bluuebunny/crossref_metadata_2025 huggingface dataset:
A study of the relationship between mineral content and mechanical properties of turkey gastrocnemius tendon
Authors: Dr. Landis William J. , Librizzi Joseph J. , Dunn Michael G. , Silver Frederick H.
Published: February 2010

The vertebrate skeletal system undergoes adaptation in response to external forces, but the relation between the skeletal changes and such forces is not understood. In this context, the variation in the amount and location of calcification has been compared with changes in mechanical properties of the normally mineralizing turkey gastrocnemius tendon using ash weight measurements, X-ray radiography, and mechanical testing. Radiographic evidence from 12- to 17-week-old birds showed calcification in only portions of gastrocnemius tendons proximal to the tarsometatarsal joint. Mechanical testing of these dissected proximal regions demonstrated an increased ultimate stress and modulus and a decreased maximum strain that appeared to parallel calcification. Further, stress–strain curves of portions of uncalcified turkey gastrocnemius tendon were shaped similar to those of other typical unmineralized tendon curves while highly calcified tendons yielded curves resembling those of bone. The proximal portions of the gastrocnemius where mineralization begins were observed to have a decreased tendon cross-sectional area compared with distal portions which do not mineralize. Based on the resultant measures of mineral content and location and mechanical properties, it is hypothesized that increased calcification is a result of increased stresses at certain locations of the tendon, perhaps the consequence of the natural forces exerted by the large leg muscles of the bird into which the gastrocnemius inserts. More specifically, tendon calcification may be the result of stress-induced exposure of charged sites on the surfaces of collagen molecules, fibrils, or fibers so that deposition of mineral and subsequent mechanical reinforcement occur in the tissue. The concept of stress-induced calcification of avian tendon is found to be consistent with other examples of biological mineralization and supports the view that vertebrate calcification is an adaptive process mediated in part by the accommodation of natural or applied forces.
Impact of soft tissue on in vivo accuracy of bone mineral measurements in the spine, hip, and forearm: A human cadaver study
Authors: Dr. Svendsen Ole L. , Hassager Christian , Skødt Vera , Christiansen Claus
Published: February 2010

The impact of soft tissue in vivo on accuracy of bone mineral density (BMD) measurements of the spine and hip by dual energy X-ray absorptiometry and of the forearm by single photon absorptiometry was assessed by use of 14 human cadavers. The in vivo accuracy errors (SEE%) were: forearm 3–5%, anteroposterior spine 5.3%, lateral spine 10–12%, and femoral greater trochanter, neck, total, intertrochanteric, and Ward's triangle 3%, 6.5%, 6.7%, 8%, and 11–13%, respectively. Except from the lateral spine and the greater trochanter, the slopes of the linear regressions of in vivo BMD against in vitro BMD were not significantly different from 1 (p > 0.05). The calculated random accuracy error of BMD measurements due to fat inhomogeneity was estimated to 3–4% for the anteroposterior spine and 9–14% for the lateral spine (from abdominal computed tomography in 26 healthy women). In conclusion, acceptable accuracy errors below 6–7% (of soft tissue in vivo) of BMD measurements were obtained in the anteroposterior spine, the forearm, and the neck, greater trochanter, and total proximal femur.
Cell-to-cell communication in osteoblastic networks: Cell line–dependent hormonal regulation of gap junction function
Authors: Dr. Donahue Henry J. , Mcleod K.J. , Rubin C.T. , Andersen J. , Grine E.A. , Hertzberg E.L. , Brink P.R.
Published: February 2010

We have characterized the distribution, expression, and hormonal regulation of gap junctions in primary cultures of rat osteoblast-like cells (ROBs), and three osteosarcoma cell lines, ROS 17/2.8, UMR-106, and SAOS-2, and a continuous osteoblastic cell line, MC3T3-E1. All cell lines we examined were functionally coupled. ROS 17/2.8 were the more strongly coupled, while ROB and MC3T3-E1 were moderately coupled and UMR-106 and SAOS-2 were weakly coupled. Exposure to parathyroid hormone (PTH) for 1 h increased functional coupling in ROB cells in a concentration-dependent manner. Furthermore, PTH (3–34), an analog of PTH with binds to the PTH receptor and thus attenuates PTH-stimulated cAMP accumulation, also attenuated PTH-stimulated functional coupling in ROB. This suggests that PTH increases functional coupling partly through a cAMP-dependent mechanism. A 1 h exposure to PTH did not affect coupling in ROS 17/2.8, UMR-106, MC3T3-E1, or SAOS-2. To examine whether connexin43 (Cx43), a specific gap junction protein, is present in functionally coupled osteoblastic cells, we characterized Cx43 distribution and expression. Indirect immunofluorescence with antibodies to Cx43 revealed that ROS 17/2.8, ROB, and to a lesser extent MC3T3-E1 and UMR-106, expressed Cx43 immunoreactivity. SAOS-2 showed little if any Cx43 immunoreactivity. Cx43 mRNA and Cx43 protein were detected by Northern blot analysis and immunoblot analysis, respectively, in all cell lines examined, including SAOS-2. Our findings suggest that acute exposure to PTH regulates gap junction coupling, in a cell-line dependent manner, in osteoblastic cells. Furthermore, Cx43 is expressed in osteoblastic cells regardless of the degree to which the cells are functionally coupled or the degree to which coupling is regulated by PTH.
The BsmI vitamin D receptor restriction fragment length polymorphism (BB) predicts low bone density in premenopausal black and white women
Authors: Dr. Fleet James C. , Harris Susan S. , Wood Richard J. , Dawson-Hughes Bess
Published: February 2010

