bluuebunny/crossref_metadata_2025 huggingface dataset:
Three-dimensional type I collagen gel system for the study of osteoblastic metastases produced by metastatic prostate cancer
Authors: Koutsilieris M. , Sourla A. , Pelletier G. , Doillon C.J.
Published: February 2010

We developed a three-dimensional type I collagen gel cell culture system that allows coculturing of human MG-63 osteoblast-like cells and various human cancer cells. Inoculation of human PC-3 metastatic prostate cancer cells into this type I collagen gel containing human MG-63 osteoblast-like cells produced an osteoblastic-like reaction that presented as an increased number of MG-63 cells and increased density of type I collagen around MG-63 cells adjacent to inoculated PC-3 cells by microscope analysis. Under identical experimental conditions, inoculation of cell-free medium, human KLE endometrial adenocarcinoma cells, and Calu-1 lung cancer cells did not produce this blastic-like reaction. In situ hybridization documented the uniform expression of insulin-like growth factor I (IGF-I) and of urokinase-type plasminogen activator (uPA) mRNA in MG-63 and PC-3 cells separately cultured in this substrata. The uniform expression of uPA was also documented by immunocytochemistry using a monoclonal and a polyclonal antihuman uPA antibody. The relative expression of uPA was higher in PC-3 cells than in MG-63, KLE, and Calu-1 cancer cells. We conclude that this novel cell culture system may become a useful model to study the pathophysiology of the osteoblastic reaction in vitro.
Posture, age, menopause, and osteopenia do not influence the circadian variation in the urinary excretion of pyridinium crosslinks
Authors: Schlemmer Annette , Hassager Christian , Pedersen Brian J. , Christiansen Claus
Published: February 2010

This study was performed to investigate whether the circadian variation in urinary pyridinium crosslinks is related to physical activity, age, the menopause, and asymptomatic osteopenia. We measured urinary pyridinoline/creatinine (Pyr/Cr) and deoxypyridinoline/creatinine (D-Pyr/Cr) in 9 healthy premenopausal women in two 27 h studies, before and at the end of 5 days of total bed rest. Both Pyr/Cr and D-Pyr/Cr showed highly significant circadian variations, with the peak at night and the nadir during the day (p < 0.001). The 5 days of complete bed rest produced no changes in the circadian pattern, but a general increase of 28% was observed in pyridinium crosslinks. A group of 12 healthy, early postmenopausal women (aged 55 + 2 years), 12 healthy, elderly postmenopausal women (aged 73 + 1 years), and 12 elderly osteopenic but otherwise healthy women (aged 73 + 1 years) were also studied for 27 h. All three groups showed highly significant (p ≤ 0.001) circadian variations in the urinary excretion of pyridinium crosslinks. As expected, both Pyr/Cr (p < 0.05) and D-Pyr/Cr (p < 0.001) increased at the time of menopause, but the circadian variations in Pyr/Cr and D-Pyr/Cr were similar in all groups studied. We conclude that the circadian variation in the urinary excretion of pyridinium crosslinks is independent of physical factors. Furthermore, the circadian variation in pyridinium crosslinks was not related to age, menopausal status, or asymptomatic osteopenia.
Fractal organization of trabecular bone images on calcaneus radiographs
Authors: Dr. Benhamou C.L. , Lespessailles E. , Jacquet G. , Harba R. , Jennane R. , Loussot T. , Tourliere D. , Ohley W.
Published: February 2010

Bone density is not the unique factor conditioning bone strength. Trabecular bone microarchitecture also plays an important role. We have developed a fractal evaluation of trabecular bone microarchitecture on calcaneus radiographs. Fractal models may provide a single numeric evaluation (the fractal dimension) of such complex structures. Our evaluation results from an analysis of images with a varying range of gray levels, without binarization of the image. It is based on the fractional brownian motion model, or more precisely on the analysis of its increment, the fractional gaussian noise (FGN). The use of this model may be considered validated if two conditions are fulfilled: the gaussian repartition and the self-similarity of our data. The gaussian repartition of intermediate lines of these images was tested on a sample of 32,800 lines from 82 images. Following a chi-square goodness-of-fit test, it was checked in 86% of these lines for α = 0.01. The self-similarity was tested on 20 images by two estimators, the variance method of Pentland and the spectrum method of Fourier. Self-similarity is defined by lined-up points in a log-log plot of the FGN spectrum or of the variance as a function of the lag. We found two self-similarity areas between scales of analysis ranging from 105 to 420 μm, then above 900 μm, where linear regression produced high mean correlation coefficients (r ≥ 0.97). Following this validation, we studied the reproducibility of this new technique. Intra- and interobserver reproducibility, influence of transferring the region of interest, and long-term reproducibility were assessed and given CV of 0.61 ± 0.15, 0.68 ± 0.47, 0.53 ± 0.16, and 2.07 ± 0.84%, respectively. These data have allowed us to validate the use of this fractal model by checking the fractal organization of our radiographic images analyzed by the model. The good reproducibility of successive x-rays in the same subject allows us to undertake population studies and to envisage longitudinal series.
Bone morphogenetic protein 2 increases insulin-like growth factor I and II transcripts and polypeptide levels in bone cell cultures
Authors: Canalis Ernesto , Gabbitas Bari
Published: February 2010

