bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered trabecular architecture induced by Corticosteroids: A Bone Histomorphometric Study
Authors: Chappard D. , Legrand E. , Basle M.F. , Fromont P. , Racineux J.L. , Rebel A. , Audran M.
Published: February 2010

Prolonged corticosteroid (CS) therapy induces osteoporosis and fractures. Osteoporosis is characterized at the histomorphometric level by reduced bone volume (BV/TV) and disruption of the three-dimensional (3D) trabecular architecture. Several stereological methods have been proposed to characterize these alterations: measurements of trabecular thickness and trabecular number, star volumes, interconnectivity index (ICI) of the bone marrow spaces, and trabecular bone pattern factor (TBPf). These methods were computerized with a single program running on an image analyzer to evaluate the bone changes in a series of iliac biopsies performed on 31 male patients. All of them were asthmatic and had received CS for a long period of time. BV/TV was reduced when compared with age-matched controls. In the CS-treated population, exponential relationships were obtained between bone volume and the different connectivity parameters. The various methods used to measure connectivity were well correlated. When the population was divided into two groups (BV/TV greater or less than an 11% threshold), the architectural disturbances were found to imply two mechanisms. A progressive decline in trabecular thickness was noted in both groups versus controls. Trabecular perforations were not established in the group with BV/TV > 11% with the star volume or ICI, although some alterations were detected by trabecular bone pattern factor measurement. However, perforations were revealed in the group with BV/TV < 11% by all the different methods. Perforations seemed to occur when the trabecular thickness was below 70 μm. This strongly suggests that bone histomorphometry should take into consideration bone volume in combination with detailed 3D descriptors of the trabecular architecture. Several histological methods need to be used in combination to appreciate the 3D architecture of trabecular bone.
Characterization of a collagenous cementum-derived attachment protein
Authors: Wu Dayang , Ikezawa Kazuhiko , Parker Todd , Saito Masahiro , Narayanan A. Sampath
Published: February 2010

We report further characterization of a cementum-derived protein that promotes the adhesion and spreading of periodontal cells. The cementum attachment protein (CAP) was extracted from bovine cementum, separated by diethylamino ethyl (DEAE)-cellulose chromatography, and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and C18 reverse phase high performance liquid chromatography. The purified preparation contained a single protein band migrating with Mr 56,000. It did not cross-react with polyclonal antibodies to osteopontin, vitronectin, or other attachment proteins. The attachment activity was resistant to chondroitinase ABC digestion. An internal amino acid sequence of six peptides was determined by microsequencing, and the peptide sequences were not present in other attachment proteins described in cementum. Four sequences contained Gly-X-Y repeats typical of collagen helix. One 17 amino acid peptide had 82% homology with a type XII collagen domain. However, bovine type XII collagen did not promote fibroblast attachment. Although another 19-amino-acid-long peptide had 95% homology to bovine α1[I], two other peptides were only 74% and 68% homologous, and the CAP was not recognized by anti-type I collagen antibody. The attachment activity of CAP was susceptible to bacterial collagenase. The CAP did not cross-react with antibodies to type V, XII, and XIV collagens. These data and our previous immunostaining data indicate that the CAP is not related to other collagens or attachment proteins and that it is a collagenous attachment protein localized in cementum.
Osteomalacia and osteoporosis in a woman with ankylosing spondylitis
Authors: Kohlmeier Lynn A. , Federman Micheline , Leboff Meryl S.
Published: February 2010

Three months postpartum, a 33-year-old woman with ankylosing spondylitis (AS) suffered multiple vertebral fractures. Bone mineral density was 61–67% of age-matched normal values at the lumbar spine and proximal femur, and an initial iliac crest bone biopsy revealed osteoporosis and osteomalacia. Secondary causes of bone disease were excluded, and the patient was treated with calcium, vitamin D, and nasal spray calcitonin (400 μ/day). Over 4 years, she has shown partial recovery of bone mass and almost complete resolution of osteomalacia. Osteoporosis and fracture occur in patients with AS, yet this case represents a rare association between AS and both osteomalacia and postpregnancy spinal osteoporosis.
A new role for the chondrocyte in fracture repair: Endochondral ossification includes direct bone formation by former chondrocytes
Authors: Miss Scammell Brigitte E. , Roach Helmtrud I.
Published: February 2010

We studied the endochondral ossification that occurs during the transition of soft to hard callus during fracture healing in the rabbit. During this process, parts of the cartilaginous soft callus are invaded by capillaries, and new bone is laid down onto the central unresorbed cartilage struts. We found that the chondrocytes within these cartilage struts changed phenotype and became bone-forming cells which directly replaced the central cartilage core with bone matrix. We have termed this bone “lacunar” bone to distinguish it from the “vascular” bone laid down by osteoblasts. With time the lacunar bone spread beyond the confines of the lacunae and gradually replaced all the cartilage matrix that was originally present in the early endochondral spicules. The lacunar bone could still be distinguished from the vascular bone as follows: (1) it was woven bone, whereas vascular bone was lamellar bone; (2) it contained acid phosphatase activity, whereas vascular bone did not; and (3) it had strong antigenicity for bone sialoprotein, whereas this noncollagenous protein was undetectable in vascular bone. Eventually the hard callus was resorbed and remodeled, but at an interim period of endochondral ossification the direct replacement of cartilaginous callus by the formation of lacunar bone is a rapid mechanism by which the mechanical strength of fracture callus is increased.
Relationship between body composition and bone mass in women
Authors: Khosla Sundeep , Atkinson Elizabeth J. , Riggs B. Lawrence , Melton L. Joseph
Published: February 2010

