bluuebunny/crossref_metadata_2025 huggingface dataset:
Episodic Secretion of Parathyroid Hormone in Postmenopausal Women: Assessment by Deconvolution Analysis and Approximate Entropy
Authors: Dr. Samuels M. H. , Veldhuis J. D. , Kramer P. , Urban R. J. , Bauer R. , Mundy G. R.
Published: April 2006

In healthy young subjects, parathyroid hormone (PTH) is secreted presumptively in a dual fashion, with low amplitude pulses apparently superimposed on tonic secretion. In contrast, PTH secretion has not been as well characterized in postmenopausal women, and relationships among bone density, estrogen status, and PTH release have not been explored. It is possible that a pulsatile pattern of PTH secretion is important for bone remodeling, since exogenous PTH administered in a pulsatile manner stimulates bone formation. To assess the importance of pulsatile PTH secretion as a determinant of bone mass, we measured PTH in blood sampled every 2 minutes for 6 h in four groups of older women: (1) high bone density receiving estrogen (n = 6), (2) high bone density not receiving estrogen (n = 5), (3) low bone density receiving estrogen (n = 6), and (4) low bone density not receiving estrogen (n = 8). The plasma PTH release profiles were subjected to deconvolution analysis, which resolves measured hormone concentrations into secretion and clearance components, and to an approximate entropy (ApEn) estimate, which provides an ensemble measure of the serial regularity or orderliness of the release process. In postmenopausal subjects, PTH was secreted in a fashion similar to that observed in young adults, with significant tonic secretion and PTH pulse occurrences averaging every 18–19 minutes. Pulsatile PTH secretion accounted for approximately 25% of the total secreted PTH. There were no differences in the amplitude or frequency of pulsatile PTH secretory parameters or in ApEn values among the four groups or compared with young controls. We conclude that in postmenopausal women, PTH secretory patterns and temporal organization are similar to those in healthy young subjects and are not altered in states of low bone density or estrogen deficiency. This suggests that abnormalities in orderly pulsatile PTH secretion are unlikely to play a major role in established postmenopausal osteoporosis.
A New Trabecular Region of Interest for Femoral Dual X-Ray Absorptiometry: Short-Term Precision, Age-Related Bone Loss, and Fracture Discrimination Compared with Current Femoral Regions of Interest
Authors: Takada Masahiko , Grampp Stephan , Ouyang Xiaolong , Engelke Klaus , Genant Harry K.
Published: April 2006

We defined a new region of interest (ROI) for femoral Ward's triangle, centered on the femoral neck axis including comparatively trabecular-rich bone. Forty-seven premenopausal, 39 healthy postmenopausal, and 35 osteoporotic postmenopausal women with vertebral fractures were evaluated comparing the new with the standard femoral ROIs, using a Hologic QDR-2000. Additionally, spinal dual X-ray absorptiometry (DXA) was performed. The short-term precision error of the new ROI expressed as the root mean square of the coefficient of variations was 1.34% for premenopausal women, 1.69% for healthy postmenopausal women, and 2.46% for osteoporotic postmenopausal women. Bone mineral density (BMD) values of the new ROI correlated highly with those of the standard femoral ROIs (r = 0.91 − 0.96) and the spinal BMD (r = 0.74). Age-related bone loss of the new ROI was 0.75% per year (r = 0.66) in healthy women, which was approximately 1.5 times higher than the bone loss of the standard femoral ROIs, except for Ward's triangle. Regarding the intergroup discrimination, the t-value of the new ROI was similar to the t-value of Ward's triangle, and the intergroup percent decrements in BMD of the new ROI approximated those of Ward's triangle. For discriminating women with vertebral fractures, the new ROI demonstrated odds ratios of 1.6 similar to most ROIs but lower than that of the trochanteric region. The new, substantially trabecular ROI appears to be an alternative to the Ward's ROI traditionally used in femoral DXA having improved short-term precision and comparable sensitivity.
Synovial Fluids from Patients with Osteoarthritis and Rheumatoid Arthritis Contain High Levels of Parathyroid Hormone–Related Peptide
Authors: Kohno Hiroaki , Shigeno Chohei , Kasai Ryuichi , Akiyama Haruhiko , Iida Hirokazu , Tsuboyama Tadao , Sato Kanji , Konishi Junji , Nakamura Takashi
Published: April 2006

High levels of immunoreactive and biologically active parathyroid hormone–related peptide (PTHrP) were detected in synovial fluids from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The levels of PTHrP immunoreactivity in synovial fluids, measured by a two-site immunoradiometric assay (IRMA) which detects hPTHrP(1–72) or longer peptides and a radioimmunoassay (RIA) specific to the carboxy-terminal portion of hPTHrP, were 3.2 ± 0.3 pmol of hPTHrP(1–86)/l and 61 ± 7.0 pmol of hPTHrP(109–141)/l in OA patients (mean ± SE, n = 23), and 4.8 ± 0.8 pmol of hPTHrP(1–86)/l and 164 ± 30 pmol of hPTHrP(109–141)/l in RA patients (n = 26). Synovial fluid PTHrP levels distributed above the normal plasma reference ranges in each assay (0.7–2.6 pmol of hPTHrP(1–86)/l; 16–60.6 pmol of hPTHrP(109–141)/l). After concentration using sequential cation-exchange and reverse-phase chromatography, synovial fluid exhibited the activity that stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblastic ROS 17/2.8 cells expressing PTH/PTHrP receptors. The cAMP accumulation activity in synovial fluid was sensitive to coincubation with excess hPTHrP(3–40), a PTH/PTHrP receptor antagonist, and was completely neutralized by preincubation with a monoclonal antibody specific to hPTHrP but not PTH. Immunohistochemical analysis of RA synovium revealed that PTHrP was localized in fibroblast-like cells in the synovial pannus invading articular cartilage. Our data show that PTHrP is produced locally by the diseased synovial tissue and released into synovial fluid at high concentrations, allowing us to hypothesize that PTHrP plays a novel role as a paracrine/autocrine factor in the pathology of OA and RA.
Altered Regulation of Parathyroid Hormone Secretion by Calcium in Pregnant and Lactating Rats
Authors: Schultz Vijitha L. , Boass Agna , Garner Sanford C. , Toverud Svein U.
Published: April 2006

