bluuebunny/crossref_metadata_2025 huggingface dataset:
X-linked hypophosphatemic rickets: A study (with literature review) of linear growth response to calcitriol and phosphate therapy
Authors: Petersen Deborah J. , Boniface Anne M. , Schranck Francine W. , Rupich Reta C. , Whyte Michael P.
Published: February 2010

Not all children with X-linked hypophosphatemia (XLH) have demonstrated improved linear growth with calcitriol [1,25-(OH)2D3] and inorganic phosphate (Pi) therapy. To assess which factors are associated with a favorable growth response during this treatment, we retrospectively compared demographics and biochemical parameters of bone metabolism to the linear growth patterns of 20 children with XLH who were prepubertal and had not required osteotomy. A total of 15 patients had family histories consistent with XLH; 5 appeared to be sporadic cases. During 3 years of therapy, the growth velocities of 12 patients had been at or above the mean for age (good growers) and those of 8 patients had been below the mean (poor growers). Data from the two groups were contrasted. We found no difference between the good growers and poor growers before or after the 3 year period of therapy in mean age, dietary calcium, calcitriol dose or compliance, or Pi dose or compliance. Both groups increased their mean fasting serum Pi levels with treatment. The TmP/GFR (x + SEM) of the good growers improved with therapy (1.9 + 0.2 to 2.6 + 0.2 mg/dl, p = 0.01), and their posttreatment value was higher compared to that of the poor growers (2.6 + 0.1 versus 2.2 + 0.1 mg/dl, p = 0.02). However, their enhanced TmP/GFR was not associated with a reduction in serum iPTH levels (before, 693 + 50; after, 688 + 76 pg/ml; p = 0.9). The Z test for binomial proportions showed that the group that grew well contained a disproportionate number of girls (10 of 12, p = 0.04). Our findings suggest that calcitriol may exert a direct effect on the renal tubule to improve Pi reclamation in XLH. The observation that heterozygous girls appear to respond better than hemizygous boys to calcitriol and Pi therapy provides evidence for a gene dosage effect in the expression of this X-linked dominant disorder.
Age-related decrements in bone mineral density in women over 65
Authors: Steiger Peter , Cummings Steven R. , Black Dennis M. , Spencer Nan E. , Genant Harry K.
Published: February 2010

Age-related changes in bone density contribute to the risk of fractures. To describe the relationship between age and bone mass in elderly women, we studied a large cohort of women over age 65 years who were recruited from population-based lists in four cities in the United States. Bone density in g/cm2 was measured by single-photon absorptiometry (SPA) and dual x-ray absorptiometry (DXA) at the distal and proximal radius, the calcaneus, the lumbar spine, and the proximal femur. Centralized data collection was used to control data quality and consistency. We found a strong inverse relationship between bone density and age for most sites. Decrements in bone density between women aged 65–69 years and women 85 years and older exceeded 16% in all regions except the spine, where the difference between the two age groups was 6%. Ward's triangle and the calcaneus exhibited the largest decrements, with 26 and 21%, respectively. The estimates of annual changes in bone mineral density by linear regression at sites other than the spine ranged from -0.82% at the femoral neck and trochanter to -1.30% at Ward's triangle. Correlations between the different regions ranged from r = 0.51 between the proximal radius and Ward's triangle to r = 0.66 between the distal radius and calcaneus. We conclude that the inverse relationship between age and bone mass measured by absorptiometry techniques in white women continues into the ninth decade of life. The relationship is strongest for bone density of Ward's triangle and the calcaneus and weakest for the spine.
Axial and appendicular bone density predict fractures in older women
Authors: Black Dennis M. , Cummings Steven R. , Genant Harry K. , Nevitt Michael C. , Palermo Lisa , Browner Warren
Published: February 2010

To determine whether measurement of hip and spine bone mass by dual-energy x-ray absorptiometry (DEXA) predicts fractures in women and to compare the predictive value of DEXA with that of single-photon absorptiometry (SPA) of appendicular sites, we prospectively studied 8134 nonblack women age 65 years and older who had both DEXA and SPA measurements of bone mass. A total of 208 nonspine fractures, including 37 wrist fractures, occurred during the follow-up period, which averaged 0.7 years. The risk of fracture was inversely related to bone density at all measurement sites. After adjusting for age, the relative risks per decrease of 1 standard deviation in bone density for the occurrence of any fracture was 1.40 for measurement at the proximal femur (95% confidence interval 1.20–1.63) and 1.35 (1.15–1.58) for measurement at the spine. Results were similar for all regions of the proximal femur as well as SPA measurements at the calcaneus, distal radius, and proximal radius. None of these measurements was a significantly better predictor of fractures than the others. Furthermore, measurement of the distal radius was not a better predictor of wrist fracture (relative risk 1.64: 95% CI 1.13–2.37) than other sites, such as the lumbar spine (RR 1.56; CI 1.07–2.26), the femoral neck (RR 1.65; CI 1.12–2.41), or the calcaneus (RR 1.83; CI 1.26–2.64). We conclude that the inverse relationship between bone mass and risk of fracture in older women is similar for absorptiometric measurements made at the hip, spine, and appendicular sites.
Adaptive regulation of ascorbate transport in osteoblastic cells
Authors: Dixon S. Jeffrey , Wilson John X.
Published: February 2010