Increasing body weight is associated both with higher bone mass and with lower rates of bone loss. Whether the effects of body weight are mediated by lean body mass (LBM) or fat body mass (FBM) is, however, uncertain because different studies have used different measures of bone mass and arrived at contradictory conclusions. The parameter actually measured is bone mineral content (BMC). Bone mineral density (BMD), bone mineral apparent density (BMAD), and the BMD/height attempt to “correct” BMC for differences in bone or body size, but these corrections may bias the analysis of the effects of body composition on the skeleton. To resolve this issue, we measured BMC at the total body, lumbar spine, proximal femur, and forearm using dual energy X-ray absorptiometry (DXA) in a population-based sample including 138 premenopausal women (age range 21–54 years, mean 35 years) and 213 postmenopausal women (age range 34–94 years, mean 68 years). BMD, BMAD, and BMD/ height were also calculated for each site. LBM and FBM were determined from the DXA whole body scan. In a multivariate analysis that included age and height, both LBM and FBM predicted total body BMC in pre- and postmenopausal women (p < 0.002 for LBM and FBM in both groups). LBM had a dominant effect on spine and forearm BMC in both groups (p < 0.004) and hip BMC in premenopausal women (p < 0.001), whereas both LBM and FBM predicted hip BMC in postmenopausal women (p < 0.001). However, as BMC was adjusted for bone or body size using BMD, BMAD, or BMD/height, FBM tended to become more important than LBM in the analysis. This was, in part, due to the fact that each of the correction factors in the BMD and BMAD calculations, as well as height, were highly correlated with LBM (r = 0.57 and 0.52 for height versus LBM in pre- and postmenopausal women, respectively [p < 0.001]), and weakly or not at all with FBM (r = 0.08 and 0.11, respectively). Therefore, dividing BMC by these correction factors tended to bias the analysis against potential effects of LBM on bone mass. Thus, the relationship between body composition and bone mass is critically dependent on which bone mass parameter is used in the analysis. Both LBM and FBM have important effects on bone mass, depending on the bone mass parameter used, the skeletal site measured, and menopausal status.
Ca2+ oscillations and intercellular Ca2+ waves in ATP-stimulated articular chondrocytes
Authors: Dr. D'Andrea R. P. , Vittur F.
Published: February 2010

Cytosolic Ca2+ oscillations are known to occur in many cell types stimulated with agonists linked to the phosphoinositide signaling pathway. Trains of repetitive short-lasting Ca2+ spikes could be induced in articular chondrocytes by extracellular ATP, an agonist potently effective in stimulating cartilage resorption. The mechanism of these Ca2+ oscillations was studied by computerized video imaging on primary cultures of articular chondrocytes. Few cycles of oscillatory activity could be evoked in the absence of extracellular Ca2+, while, for oscillations to be sustained, Ca2+ influx was required. Thapsigargin irreversibly blocked Ca2+ oscillations, thus demonstrating the crucial involvement of intracellular stores in triggering the rhythmic activity. Apart from activating intracellular Ca2+ release, extracellular ATP also induced a noncapacitive Ca2+ influx in these cells. This ATP-mediated influx modulates both the oscillation frequency and intracellular stores refilling. In monolayers of confluent cells, Ca2+ oscillations spread from cell to cell in the form of intercellular waves. Propagating waves could also be observed in the absence of extracellular Ca2+, demonstrating that Ca2+ itself is not required for signal coordination. These results demonstrate that complex spatiotemporal pathways of Ca2+ oscillations and intercellular Ca2+ waves could be activated in articular chondrocytes during degenerative diseases. (J Bone Miner Res 1996;11:946-954)
Vitamin D receptor alleles, bone mineral density and turnover in premenopausal Japanese women
Authors: Dr. Tokitan Akifumi , Matsumoto Hiroshi , Morrison Nigel A. , Tawa Toshiya , Miura Yuko , Fukamauchi Kazutaka , Mitsuhashi Naoki , Irimoto Masahiro , Yamamori Syunji , Miura Masakazu , Watanabe Takako , Kuwabara Yoshihiro , Yabuta Keijiro , Eisman John A.
Published: February 2010

Recent studies have shown that genetic effects on bone mineral density (BMD) and bone turnover are related to allelic variation in the vitamin D receptor (VDR) gene. We examined allelic influences of the VDR gene on bone turnover and density in 202 normal healthy premenopausal Japanese women (age 30.1 ± 1.2, mean ± SEM). The VDR effect on BMD and turnover is similar to that observed in Caucasian women; however, there are major differences in allele frequency. The B allele by Bsm I restriction fragment length polymorphisms (RFLPs), associated with low BMD and high bone turnover, is found in only 12% of Japanese women (1.4% homozygote BB), compared with 41% of Caucasians (16.7% homozygote BB). In comparing the two most frequent genotypes, Bb heterozygotes (21.5%) and bb homozygotes (77.1%), BMD is 5.3% lower in Bb heterozygotes, and levels of bone formation markers including osteocalcin and bone-specific alkaline phosphatase are 20–32% higher with lower serum calcium (2.30 ± 0.02 vs 2.35 ± 0.01 mmol/1) and higher 1,25-dihydroxyvitamin D (95 ± 4.8 vs. 76 ± 3.8 pmol/1). Further discrimination of the genotype was achieved using two additional RFLPs (Apa I, A and Taq I, T); the lumbar spine BMD of the common genotype BbAATt was 9.3% (0.94 SD) lower than in the bbaaTT genotype in premenopausal Japanese women. These data confirm that VDR RFLPs affect bone mineral metabolism regardless of racial differences. Moreover, the VDR genotypes based on haplotype analysis should yield useful insights into the potential prevention of osteoporosis. (J Bone Miner Res 1996;11:1003-1009)
A prospective study of proximal femur bone mass following cemented and uncemented hip arthroplasty
Authors: Marchetti Michael E. , Steinberg Gerald G. , Greene James M. , Jenis Louis G. , Baran Daniel T.
Published: February 2010