Insulin-like growth factors (IGF) I and II are among the most prevalent growth factors secreted by bone cells and are presumed to act as autocrine regulators of bone formation. Certain growth factors, synthesized by skeletal cells and known to stimulate the replication but not the differentiated function of cells of the osteoblastic lineage, have been shown to inhibit skeletal IGF-I and II synthesis. We postulated that growth factors with limited mitogenic activity and with differentiation-inducing properties, such as bone morphogenetic protein (BMP) 2, have the opposite effect and enhance IGF-I and II synthesis. We tested the effects of BMP-2 on IGF-I and II mRNA expression and polypeptide concentrations in cultures of osteoblast-enriched (OB) cells from 22 day fetal rat calvariae. Steady-state IGF-I and II mRNA levels were determined by northern blot analysis, and IGF-I and II concentrations were determined in acidified and fractionated culture medium by a specific radioimmunoassay. After 24-48 h of treatment, BMP-2 at 3.3 nM increased IGF-I and II transcripts by up to twofold and polypeptide levels by up to fourfold. BMP-2 was a more potent stimulator of IGF-II synthesis, and it was active at doses as low as 0.03 nM for IGF-II mRNA and 0.3 nM for IGF-II protein, whereas a dose of 3.3 nM was required to observe the effect on IGF-I synthesis. The effects of BMP-2 on IGF-I and II transcripts and polypeptide levels were dependent on protein synthesis and decreased in the presence of cycloheximide at 3.6 μM. In conclusion, BMP-2 increases skeletal IGF-I and II synthesis by increasing IGF-I and II transcript levels, and this effect may contribute to its actions on selected aspects of OB cell differentiated function.
Alterations in calcium intake on peak bone mass in the female rat
Authors: Peterson Catherine A. , Eurell Jo Ann C. , Erdman John W.
Published: February 2010

This study compared the effect of a calcium deficit or surfeit on bone growth and development in the early phase of peak bone mass attainment with the late phase of peak bone mass attainment using the female Sprague-Dawley rat as a model. Groups of weanling animals were fed one of three nutritionally complete but calcium-altered diets (0.25%, 0.5%, or 1.0% calcium) for 8 weeks. Animals within each diet group were then rerandomized into one of the above diets and fed until 37 weeks of age. Each group contained five rats. In addition, three groups that received the 0.25% calcium diet for the first 8 weeks remained on the diet until week 20 when they were further randomized into one of the three diet groups and fed until 37 weeks of age. Results of this experiment indicate that increasing the calcium intake after adolescence (12-weeks-old) of those female rats consuming a low calcium diet will not substantially alter the adult bone volume of the metaphyseal region of the proximal tibia. Further, low calcium intakes through adolescence retard and prolong longitudinal bone growth. In contrast, however, those rats fed a diet providing calcium either at (0.5%) or twice the National Research Council's requirement level through adolescence had greater tibial bone volume as an adult when fed diets containing 1.0% calcium after this time period. It appears that the mechanism for this increase involves both a protection from resorption and an increase in bone formation/mineralization. This study is the first to show that low calcium intakes through adolescence have a nonreversible, deleterious effect on peak bone mass, whereas higher intakes promote greater peak bone mass and provide potential protection from age-related bone loss.
AzaThioprine alone is bone sparing and does not alter cyclosporin A-induced osteopenia in the rat
Authors: Bryer Haldon P. , Isserow Jonathan A. , Armstrong Eamon C. , Mann Gary N. , Rucinski Boguslaw , Buchinsky Farrel J. , Romero David F. , Epstein Solomon
Published: February 2010

The immunosuppressant agent cyclosporin A (CsA) induces a high turnover osteopenic state, while the effect on bone of the antimetabolite azathioprine, a drug often used in conjunction with CsA in transplant patients, is less clear. This study was therefore designed to investigate the outcome of azathioprine administration, with reference to CsA, on bone mineral metabolism using the rat model. Four groups of 10-week-old male Sprague-Dawley rats (12 per group) were randomly allocated to receive by daily gavage for a 28-day period: (1) no treatment (control group); (2) azathioprine 1.5 mg/kg bw; (3) CsA 15 mg/kg bw; and (4) a combination of azathioprine and CsA, as described above. Rats were weighed and blood assayed serially for osteocalcin, ionized calcium, 1,25-dihydroxyvitamin D (1,25(OH)2VitD), and parathyroid hormone (PTH). Tibiae were removed following sacrifice on day 28 after double calcein labeling for histomorphometric analysis. Immunosuppressant groups were compared with nontreated control. We confirmed our previous findings that CsA induces a state of high turnover bone loss which is accompanied by a diminished gain in body weight (p < 0.01) and elevated serum osteocalcin (p < 0.001) and 1,25(OH)2VitD levels (p < 0.001). Azathioprine treatment alone did not alter ionized calcium, 1,25(OH)2VitD, or PTH levels. However, there was biochemical evidence of impaired osteoblastic activity as seen by decreased osteocalcin values on days 14 and 28 (p < 0.001). Azathioprine caused no loss of bone volume nor any deviation from the norm in mineral apposition rate, bone formation rate, or longitudinal bone growth. All three treatment groups showed an increased recruitment of osteoclasts to the bone surface. In summary, this report demonstrates that azathioprine, despite appearing to suppress osteoblastic activity and encourage osteoclast recruitment, maintains bone volume in the rat and does not alter the osteopenic effects brought about by CsA administration.
Matrix mineralization and the differentiation of osteocyte-like cells in culture
Authors: Mikuni-Takagaki Yuko , Kakai Yoshio , Satoyoshi Masanori , Kawano Eiji , Suzuki Yoshihisa , Kawase Toshio , Saito Shigeru
Published: February 2010

