bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma levels of parathyroid hormone-related peptide are elevated in hyperprolactinemia and correlated to bone density status
Authors: Stiegler Claudia , Leb Georg , Kleinert Reinhold , Warnkross Helga , Ramschak-Schwarzer Sigrid , Lipp Rainer , Clarici Georg , Krejs Guenter J. , Dobnig Harald
Published: February 2010

Osteopenia is an important clinical manifestation of hyperprolactinemia. Bone loss in these patients has mainly been attributed to concomitant deficiency of gonadal hormones rather than to hyperprolactinemia per se. Parathyroid hormone-related peptide (PTHrP) is expressed in human mammary tissue, and elevated circulating PTHrP levels as well as concomitant hypercalcemia have been described during lactation. We sought to determine circulating PTHrP levels in patients with long-standing hyperprolactinemia and whether PTHrP may exert possible systemic effects on bone and mineral metabolism. We studied 45 patients (30 women and 15 men) with persisting hyperprolactinemia 6 ± 4 years (mean ± SD) after trans-sphenoidal surgery for prolactin-producing pituitary adenomas. PTHrP levels in 117 healthy controls were 10.6 ± 73 pmol-eq/l (mean ± SD). In hyperprolactinemic patients, plasma PTHrP was elevated to 303 ± 13.4 pmol-eq/l (p < 0.001, n = 45), and in patients with humoral hypercalcemia of malignancy PTHrP levels were 52.9 ± 29.6 (p < 0.001 to controls and hyperprolactinemic patients). Fifty-three percent of hyperprolactinemic patients (n = 24) had clearly elevated PTHrP levels (>2 SD). Retrospective immunocytochemical studies of the removed pituitary adenomas from 19 patients generally showed a higher degree of immunoreactivity for PTHrP (1–34) in all but one case when compared with normal pituitary tissue. Patients with elevated circulating PTHrP levels showed in most instances strong immunoreactivity to PTHrP in 70–100% of tumor cells. PTHrP was significantly correlated to blood pressure (systolic: r = –0.42, p < 0.005; diastolic: r = –0.41, p < 0.01), serum calcium (r = 0.40, p < 0.01), parathyroid hormone (r = –0.43, p < 0.005), and bone density measurements (r = –0.41, p < 0.005). Patients taking low doses of bromocriptine (n = 15) had similar reductions in bone mineral density despite lower prolactin levels (p < 0.005), and there was no correlation between either prolactin levels or estrogen status and bone mineral density measurements (r = –0.12 and r = –0.28, respectively). Our data demonstrate that circulating PTHrP levels are clearly elevated in approximately 50% of patients with pituitary hyperprolactinemia and suggests the pituitary adenomas to be the major source of PTHrP production. Independent of gonadal function and the degree of hyperprolactinemia, PTHrP functions as an active hormone and contributes substantially to bone loss seen in these patients.
Ethnic differences in regional bone density, hip axis length, and lifestyle variables among healthy black and white men
Authors: Nelson D.A. , Jacobsen G. , Barondess D.A. , Parfitt A.M.
Published: February 2010

There are few published data on bone mass, measured by dual-energy X-ray absorptiometry (DXA), in healthy white or black men. Similarly, a recently described predictor of hip fracture among white women, hip axis length (HAL), has not been studied in men. We recruited 160 white and 34 black men, aged 23–80 years, and screened for diseases and drug exposures that adversely affect skeletal health. We measured bone mineral density (BMD) in the lumbar spine, femoral neck, and radial shaft by DXA; height and weight; skin color by reflectometry; and hip axis length both directly from DXA output and using automated software in a subsample. We also obtained historical data on education, smoking, exercise, and fractures. There were no significant black/white differences in mean weight, height, body mass index (BMI), or HAL. The black men had higher BMDs than did the white men at every site (5% for the radius, 10% for the lumbar spine, and 20% for the femoral neck). Skin pigmentation and BMD were not significantly correlated in either group (p > 0.38). Among the white men, smoking was associated with lower lumbar BMD, but there was no significant relationship between BMD and exercise frequency in either group. There was no significant ethnic difference in fracture experience. We conclude that: (1) the higher BMD in black men than in white men is not due to greater body size, (2) the lower hip fracture risk reported for black men than for white men is not due to a difference in hip axis length; (3) skin color is not related to BMD in either sex.
Carboxy-terminal propeptide of human type I collagen and pyridinium cross-links as markers of bone growth in infants 1 to 18 months of age
Authors: Lieuw-A-Fa Mariska , Sierra Rosa I. , Dr. Specker Bonny L.
Published: February 2010