Cross-sectional studies have demonstrated significant decreases in bone mass in femoral cortical bone adjacent to prostheses. Thirty-six patients who had undergone 31 cemented and 9 uncemented primary total hip arthroplasties (THA) were prospectively studied to define further the natural history of this femoral cortical bone loss. Dual-energy X-ray absorptiometry (DXA) was employed to quantify bone mineral density (BMD) changes in four subregions around the femoral prostheses. Femoral BMD loss (average 12.3%) was observed in the three proximal subregions 2 months postoperatively. This loss increased to 21.2% by 6 months postoperatively, and by 2 years postoperatively it averaged 25.5% in the same three subregions. There were no significant BMD changes in the subregion distal to the prosthesis tip or in the contralateral hip. Subgroups were compared based on prosthesis size and cement use. Statistically significant differences in BMD loss were observed between the large cemented cobalt chrome prosthesis group (n = 8) and the large uncemented titanium prosthesis group (n = 8). These differences were only present in the most proximal medial subregion where the large cemented groups had twice the bone loss in this area as compared with the large uncemented group. The data indicate that bone loss occurs adjacent to femoral prosthesis within 2 months of surgery and that DXA is a useful technique to quantify prospectively femoral cortical bone loss following THA. (J Bone Miner Res 1996;11:1033–1039)
Evaluation in vivo of a potent parathyroid hormone antagonist: [Nle8,18, D-Trp12, Tyr34]bPTH(7–34)NH2
Authors: Dresner-Pollak Rivka , Yang Qui-Ming , Behar Vered , Nakamoto Chizu , Dr. Chorev Michael , Rosenblatt Michael
Published: February 2010

In an effort to design and select potent parathyroid hormone (PTH) antagonists suitable for clinical utility, a PTH analog was evaluated in vivo in an animal model to assess its properties in preparation for human studies. The previously described PTH antagonist, [Nle8,18, D-Trp12, Tyr34]bPTH(7–34)NH2, which is highly active in vitro, was documented in these studies to be an effective antagonist of the PTH-stimulated calcemic response in vivo. In thyroparathyroidectomized (TPTX) rats, the efficacy of the antagonist was demonstrated to be dose-dependent. Inhibition was demonstrated when intravenous administration of antagonist started 1 h prior to coinfusion with the PTH agonist [Nle8,18,Tyr34]bPTH(1–34)NH2. Maximal inhibition by antagonist (an 84% decline in serum calcium levels compared with agonist alone) of the calcemic response was observed when a 200-fold molar excess of antagonist (12 nmol/h) was administered. At dose ratios of antagonist:agonist as low as 10:1, a 40–50% inhibition of PTH-stimulated calcemic response is evident, provided a longer (2 h) lead time for antagonist infusion is allowed. Based on these and related studies, the antagonist [Nle8,18,D-Trp12,Tyr34]bPTH(7–34) NH2 has displayed sufficient potency to obtain approval from the appropriate institutional and regulatory agencies for clinical trials in hypercalcemic states of parathyroid and tumor origin.
Broadband ultrasound attenuation imaging: A new imaging method in osteoporosis
Authors: Roux Christian , Fournier Bruno , Laugier Pascal , Chappard Christine , Kolta Sami , Dougados Maxime , Berger Geneviève
Published: February 2010

The purpose of this study was to evaluate the usefulness in osteoporosis of a new ultrasound imaging device able to create a parametric image of broadband ultrasound attenuation (BUA) at the os calcis. Three regions of interest were located in the great tuberosity of the os calcis. Precision was evaluated in 37 patients. Calcaneal bone mineral density (BMD) and BUA were compared in 33 patients. In 236 patients (including 77 with osteoporotic fractures), BUA and lumbar and femoral BMD measurements were performed. The measurements were compared using correlation coefficients. Their clinical value was estimated by comparisons of the results between patients with fractures and age-matched controls, using comparisons of the means, areas under the ROC curves, and logistic regression. Precision was in a 1.4-3.3% range. Local BUA and BMD were highly correlated (r = 0.88). Significant correlations were found between BUA and lumbar (r = 0.56) and femur (r = 0.66) BMD. In multiple regression, years since menopause and weight were significant predictors of BUA. Patients with fractures had lower BUA and BMD than age-matched controls. BUA showed the largest difference between the two populations (13-16%). Areas under the ROC curves were similar for BUA and BMD. Logistic regression after adjustment for confounding factors showed that BUA discriminated between fracture and nonfracture subjects. Broadband ultrasound attenuation imaging improves the reproducibility of ultrasound measurements. It may be useful in osteoporosis, due to its good discriminating value.
The role of type I collagen in the regulation of the osteoblast phenotype
Authors: Shi Songtao , Kirk Martin , Kahn Arnold J.
Published: February 2010

