bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of ipriflavone on expression of markers characteristic of the osteoblast phenotype in rat bone marrow stromal cell culture
Authors: Notoya Kohei , Yoshida Keiji , Tsukuda Ryoichi , Taketomi Shigehisa
Published: February 2010

The effects of ipriflavone on cellular proliferation and differentiation of osteoblasts were investigated using stromal cells isolated from the femoral bone marrow of young rats. To induce the formation of mineralized bone-like tissue in vitro, the cells were cultured in the presence of β-glycerophosphate and dexamethasone. Ipriflavone was added when subculturing was started. After 14 days of culturing with ipriflavone (10−7-10−5 M), increases in both the alkaline phosphatase activity and the hydroxyproline content per culture dish and a slight decrease in the saturated cell density were observed. Furthermore, continuous treatment with ipriflavone for 14–33 days resulted in an increase in the area of bone-like mineralized tissue accompanied by an increase in the secretion of osteocalcin. When culture medium lacking dexamethasone was used, rat bone marrow stromal cells neither differentiated into osteoblasts nor formed bone-like tissue, and under these conditions, ipriflavone had no effect on the proliferation or the phenotypic expression of the cells. These results suggest that ipriflavone directly stimulates markers of the osteoblast phenotype at a certain stage in bone formation without affecting undifferentiated cells that have not been committed to the osteogenic lineage.
Macrophage colony-stimulating factor restores bone resorption in op/op bone in vitro in conjunction with parathyroid hormone or 1,25-dihydroxyvitamin D3
Authors: Morohashi Tomio , Corboz Véronique Antonioli , Fleisch Herbert , Cecchini Marco G. , Felix Rolf
Published: February 2010

The in vivo administration of macrophage colony-stimulating factor (M-CSF) restores osteoclastogenesis and bone resorption in the op/op murine osteopetrosis. In vitro, exogenous M-CSF has been shown to be necessary for the generation of osteoclast-like cells in cocultures of hematopoietic and mesenchymal cells obtained from this mutant. In this study we investigated the capacity of M-CSF and other cytokines and hormones, alone or in combination, to induce bone resorption in explants of op/op metatarsals and metacarpals prelabeled with 45Ca. The effect on bone resorption was verified by counting the number of osteoclasts generated in the mineralized matrix. No osteoclast formation and no bone resorption were observed in the absence of M-CSF. M-CSF alone had only a slight effect at the high concentration of 104 units/ml. Addition of PTH or 1,25-(OH)2D3 together with M-CSF induced both osteoclastogenesis and bone resorption. The release of 45Ca was linear with time up to 15 days. PTH or 1,25-(OH)2D3 could not be substituted by TNF-α or IL-1, whereas IL-6 had a weak effect. M-CSF could not be replaced by GM-CSF. This study further emphasizes the role of M-CSF, PTH, and 1,25-(OH)2D3 in osteoclastogenesis.
Effect of growth hormone, insulin-like growth factor I, basic fibroblast growth factor, and transforming growth factor β on cell proliferation and proteoglycan synthesis by avian postembryonic growth plate chondrocytes
Authors: Rosselot Gaston , Vasilatos-Younken Regina , Leach Roland M.
Published: February 2010

We examined the in vitro effects of pituitary-derived chicken growth hormone (cGH), recombinant human insulin-like growth factor-I (rhIGF-I), recombinant human basic fibroblast growth factor (rhbFGF), and porcine transforming growth factor β (pTGF-β) on proliferation ([3H]thymidine uptake) and matrix proteoglycan synthesis (35SO4 incorporation) by chicken epiphyseal growth plate chondrocytes. Factorial experiments were used to study the effect of these substances in a serum-free culture system. Basic FGF had to be present in the culture medium for mitogenesis to take place. In the presence of this peptide, TGF-β, TGF-β + IGF-I, and newborn calf serum (NCS) stimulated mitogenesis. The mitogenic activity of NCS could be duplicated by adding platelet-derived growth factor (PDGF) to the culture medium. For matrix synthesis, IGF-I was the key factor, with the addition of TGF-β, TGF-β + bFGF, or serum producing further stimulation in matrix synthesis. Using this culturing system, homologous cGH did not stimulate cell proliferation or proteoglycan synthesis. The lack of stimulatory activity of cGH was consistent, regardless of the age of the birds from which the chondrocytes were isolated, the zone of the growth plate, or the level of cGH used. None of the growth factors used in this study or several other systemic hormones were found to be permissive factors for GH to be active. Either other factors must be present for a direct effect of GH on growth plate chondrocytes, or the avian species differ from their mammalian counterpart.
Precision of measurement by dual-energy X-ray absorptiometry of bone mineral density and content in rat hindlimb in vitro
Authors: Sievänen Harri , Kannus Pekka , Järvinen Markku
Published: February 2010

Dual-energy x-ray absorptiometry (DXA) is a precise and effective method for measuring bone mineral density (BMD) and content (BMC) at various skeletal sites in the human, but its precision for measuring low BMD and BMC in tiny rat bones has scarcely been evaluated. The short-term precision of DXA (Norland XR-26) for eight regions of interest (ROI) was determined by measuring dissected, not totally defleshed hindlimbs from four rats of differently weights 10 times consecutively. The accuracy of the BMD (area) measurement was also evaluated. The following average precision values (coefficients of variation) with negligible reanalysis error were obtained for BMD and BMC measurements: 0.7 and 1.2% (total); 0.9 and 1.2% (femur); 1.3 and 2.8% (tibia); 1.3 and 2.1% (foot); 2.1 and 4.7% (proximal femur); 1.4 and 2.9% (femoral shaft); 1.8 and 5.6% (distal femur); and 2.7 and 7.6% (proximal tibia). As for the accuracy, BMD measured in tiny bones is likely to be markedly underestimated. We conclude that the standard DXA technique without additional modification for small animal measurements is a precise method for BMD and BMC measurements at multiple sites in rat hindlimb in vitro.
Effects of mild asymptomatic primary hyperparathyroidism on bone mass in women with and without estrogen replacement therapy
Authors: McDermott Michael T. , Perloff Jan J. , Kidd Gerald S.
Published: February 2010

