bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of Bone-Resorbing Osteoclasts from B220+ Cells: Its Role in Accelerated Osteoclastogenesis due to Estrogen Deficiency
Authors: Sato Takuya , Shibata Takeshi , Ikeda Kyoji , Watanabe Ken
Published: April 2006

Estrogen deficiency stimulates both osteoclastic bone resorption and pre-B lymphopoiesis, the interrelationships between which remain unknown. To investigate the involvement of an increase in the number of B220+ cells in accelerated osteoclastogenesis after estrogen deficiency, we first examined whether ovariectomy (OVX) increased the frequency of clonogenic osteoclast precursors in bone marrow. The results were that after OVX, the frequency of clonogenic osteoclast precursors is increased in bone marrow, suggesting that accumulated osteoclast precursors contribute to accelerated osteoclastogenesis. Further, we found that cocultures of B220+ cells purified from bone marrow cells and stromal ST2 cells in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] gave rise to osteoclasts that can resorb bone and express calcitonin receptors. When the frequencies of clonogenic osteoclast precursors in the purified B220+ and B220− cell fractions were compared, it was found that the fractions gave rise to osteoclasts at similar frequencies, which rules out the possibility of cross-contamination and suggests that the two fractions contain comparable numbers of osteoclast precursors. Furthermore, we identified cells that are positive for both tartrate-resistant acid phosphatase (TRAP) and B220, not only in cocultures of B220+ and ST2 cells, but also in freshly isolated unfractionated bone cells. Therefore, it is concluded that at least a subfraction of B220+ cells are capable of generating osteoclasts and that the increase in the number of B220+ cells caused by estrogen deficiency may contribute to accelerated bone resorption by this novel osteoclastogenesis pathway.
Conditional Ablation of the Osteoblast Lineage in Col2.3Δtk Transgenic Mice
Authors: Visnjic D. , Kalajzic I. , Gronowicz G. , Aguila H. L. , Clark S. H. , Lichtler A. C. , Rowe D. W.
Published: April 2006

Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragment of the rat α1 type I collagen promoter driving a truncated form of the herpes thymidine kinase gene (Col2.3Δtk). Expression of the transgene was found in osteoblasts coincident with other genetic markers of early osteoblast differentiation. Mice treated with ganciclovir (GCV) for 16 days displayed extensive destruction of the bone lining cells and decreased osteoclast number. In addition, a dramatic decrease in bone marrow elements was observed, which was more severe in the primary spongiosum and marrow adjacent to the diaphyseal endosteal bone. Immunostaining for transgene expression within the bone marrow was negative and marrow stromal cell cultures developed normally in the presence of GCV until the point of early osteoblast differentiation. Our findings suggest that the early differentiating osteoblasts are necessary for the maintenance of osteoclasts and hematopoiesis. Termination of GCV treatment produced an exaggerated response of new bone formation in cortical and trabecular bone. The Col2.3Δtk mouse should be a useful model to define the interrelation between bone and marrow elements as well as a model to analyze the molecular and cellular events associated with a defined wave of osteogenesis on termination of GCV treatment.
Arthrodesis of Lumbar Spine Transverse Processes Using Nacre in Rabbit
Authors: Lamghari Meriem , Antonietti Pierre , Berland Sophie , Laurent Alex , Lopez Evelyne
Published: April 2006