We have previously shown that the serum parathyroid hormone (PTH) concentration in lactating (L) rats is not suppressed by high serum Ca2+ to the same extent as in nonmated (NM) rats. To investigate further Ca2+ regulation of PTH secretion, parathyroid cells from NM rats and rats in late pregnancy and at peak lactation were dispersed and incubated for 2 h in medium containing 0.52–2.05 mM Ca2+. Medium PTH was assayed with a homologous immunoradiometric assay (IRMA). At the two highest Ca2+ levels (1.81 and 2.05 mM), medium PTH was significantly higher (p = 0.031) for cells from L rats than for cells from NM rats. In contrast, significantly less (p < 0.001) PTH was secreted for the L group versus the NM group at medium Ca2+ values of 1.27 and 1.46 mM. Estimated set points for L and NM groups were 1.17 mM and 1.35 mM, respectively, corresponding closely to the prevailing serum Ca2+ for these two groups. Consistent with the present in vitro data, high serum PTH (>40 pg/ml) in L rats occurred only at serum Ca2+ values below 1.27 mM. Elevated serum PTH at lower serum Ca2+ levels was also seen in pregnant rats. Dispersed parathyroid cells from 20− to 21-day pregnant rats secreted significantly more PTH (p = 0.028) than cells from NM rats at all Ca2+ levels tested (1.1–1.6 mM). In conclusion, the relationship between extracellular Ca2+ and PTH secretion is altered in rats during late pregnancy and at peak lactation, perhaps as part of the adaptation to the demands for calcium for pre- and postnatal growth.
Electrophysiological Responses of Human Bone Cells to Mechanical Stimulation: Evidence for Specific Integrin Function in Mechanotransduction
Authors: Salter D. M. , Robb J. E. , Wright M. O.
Published: April 2006

Bone cells respond to mechanical stimuli, but the transduction mechanisms responsible are not fully understood. Integrins, a family of heterodimeric transmembrane glycoproteins, which link components of the extracellular matrix with the actin cytoskeleton, have been implicated as mechanoreceptors. We have assessed the roles of integrins in the transduction of cyclical mechanical stimuli to human bone cells (HBCs), which results in changes in membrane potential. HBC showed membrane depolarization following 0.104 Hz mechanical stimulation and membrane hyperpolarization following stimulation at 0.33 Hz. The membrane depolarization response involved tetrodotoxin-sensitive sodium channels and could be inhibited by antibodies against αV, β1, and β5 integrins. In contrast, the hyperpolarization response was inhibited by gadolinium and antibodies to the integrin-associated protein (CD47), α5 and β1 integrin. Both responses could be abrogated by Arg-Gly-Asp (RGD)-containing peptides, inhibition of tyrosine kinase activity, and disruption of the cytoskeleton. These results demonstrate differential electrophysiological responses of HBC to different frequencies of mechanical strain. Furthermore, they suggest that integrins act as HBC mechanoreceptors with distinct signaling pathways being activated by different frequencies of mechanical stimuli.
Differential Effects of Nonsteroidal Anti-Inflammatory Drugs on Constitutive and Inducible Prostaglandin G/H Synthase in Cultured Bone Cells
Authors: Pilbeam Carol C. , Fall Pamela M. , Alander Cynthia B. , Raisz Lawrence G.
Published: April 2006

The production of prostaglandins by osteoblasts is an important mechanism for the regulation of bone turnover. Bone cells contain both inducible and constitutive prostaglandin G/H synthase (PGHS-2 and PGHS-1) and these are differentially regulated. Nonsteroidal anti-inflammatory drugs (NSAIDs), which selectively inhibit one of these enzymes, would be useful in assessing their relative roles in bone metabolism. By Northern analysis, only PGHS-2 is expressed by the immortalized rat osteoblastic cell line, Py1a, while only PGHS-1 is expressed by the rat osteosarcoma cell line, ROS 17/2.8. We tested the relative inhibitory potency (IC50) of seven different NSAIDs on these two cell lines. A recently described selective inhibitor of PGHS-2, NS-398, was approximately 30 times more potent in inhibiting PGHS-2 than PGHS-1, and diclofenac was approximately 10 times more potent. Both had IC50's of approximately 3 nM for PGHS-2 in Py1a cells. Indomethacin, flurbiprofen, naproxen, and piroxicam were relatively nonselective with IC50's ranging from 30 nM to 1 μM, while 6-methoxy-2 naphthyl acetic acid, the active metabolite of nabumetone, was inhibitory only at concentrations greater than 1 μM. These results indicate that the presently available NSAIDs are unlikely to distinguish completely between effects mediated by PGHS-2 or PGHS-1. However, the cell systems employed could provide a model for the analysis of new compounds with greater selective activity.
Effects of a New Positioner on the Precision of Hip Bone Mineral Density Measurements
Authors: Hans D. , Duboeuf F. , Schott A. M. , Horn S. , Avioli L. V. , Drezner M. K. , Meunier P. J.
Published: April 2006

