bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical parameters associated with low bone density in healthy men and women
Authors: Dr. Sherman Sheryl S. , Tobin Jordan D. , Hollis Bruce W. , Gundberg Caren M. , Roy Tracey A. , Plato Chris C.
Published: February 2010

A causal role in age-related bone loss has been attributed to alterations in vitamin D status, the bone mineral regulating hormones, and/or renal function. We assessed biochemical parameters of bone metabolism and renal function in healthy subsets of young and old men (n = 191) and women (n = 120) and evaluated the relationships between these parameters and bone mineral density (BMD) in the radius, spine, and femur. There were no significant associations between BMD at any site and serum 25-OHD, 1,25-(OH)2D, PTH, or creatinine clearance in either young men or in young or old women, after controlling for age. In old men, however, lower radius BMD was significantly related to higher PTH and higher 1,25-(OH)2D and marginally related to lower 25-OHD values. In young men, there were unexpected but significant associations between lower femoral neck BMD and higher serum osteocalcin and urinary calcium/creatinine excretion after age adjustment. In old women, lower spine and radius BMD was also significantly correlated with higher serum osteocalcin. In this healthy, vitamin D-replete population, there were significant cross-sectional declines in BMD in the femur in young and old men and at all sites in old women. Elevated remodeling may be an important feature that contributes to reduced femoral BMD in young men and reduced spine and radius BMD in old women. However, compromised renal function or levels of 1,25-(OH)2D or elevated PTH appear to be neither necessary nor relevant as determinants of osteopenia in the spine or femur in these normal, healthy men and women.
Transcriptional activation of c-fos and c-jun protooncogenes by serum growth factors in osteoblast-like MC3T3-E1 cells
Authors: Okazaki Ryo , Ikeda Kyoji , Sakamoto Akemi , Nakano Toshiaki , Morimoto Kyoko , Kikuchi Tomoko , Urakawa Kazumi , Ogata Etsuro , Matsumoto Toshio
Published: February 2010

The present study was undertaken to clarify the relationship between c-fos and c-jun protooncogene expression and the differentiation and/or proliferation of osteoblasts, using osteoblast-like MC3T3-E1 (E1) cells. c-fos mRNA was barely detectable, whereas c-jun mRNA was constitutively expressed in E1 cells after serum deprivation for 24–72 h. When serum was added, a rapid and transient induction of c-fos and c-jun mRNAs was observed. The c-fos and c-jun mRNAs reached peak levels at 30 minutes, with a rapid disappearance of c-fos mRNA within 3 h and a much slower decrease in c-jun mRNA. The addition of serum together with cycloheximide, an inhibitor of protein synthesis, resulted in the superinduction of both c-fos and c-jun mRNAs. Among various growth factors, PDGF, EGF, and bFGF mimicked the serum effect, whereas IGF-I and TGF-β failed to induce c-fos and c-jun mRNA. The effects of PDGF, EGF, and bFGF were completely abolished by pretreatment with actinomycin D, an inhibitor of RNA synthesis, suggesting a transcriptional mechanism. Nuclear runoff experiments showed that the transcription rate of c-fos and c-jun protooncogenes was increased by serum and growth factors. The effects of PDGF, EGF, and bFGF were inhibited by H-7 or staurosporine, inhibitors of protein kinase C (PKC), but not by HA1004 with a much weaker inhibitory activity, suggesting the involvement of PKC for the activation of the protooncogenes. PDGF, EGF, and bFGF, which induced the expression of c-fos and c-jun protooncogenes, stimulated the proliferation of E1 cells, whereas IGF-I and TGF-β, which failed to induce the expression of the protooncogenes, had no effect or an inhibitory effect on the proliferation of E1 cells, respectively. In addition, when the protooncogene induction was inhibited by H-7, but not by HA1004, the proliferative responses to the growth factors were also completely abolished. Although E1 cells are known to develop osteoblastic phenotypes during prolonged culture period, the expression of c-fos and c-jun mRNAs was not altered during 2–28 days of culture at various stages of differentiation. These results suggest that the expression of c-fos and c-jun protooncogenes may play a role as an immediate early event in the proliferative responses of osteoblasts to various growth factors.
Mechanical properties of trabecular bone within and adjacent to osseous metastases
Authors: Hipp John A. , Rosenberg Andrew E. , Hayes Wilson C.
Published: February 2010

Despite radiographic and histologic evidence of trabecular bone density changes within and adjacent to osseous metastases, there currently exist no data to demonstrate whether these changes are important in predicting the risk of fracture. To determine if these density changes result in significant reductions in mechanical properties, trabecular bone specimens were prepared from lower thoracic and lumbar vertebrae from two cadavers with radiographic, gross, and histologic evidence of lytic and/or blastic osseous metastases. Each specimen was classified as normal, lytic, or blastic based on appearance in fine-grain radiographs of 8–9 mm thick coronal plane sections. Specimens were tested to failure in uniaxial compression, and tissue and apparent densities were measured. Mean tissue densities were within normal ranges. The mean apparent density for all specimens combined was within the normal range for human vertebrae, and the mean apparent density for radiographically normal (0.131 g/ml) and lytic (0.111 g/ml) specimens was less than the mean apparent density of blastic (0.182 g/ml) specimens (p < 0.02). The moduli of lytic and blastic specimens were less than for normal specimens (p < 0.025). The strength of lytic specimens was less than normal (p = 0.057), but the strength of blastic specimens was not (p > 0.1). Apparent density explained significant fractions of the variations in both modulus (p < 0.001) and strength (p < 0.001). The data suggest that blastic changes associated with osseous metastases to trabecular bone disrupt the normal dependence of trabecular mechanical properties on apparent density, but lytic changes do not. These data also suggest that fracture risk predictors that utilize bone density to estimate stiffness or strength should adjust for the effects of metastases.
Effects of endothelin-1 on signal transduction in UMR-106 osteoblastic cells
Authors: Tatrai Agnes , Lakatos Peter , Thompson Sharron , Stern Paula H.
Published: February 2010