Serum carboxy-terminal propeptide of human type I collagen (PICP) concentrations, as a marker for bone formation, and urinary pyridinium (Pyd) cross-link concentrations, as a marker of bone resorption, were determined in 66 healthy infants aged 1–18 months who are being studied longitudinally. We hypothesized that there would be a positive correlation of growth velocity, increase in bone area, and bone mass accretion rates with PICP and Pyd cross-link concentrations. Since osteocalcin is currently used as a marker of bone formation, serum osteocalcin concentrations were also measured. Mean serum PICP and urinary Pyd cross-link concentrations were significantly greater than adult concentrations. Future growth velocity, increase in bone area, and bone mass accretion rates were not associated with PICP, Pyd cross-link, or osteocalcin concentrations. Growth velocity during the 3 months preceding sample collection correlated with serum PICP, Pyd/kg, and osteocalcin concentrations (r = 0.474, p < 0.001; r = 0.379, p < 0.001; and r = 0.516, p < 0.001, respectively). Previous increase in bone area correlated with serum PICP concentrations (r = 0.359, p = 0.01). The relationship between the infant's previous bone mass accretion rate and PICP was of borderline significance (r = 0.281, p = 0.055). In summary, normative data for PICP, Pyd cross-link concentrations, and parameters of bone growth are provided for infants 1–18 months of age and indicate that these markers reflect past and current bone metabolism and may be helpful in monitoring bone disorders in infants.
Effects of methotrexate on human osteoblasts in vitro: Modulation by 1,25-dihydroxyvitamin D3
Authors: Dr. Scheven Ben A.A. , van der Veen Maaike J. , Damen Cora A. , Lafeber Floris P.J.G. , van Rijn Herman J.M. , Bijlsma Johannes W.J. , Duursma Sumen A.
Published: February 2010

This study was designed to investigate whether methotrexate (MTX), used in the treatment of rheumatoid arthritis (RA), affects proliferation and differentiation of human osteoblasts in culture. The effects of MTX were assessed by analyzing markers of proliferation and differentiation of human trabecular bone–derived osteoblast-like cells cultured in the presence or absence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). Treatment of the osteoblastic cells with MTX resulted in a strong dose-dependent inhibition of cell proliferation with half maximal response at a dose of 30 nM. MTX did not interfere with cellular alkaline phosphatase (AP) activity, the number of cells expressing cytochemical AP, or basal osteocalcin production. Addition of 1,25(OH)2D3 to the cultures caused an enhanced AP expression and osteocalcin production coinciding with a decreased osteoblast proliferation. Coincubation of 1,25(OH)2D3 with MTX in doses ≥100 nM further inhibited osteoblast growth and induced a significant stimulation of AP expression and activity, and production of osteocalcin above the values reached in the 1,25(OH)2D3 cultures. In conclusion, MTX proved to be a potent inhibitor of osteoblast proliferation but did not affect basal osteoblastic phenotypic expression. In the presence of the osteoblast differentiation-promotor, 1,25(OH)2D3, MTX further inhibited cell growth which was associated with enhanced AP activity and osteocalcin production. Thus, MTX may have profound effects on bone metabolism and remodeling by interfering with bone cell turnover.
A comparison of the effects of parathyroid hormone and parathyroid hormone-related protein on osteocalcin in the rat
Authors: Gundberg Caren M. , Fawzi Mervat I. , Clough Mary E. , Calvo Mona S.
Published: February 2010

We compared the effects of parathyroid hormone (PTH1–34) and parathyroid hormone-related protein (PTHrpl–34) on osteocalcin release in the isolated rat hindlimb and in intact and thyroparathyroidectomized (TPTX) rats. PTH1–34 suppressed osteocalcin release from perfused rat hindquarters, while PTHrp1–34 had no effect on osteocalcin release, despite comparable stimulation of cAMP production. Similarly, serum osteocalcin declined in the intact and TPTX animals by 5 h after a single dose of PTH1–34, while there was no response to PTHrpl–34. Chronic administration of PTH1–34 or PTHrpl–34 produced comparable hypercalcemia and hypophosphatemia in sham-operated and TPTX animals. Chronic PTH1–34 administration produced significant increases in serum osteocalcin both in the sham-operated rats and in the TPTX animals. However, in animals chronically treated with PTHrp1–34, there was no change at any time point in osteocalcin in either sham-operated or TPTX rats. These differences could be inherent or merely due to potency differences between the two peptides.
Serum pyridinoline as a specific marker of collagen breakdown and bone metabolism in hemodialysis patients
Authors: Ureńa Pablo , Ferreira Aníbal , Kung Viola T. , Morieux Caroline , Simon Pierre , Ang Kim S. , Souberbielle Jean Claude , Segre Gino V. , Drüeke Tilman B. , de Vernejoul Marie Christine
Published: February 2010

Type I collagen represents more than 90% of bone matrix. Quantitative analysis of collagen cross‐link molecules such as pyridinoline (PYD) provides valuable information on bone resorption rate. We have studied 37 hemodialysis patients who underwent a systematic transiliac bone biopsy for histomorphometry study. Eighteen of them had tetracycline double labeling, allowing to determine dynamic, in addition to static bone parameters. Measurement of serum‐free PYD was performed using a new competitive enzyme immunoassay. Serum PYD values were compared with those of three other serum markers of bone metabolism, namely intact PTH (iPTH), bone‐specific alkaline phosphatase (bAP), and osteocalcin, for the correlations with bone histomorphometric parameters. Serum PYD levels (mean ± SD) were significantly higher in dialysis patients than in normal individuals, 90.6 ± 99.6 nM versus 1.9 ± 0.4 nM, respectively. Patients with high turnover bone disease had significantly higher serum PYD levels than patients with normal or low bone turnover, 108.8 ± 108.0 nM versus 34.1 ± 12.8 nM, respectively. Serum PYD levels were positively correlated with bone resorption parameters including osteoclast surface (r = 0.59, p < 0.0001) and osteoclast number/mm2 (r = 0.61, p < 0.0001), and also with bone formation parameters, osteoblast surface (r = 0.43, p < 0.008), double‐labeled surface (r = 0.81, p < 0.001), and BFR (r = 0.91, p < 0.0001). The BFR was better correlated with serum PYD levels than with either serum iPTH or osteocalcin concentrations. However, correlation with serum bAP was comparable. Serum cross‐linked type I carboxy‐terminal telopeptide (ICTP) and type I carboxy‐terminal extension peptide (PICP) levels were also significantly increased in both groups of dialysis patients but they did not correlate with any of the bone turnover parameters. In conclusion, PYD, which is a specific serum marker of collagen breakdown, provides valuable information on bone turnover in hemodialysis patients.
Extra cancellous bone induced by combined prostaglandin E2 and risedronate administration is maintained after their withdrawal in older female rats
Authors: Dr. Jee Webster S. S. , Lin Bai Yun , Ma Yan Fei , Ke Hua Zhu
Published: February 2010

