bluuebunny/crossref_metadata_2025 huggingface dataset:
Mild versus definite osteoporosis: Comparison of bone densitometry techniques using different statistical models
Authors: Heuck Andreas F. , Block Jon , Glueer Claus-Christian , Steiger Peter , Dr. Genant Harry K.
Published: February 2010

The purpose of this investigation was to determine the ability of three bone densitometry techniques to discriminate subjects with mild vertebral deformities from those with definite compression fractures. We determined bone mineral density (BMD) in 68 postmenopausal women by quantitative computed tomography (QCT) and dual-photon absorptiometry (DPA) of the spine, as well as single-photon absorptiometry (SPA) of the radius. Forty four individuals were classified as having mild deformities of the spine and 24 were considered to have definite vertebral compressions. Several statistical approaches were used to compare these subgroups and to estimate the relative risk of vertebral fracture. Included among these were percent decrements and z-scores, ROC curves, odds ratio estimations, and logistic regression analysis. Individuals with definite vertebral fractures had lower bone mineral density at all sites, but measurement of radial compact bone by SPA failed to reach significance. Using ROC analysis to distinguish mild deformities from true compressions, we found that measurement of spinal trabecular bone by QCT to be the most sensitive discriminator; although measurement of spinal integral bone by DPA also gave satisfactory discrimination, whereas assessment of radial compact bone did not adequately differentiate patients with mild deformities from those with definite compressions. Likewise, we found determination of spinal trabecular bone to be the most robust predictor of relative risk of definite fracture using either odds ratios or logistic regression analysis. Measurement of BMD in the peripheral cortical skeleton offered no predictive power for true vertebral fracture. We concluded that direct assessment of the spine, particularly of the trabecular portion, offered the strongest discrimination and relative risk prediction for definite osteoporotic fractures compared with milder forms of this condition.
Aminohydroxypropylidene bisphosphonate (APD) treatment for tumor-associated hypercalcemia: A randomized comparison between a 3-day treatment and single 24-hour infusions
Authors: Body J. J. , Magritte A. , Seraj F. , Sculier J. P. , Borkowski A.
Published: February 2010

Intravenous aminohydroxypropylidene bisphosphonate (APD) normalizes serum calcium in most hypercalcemic cancer patients, however the optimal therapeutic scheme has not been established. We compared in a randomized prospective trial the efficacy and the tolerance of APD given as a 3-day treatment of daily 2-h infusions of 0.5 mg/kg·d in 250 ml of saline (group A) with single 24-h infusions of 1.5 mg/kg (group B) or of 0.5 mg/kg in 1 liter of saline (group C). Thirty-three cancer patients remaining hypercalcemic after a 48-h rehydration period were included and monitored daily until normocalcemia or treatment failure was documented. Serum calcium became normal in all but 1 patient (in group C) but remained normal for only 1 or 2 days in 4 other patients (1 in A, 1 in B, 2 in C). The decline in total or ionized serum calcium was slightly less marked in group C than in the two other groups, but the differences were not significant. The fall of fasting urinary calcium excretion was however significantly less rapid in group C (p < 0.05 from day 1 to day 4). Serum concentrations of iPTH and 1,25-dihydroxyvitamin D [1,25-(OH)2D] increased significantly in the three groups. Serum magnesium concentrations fell slightly from 1.41 ± 0.05 to 1.28 ± 0.04 mEq/liter (p < 0.001) after rehydration but returned to normal after APD administration (day 5, 1.52 ± 0.04 mEq/liter, p < 0.001 versus day 0). On the other hand, APD induced a fall in serum phosphate levels, from 2.9 ± 0.1 to 2.3 ± 0.1 mg/dl on day 4 (p < 0.001), without any significant change in TmP/GFR. Treatment was very well tolerated; we only observed 3 cases of drug-induced fever, 1 in each group. Based on this and other dose-response trials, we recommend a total dosage of 1.0 to 1.5 mg of APD/kg body weight for the therapy of tumor-associated hypercalcemia; this treatment can be safely and efficiently given as a single 24 h infusion or as daily 2 h infusions for 3 days.
The synthetic human parathyroid hormone-related protein is inhibited by a parathyroid hormone antagonist in rats in vivo
Authors: Horiuchi Noboru , Hongo Takashi , Clemens Thomas L.
Published: February 2010

The structure of a novel protein, parathyroid hormone-related protein (PTHrP), secreted by human tumors associated with hypercalcemia has recently been determined. Administration of a synthetic fragment of this protein in vivo reproduces features of the clinical paraneoplastic syndrome of humoral hypercalcemia of malignancy and produces biologic responses closely similar to those obtained with parathyroid hormone (PTH). A PTH antagonist designed to reversibly occupy PTH receptors inhibited major actions of the tumor peptide in vivo, including phosphaturia, urinary cAMP excretion, and increased serum ionized calcium. These studies indicate that PTHrP and PTH mediate their bioactivities through shared receptors in vivo and establish a potential specific mechanism-based approach utilizing PTH antagonists for the therapy of tumor-associated hypercalcemia.
1,25-dihydroxyvitamin D3 metabolism in a human osteosarcoma cell line and human bone cells
Authors: Miller Barbara E. , Chin David P. , Jones Glenville
Published: February 2010