This study compares the osteogenic effects of nacre and autogenous bone grafts in a rabbit model of lumbar spine transverse process arthrodesis. A total of 15 rabbits were processed for arthrodesis between the fifth and sixth lumbar vertebrae using nacre powder mixed with autologous blood or autogenous iliac crest bone. Control rabbits were sham operated. Sample vertebrae were removed from the nacre-implanted rabbits at 2, 5, and 11 weeks postsurgery. The autogenous bone graft and sham-operated groups were processed for histological study 11 weeks postsurgery. The results for the three groups were compared at 11 weeks. The nacre-implanted samples taken at 2 weeks showed that the nacre was well tolerated by the host tissue. Endochondral bone formation was seen in the region of the dissolving nacre particles by 5 weeks. The newly formed bone formed a solid fusion between the transverse processes in one-third of the rabbits. There was still new bone formation at 11 weeks at the nacre implant site. Two-thirds of the rabbits had formed a solid fusion. Light microscopy also showed new bone formation 11 weeks after the autologous bone graft. All rabbits had a solid fusion. This initial study indicates that nacre can induce spinal fusion in an acceptable percentage of cases.
Identification of the Calcium-Sensing Receptor in the Developing Tooth Organ
Authors: Mathias Robert S. , Mathews Catharina H. E. , Gao Cen , Machule Darren , Li Wu , Denbesten Pamela K.
Published: April 2006

Calcium (Ca2+) is a critical component of tooth enamel, dentin, and the surrounding extracellular matrix. Ca2+ also may regulate tooth formation, although the mechanisms for such action are poorly understood. The Ca2+-sensing receptor (CaR) that is expressed in the parathyroid gland, kidney, bone, and cartilage has provided a mechanism by which extracellular Ca2+ can regulate cell function. Because these tissues play an important role in maintaining mineral homeostasis and because Ca2+ is hypothesized to play a crucial role in tooth formation, we determined whether the CaR was present in teeth. In this study, using immunohistochemistry, CaR protein was detected in developing porcine molars localized in the predentin (pD), early secretory-stage ameloblasts, maturation-stage smooth-ended ameloblasts (SA), and certain cells in the stratum intermedium. CaR protein and messenger RNA (mRNA) were detected also in an immortalized ameloblast-like cell line (PABSo-E) using immunofluorescence, reverse-transcription polymerase chain reaction (RT-PCR), and Northern analysis. Based on the observation that the CaR is expressed in cultured ameloblasts, we determined whether increments in medium Ca2+ concentration could activate the intracellular Ca2+ signal transduction pathway. In PABSo-E cells, increasing extracellular Ca2+ in the medium from 0 (baseline) to 2.5mM or 5.0 mM resulted in an increase in intracellular Ca2+ above baseline to 534 ± 69 nM and 838 ± 86 nM, respectively. Taken together, these results suggest that the CaR is expressed in developing teeth and may provide a mechanism by which these cells can respond to alterations in extracellular Ca2+ to regulate cell function and, ultimately, tooth formation.
Measurement of Midfemoral Shaft Geometry: Repeatability and Accuracy Using Magnetic Resonance Imaging and Dual-Energy X-ray Absorptiometry
Authors: Woodhead Helen J. , Kemp Allan F. , Blimkie Cameron J. R. , Briody Julie N. , Duncan Craig S. , Thompson Madeleine , Lam Albert , Howman-Giles Robert , Cowell Christopher T.
Published: April 2006