Prostaglandin E2 (PGE2) has been recognized for its marked anabolic effect on bone, but the bone gain is lost after the cessation of PGE2 treatment. In previous studies, we were successful in maintaining the new bone by administering a bisphosphonate after the withdrawal of PGE2 treatment. The objective of this study was to determine the fate of the extra bone induced by a combination with PGE2 and risedronate after discontinuing treatment. Ninety-six 9-month-old virgin female Sprague-Dawley rats were treated with 1 or 5 μg of risedronate/ kg/twice weekly, 6 mg of PGE2/kg/day alone or 6 mg of PGE2/kg/day plus 1 or 5 μg of risedronate/kg/twice weekly for 60 days (day 0) and followed by 60 days without treatment (day 60). We have reported the results from the groups treated for 60 days previously. This report is restricted to the histomorphometric findings on the secondary spongiosa of the proximal tibial metaphysis in the groups after withdrawal for 60 days. We found that the only group that maintained the PGE2 induced new bone after withdrawal was the group treated with 6 mg of PGE2/kg/day plus 5 μg of risedronate/kg/twice a week. Withdrawal of this combined treatment depressed bone turnover (bone-based bone formation rate, activation frequency) and bone resorption (percent eroded perimeter). The tissue mechanisms responsible for the protection drew from the previously deposited risedronate.
Nitric oxide: A cytokine-induced regulator of bone resorption
Authors: Ralston Stuart H. , Ho Ling-Pei , Helfrich Miep H. , Grabowski Peter S. , Johnston Peter W. , Benjamin Nigel
Published: February 2010

Nitric oxide (NO) has been reported to inhibit osteoclastic bone resorption, yet potent stimulators of bone resorption, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), are known to stimulate NO production. This paradox prompted us to reinvestigate the relationship between NO production and bone resorption in mouse calvarial organ cultures. Control cultures and those stimulated with calciotropic hormones and individual cytokines produced little NO, and under these conditions the NO synthase inhibitor, L-NG-monomethyl arginine (LMMA), had no significant effect on bone resorption. Cytokine combinations were much more potent stimulators of NO production than individual cytokines. Dramatic stimulation of NO production and inhibition of bone resorption resulted when gamma-interferon (IFN) was combined with IL-1 or TNF and these effects were reversed by LMMA. IFN had no effect on bone resorption and little effect on NO production when used alone or in combination with calciotropic hormones, however, suggesting that IFN selectively inhibits cytokine-induced bone resorption by generating large amounts of NO. IL-1 and TNF acted together to stimulate NO production but to a lesser degree than when combined with IFN. LMMA inhibited bone resorption induced by IL-1 and TNF, suggesting that lower concentrations of NO stimulate bone resorption. Experiments with the pharmacological NO donor S-nitroso-acetyl-penicillamine (SNAP) supported this view in showing generalized suppression of bone resorption at high SNAP concentrations, but potentiation of IL-1 induced bone resorption at lower SNAP concentrations. We conclude that cytokines are potent inducers of NO in bone and that cytokine-induced NO production has biphasic effects on bone resorption. While the mechanisms of action of NO in bone will require further research, these data raise the possibility that local production of NO may be involved in the regulation of bone remodeling, especially in diseases of cytokine activation such as rheumatoid arthritis.
Normalization of mineral homeostasis after reversal of osteopetrosis
Authors: Nesbitt Teresa , Drezner Marc K. , Marks Sandy C. , Jackson Mary E. , Mackay Carole A.
Published: February 2010

Whether a radiographic and histologic cure of osteopetrosis includes normalization of mineral homeostasis remains unknown. Thus, we explored the extent of defective mineral metabolism in the microphthalmic (mi/mi) mouse before and after cure. Under basal conditions mi mutants exhibit normocalcemia, hypophosphatemia, and elevated renal 25-hydroxyvitamin D-1-hydroxylase activity. However, administration of PTHrP (3 μg/h × 24 h) further stimulated enzyme activity in mi mutants with active disease, to a level no different than that in treated normals. Serum phosphorus levels also declined in mi/mi mice following PTHrP, suggesting a normal renal response to this hormone. In contrast, failure to suppress enzyme function in mi/mi mice following prolonged calcitriol infusion indicates that the observed enhancement of 1,25-dihydroxyvitamin D production occurred secondary to autonomous parathyroid function and/or nonparathyroid hormone-related stimuli. Although an increased fractional excretion and decreased tubular reabsorption of phosphate were demonstrated in mi/mi mice, serum PTH levels were no different in mi mutants compared with normal littermates. Following skeletal cure, the mi/mi mice surprisingly display normal serum phosphorus levels and renal enzyme activity. Moreover, treatment restored normal responsiveness to calcitriol suppression and maintained normal PTHrP responsiveness of enzyme activity. These data indicate that the cure of osteopetrosis in the mi mutant is universal and includes normalization of serum phosphorus and renal 25-hydroxyvitamin D-1-hydroxylase. Furthermore, these data suggest that phosphate depletion of unknown origin is the likely cause of elevated enzyme activity in this murine osteopetrotic mutant.
Degradation of subcutaneous implants of bone particles from normal and warfarin-treated rats
Authors: Serre C. M. , Price P. , Delmas P. D.
Published: February 2010