Evidence from a variety of sources indicates that the extracellular matrix forms an important part of a feedback loop governing the migration, proliferation, and differentiation of the cells that produce it. In keeping with this, we showed previously that the extracellular matrix of a multipotential mesenchymal clonal cell line (ROB-C26) induced to differentiate into a more osteoblastic cell type by the addition of exogenous retinoic acid produces an extracellular matrix capable of osteoinductive activity in vivo and of stimulating alkaline phosphatase activity in vitro. Since type I collagen is the major structural component of this extracellular matrix, we sought to determine whether and to what extent this protein is responsible for the previously observed inductive/stimulatory activity. To this end, C26 cells are cultured on plastic, in the presence of retinoic acid, on a type I collagen film, or on an extracellular matrix from retinoic acid-treated C26 cells, and cell differentiation is assessed by measuring changes in the abundance of a number of osteoblast-related mRNAs. These determinations are made by RNAse protection assay after 3 or 6 days of incubation and include measurements of the RNAs for type I collagen, alkaline phosphatase, osteopontin, transforming growth factor α1 and β2, and Vgr-1/BMP-6. In addition, C26 cells are incubated in the presence of retinoic acid and several established inhibitors of the synthesis or assembly of extracellular matrix components and the effects on induced alkaline phosphatase activity determined. Our data show that while the collagen substrate mimics some of the effects of retinoic acid and the extracellular matrix, it cannot reproduce all of them. Specifically, while the latter two culture conditions increase the abundance of all six mRNAs, type I collagen film increases the levels of only three of the six (collagen I, alkaline phosphatase, and osteopontin). Moreover, while type I collagen film produces an increase in alkaline phosphatase message, it fails to produce a similar change in alkaline phosphatase activity, an effect seen with both retinoic acid and extracellular matrix. However, interruption of collagen I synthesis by cis-4-hydroxy-L-proline blocks the increase in alkaline phosphatase activity associated with retinoic acid treatment. Thus, it appears likely that type I collagen is a necessary but, by itself, insufficient factor to elicit the comprehensive expression of the osteoblastic phenotype in immature mesenchymal cells.
Bone resorption and response to calcium-regulating hormones in the absence of tissue or urokinase plasminogen activator or of their type 1 inhibitor
Authors: Leloup Gaetane , Lemoine Pascale , Carmeliet Peter , Dr. Gilbert Vaes
Published: February 2010

Plasminogen activators (PA) are implicated in cell migration and tissue remodeling, two componets of the bone resorption processes. Uning mice with inactivated tissue PA (tPA), urokinase PA (uPA), or type 1 PA inhibitor (PAI-1) genes, we evaluated whether these processes, or their stimulation by parathyroid hormone (PTH) or 1,25-dihydroxyvitamin (1,25{oH}2D3) are dependent on these genes. Two culture models were used, one involving 19-day fetal calvariae, to evaluate the direct resorptive activity of osteoclasts, and the other involving 45Ca-labeled 17-day fetal metatarsals, in which this activity depends on preliminary (pre)osteoclast migration. PTH similarly increased (about 10-fold) PA activity in calvariae from wild-type tPA+/+ and uPA+/+ or deficient uPA−/− and PAI−/− mice; it affected only tPA, not uPA. In tPA−/− bones, the low PA levels, due to uPA, were not influenced by PTH. Calcitonin did not affect PA responses to PTH. No differences were observed between tPA+/+, tPA−/−, uPA+/+, and uPA−/− calvariae for any parameter related to bone resorption (development of lacunae, release of calcium and lysosomal enzymes, accumulation of collagenase, loss of hydroxyproline), indicating similar responses to PTH or calcitonin. The progressive 45Ca release was largely similar in cultures of tPA+/+, tPA−/−, uPA+/+ uPA−/−, PAI+/+, or PAI−/− metatarsals and it was similarly enhanced by PTH or 1,25(OH)2D3. However, uPA−/− metatarsals released 45Ca at a slower rate at the beginning of the cultures, suggesting an impaired recruitment of the (pre)osteoclasts, which migrate at that time from the periosteum into the calcified cartilage. Thus, it appears that the direct resorptive activity of the osteoclasts does not necessitate the presence of either tPA or uPA, but uPA is likely to facilitate the migration of the (pre)osteoclasts toward the mineralized surfaces. Although considerably enhanced by PTH, tPA does not mediate the actions of PTH (nor of 1,25{OH}2D3) evaluated in these models.
Epitope mapping of nine monoclonal antibodies against osteocalcin: Combinations into two-site assays affect both assay specificity and sample stability
Authors: Hellman Jukka , Käkönen Sanna-Maria , Matikainen Marja-Terttu , Karp Matti , Lövgren Timo , Väänänen H. Kalervo , Pettersson Kim
Published: February 2010