We conducted a study to determine whether a recently described restriction fragment length polymorphism in the vitamin D receptor gene (VDR-RFLP) predicts bone mineral density (BMD) in unrelated, premenopausal women as well as to determine the racial contribution to any genotypic influences on BMD. White (n = 83) and black (n = 72) women between 20 and 40 years of age were genotyped based on the presence (b) or absence (B) of a Bsm1 restriction enzyme site in the VDR gene, and BMD in the lumbar spine and femur neck was determined for each subject. There were 16 BB, 73 Bb, and 66 bb women. No significant difference was observed in genotypic distribution between the racial groups. The interaction of race by genotype on age- and body mass index (BMI)-adjusted BMD was not significant at either site. Age- and BMI-adjusted BMD was higher in black women at the spine (by 7.2%, p = 0.046) and femur neck (7.3% higher, p = 0.004). In the group as a whole, mean BMD in the femur neck was lower in the BB women than the bb (by 8.1%, p = 0.034) or Bb women (by 9.3%, p = 0.015) after controlling for age, BMI, race, and the race by genotype interaction. Adjusted lumbar spine BMD was lower in the BB women than the Bb women (6.4% lower, p = 0.036) in the group as a whole. No differences were detected between Bb and bb women at either site. A similar pattern of low BMD at the femur neck and the lumbar spine was seen in BB women of both races. These data provide support for an association between low bone density and the BB genotype in a racially mixed, premenopausal population and suggests that this genotype may limit peak bone mass. Furthermore, racial differences in BMD appear to be independent from the VDR genotype.
Characterization of MCSF-induced proliferation and subsequent osteoclast formation in murine marrow culture
Authors: Biskobing Diane M. , Fan Xian , Rubin Janet
Published: February 2010

To clarify events involved in 1,25(OH)2D3-stimulated osteoclast-like cell (OCLC) formation in primary murine marrow culture, we have characterized kinetics of precursor proliferation and fusion and their dependence on macrophage colony-stimulating factor (MCSF). 3H-thymidine nuclear incorporation in tartrate-resistant acid phosphatase positive multinucleated cells (TRAP+ MNCs) was assessed: 3H-thymidine incorporation was greatest when tracer was added during day 4 or 5, with labeled nuclei in 81% (day 4) and 90% (day 5) of the TRAP+ MNCs counted at the end of day 7. The percentage of total nuclei labeled was highest when 3H-thymidine was dosed on day 4 (58%), decreasing to 2% by day 7. Final TRAP+ MNC numbers were depleted by 80% when treated for 24 h with hydroxyurea on either day 3 or 4; this inhibition dropped to 57% and 12% when hydroxyurea was pulsed during days 5 or 6, respectively. The absence of 1,25(OH)2D3 during days 1–4 caused 70% attenuation of TRAP+ MNC formation; however, exposure to 3H-thymidine during day 4 in this experiment resulted in subsequent labeling of 81% of the TRAP+ MNCs formed, indicating that precursor proliferation occurred in the absence of 1,25(OH)2D3. To demonstrate that proliferation required MCSF, cultures were exposed to a monoclonal anti-MCSF antibody during days 3, 4, 5, 6, or 7. Inhibition of TRAP+ MNC formation was 85% when antibody was added during day 3. Antibody treatment after day 5 had little effect on the OCLC number. Fusion of precursors showed steady progression with OCLCs containing 4.8 ± 03 nuclei at the end of day 4, 8.3 ± 0.5 nuclei after day 5, 12.0 ± 1.3 after day 6, and 13.7 ± 1.5 at the end of day 7. This steady accretion of nuclei was unaffected by doses of MCSF antibody which blocked proliferation. In conclusion, we have shown that OCLCs arise from an MCSF-dependent expansion of the precursor pool occurring during days 3 and 4. Fusion of these precursors, which begins as proliferation diminishes, is able to progress in the presence of anti-MCSF antibody. These results should help refine the analysis of factors affecting proliferation and fusion of osteoclasts in murine marrow culture.
Influence of high glucose on 1,25-dihydroxyvitamin D3-induced effect on human osteoblast-like MG-63 cells
Authors: Inaba Masaaki , Terada Makoto , Koyama Hidenori , Yoshida Osamu , Ishimura Eiji , Kawagishi Takahiko , Okuno Yasuhisa , Nishizawa Yoshiki , Morii Hirotoshi , Otani Shuzo
Published: February 2010

Impaired bone formation due to defective osteoblast function, as reflected in a decreased serum osteocalcin (OC) concentration in the patients with diabetes, has been implicated in the development of diabetic osteopenia. The role of hyperglycemia in this decrease in serum OC concentration was investigated. 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), an active form of vitamin D3, stimulated OC secretion from the human osteosarcoma cell line MG-63 in a dose-dependent manner. Exposure of the cells to high concentrations of glucose for 7 days significantly impaired 1,25(OH)2D3-induced OC secretion as compared with that observed with cells maintained under normal glucose (5.5 mM) or high mannitol conditions. The inhibitory effect of glucose was in a dose-dependent manner up to 55 mM. High glucose (55 mM) also attenuated the 1,25(OH)2D3-induced increase in OC mRNA abundance in MG-63 cells, suggesting that the inhibition of the 1,25(OH)2D3-induced increase in OC secretion by exposure to a high concentration of glucose was, at least in part, mediated at the transcriptional level. High glucose significantly decreased the number of 1,25(OH)2D3 receptors in MG-63 cells, without any change in the dissociation constant for 1,25(OH)2D3; this effect was not mimicked by high mannitol, indicating specificity for glucose. These observations suggest that a high glucose concentration significantly impairs the ability of osteoblastic cells to synthesize OC in response to 1,25(OH)2D3 by reducing 1,25(OH)2D3 receptor number, and that impaired cell function caused by sustained exposure to high glucose contributes to the defect in bone formation observed in the patients with diabetic osteopenia. (
The effects of calcium supplementation (milk powder or tablets) and exercise on bone density in postmenopausal women
Authors: Prince Richard , Devine Amanda , Dick Ian , Criddle Arthur , Kerr Deborah , Kent Neil , Randell Andrew , Price Roger
Published: February 2010