Osteoclasts are multinucleated cells specific to bone tissue and of hemopoietic origin. They are formed by fusion of mononucleated cells in a manner related to the formation of macrophage polykarions. Subcutaneous implantation of mineralized bone particles induces multinucleated giant cell recruitment. There is controversy, however, about the nature of these cells. Although subcutaneous implantation of bone particles derived from warfarin-treated animals has been applied as an in vivo model to study the role of osteocalcin in bone resorption, the exact nature of multinucleated cells elicited in this model is still unclear. In this paper, subcutaneous implants of bone particles from normal and warfarin-treated rats were implanted in Sprague-Dawley rats. Resorption was assessed in 12 and 16 day implants by chemical analysis (calcium content) and by histomorphometric measurement of the bone particle area and the number of multinucleated and tartrate-resistant acid phosphatase-positive cells. No significant difference in calcium content and bone area were observed, after 12 or after 16 days of implantation, between implants from normal and warfarin-treated rats. The number of tartrate-resistant acid phosphatase-positive cells elicited by bone particles represented less than 25% of the number of multinucleated cells and did not differ between bone particles from normal and warfarin-treated rats. By electron microscopy, a majority of multinucleated cells did not show a ruffled border in contact with bone particles, and their morphological features were suggestive of a foreign body giant cell reaction. In our experience this model appears to elicit only a few osteoclasts among multinucleated macrophagic cells and may not be the most appropriate one for the study of resorption of normal or osteocalcin-depleted bone.
Skeletal unloading induces selective resistance to the anabolic actions of growth hormone on bone
Authors: Halloran Bernard P. , Bikle Daniel D. , Harris Jonathan , Autry Christopher P. , Currier Patricia A. , Tanner Sharon , Patterson-Buckendahl Patricia , Morey-Holton Emily
Published: February 2010

Loss of skeletal weight bearing or physical unloading of bone in the growing animal inhibits bone formation and induces a bone mineral deficit. To determine whether the inhibition of bone formation induced by skeletal unloading in the growing animal is a consequence of diminished sensitivity to growth hormone (GH) we studied the effects of skeletal unloading in young hypophysectomized rats treated with GH (0, 50, 500 μg/100 g body weight/day). Skeletal unloading reduced serum osteocalcin, impaired uptake of 3H-proline into bone, decreased proximal tibial mass, and diminished periosteal bone formation at the tibiofibular junction. When compared with animals receiving excipient alone, GH administration increased bone mass in all animals. The responses in serum osteocalcin, uptake of 3H-proline and 45Ca into the proximal tibia, and proximal tibial mass in non-weight bearing animals were equal to those in weight bearing animals. The responses in trabecular bone volume in the proximal tibia and bone formation at the tibiofibular junction to GH, however, were reduced significantly by skeletal unloading. Bone unloading prevented completely the increase in metaphyseal trabecular bone normally induced by GH and severely dampened the stimulatory effect (158% vs. 313%, p < 0.002) of GH on periosteal bone formation. These results suggest that while GH can stimulate the overall accumulation of bone mineral in both weight bearing and non-weight bearing animals, skeletal unloading selectively impairs the response of trabecular bone and periosteal bone formation to the anabolic actions of GH.
Expression of bone sialoprotein mRNA during bone formation and resorption induced by colchicine in rat tibial bone marrow cavity
Authors: Arai N. , Ohya K. , Kasugai S. , Shimokawa H. , Ohida S. , Ogura H. , Amagasa T.
Published: February 2010

In the rat tibial bone marrow cavity, following colchicine injection, there is a phase of osteogenesis in which bone trabeculae replace the necrotic bone marrow tissues and fill the marrow cavity. The newly formed bone is subsequently resorbed by osteoclasts and normal bone marrow is restored. In this study, we correlated these morphologic events with the pattern of gene expression of bone sialoprotein (BSP), an extracellular matrix protein in mineralized tissues, to elucidate the possible functions of BSP in bone formation and resorption in vivo. The expressions of osteopontin (OPN) and type I collagen were also examined. Northern hybridization of the tibia demonstrated that OPN mRNA was gradually increased and expressed at a maximal level 10 days after colchicine injection (during the bone resorption process), while BSP mRNA expression already reached a maximal level at day 6 (during the initial process of bone formation). Its expression was, thus, quite temporary at the beginning of bone formation and different from that of type I collagen, which was continually elevated from days 6 to 10. In situ hybridization of the newly formed bone induced in the tibia revealed that BSP mRNA was evenly expressed in most osteoblasts and osteocytes, moreover in interconnecting colonies of spindle-shaped cells, possibly preosteoblasts, at day 6. At day 10, however, its expression became restricted to some cells on the bone surfaces, some osteoblasts, and most osteoclasts. These observations suggest that BSP may play an important role mainly in the initiation of bone formation and is also associated with the functions of osteoclast in vivo.
Serum IGF 1 is low and correlated with osteoblastic surface in idiopathic osteoporosis
Authors: Reed Berenice Y. , Zerwekh Joseph E. , Sakhaee Khashayar , Breslau Neil A. , Gottschalk Frank , Pak Charles Y. C.
Published: February 2010