Nine monoclonal antibodies (Mabs) were raised against human recombinant osteocalcin fusion protein (rGST-hOC) or bovine osteocalcin (bOC) and selected to develop two-site immunoassays for human osteocalcin (hOC). The detection system was based on the time-resolved measurement of the fluorescence of europium chelates conjugated to the tracer Mabs. Based on the ability of the Mabs to recognize different forms of hOC (carboxypeptidase Y-digested, alkylated hOC, thermally decarboxylated hOC, recombinant forms of hOC, and tryptic peptides derived from hOC) and the information obtained from combinations of the Mabs in two-site assays, an epitope map was created. The epitope map was useful in understanding the behavior of the two-site combinations of the Mabs with serum samples. The two-site combinations could be divided into subgroups detecting either full-length hOC or full length + large NH2-terminal fragment as simulated by the carboxypeptidase Y-digested form of hOC (it lacks four COOH-terminal residues), which with intact specific assays showed cross-reactivities ranging from 7 to 14% when compared with full-length hOC. In addition, differences were observed in the ability of the combinations to detect thermally decarboxylated hOC (lacks γ-carboxylation at residues 17, 21, and 24) with cross-reactivities ranging from 8 to 85% when compared to γ-carboxylated hOC. The analysis of human serum samples showed considerable differences in the concentration and stability of serum OC. This was attributed as the varying ability of the Mabs to detect different proteolytic fragments derived from hOC and/or differences in the degree of γ-carboxylation of hOC. The in vitro generation of the large NH2-terminal fragment during incubation of the serum samples at room temperature (RT) and during prolonged storage at −20°C in an undercooled state was detectable as loss of immunoreactivity (ranging from −42 ± 17 to −50 ± 15% in 16 h at RT, n = 3) with Mab combinations detecting only full-length hOC. Two-site combinations detecting full-length + large NH2-terminal fragment showed no loss of immunoreactivity after incubation of the serum samples at RT for 16 h. With all assays, an increase of serum OC ranging from 16 to 38% was found in postmenopausal samples (n = 24) when compared with premenopausal samples (n = 17), but the degree of statistical significance varied from not significant to p < 0.01.
Factors associated with bone mineral density in middle-aged men
Authors: Bendavid Eli J. , Shan Jun , Barrett-Connor Elizabeth
Published: February 2010

This cross-sectional population-based study examined the association of anthropometric and lifestyle risk factors with bone mineral density (BMD) in 218 white ambulatory men aged 50–64 from the Rancho Bernardo, California cohort. BMD was measured at the lumbar spine and hip using dual-energy X-ray absorptiometry and at the ultradistal wrist and midshaft radius of the forearm using single-photon absorptiometry. Body mass index (BMI) was significantly correlated with BMD at all four skeletal sites. Overall, 17.0% of men aged 55–64 were osteopenic (BMD ≥ 2 SD below the distribution for ages 50–54) at one skeletal site, 16.5% were osteopenic at two sites, and 13.6% were osteopenic at three or more sites. Men who reported regular exercise had significantly higher BMD levels at the spine and hip. Men meeting the recommended daily allowance (RDA) for calcium intake (≥800 g/day) had significantly higher BMD levels at the spine and wrist. Alcohol intake and smoking were associated with differences of borderline significance in BMD at the spine. In analyses adjusted for BMI, weight change, exercise, smoking, drinking, and calcium intake, there was a significant independent age-related decline in BMD at the hip (0.008 g/cm2/year; p = 0.001), at the wrist (0.004 g/cm2; p < 0.01), at the forearm (0.006 g/cm2; p < 0.01), but not at the spine (0.005 g/cm2). These data, although cross-sectional, strongly suggest that age-related bone loss occurs in middle-aged men and that both physical activity and an adequate calcium intake are associated with better bone density.
Abnormal modulation of serum osteocalcin by dietary phosphate and 1,25-dihydroxyvitamin D3 in the hypophosphatemic mouse
Authors: Tsuji H. , Cawthorn C. , Ecarot B.
Published: February 2010

We evaluated in normal and hypophosphatemic (Hyp) mice whether changes in serum levels of osteocalcin in response to dietary phosphate supplementation, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) administration were related to perturbations in calcium phosphate homeostasis. In normal mice, serum osteocalcin levels were not altered by phosphate supplementation. In contrast, phosphate supplementation in Hyp mice led to a 2-fold decrease in serum osteocalcin to normal levels after 3 days and to an increase in osteocalcin levels after 14 days. The decrease in osteocalcin was associated with normophosphatemia, severe hypocalcemia, and marked increases in circulating 1,25(OH)2D3 levels, whereas the increase in osteocalcin levels was associated with normophosphatemia and no change in serum calcium and 1,25(OH)2D3. Administration of PTH decreased serum osteocalcin in both genotypes. Infusion of 1,25(OH)2D3 for 3 days elicited increases in serum osteocalcin and calcium levels in normal mice, whereas in Hyp mice it produced significant decreases in osteocalcin levels and no change in serum calcium. However, with a more prolonged infusion of 1,25(OH)2D3, hypercalcemia and increases in serum osteocalcin were induced in mutant mice. Our results suggest that the abnormal osteocalcin response of Hyp mice is not directly attributable to an osteoblast dysfunction but is secondary, at least in part, to perturbations in factors that modulate the osteoblast activity, especially serum calcium and/or PTH.
Anabolic effects of prostaglandins in cultured fetal rat calvariae: Structure-activity relations and signal transduction pathway
Authors: Woodiel Florence N. , Fall Pamela M. , Raisz Lawrence G.
Published: February 2010

The structure-activity relations and signal transduction pathways for the anabolic effects of prostaglandins were examined in cultured fetal rat calvariae. In the presence of cortisol prostaglandins of the E and F series (10−9 to 10−5 M) produced a dose-related increase in [3H]thymidine incorporation up to 4-fold at 24 h. Prostaglandin E2 (PGE2) was also effective in the absence of cortisol. Butaprost (10−6 M), a selective EP-2 receptor agonist, produced partial stimulation. Prostaglandin D2, prostacyclin, sulprostone, an EP-1 and EP-3 receptor agonist, and fluprostenol, an FP receptor agonist, were ineffective. Forskolin (10−4 M) increased [3H] thymidine incorporation 3-fold, while phorbol myristate acetate (PMA) (10−6 M) produced a 1.8-fold increase. Isobutylmethylxanthine (IBMX) increased [3H] thymidine incorporation in control cultures, in the absence of cortisol, and increased the response to PGE2 in control and cortisol-treated cultures. [3H] proline incorporation into collagen and noncollagen protein was measured in the continuous presence of prostaglandins and cortisol for 72–96 h (continuous model) or when prostaglandins and cortisol were applied for 24 h, followed by culture for 48 h in control medium (on/off model). The effects on collagen were greater than on noncollagen proteins, so that the percent of collagen synthesis increased. The effects of prostaglandins and forskolin paralleled their mitogenic effects. PMA increased only noncollagen protein. Indomethacin did not diminish the anabolic response, while aphidicolin produced only partial inhibition. We conclude that the anabolic effects of prostaglandins on replication and differentiation of osteoblasts are likely to be mediated by an EP-2 receptor that stimulates adenylate cyclase.
Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-β1 or PDGF-BB as assayed in vivo and in vitro
Authors: Cassiede Pierre , Dennis James E. , Ma Felix , Caplan Arnold I.
Published: February 2010