Osteoblasts possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na+-dependent ascorbate transporter located in the plasma membrane. The transporter is specific for ascorbate and stereoselective for L-ascorbate over D-isoascorbate. The present study examined the effects of ascorbate supplementation and deprivation on the activity of this transport system. L-ascorbate transport activity was determined by measuring uptake of the vitamin by ROS 17/2.8 osteosarcoma cells during 1 minute incubations with 5 μM L-[14C]ascorbate. The initial rate of L-[14C]ascorbate uptake by ROS 17/2.8 cells grown for 18 h in L-ascorbate-replete medium was 89 + 8 nmol/g protein per minute. Following removal of L-ascorbate from the growth medium, the initial rate of uptake increased within 6 h to 126 + 13 nmol/g protein per minute. Conversely, the initial rate of uptake by cells grown in ascorbate-free medium decreased following the addition of L-ascorbate, but not D-isoascorbate, to the medium. The effect of ascorbate pretreatment was specific for ascorbate transport in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-glucose. Kinetic analysis revealed that modulation of ascorbate transport arose from changes in the apparent maximum rate of transport (Vmax) without changes in the affinity of the transport system for L-ascorbate. These experiments are the first to show that ascorbate transport by osteoblastic cells responds to vitamin C deprivation and supplementation. Adaptation of transport activity to substrate availability may play an important role in the physiological regulation of intracellular ascorbate levels.
Isolation and characterization of a cDNA for osteopontin-k: A kidney cell adhesion molecule with high homology to osteopontins
Authors: Crivello Joseph F. , Delvin E.
Published: February 2010

Screening of a bovine renal cDNA library with MAbs(1) resulted in the isolation of a 1447 bp cDNA. This cDNA (pBk2.1) was sequenced and shown to contain an open reading frame with a putative protein of 261 amino acids, with a molecular weight of 29,573 (minute leader sequence) and a hydrophobic leader sequence of 16 amino acids. pBk2.1 was shown to share a high level of nucleic acid sequence homology over portions of its sequence to human, porcine, mouse, and rat osteopontins (40–60%). The peptide (osteopontin-k) had a potential glycosylation site (Asn-X-Ser/Thr), a GRGDS receptor binding region, a high level of asparagine residues, and a high abundance of acid amino acids characteristic of osteopontin-like cell adhesion molecules. The N-terminal amino acid region of pBk2.1 (the first 82 amino acids) and 42 amino acids at the C terminus had the highest level of homology with the osteopontins at 86%. The middle portion of the peptide had greatly reduced homology, ranging from 50% (amino acids 83–174) to 12% (amino acids 175–219). There were also deletions and additions of sequence in osteopontin-k that were not found in the other osteopontins. The homologies suggest that these proteins are highly related and may be derived from a common gene by alternative splicing. A 678 bp cRNA probe constructed from pBk2.1, containing a region with low homology to the osteopontins (amino acids 183–219 with <20% homology, plus amino acids 220–261 and untranslated sequence), was used in northern blots and RNAse protection assays. In a northern blot, this cRNA probe detected a 1.8–2.0 kb mRNA in bovine kidney. The greatest signal was seen in kidney, followed by liver and heart. Normal trabecular bone had a weak signal, with no signal in pancrease or skeletal muscle. For more accurate quantitation of mRNA levels, RNAse protection assays were used. These experiments demonstrated that this mRNA is expressed at highest levels in bovine kidney but at very low levels in osteosarcoma cells and at lower levels in liver, heart, and skeletal muscle. Treatment of bovine proximal tubule cells with 100 nM 1,25-(OH)2D3 resulted in a increase in osteopontin-k mRNA expression (2.7-fold). PTH had no effect on the expression of osteopontin-k mRNA. These results suggest that pBk2.1 represents a predominantly renal-specific cell adhesion molecule within the family of osteopontins that may be under hormonal regulation by the vitamin D system.
Factors influencing synthesis and mineralization of bone matrix from fetal bovine bone cells grown in vitro
Authors: Whitson S. William , Whitson Marcia A. , Bowers Daniel E. , Falk Michael C.
Published: February 2010

This study of the in vitro synthesis and mineralization of bovine bone demonstrates that sheets of mineralized matrix can be produced consistently within 18–24 days of cell isolation. Mineralization surpasses that achieved by other systems with other species: The deposition of mineral extends beyond nodules to form branching trabeculae and then solid wafers of bone. Comparison of the fetal age of the bone source, enzyme digestion methods, seeding density, culture surface, nutritive media, and concentration of fetal calf serum and other additives, including insulin and ascorbic acid, has yielded a set of optimal culture conditions. In the presence of ascorbic acid and β-glycerol phosphate, insulin has a dose-dependent effect on the morphology of the mineralized bone matrix produced. Quantitative analysis shows that in these cultures calcium accumulates most rapidly between days 6 and 10 after the introduction of mineralization medium but that mineral accretion continues throughout 14–16 days of culture. Alkaline phosphatase levels rise up to 200-fold, concomitant with a rapid increase in the number of cells per culture during the early mineralization phases; both fall as mineralization proceeds. This system has been used to study the induction of mRNA of type I collagen, alkaline phosphatase, and several noncollagenous bone proteins during the course of mineralization. Because of the degree of mineralization achieved with this system, it has many potential applications.
Bone matrix mRNA expression in differentiating fetal bovine osteoblasts
Authors: Ibaraki Kyomi , Termine John D. , Whitson S. William , Young Marian F.
Published: February 2010

