bluuebunny/crossref_metadata_2025 huggingface dataset:
Family history of osteoporosis and bone mineral density at the axial skeleton: The rancho bernardo study
Authors: Soroko Sharon B. , Barrett-Connor Elizabeth , Edelstein Sharon L. , Kritz-Silverstein Donna
Published: February 2010

To determine whether a family history of osteoporosis identifies individuals with low bone mineral density (BMD), we studied 1477 white elderly (aged 60–89 years), noninstitutionalized ambulatory men (n = 600) and women (n = 877) from the Rancho Bernardo, California cohort. Family history data on biologic parents and full sisters were obtained by questionnaire. BMD of the lumbar spine and hip was measured using dual-energy x-ray absorptiometry. After adjustment for age, body mass index, history of cigarette smoking, thiazide use, and estrogen use, men and women with a family history of osteoporosis had lower BMD than those with a negative family history. In men, a positive family history was associated with lower BMD at the hip (p = 0.01), whereas in women a significant association was observed for the spine (p = 0.02). BMD decreased in a stepwise fashion with an increasing number of family members with a history of osteoporosis. Analysis of the effect of parental history of osteoporosis on BMD showed a significant relation between paternal (but not maternal) history and lumbar spine BMD in both sexes and a significant relation between maternal (but not paternal) history and hip BMD only in men. The relative risk of having categoric osteopenia was highest in those whose fathers had a history of osteoporosis (RR 2.16, 95% CI = 1.38–3.37). A similar association was found for subjects with fractures. These results were not explained by differential awareness of family history in individuals with known osteoporosis, because the prevalence of family history was unrelated to personal history of osteoporosis in men and only weakly related in women. The positive predictive value of family history as an indicator of categorically defined low bone density was 22% in men and 24% in women, although in women this value increased to 33% when father's history alone was considered. The negative predictive value of overall family history was 65% in men and 81% in women. Overall, these data suggest that clinicians who ask patients about family history of osteoporosis should ask about both parents.
Stimulation of prostaglandin e2 production by interleukin-1α and transforming growth factor α in osteoblastic MC3T3-E1 cells
Authors: Harrison J.R. , Lorenzo J.A. , Kawaguchi H. , Raisz L.G. , Pilbeam C.
Published: February 2010

The mechanism by which interleukin-1 (IL-1) and transforming growth factor α (TGF-α) regulate prostaglandin synthesis has been examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in DMEM containing 10% fetal calf serum. Prostaglandin E2 (PGE2) production was determined by radioimmunoassay or by prelabeling cells with [H]arachidonic acid, followed by high-performance liquid chromatography (HPLC) analysis of the labeled products released into the medium. Prostaglandin G/H synthase (PGHS) mRNAs were quantified by northern blot analysis using [32P]labeled cDNA probes. By HPLC, PGE2 was the major prostanoid produced under basal or stimulated conditions. No release of thromboxane or 6-keto-PGF1α into the medium was detected. PGE2 production was stimulated approximately 7- to 14-fold by IL-1 (1 ng/ml) and 3- to 8-fold by TGF-α (30 ng/ml) after 24 h. In combination, however, IL-1 and TGF-α caused a synergistic 37- to 71-fold increase in PGE2 accumulation. PGHS-1 mRNA levels were maximally increased approximately 2- to 3-fold by IL-1 and 1.5 to 2.5-fold by TGF-α after 24 h; the combination of IL-1 and TGF-α produced only an additive 3- to 6-fold increase. Western blotting revealed a corresponding 3-fold increase in immunoreactive PGHS-1 protein in response to combined IL-1 and TGF-α. PGHS-2 mRNA was increased 1.4-fold by TGF-α at 1 h, and the combination of IL-1 and TGF-α caused a 1.7-fold increase. After 3.5 h, IL-1 caused a dramatic induction of PGHS-2 mRNA levels but TGF-α alone no longer had an effect. However, the combination of IL-1 and TGF-α produced an increase in PGHS-2 mRNA levels that was twice that of IL-1 alone. The effects of IL-1 and TGF-α on the release of preincorporated [H]arachidonic acid from membrane phospholipid stores were examined at early time points in the presence of indomethacin. After 1 h, arachidonic acid release was enhanced 3-fold by IL-1, 1.5-fold by TGF-α, and 12-fold by IL-1 and TGF-α in combination. In conclusion, the synergistic actions of IL-1 and TGF-α on PGE2 synthesis in MC3T3-E1 cells involve multiple regulatory sites, including stimulation of de novo PGHS-1 and PGHS-2 synthesis and an early mobilization of arachidonic acid from phospholipid stores.
Immunolocalization of osteopontin, osteocalcin, and dentin sialoprotein during dental root formation and early cementogenesis in the rat
Authors: Bronckers Antonius L.J.J. , Farach-Carson Mary C. , Van Waveren Erwin , Butler William T.
Published: February 2010

Using immunohistochemical methods we studied the tissue localization of the extracellular matrix proteins osteopontin (OPN), osteocalcin (OC), and dentin sialoprotein (DSP) during the formation of acellular and cellular cementum in newly born rats. In the layer of acellular cementum of developing incisor and molar teeth we found a very strong staining for OPN but not for DSP or OC. Many cells immediately adjacent to acellular cementum and PDL cells were also positive for OPN but not for DSP or for OC. In contrast, cellular cementum in molar teeth stained strongly for OPN and OC but not for DSP. Consistent with these observations, the cells engaged in the formation of cellular cementum (cementoblasts and cementocytes) reacted strongly for OPN and OC but not for DSP. In advanced stages of dentinogenesis, both crown and root odontoblasts and dentin stained for OPN, OC, and DSP. Cells and matrices of surrounding alveolar bone stained for OPN and OC but not for DSP. We conclude that cementoblasts and cementocytes of cellular cementum produce OPN and OC but not DSP and thus express an osteoblast-like, not an odontoblast-like, phenotype. The cells responsible for the production of acellular cementum are likely cells of the PDL in close contact with the dental root surface. These fibroblast-like cells express OPN but not OC or DSP and accordingly express only a partial osteoblastic phenotype.
Studies on the regulation of insulin-like growth factor binding protein 3 secretion in human osteosarcoma cells in vitro
Authors: Nakao Y. , Hilliker S. , Baylink D.J. , Mohan S.
Published: February 2010