Mesenchymal progenitors cells can be isolated from rat bone marrow and mitotically expanded in vitro. When these cells, which we operationally call mesenchymal stem cells (MSCs), are placed in an appropriate environment, they have the capacity to differentiate into bone and/or cartilage. This capacity is called osteochondrogenic potential. In this study, preconfluent MSCs were exposed in vitro to 5 ng/ml transforming growth factor-β1 (TGF-β1) or platelet-derived growth factor, isoform BB (PDGF-BB) for a pulse of 48 h and assayed for cell proliferation, alkaline phosphatase activity, and osteochondrogenic potential; untreated MSC's served as controls. In these cell culture conditions, TGF-β1 or PDGF-BB had similar effects on proliferation and alkaline phosphatase activity. Both growth factors increased cell proliferation and decreased alkaline phosphatase activity of MSCs. Sister cultures of TGF-β1- or PDGF-BB-treated MSCs and untreated MSCs were trypsinized. For each type of culture, the trypsinised MSCs were split in two parts: one part was replated in an osteogenic medium to assess its in vitro osteogenic potential, whereas the other part was seeded into porous calcium phosphate ceramics and implanted subcutaneously in syngeneic rats to assess its in vivo osteochondrogenic potential. PDGF-pretreated MSCs showed no difference in in vivo and in vitro osteochondrogenesis from that of control MSCs, while TGF-β1 pretreatment blocked the osteochondrogenic potential of MSCs when assayed in vitro for bone nodule formation. However, when tested in vivo, TGF-β1-pretreated MSCs were able to form bone and cartilage. These data show that measurements of proliferation and alkaline phosphatase activity of preconfluent MSCs immediately after exposure to growth factor were not predictive of their subsequent osteochondrogenic potential. Moreover, the variation of the osteochondrogenic potential of MSCs after exposure to growth factor was further modulated by the environment in which the MSCs were assayed.
Mineral anisotropy in mineralized tissues is similar among species and mineral growth occurs independently of collagen orientation in rats: Results from acoustic velocity measurements
Authors: Takano Yuichi , Turner Charles H. , Burr David B.
Published: February 2010

It has been reported that the mineral crystals in long bones have their c-axis aligned with the bone axis, presumably because collagen fibrils in bone also align with the bone axis. However, the predominant collagen orientation in bone often does not appear to be aligned with the mineral crystals, especially in rat primary bone. We hypothesized that mineral orientation in bone is not necessarily related to collagen orientation. An acoustic microscope was used to measure elastic constants of mineralized tissues from rat, cow, monkey, and human bone, and mineralized turkey leg tendon (MTLT). Measurements were made before and after demineralization with 10% ethylenediaminetetraacetic acid (EDTA) or decollagenization with 7% sodium hypochlorite. The elastic anisotropy ratio (AR) was defined as the ratio of the elastic coefficient in the longitudinal direction to the elastic coefficient in the transverse direction. Anisotropy ratios of mineralized tissues were not affected by formalin fixation or plastic embedding. An evaluation of tissues from the different species showed that the AR after decollagenization was not significantly different (p > 0.4, analysis of variance) among the groups, while AR after demineralization varied from 1.04 (rat bone) to 1.51 (MTLT). There was no correlation between AR after demineralization and AR after decollagenization (r = 0.13, p = 0.5). This showed that the elastic anisotropy of collagen is more variable than mineral anisotropy in bone and MTLT. Another experiment showed that mineralization of turkey leg tendon changes the elasticity of the collagen matrix, making it less anisotropic. A final, prospective experiment was performed in which tibiae of rats were subjected to mechanical loading for 16 weeks. After 12 days, new periosteal woven bone was observed on the tibiae and, after 16 weeks, this new bone was consolidated and mineralized. Mineral in the newly formed woven bone was virtually isotropic (AR = 1.07) after 12 days of loading, then became more anisotropic (AR = 1.52) after 16 weeks of mechanical loading, as the mineral density of the new bone increased. This increase in anisotropy of bone mineral occurred even though the collagen matrix was woven and had no measureable fibril orientation. We conclude that (1) collagen anisotropy and mineral anisotropy are not necessarily correlated in mineralized tissues, (2) mineralization can affect the collagen matrix elasticity of mineralized tissues, and (3) an organized mineral structure can form in the absence of an organized collagen matrix.
Strontium distribution and interactions with bone mineral in monkey iliac bone after strontium salt (S 12911) administration
Authors: Boivin Georges , Deloffre Pascale , Perrat Brigitte , Panczer Gérard , Boudeulle Micheline , Mauras Yves , Allain Pierre , Tsouderos Yannis , Meunier Pierre J.
Published: February 2010