Osteocyte-like cells were prepared by sequentially treating calvaria from newborn rats with collagenase and chelating agents. On a reconstituted gel of basement membrane components, cells from the third collagenase digest displayed a round shape and expressed the highest level of alkaline phosphatase with minimal osteocalcin deposition into the matrix. On the other hand, cells derived from the interior after EDTA treatment exhibited well-developed dendritic cell processes and expressed essentially no alkaline phosphatase. The latter population also showed quite distinct characteristics such as higher extracellular activities of casein kinase II and ecto-5′-nucleotidase and the extracellular accumulation of a large amount of osteocalcin associated with mineral. These diverse phenotypic and protein expressions as well as the sites from which each population of cells were recovered strongly suggest that we have isolated osteoblastic and osteocytic cells. Bone sialoprotein II was extracellularly phosphorylated by casein kinase II in osteocytic cells but not in osteoblastic cells. We discuss the possibility that differentiation of young osteocytes from osteoblasts may facilitate the biochemical sequence of mineral deposition in the bone matrix.
In vivo, three-dimensional microscopy of trabecular bone
Authors: Dr. Kinney J.H. , Lane N.E. , Haupt D.L.
Published: February 2010

Osteoporosis, which is correlated with low bone mass and increased bone fragility, is responsible for about 1.2 million fractures per year in the United States. We have used the three-dimensional (3-D), X-ray tomographic microscope to image the trabecular bone architecture of the proximal tibias of six Sprague—Dawley rats in vivo. Three of these rats were then ovariectomized to induce estrogen depletion, and three remained as controls. Five weeks later, the tibias were reimaged. The ovariectomized rats lost approximately 65% of their trabecular bone volume as compared with an insignificant change in the control rats. The connectivity density of the trabecular bone, as measured by the Euler characteristic, was linearly correlated with trabecular bone volume, even in the ovariectomized rats. Hypoestrogenemic bone loss manifested itself in greatly reduced connectivity and fewer trabecular elements, but not in thinning of trabeculae. The ability to microscopically image sequential changes in the 3-D architecture of trabecular bone in vivo will allow exploration of the earliest stages of hypoestrogenemic bone loss and to more rapidly test the effectiveness of new clinical treatments for this major public health problem.
Ethnic differences in bone density in female South African nurses
Authors: Dr. Daniels Elvis D. , Pettifor John M. , Schnitzler Christine M. , Russell Sheila W. , Patel Deepak N.
Published: February 2010

In the United States, the higher prevalence of osteoporosis and the higher incidence of fractures in whites than in blacks may be attributed to the finding of lower bone density (BD) in both white children and adults. In South Africa, osteoporosis and fractures also occur more frequently in whites than in blacks. Appendicular BD has been found to be similar in black and white children in South Africa, but there is little information available on BD of adults in South Africa. This cross-sectional study aimed to assess changes in BD with age in adult females in South Africa and to assess possible differences in peak BD and in the rate of postmenopausal bone loss between blacks and whites. Data for 180 black and 184 white female nurses aged 20–64 years were analyzed. The distal radius bone density (RBD) was measured by single photon absorptiometry. The lumbar spine bone density (SBD) and the femur bone density (FBD) were measured by dual-energy X-ray absorptiometry. Blacks were shorter than whites (p = 0.0001), and blacks' weight, body mass index, and skinfold thickness increased with age. Peak SBD and RBD were similar in blacks and whites, but peak FBD was higher in blacks (p = 0.0001). This ethnic difference in peak FBD became apparent in the fourth decade. Peak FBD was similar in black and white subjects with normal body mass indices (p = 0.09), but in overweight subjects peak FBD was higher in blacks than in whites (p = 0.0001). SBD (p = 0.03), RBD (p = 0.0002), and FBD (p = 0.04) in whites, but not in blacks, decreased after the menopause. Weight had a positive association with FBD in premenopausal blacks (R = 0.24, p = 0.0001) but not in whites. In the postmenopausal group, adiposity had a positive association with SBD and RBD in blacks and with FBD in both blacks and whites. These results suggest that weight-bearing is important in the attainment of peak FBD and that adiposity may be important in decreasing postmenopausal bone loss. The finding of higher FBD in blacks than in whites may explain the lower rate of femoral fractures in black South Africans. However, the similarity in SBD between blacks and whites in this study suggests that factors other than bone density might contribute to the lower vertebral fracture rate in black compared with white South Africans.
Physicochemical effects of a new slow-release potassium phosphate preparation (UroPhos-K) in absorptive hypercalciuria
Authors: Breslau Neil A. , Padalino Paulette , Kok Dirk Jan , Kim Young Gon , Pak Charles Y.C.
Published: February 2010

A new slow-release, neutral potassium phosphate salt (UroPhos-K) has been formulated in order to minimize gastrointestinal side effects and avoid sodium-induced calciuria. It was tested in a prospective randomized, double-blind trial in a group of 21 kidney stone patients with absorptive hypercalciuria type I (AH). Twelve patients allocated to the UroPhos-K group received four tablets twice daily with breakfast and an evening snack providing 1240 mg of phosphorus and 63.5 mEq of potassium daily. Nine patients assigned to the placebo group received placebo tablets of the same appearance containing excipient only. Subjects were studied during a 3-day period in the hospital while consuming a constant metabolic diet containing 400 mg Ca, 100 mEq Na, and 800 mg P per day before and after 3 months of treatment. Treatment with UroPhos-K did not cause any significant gastrointestinal side effects; nor did it raise fasting serum K or phosphorus, or reduce hemoglobin or creatinine clearance. It was associated with a rise in urinary K from 46 ± 7 to 98 ± 9 mEq per day and phosphorus from 744 ± 185 to 1535 ± 112 mg per day (p < 0.001 each). UroPhos-K treatment reduced urinary Ca from 288 ± 63 to 171 ± 49 mg/day (p < 0.001), without altering oxalate excretion. It reduced the urinary saturation of calcium oxalate without altering that of brushite. Moreover, by increasing urinary excretion of inhibitors (citrate and pyrophosphate), it reduced the propensity for spontaneous nucleation of brushite (increased formation product of brushite) and inhibited crystal agglomeration of calcium oxalate. None of these changes were seen in the placebo-treated group. It is concluded that treatment with UroPhos-K reduces urinary stone-forming potential without causing gastrointestinal side effects.
Autoregulation of inducible prostaglandin G/H synthase in osteoblastic cells by prostaglandins
Authors: Pilbeam Carol C. , Raisz Lawrence G. , Voznesensky Olga , Alander Cynthia B. , Delman Bradley N. , Kawaguchi Hiroshi
Published: February 2010