The metabolism of 1,25-dihydroxy vitamin D3 [l,25-(OH)2D3] by a human osteoblastic sarcoma cell line, U-2 OS, and by primary cultures of human bone-derived cells was examined at physiologic (5 x 10−11 M) and pharmacologic (3.5 x 10−7 M) substrate concentrations. For metabolite identification purposes, cells nearing confluency were incubated for 18 h with 3.5 x 10−7 M l,25-(OH)2D3 in serum-free medium. The putative vitamin D metabolites produced during this incubation were isolated from a total lipid extract of cells and medium. Identification of the metabolites was achieved by comigration with authentic standards on three high-performance liquid chromatography systems, UV spectral analysis, mass spe trometry, and chemical modification by sodium borohydride and sodium metaperiodate. The identified metabolites produced from l,25-(OH)2D3 by the human osteosarcoma cells include 1,24,25-trihydroxyvitamin D3; 24-oxo-l,25-dihy-droxyvitamin D3; 24-oxo-l,23,25-trihydroxyvitamin D3; and 24,25,26,27-tetranor-l,23-dihydroxyvitamin D3. Evidence is presented that (1) l,25-(OH)2D3 metabolism occurs constitutively in U-2 OS osteosarcoma cells at a physiologic substrate concentration (5 x 11−11 M), (2) the pathway can be further induced by pharmacologic 1,25-(OH)2D3 concentrations (10−7 M), and (3) this pathway is present in primary cultures of normal human bone-derived cells.
A short course of recombinant human growth hormone treatment stimulates osteoblasts and activates bone remodeling in normal human volunteers
Authors: Dr. Brixen Kim , Nielsen Henning K. , Mosekilde Leif , Flyvbjerg Allan
Published: February 2010

The effects of recombinant human growth hormone (rhGH) on biochemical markers of bone turnover and bone mineral content (BMC) were investigated in 20 normal male volunteers (aged 22-31 years) randomized to treatment for 7 days with either rhGH (0.1 IU/kg subcutaneously twice a day) or placebo. Serum somatomedin C rose during treatment (p < 0.001) but was not significantly different from baseline at day 14. The fasting urinary hydroxyproiine/creatinine (p < 0.001) and calcium/creatinine ratios (p < 0.01) increased during treatment and remained elevated for 4 and 2 weeks, respectively. Serum bone 7-carboxyglutamic acid-containing protein (BGP) increased during treatment (p < 0.001) and remained elevated for 6 months (p < 0.02). Serum bone alkaline phosphatase (B-AP), after an initial fall in the treatment period (p < 0.001), increased slightly in the following months (p < 0.01). In the rhGH group BMC was significantly higher than the prestudy value at day 14 (p < 0.05) but was unaltered at the end of study. The simultaneous increase in markers of bone resorption and formation during rhGH treatment followed by a decline in resorption parameters within a few weeks and the prolonged effect on BGP and B-AP demonstrate that rhGH treatment stimulates osteoblasts and activates bone remodeling.
Prevention of glucocorticoid-induced osteoporosis
Authors: Dr. Reid Ian R. , Schooler Beth A. , Stewart Alistair W.
Published: February 2010

There is increasing evidence that pamidronate and related compounds are effective in the prevention and treatment of osteoporosis. It is therefore of relevance to document the time course and mechanism of bis-phosphonate action in this condition. To this end, the present study describes the biochemical responses to prophylactic treatment with oral pamidronate (APD, 150 mg/day) in 16 glucocorticoid-treated patients and contrasts them with those in 19 steroid-treated control subjects. Measurements were made over a period of 12 months.

The treated patients showed a fall in urine hydroxyproline excretion at 6 weeks associated with a reduction in serum ionized calcium concentration, a rise in serum 1,25-(OH)2D3, and a nonsignificant rise in serum bone gla protein (BGP). In contrast to BGP, serum alkaline phosphatase activity declined at 6 weeks, falling further at 3 months. Between 3 and 12 months, BGP levels paralleled those of alkaline phosphatase and hydroxyproline, all these being significantly below their initial values, and the other parameters returned to baseline. There was a gradual increase in plasma phosphate concentrations in the treated group over the 12 month period.

It is concluded that pamidronate produces an acute and sustained inhibition of bone resorption followed by a more gradual reduction in bone formation. This transient dissociation results in a reduction in serum calcium, leading to a rise in serum 1,25-(OH)2D3, which in turn stimulates BGP production. Thereafter, indices of bone turnover remain subnormal but serum calcium and 1,25-(OH)2D3 return to baseline.
Long-term estrogen replacement therapy in postmenopausal women sustains vertebral bone mineral density
Authors: Moore Marisa , Bracker Mark , Sartoris David , Saltman Paul , Strause Linda
Published: February 2010

The cross-sectional relationship between long-term estrogen use and vertebral (L2-4) bone mineral density (BMD) was determined in 65 postmenopausal white women between 55 and 75 years who were at least 10 years from their menopause. Long-term estrogen users began therapy within 5 years of menopause and continued for a duration of at least 10 years. The mean duration of use was 19.8 years. Controls used estrogen for less than 1 year. There was a significant difference (p < 0.02) in mean spinal BMD between estrogen users (1.219 g/cm2) and controls (1.092 g/cm2). There was no significant difference in age, height, weight, or dietary calcium (Ca) intake between the two groups. The statistical difference in BMD was retained when (1) 23 estrogen users were paired with age-matched controls, (2) only women with a natural menopause or history of bilateral oophorectomy were included, and (3) only women with a natural menopause were compared. A spinal BMD below the estimated fracture threshold of 0.965 g/cm2 was found in 11 of 40 controls and only 2 of 25 estrogen users. Comparison of estrogen users with a natural menopause to those with bilateral oophorectomy revealed no significant difference in BMD. These data confirm the salutary effect of long-term estrogen use in the maintenance of vertebral bone mass in postmenopausal women.
Changes with age in the urinary excretion of lysyl- and hydroxylysylpyridinoline, two new markers of bone collagen turnover
Authors: Beardsworth Lindsay J. , Eyre David R. , Dickson Ian R.
Published: February 2010