Endothelin-1 is now recognized to affect the functions of a number of tissues and to activate calcium/phospholipid second messenger pathways in target cells. In the present study, we characterized its effects on signal transduction in UMR-106 cells. To study calcium transients elicited by endothelin-1, cells were loaded either with fluo-3 (for the measurement of cytosolic free calcium) or chlortetracycline (for the measurement of intracellularly stored calcium) as fluorescent probes. Intracellular production of inositol phosphates and cyclic AMP was also measured. Endothelin-1 elicited dose-dependent cytosolic calcium transients with an ED50 of 20 nM. This effect was also seen in EGTA-containing or calcium-free medium; however, the signals were reduced in magnitude. The dihydropyridine calcium channel antagonist nifedipine did not affect the response. Repeated administration of endothelin-1 resulted in homologous desensitization of the response. A 4 minute pretreatment with phorbol ester reduced the initial response to endothelin-1 in both calcium-containing and calcium-free media. A 24 h pretreatment with indomethacin had no effect on response. Using chlortetracycline as an indicator, a significant reduction in intracellularly stored calcium by endothelin-1 was observed. This was prevented by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate, a blocker of calcium release from internal stores. Endothelin-1 also stimulated the dose-dependent production of inositol phosphates by UMR-106 cells. Indomethacin was also without effect on this process. The increase in inositol trisphosphates was seen within the same time frame as the increase in cytosolic calcium. Endothelin-1 did not influence cyclic AMP production over 5 minutes in these cells. In conclusion, endothelin-1 has a significant effect on UMR-106 osteoblastic cells to activate the calcium and inositol phosphate second messenger systems. Our findings raise the intriguing question of the physiologic role of endothelin-1 in bone metabolism.
Excessive L-thyroxine therapy decreases femoral bone mineral densities in the male rat: Effect of hypogonadism and calcitonin
Authors: Ongphiphadhanakul Boonsong , Alex Sharon , Braverman Lewis E. , Baran Daniel T.
Published: February 2010

Excess thyroid hormone decreases bone mineral density (BMD), a potential problem in managing patients with differentiated thyroid carcinoma and nontoxic goiter who require lifelong TSH-suppressive doses of thyroid hormone. We studied the effect of thyroid hormone excess on vertebral and femoral BMD and the role of hypogonadism in modulating this effect in a rat model. The potential role of calcitonin (CT) in preventing thyroid hormone-associated bone loss was also investigated. A total of 40 male Sprague-Dawley rats were divided into four groups. Groups 1 and 2 were orchidectomized (ORX); groups 3 and 4 were sham operated (SO). Groups 1 and 3 received 20 μg intraperitoneal L-thyroxine (L-T4) per 100 g body weight daily for 3 weeks; groups 2 and 4 received vehicle IP. Another 40 rats were divided into four groups. Groups 1 and 2 received L-T4, and groups 1 and 3 received CT, 2.5 U per 100 g body weight, subcutaneously (SC) daily for 3 weeks. BMD of the L4 and 5 and the right femur were measured by dual-energy x-ray absorptiometry at baseline and at the end of the study. Orchidectomy decreased femoral (P < 0.05) but not lumbar BMD. The administration of excess L-T4 decreased femoral (cortical) BMD in both SO (P < 0.05) and ORX rats (P < 0.05) without affecting lumbar (trabecular) BMD. CT increased lumbar BMD in both vehicle (P < 0.001) and L-T4-treated rats (P < 0.001). However, CT did not affect femoral BMD in vehicle-treated rats and did not prevent the L-T4-induced femoral bone loss. Serum tartrate-resistant acid phosphatase (TRAP) was increased in the L-T4-treated (P < 0.001) and the orchidectomized (P < 0.05) rats. CT had no effect on TRAP activity and did not prevent the L-T4-induced increase in TRAP. Neither excess L-T4 nor orchidectomy affected osteocalcin concentrations. CT decreased serum osteocalcin concentrations, alone (P < 0.05) and in the presence of excess L-T4 (P < 0.05). We conclude that large doses of L-T4 administered to the rat preferentially decrease femoral BMD. Short-term hypogonadism decreases femoral but not lumbar BMD and does not make the lumbar spine more susceptible to the potential thyroid hormone-induced bone loss. CT increases lumbar BMD but does not prevent the thyroid hormone-induced decrease in femoral BMD. This rat model can be utilized to evaluate different therapies to prevent bone loss induced by excess thyroid hormone administration.
Regulation of calcitonin gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat
Authors: Naveh-Many Tally , Raue Friedhelm , Grauer Andreas , Silver Justin
Published: February 2010

