo was found between embryonic day 11 (E11) and E15 during mouse embryonic development, coinciding with stages of active organ formation. The expression of Zfp60 mRNA and protein was analyzed further in mouse embryos during skeletal development. The most prominent expression was found in prehypertrophic chondrocytes, where it coincides with the expression of key regulators of chondrocyte maturation, Indian hedgehog (Ihh), and the parathyroid hormone-related peptide (PTHrP) receptor. Zfp60 mRNA was also found transiently expressed during chondrogenesis of C1 cells in vitro, preceding collagen type X expression and cellular hypertrophy. Overexpression of Zfp60 inhibited cartilage differentiation in the chondrogenic ATDC5 cell line. These results suggest a role for Zfp60 as a negative regulator of gene transcription, specifically during the development and/or differentiation of chondrocytes.
Tissue Transglutaminase and Its Substrates in Bone
Authors: Kaartinen Mari T. , El-Maadawy Sherif , Räsänen Niina H. , McKee Marc D.
Published: April 2006

Tissue transglutaminase (tTG) is an intra- and extracellular, protein-cross-linking enzyme that has been implicated in apoptosis, matrix stabilization, and cell attachment in a variety of tissues. This study provides in vivo evidence in bone of TG activity, its tissue localization, and identification of its substrates. In microplate- and blotting-based activity assays using biotinylated primary amine as a probe, we show TG activity in protein extracts from the mineralized compartment of intramembranous rat bone. Avidin affinity purification of bone extract labeled with biotinylated primary amine in the presence of tTG, in conjunction with Western blotting, permitted identification of three major noncollagenous TG substrates in bone: osteopontin (OPN), bone sialoprotein (BSP), and α2 HS-glycoprotein (AHSG), of which the latter two are novel substrates. Cross-linking and labeling of purified proteins confirmed their ability to serve as TG substrates, because they readily incorporated biotinylated primary amine and formed large protein aggregates in the presence of tTG. All three proteins were also identified in the high molecular weight complexes extractable from the mineralized compartment of bone. Two-dimensional (2D) gel electrophoretic analysis combined with Western blotting indicated that the proteins are not cross-linked to each other, but form distinct homotypic polymers. In the extracellular matrix of bone, tTG and isopeptide bonds were localized by immunohistochemistry in the osteoid and in the pericellular matrix surrounding osteocytes. At the cellular level, osteoblasts and osteocytes were immunostained for tTG. Collectively, these data suggest a role for tTG and its covalently cross-linked substrates in cell adhesion and possibly also in bone matrix maturation and calcification.