Two intermolecular cross-linking amino acids, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), are promising markers in urine of collagen resorption because their levels in urine should reflect only collagen resorption and, unlike hydroxyproline, should not be influenced by degradation of either newly synthesized collagen molecules or noncollagenous proteins. Changes with age in the urinary excretion of HP and LP were studied in 24 h collections of urine from a group of 22 male and 27 female healthy subjects aged from 2 to 70 years. The pyridinolines were quantitated, utilizing their natural fluorescence, after resolution by reversed-phase HPLC. Levels of both pyridinolines were higher in children than in adults, but in adults no evidence of age or sex variations were observed except in the 20–30 year age group. Mean values of HP/Cr and LP/Cr in 37 adults (21–70 years) were 27.2 ± 1.9 and 8.8 ± 0.8 μol/mol, respectively; in the 12 children (2–15 years) the mean values were 14.4 and 12.4 times higher than the respective adult values. Making certain assumptions, the mean amount of bone resorbed in normal adults was tentatively estimated at 1.9 g per 24 h. The finding that differences between children and adults in these relatively specific markers were greater than with hydroxyproline suggests that hydroxyproline values may considerably underestimate the actual amount of bone turnover occurring in growing children or overestimate the adult turnover rate.
Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-β in human dentin
Authors: Dr. Finkelman Richard D. , Mohan Subburaman , Jennings John C. , Taylor Arch K. , Jepsen Soren , Baylink David J.
Published: February 2010

Human bone matrix is known to contain a battery of polypeptide growth factors. Since dentin is a mineralized tissue similar to bone in composition and perhaps in formation, human dentin was assayed for the presence of similar growth factors.

Root dentin proteins were extracted by demineralization in 4 M guanidine hydrochloride (Gu) and 30 mM Tris (pH 7.4) containing 20% EDTA and proteinase inhibitors. Gu-EDTA extracts were desalted and used for the following assays: (1) bone cell proliferation in chick calvarial cell mitogenic assay using the incorporation of [3H]thymidine into TCA-insoluble material; (2) osteocalcin by radioimmunoassay (RIA); (3) insulinlike growth factor I (IGF-I) by RIA; (4) skeletal growth factor/insulinlike growth factor II (SGF/IGF-II) by radioreceptor assay; and (5) transforming growth factor beta (TGF-β) by bioassay.

Gu-EDTA extracts stimulated bone cell proliferation. At 10 μg/ml, dentin proteins increased the incorporation of [3H]thymidine by calvarial cells to 320% of that by BSA-treated control cells. Consistent with the presence of mitogenic activity, growth factors were found in dentin in the following concentrations (ng/μg Gu-EDTA protein): (1) IGF-I, 0.06; (2) SGF/IGF-II, 0.52; and (3) TGF-β, 0.017. All three growth factors were present in concentrations lower than that found in human bone. Osteocalcin was detected at a concentration of 3.0 mg/g Gu-EDTA protein, also much lower than that in bone.
Regulation by calcium and 1,25-(OH)2D3 of cell proliferation and function of bovine parathyroid cells in culture
Authors: Ishimi Yoshiko , Russell John , Sherwood Louis M.
Published: February 2010

The effects of high calcium and 1,25-(OH)2D3 on parathyroid cell growth, PTH secretion, and steady-state levels of pre-proPTH mRNA in proliferating bovine parathyroid cells were examined. Cells were established in primary tissue culture and then tested in passages 2 and 5. Cell proliferation was suppressed by 10−9-10−7 M 1,25-(OH)2D3 but not by high calcium (2.5 mM). Cells at passages 2 and 5 were grown to subconfluence and then exposed for 72 h to 2.5 mM calcium or 10 −7 M 1,25-(OH)2D3. Pre-proPTH mRNA was decreased to approximately 50% of control by 2.5 mM calcium compared with 0.3 and 1.0 mM calcium. PTH secretion, as tested by low calcium stimulation for 1 h at the end of 72 h incubation, was inhibited by 50% in cells that had been exposed to high calcium compared with control. Incubation with 10−7 M 1,25-(OH)2D3 caused a decrease in the levels of pre-proPTH mRNA and PTH release to 50% of control at 72 h. These results suggest that cultured bovine parathyroid cells, at least in early passages, have responses to high calcium and l,25-(OH)2D3 similar to those in primary nonproliferating cultures studied earlier and that 1,25-(OH)2D3 inhibits the proliferation of parathyroid cells in a dose-responsive fashion.
Insulinlike growth factor 1 regulates mRNA levels of osteonectin and pro-α1(I)-collagen in clonal preosteoblastic calvarial cells
Authors: Thiebaud Daniel , Ng Kong Wah , Findlay David M. , Harker Michael , Prof. Martin T. John
Published: February 2010

A nontransformed rat clonal cell line (UMR-201) with phenotypic characteristics of osteoblastic precursor cells was found to respond to insulinlike growth factor 1 (IGF-1) by increased osteonectin and pro-α1 (1)-collagen mRNA expression. Cells were treated for 24 h with insulin, growth hormone, or IGF-1 to study the regulation of messenger RNA for osteonectin and pro-α1(I)-collagen using Northern blot hybridization.

UMR-201 cells possess specific high-affinity receptors for growth hormone, although there were no significant effects of growth hormone (10−9-10−7 M) or insulin (10−9-10−6 M) on mRNA species for osteonectin or pro-α(I)-collagen. However, IGF-1 increased both mRNA species from a concentration of 10−9 M. The effect on osteonectin mRNA expression was likely due to increased transcription; when 5′ flanking osteonectin (ON) genomic fragments were linked to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) and introduced by transfection into UMR-201 cells, the transcriptional activity of the ON-CAT construct was increased 235 and 270% by 10−8 and 10−7 M IGF-1, respectively. In contrast, growth hormone did not change the transcriptional activity of the ON-CAT construct.