Although macroscopic geometric architecture is an important determinant of bone strength, there is limited published information relating to the validation of the techniques used in its measurement. This study describes new techniques for assessing geometry at the midfemur using magnetic resonance imaging (MRI) and dual-energy X-ray absorptiometry (DXA) and examines both the repeatability and the accuracy of these and previously described DXA methods. Contiguous transverse MRI (Philips 1.5T) scans of the middle one-third femur were made in 13 subjects, 3 subjects with osteoporosis. Midpoint values for total width (TW), cortical width (CW), total cross-sectional area (TCSA), cortical cross-sectional area (CCSA), and volumes from reconstructed three-dimensional (3D) images (total volume [TV] and cortical volume [CVol]) were derived. Midpoint TW and CW also were determined using DXA (Lunar V3.6, lumbar software) by visual and automated edge detection analysis. Repeatability was assessed on scans made on two occasions and then analyzed twice by two independent observers (blinded), with intra- and interobserver repeatability expressed as the CV (CV ± SD). Accuracy was examined by comparing MRI and DXA measurements of venison bone (and Perspex phantom for MRI), against “gold standard” measures made by vernier caliper (width), photographic image digitization (area) and water displacement (volume). Agreement between methods was analyzed using mean differences (MD ± SD%). MRI CVs ranged from 0.5 ± 0.5% (TV) to 3.1 ± 3.1% (CW) for intraobserver and 0.55 ± 0.5% (TV) to 3.6 ± 3.6% (CW) for interobserver repeatability. DXA results ranged from 1.6 ± 1.5% (TW) to 4.4 ± 4.5% (CW) for intraobserver and 3.8 ± 3.8% (TW) to 8.3 ± 8.1% (CW) for interobserver variation. MRI accuracy was excellent for TV (3.3 ± 6.4%), CVol (3.5 ± 4.0%), TCSA (1.8 ± 2.6%), and CCSA (1.6 ± 4.2%) but not TW (4.1 ± 1.4%) or CW (16.4 ± 14.9%). DXA results were TW (6.8 ± 2.7%) and CW (16.4 ± 17.0%). MRI measures of geometric parameters of the midfemur are highly accurate and repeatable, even in osteoporosis. Both MRI and DXA techniques have limited value in determining cortical width. MRI may prove valuable in the assessment of surface-specific bone accrual and resorption responses to disease, therapy, and variations in mechanical loading.
Can Metacarpal Cortical Area Predict the Occurrence of Hip Fracture in Women and Men Over 3 Decades of Follow-Up? Results From the Framingham Osteoporosis Study
Authors: Kiel Douglas P. , Hannan Marian T. , Broe Kerry E. , Felson David T. , Cupples L. Adrienne
Published: April 2006

The purpose of this study was to determine if a single measurement of metacarpal cortical area could predict the subsequent risk of hip fracture over a long-term follow-up period. Thirteen hundred eighty-six women and 1014 men (mean age [±SD] 61 ± 8 years) underwent posteroanterior hand radiography between 1966 and 1970 as part of the Framingham Study. Measurements of cortical bone width (external width and medullary width) were made at the midpoint of the second metacarpal with a digital caliper to the nearest 0.1 mm. Hip fracture occurrence was ascertained on all survivors through December 1995. Surprisingly, in women, there was no significant increase in hip fracture according to metacarpal cortical area measurements (per SD decrease) in either age-adjusted (hazard ratio [HR] = 1.13; 95% CI, 0.94–1.35) or multivariate-adjusted models (HR = 1.06; 95% CI, 0.88–1.27). The same results were seen when considering only those women who were ≥65 years of age at the time of their X-ray or when considering only the first 10 years of follow-up. When the type of hip fracture was considered in women, after adjustment for other risk factors, there appeared to be an association between metacarpal cortical area and intertrochanteric fracture risk (HR = 1.24; 95% CI, 0.91–1.71) but not femoral neck fracture risk (HR = 0.93; 95% CI, 0.71–1.22). In men, the age-adjusted risk of hip fracture was increased modestly per SD decrease in metacarpal cortical area (HR = 1.38; 95% CI, 1.02–1.87), and this remained true after adjustment for potential confounders. In this prospective cohort study with up to 30 years of follow-up, metacarpal cortical area in men predicted hip fracture risk. In women, the only association between metacarpal cortical area and fracture risk was observed for intertrochanteric fractures and was not significant when adjusting for multiple potential confounders. We conclude that this peripheral measure of bone status is not a potent predictor of hip fracture over a long period of follow-up.
High Serum Retinyl Esters Are Not Associated with Reduced Bone Mineral Density in the Third National Health and Nutrition Examination Survey, 1988–1994
Authors: Ballew Carol , Galuska Deborah , Gillespie Cathleen
Published: April 2006