Previous studies demonstrated that insulin-like growth factors (IGFs) are important autocrine and paracrine mitogens for human bone cells in vitro and that IGF binding proteins (IGFBPs) are important regulators of the biologic actions of IGFs. Thus, the actions of IGFs may be determined not only by their concentrations but also by the type and amount of IGFBPs produced by human bone cells at a local site in bone. In this study, we sought to determine the effects of dexamethasone, 1,25-(OH)2 D3, and parathyroid hormone (PTH) on the secretion of IGFBP-3 in human osteosarcoma cell lines. Serum-free cultures of low- and high-alkaline phosphatase (ALP) SaOS-2, MG-63, and TE89 human osteosarcoma cells were treated for 24 or 48 h with the effectors and the conditioned media used for determination of IGFBP-3 using a radioimmunoassay. We report that (1) the basal rate of IGFBP-3 secretion (ng/mg cellular protein) was dependent upon cell type, with TE89 > low-ALP Saos-2 > MG-63 > high-ALP SaOS-2 cells, and did not correlate with either basal cell proliferation or basal cellular ALP activity; (2) dexamethasone (10−12-10−7 M) inhibited IGFBP-3 secretion in a dose-dependent manner in low-ALP SaOS-2, MG-63, and TE89 cells but not in high-ALP SaOS-2 cells; (3) 1,25-(OH)2D3 (10−11-10−8 M) stimulated IGFBP-3 secretion in a dose-dependent manner in MG-63, low-ALP SaOS-2, and high-ALP SaOS-2 cells, and the coaddition of TGF-β and 1,25-(OH)2D3 increased synergistically IGFBP-3 secretion and cellular ALP activity in MG-63 cells; and (4) human PTH-(1–34) (0.1–100 ng/ml) had no significant effect on IGFBP-3 secretion in MG-63, low-ALP SaOS-2, or high-ALP SaOS-2 cells. We conclude that such agents as dexamethasone, 1,25-(OH)2D3, and PTH differentially regulate IGFBP-3 secretion in human osteosarcoma cells in vitro.
Analysis of gene expression in human bone biopsies by polymerase chain reaction: Evidence for enhanced cytokine expression in postmenopausal osteoporosis
Authors: Ralston Stuart H.
Published: February 2010

It has been suggested that one of the mechanisms by which estrogen protects against postmenopausal osteoporosis is by modulating the production of cytokines, such as interleukin-1 (IL-1), tumor necrosis factors (TNF), and interleukin-6 (IL-6), in the bone microenvironment. In this study, reverse transcription/polymerase chain reaction (RT/PCR) was used to analyze expression of the mRNAs encoding these cytokines in freshly isolated human bone biopsy samples. Marked differences were found in the prevalence with which certain cytokines were expressed in different patient subgroups. Specifically, IL-1α, IL-1β, and IL-6 mRNAs were expressed significantly more often in bone samples from postmenopausal women with osteoporotic fractures than in postmenopausal women with normal bone density or postmenopausal women on hormone replacement therapy (HRT). The prevalence of IL-1α expression was 54% in bone samples from patients with osteoporotic fractures (n = 22), compared with 30% in nonosteoporotic postmenopausal patients (n = 10) and 10% in postmenopausal patients on HRT (difference between groups by χ test = 7.0; DF = 2,p < 0.05). Corresponding figures for IL-1β were 54 versus 30 versus 0% (χ = 8.6; DF = 2, p < 0.01) and, for IL-6, 94 versus 51 versus 40%; χ = 13.5; DF = 2, p < 0.01). TNF-α was expressed in a similar proportion of osteoporotic (63%) and normal postmenopausal patients (60%), whereas only 10% of HRT-treated patients showed expression of TNF-α (χ = 8.2; DF = 2, p < 0.05). TNF-β was less commonly expressed in osteoporosis (18%) and was not detected at all in normal postmenopausal or HRT-treated patients, but this difference was not significant. Cytokine expression in bone samples from young healthy individuals showed sex-dependent differences in cytokine expression. In males, the pattern was similar to that observed in normal postmenopausal patients, with IL-1α expression in 36%, IL-1β in 36%, TNF-α in 63%, TNF-β in 9%, and IL-6 in 54% of cases. In young females (n = 7), however, the patterns of expression were similar to those in the HRT group, with IL-1α expression in 28%, IL-1β in 14%; TNF-α in 43%, TNF-β in 6%, and IL-6 in 28%. These experiments demonstrate that several cytokine mRNAs are expressed in the bone microenvironment. The high proportion of osteoporotic biopsies that showed expression of IL-1α, IL-1β, TNF-α, and IL-6 mRNAs lends support to the hypothesis that activation of cytokine expression in bone is a feature of postmenopausal osteoporosis and raises the possibility that local release of these cytokines contributes to bone loss in this condition.
Reversal of vertebral deformities in osteoporosis: Measurement error or “rebound”?
Authors: Nelson D.A. , Kleerekoper M. , Peterson E.L.
Published: February 2010