In an attempt to reduce patient positioning errors, the authors tested the use of a new hip-specific positioning tool, OsteoDyne's Hip Positioner System (HPS). The HPS is an “A” frame splint designed to abduct both legs approximately 15° to hold them in full extension at the hips and knees and to lock the feet in a neutral position. Seventy volunteer women aged 35–82 years were randomly assigned in two age-matched groups (mean age 56 years). Each group underwent two consecutive femur dual X-ray absorptiometry (DXA) scans with intermediate repositioning using the HPS system and two others utilizing the standard hip positioner provided with Hologic and Lunar scanners. One technician performed all scans using a Hologic QDR 1000-Plus and Lunar DPX-Plus densitometer. One hundred and fifty volunteer women aged 50–84 years (mean age, 64 years) were recruited in a multicenter study for the assessment of precision. Each subject underwent three consecutive femur DXA scans with intermediate repositioning using the HPS system. The coefficient of variation (CV) was significantly improved at the femoral neck by the use of the HPS with 0.7 versus 1.2 with the Hologic densitometer but only moderately altered at other sites. Similar results were found with the Lunar densitometer with improvement of precision at the femoral neck, 0.8 versus 1.8 with a similar trend but no significant difference at the other regions. No statistical difference was noted between the femoral neck BMD measured with the HPS system and with the standard positioners in either group. The mean precision of data obtained on the QDR 1000+ was 0.8% (range 0.1–1.4) for the femoral neck BMD, 1.1% (range 0.1–3.0) for the trochanter BMD, 2.3% (range 0.2–5.2) for Ward's triangle BMD, and 0.8% (range 0.1–1.9) for the total femur BMD. The mean precision of data obtained on the QDR 2000 was 0.7% (range 0.1–2), 1% (range 0.1–4.9), 2.6% (range 0.3–5.7), and 0.7% (range 0.1–1.8), respectively. In conclusion, data obtained with the new OsteoDyne's HPS seem capable of reducing patient positioning errors for the hip measurement. Its use is likely to improve confidence in hip bone mineral density measurements.
Mechanical Stimulation of Tissue Repair in the Hydraulic Bone Chamber
Authors: Guldberg R. E. , Caldwell N. J. , Guo X. E. , Goulet R. W. , Hollister S. J. , Goldstein S. A.
Published: April 2006

A hydraulically activated bone chamber model was utilized to investigate cellular and microstructural mechanisms of mechanical adaptation during bone repair. Woven trabecular bone and fibrotic granulation tissue filled the initially empty chambers by 8 weeks postimplantation into canine tibial and femoral metaphyses. Without mechanical stimulation, active bone remodeling to lamellar trabecular bone and reconstitution of marrow elements were observed between 8 and 24 weeks. In subsequent loading studies, the hydraulic mechanism was activated on one randomly chosen side of 10 dogs following 8 weeks of undisturbed bone repair. The loading treatment applied an intermittent compressive force (18 N, 1.0 Hz, 1800 cycles/day) for durations of a few days up to 12 weeks. Stereological analysis of three-dimensional microcomputed tomography images revealed an increase in trabecular plate thickness and connectivity associated with the loaded repair tissue microstructure relative to unloaded contralateral controls. These microstructural alterations corresponded to an over 600% increase in the apparent modulus of the loaded bone tissue. A significant increase in the percentage of trabecular surfaces lined by osteoblasts immunopositive for type I procollagen after a few days of loading provided further evidence for mechanical stimulation of bone matrix synthesis. The local principal tissue strains associated with these adaptive changes were estimated to range from approximately −2000 to +3000 μstrain using digital image-based finite element methods. This study demonstrates the sensitivity of bone tissue and cells to a controlled in vivo mechanical stimulus and identifies microstructural mechanisms of mechanical adaptation during bone repair. The hydraulic bone chamber is introduced as an efficient experimental model to study the effects of mechanical and biological factors on bone repair and regeneration.
The Effects of Smoking on Bone Mass and the Rates of Bone Loss Among Elderly Japanese-American Men
Authors: Vogel John M. , Davis James W. , Nomura Abraham , Wasnich Richard D. , Ross Philip D.
Published: April 2006

Bone density and bone loss rates were examined among Japanese-American men categorized as current cigarette smokers, past smokers, and nonsmokers. The design included a retrospective study of smoking and bone density and a prospective study of current smoking and bone loss rates. The mean length of follow-up was 5 years; the setting was the island of Oahu. The subjects included 1303 men in the Hawaii Osteoporosis Study, 51–82 years old at their initial examination. Twenty percent were current smokers, 45% past smokers, and 35% had never smoked. Their bone density was measured at the distal and proximal radius and calcaneus using single photon absorptiometry. Compared with never smokers, current and past smokers had significantly lower bone density, especially in the predominantly cancellous calcaneus (4.8 and 4.3% lower, respectively) and partially trabecular distal radius (1.8 and 3.3% lower, respectively). The magnitude of the smoking effect was linked strongly to the duration of smoking and also to the number of cigarettes smoked. Bone loss rates subsequent to the initial measurement were greater in the current smokers than the never smokers (20.5, 27.2, and 9.7% greater at the calcaneus, distal, and proximal radius, respectively) but the differences did not achieve significance. Smokers of more than one pack per day had 32.0, 77.6, and 30.7% greater loss rates than never smokers in these same sites; the difference achieved significance at the distal radius. The results from the distal radius suggest that these smokers may increase their fracture risk 10–30% per decade of smoking. The adverse effects of smoking appeared to be greater in cancellous than cortical bone.
Short-Term and Long-Term Fracture Prediction by Bone Mass Measurements: A Prospective Study
Authors: Dr. Huang Chun , Ross Philip D. , Wasnich Richard D.
Published: April 2006