High calcium leads to the secretion of calcitonin, and the administration of 1,25-dihydroxyvitamin D3 leads to a decreased transcription of the calcitonin gene. We now report the effect of chronic hypercalcemia, hypocalcemia, and vitamin D deficiency on calcitonin gene expression in vivo in the rat. Hypercalcemia was created by calcium infusions for 6 h, a high-calcium diet given to weanling rats for 3 weeks, and the transplantation of the Walker carcinosarcoma 256 cell line. Despite serum calcium as high as 22 mg/dl, there was no difference in calcitonin mRNA levels among these rats. The control genes studied, actin and somatostatin, which is specific for C cells in the thyroparathyroid tissue, also did not differ among the different groups of rats. Injected 1,25-(OH)2D3 decreased calcitonin mRNA levels at 6 h, as previously reported. Hypocalcemia, created by feeding diets deficient in calcium and vitamin D to weanling rats for 3 weeks, had no effect on calcitonin mRNA levels, in contrast to the large increases in PTH mRNA levels. These results demonstrate that calcitonin gene expression in vivo in the rat is regulated by administered 1,25-(OH)2D3 but not by changes in serum calcium.
Bone mineral density, muscle strength, and recreational exercise in men
Authors: Snow-Harter Christine , Whalen Robert , Myburgh Kathy , Arnaud Sara , Marcus Robert
Published: February 2010

Muscle strength has been shown to predict bone mineral density (BMD) in women. We examined this relationship in 50 healthy men who ranged in age from 28 to 51 years (average 38.3 years). BMD of the lumbar spine, proximal femur, whole body, and tibia were measured by dual-energy x-ray absorptiometry (Hologic QDR 1000W). Dynamic strength using one repetition maximum was assessed for the biceps, quadriceps, and back extensors and for the hip abductors, adductors, and flexors. Isometric grip strength was measured by dynamometry. Daily walking mileage was assessed by 9 week stepmeter records and kinematic analysis of video filming. Subjects were designated as exercisers and nonexercisers. Exercisers participated in recreational exercise at least two times each week. The results demonstrated that BMD at all sites correlated with back and biceps strength (p < 0.01 to p = 0.0001). Body weight correlated with tibia and whole-body BMD (p < 0.001); age negatively correlated with Ward's triangle BMD (p < 0.01). In stepwise multiple regressions, back strength was the only independent predictor of spine and femoral neck density (R2 = 0.27). Further, back strength was the most robust predictor of BMD at the trochanter, Ward's triangle, whole body, and tibia, although biceps strength, age, body weight, and leg strength contributed significantly to BMD at these skeletal sites, accounting for 35–52% of the variance in BMD. Exercisers and nonexercisers were similar for walking (3.97 versus 3.94 miles/day), age (37.8 versus 38.5 years), and weight (80.0 versus 77.7 kg). However, BMD and muscle strength were significantly greater in exercisers than in nonexercisers. In conclusion, in young to middle-aged men, (1) muscle strength makes important contributions to bone mineral density; (2) strength of back extensors more powerfully predicts BMD than age, body weight, or strength of other muscle groups; and (3) recreational exercise is a better predictor of BMD than habitual daily walking.
Regulation of cytoplasmic calcium concentration in tetracycline-treated osteoclasts
Authors: Donahue Henry J. , Iijima Kazumoto , Goligorsky Michael S. , Rubin Clinton T. , Rifkin Barry R.
Published: February 2010

The ability of low-dose tetracyclines to inhibit collagenase activity and inactivate osteoclasts suggests that these compounds have great potential as a prophylaxis for metabolic bone disease. However, the cellular mechanism by which tetracyclines interact with skeletal tissue is not yet clear. To better understand the effects of tetracyclines on bone metabolism, we examined their effect on osteoclast activity in vitro. Because tetracyclines can enter the cell and bind calcium and have been reported to directly interact with osteoclasts, we postulated that exposure to either of two tetracyclines, minocycline or doxycycline, would alter cytosolic Ca2+ regulation in rat osteoclasts. [Ca2+]i was measured in single rat osteoclasts utilizing fura-2. Addition of extracellular Ca2+ (5 mM CaCl2), a potent osteoclast inhibitor, increased [Ca2+]i in all osteoclasts, but 10−6 M salmon calcitonin (sCT) did so only in a subpopulation of osteoclasts. Neither minocycline nor doxycycline (10 μg/ml) altered steady-state osteoclast [Ca2+]i. Further, neither minocycline nor doxycycline pretreatment affected the sCT-mediated increases in [Ca2+]i. However, tetracycline pretreatment significantly decreased the cytosolic Ca2+ response to extracellular CaCl2. Our results strongly suggest that tetracyclines have a specific effect on extracellular Ca2+-stimulated cytosolic Ca2+ mobilization in osteoclasts, which is not solely dependent on their ability to buffer Ca2+. Furthermore, these results point to the potential use of tetracyclines as probes to study cytosolic Ca2+ regulation. However, that tetracyclines attenuate a signal response associated with decreased osteoclastic resorption suggests that the reported antiresorptive attributes of tetracyclines must be achieved independently of an effect on osteoclastic cytosolic Ca2+.
The effect of transforming growth factor β on the plasminogen activator activity of normal human osteoblast-like cells and a human osteosarcoma cell line MG-63
Authors: Dr. Fawthrop Fiona W. , Oyajobi Babatunde O. , Bunning Rowena A.D. , Russell R. Graham G.
Published: February 2010