In the accompanying study, we report an in vitro culture system from bovine bone cells that can be applied to investigate bone cell growth and differentiation. In this system, bovine bone cells placed in mineralization medium formed multilayers (days 2–3), began deposition of mineral (days 5–6), and eventually acquired a mineralized matrix sheet (days 14–20) through the stages of mineralizing nodules and trabecular-like structure. In the current study we used this system to investigate the relative expression of bone matrix genes that may play an important role in bone development and metabolism. α1(I)-collagen, alkaline phosphatase, osteonectin, biglycan (PgI), decorin (PgII), osteopontin, and bone sialoprotein mRNA gene expression were measured on days 0, 2, 6, 10, and 20 (date when the cells were placed in mineralization medium as day 0). Total RNA was purified and analyzed by northern blot using radiolabeled cDNA encoding these genes. To comprehend the relationship between gene expression and mineralization, total calcium content in the cultures was also measured. During the culture period we observed several very different gene expression profiles. The expression of both α1(I)-collagen and biglycan increased 3- to 4-fold by day 6 and then returned to basal levels by day 20. The osteonectin gene was highly expressed throughout the culture, with no significant increase in induction found during any time of culture. A significant induction of alkaline phosphatase (13.8-fold) gene expression was observed by day 6. Osteopontin showed a similar profile to that of alkaline phosphatase but had a much greater level of relative expression (26-fold) compared to day 0. Interestingly, downregulation during mineral accumulation seemed a common occurrence among many of the genes measured. In contrast, the bone sialoprotein gene showed a significant and distinct expression pattern, increasing rapidly after the onset of mineralization on day 6 and ultimately reaching 140-fold that of day 0. Decorin (Pg II) showed an increasing pattern, with the final relative level of induction 5-fold on day 20. These data suggest that the development of the mature osteoblastic phenotype, complete with the ability to produce a thick mineralized matrix, requires the differential regulation of a series of genes and their gene products over the culture period.
Bone mineral density in the proximal femur and hip fracture type in the elderly
Authors: Nakamura Norimasa , Kyou Takeshi , Takaoka Kunio , Ohzono Kenji , Ono Keiro
Published: February 2010

Bone mineral density (BMD) in the contralateral proximal femur in 100 female elderly patients with hip fracture and the 35 controls without hip fracture were investigated using dual-photon absorptiometry. The hip fracture patients were divided into intracapsular fracture (n = 53) and extracapsular fracture (n = 47) groups, and these two groups were further divided into five subgroups according to fracture site: intracapsular fracture type 1 (transcervical fracture, n = 29) and type 2 (subcapital fracture, n = 24); extracapsular fracture type 1 (intertrochanteric line fracture, n = 13), type 2 (pertrochanteric fracture, n = 28), and type 3 (combined type of pertrochanteric and subtrochanteric fracture, n = 6). The intracapsular fracture group showed BMD values similar to those of controls; the extracapsular fracture group showed significantly lower BMD values than controls. When these two were subclassified into five subgroups, different results were seen in terms of BMD value in the proximal femur and fracture types; intracapsular fracture type 1 showed BMD values equivalent to those of controls; on the other hand, type 2 showed significantly lower BMD value than controls, and the BMD distribution in the proximal femur among the extracapsular fracture subgroups 1–3 differed, although all of them showed significantly lower BMD values than controls. The degree of trauma causing the fractures was also assessed according to available anamnestic data, but no significant difference was found in trauma tendency between the intra- and the extracapsular fracture group or among the subgroups in each group. Based on these results, it is suggested that BMD distribution in the proximal femur is involved in determining hip fracture type in the elderly.
Establishment of the primary structure of the major lipid-dependent Ca2+ binding proteins of chicken growth plate cartilage matrix vesicles: Identity with anchorin cii (annexin V) and annexin II
Authors: Genge Brian R. , Cao Xu , Wu Licia N. Y. , Buzzi William R. , Showman Richard W. , Arsenault A. Larry , Ishikawa Yoshinori , Dr. Wuthier Roy E.
Published: February 2010

Electron microscopic studies of calcifying vertebrate tissues reveal the locus of de novo mineral formation within matrix vesicles (MV). The direct involvement of MV in the initiation of mineral formation is supported by the fact that MV isolated from avian growth plate cartilage rapidly accumulate large amounts of Ca2+ and Pi and induce mineral formation. Exploration of the constituents of MV has revealed two major protein components, a 33 and a 36 kD protein, the former of which binds to cartilage-specific collagens. These annexin-like proteins bind to acidic phospholipids in the presence of submicromolar levels of Ca2+. Antibodies raised against both the purified 33 and the 36 kD MV annexin do not cross-react with the other, indicating that they are distinct proteins. Reported here are studies elucidating the primary structure of both MV proteins using both conventional protein and molecular biologic methods. These studies establish that the 33 kD protein is nearly identical to anchorin CII (annexin V) and that the 36 kD protein is identical to avian annexin II. Immunolocalization studies show that hypertrophic chondrocytes at the calcification front of avian growth plate contain the highest level of these annexins. Further, immunogold labeling indicates that the annexins are localized within MV isolated from the growth plate. Recent studies indicate that annexin V is a new type of ion-selective Ca2+ channel protein that possesses selective collagen binding properties. Since MV are tightly associated with the collagen- and proteoglycan-rich matrix, it is tempting to speculate that this MV protein may be a component of stretch-activated ion channels that enhance Ca2+ uptake during mechanical stress.
Impact of short-term estrogen administration on growth hormone secretion and action: Distinct route-dependent effects on connective and bone tissue metabolism
Authors: Dr. Ho Ken K.Y. , Weissberger Andrew J.
Published: February 2010