Prospective and cross-sectional studies have demonstrated that bone mass predicts fracture risk. However, most prospective studies have been limited to a few years of follow-up. We investigated the long-term associations of bone mass with vertebral fractures using longitudinal data collected from more than 500 postmenopausal Japanese-American women in the Hawaii Osteoporosis Study. New vertebral fractures were identified during an average of 2.7 years between 1992 and 1995. Short-term fracture prediction was evaluated using bone mass (spine, calcaneus, distal radius, and proximal radius) measured at the beginning of follow-up. Long-term prediction was evaluated using bone mass measured before the follow-up period (11 years earlier for nonspine bone mass and 8 years earlier for spine). All four bone mass measurements were significant predictors of vertebral fractures identified during the subsequent 2.7 years (short-term prediction), with odds ratios (ORs) ranging from 1.5 to 1.9. The ORs for long-term prediction were slightly lower in magnitude, but the confidence intervals overlapped the short-term ORs considerably, suggesting that both long-term and short-term associations are similar in magnitude. Furthermore, cross-sectional analyses based on bone mass measurements performed at the end of follow-up (after fractures had occurred) yielded results similar to those based on prospective data (bone mass measured prior to fractures), suggesting that the relatively quick and inexpensive cross-sectional studies are useful for preliminary evaluations of new bone mass measurement techniques. The results suggest that bone mass measurements made up to 11 years earlier can predict vertebral fractures almost as well as measurements made more recently.
Effect of Locally Infused IGF-I on Femoral Gene Expression and Bone Turnover Activity in Old Rats
Authors: Wakisaka Atsuhiko , Tanaka Hiroshi , Barnes Janice , Liang C. Tony
Published: April 2006

Previously, we showed that the age-dependent deficit in bone formation activity can be attributed in part to a decline in local expression of insulin-like growth factor I (IGF-I) and altered mitogenic response of old osteoprogenitor cells to IGF-I. To establish the cellular basis for using IGF-I as a possible therapeutic agent for osteoporosis, we examined the effect of locally infused (50 ng/day for 14 days) on the expression of osteoblast-related genes in femurs of old rats. Northern and dot blot analyses showed that the expression of procollagen (I), osteopontin, alkaline phosphatase, and osteocalcin was increased 0.4- to 1.5-fold in IGF-1–treated femurs as compared with control femurs. Histomorphometric analyses were carried out in parallel experiments to assess the changes in bone remodeling activity. Trabecular bone volume, trabecular number, and trabecular thickness were increased 56%, 29%, and 23%, respectively, whereas trabecular separation was reduced 26% by IGF-1 treatment. IGF-I treatment increased significantly the osteoid volume, osteoid surface, osteoblast number, and osteoblast surface. Mineralizing surface and mineral apposition rate, kinetic indices of bone formation, were also stimulated by IGF-I treatment. The bone formation rate was stimulated 81% in IGF-I–treated femurs as compared with control femurs. In contrast, eroded surface and osteoclast surface, parameters associated with bone resorption, were not affected by IGF-I treatment. These findings suggest that local administration of IGF-I into femurs of old rats can stimulate the expression of matrix proteins and improve trabecular bone status by stimulating bone formation without any appreciable effect on bone resorption.
Modulation of Osteoclast Function by Adenovirus Vector-Induced Epidermal Growth Factor Receptor
Authors: Tanaka Sakae , Takahashi Tokiharu , Takayanagi Hiroshi , Miyazaki Tsuyoshi , Oda Hiromi , Nakamura Kozo , Hirai Hisamaru , Kurokawa Takahide
Published: April 2006

We have explored the use of adenovirus vector-mediated gene transfer to introduce foreign genes into osteoclasts, terminally differentiated cells responsible for bone resorption. A replication-deficient adenovirus vector that contains a reporter gene encoding β-galactosidase efficiently infected human osteoclast-like cells (OCLs) derived from human giant cell tumors and mouse OCLs formed in vitro. We then constructed an adenovirus vector carrying human epidermal growth factor receptor (EGFR) cDNA (Ax1CAhEGFR) and introduced the EGFR gene into mouse OCLs. Clear induction of EGF receptor was detected in Ax1CAhEGFR-infected OCLs (EGFR-OCLs) by immunocytochemistry and immunoblotting, and EGF stimulation induced rapid tyrosine phosphorylation of several proteins including EGF receptor itself. Large vacuoles appeared in EGFR-OCLs in response to EGF treatment, and pit-forming activity by EGFR-OCLs was dose-dependently suppressed by recombinant human EGF. In addition, survival of EGFR-OCLs was prolonged by EGF. No expression of EGF receptor or effects of EGF were observed in noninfected OCLs or control vector-infected OCLs. These results suggest that adenoviral vectors are useful for modulating osteoclast function by introducing foreign genes into osteoclasts and that they will be a good means of gene therapy of metabolic bone diseases.
The Reproducibility of Ultrasound Bone Measures in a Triethnic Population of Pregnant Adolescents and Adult Women
Authors: Sowers M. , Jannausch M. , Scholl T. , Schall J.
Published: April 2006