The etiology of age-related bone loss is unclear but both lack of exercise and dietary calcium deficiency have been implicated in its causation. This 2-year randomized placebo-controlled study was designed to examine the effects of increased dietary calcium and exercise in 168 women who were more than 10 years postmenopausal. The subjects were randomized into one of 4 groups: placebo, milk powder containing 1 g of calcium, calcium tablets 1 g/night, and calcium tablets 1 g/night and an exercise regimen. The exercise group aimed to undertake 4 h of extra weight-bearing exercise per week and were undertaking 10% more activity than other groups at 2 years. Bone mineral density at the lumbar spine, three hip sites, and two sites of the tibia close to the ankle joint were measured at 6 month intervals. Dietary intake was evaluated by a weighed food record, exercise was evaluated by an exercise diary, and blood and urine samples were obtained to examine effects on calcium homeostasis. Individual data points were compared using repeated measures ANOVA and least squares regression. Calcium supplementation by either the calcium tablets or the milk powder resulted in cessation of bone loss at the intertrochanteric hip site (placebo, calcium tablets, calcium and exercise, milk powder −0.81, +0.17, +0.23, and +0.07% per year, respectively; p < 0.05 for all supplementation groups compared with placebo) with similar results at the trochanteric hip site. The calcium and exercise group had less bone loss at the femoral neck site when compared with calcium supplementation alone (placebo, calcium tablets, calcium and exercise, milk powder −0.67, −0.18, +0.28, and −0.18% per year, respectively; p < 0.05 for calcium and exercise compared with calcium alone). There was a significant reduction in the rate of bone loss at the ultradistal site of the tibia (placebo, calcium tablets, calcium and exercise, milk powder −2.5, −1.6, −1.0, and −1.5% per year, respectively; p < 0.05 for all supplementation groups compared with placebo). There was no significant bone loss at the spine site in any group. These data support the implementation of a simple public health regimen to prevent age-related bone loss with calcium supplementation either by calcium tablet or by milk powder. The extra effect of exercise plus calcium at the femoral neck site suggests a site-specific effect of physical activity on bone density in addition to its possible effect to prevent fracture by maintenance of muscle strength and coordination. (
Maxillary molar extraction causes increased bone loss in the mandible of ovariectomized rats
Authors: Elovic Rebecca P. , Hipp John A. , Hayes Wilson C.
Published: February 2010

Although osteoporosis is a major public health concern, its effect on oral bone has not been determined. More important may be the effect of estrogen depletion on the response of oral bone to dental treatments such as tooth extraction or pathologic processes such as periodontal disease. Our objective was to determine if maxillary molar extraction increases mandibular bone loss in the ovariectomized compared with a sham-operated control. Fifty-three ovariectomized and 53 sham-operated 6-month-old Sprague-Dawley rats were randomly assigned to the following groups: (1) ovariectomized, adult; (2) sham-operated, adult; (3) ovariectomized, adult, extraction; (4) sham-operated, adult, extraction; (5) ovariectomized, old; (6) sham-operated, old; (7) ovariectomized, old, extraction; and (8) sham-operated, old, extraction. Fourteen days following ovariectomy, the extraction groups had their bilateral maxillary molars extracted. The adult and old rats were sacrificed 114 and 200 days postovariectomy, respectively. The right mandible was tested to failure in three point bending. The bone mineral density (BMD) of the left mandible was measured with high resolution dual energy X-ray absorptiometry. The area fraction and area moment of inertia of mandible sections were determined using image processing software. In the ovariectomized rats, maxillary molar extraction resulted in decreases (p < 0.05) in the failure load (21%), stiffness (39%), BMD (3%), and bone area fraction (8%) of the mandible. However, in the sham-operated rats, these decreases following maxillary molar extraction were less (p < 0.05) than those in the ovariectomized rats and only present in the mandibles of the aged rats. Therefore, maxillary molar extraction resulted in a more rapid decrease in structural properties and BMD and a larger decrease in bone area fraction of the mandible in ovariectomized rats compared with those in sham-operated rats. These findings indicate that oral bone loss that occurs as a result of pathologic conditions or dental treatments may be accelerated in individuals who have estrogen depletion. (
Extracellular matrix stoichiometry in osteoblasts from patients with osteogenesis imperfecta
Authors: Fedarko Neal S. , Robey Pamela Gehron , Vetter Ulrich K.
Published: February 2010