Primary hyperparathyroidism (HPT) presents most commonly as a mild elevation of the serum calcium concentration in an asymptomatic individual. There are conflicting data regarding the effects of mild primary HPT on bone mass. This cross-sectional study was conducted to examine this question further and to determine whether estrogen replacement therapy (ERT) in postmenopausal women with primary HPT might be beneficial. We measured bone mass in 59 women with mild asymptomatic primary HPT, of whom 43 (HPT) had never taken and 16 (estrogen-replaced HPT) were currently taking ERT. We also studied 84 healthy normocalcemic women who were not on ERT (controls) and 45 who were on ERT (estrogen-replaced controls). After adjustment for age, height, and weight, mean bone mass values in the HPT group were significantly reduced in the midradius (20%), distal radius (20%), lumbar spine (17%), and femoral neck (11%) compared with the controls. The estrogen-replaced HPT group had mean bone mass values greater than those in the HPT group, similar to those in the controls, and lower than those in the estrogen-replaced controls. Mild asymptomatic primary HPT results in bone loss from both the appendicular and axial skeleton, and ERT in postmenopausal women with primary HPT may ameliorate this loss.
Bone functional changes in intact, ovariectomized, and ovariectomized, hormone-supplemented adult cynomolgus monkeys (Macaca fascicularis) evaluated by serum markers and dynamic histomorphometry
Authors: Dr. Jerome Christopher P. , Carlson C.S. , Register T.C. , Bain F.T. , Jayo M.J. , Weaver D.S. , Adams M.R.
Published: February 2010

Several parameters of bone mass and function were investigated in three experiments involving intact, ovariectomized, or hormone-supplemented ovariectomized female cynomolgus monkeys. Ovariectomized animals had increased serum levels of alkaline phosphatase and acid phosphatase compared with intact and hormone-supplemented animals. Vertebral bone mass measured ex vivo by dual-photon absorptiometry was reduced by 11–19% in ovariectomized animals compared with intact and hormone-supplemented animals. The most dramatic effects observed with ovariectomy were markedly increased (30–60%) bone formation rates in vertebral cancellous bone, primarily caused by higher activation frequency of basic multicellular units of bone. In addition, combined resorption and reversal periods were decreased and formation period increased in untreated ovariectomized animals. Changes in static histomorphometry parameters were less dramatic, cancellous bone volume being 1–14% lower in ovariectomized animals compared with intact or ovariectomized hormone-supplemented animals. The data indicate that changes in bone resorption are primarily responsible for the lower bone mass of estrogen deficiency and increased bone mass in hormone-supplemented animals. Bone changes in ovariectomized cynomolgus monkeys resemble those in women after menopause and similarly respond positively to hormone supplementation. As such, cynomolgus monkeys are an excellent model for studying the basic mechanisms of osteoporosis and for the development of suitable therapeutic regimens.
Isolation and sequence of the vitamin K-dependent matrix Gla protein from the calcified cartilage of the soupfin shark
Authors: Rice Jeffrey S. , Williamson Matthew K. , Dr. Price Paul A.
Published: February 2010

High levels of the vitamin K-dependent matrix Gla protein (MGP) have been found in the calcified costal cartilage of the cow and the calcified vertebral cartilage of the soupfin shark (Galeorhinus galeus). In both species, MGP accounts for 35–40% of the total protein in the acid demineralization extract of calcified cartilage, and the mineral content of calcified cartilage is comparable to that of bovine cortical bone. Shark and bovine MGP are both nearly insoluble in neutral buffers, a conserved property that indicates that self-aggregation could be important to the as yet unknown function of MGP. The complete amino acid sequence of shark MGP was determined to compare the structure of the elasmobranch protein to the several currently known mammalian MGP sequences. Shark MGP contains 4 residues of the vitamin K-dependent amino acid γ-carboxyglutamic acid in its 102 residue sequence and has a calculated molecular weight = 12,770 daltons. The first 76 residues of shark MGP are homologous in sequence to mammalian MGPs, with 37% sequence identity, but the C-terminal 23 residues of the shark protein have no counterpart in the mammalian MGPs. This C-terminal segment of shark MGP contains 8 basic residues and no acidic residues. Among the features conserved in shark MGP, in all mammalian MGPs, and in all other currently known vitamin K-dependent mammalian proteins are a 15-residue region of sequence homology that has been shown to function as the γ-carboxylase recognition sequence and an invariant sequence of unknown function, Gla-Xaa-Xaa-Xaa-Gla-Xaa-Cys.
Risk of hip fracture in women with vertebral fracture
Authors: Kotowicz Mark A. , Dr. Melton L. Joseph , Cooper Cyrus , Atkinson Elizabeth J. , O'Fallon W. Michael , Riggs B. Lawrence
Published: February 2010