We used bone ultrasound technology with its measurement of attenuation (broadband ultrasound attenuation [BUA] as dB/MHz) and sound velocity (speed of sound as m/s) for assessing the quantitative ultrasound index (QUI) summary measure in a triethnic population of 280 pregnant women. The study purpose was to describe the reproducibility of the ultrasound technology and determine if the correlations of age, weight, and ethnicity with the bone status measures in this population are consistent with the correlations of age, weight, and ethnicity that have been reported with other technologies that measure bone mass. We evaluated the first 280 women enrolled in our longitudinal study of lead turnover from maternal bone during pregnancy and lactation. Enrollees were pregnant, aged 12–29 years, and self-classified as black, white, or Hispanic. Bone ultrasound was measured twice at entry to prenatal care, which, on average, was at 14 weeks gestation. Reproducibility was described with intraclass correlations and the standard error of measurement. Age, weight, and ethnicity were associated with bone status measures using Spearman correlations and generalized linear models. The reproducibility of the summary bone measure, QUI, was high (96–97%). Variation in age and ethnicity did not alter reproducibility; however, the reproducibility of the attenuation measure (BUA as dB/MHz) lessened with increasing weight, declining from 95% to 89%. Since this attenuation is included in the summary QUI measure, there was a slight, and nonsignificant, decline in QUI reproducibility (from 97% to 96%) as women increased in size. There were no statistically significant differences in mean bone ultrasound measures according to age, where ages ranged from 12–29 years. Women who categorized themselves as black had, on average, an 8.5% greater QUI than did women who classified themselves as Hispanic or white. There were no significant pair-wise differences in mean ultrasound measures of bone between women classifying themselves as Hispanic or white. The use of ultrasound is a highly reproducible measure to assess bone characteristics in a population of pregnant adolescent and young adult women and its summary measure of bone mass is correlated with ethnic as well as body size characteristics.
Parathyroid Hormone Induces Expression of the Inducible cAMP Early Repressor in Osteoblastic MC3T3-E1 Cells and Mouse Calvariae
Authors: Tetradis Sotirios , Nervina Jeanne M. , Nemoto Ken , Dr. Kream Barbara E.
Published: April 2006

Parathyroid hormone (PTH) regulates gene expression in skeletal osteoblasts mainly through the cAMP–protein kinase A (PKA) pathway. In neuroendocrine cells, activation of the cAMP–PKA signaling pathway leads to induction of the inducible cAMP early repressor (ICER), which is transcribed from an intronic promoter of the CREM gene and acts as a transcriptional repressor. To investigate whether PTH induces ICER expression in osteoblastic cells, RNA from MC3T3-E1 cells was subjected to reverse transcriptase-polymerase chain reaction using primers spanning the ICER sequence. Amplified products were subcloned, sequenced, and used as a probe for Northern blot analysis. In MC3T3-E1 cells, PTH induced ICER mRNA levels, which peaked at 2 h and declined to baseline by 8 h. Cycloheximide caused superinduction of ICER mRNA in response to PTH. In cultured mouse calvariae, PTH also induced ICER mRNA accumulation, which peaked at 2 h and returned almost to baseline by 10 h. Overexpression of ICER IIγ decreased both baseline and PTH-stimulated prostaglandin G/H synthase 2 promoter activity in MC3T3-E1 cells. The induction of ICER represents a novel mechanism by which PTH regulates gene expression in osteoblastic cells.
Inhibition of BMP Receptor Synthesis by Antisense Oligonucleotides Attenuates OP-1 Action in Primary Cultures of Fetal Rat Calvaria Cells
Authors: Yeh Lee-Chuan C. , Betchel Karen P. , Lee John C.
Published: April 2006

Osteogenic protein-1 (OP-1 or bone morphogenetic protein-7 [BMP-7]) stimulates osteoblast differentiation in vitro and induces bone formation in vivo. BMPs exert their effects through complex formation with a heterodimeric receptor composed of a type I and a type II polypeptide. In the present study, mRNAs for three BMP subtype I receptors (ActR-I, BMPR-IA, and BMPR-IB) and one BMPR-II receptor were detected by Northern analysis in two human osteosarcoma cell lines (SaOS-2 and TE85) and in the primary cultures of fetal rat calvaria (FRC) cells. OP-1 affected the steady-state mRNA levels of these receptors differently among these cell types. To study the role of each receptor type in OP-1 action in FRC cells, receptor synthesis was inhibited by antisense oligonucleotides. Inhibition of receptor synthesis was confirmed by immunoprecipitation of radiolabeled cellular proteins with specific antibodies. The osteogenic action of OP-1 was measured by alkaline phosphatase (ALP) activity and mineralized bone nodule formation in FRC cells. Results showed that inhibition of synthesis of a single subtype I receptor alone did not affect significantly the OP-1–stimulated ALP activity. Inhibition of BMPR-II synthesis reduced the OP-1–stimulated ALP activity by about 50%. Inhibition of synthesis of any one of the type I receptor plus the BMPR-II receptor did not reduce the OP-1–stimulated ALP activity significantly beyond that observed by inhibition of BMPR-II alone. Under these conditions, nodule formation was affected similarly, thus supporting the observations made with the ALP measurements. The present results suggest that the ActR-I, BMPR-IA, and BMPR-IB receptors and the BMPR-II receptor are expressed and functional for OP-1 in FRC cells and that regulation of synthesis of these receptors may be a mechanism by which a specific cell type responds to OP-1. The turnover rate of these receptor proteins might be relatively long and another type II receptor(s) for OP-1 might be functional in FRC cells.
Contortrostatin, a Homodimeric Snake Venom Disintegrin, Is a Potent Inhibitor of Osteoclast Attachment
Authors: Mercer B. , Markland F. , Minkin C.
Published: April 2006