In confirmation of other work, transforming growth factor β (TGF-β, 0.1-2.5 ng/ml) increased mRNA for osteonectin and pro-α1(I)-collagen in a dose-dependent manner. Transforming growth factor α (TGF-α) at 0.1-10 ng/ml had no consistent effects in repeated experiments on osteonectin and pro-α1(I)-collagen mRNA.

The positive stimulatory effect of both IGF-1 and TGF-β provides insights on the regulation of bone matrix proteins and suggests a major role of these growth factors in the remodeling process of bone.
Failure of isolated rat tibial periosteal cells to 5α reduce testosterone to 5α-dihydrotestosterone
Authors: Turner Russell T. , Bleiberg Batia , Colvard Douglas S. , Keeting Philip E. , Evans Glenda , Spelsberg Thomas C.
Published: February 2010

Periosteal cells were isolated from tibiae of adult male rats after collagenase treatment. Northern blot analysis of total cytoplasmic RNA extracted from the isolated periosteal cells was positive for expression of genes encoding the osteoblast marker proteins osteocalcin (BGP) and pre-pro-α2(I) chain of type 1 procollagen. The isolated periosteal cells were incubated with 1 nM [3H]testosterone ([3H]T) for up to 240 minutes and the reaction products separated by high-performance liquid chromatography. [3H]5α-dihydrotestosterone ([3H]DHT) was not detected in extracts of periosteal cell incubations. In contrast, [3H]DHT was produced in a time-dependent manner by cells from seminal vesicles. These results suggest that testosterone 5α-reductase activity is not expressed by osteoblasts in rat tibial periosteum and that the anabolic effects of androgens in this tissue are not mediated by locally produced DHT.
Regulation of 25-hydroxyvitamin D3 metabolism in cultures of osteoblastic cells
Authors: Letton Robert W. , Fanti Paolo , Malluche Hartmut H.
Published: February 2010

This study was designed to investigate the mechanisms involved in the regulation of the conversion of 25-hydroxyvitamin D3 (25-OHD3) to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] in primary cultures of osteoblastlike cells from neonatal mouse calvariae. These cells, when incubated with tritiated 25-OHD3 ([3H]25-OHD3), spontaneously synthesized [3H]24,25-(OH)2D3 20-50 times more efficiently than [3H]1,25-(OH)2D3 at a rate of conversion that was substrate dependent and linear from 1 to 36 h. Gas chromatography-mass spectrometry verified the identity of the dihydroxylated metabolites. The calcium ionophore A23187 (5 μM) consistently stimulated the synthesis of 1,25-(OH)2D3 while suppressing the production of 24,25-(OH)2D3. This effect was sustained for 36 h and was dose dependent for concentrations from 0.05 to 10 μM. Furthermore, A23187 stimulated cAMP production and indomethacin (50 ng/ml) blocked the A23187-induced production of cAMP and 1,25-(OH)2D3 but had no effect on the suppression of 24,25-(OH)2D3 by A23187. This led to other experiments to find out whether the stimulative effect of A23187 on 1,25-(OH)2D3 synthesis is mediated by prostaglandins or cAMP, or both. PGE2 (10−8-10−6 M) increased the production of 1,25-(OH)2D3 and of 24,25-(OH)2D3. Forskolin (0.01-10 μM) and dibutyryl cAMP (0.1-10 mM) increased the production of both metabolites but to a lesser degree than PGE2. These data suggest that osteoblastlike cells are stimulated by A23187 to increase the synthesis of 1,25-(OH)2D3 through mechanisms involving prostaglandins and cAMP. The synthesis of 24,25-(OH)2D3 is suppressed by A23187 through different mechanisms.
Subclones of a rat parathyroid cell line (PT-r): Regulation of growth and production of parathyroid hormone-related peptide (PTHRP)
Authors: Sakaguchi Kazushige , Ikeda Kyoji , Curcio Francesco , Aurbach Gerald D. , Brandi Maria Luisa
Published: February 2010

Four subclones from a rat parathyroid cell line (PT-r cell) have been isolated, and morphological and functional characteristics have been examined. Subclones 1 and 2 display a polygonal shape, show growth and secretory responses to calcium (half-maximal suppressions at 1.2 and 1.7 mM, respectively), and respond to secretin with cAMP production (14.5-fold and 16.9-fold over basal) and hormone secretion (41 and 58% over basal). Subclone 4 is elongated in form and does not respond to calcium or secretin. Subclone 3 shows mixed morphology, elongated and polygonal shapes, with moderate response to calcium (half-maximal suppression at 1.7 mM) and secretin (cAMP, 3.2-fold increase and hormone secretion, 50% increase over basal). The clones were tested for content of messenger RNA (mRNA) representing parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP). Only PTHRP mRNA was found. The peptide released is virtually all PTHRP. PTH mRNA was not detected even with a sensitive RNA probe. The amount of mRNA for PTHRP closely paralleled the amount of PTH-like bioactivity released into the medium from each clone (144.7 ± 12.1, 110.0 ± 12.9, 68.0 ± 5.6, and 39.9 ± 2.4 pgEq of rat PTH-(-34) per 107 cells per 12 h in a medium with 0.7 mM ionized calcium, from subclones 1, 2, 3, and 4, respectively). Culture conditions, low-density passage (less than 1:50 split ratio) or high-density passage (greater than 1:10 split ratio), affected morphology and function of the clones 1 and 2. They became elongated and functionally dedifferentiated like subclone 4 after 3 months of high-density culture. Upon dedifferentiation, morphological and functional properties were not reversible with high calcium, 1,25-(OH)2D3, or retinoic acid. These clones may be useful for studies of phenotypic expression, calcium regulation, and gene expression and interrelationships among these functions.
Rapid publication a chromogranin a-derived peptide differentially regulates the secretion of calcitonin gene products
Authors: Dr. Deftos Leonard J. , Hogue-Angeletti Ruth , Chalberg Cheryl , Tu Su
Published: February 2010