We recently reported that estrogen exerts distinct effects on the GH/IGF-1 axis that are dependent on the route of delivery, probably reflecting a first-pass effect on hepatic IGF-1 production. Oral administration reduces IGF-1 and increases GH levels; transdermal administration elevates IGF-1 without changing GH concentrations. Since mesenchymal tissue is a target for GH and IGF-1 action, we studied changes in the GH/IGF-1 axis following oral (ethinyl estradiol, 20 μg/day) versus transdermal (Estraderm 100 TTS, Ciba Geigy, 100 μg 17β-estradiol per day) estrogen delivery and compared corresponding effects on connective and bone tissue metabolism. Mean 24 h GH levels, IGF-1, markers of fibroblast (procollagen III) and osteoblast (procollagen I, osteocalcin) function, and indices of bone turnover (fasting urinary hydroxyproline and calcium to creatinine ratios, UOHPr/Cr and UCa/Cr) were measured before and after 2 months of either oral or transdermal therapy in two groups of postmenopausal women. Transdermal estrogen administration significantly (p < 0.05) increased IGF-1, procollagen III, procollagen I, osteocalcin, and UOHPr/Cr. In contrast, oral estrogen administration had a suppressive effect; the levels of IGF-1 (p = 0.001), procollagen III (p = 0.018), procollagen I (p = 0.002), osteocalcin (p = 0.015), and UOHPr/Cr (p = 0.004) were significantly different from those measured during transdermal administration. Both treatments significantly reduced UCa/Cr (p < 0.015). IGF-1 changes during estrogen therapy were significantly related (p < 0.05) to changes in procollagen III, procollagen I, osteocalcin, and UOHPr/Cr. Transdermally delivered estrogen stimulates IGF-1 production, increases osteoblastic function, and stimulates bone and nonbone collagen synthesis. When delivered orally, estrogen reduces IGF-1 and inhibits osteoblastic function; both routes suppress urinary calcium loss. Estrogen administration confers distinct route-dependent effects on connective and bone tissue metabolism; the specific effects on fibroblast and osteoblast function suggest IGF-1 dependency. The divergent effects on indices of bone turnover and connective tissue metabolism suggest that the route of estrogen replacement therapy may have different effects on the integrity of structural tissue in the menopause.
Detection of canine distemper virus in bone cells in the metaphyses of distemper-infected dogs
Authors: Mee Andrew P. , Webber David M. , May Christopher , Bennett David , Dr. Sharpe Paul T. , Anderson David C.
Published: February 2010

In the light of recent evidence implicating canine distemper virus (CDV) as a possible etiologic agent in Paget's disease of bone, we thought that it would be of interest to examine distemper-infected bone in the natural host. Samples from the long bones, spleen, and bladder of four distemper-infected and three uninfected dogs were examined for the presence of CDV nucleocapsid and phosphoprotein genes and the measles virus (MV) nucleocapsid gene using the technique of in situ hybridization with radioactively labeled riboprobes. Two of the four distemper-infected dogs showed strongly positive hybridization with both of the CDV probes. The signal was present in marrow cells, in osteoblasts, in osteocytes, and particularly in osteoclasts. No hybridization was seen over the cartilage cells of the growth plate, and there was a clear line of demarcation at the point of invasion of osteoclasts and vascularization. The spleen and bladder samples from infected dogs also showed positive hybridization. There was no hybridization with the MV probe in any of the distemper-infected tissue. Samples from the uninfected dogs showed no evidence of hybridization with either the CDV or MV probes. These results show that CDV can infect bone cells of the natural host and provide further support for the theory that CDV may play a role in human Paget's disease of bone.
Enhancement of osteoinduction by vitamin D metabolites in rachitic host rats
Authors: Atkin Isaac , Dean David D. , Muniz Ofelia E. , Agundez Agueda , Castiglione Greg , Cohen Galit , Howell David S. , Ornoy Asher
Published: February 2010

Diaphyseal bone from normal Sprague-Dawley rats was delipidated in chloroform-methanol and demineralized in 0.6 N HCl at 4°C. The bones were then implanted for 7–28 days into rats made rachitic by a low-phosphate, vitamin D-deficient diet (VDP−) for 3 weeks. Bones from VDP− and normal rats were also implanted into normal hosts. When normal rats were used as the host environment, a consistent sequence of cartilage induction and bone formation was observed. Demineralized rachitic bone (RB) implanted into normal host rats resulted in cartilage and bone induction similar to that seen for normal bone (NB) implants. Transmission electron microscopy of RB in normal hosts revealed morphologically normal chondrocytes and cartilage matrix with normal mineralization. In contrast, implantation of NB in VDP− hosts resulted in delayed chondrogenesis and lack of calcification. Furthermore, similar results were observed when RB was implanted into VDP− hosts. Treatment of VDP− hosts with either 1α-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 did not accelerate the sequential appearance of precartilage or cartilage. However, 24,25-(OH)2D3 administered alone or in combination with 1α-OHD3 significantly increased the amount of calcified cartilage observed at 2 weeks postimplantation compared to implants from either untreated VDP− hosts or those treated only with 1α-OHD3. New bone formation was observed at 4 weeks postimplantation in all vitamin D-treated groups as determined by von Kossa staining or direct electron microscope examination. There was no apparent difference in the quantitative or qualitative bone formed within the various vitamin D-treated groups. Serum calcium and phosphorus levels were lower and alkaline phosphatase levels were higher in VDP− hosts compared with normal animals or those treated with vitamin D metabolites. The results of this study show a reduction in the capacity of progenitor cells in VDP− rat hosts to respond to osteoinductive factor(s). This impaired response appears to be corrected by vitamin D metabolites.
Evidence for the formation of a complex between osteopontin and osteocalcin
Authors: Ritter Nadine M. , Farach-Carson Mary C. , Butler William T.
Published: February 2010