The analysis of the interaction of strontium (Sr) with bone mineral is of interest because a new agent containing Sr (S 12911) has shown positive effects on bone mass in various animal models of osteoporosis and is currently being developed for preventive and curative treatment of postmenopausal osteoporosis. Iliac bone samples were obtained from 20 male monkeys: 4 untreated control animals, 12 animals sacrificed at the end of a 13-week treatment with high dose levels of S 12911 (750, 275, or 100 mg/kg/day orally), and 4 animals sacrificed 6 weeks after the end of a 13-week treatment with S 12911 (750 or 100 mg/kg/day orally). The distribution of Sr was determined and quantified by X-ray microanalysis. Changes at the crystal level were evaluated by X-ray diffraction and Raman microspectrometry. In the control animals, traces of Sr were found to be homogeneously distributed throughout the bone tissue. In the treated monkeys, Sr could only be detected in calcified matrix. In monkeys sacrificed at the end of the treatment, Sr was found to be dose-dependently incorporated into the mineral substance of the compact and cancellous bone. Sr was heterogeneously distributed with three to four times more Sr in new than in old compact bone, and approximately two and a half times more Sr in new than in old cancellous bone. The bone Sr content dramatically decreased in the animals sacrificed 6 weeks after the end of the treatment. Diffraction showed no significant changes in the characteristics of the crystal lattice. Sr appeared to be easily exchangeable from bone mineral and was slightly linked to mature crystals through ionic substitutions. Even at the highest dose level tested, less than 1 calcium ion out of 10 was substituted by 1 Sr ion in each crystal. In conclusion, taken up by bone, Sr was heterogeneously distributed with a higher concentration in new than in old bone but induced no major modifications of the bone mineral (crystallinity, crystal structure) at the crystal level. As a result, a treatment with S 12911 Sr salt should not induce any alteration of bone mineral.
The effect of weight-bearing exercise on bone mineral density: A study of female ex-elite athletes and the general population
Authors: Etherington J. , Harris P. A. , Nandra D. , Hart D. J. , Wolman R. L. , Doyle D. V. , Dr. Spector T. D.
Published: February 2010

The aim of this retrospective cohort study was to estimate the changes in bone mineral density (BMD) as a consequence of exercise in female ex-athletes and age-matched controls. Eighty-three ex-elite female athletes (67 middle and long distance runners, 16 tennis players, currently aged 40–65) were recruited from the original records of their sporting associations. Controls were 585 age-matched females. The main outcome measures were BMD of lumbar spine (LS), femoral neck (FN), and forearm, estimated by dual-energy X-ray absorptiometry (DXA) scan. Levels of physical activity were assessed using a modified Allied Dunbar Fitness Survey scale and classified as (a) ex-athletes, (b) active controls (≥1 h of vigorous physical activity currently and in the past), (c) low activity controls with inconsistent or intermediate levels of activity, and (d) inactive controls (< 15 minutes of exercise per week). After adjustment for differences in age, weight, height, and smoking, athletes had greater BMDs than controls: 8.7% at the LS (95% confidence interval [CI] 5.4-12.0; p < 0.001) and 12.1% at FN (CI 9.0-15.3; p < 0.001). The benefits of exercise appeared to persist after cessation of sporting activity. Active controls (n = 22) had greater BMDs than the inactive group (n = 347): 7.9% LS (CI 2.0-13.8; p = 0.009) and 8.3% FN (CI 2.7-13.8; p = 0.004). The low activity controls (n = 216) had an intermediate BMD. Tennis players had greater BMDs compared with runners: 12.0% LS (CI 5.7-18.2; p = 0.0004) and 6.5% FN (CI −0.2-13.2; p = 0.066). The BMD of tennis players' dominant forearms were greater than their nondominant forearms. In conclusion, regular vigorous weight-bearing exercise of 1 h or more per week is associated with an increase in BMD within a normal population. This study confirms long-term weight-bearing exercise as an important factor in the regulation of bone mass and fracture prevention.
Predictors of ankle and foot fractures in older women
Authors: Seeley Dana G. , Kelsey Jennifer , Jergas Michael , Nevitt Michael C.
Published: February 2010

To determine risk factors for ankle and foot fractures, data collected from 9704 women 65 years of age or older from four areas of the United States were analyzed. Self-reported baseline questionnaires covered areas such as lifestyle factors (physical activity, diet, and smoking habits) and functional impairment (history of fracture, falling, and other diseases). Bone mineral density (BMD) and performance on neuromuscular tests were also measured. During 5.9 years of follow-up, 191 women fractured an ankle and 204 women fractured a foot. Proportional hazard models were used to estimate relative risks. In multivariable models, factors associated with ankle fracture included one or more falls in the year prior to baseline (relative risk [RR] 1.5; 95% confidence interval [CI] 1.1-2.1), greater vigorous physical activity (RR per 2 times/week, 1.2; CI 1.1-1.3), weight gain since age 25 (RR per 20% gain, 1.4; CI 1.2-1.5), self-reported osteoarthritis (RR 0.5; CI 0.3-0.8), a sister's history of hip fracture after age 50 (RR 1.7; CI 1.0-3.0), out of house ≥ 1 per week (RR 3.0; CI 1.4-6.6), and low distal radius BMD (RR per −0.1 g/cm2, 1.2; CI 1.0-1.4). Factors associated with foot fracture included insulin-dependent diabetes (RR 2.9; CI 1.2-7.2), use of seizure medications (RR 2.3; CI 1.0-5.7) or of benzodiazepines (RR 1.5; CI 1.1-2.2), history of hyperthyroidism (RR 0.5; CI 0.3-1.0), poor far depth perception (RR 0.7; CI 0.5-1.0), and low distal radius BMD (RR per −0.1 g/cm2, 1.3; CI 1.1-1.5). Ankle and foot fractures have different profiles of risk factors that are largely independent of low bone mass.
Parathyroid hormone regulates osteoblast differentiation positively or negatively depending on the differentiation stages
Authors: Isogai Yukihiro , Akatsu Takuhiko , Ishizuya Toshinori , Yamaguchi Akira , Hori Masayuki , Takahashi Naoyuki , Dr. Suda Tatsuo
Published: February 2010