Transforming growth β (TGF-β) has been proposed to have a role in bone remodeling by affecting the differentiation and activity of osteoblasts and osteoclasts and by inhibiting the production of proteinases, such as plasminogen activators (PAs). Studies on PAs have largely been based on data from nonhuman and fetal cell lines, however. The purpose of this study was to investigate the effect of TGF-β on the PA activity of normal human osteoblast-like cells and to compare this with its action on the human osteosarcoma cell line MG-63. The action of interleukin-1β (IL-1β) was also assessed because it has been shown to increase PA activity in other connective tissue cell types. Normal osteoblast-like cells had low to undetectable basal urokinase (uPA) and tissue plasminogen activator (tPA) activity, which was significantly stimulated by TGF-β1. This action was shown to be dependent on transcription and new protein synthesis. TGY-β2 had a similar action. IL-1β did not stimulate PA activity. In contrast, the MG-63 cell line had high basal tPA and uPA activities. TGF-β1 decreased basal PA activity, the effect being most marked for uPA activity. IL-1β stimulated uPA and tPA activity. TGF-β1 inhibited IL-1β-stimulated uPA activity, but the effect on tPA was more variable. This study has shown that TGF-β has opposite effects on the PA activity of the two osteoblast-like cell types studied. Care must therefore be used before extrapolating data from one cell type to another. It is suggested that under certain circumstances TGF-β may be involved in bone resorption by modulating PA activity.
Bone mass measurement by DXA: Influence of analysis procedures and interunit variation
Authors: Trevisan Carlo , Gandolini Giorgio G. , Sibilla Paolo , Penotti Mauro , Caraceni Maria Pia , Ortolani Sergio
Published: February 2010

Interunit variability among bone densitometers is due to different factors, including different calibration procedures and algorithms and variability in photon source energies and/or intensities. Other factors, such as the choice of scan parameters or the analysis procedures, can also introduce variability. The new generation of dual-energy x-ray absorptiometry (DXA) has partially improved this situation. The aim of this study was to investigate the operator-dependent analysis procedures that can affect scan results and to evaluate the phantom and in vivo interunit variation of some DXA instruments. Four DXA instruments (QDR 1000 and 1000/W, Hologic, Inc.) were used. Potential sources of variability in the analysis procedures of anteroposterior lumbar spine and hip scans were considered: in most cases these procedures significantly influenced scan results. On lumbar spine, an enlargement of the scan window of less than 3 cm was responsible for an average increase in bone mineral density (BMD) of about 3%. On the hip, lowering the scan window by about 1 cm accounted for an increase in the whole-segment BMD of about 4%. After standardization of analysis procedures, interunit and intraunit coefficients of variation and percentage differences among instruments were less than 1% for all the parameters considered (area and bone mineral content and density) with both an anatomic and a geometric phantom, and in nine subjects scanned by two different devices the percentage difference in BMD was greater than 2%. This study shows that present interunit variability allows comparisons among laboratories, but only if highly standardized analysis procedures are used.
Osteoporosis and chronic back pain: A study with single-photon emission computed tomography bone scintigraphy
Authors: Dr. Ryan P.J. , Evans P. , Gibson T. , Fogelman I.
Published: February 2010

A group of 26 patients aged 47–81 years (average 67) with spinal osteoporosis and back pain for more than 6 months were investigated with planar and SPECT bone scintigraphy and anterior and lateral x-rays of the thoracolumbar spine. Abnormal activity on bone scintigraphy was found in all patients, of whom 22 had multiple lesions. A total of 17 (65%) patients had abnormal activity associated with collapsed vertebral bodies, 5 (19%) had degenerative disk disease, and 21 (81%) had facetal joint disease. Of 112 lesions identified by SPECT, 60 (54%) were localized to the apophyseal joints. Facetal lesions were commoner in those patients with more collapsed vertebrae, and 32 facetal lesions (54%) were associated with collapse of the vertebra immediately above or below. Bone scintigraphy findings suggest that in some individuals with osteoporosis and chronic back pain, collapse of the vertebral body or degenerative disk disease are causes of pain. However, the high frequency of increased apophyseal joint activity suggests that the facet joints may also be an important site of origin of pain in these individuals. Bone scintigraphy may identify a subgroup of osteoporotic patients with chronic back pain who would benefit from treatment to the facet joints.
Journal of Bone and Mineral Research
Authors: Shelton Richard M. , El Haj Alicia J.
Published: February 2010

Periosteal cells grown in macroporous microcarrier bead columns were cyclically loaded with compressive force using a newly developed model. In response to 1/2 h of cyclic compression, RNA synthesis increased significantly by twofold, from 113.4, 19.6 to 260.7 + 363 (p lt; 0.01) after 16 h, whereas DNA synthesis did not increase significantly after 24 h. The microcarrier bead model was calibrated using a linear volume displacement transducer with a range of strain magnitudes applied across the column of 1000–6000 μstrain.
Journal of Bone and Mineral Research
Authors: Lozupone Enrico , Favia Angela , Grimaldi Antonella
Published: February 2010