Digitized morphometry of vertebral bodies on lateral spine films is used to identify and quantify vertebral deformities or fractures. One problem associated with this method is the phenomenon of “disappearing fractures,” which results from the apparent increase in vertebral body heights of previously deformed vertebrae on subsequent radiographs. These have been considered biologically implausible and therefore a result of measurement error. Measurement error is unlikely to be unidirectional, so that a proportion of fractures identified by morphometry is also the result of measurement error. Since some vertebral deformities are real events, some disappearances of deformities detected by morphometry may be real events. In this report, we examine the data from our clinical trial of sodium fluoride in spinal osteoporosis to assess critically the plausibility of two hypotheses: (1) The “rebound” phenomenon results from measurement error. If this is the case, then some fractures of the same magnitude as the rebound must also represent measurement error. (2) Some deformed vertebrae in fact rebound toward their original shape and size, displaying an elastic response to deformation. If this occurs, then some vertebral deformities are transient events, not true fractures. We conclude that the variability inherent in morphometric data obtained from serial spine x-rays results in both disappearing fractures and a high false positive fracture rate. The use of more stringent criteria for defining significant deformities, or true fractures, will minimize these problems. We cannot exclude the second hypothesis, that some vertebral deformities may be transient events, but this needs further study.
Bone resorption assessed by immunoassay of urinary cross-linked collagen peptides in patients with osteogenesis imperfecta
Authors: Dr. Brenner Rolf E. , Vetter Ulrich , Bollen Anne-Marie , Mörike Martin , Eyre David R.
Published: February 2010

Urinary excretion of type I collagen cross-linked N-telopeptides was studied in 52 children and adolescents with osteogenesis imperfecta (OI) and found to be above the 75th percentile of controls in 44 of the patients. OI patients suffering from fractures during the preceding 6 months had significantly higher values (p < 0.05). In contrast, patients with better motor performance tended to have lower values (p = 0.059). The concentration of urinary type I collagen cross-linked N-telopeptides was positively correlated with urinary calcium excretion (p < 0.05), which was found to be elevated in 20 of the patients. Our results show that during childhood and adolescence in OI not only the synthesis but also the turnover of mature cross-linked type I collagen is disturbed and provide evidence that bone resorption rates are elevated.
Geometric structure of the femoral neck measured using dual-energy X-ray absorptiometry
Authors: Yoshikawa T. , Turner C.H. , Peacock M. , Slemenda C.W. , Weaver C.M. , Teegarden D. , Markwardt P. , Burr D.B.
Published: February 2010

An algorithm was developed to estimate the strength of the femoral neck from data generated by the dual-energy x-ray absorptiometry (DXA). This algorithm considers shape of the proximal femur as well as cross-sectional moment of inertia (CSMI) in the estimate. Proximal femora (10) from cadavers of white adults and an aluminum step wedge were scanned with the Lunar DPX to validate the calculation of CSMI. After scanning, each femoral neck was sectioned at its narrowest portion for direct measurement of CSMI. Three healthy young women were scanned five times each to evaluate the reproducibility of geometric measurements using DXA. There was a strong linear association between the CSMI measured directly and using DXA in both cadaver bones (r2 = 0.96) and the aluminum step wedge (r2 = 0.99). The coefficient of variation for CSMI from repeated measurements using DXA was less than 3%. This indicates that it is possible to estimate reproducibly the bending rigidity of bone from DXA measurements. The data from 306 normal subjects were analyzed to investigate geometric changes in the femoral neck with age. Although there was no strong correlation between CSMI and age in normal subjects of either sex, safety factor (SF, an index of strength of the femoral neck during walking) and fall index (FI, an index of the strength of the femoral neck during a fall) decrease with age in both sexes. We observed an alteration of the geometric structure of the femoral neck with age that may increase the stress on the femoral neck and decrease SF and FI.
Cellular and matrix changes before and at the time of calcification in the growth plate studied in vitro: Arrest of type X collagen synthesis and net loss of collagen when calcification is initiated
Authors: DR. Alini Mauro , Carey Dennis , Hirata Soichiro , Grynpas Marc D. , Pidoux Isabelle , Poole A. Robin
Published: February 2010

To understand the growth, maturation, and regulation of growth plate chondrocytes, it is necessary to isolate the different chondrocytes into distinct subpopulations of maturational development. Five subpopulations (A—E) of bovine fetal growth plate chondrocytes were separated by discontinuous gradient centrifugation. Four subpopulations (B, C, D, and E, from low to high density) with good viability were cultured at high density in microwells for up to 30 days. They all established an extensive extracellular matrix composed of proteoglycan and collagen. The largest and least dense cells in subpopulation B were the first to synthesize (at days 5–6) type X collagen and to calcify this matrix. Matrix calcification (formation of hydroxyapatite in the presence of sodium β-glycerophosphate) always followed the initiation of type X synthesis. All the other subpopulations synthesized type X collagen and calcified their extracellular matrix. Although these events occurred in the same order, they were delayed according to the order of increasing cell size. These observations indicate that these subpopulations represent different stages in cellular maturation that lead to expression of the hypertrophic phenotype. Once mineral formation was well established, there was an increase in the matrix content of the C-propeptide of type II collagen (which is known to bind to hydroxyapatite and accumulate in calcifying extracellular matrix). This was accompanied by a reduction in the total collagen content, which accompanied an abrupt reduction in type X collagen synthesis, whereas type II collagen synthesis was largely maintained. These reductions in collagen content and type II collagen synthesis were not observed in the absence of calcification (β-glycerophosphate omitted from culture). This new culture system recreates many of the sequential cellular and extracellular changes exhibited in situ during the development of the physis and provides new information about cellular and extracellular matrix changes that occur before and at the time of calcification.
High-dose glucocorticoids in multiple sclerosis patients exert direct effects on the kidney and skeleton
Authors: Dr. Cosman F. , Nieves J. , Herbert J. , Shen V. , Lindsay R.
Published: February 2010