In previous work, we compared the steady-state levels of specific matrix components in human bone cells derived from patients with osteogenesis imperfecta (OI) to those of age-matched controls. A remarkable finding was the observation that there was a reduction not only in the total levels of collagen, but also in osteonectin and three proteoglycans (a large chondroitin sulfate proteoglycan, biglycan, and decorin). This pattern was observed in patients with and without detectable collagen defects. More recent analysis of extracellular matrix composition have yielded that, compared with age-matched controls, bone cells from OI patients produced higher steady-state levels of fibronectin and thrombospondin. The percentage of these two proteins incorporated into the cell layer pool was also higher in OI than in age-matched controls. In addition, the steady-state levels of hyaluronan and a heparan sulfate proteoglycan were analyzed in both OI and age-matched controls. Although the total (medium + cell layer) steady-state levels of hyaluronan were reduced by 1/3, the percentage of the hyaluronan in the cell layer pool of patients with OI increased between 100–250% of age-matched control. Thus the matrix elaborated by human OI bone cells is not only quantitatively different but also qualitatively distinct from that of age-matched controls. Not only have specific bone cell matrix components (collagen, osteonectin, the large chondroitin sulfate proteoglycan, biglycan, and decorin) been found to be present in reduced levels in OI bone cells, but some matrix components (thrombospondin, fibronectin, and hyaluronan) have also been found to be present in elevated levels in the matrix of OI cells. These results suggest that putative mutations in type I collagen (present in OI bone cells) give rise to an altered gene expression of other matrix components.
Identification and cloning of human P2U purinoceptor present in osteoclastoma, bone, and osteoblasts
Authors: Bowler W.B. , Birch M.A. , Gallagher J.A. , Bilbe G.
Published: February 2010

Extracellular ATP acting through purinoceptors may be an important factor in the modulation of bone turnover. In this study we cloned and sequenced the P2U purinoceptor from osteoclastoma, confirming the recently published human sequence. Furthermore, by the reverse transcriptase-linked polymerase chain reaction (RT-PCR) and Southern blotting we demonstrated expression of P2U receptor mRNA in bone, primary cultures of human bone-derived cells, and two osteosarcoma cell lines, Saos2 and Te85. P2U receptor transcripts were identified in alkaline phosphatase-positive human bone-derived cells isolated by flow cytometry providing strong evidence for the expression of the P2U purinoceptor in mature osteoblasts. P2U receptor transcripts were also detected in a purified giant cell population isolated from osteoclastoma, indicating that this receptor is also expressed by osteoclasts. These data suggest that purinergic agonists may play a role in the regulation of bone metabolism.
Expression of the 1,25-dihydroxyvitamin D3-24-hydroxylase gene in rat intestine: Response to calcium, vitamin D3 and calcitriol administration in vivo
Authors: Lemay Jacques , Demers Christian , Hendy Geoffrey N. , Delvin Edgard E. , Gascon-Barré Marielle
Published: February 2010

The 25(OH)D3/1,25(OH)2D3 24-hydroxylase (24-hydroxylase) displays an induction profile responsive to vitamin D (D) abundance and is hence only observed in normal extracellular Ca2+ concentrations. However, the participation of Ca2+ in the expression of the 24-hydroxylase gene in vivo is not known. The present studies investigate the role played by the circulating Ca2+ and the D3 and/or 1,25(OH)2D3 status on the 1,25(OH)2D3-mediated inducibility of the 24-hydroxylase gene in rat duodenum. Hypocalcemic D-depleted rats were supplemented with calcium alone to normalize serum Ca2+ without normalizing the D3 status or were acutely or chronically supplemented with D3 or 1,25(OH)2D3. Messenger RNA for the 24-hydroxylase was undetectable in the intestine of hypocalcemic D-depleted rats, and short- or long-term calcium supplementation was completely unsuccessful in inducing its expression. By contrast, acute 1,25(OH)2D3 administration led to significant increases in the levels of expression of the gene which was independent of the calcium intake, the prevailing circulating Ca2+, and the D3 or 1,25(OH)2D3 status. Moreover, 24-hydroxylase gene expression was only found to respond to acutely administered 1,25(OH)2D3, the mRNA levels being unaltered following continuous exposure to physiological or pharmacological doses of the hormone for 7 days. Time-course studies revealed, however, that induction of the gene was clearly apparent early in the 1,25(OH)2D3 supplementation course but gradually faded by 3 days to return to basal uninduced levels by 7 days, suggesting the presence of intestinal adaptation mechanism(s) which down-regulated the responsiveness in the continuous presence of 1,25(OH)2D3. Our data show the lack of effect of calcium alone or in combination with 1,25(OH)2D3 on the in vivo induction of the 24-hydroxylase gene expression in rat intestine. By rapidly reacting to surges in 1,25(OH)2D3 concentrations, the 24-hydroxylase efficiently controls the amount of 1,25(OH)2D3 in intestine as the first step in the biotransformation pathway aimed at the irreversible clearance of the secosteroid hormone. (J Bone Miner Res 1995;10:1148–1157)
Vitamin D status and sex hormone binding globulin: Determinants of bone turnover and bone mineral density in elderly women
Authors: Ooms Marcel E. , Lips Paul , Roos Jan C. , van der Vijgh Wim J. F. , Popp-Snijders Corrie , Bezemer P. Dick , Bouter Lex M.
Published: February 2010