We have previously reported that bone formation is impaired on histomorphometric analysis of bone in patients with idiopathic osteoporosis. In the present study, 30 patients with idiopathic osteoporosis (18 men and 12 women mean age 44 ± 12 years) and spinal and/or appendicular fractures were studied. Compared with control values, bone biopsy analysis demonstrated reduced bone volume (13.0 ± 4.4 vs. 23.2 ± 4.4, p < 0.0001), osteoid volume (0.13 ± 0.13 vs. 0.32 ± 0.19, p = 0.001), osteoid surface (5.9 ± 4.3 vs. 12.1 ± 4.6, p = 0.0004), and diminished double-labeled mineralizing surface (MS/BS 2.0 ± 2.1 vs. 5.1 ± 2.7%, p = 0.0001) in the patients. Since insulin-like growth factor 1 (IGF-1) is one of the major determinants of bone growth and remodeling, we measured the circulating level of this growth factor in these patients. The mean serum IGF-1 concentration was lower in patients as compared with 33 healthy age-matched controls (193 ± 59 SD ng/ml vs. 232 ± 79). A significant difference was noted between the two groups only in subjects younger than 36 years. In patients with idiopathic osteoporosis, regression analysis of serum IGF-1 against the various histological parameters measured from the bone biopsy disclosed significant correlation's between serum IGF-1 and osteoblastic surface (r = 0.429, p = 0.032), mineralizing bone surface with a double label (r = 0.480, p = 0.015), and the bone formation rate (r = 0.457, p = 0.021). These findings suggest that in young eugonadal individuals with idiopathic osteoporosis, reduced IGF-1 concentrations may have an etiological role in the development of this disease.
The expression of cytokine activity by fracture callus
Authors: Einhorn Thomas A. , Majeska Robert J. , Rush Elisa B. , Levine Pamela M. , Horowitz Mark C.
Published: February 2010

Cytokines, a group of proteins known to regulate hemopoietic and immune functions, are also involved in inflammation, angiogenesis, and bone and cartilage metabolism. Since all of these processes occur following bone injury, or are known to contribute to wound repair mechanisms, this investigation sought to test the hypothesis that cytokines are involved in fracture healing. Two sets of 60 male Sprague-Dawley rats underwent the production of standard closed femoral fractures. The animals were then euthanized in groups of 15 on days 3, 7, 14, and 21 postfracture. A separate control group was also used for the harvesting of intact unfractured bone. At the time of euthanasia, calluses or bone specimens were explanted to organ culture and treated with either media alone or media containing the inducing agents lipopolysaccharide or concanavalin A. A titration of conditioned medium from these cultures was then added to factor-dependent clonal cell lines that are known to be specifically responsive to interleukin-1, interleukin-6, granulocyte-macrophage colony stimulating factor or macrophage-colony stimulating factor. To confirm the identities of each of these cytokines, neutralizing antibody studies were performed. The results showed that interleukin-1 is expressed at very low constitutive levels throughout the period of fracture healing but can be induced to high activities in the early inflammatory phase (day 3). Granulocyte-macrophage colony stimulating factor showed no constitutive activity but could also be induced to high activities with lipopolysaccharide. The ability of these two cytokines to be induced declined progressively as fracture healing proceeded. Interleukin-6 showed high constitutive activity early in the healing process (day 3), and treatment with inducing agent did not increase the activity of this cytokine at this timepoint. Lipopolysaccharide did increase interleukin-6 activity in day 7 and 14 fracture calluses. Although macrophage-colony stimulating factor is thought to be involved in a variety of metabolic bone conditions, it could not be detected or induced from any of the callus samples. Moreover, none of the samples of unfractured bone showed constitutive or inducible activities for any of these cytokines. A separate experiment in which calluses and samples of unfractured bone from similar cultures were examined histologically and tested for DNA or protein synthesis at two timepoints in the culture period (days 1 and 4) showed that tissue viability was maintained. Thus the inability to detect macrophage colony-stimulating factor in fracture callus or any cytokine activity in unfractured bones was not due to cell death. These findings suggest that the occurrence of a fracture results in a cascade of events that includes the production of cytokines. The time-dependent expression of specific cytokine activities, which are differentially regulated, supports the concept that fracture healing may be partially mediated by these factors.
Additive effects of weight-bearing exercise and estrogen on bone mineral density in older women
Authors: Kohrt Wendy M. , Snead David B. , Slatopolsky Eduardo , Birge Stanley J.
Published: February 2010