The structure of metatarsal bones from 18-day-old rats subjected to intermittent mechanical force in organ culture are reported. The application of mechanical force enhances the osteoid thickness and osteoblast number in the periosteum and increases the number of viable osteocytes. These results indicate that (1) the mature bone tissue survives in organ cultures; (2) the mechanical forces better preserve the structure of the osteocytes and stimulate the osteoblasts, and (3) stimulate the osteogenesis.
Journal of Bone and Mineral Research
Authors: Mueller Martina , Schilling Tobias , Minne Helmut W. , Ziegler Reinhard
Published: February 2010

The rate of remodeling in the region of a bone defect exceeds normal tissue activity. It was Frost who described this reaction as the regional acceleratory phenomenon (RAP). We previously showed that restoration of a local bone defect in the rat leads not only to RAP but also leads to a systemic acceleration of osteogenesis (systemic acceleratory phenomenon, SAP) in distant sites of the skeleton. In this study we investigated the impact of immobilization of the defect-bearing extremity on the development of SAP. A hole 1.2 mm in diameter was drilled in the diaphysis of the left tibia of female rats. In the experimental group (n = 15), a knee tenotomy was performed in the defect-bearing left hind leg. We examined both femora, both tibiae, and the fourth lumbar vertebra by computed x-ray densitometry on day 7 postoperatively. Immobilization of the defect-bearing limb led to a decrease in x-ray density not only of the immobilized (p lt; 0.0001) but also of the contralateral tibia (p lt; 0.0001). Both femora (p lt; 0.001) and the fourth lumbar vertebra (p lt; 0.025) of the experimental group also showed a significant decrease in x-ray density. We previously showed that SAP leads to an increase in x-ray density of both femora. This increase is no longer detectable in animals after immobilization of the defect-bearing limb. Thus we conclude that immobilization interferes with SAP. This suggests the possible dependence of SAP on mechanical load. Furthermore, these data suggest a possible impact of local immobilization on the rest of the skeleton.
Journal of Bone and Mineral Research
Authors: Schilling Tobias , Mueller Martina , Minne Helmut W. , Ziegler Reinhard
Published: February 2010

The mineral apposition rate (MAR) is a commonly used parameter for the characterization of bone formation and is often determined to test for experimental effects on cortical bone. We investigated whether there are physiologic variations in rat cortical MAR dependent on the side or site of measurement. In our experiment we used female rats. The animals were sacrificed on day 8, after double-fluorochrome labeling with calcein and tetracycline was performed. The MAR was calculated at 3.6, 5.4, 7.2, 9, and 10.8 mm from the epiphyseal growth plate of the lateral as well as of the medial endosteum of both right and left tibiae. We found a physiologic significant difference in the MAR between the lateral and the medial endosteal sites of the same tibia (p lt; 0.0001), especially near the epiphyseal growth plate. Regarding the same cortical side, there is a significant decrease (p lt; 0.0001) in the endosteal MAR with increasing distance from the epiphyseal growth plate. We conclude that the observed differences in endocortical MAR must be due to specific mechanical challenges. Because these differences are statistically significant, it is necessary to standardize the area of histomorphometric measurement not only with respect to the distance from the epiphyseal growth plate, but also with respect to the cortical side.
Journal of Bone and Mineral Research
Authors: Grigoriev Anatoly I. , Morukov Boris V. , Oganov Victor S. , Rakhmanov Alexander S. , Buravkova Ludmilla B.
Published: February 2010

As we enter a phase of space exploration that will involve long-duration flights, there is a need to use ground-based models to study the long-term effects of countermeasures to prevent the loss of bone mineral in microgravity. Mineral balances, hormone levels, and bone density were measured for 360 days in nine bed rest subjects treated with an exercise program used by cosmonauts. Four of these subjects received the bisphosphonate, ethane-1-hydroxy-1-disphosphonate, 900 mg daily, a drug known to inhibit bone resorption. Compared to a 120 day control period, the bisphosphonate combined with exercise reduced negative calcium balances by 50% for the first 120 days, 80% for the second 120 days, and 69% during the third 120 days. Exercise alone had no effect until the second 120 day period, when calcium balance improved 52%. Negative phosphorus balances were not affected by either treatment. Magnesium balances were negative during the first 120 days and returned to nearly normal during the last 240 days in both groups. The combined exercise and bisphosphonate treatment prevented increases in serum ionized calcium and decreases in plasma calcitonin during the first 120 days, as well as trends toward decreases in the mineral density of the femoral neck. These results suggest that bisphosphonates can be efficiently used together with exercise to reduce calcium loss and prevent some of the changes in mineral metabolism during long-term simulated microgravity.
Heritable and life-style determinants of bone mineral density
Authors: Krall Elizabeth A. , Dawson-Hughes Bess
Published: February 2010