To examine the relation of the vitamin D status and the remaining estrogen activity with bone turnover and bone mineral density (BMD) in elderly women, BMD was measured at both hips using dual-energy X-ray absorptiometry and at the distal radius using single photon absorptiometry, in 330 healthy women aged 70 and over. Vitamin D metabolites, sex hormone binding globulin (SHBG), PTH(1–84), osteocalcin, alkaline phosphatase, and hydroxyproline and calcium excretion in 2 h fasting urine were measured. Multiple linear regression was used to adjust for potential confounders. In 65% of the women, serum 25(OH)D was below 30 nmol/l. Only values below a threshold for 25(OH)D were negatively related to serum PTH(1–84) (p = 0.02, threshold at 25 nmol/l) and to osteocalcin levels (p = 0.04, threshold at 30 nmol/l). BMD of the femoral neck and trochanter was positively related to serum 25(OH)D (left neck p = 0.001) with thresholds at 30 nmol/l whereas the distal radius was not (p = 0.32). Serum PTH was negatively related to BMD at all measurement sites (all p < 0.001). Serum SHBG, an inverse measure of estrogen activity, was positively related to osteocalcin levels (p = 0.004) and the urinary hydroxyproline/creatinine ratio (p = 0.002) and negatively related to the BMD of the trochanter (left trochanter p = 0.02) and the distal radius (p = 0.001). We conclude that in elderly women, serum 25(OH)D levels below 30 nmol/l are associated with secondary hyperparathyroidism and increased bone turnover. SHBG is positively related to bone turnover. Vitamin D deficiency especially influences BMD of the femoral neck, a cortical area. SHBG mainly influences BMD at the trochanteric region and distal radius, predominantly trabecular areas, which may reflect the effects of remaining estrogen activity.
Cloning and complete coding sequence of a novel human cathepsin expressed in giant cells of osteoclastomas
Authors: Li Yi-Ping , Alexander Mary , Wucherpfennig Anne L. , Yelick Pamela , Chen Wei , Stashenko Philip
Published: February 2010

A gene encoding a possible novel human cathepsin, a cysteine proteinase that is distinct from previously characterized enzymes, has been identified by differential screening of a human osteoclastoma cDNA library. This molecule, termed cathepsin X, appears to represent the human homolog of the osteoclast-expressed rabbit cathepsin OC-2. Cathepsin X (GenBank accession number U20280) is 93.9% identical to OC-2 at the amino acid level, and is 92% identical at the nucleotide level within the coding region. Cathepsin X is 52.2 and 46.9% identical to cathepsins S and L, respectively, and is therefore clearly distinct from these enzymes. Cathepsin X mRNA was localized to multinucleated giant cells within the osteoclastoma tumor by in situ hybridization. These data strongly support the hypothesis that cathepsin X represents a novel cysteine proteinase which is expressed at high levels in osteoclasts.
Changes in bone mineral density and markers of bone remodeling during lactation and postweaning in women consuming high amounts of calcium
Authors: Cross Nanna A. , Hillman Laura S. , Allen Susan H. , Krause Gary F.
Published: February 2010

A randomized clinical intervention trial to determine effects of lactation and 1 g of calcium (Ca) on bone remodeling was conducted in 15 women (calcium = 7, placebo [P] = 8) consuming 1.3–2.4 g of Ca/day from diet + prenatal supplement. Study periods were baseline, ≤2 weeks postpartum; lactation, 3 months lactation; and postweaning, 3 months postweaning. Bone mineral density (BMD) corrected for body weight was determined by dual-energy X-ray absorptiometry (DXA). Indicators of calcium metabolism, bone turnover, and lactation were measured: calcium metabolism, parathyroid hormone (PTH), 25-hydroxyvitamin D (25[OH]D), 1,25-dihydroxyvitamin D (1,25[OH]2D); bone turnover, formation, procollagen I carboxypeptides (PICP), osteocalcin, and bone alkaline phosphatase (B-ALP), resorption, tartrate resistant acid phosphatase (TRAP); and lactation, prolactin (PRL). Mean BMD changes differed by site: baseline to lactation –4.3% (P) (p < 0.04) and –6.3% (Ca) (p < 0.01) at the lumbar spine (L2–L4) and 5.7% gains of the ultradistal (UD) radius (Ca) (p < 0.04); lactation to postweaning, –6% to –11% at all sites of the radius and ulna (Ca, P) (p < 0.04) +3% at L2–L4 (Ca) (p < 0.03); baseline to postweaning, (UD) radius –5.2% (P) (p < 0.03), UD radius + ulna –6% to –8% (Ca, P) (p < 0.04) but no significant loss of L2–L4 or total body. Bone turnover markers were higher at lactation than postweaning: PICP (+34%, p < 0.001), osteocalcin (+25%, p < 0.01), TRAP (+11%, p < 0.005) as was PRL (+81%, p < 0.001). Indicators of calcium metabolism were higher postweaning than lactation for PTH (+40%, p < 0.01) and 25(OH)D (+45%, p < 0.02) but not for 1,25(OH)2D. There were no differences between P or Ca for indices of calcium metabolism, bone turnover, or PRL. An increase in markers of bone turnover and a loss of BMD of the spine during lactation appears to be part of the physiological changes of lactation and not preventable by increasing calcium intake above the recommended dietary allowance (RDA). A return of BMD toward baseline of the spine but not the arm, was associated with an increase in PTH without an increase in 1,25(OH)2D postweaning. Loss of estrogen during lactation and a return postweaning may play an important role.
EndothelinB receptor activation enhances parathyroid hormone-induced calcium signals in UMR-106 cells
Authors: Lee Suk Kyeong , Dr. Stern Paula H.
Published: February 2010