The separate and combined effects of weight-bearing exercise and hormone replacement therapy (HRT) on bone mineral density (BMD) were studied in 32 women, 60 to 72 years of age. HRT consisted of continuous conjugated estrogens 0.625 mg/day and trimonthly medroxyprogesterone acetate 5 mg/day for 13 days. Exercise consisted of 2 months of low-intensity exercise followed by 9 months of more vigorous weight-bearing exercise ˜45 minutes/day, ≥3 days/week, at 65–85% of maximal heart rate. Lumbar spine and proximal femur BMD were significantly increased in response to exercise and to HRT, and total body BMD was significantly increased in response to HRT; neither exercise nor HRT had an effect on wrist BMD. The combination of exercise + HRT resulted in increased BMD at all sites except the wrist, with effects being additive for the lumbar spine and Ward's triangle and synergistic for the total body. Based on reductions in serum osteocalcin levels, it appears that increases in BMD in response to HRT and exercise + HRT were due to decreased bone turnover. The lack of change in serum osteocalcin and IGF-I in response to exercise alone suggests that increases in BMD were due to decreased bone resorption and not increased formation. Results indicate that weight-bearing exercise + HRT may be effective in preventing and/or treating osteoporosis. It is likely that the additive effects of weight-bearing exercise and HRT on bone mineral accretion, coupled with other adaptations to the exercise (i.e., increased strength and functional capacity), could effectively reduce the incidence of falls and osteoporotic fractures.
Treatment with growth hormone and IGF-I in growing rats increases bone mineral content but not bone mineral density
Authors: Rosen Harold N. , Chen Vicki , Cittadini Antonio , Greenspan Susan L. , Douglas Pamela S. , Moses Alan C. , Beamer Wesley G.
Published: February 2010

Human growth hormone (hGH) and insulin-like growth factor I (IGF-I) both stimulate bone formation and have been proposed as therapeutic agents for osteoporosis. We examined the effect of hGH and IGF-I alone and in combination on bone size, bone mineral content (BMC), and bone mineral density (BMD) in 10- to 12-week-old growing female Sprague-Dawley rats. Sixty rats were assigned to treatment with either placebo, hGH, IGF-I, or both for 4 weeks. After 4 weeks, the right femurs and tibias were excised, and ex vivo BMC and the area of the tibia and femur were measured by dual-energy X-ray absorptiometry (DXA); volume of these bones was measured by Archimedes' principle. In addition, proximal tibial bone density was measured directly by peripheral quantitative computerized tomography (pQCT). Bone length, area, and volume in all treated groups was greater than controls. Areal bone density by DXA (BMC/area) was higher in IGF-treated rats and lower in GH-treated rats than in controls. Volumetric bone density (BMC/volume) was lower in treated groups than in controls. Measurements by pQCT confirmed that true bone density was lower in all treated groups than in controls. We conclude that treatment with hGH or IGF-I increased bone size and mineral content but decreased bone density in growing rats. Because areal correction of BMC did not adequately correct for the increased bone volume in IGF-treated rats, results of areal bone density by DXA should be interpreted with caution when treatment causes a disparity in bone size between groups.
Chondrocytes isolated from mature articular cartilage retain the capacity to form functional gap junctions
Authors: Donahue Henry J. , Guilak Farshid , Vander Molen Monique A. , Mcleod Kenneth J. , Rubin Clinton T. , Grande Daniel A. , Brink Peter R.
Published: February 2010

The distribution, expression, and functionality of gap junctions was examined in bovine chondrocytes (BCs) isolated from mature articular cartilage. BC cells displayed immunoreactivity for connexin 43 (Cx43), a specific gap junction protein. Cx43 protein expression was confirmed by Western blot analysis, and Cx43 mRNA was detected by nuclease protection assay. Additionally, BCs were shown to be functionally coupled, as revealed by dye transfer studies, and octanol, a gap junction uncoupler, greatly attenuated coupling. Furthermore, confocal microscopy of fluo-3 loaded BC cells revealed that deformation-induced cytosolic Ca2+ ion (Ca2+) signals propagated from cell-to-cell via gap junctions. To our knowledge, this is the first evidence suggesting that chondrocytes isolated from adult articular cartilage express functional gap junctions.
Energy-shunting external hip protector attenuates the peak femoral impact force below the theoretical fracture threshold: An in vitro biomechanical study under falling conditions of thel elderly
Authors: Parkkari Jari , Kannus Pekka , Heikkilä Jussi , Poutala Jarmo , Sievanen Harri , Vuori Ilkka
Published: February 2010

The first objective of this study was to design a hip protector that would effectively attenuate and shunt away from the greater trochanter the impact energies created in typical falls of the elderly. As the shock absorption material, the protector included the 12 mm-thick Plastazote, which was found to be the most efficient energy-absorbing material in our previous in vitro biomechanical tests. With an anatomically designed semiflexible outer shield of the protector (high density polyethylene), the impact surface was increased and the impact energy shunted away from the greater trochanter. In the second phase of the study, we determined the force attenuation capacity of this device in realistic (in vitro) falling conditions of the elderly. With the impact force of 6940 N used (a typical hip impact force measured in in vitro falling tests), the trochanteric soft tissue (25 mm-thick polyethylene foam) attenuated the peak femoral impact force to 5590 N and the tested protector to 1040 N. In the second series of this experiment, the peak femoral impact force was set to be so high (13,130 N) that the protector, if effective, should prevent the hip fracture in almost all cases. The trochanteric soft tissue attenuated this peak impact force to 10,400 N and the tested protector to 1810 N. Thus, the force received by the proximal femur still remained clearly below 4170 N, the average force required to fracture in vitro the proximal femur of the elderly in a fall loading configuration. In conclusion, our test results suggest that an anatomically designed energy-shunting and energy-absorbing hip protector can provide an effective impact force attenuation in typical falling conditions of the elderly. However, the efficacy of the protector in the prevention of hip fractures can only be evaluated in randomized clinical trials.
Monoclonal antibodies recognize antigen expressed by osteoblasts
Authors: Dr. Benayahu Dafna , Efrati M. , Wientroub S.
Published: February 2010