Disintegrins are small disulfide-rich proteins containing an Arg-Gly-Asp (RGD) sequence near their carboxyl terminus. These polypeptides inhibit binding of adhesion molecules to their receptors (integrins) on the surface of cells. Osteoclasts express integrins, heterodimeric cell surface adhesion receptors, that have been shown to be involved in interactions with the extracellular matrix (ECM), including attachment to bone and bone resorption. It has recently been shown that disintegrins effectively inhibit attachment of osteoclasts to components of the ECM and also disrupt osteoclast-mediated bone resorption. Here we characterize the effects of contortrostatin (CTS), a novel homodimeric snake venom disintegrin, on osteoclast attachment. Plastic dishes coated with CTS were able to support osteoclast attachment with a high affinity (EC50,CTS = 86 ± 6.7 nM) similar to that of vitronectin (VTN; EC50,VTN = 80 ± 20 nM). Further, CTS was observed to inhibit completely osteoclast attachment to fetal bovine serum (FBS; IC50,FBS = 0.36 ± 0.04 nM) and VTN (IC50,VTN = 0.85 ± 0.13 nM). We used monoclonal antibodies directed against the β1 (monoclonal antibody [MAb] CD29) and β3 (MAb F11) integrin subunits to explore the mechanism of osteoclast attachment to immobilized CTS. Only MAb F11 inhibited attachment to immobilized CTS (IC50 = 0.41 ± 0.12 μg/ml), suggesting that binding to CTS is mediated in part by a β3 integrin, presumably the αvβ3 VTN receptor. In further support of an integrin-mediated mechanism, binding of osteoclasts to CTS is inhibited by the RGD peptide, GRGDSP. CTS was also more potent (IC50,FBS = 0.36 ± 0.04 nM) at inhibiting osteoclast attachment to FBS-coated wells than the monomeric snake venom disintegrin echistatin (IC50,FBS = 8.9 ± 1.5 nM) or VTN (IC50,FBS = 97.5 ± 25.5 nM). Taken together, these data suggest that the snake venom disintegrin CTS is a potent inhibitor of β3 integrin-mediated osteoclast attachment, presumably involving the VTN receptor (an αvβ3 integrin). Further studies of the mechanism of CTS–osteoclast interactions may aid in the design of peptide mimetics to act as antiresorptive agents for the treatment of osteoporosis and other skeletal pathology.
Effect of Two Training Regimens on Bone Mineral Density in Healthy Perimenopausal Women: A Randomized Controlled Trial
Authors: Heinonen Ari , Oja Pekka , Sievänen Harri , Pasanen Matti , Vuori Ilkka
Published: April 2006

The aim of this randomized controlled trial was to evaluate the effects of 18 months of calisthenics and endurance training regimens on bone mineral density (BMD) in perimenopausal women. Clinically healthy sedentary female volunteers (n = 105) aged 52–53 years were randomly assigned to a calisthenics (n = 36), endurance (n = 34), or control (n = 35) group. The calisthenics training (2.6 times per week on average, 50 minutes per session) consisted of rhythmic strength-endurance exercises by large muscle groups, and the endurance training (3.2 times per week, 50 minutes) consisted of walking, stair climbing, ergometer cycling, and jogging at a controlled heart rate zone corresponding to 55–75% of the individual maximal oxygen uptake (VO2max) of the subjects. The control subjects performed a light stretching program once a week. The BMD of the lumbar spine (L2-L4), right femoral neck, calcaneus, and distal radius was measured by dual-energy X-ray absorptiometry at 0, 4, 8, 10, 14, and 18 months, and the maximal isometric strength during trunk extension and flexion, leg extension, and arm flexion and the VO2max by ergospirometry were evaluated at 0, 8, 10, and 18 months of intervention. The VO2max improved significantly (p = 0.021) in the endurance group. The linear trend of the femoral neck BMD in the endurance group, as determined by generalized linear models, was significantly different (p = 0.043) from that of the control group, the trend indicating a maintenance of the prestudy BMD. In the calisthenics group, the training effect was not significant. However, the distal radius BMD of the endurance group showed a significant negative trend (p = 0.006). These results suggest that multiexercise endurance training maintains the BMD the clinically important femoral neck of perimenopausal women. This form of endurance training proved also to be feasible for healthy perimenopausal women.
Intermittent and Continuous Administration of the Bisphosphonate Ibandronate in Ovariohysterectomized Beagle Dogs: Effects on Bone Morphometry and Mineral Properties
Authors: Monier-Faugere Marie-Claude , Geng Zhaopo , Paschalis Eleftherios P. , Qi Quanle , Arnala Illka , Bauss Frieder , Boskey Adele L. , Malluche Hartmut H.
Published: April 2006

Bisphosphonates have emerged as a valuable treatment for postmenopausal osteoporosis. Bisphosphonate treatment is usually accompanied by a 3–6% gain in bone mineral density (BMD) during the first year of treatment and by a decrease in bone turnover. Despite low bone turnover, BMD continues to increase slowly beyond the first year of treatment. There is evidence that bisphosphonates not only increase bone volume but also enhance secondary mineralization. The present study was conducted to address this issue and to compare the effects of continuous and intermittent bisphosphonate therapy on static and dynamic parameters of bone structure, formation, and resorption and on mineral properties of bone. Sixty dogs were ovariohysterectomized (OHX) and 10 animals were sham-operated (Sham). Four months after surgery, OHX dogs were divided in six groups (n = 10 each). They received for 1 year ibandronate daily (5 out of 7 days) at a dose of 0, 0.8, 1.2, 4.1, and 14 μg/kg/day or intermittently (65 μg/kg/day, 2 weeks on, 11 weeks off). Sham dogs received vehicle daily. At month 4, there was a significant decrease in bone volume in OHX animals (p < 0.05). Doses of ibandronate ≥ 4.1 μg/kg/day stopped or completely reversed bone loss. Bone turnover (activation frequency) was significantly depressed in OHX dogs given ibandronate at the dose of 14 μg/kg/day. This was accompanied by significantly higher crystal size, a higher mineral-to-matrix ratio, and a more uniformly mineralized bone matrix than in control dogs. This finding lends support to the hypothesis that an increase in secondary mineralization plays a role in gain in BMD associated with bisphosphonate treatment. Moreover, intermittent and continuous therapies had a similar effect on bone volume. However, intermittent therapy was more sparing on bone turnover and bone mineral properties. Intermittent therapy could therefore represent an attractive alternative approach to continuous therapy.
High-Dose Estrogen Induces De Novo Medullary Bone Formation in Female Mice
Authors: Samuels A. , Perry M. J. , Tobias J. H.
Published: April 2006