In a population-based retrospective cohort study, Rochester women aged 35–69 years who were first diagnosed with one or more vertebral fractures in 1950–1979 were followed for the development of a subsequent hip fracture. The 336 women with no history of hip fracture at the time of their vertebral fracture experienced 52 proximal femur fractures in 4788 person-years of follow-up. The standardized morbidity ratio (SMR) of observed to expected hip fractures was 1.8 (95% CI, 1.3–2.4) and was higher for intertrochanteric than cervical femoral fractures (SMR, 2.3 versus 1.3; P = 0.07). Hip fracture risk among women with symptomatic vertebral fractures was slightly less than in those with asymptomatic vertebral fractures (SMR, 1.8 versus 2.3; not significant), and younger women had no higher risk of a subsequent hip fracture than women who were ≥60 years of age at the time of their vertebral fracture (SMR, 1.4 versus 1.8; not significant). Alternative explanations are possible, but these data are consistent with heterogeneity in the pathogenesis of different osteoporotic fractures.
Effects of increasing doses of 1α-hydroxyvitamin D2 on calcium homeostasis in postmenopausal osteopenic women
Authors: Gallagher J.C. , Bishop Charles W. , Knutson Joyce C. , Mazess Richard B. , Deluca Hector F.
Published: February 2010

This study is the first reported administration of 1α-hydroxyvitamin D2 (1α-OHD2) to human subjects. A total of 15 postmenopausal osteopenic women were given increasing oral doses of 1α-OHD2, beginning with a low dose of 0.5 μg/day. In 15 subjects, the doses were raised at weekly intervals to 1.0, 2.0, 4.0, and 5.0 μg/day, and in 5 of these subjects, the dose was further increased to 8.0 or 10.0 μg/day. Mean urine calcium ± SEM showed a dose-related increase from 134 ± 17 mg/24 h on 0.5 μg/day to 198 ± 21 mg/24 h on 4.0 μg/day (p < 0.05) and to 241 ± 35 mg/24 h on 5.0 μg/day (p < 0.05). No subjects had hypercalciuria (>350 mg/24 h, the upper limit of the laboratory normal range) at doses less than 5.0 μg/day; 5 subjects had hypercalciuria at or above 5.0 μg/day (3 at 5.0 μg/day, 1 at 8.0 μg/day, and 1 at 10.0 μg/day). Mean serum calcium increased slightly on the 4.0 μg dose only (p < 0.05) but remained well within the normal range. Mean creatinine clearance and BUN, used as measures of renal function, showed no significant changes. Routine blood and urine assays also showed no significant changes. Serum osteocalcin, a marker of osteoblast activity, showed mean ± SEM dose-related increases of 10 ± 7% on 1.0 μg/day (p < 0.05), 19 ± 7% on 2.0 μg/day (p < 0.05), 21 ± 6% on 4.0 μg/day (p < 0.05), and 28% on 5.0 μg/day (p < 0.05); in the 5 subjects receiving higher dosages, mean serum osteocalcin increased 32 ± 12% on 8.0 μg/day and 55 ± 29% on 10.0 μg/day. Mean urine hydroxyproline/creatinine ratios did not change significantly at any dose of 1α-OHD2 but trended downward with increasing dosages. These findings indicate that 1α-OHD2 is well tolerated at dosages that stimulate osteoblastic activity, as evidenced by increased serum osteocalcin, and that 1α-OHD2 may be a useful agent for treating postmenopausal osteoporosis.
Efficacy and tolerability of a new formulation of oral tiludronate (tablet) in the treatment of paget's disease of bone
Authors: Reginster J.Y. , Treves R. , Renier J.C. , Amor B. , Sany J. , Ethgen D. , Picot C. , Franchimont P.
Published: February 2010

We sought to assess efficacy and safety of a new oral formulation (tablet) of tiludronate in Paget's disease of bone. We studied 128 patients with Paget's disease in an open-label uncontrolled trial. Patients received a daily dose of 400 mg oral tiludronate (two tablets). Treatment was for 6 months. Serum alkaline phosphatase activity (SAP) and fasting urinary excretion of hydroxyproline/creatinine (OH/Cr) were measured every 3 months, as were biochemical parameters reflecting renal, hepatic, and hematologic functions. Analgesic efficacy was self-evaluated from a visual analog scale (VAS). Statistical analysis revealed a significant reduction from baseline in SAP and OH/Cr levels, as well as VAS scores. In the whole population with evaluation under treatment, there was a reduction in initial SAP activity after 3 months (47.2 ± 2.2%, mean ± SEM) and 6 months (58.3 ± 2.3%). In the population with SAP levels above twice the upper limit at inclusion and with evaluation at month 3 and month 6 (n = 96), the reduction in SAP levels was 49.3 ± 2.4% after 3 months and of 59.5 ± 2.6% after 6 months (ANOVA time effect, p = 0.0001). Aside from mild gastrointestinal disturbances, as experienced with other oral bisphosphonates, clinical tolerance was good. Exhaustive biochemical investigation failed to reveal significant toxicity of tiludronate tablets at the dose of 400 mg/day. The dose of 400 mg daily of this new formulation appears to be a satisfactory tiludronate regimen for the treatment of Paget's disease of bone.
Muscle contraction increases the in vivo structural strength to the same degree in osteopenic and normal rat tibiae
Authors: Nordsletten Lars , Kaastad Trine S. , Obrant Karl J. , Skjeldal Sigmund , Kirkeby Ole J. , Stokke Oddvar , Ekeland Arne
Published: February 2010