Familial resemblance in bone mineral density at five skeletal sites was measured among 160 adult members of 40 families. Each family included a postmenopausal mother, one premenopausal daughter, one son, and the children's father. Similarities in selected life-style factors thought to influence bone density, such as physical activity, smoking, alcohol use, and diet, were also evaluated. Bone density was measured by dual-energy (total body, femoral neck, and lumbar spine) or single-photon (radius and os calcis) absorptiometry. Correlation coefficients between the midparent Z score and offspring Z scores of bone mineral density ranged from 0.22 to 0.52 among daughters and from 0.27 to 0.58 among sons. Adjustment of bone density for age, height, weight, and significant life-style or environmental factors yielded heritability estimates for the five skeletal sites between 0.46 and 0.62. That is, 46–62% of variance in bone density was attributable to heredity. Most estimates derived from the group of daughters were similar to those from the sons. These observations provide support for a significant contribution of heredity to bone density. However, an individual's life-style may account for a potentially large proportion of the nonheritable variance in bone density.
Transient recruitment of osteoclasts and expression of their function in osteopetrotic (op/op) mice by a single injection of macrophage colony-stimulating factor
Authors: Kodama Hiroaki , Yamasaki Akira , Abe Moriaki , Niida Shumpei , Hakeda Yoshiyuki , Kawashima Hiroyuki
Published: February 2010

Severe deficiency of osteoclasts in op/op mice, caused by the absence of functional macrophage colony-stimulating factor (M-CSF), is cured by daily injections of purified recombinant human M-CSF (rhM-CSF). In this study, we found that a single injection of 5 μg rhM-CSF is enough for recruitment of osteoclasts in mutant mice. Osteoclast number increased during the period between 2 and 4 days after the single rhM-CSF injection. When YM175, a new derivative of bisphosphonate, was administered to the mice 4 days after rhM-CSF injection or later, osteoclasts disappeared by 3 days after YM175 administration. However, a significant number of osteoclasts were detected even at 3 days after YM175 administration when YM175 was administered 3 days after rhM-CSF injection or earlier. These results indicate that YM175 is cytotoxic only to functioning osteoclasts and that recruitment of osteoclasts is finished 4 days after a single rhM-CSF injection. The osteoclasts actively resorbed bone trabeculae for a prolonged period, demonstrating that M-CSF is not requisite for the functioning of mature osteoclasts.
Cytokine production and surface antigen expression by peripheral blood mononuclear cells in postmenopausal osteoporosis
Authors: Hustmyer Frank G. , Walker Edwin , Yu Xiao-Peng , Girasole Giuseppe , Sakagami Yoshiyuki , Peacock Munro , Manolagas Stavros C.
Published: February 2010

It was reported earlier that IL-1 production by cultured monocytes and the ratio of helper (CD4) to suppressor (CD8) lymphocytes in peripheral blood are different in osteoporotic compared to nonosteoporotic subjects. We examined these and several other parameters related to the biosynthetic activity and differentiation status of peripheral blood mononuclear cells (PBMC) in untreated osteoporotic postmenopausal women (age 65 ± 7, n = 46), nonosteoporotic postmenopausal women (age 55 ± 3, n = 20), and nonosteoporotic premenopausal women (age 37 ± 7, n = 8), as defined by spine density. We found that unstimulated monocytes from osteoporotics did not produce detectable IL-1β as determined by ELISA. In addition, there were no significant differences between osteoporotics and nonosteoporotics in IL-1β or IFN-γ production by PBMC stimulated with OKT3, a monoclonal antibody to the T cell-receptor complex. The proliferative response of lymphocytes to OKT3 was significantly less (p < 0.02) in osteoporotics compared to nonosteoporotic post- and premenopausal women; multiple-regression analysis, however, indicated that this difference was not due to bone density but to age. Flow cytometric analysis of PBMC revealed no difference between osteoporotics and nonosteoporotics in the distribution of 18 phenotypic subsets determined, including CD4-or CD8-positive lymphocytes or the ratio of CD4 to CD8 cells. Further, there was no correlation of the surface markers with bone density, the exceptions being the subsets expressing the CD3/CD56 and CD8/CD56 markers, which were inversely related to spine density in the osteoporotic women. Nonetheless, in the osteoporotic women, the CD8/CD56 and CD14/CD13 cells were positively correlated with the levels of plasma 1,25-(OH)2D3 (r = 0.33, p < 0.04; r = 0.39, p = 0.028, respectively), whereas the CD4 cells were negatively correlated with 1,25-(OH)2D3 (r = −0.35, p = 0.026). These data suggest that cytokine production by peripheral blood mononuclear cells and the distribution of the various subsets of these cells in the peripheral blood are not related to bone mass in osteoporosis. Distribution of PBMC subsets in the peripheral blood, however, may be influenced by circulating levels of 1,25-(OH)2D3.
Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels
Authors: Guidon Peter T. , Salvatori Roberto , Bockman Richard S.
Published: February 2010

Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor β1 (TGFβ1); however, TGF-β1 was not detected in gallium nitratetreated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.
Serum markers of type I collagen formation and degradation in metabolic bone disease: Correlation with bone histomorphometry
Authors: Eriksen Erik F. , Charles Peder , Melsen Flemming , Mosekilde Leif , Risteli Leila , Risteli Juha
Published: February 2010

Type I collagen makes up more than 90% of bone matrix. Therefore, analysis of antigens related to collagen formation and degradation in bone should provide good and specific estimates of both bone resorption and bone formation rates. In this study we measured serum levels of the pyridinoline cross-linked telopeptide domain of type I collagen (ICTP) as a marker of bone resorption and serum carboxy-terminal propeptide of type I procollagen (PICP) as a marker of bone formation. Serum levels of the two antigens were correlated to histomorphometric indices of bone resorption and bone formation calculated from iliac crest bone biopsies in a group of 18 individuals with high- and low-turnover bone disease (myxedema, primary hyperparathyroidism, and thyrotoxicosis). After logarithmic transformation the regression of S-ICTP on volume-referent resorption rate (BRs/R/BV) was significant (r = 0.61, p < 0.01, SEM/Y = 56%). S-ICTP also showed a significant regression on the volume-referent cancellous bone balance (r = −0.45, p < 0.05, SEM/Y = 412%).