The effects of acute pharmacologic steroid treatment on skeletal and mineral metabolism were assessed in 56 multiple sclerosis patients who were to receive 1 g intravenous methylprednisolone for 10 days, followed by a 4 day intravenous and 28 day oral glucocorticoid taper. Serum and urine samples were obtained at baseline and then within 3 days, 1, 2, and 3 weeks after beginning steroids. A subset of patients (n = 11) had sampling throughout the 6 weeks of steroid administration and up to 8 weeks afterward. All mean basal biochemistries were normal except 25(OH)D, which was in the “insufficient” range (25-50 nM) at 40 nM. During and after steroid administration, there were no changes in ionized calcium, 25(OH)D, urinary hydroxyproline, or pyridinoline. There was an increase in 1,25(OH)2D and a decrease in serum phosphorus, accompanied by an increase in urinary phosphate clearance, within 3 days of administration (p < 0.006). Serum osteocalcin (BGP) decreased to below assay sensitivity limits within 3 days of steroid administration (p < 0.0002), increasing thereafter but remaining at 50% of baseline by the third week. PTH(1-84) increased to a peak at week 2 (p < 0.02), after both the 1,25(OH)2D peak and the serum phosphorus nadir. Tartrate-resistant acid phosphatase, urinary calcium, and urinary cyclic AMP all increased above baseline (p < 0.05) with a pattern similar to that of PTH. To investigate further the immediate effects of steroid administration, serum samples were obtained at the same four times on both the day before and the day after the first intravenous methylprednisolone dose in a randomly chosen subset of patients (n = 9). Serum phosphorus fell (p < 0.05) by 1 h after administration, associated with an immediate drop in the tubular maximum phosphate reabsorption concentration adjusted for glomerular filtration rate (TmP/GFR). Serum 1,25(OH)2D increased (p < 0.04) and BGP decreased (p < 0.0001) within 8 h after steroid administration. The decrease in serum phosphorus, increase in 1,25(OH)2D, and depression in BGP were all independent of PTH, which did not change during this time period. The profound drop in BGP was particularly significant given the rise in 1,25(OH)2D (a known stimulator of BGP synthesis). In conclusion, these studies support the theory that high-dose glucocorticoid administration causes primary depression of bone formation as well as primary effects on the kidney, including lowering the TmP/GFR and increasing 1,25(OH)2D, all before increasing PTH levels.
Hormonal regulation of Na+-Ca2+ exchange in osteoblast-like cells
Authors: Short Cynthia L. , Monk Rebeca D. , Bushinsky David A. , Krieger Nancy S.
Published: February 2010

We proposed a role for Na-Ca exchange in hormonally mediated bone resorption and recently characterized Na-dependent Ca transport in an osteoblast-like rat osteosarcoma cell line (UMR-106). To test whether calcemic agents alter Na+-Ca2+ exchange in osteoblasts, UMR cells were treated acutely or cultured in the absence or presence of calcemic agent for 24h. Cells were then loaded with the Ca-sensitive dye fura-2 in the presence of 140 mM NaCl, no Ca, and the absence or presence of 0.3 mM ouabain. Cells were resuspended at 22°C, and the fluorescence ratio at excitation wavelength of 340 and 380 nm was measured. An outward Na gradient was generated by removing extracellular Na and maintaining isotonicity with choline chloride. Na+-Ca2+ exchange was demonstrated by enhanced Ca uptake in ouabain-treated (Na-loaded) cells after the addition of 1.5 mM Ca. Acute addition of 10−7 M PTH or 10−6 M PGE2 had no effect on Na-dependent Ca uptake. However, 24 h treatment of cells with PTH, PGE2, or 1,25(OH)2D3 caused a dose-dependent inhibition of Na+-Ca2+ exchange. Using the Na-sensitive dye, SBFI, we also demonstrated that the effect was bidirectional; PTH inhibited Ca-dependent Na uptake comparably to its inhibition of Na-dependent Ca uptake. The effects of the calcemic agents were mimicked by 24 h treatment of the cells with 1 μM forskolin or 2 μM PMA. These results suggest that regulation of Na+-Ca2+ exchange by calcemic agents occurs downstream of signal transduction messengers and that alterations in Na+-Ca2+ exchange may play an integral role in their long-term regulation of osteoblast activity.
Parathyroid hormone fragments may stimulate bone growth in ovariectomized rats by activating adenylyl cyclase
Authors: Rixon Ray H. , Whitfield James F. , Gagnon Lyne , Isaacs Richard J. , Maclean Susanne , Chakravarthy Balu , Durkin Jon P. , Neugebauer Witold , Ross Virginia , Sung Wing , Willick Gordon E.
Published: February 2010