It is well recognized that, in the mouse, high-dose estrogen induces sclerosis within the shaft of long bones, an action that is largely thought to reflect increased osteoblastic cellular activity. We undertook to characterize this response in more detail, by performing a histologic analysis of the early changes induced by high-dose estrogen in the tibial cavity of young intact female mice. Female mice were sacrificed immediately before or 4, 8, 12, or 24 days after commencing subcutaneous injections of 17β-estradiol (500 μg/animal/week), and longitudinal tibial sections were subsequently examined. Estrogen was found to cause a rapid gain in cancellous bone, with cancellous bone volume increasing by ∼50% after 8 days, and by 5-fold after 24 days. Analysis of cancellous double-labeled surfaces revealed that this gain in bone reflected the emergence of new cancellous bone formation sites within the medullary cavity, rather than the reactivation and extension of formation over pre-existing bone surfaces. Comparison of the time course of these changes between proximal and distal regions of the proximal tibial metaphysis suggested that these new cancellous formation sites appear as a rapid wave extending distally from the secondary spongiosa. Alkaline phosphatase (ALP) immunocytochemistry revealed that, by 12 days after estrogen administration, a population of strongly ALP positive cells had appeared throughout the marrow cavity. We conclude that, at the proximal tibial metaphysis of female mice, estrogen-induced medullary sclerosis largely reflects a process of de novo medullary bone formation, possibly mediated by the generation of osteoblasts from bone marrow osteoprogenitor cells. (J Bone Miner Res 1999; 14: 178–186)
Effect of Prostaglandin and Bisphosphonate on Cancellous Bone Volume and Structure in the Ovariectomized Rat Studied by Quantitative Three-Dimensional Nuclear Magnetic Resonance Microscopy
Authors: Takahashi Masaya , Wehrli Felix W. , Wehrli Suzanne L. , Hwang Scott N. , Lundy Mark W. , Hartke Jim , Borah Babul
Published: April 2006

The purpose of this work was to evaluate the potential of nuclear magnetic resonance microscopy (NMRM) in conjunction with a processing technique to monitor the effect of preventive agents in an ovariectomized (OVX) rat. Twenty-five female Sprague-Dawley rats were OVX at 6 months of age (except for the intact control group), allowed to lose bone for 60 days, and then treated for 60 days. During treatment, animals were administered vehicle, prostaglandin E2 (PGE2; 6 mg/kg), or alendronate (3 μg/kg) subcutaneously once a day. Subsequently, tibiae were harvested and the marrow removed. NMRM was carried out at 9.4 T, with the specimens immersed in 1.2 mM diethylenetriaminepentaacetic acid-gadolinium salt (Gd-DTPA) aqueous solution. A three-dimensional (3D) partial flip-angle pulse sequence was used, providing a 1283 array of (46 μm)3 isotropic voxels. Fifty of the 128 axial images in the 3D data set comprising ∼2.4 mm volume distal to the growth plate were processed from each specimen using a probability-based method for determining bone volume fraction (BVF), tubularity, contiguity, as well as the mean trabecular plate thickness and separation. PGE2 and alendronate altered BVF consistently at all tibial regions. The effect of alendronate was to keep BVF about midway between intact and OVX, whereas PGE2 returned BVF to intact levels. The other parameters showed similar responses to treatment. The strongest discriminator was trabecular BVF, which could obviously differentiate the groups. The study establishes NMRM as a nondestructive histomorphometric method for the quantitative evaluation of drug response in a rat ovariectomy model.
The Anti-Androgen Hydroxyflutamide and Androgens Inhibit Interleukin-6 Production by an Androgen-Responsive Human Osteoblastic Cell Line
Authors: Hofbauer Lorenz C. , Ten Rosa M. , Khosla Sundeep
Published: April 2006

While androgens clearly have significant skeletal effects, the paracrine mediators of androgen action on bone are at present unclear. Interleukin-6 (IL-6) is a candidate cytokine that is produced by osteoblastic lineage cells and promotes osteoclastogenesis and bone resorption. Here, we assessed constitutive as well as IL-1β– and tumor necrosis factor-α (TNF-α)–stimulated IL-6 mRNA expression by Northern analysis and protein secretion by immunoassay in a human androgen-responsive osteoblastic cell line (hFOB/AR-6) which contains ∼4000 androgen receptors (ARs)/nucleus. Treatment with 5α-dihydrotestosterone (DHT) dose-dependently inhibited constitutive and TNF-α/IL-1β–stimulated IL-6 mRNA steady-state levels in hFOB/AR-6 cells by 70–80% at 10−7 M. In addition, testosterone also suppressed TNF-α/IL-1β–stimulated IL-6 mRNA levels by 57%, while the adrenal androgen dehydroepiandrosterone had no effect. Of note, the specific AR antagonist, hydroxyflutamide, also inhibited IL-6 mRNA levels by 70%. Consistent with the Northern analyses, treatment with 5α-DHT, testosterone, and hydroxyflutamide also inhibited IL-6 protein production by 79%, 62%, and 71%, respectively (p < 0.001), while these agents had no effect on IL-6 soluble receptor levels. Finally, we demonstrated that hydroxyflutamide treatment of hFOB/AR-6 cells markedly inhibited the activation and binding of NF-κB (a known stimulator of IL-6 gene transcription) to its response element, thus providing a potential mechanism for its effect on IL-6 production by osteoblasts. These data are consistent with the hypothesis that suppression of osteoblast IL-6 production by androgens may mediate, at least in part, the antiresorptive effects of androgens on bone. Moreover, our findings also indicate that hydroxyflutamide, which is a known AR antagonist in most tissues, may function as a selective AR modulator for effects on IL-6 production by osteoblasts.
Implant Design Affects Markers of Bone Resorption and Formation in Total Hip Replacement
Authors: Qureshi Abid A. , Virdi Amarjit S. , Didonna Michael L. , Jacobs Joshua J. , Masuda Koichi , Paprosky Wayne P. , Thonar Eugene J. M. A. , Sumner Dale R.
Published: April 2006