Prostaglandins (PGs) have been postulated to amplify their own production by stimulating cyclic adenosine monophosphate activity, which in turn stimulates PG production. We examined regulation of messenger RNA levels for the inducible and constitutive prostaglandin G/H synthases, PGHS-2 and PGHS-1, in murine osteoblastic MC3T3-E1 cells, which express both PGHS-1 and PGHS-2, and in rat osteoblastic Py1a cells, which express only PGHS-2. Prostaglandins E2, F2α, and D2 induced PGHS-2 mRNA in both cell lines under serum-free conditions and stimulated small increases in PGHS-1 mRNA levels in MC3T3-E1 cells. PGE2 (1 μM) increased the transcription rate of PGHS-2 mRNA 9-fold at 2 h in serum-free cells and also induced PGHS-2 protein. In the presence of arachidonic acid or serum, PGs also increased medium PGE2. Both forskolin, a protein kinase A activator, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, have previously been shown to induce PGHS-2 mRNA in MC3T3-E1 cells, but in the present study only PMA induced PGHS-2 expression in Pyla cells. The induction of PGHS-2 mRNA in Py1a cells by PGs was inhibited by chelerythrine, a PKC inhibitor, and blocked by 24 h of pretreatment with PMA. The 2 h serum stimulation of PGHS-2 mRNA in MC3T3-E1 cells was inhibited 40–50% by three structurally unrelated nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting that endogenous PGs also amplify PG production through induction of PGHS-2. We conclude that the mechanism for autoamplification is in part transcriptional and may involve multiple pathways. We speculate that the induction of PGHS-2 by PGs may be the means for expanding PG responses to small and intermittent signals, such as mechanical strains.
Zinc is a potent inhibitor of osteoclastic bone resorption in vitro
Authors: Moonga Baljit S. , Dempster David W.
Published: February 2010

It is well established that zinc, an essential trace element, plays an important role in growth and stimulates bone formation. However, the effects of zinc on bone resorption have received little attention. We studied its effects on isolated rat osteoclasts. Unexpectedly, osteoclasts were exquisitely sensitive to zinc, with a significant decrease in bone resorption occurring at concentrations as low as 10−14 M. This effect was specific for zinc and was not observed with the other transitional or alkaline metals studied. There was no evidence of toxicity at concentrations up to 10−4 M. Zinc also completely abolished the stimulatory effect of parathyroid hormone. Zinc is therefore a highly potent and selective inhibitor of osteoclastic bone resorption in vitro. The mode of action remains to be established and may represent a novel inhibitory mechanism in the osteoclast.
Morphologie stages in lamellar bone formation stimulated by a potent mechanical stimulus
Authors: Rubin Clinton T. , Gross Ted S. , McLeod Kenneth J. , Bain Steven D.
Published: February 2010

The temporal stages of lamellar bone formation were studied using an animal model subject to up to 16 weeks of a controlled, externally applied load. The left ulnae of 15 adult male turkeys were functionally isolated via transverse metaphyseal osteotomies, while transcutaneous Steinmann pins permitted in vivo loading of the preparation via a servo-hydraulic actuator. For 5 days per week, the ulnae were exposed to 100 cycles per day of an applied load sufficient to cause a peak strain normal to the bone's longitudinal axis of 2000 microstrain (με). The contralateral limb was left surgically undisturbed and served as a baseline control. Following a loading period of 4, 8, or 16 weeks, ulnae were harvested and prepared for quantitative bone histomorphometry. Compared with each animal's contralateral ulna, the area of the experimental ulnae increased by 12.5% (±5.6%) at 16 weeks. Periosteal mineral apposition rates in the loaded ulnae were significantly increased compared with control values, with a maximum rate of 6.0 ± 3.4 μm/day at 5 weeks, slowing to 2.0 ± 0.3 μm/day by 15 weeks. At 16 weeks, new bone was composed of primary and secondary osteons as well as circumferential lamellae, with osteocyte density and organization indistinguishable from that of the original cortex. Remnants of the initial woven bone response seen at 4 weeks remained clearly visible at both 8 and 16 weeks as diffusely labeled interstitial elements within the newly formed lamellar construct. The presence of secondary osteons, circumferential lamellae, and an osteocyte density and organization similar to that seen in controls suggests that the presence of woven bone in the initial stages of the adaptive process is not necessarily a pathologic or transient reaction to injury, but instead may be a normal stage in response to a potent mechanical stimulus.
Intracellular calcium increases mediated by a recombinant human calcitonin receptor
Authors: Stroop Steven D. , Moore Emma E.
Published: February 2010