The increase in structural capacity due to muscle contraction in the lower leg was investigated in osteopenic and normal rats. Osteopenia was induced by ovariectomy combined with a low-calcium diet (0.01%). The control rats were sham operated and fed a diet containing 1.1% calcium. After 7 weeks the right lower leg of all animals were fractured in three-point ventral cantilever bending during muscle contraction induced by electrical stimulation of the ischiatic nerve. The left tibiae were resected and fractured as each animal's control. During muscle contraction in vivo, the ultimate bending moment, energy absorption, bending stiffness, and deflection were significantly lower in the osteopenic than in the sham-operated animals. However, the increase in mechanical parameters due to muscle contraction comparing the in vivo and resected tibiae in each animal were equally high in the osteopenic and sham-operated animals. Ultimate bending moment in the resected tibiae was 10% higher in the sham-operated animals compared with the ovariectomized, proving mechanically weaker tibiae in the osteopenic rats. In accordance with this, the medullary area of the osteopenic rats was 46% larger in the distal tibial diaphysis, and the ultimate stress the tibiae could withstand was 15% lower in the osteopenic compared with the sham-operated rats. The trabecular bone volume in the distal tibial metaphysis of the osteopenic rats was reduced by 70% compared with the sham operated. This study shows that muscle protection against fracture can be substantial in osteopenic tibia and that it is of the same magnitude as in rats with normal bone mass.
Dual-energy X-ray absorptiometry of raloxifene effects on the lumbar vertebrae and femora of ovariectomized rats
Authors: Dr. Sato Masahiko , McClintock Cindy , Kim John , Turner Charles H. , Bryant Henry U. , Magee David , Slemenda Charles W.
Published: February 2010

A new potential therapeutic agent for postmenopausal osteoporosis, raloxifene, previously known as keoxifene, was evaluated by x-ray densitometry and more traditional techniques in quantitating the short-term (4–5 weeks) effects of ovariectomy on bones from 6-month-old rats. A Hologic QDR 1000/W and, to a limited extent, a Lunar DPXL, was used to quantitate ovariectomy, estrogen replacement, and raloxifene effects on vertebrae, femora, and tibiae. Both instruments performed well with precisions of 1.6% (Hologic) and 0.9% (Lunar) for anesthetized rats, which improved to 0.4% (Hologic) and 0.5% (Lunar) when the same rats were frozen. The lumbar vertebrae L1–4 showed a 12% decrease in bone mineral density 4 weeks after ovariectomy, compared with a 9% decrease for femora. Tibiae were also examined, but edge-detection problems prevented reproducible analysis of this site in vivo. The decrease in bone mineral density postovariectomy, especially for femora, was found to include both an increase in the projected area and a slight but not significant decrease in the bone mineral content of L1–4 and femora. These changes in density parameters of femora were supported by a decrease in dry weight and volume and a marginal increase in the second moment of inertia I for the identical femora examined ex vivo. Examination of individual lumbar vertbrae L1–5 suggested that the bone mineral density of L3 changes most dramatically in response to ovariectomy, but present techniques lack the spatial resolution and precision to quantitate bone changes reliably in individual vertebrae. 17β-Estradiol administered at 100 μg/kg/day subcutaneously inhibited ovariectomy effects on L1–4 bone mineral density, femoral moment of inertia, dry weight, and volume and to a lesser extent, femoral bone mineral density. A nonsteroidal compound, raloxifene HC1, at 1 mg/kg/day per os, had bone effects and effects on body weight that were largely indistinguishable from those of 17β-estradiol; however, raloxifene did not produce the uterotrophic effects observed with estrogen. The half-maximal efficacious dose of raloxifene on L1–4 bone mineral density was between 0.1 and 1.0 mg/kg/day per os. These data show that dual-energy x-ray absorptiometry compares favorably with traditional methods in quantitating bone changes caused by ovariectomy in small rodents, that L1–4 is a more sensitive region than whole femora in evaluating the effect of estrogen deficiency on bone loss, and the raloxifene may have promise as a treatment for conditions characterized by excessive bone loss after ovariectomy.
Reduced femoral neck bone density in the daughters of women with hip fractures: The role of low peak bone density in the pathogenesis of osteoporosis
Authors: Dr. Seeman Ego , Tsalamandris Con , Formica Carmelo , Hopper John L. , McKay John
Published: February 2010

Low bone density in women with hip fractures (“senile” osteoporosis) may be due to excessive bone loss or low peak bone density. If excessive bone loss is responsible, then no reduction in bone density is expected in their daughters. If low peak bone density is responsible, then bone density should also be reduced in their daughters because genetic and family environmental factors influence the variability in bone density. Bone density was measured using dual-photon absorptiometry and expressed as a standardized deviation or Z score relative to 697 controls, adjusting for age, height, weight, and menopausal status. In 74 women with hip fractures, the Z score (mean ± SEM) was −0.52 ± 0.14 (P < 0.001) at the femoral neck, −1.04 ± 0.17 (P < 0.001) at the femoral shaft, and −0.43 ± 0.10 (P < 0.001) at the lumbar spine. In their 41 daughters, the Z score was −0.40 ± 0.17 (P < 0.05) at the femoral neck, −0.41 ± 0.19 (P < 0.001) at the femoral shaft, and 0.23 ± 0.13 (NS) at the lumbar spine. We conclude that daughters of women with hip fractures are likely to be at increased risk for hip fractures themselves because they have reduced femoral neck bone density. Femoral neck fractures may not be entirely attributable to trauma; reduced bone density is likely to contribute and may be caused by the attainment of a lower peak femoral neck bone density.
Preclinical pharmacology of CGP 42′446, a new, potent, heterocyclic bisphosphonate compound
Authors: Green Jonathan R. , Müller Klaus , Jaeggi Knut A.
Published: February 2010