The effects of parathyroid hormone (1-34) (PTH (1-34)) on osteoblast differentiation were investigated using primary osteoblast-like cells isolated from newborn mouse calvaria. The osteoblast-like cells cultured at low cell densities, in which the cells remained in a subconfluent state at the end of culture, were exposed for 7 days to PTH. This stimulated alkaline phosphatase (ALP) activity in a dose-dependent manner. In contrast, PTH dosedependently inhibited both ALP activity and osteocalcin production in cells inoculated at high cell densities, in which they had reached a confluent state before the end of culture. The changes of ALP activity by PTH were accompanied with the expression of ALP messenger RNA. PTH induced no changes of the hydroxyproline content in the cell layer when the cells were exposed to the hormone at a subconfluent state, but reduced the content at a postconfluent state. The stimulation of ALP activity by PTH at a preconfluent state was retained even after the removal of PTH from the culture media. The opposite effect of PTH, observed between the preconfluent and the postconfluent state, was reproduced by adding dibutyryl cyclic adenosine monophosphate (cAMP) or forskolin, but not by adding phorbol myristate acetate. In a colony-forming unit fibroblastic (CFU-F) assay, using bone marrow cells isolated from tibiae of 10-week-old mice, PTH induced no changes in the total number of CFU-Fs, but increased the proportion of ALP-positive colonies. These results indicate that PTH exerts opposite effects on the phenotypic expression of osteoblasts, depending on their differentiation stages of osteoblasts. PTH may preferentially stimulate osteoblast differentiation in immature osteoblasts but inhibit it in more mature cells.
PTH-related protein is released into the mother's bloodstream during lactation: Evidence for beneficial effects on maternal calcium-phosphate metabolism
Authors: Lippuner Kurt , Zehnder Hans-Jakob , Casez Jean-Paul , Takkinen Ritva , Jaeger Philippe
Published: February 2010

Recent studies have indicated that parathyroid hormone-related protein (PTHrP) may have important actions in lactation, affecting the mammary gland, and also calcium metabolism in the newborn and the mother. However, there are as yet no longitudinal studies to support the notion of an endocrine role of this peptide during nursing. We studied a group of 12 nursing mothers, mean age 32 years, after they had been nursing for an average of 7 weeks (B) and also 4 months after stopping nursing (A). It was assumed that changes occurring between A and B correspond to the effect of lactation. Blood was assayed for prolactin (PRL), PTHrP (two-site immunoradio-metric assay with sheep antibody against PTHrP(1-40), and goat antibody against PTHrP(60-72), detection limit 0.3 pmol/1), intact PTH (iPTH), ionized calcium (Ca2+), 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), alkaline phosphatase (alkP), as well as for creatinine (Cr), protein, phosphorus (P), and total calcium (Ca). Fasting 2-h urine samples were analyzed for Ca excretion (CaE) and renal phosphate threshold (TmP/GFR). PRL was significantly higher during lactation than after weaning (39 ± 10 vs. 13 ± 9 μg/l; p = 0.018) and so was PTHrP (2.8 ± 0.35 vs. 0.52 ± 0.04 pmol/l; p = 0.002), values during lactation being above the normal limit (1.3 pmol/l) in all 12 mothers. There was a significant correlation between PRL and PTHrP during lactation (r = 0.8, p = 0.002). Whole blood Ca2+ did not significantly change from A (1.20 ± 0.02 mmol/l) to B (1.22 ± 0.02, mmol/l), whereas total Ca corrected for protein (2.18 ± 0.02 mmol/l) or uncorrected (2.18 ± 0.02 mmol/l) significantly rose during lactation (2.31 ± 0.02 mmol/l, p = 0.003 and 2.37 ± 0.03 mmol/l, p = 0.002, respectively). Conversely, iPTH decreased during lactation (3.47 ± 0.38 vs. 2.11 ± 0.35 pmol/l, A vs. B, p = 0.02). Serum-levels of 25(OH)D3 and 1,25(OH)2D3 did not significantly change from A to B (23 ± 2.3 vs. 24 ± 1.9 ng/ml and 29.5 ± 6.0 vs. 21.9 ± 1.8 pg/ml, respectively). Both TmP/GFR and P were higher during lactation than after weaning (1.15 ± 0.03 vs. 0.86 ± 0.05 mmol/l GF, p = 0.003 and 1.25 ± 0.03 vs. 0.96 ± 0.05 mmol/l, p = 0.002, respectively) as was alkP (74.0 ± 7.1 vs. 52.6 ± 6.9 U/l, p = 0.003). CaE did not differ between A and B (0.015 ± 0.003 vs. 0.017 ± 0.003 mmol/l GF, A vs. B, NS). We conclude that lactation is accompanied by an increase in serum PRL. This is associated with a release of PTHrP into the maternal blood circulation. A rise in total plasma Ca ensues, probably in part by increased bone turnover as suggested by the elevation of alkP. PTH secretion falls, with a subsequent rise of TmP/GFR and plasma P despite high plasma levels of PTHrP.