We hypothesize that the mechanisms governing bone formation and remodeling involve the assembly of some of the components of the extracellular matrix into supramolecular complexes. We have examined the associations of osteopontin (OPN) with other proteins isolated from demineralized rat long bones. Three ligand binding techniques were used to demonstrate the formation of complexes between osteopontin and osteocalcin (OCN). Using gel overlay assays, the binding between soluble 125I-OPN and OCN immobilized in acrylamide gels was visualized. Competition for 125I-OPN-OCN complexes was demonstrated when unlabeled OCN-enriched bone extract was included in gel overlay solutions. Also, gel overlay assays showed 125I-OCN binding to OPN. Saturable binding was shown in solid-phase filter binding assays, which yielded an equilibrium binding constant of moderately high affinity (∼ 10−8 M). Specificity of OPN-OCN complex formation was confirmed by measuring binding in the presence of unlabeled OPN and OCN versus a bone-localized serum protein, α2HS-glycoprotein. Finally, the formation of soluble complexes were demonstrated in a modified Hummel-Dreyer gel filtration assay. These results indicate that OPN and OCN form complexes in vitro. The possible functions of OPN-OCN complexes in osteoclast recruitment and attachment are discussed.
The bisphosphonate, alendronate, prevents bone loss in ovariectomized baboons
Authors: Thompson D.D. , Seedor J.G. , Quartuccio H. , Solomon H. , Fioravanti C. , Davidson J. , Klein H. , Jackson R. , Clair J. , Frankenfield D. , Brown E. , Simmons H.A. , Rodan G.A.
Published: February 2010

We examined the effect of the amino bisphosphonate alendronate, administered IV every 2 weeks at 0.05 and 0.25 mg/kg for 1 year, on bone loss and parameters related to bone metabolism in ovariectomized baboons. Relative to non-OVX animals, the OVX baboons experienced increased bone turnover, reflected in biochemical and histomorphometric measurements, and bone loss assessed by dual-beam absorptiometry in the lumbar spine, which was similar to changes observed in ovariectomized women. Alendronate treatment maintained all parameters of bone turnover at control (nonovariectomized) levels and prevented the bone loss in a dose-dependent manner. We concluded that ovariectomized baboons offer a suitable model for the bone changes observed in ovariectomized women and that these changes can be prevented by sustained administration of an appropriate dose of this aminobisphosphonate.
Disodium 1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphonate (EB-1053) is a potent inhibitor of bone resorption in vitro and in vivo
Authors: van der Pluijm Gabri , Binderup Lise , Bramm Erik , van derWee-Pals Lianne , de Groot Henny , Binderup Ernst , Löwik Clemens , Papapoulos Socrates
Published: February 2010

The ability of the new nitrogen-containing bisphosphonate disodium-1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphonate (EB-1053) to inhibit osteoclastic resorption was examined in vitro and in vivo. Results were compared to those obtained with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (pamidronate or APD). In vitro, when tested in osteoclast precursor-dependent systems (fetal mouse metacarpals and a coculture system), EB-1053 suppressed 45Ca release effectively and was found to be about 10 times more potent than pamidronate (ED50 = 2.5 × 10−7 versus 2.5 × 10−6 M, respectively). The EB-1053-inhibited osteoclastic resorption could be reversed by treatment with parathyroid hormone (PTH). In vivo, daily subcutaneous injections of EB-1053 to young growing rats for 7 days increased metaphyseal bone mass in tibiae dose dependently. In these experiments EB-1053 was about 50 times more potent than pamidronate. These studies show that EB-1053 is a very potent bisphosphonate that has potential use in the treatment of skeletal disorders.
Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106–01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells
Authors: Bjurholm Anders , Kreicbergs Andris , Schultzberg Marianne , Dr. Lerner Ulf H.
Published: February 2010

The effect of four different neuropeptides and norepinephrine (NE) on cyclic AMP formation in four different osteoblastic cell lines and in isolated neonatal mouse calvarial bone cells has been examined. In the rat osteosarcoma cell line UMR-106–01, vasoactive intestinal polypeptide (VIP, 0.001–1 μM), calcitonin gene-related peptide (CGRP, 0.3–30 nM), and NE (0.1–300 μM), but not neuropeptide Y (NPY, 0.001–1 μM) or substance P (SP, 0.1–10 μM), caused a dose-dependent stimulation of cyclic AMP formation. The stimulatory effects were synergistically potentiated by forskolin (0.1–3 μM). The effects of NE and VIP were time dependent, with an optimal effect seen at 5 minutes. The amount of cyclic AMP accumulated in cells stimulated with NE and VIP was in the same range. The amplitude of the cyclic AMP response induced by CGRP was smaller than that caused by VIP and NE. In the human osteosarcoma cell line Saos-2, NE (0.1 μM) and VIP (0.3 μM) stimulated cyclic AMP formation, and the effect was synergistically potentiated by forskolin. In the absence of forskolin, no effect of CGRP (30 nM) could be seen in the Saos-2 cells, but in the presence of forskolin (3 μM) a stimulatory effect was observed. SP and NPY did not change basal cyclic AMP levels in Saos-2 cells. In the osteoblastic osteosarcoma cell line of rat, ROS 17/2.8, NE (0.1 μM) caused a significant stimulatory action on cyclic AMP formation that was synergistically potentiated by forskolin (3 μM), VIP, CGRP, and SP did not affect the cellular content of cyclic AMP in ROS 17/2.8. In the mouse calvarial cell line MC3T3-E1 and in enzymatically isolated mouse calvarial bone cells, NE (0.1 μM), VIP (0.3 μM), and CGRP (30 nM), but not SP (10 μM) or NPY (1 μM), stimulated cyclic AMP formation. In UMR-106–01 cells and in isolated bone cells, but not in Saos-2 and ROS 17/2.8 cells, NPY (1 μM) inhibited the stimulatory effect of NE (0.3 μM) on cyclic AMP formation. These data show that osteoblastic cell lines are equipped with receptors for neuropeptides and NE. Since VIP, CGRP, NPY, and NE have been shown to occur in nerves of the skeleton, the observations in the present paper suggest the possibility that neurohormones may have a role in the regulation of osteoblastic activity.
Chronic adaptation of dog parathyroid function to a low-calcium-high-Sodium-Vitamin D-deficient diet
Authors: Cloutier M. , Gascon-Barré M. , D'Amour P.
Published: February 2010