In studies of the regulation of parathyroid hormone (PTH) signal transduction, we observed that the peptide endothelin-1 (ET) added prior to PTH greatly increased the calcium transients elicited by PTH in UMR-106 osteosarcoma cells and mouse primary osteoblastic cells. Enhancement by ET also occurred in the presence of EGTA. The ETB receptor-specific agonist sarafotoxin 6c (S6c) likewise enhanced PTH-induced Ca2+ transients. Blocking the ETA receptor-mediated component of the ET signal with BQ123 failed to abolish enhancement of PTH responses by ET. The nonselective ETA/ETB receptor antagonist PD 142893 blocked both ET and S6c-induced enhancement of the PTH responses. Prostaglandin F1α (PGF1α) pretreatment also maximally potentiated PTH responses, whereas α-thrombin, epidermal growth factor (EGF), or prostaglandin E1 (PGE1) did not affect the PTH responses. Neither active phorbol ester nor forskolin mimicked the ET effect. The ET effect was not prevented by indomethacin, NG-mono-methylarginine, genistein, pertussis toxin, 4-aminopyridine, tetraethylammonium chloride, okadaic acid, or long-term treatment with phorbol-12,13-dibutyrate. ET pretreatment did not abolish the inhibition of PTH signals by PTH(3–34), although in ET-pretreated cells the suppression of the PTH signal by PTH(3–34) was not as great. ET pretreatment did not enhance the cAMP response to PTH; rather, there was a significant inhibition of the cAMP response. Thus, the calcium signal elicited by PTH is selectively modulated by activation of the ETB receptor. The effect of ET to enhance PTH-stimulated calcium signaling appears to be independent of prostaglandins, nitric oxide, protein tyrosine kinase, protein kinase A, K+ channel activation, or phorbol ester-activated protein kinase C.
Endogenous bone-resorbing factors in estrogen deficiency: Cooperative effects of IL-1 and IL-6
Authors: Miyaura Chisato , Kusano Kenichiro , Masuzawa Toshihide , Chaki Osamu , Onoe Yoshiko , Aoyagi Maki , Sasaki Takahisa , Tamura Tatsuya , Koishihara Yasuo , Ohsugi Yoshiyuki , Suda Tatsuo
Published: February 2010

Estrogen deficiency causes a marked bone loss by stimulating osteoclastic bone resorption. To explore the endogenous bone-resorbing factors involved in estrogen deficiency, we examined the bone-resorbing activity present in the supernatant fraction of mouse bone marrow collected from ovariectomized (OVX) mice. Adding bone marrow supernatants at 20–80% to organ cultures of mouse long bones dose-dependently stimulated bone resorption. The endogenous bone-resorbing activity present in bone marrow supernatants from OVX mice was much higher than that from sham-operated mice 2–4 weeks after surgery, and it was significantly diminished by indomethacin in vitro. Anti-IL-1α antibody completely neutralized the bone-resorbing activity present in bone marrow supernatants from OVX mice. Antibodies against IL-1β, IL-6, and IL-6 receptors also neutralized it, but partially. The concentration of IL-1α measured by ELISA was much higher in bone marrow supernatants than in sera, but it was not appreciably changed before or after OVX. The concentration of IL-1β in bone marrow supernatants from OVX mice was less than the detection limit. OVX stimulated IL-1 activity in bone marrow supernatants measured by means of the proliferation of thymocytes. However, the level of IL-1α present in bone marrow supernatants from OVX mice was insufficient to stimulate bone resorption. Compared with the serum concentration, bone marrow supernatants contained a much higher level of IL-6 as well, and it was further increased by OVX. However, IL-6 alone present in bone marrow supernatants from OVX mice again did not stimulate bone resorption. The concurrent addition of IL-1 (50 pg/ml), IL-6 (0.2 ng/ml), sIL-6 receptor (1 ng/ml), and PGE2 (7 ng/ml), which equaled the endogenous concentrations in bone marrow supernatants from OVX mice, co-operatively induced bone resorption. These results suggest that the enhanced bone resorption that occurs during estrogen deficiency is due to multi-factors rather than to a single factor.
Regulation of plasminogen activation, matrix metalloproteinases and urokinase-type plasminogen activator-mediated extracellular matrix degradation in human osteosarcoma cell line MG63 by interleukin-1 alpha
Authors: de Bart Anton C. W. , Dr. Quax Paul H.A. , Löwik Clemens W.G.M. , Verheijen Jan H.
Published: February 2010

Plasmin-mediated extracellular proteolysis has been implicated in the degradation of bone in normal and pathological conditions. Normal and malignant osteoblasts can produce both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). We have used the osteosarcoma cell line MG63 to address the question of whether the enhanced bone turnover in osteosarcomas is mediated by t-PA or by u-PA and to study the effect of the cytokine interleukin-1α (IL-1α), known to influence bone degradation, on the plasminogen activator production and extracellular matrix degradation in malignant osteoblastic cells. Furthermore, the effect of IL-1α on the synthesis of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) was analyzed. u-PA production by MG63 was high (approximately 180 ng/106 cells/24 h). Also t-PA and PAI-1 production was observed. u-PA production was rapidly increased in MG63 by IL-1α (10 ng/ml), whereas an effect on t-PA production was only found after a prolonged incubation and hardly any effect of IL-1α on PAI-1 production was observed. mRNA analysis revealed similar effects. u-PA receptor (u-PAR) mRNA was detectable in MG63 cells and could be increased by IL-1α after 24 h. In MG63, u-PA-mediated extracellular matrix degradation was detectable, and IL-1α increased the u-PA-mediated matrix degradation (approximately 2-fold). Under control conditions in MG63, only MMP-2, TIMP-1, and TIMP-2 mRNA could be observed. After the addition of IL-1α, a very rapid increase in MMP-1 and MMP-3 mRNA could be observed as well as a moderate increase in TIMP-1 mRNA. The presence of MMP-2 was demonstrated by gelatin zymography. These results show that IL-1α can stimulate u-PA production and can regulate extracellular proteolytic activity mainly via u-PA induction in the MG63 osteosarcoma cell line. Furthermore, IL-1α has a strong stimulating effect on the production of MMP-1 and MMP-3. These findings suggest that u-PA and possibly MMP-1 and MMP-3 play an important role in the process of bone turnover in osteosarcomas.
Microcallus formations of the cancellous bone: A quantitative analysis of the human spine
Authors: DipL. Ing. BMT Hahn Michael , Vogel M. , Amling M. , Ritzel H. , Delling G.
Published: February 2010