We have investigated the pharmacologic effects of a new bisphosphonate compound, CGP 42′446 [2-(imidazol-1-yl)-1-hydroxyethylidene-1,1-bisphosphonate], on bone metabolism. The compound exhibited potent inhibitory activity on the bone resorption induced by 1,25-dihydroxyvitamin D3 both in vivo in the thyroparathyroidectomized rat (ED50 0.072 μg/kg SC) and in vitro in mouse calvarial cultures (IC50 0.002 μM). A comparison of the in vivo and in vitro inhibitory potencies of a total of nine bisphosphonates revealed an excellent correlation between the two assays (r = 0.97). CGP 42′446 also potently inhibited calvarial bone resorption induced by parathyroid hormone (1–34), parathyroid hormone-related protein (1–34), and recombinant human interleukin-1β. Short-term treatment of growing rats with CGP 42′446 dose-dependently increased the radiographic density of the tibial proximal metaphysis (ED50 1.7 μg/kg SC) as well as increasing the calcium and hydroxyproline content of femoral trabeculae (ED50 values 0.17 and 1.1 μg/kg SC, respectively), but there was no detectable effect on cortical bone. On a molar basis in this range of in vivo screening assays, CGP 42′446 was between 940-fold (thyroparathyroidectomized rat) and 87-fold (rat femoral trabecular calcium content) more potent than pamidronate. It is concluded that CGP 42′446 is a promising new, highly potent bisphosphonate for the suppression of the increased bone resorption associated with various diseases.
Interleukin-6 antisense deoxyoligonucleotides inhibit bone resorption by giant cells from human giant cell tumors of bone
Authors: Reddy Sakamuri V. , Takahashi Shunji , Dallas Mark , Williams Ronald E. , Neckers Leonard , Roodman G. David
Published: February 2010

The effects of antisense constructs to IL-6 on the bone-resorbing capacity of purified giant cells from giant cell tumors of bone were examined to further define the role of IL-6 in human osteoclastic bone resorption. In addition, we wanted to determine the utility of antisense constructs to cytokines produced by osteoclasts as probes to identify the molecular events responsible for the bone-resorptive process. Giant cells were cultured on sperm whale dentin for 24 h in the presence of fluoresceinated antisense or scrambled antisense deoxyoligonucleotides complementary to IL-6 mRNA. The giant cells actively incorporated these oligonucleotides, as evidenced by their intense fluorescence. The number of resorptive lacunae formed and the area of the dentin resorbed were significantly decreased in cultures of giant cells treated with antisense IL-6 constructs compared with control cultures treated with scrambled antisense constructs to IL-6 (60 ± 13 versus 12 ± 6 lacunae and 1.2 ± 0.3 versus 0.26 ± 0.1 × 105 μm2). IL-6 levels in conditioned media from giant cell cultures treated with IL-6 antisense constructs were fourfold lower than those in control media obtained from giant cells treated with scrambled antisense constructs to IL-6. These data confirm the capacity of IL-6 antisense oligomers to block IL-6 production by these cells. These observations show that IL-6 plays an important role in the bone-resorptive process of human osteoclasts and suggest that antisense constructs to cytokines produced by bone cells may be useful for determining the molecular events occurring during bone resorption.
Isolation and characterization of gap junctions in the osteoblastic MC3T3-E1 cell line
Authors: Yamaguchi Dean T. , Ma Defang , Lee Austin , Huang Jason , Gruber Helen E.
Published: February 2010

Gap junctions are channels connecting cells that function in cell-to-cell communication. Gap junctions are abundant in osteoblastic cells. Membranes enriched for gap junction plaques were obtained by differential centrifugation, followed by treatment of the membranes with potassium iodide and sarkosyl before sucrose density gradient centrifugation. Electron microscopy showed that the preparation was enriched for electrondense membranes consistent with gap junctions. Coomassie Blue staining of SDS-PAGE preparations revealed a prominent band at approximately 41 kD. Western analysis with a site-directed antibody, CT-360 (D. Laird, California Institute of Technology, Pasadena, CA), to the C-terminal portion of the rat heart connexin 43 molecule was positive in the MC3T3-E1 cell line, a phenotypic osteoblastic cell line derived from normal neonatal mouse calvariae. Western analysis using a monoclonal antibody, R5.21C, to rat liver connexin 32 was negative. Additionally, a prominent band at 59 kD was detected by CT-360 in both gap junction-enriched preparations and cell lysates. Treatment of diluted samples of gap junction-enriched preparations with sulfhydryl reducing agents in combination with detergents resulted in the enhancement and diminution of the 41 and 59 kD bands, respectively. Immunoprecipitation following [35S]methionine/[35S]cysteine labeling revealed a significant band detected at 122 kD in addition to the 41 kD band. To demonstrate functional gap junctions, transfer of lucifer yellow dye to surrounding cells was monitored after microinjection of a target cell. Between passages 10 and 25 in culture, functional cell coupling was found in approximately 70% of injected cells. Coupling was detected within 1–2 minutes after injection. Simultaneous microinjection of the CT-360 antibody with lucifer yellow resulted in the decoupling of cells. In conclusion, (1) MC3T3-E1 cells possess a 41 kD protein that is recognized by connexin 43 antibody to rat heart gap junction; (2) multimers of the MC3T3-E1 gap junctions occur in the preparation; and (3) functional coupling demonstrated by dye transfer may be regulated by region(s) in the C terminus of the connexin molecule.
Alcohol-induced bone disease in the absence of severe chronic liver damage
Authors: DR. Díez Adolfo , Puig Jordi , Serrano Sergi , Marin̄oso Maria-Lluisa , Bosch Jaume , Marrugat Jaume , Mellibovsky Leonardo , Nogués Xavier , Knobel Hernando , Aubía Jaume
Published: February 2010