The development of secondary hyperparathyroidism was studied in relation to changes in serum ionized Ca (Ca2+), 25-OHD, and 1,25-(OH)2D concentrations in six dogs maintained on a low-Ca (0.05%), high-Na (1.6%), and vitamin D-deficient diet for 91 weeks. Blood samples and evaluations of the parathyroid function were obtained before and after 3, 12, 24, 36, and 91 weeks of diet. Serum iPTH was measured by an intact hormone (I) and a carboxy-terminal (C) assay. The sigmoidal relationship between ionized Ca and iPTH values was evaluated mathematically. Results are means ± SD. Statistically significant changes over a time period were evaluated by an ANOVA for repeated measurements. Over the first 3 weeks, serum Ca2+, 25-OHD, and 1,25-(OH)2D did not change but stimulated I-iPTH increased 84.3 ± 39.9% (p < 0.005) and C-iPTH only 25.3 ± 12.2% (p < 0.01), a significant difference (p < 0.02). The increase in stimulated I-iPTH reached 487.4 ± 139.6% (p < 0.0001) and 418.4 ± 76.9% (p < 0.0001) for C-iPTH by the end of the study. Similar significant increases were seen in basal and nonsuppressible iPTH at or after week 12. Serum 25-OHD concentration had decreased significantly at 12 weeks (0 = 99.9 ± 28.3 to 12 = 40 ± 6.8 nM, p < 0.005) and even more so at 91 weeks (4.1 ± 0.3 nM, p < 0.0005); this could be related to stimulated I-iPTH at 12 weeks (r = −0.5472, p < 0.02) and thereafter, even though 25-OHD does not have a direct effect on the parathyroid glands. Even if serum Ca2+ had only decreased at week 36 (0 = 1.36 ± 0.02 to 36 = 1.32 ± 0.02 mM, p < 0.05), it could be inversely related to stimulated I-iPTH from week 24 (r = −0.4290, p < 0.05). Serum 1,25-(OH)2D increased up to 24 weeks (0 = 109.7 ± 20.7 to 24 = 173.4 ± 28.7 pM, p < 0.0005) at which time it was positively related to stimulated I-iPTH (r = 0.506, p < 0.02); it then decreased to slightly below basal level by week 91 (82.7 ± 8.6 pM). Despite these two added stimuli, progression of the parathyroid function did not accelerate after week 24. In conclusion, dietary changes appear to be the main factor stimulating secondary hyperparathyroidism in this model. The addition of a low serum Ca2+ and a relatively low level of 1,25-(OH)2D did not enhance the effect of the diet.
Assessment of spinal and femoral bone density by Dual X-Ray absorptiometry: Comparison of lunar and hologic instruments
Authors: Dr. Pocock Nicholas A. , Sambrook Philip N. , Nguyen Tuan , Kelly Paul , Freund Judith , Eisman John A.
Published: February 2010

Clinical application of techniques for assessing bone mineral density (BMD) requires accurate and precise measurements that can be related to clearly defined normal ranges. In this study we investigated the clinical interpretation of BMD values in a group of individuals measured on the same day with two different dual-energy x-ray densitometers (Lunar DPX and Hologic QDR 1000). The BMD results were analyzed as absolute values in g/cm2 and with respect to young and age-specific normals as defined by each manufacturer. Absolute BMD values measured by the two instruments were highly correlated (lumbar spine r = 0.98, femoral neck r = 0.95; p < 0.0001). In the lumbar spine, the two instruments assigned almost identical values when expressed as a percentage of age-matched values and as a percentage of young normals, despite a small but systematic difference between the values assigned for the latter index. In the femoral neck, however, there were significant differences in assignments between instruments, expressed both as a percentage of young normal (mean difference 6.2%) and with respect to age-matched values (mean difference 3.3%). In particular, in premenopausal subjects femoral neck values with the Hologic instrument were assigned significantly lower values. This study shows effective comnparability between these two instruments for absolute and relative values for the lumbar spine, as well as for absolute values at the femoral neck, but important differences for normality assignments at the femoral neck. These latter differences may produce bias in the “diagnosis” of femoral neck osteoporosis and may have important implications for clinical decision making. Until these differences are resolved, clinicians should not rely solely on femoral neck BMD measurements in clinical decision making.
Restoring and maintaining bone in osteopenic female rat skeleton: I. Changes in bone mass and structure
Authors: Tang Li Ya , Jee Webster S.S. , Ke Hua Zhu , Kimmel Donald B.
Published: February 2010