A marrow stromal osteogenic cell line (MBA-15) was used to create monoclonal antibodies (MoAbs). In this study, we describe a series of MoAbs for mouse marrow stroma (MMS) (MMS-25/17, MMS-85/12, MMS-302/40, and MMS-319/4) that recognized antigens expressed by stromal cells including osteoblastic cells. The MoAbs were screened against various cell and tissue types. MMS-85/12 was positive in detecting an antigen that was highly abundant in osteoblastic cells and primary adherent bone marrow cultures (BMC) but was negative for the marrow adipocytes copartner. The MMS-85/12 MoAb is an IgGl immunoglobulin. The immunohistochemical staining pattern is suggestive of the antigen being associated with the osteoblasts' plasma membrane and with the extracellular matrix constituent secreted by these cells. Western blotting and immunoprecipitation indicated that the antigen that was recognized by MMS-85/12 apparently had a molecular weight of 84 kD.
In vitro and Ex vivo evidence that estrogens suppress increased bone resorption induced by ovariectomy or PTH stimulation through an effect on osteoclastogenesis
Authors: Most Werner , Schot Lodewijk , Ederveen Antwan , van der Wee-Pals Lianne , Papapoulos Socrates , Dr. Löwik Clemens
Published: February 2010

The mechanism of bone loss following cessation of ovarian function is still unclear. Several studies have shown an increase in bone turnover following natural or surgical menopause which could be prevented by estrogen administration. However, a direct effect of estrogen on osteoclast-mediated bone resorption has been difficult to demonstrate in vitro. Recent evidence suggested that estrogen withdrawal stimulates the production of bone resorbing cytokines, (e.g., interleukin-6, IL-6), which regulate osteoclast formation in the bone marrow microenvironment. We studied the effects of 17β-estradiol on osteoclastic resorption, measured as 45calcium release, in vitro using cultures of fetal mouse long bone explants in which different stages of osteoclast development and activity are represented. 17β-estradiol (10−12-10−8 M) had no effect on basal or parathyroid hormone (PTH)-stimulated resorption of bone explants in which mature osteoclasts (radii/ulnae) or osteoclast precursors/progenitors (metacarpals) are present. 17β-estradiol, however, inhibited significantly the PTH-stimulated resorption of osteoclast-free metacarpals cultured together with mouse fetal liver as a source of early osteoclast progenitors; basal resorption was also not inhibited in this system. In ex vivo studies we further examined the effects of culturing bone marrow cells from ovariectomized (OVX) or sham-operated mice as an osteoclastic source together with osteoclast-free metacarpals on 45calcium release and bone histology. Cocultures of the bone marrow cells from OVX mice with osteoclast-free metacarpals increased significantly the osteoclast formation and subsequent osteoclastic resorption compared with control cocultures. This increase in resorption was prevented by either treatment of the OVX animals with estrogen for 1 week starting immediately after OVX or injection of the OVX animals with an IL-6 neutralizing antibody. We conclude that estrogens suppress the increased osteoclastic resorption induced by PTH or OVX through an effect on hematopoietic progenitor cells of the osteoclast lineage. Furthermore our data suggest that IL-6 is involved in the increase in osteoclastic resorption following OVX.
Aging changes mechanical loading thresholds for bone formation in rats
Authors: Turner Charles H. , Takano Y. , Owan I.
Published: February 2010

The effect of aging on the mechanical loading thresholds for osteogenesis was investigated in rats. We applied mechanical loads varying from 30 to 64 N to the tibiae of 43 19-month-old rats using a four-point bending apparatus. Bone formation rates were measured on the periosteal and endocortical surfaces of the tibial midshaft using double-label histomorphometry. Bone formation rates from the old rats were compared with results from adult (9-month-old) rats that we reported earlier.(4) Bone formation on the periosteal surface of the old rats was predominantly woven-fibered. Periosteal bone formation was observed in a lower percentage of the old rats compared with the younger adult rats for applied loads of 40 N and greater (59% old, 100% adult). However, in the old rats that formed woven bone there were no significant differences in woven bone area (p = 0.1) or surface (p = 0.24) compared with younger adult rats. Therefore, the periosteum of old rats had a higher threshold for activation by mechanical loading, but after activation occurred, the cells had the same capacity to form woven bone as younger adult animals. On the endocortical surface, relative bone formation rates in old rats showed a marginal (p = 0.06) increase in response to an applied load of 64 N but was not increased at lower loads. The relative bone formation rate in the old rats was over 16-fold less than that reported for the younger adult rats at an applied load of 64 N and the relative bone forming surface in old rats in this study was 5-fold less than it was in younger rats under similar loading conditions. In the younger adult rats, a mechanical threshold for lamellar bone formation of 1050 μstrain was calculated for the endocortical bone surface. The old rats required over 1700 μstrain on the endocortical surface before bone formation was increased. The data suggest that both the periosteal and endocortical surfaces of the tibiae of older rats are less responsive to mechanical stimuli.
Bone mineral metabolism in T lymphocyte–deficient and –replete strains of rat
Authors: Buchinsky Farrel J. , Ma Yanfei , Mann Gary N. , Rucinski Boguslaw , Bryer Haldon P. , Paynton Barbara V. , Jee Webster S.S. , Hendy Geoffrey N. , Dr. Epstein Solomon
Published: February 2010