We studied the regulation of the secretion of CT and CGRP by chromogranin A (CgA)-derived peptides in a human lung tumor cell line. The amino-terminal peptide of CgA, CgA1-40, stimulated the secretion of CGRP and inhibited the secretion of CT; both effects occurred in a dose-dependent manner. These studies demonstrate a differential effect of a CgA-derived peptide on two products of the CT gene, CT itself and CGRP. CgA may be processed at its multiple dibasic sites to peptides that specifically regulate the secretion of its coresident hormones, in this case two calcitonin gene products that are present in the same secretory vesicle. This novel mechanism represents a new pathway for the control of calcium regulating hormones.
Metabolic modulation of disuse osteopenia: Endocrine-dependent site specificity of bone remodeling
Authors: Bain Steven D. , Rubin Clinton T.
Published: February 2010

The remodeling response of bone tissue to disuse in four normal adult male turkeys and four adult males metabolically altered by castration was compared by functionally isolating the left ulna of each animal via transverse epiphyseal osteotomies. The right ulna in each animal was left intact and served as a control. After 8 weeks, the animals were euthanized, the ulnae harvested, and 100 μm undecalcified cross sections of the midshaft microradiographed. Areal properties, osteon mineral apposition rates from in vivo fluorochrome labels, and the number and ratios of bone-forming and bone-resorbing foci were quantitated. Compared to their control ulnae, the magnitude of bone resorbed from the functionally isolated ulnae of normal versus castrated males was not significantly different (-12.8 ± 3.7 versus −10.7 ± 3.5%, respectively). However, in the functionally isolated ulnae of normal birds, 94% of the total bone loss resulted from expansion of the corticoendosteal envelope, and 97% of the decrease in cross-sectional areas of the ulnae in the castrated birds was due to intracortical porosity. Furthermore, there was a significant interaction between disuse and castration, increasing the total number of intracortical remodeling events (9.4 ± 0.9) when compared to disuse alone (4.7 ± 1.4, p < 0.01), or to the intact ulnae of castrated (2.1 ± 0.5) and normal adult males (2.0 ± 1.1). This work emphasizes that the manner in which the bone tissue responds to local changes in its physical environment is directly dependent on the status of the organism's metabolic milieu.
Differential modulation of growth and phenotypic expression of chondrocytes in sparse and confluent cultures by growth factors in cartilage
Authors: Hiraki Yuji , Inoue Hiroyuki , Asada Akira , Suzuki Fujio
Published: February 2010

The growth-promoting actions of cartilage extracts (CE) on rabbit cultured chondrocytes were studied to assess the role of locally acting growth factors in the generation and expansion of highly differentiated cells. In the present study, DNA synthesis and proteoglycan synthesis in the cultured chondrocytes were monitored by flow cytofluorometry and double-isotope autoradiography by using [3H]thymidine and [35S]sulfate. We report here that actions of the same set of growth factors extracted from cartilage evokes differential cellular responses depending upon cell density. Growth factors in the optimal dose of CE (2 μg/ml) strongly supported proliferation of the cells in sparse culture, but they had little effect on cells in confluent culture. In contrast, the optimal dose of fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 40 ng/ml) did not reveal such a cell density-dependent effect on cellular proliferation. However, growth factors in CE induced proteoglycan synthesis selectively in nonproliferating and expressing cells in confluent culture.
Influence of calcium load on absorption fraction
Authors: Heaney Robert P. , Weaver Connie M. , Lee Fitzsimmons Mary
Published: February 2010

True calcium absorption was studied as a function of the size of the ingested load in healthy adult women, under meal conditions and at loads ranging from 15 to 500 mg calcium. Fractional absorption was highly inversely correlated with the logarithm of load (P < 0.001). At the lowest loads, absorption averaged 64.0% and at the highest, 28.6%. The parameters of the best-fit relationship permit reasonably precise calculation of the impact of various calcium dosing and dietary strategies.
Bone cell responsiveness to transforming growth factor β, parathyroid hormone, and prostaglandin E2 in normal and postmenopausal osteoporotic women
Authors: Lomri Abderrahim , Marie Pierre J.
Published: February 2010

We have shown previously that the decreased trabecular bone formation in osteoporotic postmenopausal women results from a reduced ability of osteoblastic cells to proliferate. In this study we have tested the possibility that bone cells from osteoporotic women with low bone formation have an abnormal responsiveness to hormonal or local mitogenic factors. Primary cultures of bone cells with osteoblastic characteristics were obtained by migration from the trabecular bone surface in osteoporotic postmenopausal women with high (n = 7) or low (n = 7) bone formation as evaluated histomorphometrically by the extent of double tetracycline-labeled surface (DLS). Control bone cells were obtained under identical conditions from eight normal age-matched postmenopausal women. Parameters of osteoblastic differentiation (alkaline phosphatase activity and osteocalcin production) were found to be normal and similar in bone cells from osteoporotic women with low or high DLS. In contrast, cell replication as evaluated by [3H]thymidine into DNA was 3.4-fold lower in the low DLS group compared to the high DLS group, confirming our previous findings. Treatment of quiescent bone cells with TGF-β (0.5–1 ng/ml) for 24 h significantly stimulated DNA synthesis in osteoblastic cells from normal women and in bone cells from osteoporotic patients with low or high DLS, indicating a normal responsiveness to TGF-β in these patients. We have compared the effect of parathyroidhormone (PTH) on bone cells from normal and osteoporotic women. Basal cAMP levels and the cAMP accumulation in response to (1–34)-hPTH were similar in bone cells from patients with low or high DLS and were not different from normal values. The responsiveness of bone cells from osteoporotic women to exogenous prostaglandin E2 (PGE2) was also evaluated. PGE2 (24 h) produced a dose-related biphasic effect on DNA synthesis in bone cells from both normal and osteoporotic weomen. At low concentration (10−11 M) PGE2 increased DNA synthesis whereas at higher concentration (10−7 M) it was inhibitory. cAMP production was increased by PGE2 at doses that inhibited DNA synthesis. The responsiveness to PGE2 was not different in normal bone cells and in cells from osteoporotic women with low and high DLS. These results indicate that the reduced bone cell proliferative capacity in osteoporotic postmenopausal women with low bone formation does not result from a lower than normal responsiveness to TGF-β, PTH, and PGE2.
Presystemic 24-hydroxylation of oral 25-hydroxyvitamin D3 in rats
Authors: Dr. Vieth Reinhold
Published: February 2010