This experiment contains the crucial data for the lose, restore, and maintain (LRM) concept, a practical approach for reversing existing osteoporosis. The LRM concept uses anabolic agents to restore bone mass and architecture (+ phase) and then switches to an agent with the established ability to maintain bone mass, to keep the new bone (± phase). The purpose of this study was to learn whether switching to an agent known chiefly for its ability to maintain existing bone mass preserves new bone induced by PGE2 in osteopenic, estrogen-depleted rats. The current study had three phases, the bone loss (-), restore (+), and maintain (±) phases. We ovariectomized (OX) or sham ovariectomized (sham-OX) 5.5-month-old female rats (- phase). The OX rats were treated 5 months postovariectomy with 1–6 mg PGE2 per kg/day for 75 days to restore lost cancellous bone mass (+ phase), and then PGE2 treatment was stopped and treatment began with 1 or 5 μg/kg of risedronate, a bisphosphonate, twice a week for 60 days (± phase). During the loss (-) phase, the cancellous bone volume of the proximal tibial metaphysis in the OX rat fell to 19% of initial and 30% of age-matched control levels. During the restore (+) phase, the cancellous bone volume in OX rats doubled. When PGE2 treatment was stopped, however, and no special maintenance efforts were made during the maintain (±) phase, the PGE2-induced cancellous bone disappeared. In contrast, the PGE2-induced cancellous bone persisted when the PGE2 treatment was followed by either a 1 or 5 μg treatment of risedronate per kg given twice a week for 60 days during the maintain (±) phase. The tibial shaft demonstrated very little cortical bone loss during the loss (-) phase in OX rats. The tibial shaft cortical bone fell some 8%. During the restore (+) phase, new cortical bone in OX rats increased by 22%. When PGE2 treatment was stopped and nothing was given during the maintain (±) phase, however, all but the PGE2-induced subperiosteal bone disappeared. In contrast, when PGE2 treatment was stopped and 1 μg risedronate per kg twice a week for 60 days was administered during the maintenance (±) phase, the PGE2-induced subperiosteal bone and some of the subendocortical bone and marrow trabeculae persisted. When 5 μg risedronate per kg was given twice a week, all the PGE2-induced bone persisted. The study shows that most of the new cancellous and cortical bone induced by PGE2 can be maintained for at least 60 days after discontinuing PGE2 by administering enough of the resorption inhibitor, risedronate. The lower dose of risedronate was not adequate to save most of the PGE2-induced endocortical bone.
Demonstration of sodium/calcium exchange in rodent osteoblasts
Authors: Krieger Nancy S.
Published: February 2010

Based on the inhibition of stimulated Ca release from cultured bone by several different agents that alter Na transport, we proposed that hormonally stimulated bone resorption requires Na/Ca exchange. Calcemic hormones appear to interact primarily directly with the osteoblast, which then mediates the activation of osteoclast activity. In organ culture it is not possible to determine whether Na/Ca exchange is involved in this initiating step in the osteoblast or directly in osteoclast-mediated Ca release, and there have been no prior direct measurements of Na/Ca exchange in bone or bone cells. The purpose of this study was to demonstrate the presence of Na/Ca exchange transport in the osteoblast. Thus, we characterized Na-dependent Ca transport in osteoblast-like rat osteosarcoma cells (UMR-106) and primary bone cells isolated from neonatal mouse calvaria. Cells were loaded with the Ca-sensitive dye fura-2 in the presence of physiologic NaCl and the absence of Ca with or without 0.3 mM ouabain. Changes in free cytosolic Ca after the extracellular addition of 1.5 mM Ca were measured spectrofluorimetrically. An outward Na gradient was generated by decreasing extracellular Na while maintaining isotonicity. UMR-106 cells that were Na loaded by ouabain inhibition of Na,K-ATPase activity exhibited 30% greater Ca uptake than control cells. Similar results were obtained with primary bone cells. This uptake required extracellular Ca, was not inhibited by 200 μM verapamil, and was reversible upon reversal of the Na gradient. These data demonstrate the presence of a Na/Ca exchange transport system in osteoblasts.
Release of oxygen radicals by articular chondrocytes: A study of luminol-dependent chemiluminescence and hydrogen peroxide secretion
Authors: Rathakrishnan Chamu , Tiku Katherine , Raghavan Anuradha , Tiku Moti L.
Published: February 2010

We previously established that normal articular chondrocytes, like macrophages, express class II major histocompatibility antigens, present antigen, and induce mixed and autologous lymphocyte stimulation. In a recent study using the trapped indicator 2′,7′-dichlorofluorescein diacetate, we were able to measure levels of intracellular hydrogen peroxide within normal articular chondrocytes (J Immunol 245:690–696, 1990). In the present study, we utilized the technique of chemiluminescence and the biochemical method of quantitating hydrogen peroxide release to measure the production of reactive oxygen intermediates by articular chondrocytes. Chondrocytes, in suspension or adherent to coverslips, showed luminol-dependent chemiluminescence that was dependent on the number and viability of cells. There was a dose-dependent increase in chemiluminescence in response to soluble stimuli, such as phorbol myristate acetate (PMA), concanavalin A (ConA), and f-Met-Leu-Phe (FMLP). Azide inhibited chemiluminescence, suggesting that the light emission in chondrocytes is myeloperoxidase dependent. The antioxidant, catalase, inhibited chemiluminescence but superoxide dismutase had no effect, suggesting that luminol-dependent chemiluminescence in chondrocytes mostly measured hydrogen peroxide. Chemiluminescence was also observed in fragments of live cartilage tissue, indicating that chondrocytes that are cartilage matrix bound can generate the respiratory burst response. Using the scopoletin oxidation assay, we confirmed the release of increasing amounts of hydrogen peroxide by chondrocytes exposed to interleukin-1, rabbit interferon, and tumor necrosis factor α. Tumor necrosis factor α had both priming and enhancing effects on reactive oxygen intermediate production by chondrocytes. Reactive oxygen intermediates have been shown to play a significant role in matrix degradation. We suggest that reactive oxygen intermediates produced by chondrocytes play an important role in the degradation of matrix in arthritis.
Zeolite a increases proliferation, differentiation, and transforming growth factor β production in normal adult human osteoblast-like cells in vitro
Authors: Keeting Philip E. , Oursler Merry Jo , Wiegand Karl E. , Bonde Susan K. , Spelsberg Thomas C. , Riggs B. Lawrence
Published: February 2010