PTH is regarded conventionally as a catabolic hormone that stimulates osteoclastic resorption of bone. However, it has been known since 1932 that intermittent pulses of PTH stimulate bone formation in animals and humans. PTH independently activates two signal mechanisms: one that stimulates adenylyl cyclase and one that stimulates protein kinase C (PKC). The goal of this study was to use the 3- to 5-month-old ovariectomized (OVX) rat model to determine which of the two signal mechanisms is responsible for the anabolic action of PTH on bone. OVX triggered a large loss of trabecular bone without significantly affecting the normal slow growth of cortical bone in the distal halves of the femora. Daily injections of human hPTH(1–34) fragment (1 nmol/100 g body weight), which stimulated both adenylyl cyclase and membrane-associated PKC activity in osteoblast-like ROS 17/2 rat osteosarcoma cells, stimulated the growth of both cortical and trabecular bone in the OVX rats. Daily injections of the same dose of hPTH(1–31), which stimulated adenylyl cyclase but not PKC in ROS 17/2 cells, stimulated trabecular bone growth in the OVX rats less effectively than hPTH(1–34), but it stimulated cortical bone growth as rapidly and as dramatically as hPTH(1–34). Injections of equimolar amounts of desamino-hPTH(1–34) [N-propionyl(2–3)hPTH-amide], which stimulated PKC as strongly as hPTH(1–34) in ROS 17/2 cells but had a drastically reduced ability to stimulate adenylyl cyclase, or injections of recombinant hPTH(8–84) which stimulated PKC only in the ROS 17/2 cells, did not stimulate cortical or trabecular bone growth in the OVX animals. Thus, cyclic AMP and cyclic AMP-dependent protein kinases may be the primary mediators of the anabolic action of intermittent pulses of PTH on bone in OVX rats.
Bone morphogenetic protein 2 stimulates osteogenesis but does not affect chondrogenesis in osteochondrogenic differentiation of periosteum-derived cells
Authors: Iwasaki Motoki , Nakahara Haruhiko , Nakase Takanobu , Kimura Tomoatsu , Takaoka Kunio , Caplan Arnold I. , Ono Keiro
Published: February 2010

The effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) on osteochondrogenesis were examined in high-density cultures of periosteum-derived cells, which have the potential to differentiate into bone and hypertrophic cartilage in vitro. Proliferation of these cells was inhibited by treatment with rhBMP-2. The time course for alkaline phosphatase (ALP) expression was shortened and the mineralization of the culture was increased by supplementation with rhBMP-2. These stimulatory effects of rhBMP-2 were observed at doses of 10–100 ng/m. Bone Gla protein (BGP) was immunocytochemically detectable earlier in the culture treated with rhBMP-2, and the BGP-positive layer of the rhBMP-2-treated cultures was thicker than that of the control cultures. On the other hand, there was no difference in uronic acid content or the time course of α1 (II) collagen mRNA expression between the rhBMP-2-treated and the control cultures. These results indicate that rhBMP-2 shortens the time course of osteogenesis and increases the amount of bone formation, whereas chondrogenesis remains unaffected.
Hypertrophic chondrocytes undergo further differentiation to osteoblast-like cells and participate in the initial bone formation in developing chick embryo
Authors: Galotto Maria , Campanile Giuliano , Robino Grazia , Cancedda Fiorella Descalzi , Bianco Paolo , DR. Cancedda Ranieri
Published: February 2010

Differentiation of hypertrophic chondrocytes to an osteoblast-like phenotype occurs in vivo in the hypertrophic cartilage of chick embryo tibiae underneath early or prospective periosteum and in cartilage around vascular canals. Synthesis of type I collagen by hypertrophic chondrocytes was shown by immunolocalization of the C propeptide. By enzyme cytochemistry it was instead shown that, in vivo, further differentiating hypertrophic chondrocytes express alkaline phosphatase at the time of initial mineral deposition. Evidence that hypertrophic chondrocytes may resume proliferation was obtained by BrdU labeling. A monoclonal antibody (LA5) was isolated and characterized that recognizes a hypertrophic chondrocyte membrane protein. In addition to staining hypertrophic chondrocytes surrounded by a type II and type X collagen-stainable matrix, the LA5 antibodies also stained elongated chondrocytes at the cartilage/bone collar interface and cells incorporated in the first layer of bone and osteoid matrix.
Benefit of systemically administered rhIGF-I and rhIGF-I/IGFBP-3 on cancellous bone in ovariectomized rats
Authors: Bagi Cedo M. , Brommage Robert , Deleon Lita , Adams Steven , Rosen David , Sommer Andreas
Published: February 2010

The dose-related effects of systemically administered rhIGF-I and rhIGF-I/IGFBP-3 were monitored in the osteopenic rat skeleton at three different sites with cancellous bone: distal femoral metaphysis, epiphysis, and lumbar vertebral bodies. At the age of 16 weeks, rats were bilaterally ovariectomized (OVX) or sham operated (sham) and 8 weeks later divided into the control groups (sham OVX), and OVX groups treated with different doses of rhIGF-I or rhIGF-I/IGFBP-3 for 8 weeks. Fluorescent bone markers were given 9 and 2 days before necropsy. In addition, changes in designated bone sites as a result of ovariectomy alone were evaluated 8 and 16 weeks after surgery. High bone resorption, which dominates the postovariectomy remodeling process, resulted in a loss of cancellous bone at all measured sites. The highest trabecular bone loss was measured in the metaphyses (40%), compared with 22% in the lumbar vertebrae and 16% in the epiphyses. After 8 weeks of treatment with 7.5 mg/kg of rhIGF-I/IGFBP-3, bone formation rates were increased at all sites measured. Increased trabecular thickness was observed at the epiphyses and in the lumbar vertebral bodies. Increased bone resorption was restricted to the metaphyses of the rats that received the highest dose of rhIGF-I or rhIGF-I/IGFBP-3. Both formulations of rhIGF-I increased longitudinal bone growth similarly. This experiment demonstrates site-specific differences in cancellous bone reactions following ovariectomy. Epyphyses showed some advantages for cancellous bone histomorphometry over metaphyses and lumbar vertebral bodies. The data presented confirm the potential of rhIGF-I/IGFBP-3 complex to promote the bone formation process at various bone sites in osteoporotic rat skeleton.
Consequences of childhood-onset growth hormone deficiency for adult bone mass
Authors: de Boer H. , Blok G.J. , van Lingen A. , Teule G.J.J. , Lips P. , van der Veen E.A.
Published: February 2010