Microcallus formations are demonstrable in nearly all spongy bone by means of suitable preparation techniques. Histologically, these structures are immature fibrous bone. Their genesis, frequency, and importance are largely unknown. To address these issues, 26 normal human spines, 11 osteoporotic spines, and different parts of the skeleton (femur head, iliac crest) were investigated for microcallus using a new preparation technique—allowing a combined 2- and 3-dimensional analysis. According to our analysis, microcallus formation occurs frequently in persons older than 45 years of age. These formations are mainly localized in the lower thoracic and lumbar spine and are obviously more frequent in females than in males. In individuals with a trabecular bone volume (BV/TV) in the spine below 11%, microcallus formations occur regularly. But the number of microcallus formations depends more on the microarchitecture of the cancellous bone (trabecular bone pattern factor, TBPf), than on individual trabecular parameters (trabecular number, TbN; trabecular bone volume, BV/TV; and trabecular thickness, TbTh). In about 33% of cases microfractures are demonstrable in the center of the microcallus formation. It is unclear whether microcallus may be the result of a nontraumatic process. In therapy studies the bone mass could be misrepresented due to the amount of microcallus. Although it indicates instability of the bone structure, microcallus formation is not only a negative mechanism, but stabilizes and regenerates the bone tissue. Furthermore, complete new trabeculae can be formed due to bridges of microcallus between the remnant trabeculae. Osteoporosis is not the result of an inability to form microcallus formations.
Regulation of COL1A1 expression in type I collagen producing tissues: Identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice
Authors: Bedalov Antonio , Salvatori Roberto , Dodig Milan , Kronenberg MarkS , Kapural Belinda , Bogdanovic Zoran , Kream Barbara E. , Woody Charles O. , Clark Stephen H. , Mack Kristine , Rowe David W. , Lichtler Alexander C.
Published: February 2010

Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.
Inhomogeneity in body fat distribution may result in inaccuracy in the measurement of vertebral bone mass
Authors: Formica Carmelo , Loro Maria-Luiza , Gilsanz Vicente , Prof. Seeman Ego
Published: February 2010

When bone mineral content (BMC) is measured by dual X-ray absorptiometry (DXA), the X-ray beam is attenuated by bone and soft tissue. Since the component of the attenuation caused by the soft tissue overlying bone cannot be measured, the attenuation caused by soft tissue adjacent to bone is measured and is used in the calculation of BMC. The assumption underlying this approach is that the amount and composition of this adjacent soft tissue is the same as overlying bone. The aim of this study was to examine the validity of this assumption by determining whether fat distribution over and adjacent to bone differ and whether this introduces accuracy errors in the measurement of BMC by postero-anterior (PA) and lateral scanning. BMC (posterior processes plus vertebral body, g) of the third lumbar vertebra was 17.3 ± 0.7 by PA and 17.0 ± 0.7 by lateral scanning in 27 premenopausal women (p = NS), but 2.7 g or 20% higher by PA than lateral scanning in 27 postmenopausal women (14.4 ± 0.7, 11.7 ± 0.5, p < 0.01). Thus, the respective diminutions across age by PA scanning was about half that by lateral scanning (16.8 ± 3.9%, 31.2 ± 3.0%, p < 0.01). Percent fat in the soft tissue baseline (lateral to bone, ST-lat) used to derive BMC by PA scanning, was higher than in the soft tissue baseline (anterior to bone, ST-ant) used to derive BMC by lateral scanning by 2.6 ± 0.7% in premenopausal women and 7.5 ± 1.0% in postmenopausal women (both p < 0.01). After adjusting for these differences in percent fat, BMC by PA and lateral scanning no longer differed. Fat content (cm2) measured by quantitative computed tomography (QCT) in an additional 46 women, was higher in the soft tissue baseline (ST-lat) than anterior to bone in the 18 premenopausal (30.7 ± 4.2, 24.3 ± 3.5, p < 0.01) and the 28 postmenopausal women (41.6 ± 3.7, 34.1 ± 3.5, p < 0.01). Fat content in the soft tissue baseline region (ST-ant) was higher than on either side of bone in premenopausal women (31.3 ± 5.7, 26.5 ± 4.2, p < 0.05) but less than on either side of bone in postmenopausal women (44.7 ± 4.3, 50.6 ± 5.1, p < 0.05). In summary, the differing composition of soft tissue anterior 6and lateral to bone calls to question the validity of the assumption of tissue homogeneity needed for accurate measurement of BMC by DXA. In the elderly, BMC may be too high by PA scanning, resulting in a reduced cross-sectional diminution with age. Thus accuracy errors due to fat inhomogeneity compound those caused by osteophytes and suggests caution is needed in making inferences regarding the magnitude of age-related bone loss determined in vivo using bone densitometry.
The bone mineral status of patients with marfan syndrome
Authors: Kohlmeier Lynn , Gasner Cheryll , Bachrach Laura K. , Marcus Robert
Published: February 2010