S-PICP was significantly correlated to the mineral appositional rate (r = 0.53, p < 0.05) and volume-referent bone formation rate (r = 0.61, p < 0.01, SEM/Y = 48%). The correlation to bone turnover as expressed in the activation frequency was also highly significant (r = 0.61, p < 0.01, SEM/Y = 51%). No significant correlation with wall thickness or bone balance was demonstrable per remodeling cycle. Thus, assays employing antigens that reflect collagen formation and degradation are useful instruments for the evaluation of rates of bone remodeling in metabolic bone disease.
Autocrine regulators of MC3T3-E1 cell proliferation
Authors: Amarnani Saral , Merriman Harold L. , Linkhart Thomas A. , Baylink David J. , Mohan Subburaman
Published: February 2010

MC3T3-E1 cells, a clonal osteoblast-like mouse calvarial cell line, secrete several growth regulating factors. These regulators include insulin-like growth factor (IGF) type I, transforming growth factor β (TGF-β), and IGF-II in descending order of abundance. MC3T3-E1 cells in culture also produce two IGF binding proteins (IGFBP), Mr 25 and 32 kD, having sequence identity with IGFBP-4 and IGFBP-6, respectively. In addition, this is the first observation that osteoblast-like bone cells in culture produce IGFBP-6. To determine if growth factors produced by MC3T3-E1 cells have autocrine actions on these cells, the effects of IGF-I, IGF II, TGF-β1, and IGFBP-4 on MC3T3-E1 cell proliferation were determined. Exogenous addition of IGF-I and IGF-II stimulated MC3T3-E1 cell proliferation, but TGF-β1, and IGFBP-4 inhibited MC3T3-E1 cell proliferation. Based on these findings, we conclude that MC3T3-E1 cells in culture produce autocrine regulators of MC3T3-E1 cell proliferation and that the actions of IGFs may also be regulated by IGFBPs produced by these same cells.
Pre- and postmenopausal bone mineral density of the spine and proximal femur in Japanese women assessed by dual-energy X-ray absorptiometry: A cross-sectional study
Authors: Soda Masa-Yuki , Mizunuma Hideki , Honjo Shige-Ichirou , Okano Hiroya , Ibuki Yoshito , Igarashi Masao
Published: February 2010

To investigate the influence of menopause on bone mineral density (BMD) of Japanese women, BMD of the spine and proximal femur was measured by dual-energy x-ray absorptiometry (DXA) in 220 premenopausal and 166 postmenopausal Japanese women. The peak bone density of the spine in premenopausal women was detected between 35 and 39 years of age, and that of femur before 20. Thereafter spinal and femoral BMD showed a slight decrease, but they did not show a significant decrease until menopause or menstruation became irregular. Duration of substantial bone loss at lumbar spine continued for about 10 years after menopause, but that at the femur was much longer. To investigate the effect of early menopause on bone loss, postmenopausal women were divided into two groups according to the age at onset of menopause. The BMD of postmenopausal women whose menopause occurred before 50 was significantly less over the latter part of life than that of women whose menopause occurred after 50. These results suggest that bone loss is related to menopause or irregular menstruation rather than age per se, and early menopause should be recognized as one of the risk factors for involutional osteoporosis.
Leukemia inhibitory factor inhibits osteoclastic resorption, growth, mineralization, and alkaline phosphatase activity in fetal mouse metacarpal bones in culture
Authors: Van Beek Ermond , Van Der Wee-Pals Lianne , Van De Ruit Marjan , Nijweide Peter , Papapoulos Socrates , Löwik Clemens
Published: February 2010

Leukemia inhibitory factor (LIF) has been reported to affect bone metabolism, but results are variable. We examined the effect of mouse recombinant LIF on osteoclastic resorption in fetal bone explants representing different stages of osteoclast development. In cultures of 17-day-old fetal mouse metacarpals in which only osteoclast progenitors and precursors are present, resorption (measured as 45Ca release) was significantly inhibited to 29.2% and to 96.6% in the presence of LIF 100 and 1000 U/ml, respectively. Histologic examination of the explants treated with 1000 U/ml of LIF confirmed the biochemical findings and showed that osteoclast progenitors and precursors remained in the periosteum and did not invade the mineralized matrix. In metacarpals of older fetuses (18- and 19-day-old) in which the mineralizied cartilage has been invaded by mature osteoclasts, the inhibition of resorption by LIF (1000 U/ml) was 87.9 and 74.7%, respectively, the latter being significantly less than the inhibition observed in 17-day-old metacarpal cultures. The inhibitory effect of LIF was absent during concurrent administration of PTH or 1,25-(OH)2D3 and could be reversed by PTH. In addition, LIF was found to inhibit growth, mineralization, and alkaline phosphatase activity in metacarpals independently of osteoclastic resorption. These results suggest that LIF affects the development rather than the activity of osteoclasts, probably through an effect on the osteogenic cells. LIF may be an important endogenous regulator of bone metabolism.
Changes in bone histomorphometry after long-term treatment with intermittent, cyclic etidronate for postmenopausal osteoporosis
Authors: Storm Tommy , Steiniche Torben , Thamsborg Gorm , Melsen Fleming
Published: February 2010