The metabolism of 25-hydroxyvitamin D3 (25-OHD3) was compared following its intracardial or gastric administration. The rats were deprived of calcium and vitamin D. A mixture of radiolabeled (0.3 μCi) and stable (2 μg) 25-OHD3 was given as a single dose. After 24 h the rats given the dose by gastric tube had significantly lower serum concentrations of 25-OHD3 and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] than those injected intracardially. In contrast, serum 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] was much higher in the rats given the 25-OHD3 dose by gastric tube (6.2 nmol/liter ±1.3 SD, n = 7) compared to the intracardial group (0.9 nmol/liter ± 0.5, p < 0.001). The preceding results were based on specific radioactivity of metabolites. The same findings were obtained by reanalyzing the samples using conventional competitive binding assays for 25-OHD3, 1,25-(OH)2D3, and 24,25-(OH)2D3. The results show that orally administered 25-OHD3 is partly metabolized to 24,25-(OH)2D3 presystemically.
Desensitization of parathyroid hormone receptors on cultured bone cells
Authors: Dr. Pun Kin-Kee , Ho Patricia W. M. , Nissenson Robert A. , Arnaud Claude D.
Published: February 2010

Administration of excessive amounts of parathyroid hormone (PTH) in the treatment of osteoporosis can reverse the beneficial effects of a low-dose, intermittent regime. To investigate the direct actions and the possible cellular mechanisms of PTH in inducing desensitization of PTH receptors, we studied the effects of desensitization on rat osteoblastic UMR-106 cells. When the osteoblasts were preincubated with bPTH-(1–34), complete refractoriness to a subsequent challenge with the hormone developed within 1 h and at hormone concentrations as low as 5 nM. When osteoblasts thus desensitized were incubated in hormone-free medium, recovery of the cAMP responses began within 2 h and reached maximum after 16 h. Cycloheximide did not affect the process of desensitization. [NIe8, NIe18, Tyr34]bPTH-(3–34)amide significantly impaired the desensitization process by PTH-(1–34) but did not have stimulatory effect on cAMP responses. No significant heterologous desensitization was obvious after preincubation with isoprenaline (50 μM), prostaglandin E, (50 μM), or prostaglandin E2 (50 μM) for 2 h. Binding experiments with [125I]PLP-(1–36)amide after desensitization revealed that there was an approximate twofold decrease in receptor affinities as analyzed by Scatchard analysis, showing that the decrease in affinity was prominent in the process of desensitization. When the cells were treated with monensin during desensitization, PTH challenge after desensitization produced significantly lower cyclic AMP responses. Recovery after desensitization occurred over a period of 16 h. Inclusion of monensin, but not cycloheximide, impaired the recovery. The results show that homologous desensitization of rat osteoblasts to PTH is brought about by the occupancy of receptors by PTH-(1–34) but not by cAMP generation itself. It is not primarily dependent on protein synthesis or on activation of adenylate cyclase. A decrease in receptor affinity is important in the process of desensitization. Desensitization of parathyroid hormone receptors is potentiated and recovery impaired by monensin, an inhibitor of internalization and recycling, showing that both processes are important in the regulation of PTH receptors.
Identification of Ca2+-activated K+ channels in cells of embryonic chick osteoblast cultures
Authors: Ravesloot Jan H. , van Houten Ron J. , Ypey Dirk L. , Nijweide Peter J.
Published: February 2010

Primary cultures of embryonic chick osteoblasts consist of a heterogeneous cell population. Patch clamp measurements were done on 1- to 5-day-old osteoblasts, osteocytes, fibroblastlike cells, and cells that could not be classified on morphologic criteria. The measurements showed the omnipresence of depolarization-activated high-conductance channels in cell-attached patches. The whole-cell experiments showed an outward rectifying conductance activating at positive membrane potentials. Channels underlying the latter conductance were found to be K+ conducting in outside-out membrane patches. The activation potential of the outward rectifying K+ conductance shifted to negative membrane potentials upon increasing the intracellular Ca2+ concentration within the range of 10−8–10−3.2 M. The same happened with the activation potential of the K+ channels found in outside-out patches. Finally, inside-out patch experiments directly demonstrated the dependency of the activation potential of K+ channels on Ca2+ ions. Thus the identity and main characteristics of Ca2+-activated K+ channels expressed by the various cell types present in chick osteoblast cultures have now been established. Decreased input resistances were found in cells of cultures more than 2 days old. This is consistent with the establishment of electrical coupling between the cells. Functions in which Ca2+-activated K+ channels could play a role are discussed.
Proportion of human vertebral body bone that is cancellous
Authors: Eastell Richard , Mosekilde Li , Hodgson Stephen F. , Dr. Riggs B. Lawrence
Published: February 2010