Osteopenia at the hip and low total body calcium content have been reported in women with Marfan syndrome. Using dual X-ray absorptiometry (DXA), we evaluated the lumbar spine L2-L4 and proximal femur bone mineral density (BMD, g/cm2) in 32 women and 16 children with Marfan syndrome. The women were 38 ± 10 (SD) years old (23–58 years); their mean height was 178.6 ± 8 cm. The children (9 boys and 5 girls) were 9.9–17.5 years old. Children were tall for their ages but of normal weight. All subjects were moderately active, without previous nontraumatic fracture. In the women, BMD was reduced at L2–L4, femoral neck (fnk), trochanter (tr), and intertrochanter (intr) (p < 0.0001–0.006), compared with age-predicted values. Z scores for L2–L4 and for the fnk, tr, and intr, were –0.59 ± 1.06, –1.25 ± 0.99, –1.03 ± 1.10, and –1.31 ± 0.91, respectively. The average hip axis length (HAL) of 11.5 ± 0.093 cm was at the 80th percentile for women. No significant change was observed in 1 year follow-up BMD measurements in 13 women (fnk = –0.23 ± 23%/year, L2–L4 = –0.43 ± 1.57%/year). In Marfan children, BMD correlated with age, height, and pubertal development. Femoral neck BMD was reduced (Z = –0.74 ± 1.22, p < 0.05) with a nonsignificant trend toward decreased BMD at L2–L4 (Z = –0.33 ± 1.48). Resorption markers in Marfan women were normal and did not correlate with bone status. We conclude that women with Marfan syndrome have both axial and peripheral osteopenia as well as an increased HAL. This combination of findings likely increases substantially their long-term risk for hip fracture. Presence of osteopenia in Marfan children indicates that the skeletal deficits of Marfan syndrome may reflect inadequate bone acquisition.
Osteosarcoma hybrids can preferentially target alkaline phosphatase activity to matrix vesicles: Evidence for independent membrane biogenesis
Authors: Leach R.J. , Schwartz Z. , Johnson-Pais T.L. , Dean D.D. , Luna M. , Boyan B.D.
Published: February 2010

Alkaline phosphatase is the marker enzyme for matrix vesicles, extracellular organelles that play a major role in primary bone formation and calcification. Recently, we developed osteosarcoma × fibrosarcoma hybrids in which alkaline phosphatase expression was greatly reduced, a phenomenon known as extinction. In the present study, we used two cell hybrids, LTA-1 and LTA-5, constructed from a human osteoblast-like osteosarcoma, TE85, and a mouse fibrosarcoma, La−t−, to examine the differential distribution of alkaline phosphatase between matrix vesicles and the plasma membrane, postulated to be the parent membrane from which matrix vesicles are derived. While alkaline phosphatase in plasma membranes was extinguished, enzyme activity in matrix vesicles from LTA-1 hybrid cells was 34.2% of that present in matrix vesicles from the TE85 parent cells and 200 times that found in La−t− matrix vesicles. Matrix vesicles from LTA-5 had alkaline phosphatase levels similar to La−t−. When other membrane enzymes (phospholipase A2, 5′-nucleotidase, and Na+/K+ ATPase) were examined, hybrid matrix vesicle and plasma membrane levels were similar to those of TE85 and significantly higher than in La−t− membrane fractions. Northern analysis detected mRNA for alkaline phosphatase in TE85 cells, but not in the hybrids or La−t− cells. In contrast, reverse transcription-polymerase chain reaction (RT-PCR) revealed alkaline phosphatase mRNA in the hybrid cells, but at very low levels. Taken together, the data indicate that regulation of plasma membrane and matrix vesicle alkaline phosphatase is independent and suggest that matrix vesicle biogenesis is independent and distinct from that of plasma membrane biogenesis. Analysis of 1B- and 1L-type alkaline phosphatase mRNA by RT-PCR showed that alternate promoter usage of the alkaline phosphatase gene was not responsible for the differential localization of this enzyme in matrix vesicle. Thus, it is likely that matrix vesicle and plasma membrane alkaline phosphatase are regulated differently at a post-transcriptional level.
Effects of a one-year high-intensity versus low-intensity resistance training program on bone mineral density in older women
Authors: Pruitt Leslie A. , Taaffe Dennis R. , Marcus Robert
Published: February 2010

The purpose of this study was to determine the effects of a 12-month resistance training program, of two different intensities, on bone mineral density (BMD) in healthy, older women. Twenty-six Caucasian women (aged 65–79 years) completed the study. Subjects were randomly assigned to one of three groups: high-intensity (HI; n = 8), low-intensity (LI; n = 7), and control (CON; n = 11). The active groups performed 10 exercises, 3 days/week under supervision. Exercise intensity was maintained at 80% of one-repetition maximum (1-RM) for the HI group, and at 40% 1-RM for the LI group. The volume of work was maintained constant between the two groups by assigning the LI group twice as many repetitions for each exercise. Maximal muscular strength and BMD of the lumbar spine and total hip were measured at baseline and at 12 months. Strength was evaluated using the 1-RM method, and BMD was determined by dual-energy X-ray absorptiometry. Exercise session attendance was similar for the two groups (81.0% HI; 76.8% LI). Muscular strength improved in the exercisers compared with the CON group (p ≤ 0.05). Percentage change in lumbar spine BMD was 0.7 ± 1.9%, 0.5 ± 2.4%, and -0.1 ± 2.3% for the HI, LI, and CON groups, respectively. Percentage change in total hip BMD was 0.8 ± 2.3% (HI), 1.0 ± 1.7% (LI), and 0.9 ± 1.3% (CON). Group differences in BMD change were not significant (p > 0.05). These findings suggest that high-intensity and low-intensity resistance training regimens effectively increase muscular strength, but not lumbar spine or total hip BMD, in healthy, older women.