Concentrations of the bone resorption markers pyridinoline and deoxypyridinoline and the bone formation marker osteocalcin were measured in 24-h urine collections from 30 subjects who underwent unilateral total hip replacements for monoarticular symptomatic osteoarthrosis and 10 controls. The patient groups were divided based on the femoral implant type (cemented cobalt alloy stem, cementless porous coated cobalt alloy stem, and cementless porous coated titanium alloy stem). Urine collections were performed before surgery and then at 3, 6, 12, 24, and 36 months. There were significant changes over time in the three patient groups for pyridinoline, deoxypyridinoline, and the ratio of osteocalcin to deoxypyridinoline (p ≤ 0.01), but the control group values did not change over time. The resorption markers tended to peak at 3 months and the osteocalcin to deoxypyridinoline ratio was more variable, having depressed values in the cementless cobalt alloy group and elevated values in the other two groups compared with baseline. The cementless cobalt alloy group had higher resorption marker levels than the cemented cobalt alloy group at 6, 12, 24, and 36 months and higher levels than the cementless titanium alloy group at all postoperative times (p < 0.05). The osteocalcin to deoxypyridinoline ratio was lower in the cementless cobalt alloy group than in the cemented cobalt alloy group at 3, 6, and 24 months and the cementless titanium alloy group at 6, 12, and 24 months (p < 0.05). For the cemented cobalt chrome group, the baseline-normalized resorption marker values at 3 months and 6 months were correlated with the severity of radiographically assessed bone loss at 36 months (0.749 < r < 0.840; p < 0.05). For the cementless titanium alloy group, baseline-normalized osteocalcin/deoxypyridinoline ratios at 3 months and 6 months were related inversely to radiographic bone loss at 36 months (0.687 < r < 0.749; p < 0.05). Thus, body fluid markers of bone metabolism change after total hip replacement. In addition, the changes in the marker concentrations were sensitive to implant design and were correlated with subsequent stress-shielding-induced bone loss.
Mineralization of Developing Mouse Calvaria as Revealed by Raman Microspectroscopy
Authors: Tarnowski Catherine P. , Ignelzi Michael A. , Morris Michael D.
Published: April 2006

Raman microspectroscopy is a nondestructive vibrational spectroscopic technique that permits the study of organic and mineral species at micron resolution, offers the ability to work with hydrated and dehydrated specimens in vivo or in vitro, and requires minimal specimen preparation. We used Raman microspectroscopy to determine the composition of the mineral environments present in mouse calvaria, the flat bones that comprise the top of the skull. We have acquired Raman transects (lines of point spectra) from mouse calvaria during a developmental time course ranging from embryonic day 13.5 (E13.5; 6 days before birth) to 6 months of age. Exploratory factor analysis (FA) reveals the presence of a variety of apatitic mineral environments throughout the tissue series. The earliest mineral is observed in the fetal day 15.5 (F15.5) mice and is identified as a carbonated apatite. The presence of a heterogeneous mineralized tissue in the postnatal specimens suggests that ionic incorporation and crystal perfection in the lattice vary as the mouse develops. This variation is indicative of the presence of both recently deposited mineral and more matured remodeled mineral. Band area ratios reveal that the mineral/matrix ratio initially increases, reaches a plateau, and then increases again. The carbonate/phosphate band area ratio remains constant from F18.5 to postnatal day 3 (PN3) and then increases with age. Insights into the chemical species, the degree of mineralization, and the multiple mineral environments that are present in normal calvarial tissue will enable us to better understand both normal and abnormal mineralization processes.
Dissociation of Angiogenesis and Osteoclastogenesis During Endochondral Bone Formation in Neonatal Mice
Authors: Deckers Martine M. L. , Van Beek Ermond R. , Van Der Pluijm Gabri , Wetterwald Antoinette , Van Der Wee-Pals Lianne , Cecchini Marco G. , Papapoulos Socrates E. , Löwik Clemens W. G. M.
Published: April 2006

Invasion of the mineralized matrix by endothelial cells and osteoclasts is a key event in endochondral bone formation. To examine the putative role of osteoclast activity in the angiogenic process, we used two in vivo models of suppressed bone resorption: mice treated with the bisphosphonate clodronate and in osteoclast-deficient, osteopetrotic mice. Angiogenesis was assessed in caudal vertebrae of these neonatal mice. This model enables us to study the interaction between osteoclasts and endothelial cells during endochondral bone formation. In control conditions, sinusoid-like structures were detected in the vicinity of tartrate resistance acid phosphatase positive (TRAcP+) osteoclasts. Treatment with clodronate completely abolished osteoclastic bone resorption, whereas angiogenesis remained unaffected. In line with these observations, in the osteopetrotic mouse mutants c-fos knockout mice and op/op mice, capillaries invaded the calcified cartilage in the absence of osteoclasts. In conclusion, our data strongly suggest that during endochondral bone formation, vascular invasion can occur in the absence of osteo(chondro)clastic resorption. In addition, bisphosphonates show no apparent effect on angiogenesis in this in vivo model. These findings may have important clinical implications in the management of skeletal disorders such as metastatic bone disease, in which both osteoclastic bone resorption and angiogenesis contribute to tumor growth. On the other hand, our results confirm that bisphosphonates can be used safely in the treatment of disorders that affect the growing skeleton, such as in juvenile osteoporosis.