To define and identify metabolic bone disease and mineral alterations induced by chronic heavy alcoholism in patients without severe liver damage, we studied a prospective series of unselected patients admitted to a 300-bed general hospital in Barcelona (Spain). A total of 26 chronic heavy drinkers of more than 150 g/day for at least 3 years were included. A general analytic and hormonal study, including liver biopsy in cases with any abnormality in liver function tests, and plasma and urine biochemistry with calcium regulating hormones and osteocalcin levels were determined. A transiliac bone biopsy after double-tetracycline labeling, with histomorphometric study of undecalcified bone, was performed. Statistical analysis was adjusted by age and sex by means of logistic regression. A total of 26 (20 men and 6 women) chronic alcohol abusers were studied. After adjustment for age and sex, alcoholic patients showed slight but significantly increased concentrations of plasma calcium (9.56 ± 0.56; OR = 17.93; 95% CI 3.17–101.48) and decreased cPTH (0.36 ± 0.11; OR = 0.097;95% CI 0.018–0.528) compared with controls. Osteocalcin values were low (1.49 ± 0.89, normal range 1.8–6.6). There was a significant decrease in bone volume, BV/TV (12.56 ± 5.29; OR = 0.06; 95% CI 0.01–0.34), with increased resorption surfaces, ES/BS (4.28 ± 2.43; OR = 9.86; 95% CI 2.16–45.07), and increased osteoclast number, N.Oc/TA (0.21 ± 0.37; OR = 6.41; 95% CI 1.27–32.25). Dynamic parameters were abnormal (Fisher's exact test), as follows: decreased BFR/BS (0.023 ± 0.028 versus 0.049 ± 0.020; p = 0.013), MAR (0.309 ± 0.269 versus 0.771 ± 0.529; p = 0.029), MS/BS (3.743 ± 4.433 versus 5.890 ± 2.882; p = 0.008, and OMR (2.402 ± 2.833 versus 3.057 ± 0.083; p = 0.011) and increased MLT (175.80 ± 405.49 versus 34.53 ± 8.117; p = 0.008). We conclude that chronic alcohol consumption induces osteopenia with low turnover and increased osteoclast number and resorption surfaces. The increase in plasma calcium levels and decreased PTH suggests a primary effect of alcohol on bone, resulting in bone loss and calcium infusion from bone into plasma. These effects were observed in the absence of significant liver damage.
Characterization of the osteogenic stromal cell line MN7: Identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting, and microsequencing
Authors: Mathieu E. , Meheus L. , Raymackers J. , DR. Merregaert J.
Published: February 2010

Proteins secreted by the osteogenic stromal cell line MN7 were analyzed using two-dimensional polyacrylamide gel electrophoresis (PAGE), western blotting, immunodetection, and microsequencing. Trichloroacetic acid-precipitated proteins from the conditioned medium of MN7 cell cultures, harvested at different times of growth, were dissolved in denaturing and reducing sample buffer and separated in the first dimension according to isoelectric point and in the second dimension according to molecular weight. Protein patterns were visualized using silver staining. Among the 350 separated protein spots, we identified type I collagen, bone sialoprotein, osteonectin, and cathepsin B by western blotting and immunodetection using polyclonal antibodies. Osteocalcin could not be detected in the conditioned medium of MN7 cells. Furthermore, 15 MN7-specific protein spots were localized after comparison with two-dimensional PAGE patterns from the conditioned medium of the nonosteogenic stromal cell lines MM1 and MV1. Microsequencing of the internal peptides of five selected spots revealed three known proteins, namely the carboxyl-terminal propeptide of the α2 chain of collagen type I, cathepsin L, and the tissue inhibitor of metalloproteinases-2, an 18 kilodalton peptide fragment from osteopontin that has not previously been described, and a novel glycosylated 85 kD protein with an average isoelectric point of 5.7. All identified proteins did not vary in presence between the different time points analyzed by two-dimensional PAGE. The use of two-dimensional PAGE to investigate the secreted proteins of MN7 cells will enable us to establish a complete protein data base of extracellular osteoblast-specific proteins. Furthermore, two-dimensional PAGE in combination with other techniques is a fast and accurate method for the identification of novel proteins that could function as markers in osteoblast differentiation and/or bone formation.
Further definition of the protein kinase C activation domain of the parathyroid hormone
Authors: Jouishomme Hervé , Whitfield James F. , Gagnon Lyne , MacLean Susanne , Isaacs Richard , Chakravarthy Balu , Durkin Jon , Neugebauer Witold , Willick Gordon , Rixon Raymond H.
Published: February 2010

The protein kinase C (PKC) activation domain of the parathyroid hormone (PTH) was believed to be the 28–34 region of the molecule. We have now shown that PTH-(29–32) is the smallest PTH fragment that can stimulate significantly membrane-associated PKC activity in ROS 17/2 rat osteosarcoma cells. As was previously shown for full-length PTH-(1–84) and the fully bioactive PTH-(1–34) fragment, there were two peaks in the PKC response to PTH-(29–32): one peak was obtained with low picomolar concentrations and the other with much higher nanomolar concentrations of the fragment. The PKC-activating ability was unaffected by the loss of Asn33 and Phe34, but it was abolished by removing His32. Thus, the PTH-(28–31) and PTH-(29–31) fragments did not stimulate membrane-associated PKC activity. The much larger PTH-(1–31) fragment also did not stimulate membrane-associated PKC activity, although it stimulated adenylyl cyclase as strongly as PTH-(1–34). This functional sensitivity to the loss of the polar His32 was not caused by a specific need for His or another polar amino acid in this position because replacing it with the apolar Leu did not abolish adenylyl cyclase or PKC activation. It is concluded that the minimum, fully functional PKC activation domain of the PTH molecule is Gln29-Asp30-Val31-His32.
Quality control of bone densitometry in a national health survey (NHANES III) using three mobile examination centers
Authors: Wahner Heinz W. , Looker Anne , Dunn William L. , Walters Lana C. , Hauser Mary F. , Novak Catherine
Published: February 2010