Intermittent, cyclic etidronate therapy (400 mg/day for 2 weeks followed by 13 weeks free from study drug administration) resulted in a significant increase in lumbar bone mineral content and a significant decrease in the rate of new vertebral fractures in patients with postmenopausal osteoporosis. To investigate the effect of the treatment on bone histomorphometry, transiliac crest bone biopsy samples were obtained in this study before treatment and after 60 and 150 weeks of treatment with either intermittent, cyclic etidronate (n = 33) or placebo (n = 33). After 60 weeks of etidronate therapy, significant decreases in activation frequency (from 0.55 to 0.09 year,−1P < 0.01) and resorption depth (from 53.2 to 37.8 μm, P < 0.05) were observed, leading to a positive balance per remodeling cycle. In the placebo group, no significant changes were seen. The 150 week bone biopsy samples were suboptimal for analysis, probably as a result of a regional acceleratory phenomenon. Our results suggest that, as a result of reductions in both activation frequency and resorption depth, intermittent, cyclic etidronate therapy may protect the trabecular network against fortuitous perforations and thereby maintain the strength of the bony tissue.
Role of bone matrix in osteoclast recruitment in cultured fetal rat calvariae
Authors: Braidman Isobel P. , Anderson David C.
Published: February 2010

In cultured 19 day fetal rat calvaria, osteoclasts first appear after 48 h, more rapidly than with other cultured embryonic long bone rudiments. This may be because the calvarial osteoclast precursors are more differentiated or intramembraneous bone is a more powerful stimulus for osteoclast maturation than endochondral bone. To investigate this further, 19 day calvariae were stripped of their endocranial membranes, devoiding them of osteoclast precursors, and cocultured with the membranes or with other sources of these cells, such as bone marrow, fetal liver, spleen, and blood. There was similar recruitment of mature osteoclasts onto the surface of the “stripped” calvariae from the endocranial membranes and from the hematopoietic tissues after 48 h culture. Intact 19 day fetal calvariae were cultured with human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) or with 1,25-dihydroxyvitamin D3, [1,25-(OH)2D3], each thought to influence different stages of osteoclast maturation. They stimulated osteoclast recruitment, although 1,25-(OH)2D3 was effective only in the first 24 h of culture. They also increased osteoclast recruitment from fetal liver onto stripped calvariae. When intact bones were cultured with hrGM-CSF and 1,25-(OH)2D3 together, osteoclast number decreased but their area increased. Calvariae therefore appear to contain osteoclast precursors at earlier (GM-CSF-sensitive) and later [1,25-(OH)2D3-sensitive] stages. As recruitment onto stripped calvariae was similar whichever source of precursors was used, it is likely that calvarial bone matrix is an important influence on rapid osteoclast maturation in these bones in vitro.
Attachment to extracellular matrix molecules by cells differing in the expression of osteoblastic traits
Authors: Majeska Robert J. , Port Michael , Einhorn Thomas A.
Published: February 2010

Two sets of clonal cell populations differing in the expression of osteoblastic traits, the rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 and the immortalized fetal rat calvarial cell lines RCT-1 and RCT-3, were compared for their ability to attach to a series of extracellular matrix (ECM) constituents in vitro. Both osteoblastic (ROS 17/2.8, RCT-3) and nonosteoblastic (ROS 25/1, RCT-1) cell lines attached in a time- and concentration-dependent manner to plates coated with fibronectin (FN), osteopontin (OP), type I collagen (Col I), type IV collagen (Col IV), and laminin (LN) but only weakly to osteocalcin (OC) and thrombospondin (TSP). In both systems, the osteoblastic and nonosteoblastic clones attached identically to FN. Both ROS 17/2.8 and ROS 25/1 attached to similar molar amounts of substrate with the same preference order: FN > LN > Col I ≥ Col IV. Maximal ROS 17/2.8 attachment to OP was ≥ Col I but required approximately 2.5 times more substrate. ROS 25/1 attached less effectively than ROS 17/2.8 to most non-FN substrates. RCT-3 cells attached similarly to ROS 17/2.8 except that the preference order for Col I and LN was reversed and attachment to OP was lower than for ROS 17/2.8 RCT-1 cells attached best to Col I rather than FN, and equaled or surpassed RCT-3 in attachment to other non-FN substrates. Thus in these experimental systems, cells expressing an osteoblast-like phenotype exhibited generally similar ECM attachment properties. Their nonosteoblastic counterparts recognized the same spectrum of ECM constituents but differed from the osteoblastic cells and from each other in the effectiveness of their attachment to substrates other than FN.