To assess the implications of prolonged growth hormone deficiency (GHD) for the acquisition and maintenance of bone mass, bone mineral density (BMD) was measured in 70 adult males (mean age 26.7 ± 4.5 years) with childhood-onset GHD, 7.4 ± 4.2 years after discontinuation of previous GH therapy. Because most of these patients were short (mean height 165.8 ± 6.6 cm), the influence of body height on standard BMD measurements, conventionally reported as the areal density (BMDarea, expressed in g/cm2), was analyzed in a group of age-matched healthy males. In GHD patients, BMDarea was significantly reduced at the lumbar spine (Z score -1.59 ± 1.08, p > 0.001) as well as at the nondominant hip (Z score -1.18 ± 0.95, p > 0.001). The reduction in BMDarea was similar for patients with isolated GHD (N = 25) and those with combined deficiencies of GH and luteinizing hormone (N = 40). In patients and controls, BMDarea was positively correlated with body height, a relation that was attributed to skeletal size. Bone dimensions were significantly smaller in patients than in controls, and therefore it was hypothesized that the difference in areal density between patients and controls might be confounded by differences in bone size. Measured bone mineral content corrected for the estimated bone volume (BMDvolume, expressed in g/cm3) remained significantly reduced (Z score: lumbar spine, -0.90 ± 1.08, p > 0.001; femoral neck, -0.74 ± 1.00, p > 0.001), but the differences between GHD patients and controls were less than indicated by BMDarea (p > 0.01). We conclude that the low BMDarea in our patient population can be explained in part by their subnormal body height. However, after correction for the effect of body height-related differences in bone volume, bone density was still significantly reduced. This indicates that in adult males with childhood-onset GHD a moderate degree of osteopenia is present. Insufficient bone acquisition during childhood years is considered the primary cause of the observed reduction in bone density.
Effects of tiludronate on bone mass, structure, and turnover at the epiphyseal, primary, and secondary spongiosa in the proximal tibia of growing rats after sciatic neurectomy
Authors: Murakami Hiroshi , Nakamura Toshitaka , Tsurukami Hiroshi , Abe Mitsuhiro , Barbier Alain , Suzuki Katsumi
Published: February 2010

To evaluate the effects of tiludronate on the mass, structure, and turnover of cancellous bone regions in immobilized rat tibiae, we performed a 4 week dosing experiment. The right hindlimbs of 84 Sprague-Dawley rats (5 weeks old) were neurectomized or sham operated. Animals were assigned to seven groups (n = 12 each); group 1 was sham operated, and groups 2-7 were neurectomized. Groups 1 and 2 were given vehicle only (distilled water), and groups 3, 4, and 5 were given tiludronate orally at doses of 25, 50, and 100 mg/kg body weight (BW)/day, respectively, throughout the experimental period. Group 6 was given 100 mg/kg BW/day of the agent for the first 2 weeks only, and group 7 received vehicle only for the first 2 weeks and then 100 mg/kg BW/day of the agent for the last 2 weeks. After tetracycline labeling was performed, the right tibiae were removed from the animals and processed to yield undecalcified sections. Histomorphometry was performed in the epiphyseal, primary, and secondary spongiosa of the proximal tibia. In group 2, trabecular bone volume (BV/TV) and trabecular number (Tb.N) were significantly decreased in the primary and secondary spongiosae, but this did not occur in the epiphyseal spongiosa. Osteoid surface (OS/BS) was decreased and osteoclast surface (Oc.S/BS) was increased in the secondary spongiosa. Tiludronate increased BV/TV and Tb.N in the primary spongiosa by reducing the values for the parameters of osteoclast surface (Oc.S/BS) and osteoclast number (Oc.N/BS). Osteoid surface in this region was not decreased by the agent. In groups 4 and 5, tiludronate prevented bone loss in the secondary spongiosa by reducing both OS/BS and Oc.S/BS. In group 6, BV/TV in the primary spongiosa was maintained at the level of group 1, but Oc.S/BS and Oc.N/BS were elevated. In the secondary spongiosa, bone mass was preserved and the reduction in these parameters was maintained. In group 7, however, BV/TV was increased in the primary spongiosa as a result of a reduction in osteoclastic resorption; in the secondary spongiosa, however, BV/TV was decreased and trabecular turnover was not reduced at the end of the experiment in these growing animals. Mineral apposition rates were not reduced by tiludronate. This study clearly demonstrated that this agent prevented immobilization bone loss by inhibiting resorption. The histomorphometric data suggest that (1) tiludronate begins to act early after administration in the primary spongiosa and rather late in the secondary spongiosa, and (2) the bone matrix is involved in the antiresorbing action of this bisphosphonate.
Cancellous bone turnover in growing rats: Time-dependent changes in association between calcein label and osteoblasts
Authors: Turner Russell T.
Published: February 2010