A quality control (QC) program for bone mineral measurements at the proximal femur by dual-energy x-ray absorptiometry (DXA) was designed for the osteoporosis component of the Third National Health and Nutrition Examination Survey (NHANES III). Major elements of the QC program are (1) a QC center for review of all scans, (2) setup procedures for and continuous monitoring of daily QC procedures, (3) reference standards for cross-calibration, (4) longitudinal studies for assessment of instrument stability, (5) monitoring of technologist performance, and (6) training. This report describes the results of the QC program of the first half of this 6 year study, which began in 1988. Measurements were performed on 7376 subjects in three mobile examination centers, which traveled to a new location about every 3 months, a total of 44 locations. A small percentage (3.5%) of all scans were rejected, mostly because of patient motion during scanning, but 33% of the remaining scans required reanalysis at the QC center to refine the location of the regions of interest. Precision in spine and hip phantoms was below 1% at all ROIs. In 535 subjects with duplicate scans, age 20–91 of both sexes, examined in a blinded review, precision for BMD at the femur neck ROI was 3.2% (CV) and 5.1% for Ward's triangle BMD. The central review improved scan quality, increased the number of usable scans, and reduced significantly the range of the mean percentage difference in the duplicate scans. Minor alterations in machine function were observed and corrected. QC results on phantoms were similar to those obtained with stationary instruments in dedicated laboratories. Retrospective analysis of the regression slopes from QC records did not show a need for correction of the data base, indicating that the instruments were stable during the course of the study.
Long-term effect of calcium supplementation on bone loss in perimenopausal women
Authors: Elders Petra J. M. , Lips Paul , Netelenbos J. Coen , van Ginkel Floris C. , Khoe Edwin , van der Vijgh Wim J. F. , van der Stelt Paul F.
Published: February 2010

We observed in a controlled 2 year longitudinal trial in 248 perimenopausal women that a daily calcium supplement of either 1000 or 2000 mg Ca2+ significantly reduced lumbar bone loss and bone turnover in the first year of calcium supplementation. In the second supplementation year the rate of lumbar bone loss in the treated subjects was not significantly different from that in the control group, although two of the three biochemical parameters of bone turnover remained decreased throughout the study. To quantify further the long-term effect of calcium supplementation, we extended the study for another year in 214 women. In the women of the control group who were menstruating until the last year of the trial, the mean change in lumbar bone mineral density after 3 years was –3.2% of the initial value versus 1.6% in the calcium-supplemented groups (p < 0.01). The decrease in lumbar bone loss in these supplemented premenopausal and early perimenopausal women remained statistically significant in the second and third years of supplementation. In the women who stopped menstruating before or during the study, the long-term reduction in lumbar bone loss was not significant (mean difference between control and treatment groups <0.6% points after 3 years). The decrease in metacarpal cortical thickness (MCT) in the treated subjects during 3 years was on average –3.0% of the initial value in the control versus–2.0% in the supplemented subjects (P < 0.01). The effect of calcium supplementation on MCT was not significantly related to the menopausal status of the subjects. Serum alkaline phosphatase, osteocalcin, and urinary hydroxyproline excretion decreased after calcium supplementation in all menopausal groups. These parameters remained decreased throughout the trial, with exception of alkaline phosphatase in the 1000 mg calcium group. We conclude that calcium supplementation substantially reduces cortical and trabecular bone loss in the years immediately preceding menopause. Although it reduces postmenopausal cortical bone loss to some extent, it does not prevent the menopause-related lumbar bone loss.
Paracrine interactions among parathyroid cells: Effect of cell density on cell secretion
Authors: Sun Feng , Maercklein Peter , Fitzpatrick Lorraine A.
Published: February 2010

Cell-cell interactions are important in the regulation of endocrine cell secretion. To investigate the possibility that cell communication may alter the regulation of parathyroid cell secretion, we utilized the reverse hemolytic plaque assay (RHPA) to measure parathyroid hormone (PTH) release from individual cells. Bovine parathyroid cells were dispersed and plated with protein A-conjugated erythrocytes at cell densities ranging from 0.9 to 36 X 102 cells/cm2 in 0.2 mM calcium. Cell populations were greater than 98% homogeneous as determined by immunocytochemistry and in situ hybridization for PTH mRNA. Plaques were developed and data analyzed for the amount of PTH per cell released (plaque area in μm2 X 104) and the determination of cell recruitment (% plaques formed). A positive correlation existed between parathyroid cell density and the amount of PTH released. As the distance between cells increased, the plaque area (amount of PTH released per cell) decreased (ranging from 1.0 X 1044 μm2 at 0.9 X 102 cells/cm2 versus 1.6 X 104 μm2 at 36 X 102 cells/cm2). The percentage of cells releasing PTH (recruitment) also decreased (16% at 0.9 X 102 cells/cm2 versus 47% at 36 X 102 cells/cm2). These data suggest that parathyroid cells in close proximity are stimulated to secrete more hormone than those at lesser densities. In addition, parathyroid cells are recruited to secrete PTH when plated at high density. Factor(s) released by the parathyroid cell may increase cell responsiveness and stimulate secretion in a paracrine fashion.
Correlation between low levels of estrogen receptors and estrogen responsiveness in two rat osteoblast-like cell lines
Authors: Davis Vicki L. , Couse John F. , Gray T. Kenney , Korach Kenneth S.
Published: February 2010

With the knowledge that estrogen replacement therapy can circumvent postmenopausal osteoporosis and with the discovery of estrogen receptors (ER) in cultures of normal osteoblast-like cells, extensive investigations have been directed toward understanding the role of the ER in normal bone homeostasis. ROS 17/2.8 and UMR-106-01, two established osteoblast-like cell lines derived from rat osteosarcomas, have been shown to have estrogen-regulated biologic responses. Only the ROS 17/2.8 cell line has been reported to contain ER. In this study, high-affinity, saturable binding sites characteristic of the ER were detected in UMR-106-01 cells by binding assays with the high-affinity ligand, [125I]17β-estradiol. An initial immunoconcentrtion step before western blot analysis also allowed detection of the full-length ER protein. In addition, northern blot analysis indicated that the entire ER transcript was expressed and that the half-life of the ER message was increased following cycloheximide treatment. Message levels were also regulated by removal of serum and treatment with estradiol. An estrogen-regulated reporter vector, ERET81CAT, was transfected into the UMR-106-01 cells to determine whether the detected level of ER was transcriptionally functional. Using this assay, estrogen responsiveness was evident; however, the response was inconsistent. Multiple factors, such as serum, estradiol, and cell density, influence the ER levels in these cells and probably cause fluctuations in the abundance of receptors available to induce the CAT response. When the cells are responsive, the ICI 164,384 antagonist could block the estrogen-induced activation of CAT.