The concept that vertebral fractures are caused by excessive loss of cancellous bone has been challenged by a recent study (J Bone Min Res 2:221, 1987) suggesting that vertebral bodies are composed mainly of cortical bone rather than cancellous bone. To resolve disagreement we used two independent methods to quantify the proportions of cortical and cancellous bone in 400 μm thick sections of the bodies of the second lumbar vertebrae from six men (aged 21–58 years) and seven women (aged 25–58 years). Based on the ash weight of the manually dissected components, 80% of the total bone in men and 72% in women was cancellous bone. Based on computer-assisted scanning of sections with a microdensitometer, 81% of the total bone in men and 71% in women was cancellous bone. We conclude that the traditional concept is correct: the vertebral body is composed mainly of cancellous bone.
The effects of extracellular pH on bone resorption by avian osteoclasts in vitro
Authors: Walsh C.A. , Dawson W.E. , Birch M.A. , Gallagher J.A.
Published: February 2010

It has previously been reported that low extracellular pH stimulates the excavation of resorption lacunae by rodent osteoclasts in vitro. Using avian bone cells in a similar in vitro assay we have demonstrated that osteoclast activity is optimal at pH 7.20–7.40 and is inhibited at extremes of pH (<7.10 and >7.60). Over the first 24 h of incubation at low pH there may be an increase in osteoclastic resorption but to a lesser extent than that reported for rodent cells. However, after 24–30 h in culture there is little or no further increase in bone resorption, presumably due to a cytotoxic effect of low pH acting either on the osteoclast directly or via nonosteoclastic bone cells. In contrast to a previous report, in which preincubation of wafers for 24 h had no effect on bone resorption, we found that preincubation of bone substrates at pH 6.50 for longer periods enhances subsequent resorption at pH 7.20.
Effects of testosterone and venesection on spinal and peripheral bone mineral in six hypogonadal men with hemochromatosis
Authors: Dr. Diamond Terrence , Stiel Daniel , Posen Solomon
Published: February 2010

To measure the effect of testosterone replacement and venesection on spinal and peripheral bone mineral we prospectively studied six hypogonadal men and six eugonadal men with idiopathic hemochromatosis for 24 months. Venesections were performed every week on all patients, and intramuscular injections of testosterone were administered every 3 weeks to the hypogonadal men only. Bone mineral was measured by quantitative computed tomography in the spine and by single-photon absorptiometry in the forearm.

During the 24 month period of observation serum testosterone concentrations and serum ferritin levels became normal. In the hypogonadal men mean lumbar spine bone mineral increased by 13.1 ± 4.9% (95% CI, 0.5–25.6) and mean forearm bone mineral increased by 4.7 ± 3.8% (95% CI, −5.1 to 14.6). In contrast in the eugonadal men treated over the same period, mean lumbar spine bone mineral decreased by 3.5 ± 2.8% (95% CI, −10.9 to 3.8, P < 0.01) and mean forearm bone mineral remained virtually unchanged (0.07 ± 0.9%; 95% CI, −1.7 to 3.1, P < 0.05).

These data suggest that bone mineral increases in the lumbar spine and in the forearm in hypogonadal men with hemochromatosis treated by testosterone replacement and venesection.
Lower serum osteocalcin in ethanol-fed rats
Authors: Peng Tai-Chan , Lian Jane B. , Hirsch Philip F. , Kusy Robert P.
Published: February 2010

Serum osteocalcin was remarkably and significantly (-34 and −41% in two separate experiments; p < 0.001) lower in rats fed an 8% (w/v) ethanol liquid diet (ELD) for 1 week than in rats fed an isocaloric control liquid diet (CLD). In a longer experiment that spanned 4 weeks, the ELD rats were given 6% ethanol on day 4, increased stepwise to 8% by day 9, and then maintained at 8% until day 28, when the experiment was terminated. Again, serum osteocalcin was much lower (-32%, p < 0.001) in the ELD-fed rats than in CLD-fed rats. Even in rats fed only a 6% ELD for 12 days, serum osteocalcin was lower (-33%, p < 0.001) than in controls. Also, the femora were weaker, more compliant, and more ductile in ELD- than in CLD-fed rats, findings that confirmed our earlier, related work. The fall in serum osteocalcin in ELD-fed rats is associated with a fall in femur ash weight and bone strength. There were significant correlations between serum osteocalcin and bone strength (r = 0.80; p < 0.001) and between serum osteocalcin and bone stiffness (r = 0.83; p < 0.001). Serum ionized calcium, like osteocalcin, was consistently lower in rats given ethanol for 1 or 4 weeks than in controls. From these experiments we conclude that excessive ethanol consumption inhibits osteoblastic activity as indicated by the reduced serum osteocalcin. The inhibition is also associated with other deleterious effects of ethanol on bone, including ash weight, bone strength, and bone stiffness. These results in rats suggest that serum osteocalcin may be a useful biologic marker of bone status in alcoholic patients.
Human osteoblastlike cells do not respond to interleukin-6
Authors: Littlewood A.J. , Aarden L.A. , Evans D.B. , Russell R.G.G. , Gowen M.
Published: February 2010

Interleukin 6 (IL-6) exerts well-established effects on cells of the immune system as well as on various other cell types. It has been implicated in the control of connective tissue cells in such conditions as rheumatoid arthritis and osteoporosis. We have investigated the effects of recombinant human interleukin-6 (rhIL-6) on human osteoblastlike cells derived from explants of trabecular bone. ROS 17/2.8 cells were used as an additional osteoblastlike cell model system.