Calcein was given to growing male rats to label mineralizing bone, and groups of animals were sacrificed from 6 h to 12 days later. Fluorochrome-labeled perimeter and osteoblast perimeter were determined in a growth-adjusted sampling site in the secondary spongiosa of the proximal tibial metaphysis. The best correspondence between calcein-labeled perimeter and osteoblast-lined perimeter was during the initial 24 h following administration of the label. This association decreased progressively thereafter because of (1) cessation of bone formation on fluorochrome-labeled surfaces and (2) initiation of new foci of bone formation. The time-dependent decline in osteoblast-lined trabeculae with adjacent calcein label exceeded the increase in osteoblast perimeter on nonfluorochrome-labeled trabeculae, indicating that there was a net decrease in osteoblast number. Fluorochrome-labeled perimeter and bone area showed parallel decreases with time after labeling, as a result of bone resorption. Interestingly, the decrease in cancellous bone was caused exclusively by a reduction in trabecular number. There was no change in trabecular thickness. The findings suggest that the decreased osteoblast number and progressive bone resorption resulted in complete erosion of trabeculae most distal to the growth plate. As a result of the nearly equal growth and destruction of trabeculae, there was no change in the length of the cancellous metaphysis with time. It is concluded that cancellous bone turnover in growing rats represents a maturation process that differs fundamentally from bone turnover in adults.
Structure-activity studies of the s-echistatin inhibition of bone resorption
Authors: DR. Sato Masahiko , Garsky Victor , Majeska Robert J. , Einhorn Thomas A. , Murray John , Tashjian Armen H. , Gould Robert J.
Published: February 2010

Synthetic Arg-Gly-Asp (RGD)-containing peptides were examined in bone resorption or attachment and detachment assays with isolated mammalian osteoclasts in an effort to elucidate the mechanistic and structural basis for the inhibition of bone resorption by s-echistatin. Bone resorption was the process most sensitive to inhibition by s-echistatin, with IC50 = 0.3 nM; inhibition of attachment to bone or detachment (lamellipodial retraction) was 30- to 70-fold less sensitive, with IC50 = 10 or 20 nM, respectively. Single amino acid substitutions within the 49-residue sequence of s-echistatin showed that although the efficacy of s-echistatin is dependent on the Arg24-Gly25-Asp26 sequence, additional residues, including Asp27, Met28, and Cys39, are also critical for potent inhibition of the resorbing activity of isolated rat osteoclasts. Because of the identification of the avβ3 as the primary integrin on rat osteoclasts interacting the RGD peptides (Helfrich et al.), we examined the possibility of modeling bone resorption with other β3-mediated processes. Specifically, avβ3 endothelial cell (human or rat) attachment to vitronectin and aIIbβ3 platelet aggregation were compared with bone resorption for sensitivity to s-echistatin analogs, linear RGD peptides, and cyclic RGD peptides. Essentially no similarity in sensitivity to RGD peptides were observed between bone resorption, platelet aggregation, or endothelial cell attachment. Because rat osteoclasts and human giant cell tumors (osteoclastomas) shared similar sensitivity to s-echistatin and rat and human endothelial cells showed a similar sensitivity profile to RGD peptides, the dissimilarity of bone resorption to other β3-mediated processes cannot be explained in terms of species differences. These data show that the osteoclast avβ3 integrin is functionally distinct, despite epitopic similarity to the vitronectin receptor on other cells. Laser scanning confocal and scanning electron micrographs of rat osteoclasts on bone supported the hypothesis that resorption differs from the processes of attachment or detachment from substrates and suggest that inhibition of a minority of integrin-bone interactions by s-echistatin is sufficient to inhibit bone resorption. s-Echistatin was also found to inhibit bone resorption in an organ culture system of neonatal mouse calvariae with IC50 = 1 μM. Taken together, these data show that although it may be possible to target the osteoclast avβ3 integrin for the development of pharmaceuticals, s-echistatin itself is not useful in this capacity.
Bone mineral density of total body, spine, and femoral neck in children and young adults: A cross-sectional and longitudinal study
Authors: DR. Lu Pei Wen , Briody Julie N. , Ogle Graham D. , Morley Kate , Humphries Ian R.J. , Allen Jane , Howman-Giles Robert , Sillence David , Cowell Christopher T.
Published: February 2010

Bone mineral density (BMD) of total body (TBMD), lumbar spine (L2-4), and femoral neck was measured in 266 normal subjects (136 males) aged 4-27 years (mean 13 years) using dual-energy x-ray absorptiometry (DXA). BMD of all sites increased significantly with age until 17.5 years in males and 15.8 years in females, except for femoral neck BMD in females, which peaked at age 14.1 years. Males had higher peak TBMD, which was attributed to greater weight and lean tissue mass. In contrast, despite a later timing, peak L2-4 BMD in males was not different from that in females. Before peak BMD, weight was the best predictor of TBMD and L2-4 BMD in both sexes (r2 ranged from 0.77 to 0.88), whereas femoral neck BMD was predicted equally by height and weight. Longitudinal information collected from 53 (25 boys) of these children, aged 4-16.9 years, showed that the average annualized gain in TBMD was 0.047 g/cm2 for boys and 0.039 g/cm2 for girls. No significant difference in the association between age and BMD (slopes) was found between cross-sectional and longitudinal data for either sex. We conclude that the timing for peak BMD was consistent for total body, lumbar spine, and femoral neck for each sex. The earlier peak BMD in females is most likely related to earlier puberty. The cross-sectional normative data of this study are useful in serving as a standard for serial assessment in health and disease states.
Analysis of type II and type X collagen synthesis in cultured growth plate chondrocytes by in situ hybridization: Rapid induction of type X collagen in culture
Authors: O'Keefe R.J. , Puzas J.E. , Loveys L. , Hicks D.G. , Rosier R.N.
Published: February 2010