Stable transfectants expressing a recombinant human calcitonin receptor respond to calcitonin via increased cyclic adenosine 3′,5′ monophosphate (cAMP, EC50 = 0.06 nM salmon calcitonin [sCT]) and a transient mobilization of intracellular calcium ([Ca2+]i) coincident with turnover of inositol phosphate (IP; EC50 = 6 nM sCT). Millimolar increases in extracellular calcium ([Ca2+]o, EC50 = 8 mM) cause a rapid elevation in [Ca2+]i after a calcitonin dose-dependent pretreatment of cells (pretreatment EC50 = 0.2 nM sCT). Cells exhibit persistent sensitivity to increased [Ca2+]o up to 3 h after hormone exposure and even after multiple cycles of increased [Ca2+]o followed by wash. Calcitonin pretreatment of cells also allows apparent influx of elevated extracellular strontium and manganese, but little or no effect is observed on addition of barium, cadmium, or lanthanum. Human amylin (100 nM) causes a rapid and transient increase in [Ca2+]i comparable to that of calcitonin; however, no significant response to increased [Ca2+]o is observed after amylin addition. Human calcitonin gene-related product (hCGRP) (300 nM) and forskolin do not increase [Ca2+]i or activate a sensitivity to increased [Ca2+]o. Nevertheless, human amylin and human calcitonin gene-related product (hCGRP) activate adenylate cyclase with EC50s of 0.7 nM and 8 nM, respectively. The calcium-channel drugs verapamil, BAY K 8644, diltiazem, and nifedipine have little effect on [Ca2+]i increases. The calcitonin-induced transient mobilization of calcium is inhibited by treatment of cells with cholera toxin or 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8); whereas, the response to subsequent increased [Ca2+]o is inhibited by lanthanum chloride (200 μM) and lower pH (6.0). These studies suggest that a recombinant human calcitonin receptor activates three unique signal transduction pathways in BHK cells. Subnanomolar calcitonin persistently activates adenylate cyclase and a novel pathway coupled to calcium influx while much higher calcitonin levels increase inositol phosphate turnover and generate a transient mobilization of [Ca2+]i stores.
Solar ultraviolet B radiation and photoproduction of vitamin D3 in central and southern areas of argentina
Authors: Ladizesky Marta , Lu Zhiren , Oliveri Beatriz , Roman Nemesio San , Diaz Susana , Holick Michael F. , Mautalen Carlos
Published: February 2010

The incidence of nutritional rickets in the southern part of Argentina is 8–12 times higher than in the rest of the country. Winter 25(OH)D serum levels in normal population of southern areas are lower than in central and northern areas. To elucidate these differences, we compared the photoconversion of provitamin D3 (7-DHC) to previtamin D3 in two cities: Ushuaia (latitude 55°S) and Buenos Aires (34°S). Ampules containing 7-DHC were exposed to sunlight one day in the middle of each month either from 10:30 a.m. to 2:30 p.m. or from 8:00 a.m. to 5:00 p.m. The percentages of photoproducts formed were determined by high performance liquid chromatography (HPLC). Previous studies have proved that this is a valid model to assess “in vitro” the photoproduction of vitamin D3 in human skin. Previtamin D3 + vitamin D3 formed in Ushuaia were less (p < 0.02) than those found in Buenos Aires during all seasons: summer, (X ± SEM) 6.4 ± 0.8% vs. 13.2 ± 1.8%; autumn, 1.2 ± 0.7% vs. 63 ± 1.3%; winter, 0.8 ± 0.7% vs. 3.6 ± 0.7%; spring, 3.4 ± 0.5% vs. 9.1 ± 1.1%. The photoproducts produced from 10:30 a.m. to 2:30 p.m. were similar for each month and latitude to those formed when the ampules were exposed from 8:00 a.m. to 5:00 p.m. We conclude that in Ushuaia there is a prolonged “vitamin D winter” during which cutaneous synthesis of vitamin D is absent, leading to lower serum values of 25(OH)D and contributing to the higher incidence of rickets.
The phosphatidylinositol-glycolipid anchor on alkaline phosphatase facilitates mineralization initiation in vitro
Authors: Harrison Gerald , Shapiro Irving M. , Golub Ellis E.
Published: February 2010

Alkaline phosphatase (AP) is required for the proper mineralization of cartilage and bone. The enzyme is localized to the outer surface of cells through a phosphatidylinositol-glycolipid anchor, which is covalently attached to the carboxyl terminus of the protein. In calcifying cartilage, AP-rich matrix vesicles (MVs) are released into the matrix from chondrocytes, and apatite formation is initiated within and around these particles. In this paper we examine the role of the AP glycolipid anchor using an in vitro mineralization assay system. AP was purified to homogeneity, and the purified enzyme was used to drive mineral formation in vitro with and without the anchor. Mineral formation was initiated through phosphate release from β-glycerol phosphate (β-GP). The amount of PO−34 released was similar whether the anchor was present or absent. However, SEM and X-ray microanalysis revealed that the mineral produced by anchored AP was indistinguishable from that produced by MVs and that both of those minerals were more apatite-like than mineral formed by soluble AP or through spontaneous precipitation. Taken together, the data suggest that in addition to providing PO−34 to drive mineralization, AP influences the nature of the mineral formed. Further, AP containing its glycolipid anchor produces mineral comparable with that formed by tissue-derived MVs. Thus, in the absence of extracellular matrix, MV mineralization in vitro can be emulated by glycolipid-anchor containing AP.
A two-year program of aerobics and weight training enhances bone mineral density of young women
Authors: Friedlander Anne L. , Genant Harry K. , Sadowsky Steven , Byl Nancy N. , Glüer Claus-C.
Published: February 2010