We were unable to identify any effects of rhIL-6 (5–5000 pg/ml) on the proliferation, alkaline phosphatase activity, osteocalcin production, or release of cytokines or prostaglandins by either osteoblastlike cell model system. Since we have shown previously that these cells release IL-6 in culture, we used a sheep anti-human IL-6 antibody to investigate the possibility that (1) action of added exogenous IL-6 could be masking endogenous production, and (2) endogenous IL-6 may regulate the effects of osteotropic agents on the osteoblastlike cells. Presence of the antibody exerted no detectable effects on 1,25-(OH)2D3-stimulated alkaline phosphatase or on proliferation or TNF production enhanced by IL-1. Thus IL-6 does not appear to be involved in the regulation of osteoblast activity.
Chicken parathyroid hormone-related protein and its expression during embryologic development
Authors: Schermer D.T. , Chan S.D.H. , Bruce R. , Nissenson R.A. , Wood W.I. , Dr. Strewler G.J.
Published: February 2010

A parathyroid hormone-related protein (PTHrP) is the probable cause of humoral hypercalcemia in malignancy, but its normal physiologic role remains unknown. Since current evidence suggests that PTHrP may have a role in embryonic development, we cloned a genomic fragment that encodes chicken PTHrP (cPTHrP) and studied the expression of PTHrP in developing chick embryos. Blot hybridization of chicken genomic DNA with a cPTHrP genomic DNA probe showed a single band, suggesting that a single-copy gene encodes cPTHrP. By screening a chicken genomic library with the human probe an open reading frame was identified that corresponds to the human PTHrP (hPTHrP) exon IV. Compared to the human sequence the 5′ splice junction is highly conserved and the two predicted propeptide residues are identical. The sequence predicts a mature peptide of 139 amino acids; all of the first 21 and 94 of the first 112, but only 8 of the final 27 residues of cPTHrP are identical to the human sequence. The structural features required for PTH receptor binding and activation are highly conserved between chicken and hPTHrP. Poly(A)-enriched RNA from 3–15 day chicken embryos was surveyed by hybridization to the chicken probe. A hybridizing band of 1.45 kb was found in tissues derived from all three germ layers, including brain, heart, lung, liver, gizzard, intestine, chorioallantoic membrane, yolk sac, and skeletal muscle. An additional 1.2 kb hybridizing band was found in some tissues. The conservation of the PTHrP sequence between chicken and mammals supports the view that PTHrP has an important physiologic role. The presence of PTHrP mRNA in early embryos suggests that this role may be in embryonic development.
Intertissular variations in osteonectin: A monoclonal antibody directed to bone osteonectin shows reduced affinity for platelet osteonectin
Authors: Malaval Luc , Darbouret Bruno , Preaudat Corinne , Jolu Jean-Pierre , Dr. Delmas Pierre D.
Published: February 2010

Osteonectin, a major noncollagenous protein of bone, is also synthesized and secreted by various nonmineralized tissues and by platelets. To establish whether there are structural specificities of osteonectin according to its tissular origin, we raised 12 monoclonal antibodies against bovine bone osteonectin and screened them for their ability to recognize bone and platelet osteonectin. When hybridoma culture media were radio-immunoassayed all MAbs showed the same titer for [125I]human platelet osteonectin and for [125I]bovine bone osteonectin, except MAb 2, which poorly bound platelet osteonectin. Immunoprecipitation and immunoblotting experiments were performed on human bone protein extracts and on material secreted by human platelets upon thrombin stimulation; in these experiments MAb 2 recognized human bone osteonectin and only faintly human platelet osteonectin. A “sandwich” immunoradiometric assay was devised in which osteonectin bound to a solid phase by a first MAb was recognized by a 125I-labeled second MAb. In this assay MAb 2, used as a tracer, showed a 100-fold lower affinity for purified human platelet osteonectin than for purified human bone osteonectin. These results suggest the existence of structural variations in osteonectin obtained from bone and platelets. Whether these variations result from differences in sequence, post-translational processing, or postsecretional fate remains to be established.
The bisphosphonate alendronate (MK-217) inhibits bone loss due to ovariectomy in rats
Authors: Seedor J. Gregory , Quartuccio Helen A. , Thompson David D.
Published: February 2010

Estrogen deficiency in mammals is known to increase bone turnover and result in reduced bone mass. The bisphosphonate, 4-amino-1-hydroxybutylidene-1,1-bisphophonic acid disodium salt, alendronate (MK-217), is a potent inhibitor of bone resorption and was evaluated in this study for its ability to inhibit bone loss following ovariectomy in rats. Alendronate was administered sc in doses of 0.0, 0.056, 0.28, 1.40, and 7.0 mg P/kg/month, divided into two, four, or eight monthly subcutaneous injections for each dose, to female Sprague-Dawley rats (250-280 g) that underwent bilateral ovariectomy. Rats were sacrificed 12 weeks postovariectomy, the femora ashed, and the tibiae prepared for static and dynamic histomorphometric analyses.

Femoral bone mass in vehicle-treated rats was reduced by 12% 12 weeks after ovariectomy compared to the nonovariectomized control group. In MK-217-treated rats femoral bone mass was significantly increased in a dose-dependent manner compared to either ovariectomized or nonovariectomized controls. Histomorphometric analysis showed significant increases in tibial trabecular bone volume with no decrease in osteoclast number. Doses delivered twice per month or eight times per month were equally effective in achieving the peak bone volume 12 weeks after ovariectomy.

In conclusion, alendronate (MK-217) was effective in inhibiting bone loss due to estrogen deficiency in rats, and the magnitude of its effect was related primarily to the total amount of compound administered rather than the frequency of its administration.