The immune and skeletal systems are known to interact. We have repeatedly shown that in contrast to in vitro data, the administration of T lymphocyte immunosuppressants, such as cyclosporin A, leads to an increase in bone resorption and a high turnover osteopenia. The purpose of this study was to characterize the bone metabolism of the T lymphocyte deficient Rowett athymic homozygous (rnu/rnu) nude rat. We wished to determine whether these rats share the bone abnormalities of cyclosporin A–treated rats. Eleven 10-week-old Sprague–Dawley rats and 12 similarly aged nude rats were studied over a 4-week period. Metaphyseal cancellous bone histomorphometry was similar in the two groups of rats and only differed with regard to percentage eroded perimeter (lower in nude rats, p = 0.008) and longitudinal growth rate (49% lower in nude rats, p < 0.001). The nude rats had less body mass (p < 0.001) but nevertheless gained the same percentage of their body weight over the study period. The athymic rats had lower levels of serum 1,25-dihydroxyvitamin D (p < 0.014) and serum osteocalcin (p < 0.009), and at the age of 14 weeks the nude rats had lower concentrations of serum creatinine (p = 0.001) and blood ionized calcium (p = 0.0002), yet serum PTH was similar throughout. RNA isolated from the contralateral tibias revealed that the nude group had lower steady-state levels of osteocalcin mRNA despite similar rates of bone formation. In its entirety, the data suggest that T cell deficiency per se is not necessarily associated with high turnover osteopenia.
Isolation of calcium-phosphate crystals of bone by non-aqueous methods at low temperature
Authors: Kim Hyun-Man , Rey Christian , Dr. Glimcher Melvin J.
Published: February 2010

We have developed low temperature nonaqueous solution methods as well as low power plasma ashing for the degradation of the organic matrix of bone powder which have permitted us to obtain bone crystals essentially free of organic matrix constituents without any significant change in their composition, overall structure, or internal short-range order. We have also been able to disperse the crystals, which has made it possible to examine the isolated crystals by X-ray diffraction and resolution-enhanced Fourier transform infrared (FTIR) spectroscopy and isolated single crystals by high resolution transmission electron microscopy (TEM) and electron diffraction. TEM of isolated single crystals of fish, chicken, mouse, and bovine bone have clearly demonstrated that the crystals are very thin plates. No rod or needle-like crystals were observed in any of the bone samples in the four species studied including the earliest crystals deposited. Although there were variations in the size distribution of the crystals in the different species studied, in general the average crystal dimensions were very similar.
Mineralization of bone-like extracellular matrix in the absence of functional osteoblasts
Authors: Marsh Mary E. , Munne Anna M. , Vogel James J. , Cui Yingqi , Dr. Franceschi Renny T.
Published: February 2010

When grown in medium containing ascorbic acid and β-glycerol phosphate, mouse MC3T3-E1 cells express an osteoblast phenotype and produce a highly mineralized extracellular matrix. The purpose of this study was to independently examine the role of the collagenous matrix and functional osteoblasts on the mineralization process. Cultures with and without an extensive collagenous matrix were prepared by growing MC3T3-E1 cells in the presence and absence of ascorbic acid. Matrix-rich cultures mineralized at much lower calcium phosphate ion products than nonmatrix cultures. At higher ion products, spontaneous precipitation in the medium and cell layers of nonmatrix cultures were observed. In contrast, mineral in matrix-rich cultures was still exclusively associated with collagen fibrils and not with ectopic sites in the cell layer or medium. To examine the effect of cell viability on matrix mineralization, cells were grown 8 or 16 days in the presence of ascorbic acid, then killed and incubated in a mineralizing medium. Significant mineralization was not observed in the collagenous matrix of 8-day killed cultures or age-matched controls. At 16 days mineral was associated with collagen fibrils at specific foci in the matrix of both viable and killed cultures. This observation is consistent with the concept that collagenous matrices must undergo a maturation process before they can support mineral induction and growth. It further shows that osteoblast-like cells are not required for mineralization of mature matrices, but are required for matrix maturation.
Leukemia inhibitory factor as a mediator of JAK/STAT activation in murine osteoblasts
Authors: Dr. Lowe Carolyn , Gillespie Gerald A.J. , Pike Wesley J.
Published: February 2010

A number of cytokines have been shown to exert their effects via a recently discovered signaling cascade. One step in this pathway is mediated by a family of nonreceptor protein tyrosine kinases, the Janus kinases or JAK kinases, which become phosphorylated upon ligand-receptor binding and receptor phosphorylation. This in turn is followed by phosphorylation of certain members of a family of latent transcription factors, called signal transducers and activators of transcription (STATs), which subsequently enter the nucleus, bind to DNA in a sequence-specific fashion, and modulate transcription. In view of the apparent role of leukemia inhibitory factor (LIF) in bone remodeling, we sought to determine which, if any, of the JAK/STAT family members are involved in mediating the actions of LIF using the MC3T3-E1 cell line (a spontaneously immortalized osteoblast) and normal murine calvarial osteoblasts. We report here rapid and transient phosphorylation of the LIF receptor, and similarly, we detect phosphorylation of predominantly JAK1 and to a minor extent JAK2 in response to LIF treatment in MC3T3-E1 cells. In these experiments we also detect phosphorylation of STAT1 and to a much lesser degree STAT3 upon addition of LIF. Phosphorylation of the STAT proteins correlates directly with their ability to bind DNA in a gel mobility shift assay in MC3T3-E1 cells and in normal calvarial osteoblasts. These studies suggest that LIF action in these cells, as in other cell types, is mediated in part via specific members of the JAK/STAT pathway.