Previous research suggests that physical activity may have a beneficial effect on bone mineral density (BMD) in women. This relationship was explored in a 2-year, randomized, intervention trial investigating the efficacy of exercise and calcium supplementation on increasing peak bone mass in young women. One hundred and twenty-seven subjects (ages of 20–35 years) were randomly assigned either to an exercise program that contained both aerobics and weight training components or to a stretching program. Calcium supplementation (up to 1500 mg/day including dietary intake) or placebo was given in a double-blinded design to all subjects. Spinal trabecular BMD was determined using quantitative computed tomography (QCT). Spinal integral, femoral neck, and trochanteric BMD were measured by dual X-ray absorptiometry (DXA) and calcaneal BMD by single photon absorptiometry (SPA). Fitness variables included maximal aerobic capacity (VO2max), and isokinetic muscle performance of the trunk and thigh. Measurements were made at baseline, 1 year, and 2 years. Sixty-three subjects (32 exercise, 31 stretching) completed the study, and all the measured bone parameters indicated a positive influence of the exercise intervention. There were significant positive differences in BMD between the exercise and stretching groups for spinal trabecular (2.5%), femoral neck (2.4%), femoral trochanteric (2.3%), and calcaneal (6.4%) measurements. The exercise group demonstrated a significant gain in BMD for spinal integral (1.3 ± 2.8%, p < 0.02), femoral trochanteric (2.6 ± 6.1%, p < 0.05), and calcaneal (5.6 ± 5.1, p > 0.01) measurements. In contrast to exercise, the calcium intervention had no positive effect on any of the bone parameters. In regard to fitness parameters, the exercise group completed the study with significant gains in VO2max and isokinetic (peak torque) values for the knee flexion and extension and trunk extension. This study indicates that over a 2-year period, a combined regimen of aerobics and weight training has beneficial effects on BMD and fitness parameters in young women. However, the addition of daily calcium supplementation does not add significant benefit to the intervention.
Oral 1,25-dihydroxyvitamin D administration in osteoporotic women: Effects of estrogen therapy
Authors: Dr. Cosman Felicia , Nieves Jeri , Shen Victor , Lindsay Robert
Published: February 2010

Estrogen has been shown to modify calcium and skeletal homeostasis. In this study, we tested the ability of estrogen to influence the effects of short-term 1,25(OH)2D administration on biochemical indices of bone formation and resorption in a cross-sectional analysis of untreated (n = 10) and estrogen-treated (n = 14) osteoporotic women. Patients were given oral 1,25(OH)2D (Rocaltrol) 0.5 μg twice a day for 5 days. Serum and urine were sampled at baseline and then 1 h after the first daily Rocaltrol dose for the 5 days of the study. 1,25(OH)2D levels rose similarly in both groups with plateaus reached by the third day of the investigation. Serum PTH levels decreased by the first sampling period (1 h after first Rocaltrol dose; p < 0.008 both groups) and continued to fall gradually in both groups. There were no changes in serum calcium but serum phosphorus rose by the second day (p < 0.05 both groups) and remained elevated throughout the remainder of the protocol. Serum bone GIa protein increased approximately 40% (p < 0.05) with no group differences. In contrast, total alkaline phosphatase and carboxy-terminal propeptide of type I collagen did not increase in either group. Furthermore, there were no significant increments in any bone resorption indicators, including serum tartrate-resistant acid phosphatase and cross-linked carboxy-terminal telopeptide of type I collagen, as well as urine hydroxyproline and pyridinoline. Serum IGF-1 levels also remained unchanged in both groups. We conclude that oral 1,25(OH)2D administration decreased 1–84PTH levels, probably due to a suppression of parathyroid production, and did not stimulate bone resorption. Since only bone GIa protein increased, it is unclear whether or not bone formation was actually stimulated. Estrogen treatment did not modify the skeletal response to low levels of oral 1,25(OH)2D stimulation in osteoporotic women.
Different effects of bisphosphonate and estrogen therapy on free and peptide-bound bone cross-links excretion
Authors: Garnero Patrick , Gineyts Evelyne , Arbault Patrice , Christiansen Claus , Delmas Pierre D.
Published: February 2010

We have measured the free and peptide-bound type I collagen cross-link excretions in normal women and in patients with metabolic bone disease using the HPLC technique and immunoassays recognizing specifically the free or peptide-bound forms of pyridinoline (Pyr). After menopause, free deoxypyridinoline (free D-Pyr) excretion measured by HPLC without urine hydrolysis and expressed as a fraction of the total excretion was lower than in premenopausal women (45 ± 15% vs. 59 ± 12%, p > 0.005), whereas the fraction of free Pyr was not changed. In normal pre- and postmenopausal women (n = 43), the fraction of free D-Pyr was negatively correlated with bone turnover rate as assessed by the total urinary excretion of Pyr (r = –0.64, p > 0.001). In patients with a variety of metabolic bone diseases characterized by increased bone turnover (osteoporosis, Paget's disease, and hyperthyroidism), the fractions of free Pyr and free D-Pyr were significantly lower than in premenopausal controls (p > 0.001 for all diseases). After 3 days of intravenous (iv) treatment with the bisphosphonate pamidronate in patients with Paget's disease and osteoporosis, the urinary excretion of cross-linked peptides measured by high performance liquid chromatography (HPLC) or enzyme-linked immunoassay (ELISA) (NTX and CrossLaps) was markedly decreased (–52% and –85% for NTX, –71% and –93% for CrossLaps™ in Paget's disease and osteoporosis, respectively). The excretion of total cross-links was decreased to a lesser extent after treatment (–25% and –25% for total Pyr, –37% and –45% for total D-Pyr) and, surprisingly, free cross-links measured either by HPLC without urine hydrolysis or with an ELISA specific for free D-Pyr were unchanged after treatment. In contrast to bisphosphonate therapy, estrogen treatment of postmenopausal women decreased not only total and cross-linked peptides but also the free cross-link excretion that was reduced by about 30–40%. The different effects of bisphosphonate and estrogen therapy on the excretion of peptide-bound and free cross-link excretion were confirmed by gel filtration of the urine on Sephadex G 25 before and after treatment. In conclusion, we have shown that increased bone turnover in patients with metabolic bone disease could result in a larger increase of the urinary excretion of cross-linked peptides over the increase of free cross-links. Bisphosphonate therapy decreased markedly cross-linked peptides without significant change in free cross-link excretion contrasting with a decrease of both free and peptide-bound cross-links after estrogen therapy. These data suggest that these two antiresorptive therapies may affect differently the pattern of bone collagen degradation, an intriguing possibility that should be further investigated in vitro.
Cyclical clodronate is effective in preventing postmenopausal bone loss: A comparative study with transcutaneous hormone replacement therapy
Authors: Prof. Filipponi Paolo , Pedetti Mariano , Fedeli Leone , Cini Luisella , Palumbo Renato , Boldrini Stefano , Massoni Cristina , Cristallini Sonia
Published: February 2010