The ROS 17/2.8 cells were also analyzed in parallel with the UMR-106-01 cells to allow comparisons between these two osteoblast-like cell lines because they exhibit phenotypes for two unique stages of differentiation. ROS 17/2.8 cells were found to contain more receptor sites/cell by the 125I-E2 (estradiol) binding assays, as well as higher levels of ER-specific transcripts, than UMR-106-01 cells (two- to threefold). This level of ER was consistently able to modulate estrogen-induced stimulation of the reporter CAT vector. Therefore, functional ER is expressed in both cell types, but the higher level of receptors found in the ROS 17/2.8 cell line improves the estrogen responsiveness of these osteoblast-like cells. These data also indicate that levels of ER that are low or undetectable by conventional methods are able to mediate biologic responses through direct interactions of the ER with the specific DNA response element.
Inhibitory effects of bisphosphonates on growth of amoebae of the cellular slime molddictyostelium discoideum
Authors: Dr. Rogers Michael J. , Watts Donald J. , Russell R. Graham G. , Ji Xiaohui , Xiong Xiaojuan , Blackburn G. Michael , Bayless Allan V. , Ebetino Frank H.
Published: February 2010

Bisphosphonates are inhibitors of bone resorption and are used increasingly as therapeutic agents for treating clinical disorders of skeletal metabolism. Their mode of action is still not fully understood. The demonstration that methylenebisphosphonate, a simple methylene analog of pyrophosphate, inhibits the axenic growth of amoebae of the slime mold Dictyostelium discoideum and is incorporated into adenine nucleotides suggested that this organism might be useful in elucidating the cellular effects of bisphosphonates. We examined 24 bisphosphonates, including all those of clinical interest as inhibitors of osteoclast-mediated bone resorption in vivo, for their effects on D. discoideum. All the geminal bisphosphonates inhibited growth of Dictyostelium, although the effectiveness of individual compounds varied widely. When the bisphosphonates were ranked there was a remarkable similarity between the order of potency as inhibitors of growth of Dictyostelium and the order of potency as inhibitors of bone resorption. Thus, bisphosphonates with more complex side-chain structures, especially those containing a nitrogen group, were more potent than simple substituted bisphosphonates, some inhibiting Dictyostelium growth even at concentrations below 10 μM. It therefore appears that the mechanism by which bisphosphonates prevent Dictyostelium growth could be similar to the mechanism by which these compounds affect the activity of osteoclasts. Because the mechanisms of action of bisphosphonates on osteoclasts remains unclear, Dictyostelium may provide an additional model for studying the biochemical mode of action of bisphosphonates. Furthermore, these studies suggest that Dictyostelium may also be a convenient organism for rapid evaluation of potentially active bisphosphonates.
Effect of parathyroid hormone-related peptide supplementation of soy protein formulas in the neonatal pig model
Authors: Hillman Laura S. , Forte Leonard R. , Veum Trygve , Ru Yuxia , Howard Barbara , Rowe Angela , Hillman Helena W. , Hillman Laura S.
Published: February 2010

PTH-related peptide (PTHrP) is found in all milks, including human and pig. To define a role for PTHrP in milk, 2-day-old piglets were randomized to receive soy formula devoid of PTHrP or supplemented with 1 nM synthetic PTHrP(1–86) (n = 8 per group). The number of serum samples with detectable PTHrP by immunoassay (Incstar) and radiometric assay (Nichols) was 9 of 33 and 3 of 13 in PTHrP− and 8 of 27 and 3 of 15 in PTHrP+ formula-fed piglets and 8 of 14 and 7 of 12 in naturally suckling piglets, respectively. Serum and urine concentrations of calcium and magnesium and total and bone alkaline phosphatase were similar in both groups at 3, 6, 10, and 17 days of age. No differences were seen in bone mineral content of the tibia measured by single-photon absorptiometry (BMC 0.22 + 0.06 and 0.22 + 0.10) or dual x-ray absorption (BMC 1.43 + 0.36 and 1.31 + 0.78) either in vivo or on excised bone or by measurement of Ca, Mg, or P content or total bone ash (1.26 + 0.26 and 1.38 + 0.28 mg). Intestinal histology, serum intestinal alkaline phosphatase, and net absorption and retention of Ca, Mg, and P in balances from age 11–17 days were all similar. As in humans, however, a developmental pattern was seen for phosphorus regulation in both groups. Serum phosphorus (mg/dl) increased between day 3 and days 6 and 10 of age and then fell to published values for 8-week-old suckling pigs at day 17 associated with an increase in urine phosphorus and cyclic AMP excretion. The rise in serum phosphorus from 3 to 6 days was suggestively attenuated in the piglets receiving PTHrP (4.6 + 1.5 in –PTHrP and 2.6 + 1.3 in +PTHrP; p = 0.02 by nonpaired t-test, ANOVA for treatment effect p = 0.14). Thus, although a suggestive early effect of milk PTHrP on phosphorus regulation may be present before the development of full responsiveness to endogenous PTH, no effects could be demonstrated on mineral absorption, renal regulation, or bone mineralization. Because the synthetic PTHrP studied was only 1–86 amino acids, an effect of more distal, carboxyl-terminal, peptides cannot be excluded.