Silicon in trace amounts enhances bone formation, and the silicon-containing compound zeolite A (ZA) increases eggshell thickness in hens. In the studies reported here, treatment of nearly homogeneous strains of normal human osteoblast-like cells for 48 h with ZA at 0.1–100 μg/ml induced a dose-dependent increase (r = 0.35, P < 0.001) in DNA synthesis (n = 31) to 162 ± 16% (mean ± SEM) of control and in the proportion of cells in mitosis (n = 4) from 9.1 ± 1.8 to 27.0 ± 4.5% (r = 0.69, P < 0.005). ZA treatment also increased alkaline phosphatase activity (P < 0.05) and osteocalcin release (P < 0.05) but did not significantly affect collagen production per individual cell. The mitogenic action of ZA was dependent on cell seeding density over the range of 1250–40,000 cells per cm2, which is consistent with induction of an autocrine factors). TGF-β is a potent mitogen for osteoblasts. ZA treatment increased the steady-state mRNA levels of transforming growth factor β1 (TGF-β1) and induced the release of the latent form of TGF-β protein into the conditioned medium within 6 h. We conclude that ZA induces the proliferation and differentiation of cells of the osteoblast lineage.
Preparation and characterization of a mouse osteoclast-like multinucleated cell population
Authors: Akatsu Takuhiko , Tamura Tatsuya , Takahashi Naoyuki , Udagawa Nobuyuki , Tanaka Sakae , Sasaki Takahisa , Yamaguchi Akira , Nagata Naokazu , Dr. Suda Tatsuo
Published: February 2010

We have reported that numerous tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated cells (TRAP+ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1α25-dihydroxyvitamin D3 [1α,25-(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gelcoated dishes. The TRAP+ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+ MNCs was 20,000–40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (∼ 1000 cells per head). The purity of TRAP+ MNCs was 5.6 ± 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+ MNCs after density gradient centrifugation was 30–40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+ MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kd for TRAP+ MNCs of 8.9 ± 0.7 × 1010 M and 16.5 ± 1.5 ± 106 binding sites per cell. These results were similar to the value (3.5 × 10−10 M) and the number of binding sites (3.3 × 106 per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: salmon CT at 10−10 M, human CT at 10−8 M; ED50: salmon CT, 2.2 × 10−11 M, human CT, 1.3 × 10−9 M) in the MNC preparation. These results indicate that a large number of mouse TRAP+ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.
Interrelationship between parathyroid hormone and insulin: Effects on DNA synthesis in UMR-106–01 cells
Authors: Felsenfeld Arnold J. , Iida-Klein Akiko , Hahn Theodore J.
Published: February 2010

UMR-106–01 osteoblast-like cells respond to high concentrations of parathyroid hormone (PTH) in vitro by decreasing thymidine incorporation, a marker of DNA synthesis and cell proliferation. This response is different from in vivo conditions, such as primary and secondary hyperparathyroidism, in which high PTH levels are associated with an increased number of osteoblasts. When the response of UMR-106–01 cells to PTH is evaluated in vitro, however, these cells are exposed to only a single hormone. The present study was designed to evaluate the combined effects of two hormones, PTH and insulin, on the DNA synthesis of UMR-106–01 cells. PTH is known to decrease and insulin to increase thymidine incorporation by UMR-106–01 cells. To examine the interaction of these hormones, acute studies, defined as a 24 h exposure to hormone, and chronic studies, defined as a 7 day exposure to hormone, were performed. Both acute and chronic exposure to 10−9 M PTH decreased thymidine incorporation by UMR-106–01 cells, with suppression ranging from 27 to 81% (P < 0.05). Both acute and chronic exposure to 10−8 M insulin (INS) increased thymidine incorporation by UMR-106–01 cells; this ranged from 26 to 58% (P < 0.05). However, chronic exposure to 10−9 M PTH followed by an acute exposure to 10−8 M INS resulted in a 710% increase in thymidine incorporation (P < 0.01). Reversing the sequence by chronically exposing UMR-106–01 cells to 10−8 M INS followed by acute exposure to 10−9 M PTH resulted in a 53% decrease in thymidine incorporation (P < 0.01). When 10−9 M PTH and 10−8 M INS were administered simultaneously, the increase in thymidine incorporation produced by acute and chronic administration ranged from 35 to 56% of nontreated control values (P < 0.05). In conclusion, the timing of PTH and insulin administration may produce different responses in thymidine incorporation by UMR-106–01 cells. Whether during hyperparathyroidism in vivo, pulsatile increases in insulin secretion are important for osteoblast proliferation remains to be determined.