An investigative study was carried out for 2 years involving 124 randomly selected early postmenopausal women with spine bone mineral density (BMD) below the mean value of a normal premenopausal subject. After random division into three groups, the first 42 patients were treated with transcutaneous 17-β-estradiol (50 μg daily), the second 42 were treated with cyclical intravenous clodronate (200 mg/month iv infusion), and the third group of 40 (controls) was left untreated. After 2 years, the total drop in BMD within the control group was more than 7% as opposed to the values of –0.14% ± 0.93 in the estradiol group and 0.67% ± 0.84 in the clodronate group. A change in BMD of < 1% was considered satisfactory, and this result was obtained in 32% of the controls, in 79% of the estradiol group where the percentage change in BMD moderately correlated with serum estradiol levels (r = 0.399), and in 90% of the clodronate-treated patients, in whom the percentage change in BMD inversely correlated with basal values of markers of bone turnover. Both estrogen and clodronate prevent postmenopausal bone loss. The response to transcutaneous hormone replacement therapy may be influenced by transcutaneous absorption and by a lower sensitivity to estrogen. Response to cyclical clodronate seems to be influenced by the rate of bone turnover. An interdosage interval ranging from 2–4 weeks appears suitable for most patients.
Inhibitory effects of basic fibroblast growth factor on chondrocyte differentiation
Authors: Dr. Wroblewski J. , Edwall-Arvidsson C.
Published: February 2010

The role of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I) in cartilage growth was studied in primary cultures of rat rib growth plate chondrocytes. Growth factors effects on expression of the proto-oncogene c-fos, DNA synthesis, differentiation, and morphological changes were analyzed by in situ hybridization, 3H-thymidine incorporation, and light and fluorescence microscopy. In serum-deprived cells, bFGF induced a transient expression of c-fos with a maximal effect 15–30 minutes after stimulation. After 24 h of culture it had a slightly lower stimulatory effect on DNA synthesis than IGF-I, but became a significantly more potent mitogen than IGF-I after 48 and 72 h. The stimulatory effect of bFGF on DNA synthesis coincided with a decrease in collagen type II and IGF-II expression. In contrast, IGF-I alone stimulated expression of these genes. In bFGF-treated cultures, cell morphology and the appearance of actin filaments was changed. Polygonal chondrocytes became elongated, fibroblast-like, and the smooth actin filaments were brush-like and disrupted. Addition of IGF-I reduced these changes without affecting c-fos expression induced by bFGF. Our results suggest that bFGF stimulates cell proliferation by preventing terminal differentiation of chondrocytes. This effect is mediated by induction of c-fos expression and a decrease in the steady-state levels of transcripts for collagen II and IGF-II.
Prevalence of low femoral bone density in older U.S. women from NHANES III
Authors: Looker Anne C. , Johnston Conrad C. , Wahner Heinz W. , Dunn William L. , Calvo Mona S. , Harris Tamara B. , Heyse Stephen P. , Lindsay Robert L.
Published: February 2010

Data on the number of U.S. women with low femoral bone mineral density (BMD) are currently available only from indirect estimates. We used dual-energy X-ray absorptiometry (DXA) measurements of femoral BMD from phase 1 of the third National Health and Nutrition Examination Survey (NHANES III, 1988–1991) to estimate prevalences of low femoral BMD in women ages 50 years and older using an approach proposed recently by an expert panel of the World Health Organization (WHO). Cutpoints for low BMD were derived from BMD data of 194 non-Hispanic white (NHW) women aged 20–29 years from the NHANES III dataset. The prevalence of older U.S. women with femoral osteopenia (BMD between 1 standard deviation [SD] and 2.5 SD below the mean of young NHW women) ranged from 34–50% in four different femur regions, which corresponds to ∼12–17 million women. The prevalence with osteoporosis (BMD >2.5 SD below the mean of young NHW women) ranged from 17–20%, or ∼6–7 million women. Prevalences were 1.3–2.4 times higher in NHW women than non-Hispanic black women (NHB), and 0.8–1.2 times higher in NHW versus Mexican American (MA) women. The estimated numbers of NHW, NHB, and MA women with osteopenia were 10–15 million, 800,000–1.2 million, and 300,000–400,000, respectively; corresponding figures for osteoporosis were 5–6 million, 200,000–300,000, and 100,000 respectively. Thus, the first data on BMD from a nationally representative sample of older women show a substantial number with low femoral BMD. The majority of these women are white, but the number of minority women with low BMD is not trivial.
Quantitative analysis of trabecular microstructure by 400 MHz nuclear magnetic resonance imaging
Authors: Chung Hsiao-Wen , Wehrli Felix W. , Williams John L. , Kugelmass Steven D. , Wehrli Suzanne L.
Published: February 2010