Type X collagen is produced by hypertrophic chondrocytes and serves as a highly specific marker for chondrocyte maturation. This study was designed to compare the expression of type II and type X collagen in growth plate sections and in distinct populations of chondrocytes in culture by in situ hybridization. Growth plate sections were treated with type II and type X collagen cDNA probes. Type II collagen mRNA was present throughout the growth plate but greatest in the lower proliferating and upper hypertrophic regions. In contrast, type X collagen was expressed only in the hypertrophic region. Northern analysis confirmed the specificity of the probe for type X collagen mRNA. Chick growth plate chondrocytes were separated by countercurrent centrifugal elutriation into five distinct populations and plated in serum-containing medium. These cultures were examined at varying times after plating for the expression of type II and type X collagen mRNA. At 3 h, type II collagen was present in the majority of the cells in all fractions, and approximately 15–20% of the cells expressed type X collagen mRNA. The cells expressing type X were from the hypertrophic region. At 24 h, however, nearly all cells in culture expressed type X mRNA, and there was a decrease in expression of type II collagen mRNA. Similar results were obtained in cultures in the absence of serum, and SDS-PAGE analysis of collagen synthesis confirmed the expression of type X collagen in all populations of fractionated cells at 24 h at the protein level. Type X collagen is an important marker through which cellular matruation can be evaluated in culture. The rapid induction of type X collagen in culture and its evaluation through in situ hybridization permit the investigation of factors that enhance or delay the development of cell hypertrophy and maturation. This may have important implications for the understanding and study of endochondral ossification and fracture repair.
Isolation and localization of basic fibroblast growth factor-immunoreactive substance in the epiphyseal growth plate
Authors: Twal W.O. , Vasilatos-Younken R. , Gay C.V. , Leach R.M.
Published: February 2010

Previous research in our laboratory has shown basic fibroblast growth factor (bFGF) to be a permissive mitogen for isolated avian growth plate chondrocytes. The present study was conducted to determine whether bFGF is present in avian growth plate and, if present, to determine its localization within the tissue. Immunohistochemical studies revealed that bFGF is present in the resting proliferative and hypertrophic calcifying zones of the growth plate but is absent from the prehypertrophic zone. Basic FGF appears to be associated with the extracellular matrix of the proliferative zone, but it is predominantly intracellular in the hypertrophic and mineralizing zone chondrocytes. Partial purification of cartilage-derived bFGF was performed on crude extracts of cartilage using heparin-Sepharose affinity chromatography. The presence of bFGF in the heparin-Sepharose column fractions was confirmed by immunoblotting and radioimmunoassay. Furthermore, western blot analysis of the extracts showed multiple protein bands having bFGF immunoreactivity, in the molecular weight range 14.4–18 kD. The data support the hypothesis that bFGF has a dual role in the growth plate. In the proliferative zone it acts as a chondrocyte mitogen, whereas when released from terminal hypertrophic chondrocytes, bFGF may serve as a chemotactic signal for metaphyseal blood vessel proliferation.
Skeletal unloading induces resistance to insulin-like growth factor I
Authors: Bikle Daniel D. , Harris Jonathan , Halloran Bernard P. , Morey-Holton Emily R.
Published: February 2010

In previous studies with a hindlimb elevation model, we demonstrated that skeletal unloading transiently inhibits bone formation. This effect is limited to the unloaded bones (the normally loaded humerus does not cease growing), suggesting that local factors are of prime importance. IGF-I is one such factor; it is produced in bone and stimulates bone formation. To determine the impact of skeletal unloading on IGF-I production and function, we assessed the mRNA levels of IGF-I and its receptor (IGF-IR) in the proximal tibia and distal femur of growing rats during 2 weeks of hindlimb elevation. The mRNA levels for IGF-I and IGF-IR rose during hindlimb elevation, returning toward control values during recovery. This was accompanied by a 77% increase in IGF-I levels in the bone, peaking at day 10 of unloading. Changes in IGF binding protein levels were not observed. Infusion of IGF-I (200 μg/day) during 1 week of hindlimb elevation doubled the increase in bone mass of the control animals but failed to reverse the cessation of bone growth in the hindlimb-elevated animals. We conclude that skeletal unloading induces resistance to IGF-I, which may result secondarily in increased local production of IGF-I.
Fractal geometry and vertebral compression fractures
Authors: Weinstein Robert S. , Majumdar Sharmila
Published: February 2010

Cancellous bone in postmenopausal osteoporosis is characterized by widely spaced and disconnected trabeculae. These architectural changes may disproportionately increase bone fragility compared with the decrease in bone mass alone. To determine whether there is an independent architectural contribution to fracture risk, we applied fractal geometry to the cancellous bone in osteoporosis. Fractal objects have recurrent, branching patterns that are quantified by a fractal dimension D, that describes how the object fills space. Photomicrographs of transiliac bone biopsy specimens were digitized, and D was calculated from the negative of the straight portion of the slope of the log of the number of pixels containing boundary points versus the log of the pixel size over a range of pixel sizes. The results were compared with the cancellous bone histomorphometry in 31 individuals, aged 19–80 years, who were healthy before sudden death. D was inversely related to age (r = −0.72, p < 0.0001) and trabecular spacing (r = −0.87; p < 0.0001) and directly related to bone area (r = 0.86, p < 0.0001) and trabecular number (r = 0.83, p < 0.0001). We then examined 12 healthy volunteers and 12 patients with vertebral compression fractures as a result of osteoporosis who were matched for age and cancellous bone area. The patients with fractures had a 7.9% lower mean value for D (1.224 ± 0.085 SD versus 1.329 ± 0.125, p < 0.026). Other markers of the cancellous architecture, such as trabecular width, separation, and number, were not different between the matched groups. D was more than 1 SD below the normal mean value in 50% of the fracture cases and in only 1 of the nonfracture cases (p < 0.025). Applied to cancellous bone, fractal geometry may discriminate more effectively between fracture and nonfracture cases than measurement of bone area and help accent the impact of architecture on bone fragility. The single expression, D, greatly simplifies description of the spatial distribution of cancellous bone and thus has a potential role in the computer-assisted histologic investigation of osteoporosis.