A new approach for the quantitative analysis of trabecular microstructure, based on high-field proton nuclear magnetic resonance (NMR) imaging, is presented. NMR is ideal because it provides high contrast between the marrow proton signal and the bone, which appears with background intensity. Images from 1 cm3 defatted specimens of trabecular bone, suspended in water doped with 1 mM Gd(DTPA) to shorten T1 to about 300 ms, can be obtained at a resolution on the order of 30–50 μm and slice thickness of 150 μm, in 10 minutes at 400 MHz proton frequency. Digital image processing algorithms were designed and evaluated for the measurement of bone area fraction, perimeter length, mean trabecular thickness, and separation. Bone area fraction derived from the NMR images was found to be in excellent agreement with bone volume fraction measured independently (slope = 0.96, r2 = 0.924, p < 0.0001). Errors in the mean trabecular thickness and separation were <6%. The effects of finite imaging slice thickness and signal-to-noise ratio (SNR) were also evaluated. The data suggest a resolution of 50 × 50 × 200 μm3 and an SNR on the order of 10 to provide safe margins for precise and accurate structural analysis by means of the algorithms presented in this paper. The method allows simultaneous measurement at multiple locations within the specimen volume without the need for physical sectioning.
The effects of menopause and age on calcitropic hormones: A cross-sectional study of 655 healthy women aged 35 to 90
Authors: Assoc. Prof. Prince Richard L. , Dick Ian , Devine Amanda , Price Roger I. , Gutteridge Donald H. , Kerr Deborah , Criddle Arthur , Garcia-Webb Peter , John Andrew St.
Published: February 2010

Although women lose 30% of their skeletal mass after the menopause, the mechanism of this loss is uncertain. Clearly estrogen deficiency is important but whether this works only through direct effects on the skeleton is uncertain. To examine these mechanisms further we have evaluated calcium-related metabolic factors in 655 healthy women. Fasting blood samples were collected from all subjects who were up to 35 years past the menopause, and fasting urine and 24-h urine samples were collected in 365 women who were up to 25 years past the menopause. In the first 15 years postmenopause, there was a rise in total plasma calcium due to a rise in albumin. Bone resorption (hydroxyproline creatinine ratio), bone formation (alkaline phosphatase), and the urine calcium creatinine ratio all rose at menopause and remained elevated for the next 25 years. There was a transient further rise in bone resorption for the 10 years following menopause. Neither PTH nor the free calcitriol index changed for the first 10 years following menopause. Ten years past the menopause, although total calcitriol rose, the free calcitriol index fell due to a rise in vitamin D binding protein. PTH began to rise at 15 years past menopause. GFR fell gradually over the 25 years following menopause. Thus following menopause there is an increase in bone turnover and increased urine calcium loss independent of any effect of PTH or calcitriol, suggesting a direct effect of estrogen deficiency on bone and kidney. At 10 years postmenopause, the free calcitriol index fell and PTH began to rise, these changes being associated with persistent elevation of bone resorption perhaps due to increased PTH action on the bone. The cause of the fall in the free calcitriol ratio may be due to progressive renal deterioration and phosphate retention. The cause of the rise in PTH may be due to a negative external calcium balance due to a reduction in gut calcium absorption, a consequence of relative deficiency of 25 hydroxyvitamin D and the fall in calcitriol together with persistent renal calcium loss.
Evidence that human bone cells in culture secrete insulin-like growth factor (IGF)-II and IGF binding protein-3 but not acid-labile subunit both under basal and regulated conditions
Authors: Kanzaki Susumu , Baxter Robert C. , Knutsen Raymond , Baylink David J. , Mohan Subburaman
Published: February 2010

Insulin-like growth factors (IGFs) are found in human circulation predominantly as part of a growth hormone (GH)–dependent complex of 125–150 kD, which is composed of three subunits: IGF-I or IGF-II, an acid stable IGF binding protein (IGFBP)-3, and an acid labile subunit (ALS). Although recent studies demonstrate that a number of cell types in culture secrete IGFs and IGFBP-3, very little is known with regard to the origin of circulating ALS. To test the hypothesis that human bone cells (HBCs), which produce abundant amounts of IGF-II and IGFBP-3, also produce ALS, we measured the IGF-I, IGF-II, IGFBP-3, and ALS levels using specific radioimmunoassays (RIAs) in the conditioned medium (CM) of untransformed normal HBCs and SaOS-2 osteosarcoma cells treated with various effectors (IGF-II, osteogenic protein-1 [OP-1, bone morphogenetic protein-7] and human GH) for 48 h. No detectable levels (<3 ng/ml) of ALS were found in the CM of various HBC types under basal conditions. In contrast, CM collected from liver explants in culture contained significant amount of ALS prepared and assayed under identical conditions. The IGF-I level was also undetectable in the CM of various HBC types. In the IGF-II (3, 30 ng/ml)-treated HBC CM, the IGFBP-3 level was increased in a dose-dependent manner but neither IGF-I nor ALS could be detected. In the SaOS-2 cell culture, OP-1 (1, 100 ng/ml) increased both IGF-II and IGFBP-3 secretion but neither ALS nor IGF-I secretion. Treatment of HBCs with GH (1, 10, 100 ng/ml) had no significant effect on the secretion of either IGF-I, IGF-II, IGFBP-3, or ALS. The level of IGF-II in the CM of various HBC types correlated positively with that of IGFBP-3 (r = 0.84). From these results, we conclude that the production of ALS and IGFBP-3 are not concomitantly regulated by the same effectors in HBCs. The finding of this study together with the previous findings that GH regulates ALS secretion in liver cells suggest that the primary functions of IGFs produced in the bone and liver may be different (i.e., local versus endocrine effects).