Hypervitaminosis A is sometimes associated with abnormalities of calcium metabolism and bone mineral status. A recent study found a negative association between reported dietary vitamin A intake and bone mineral density (BMD). Some segments of the U.S. population have high fasting serum retinyl ester concentrations, a physiological marker that may reflect high and possibly excessive vitamin A intake. We examined the association between fasting serum retinyl esters and BMD in the Third National Health and Nutrition Examination Survey, 1988–1994 (NHANES III), a large, nationally representative sample of the U.S. population. BMD was measured for the femoral neck, trochanter, intertrochanter, and total hip on all nonpregnant participants aged ≥20 years; 5790 participants also had complete data on fasting serum retinyl esters and covariates including age, body mass index (BMI), smoking, alcohol consumption, dietary supplement use, diabetes, physical activity, and, among women, parity, menopausal status, and the use of oral contraceptives or estrogen-replacement therapy. The sample included non-Hispanic white, non-Hispanic black, and Mexican American men and women. We examined the association between fasting serum retinyl esters and BMD at each site, controlling for covariates with multiple linear regression. We examined the association with osteopenia and osteoporosis with multiple logistic regression. Although the prevalences of high fasting serum retinyl esters concentration and low BMD were both substantial in this sample, there were no significant associations between fasting serum retinyl esters and any measure of bone mineral status.
Mutational Analysis and Functional Correlation With Phenotype in German Patients With Childhood-Type Hypophosphatasia
Authors: Orimo Hideo , Girschick Hermann J. , Goseki-Sone Masae , Ito Masahiro , Oda Kimimitsu , Shimada Takashi
Published: April 2006

The tissue-nonspecific alkaline phosphatase (TNSALP) gene from five German family members with childhood-type hypophosphatasia (HOPS) was analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-direct sequencing method. Four novel missense mutations (T51M, R54S, L258P, and R374H) and two that had been described previously (A160T and R206W) were detected in the respective patients. Mutation A160T was detected in 3 distinct patients, and a polymorphism V505A that had been described previously was detected in the same allele as L258P mutation in 1 patient and in 2 fathers whose V505A alleles were not transmitted to the probands. No other mutations were found in 2 patients. Transient expression of the mutant proteins in COS-1 cells showed that the four novel mutations and R206W were severe alleles, whereas A160T was a moderate allele. Analysis of its enzymatic activity and genetic transmission patterns confirmed that V505A was a polymorphism. Immunoprecipitation of the transiently expressed proteins showed that levels of the 80-kDa mature form of the enzyme were diminished or absent with the severe alleles; instead, levels of high-molecular mass disulfide-linked aggregates were increased. These results suggest that in compound heterozygotes, the combination of severe and moderate alleles may combine to cause the mild phenotype seen in childhood-type HOPS.
The Release and Activation of Transforming Growth Factor β2 Associated with Apoptosis of Chick Hypertrophic Chondrocytes
Authors: Gibson G. , Lin D.-L. , Wang X. , Zhang L.
Published: April 2006

The apoptosis of hypertrophic chondrocytes at the interface between growth cartilage and invading vessels is at the center of a series of critical events in endochondral formation. We have shown that the hypertrophy and apoptosis of chick chondrocytes in culture is associated with the release and activation of transforming growth factor β2 (TGF-β2). Supplementation of the culture medium with agents that influenced the maintenance of hypertrophic differentiation also influenced the release of TGF-β2. A large proportion of the TGF-β2 released from the cells was shown to be in an active form—particularly TGF-β2 associated with the support matrix. Inhibition of apoptosis with a broad-spectrum caspase inhibitor inhibited activation of the matrix-associated TGF-β2. However, inhibition of apoptosis did not diminish the release of TGF-β2. Release of TGF-β2 by chondrocytes at a late stage of their terminal differentiation and its activation in association with apoptosis may provide a mechanism controlling the processes of vascular invasion of growth cartilage and the deposition of bone matrix on nearby cartilage remnants.
Goats as an Osteopenic Animal Model
Authors: Leung K. S. , Siu W. S. , Cheung N. M. , Lui P. Y. , Chow D. H. K. , James A. , Qin L.
Published: April 2006

A large osteopenic animal model that resembles human osteoporotic changes is essential for osteoporosis research. This study aimed at establishing a large osteopenic animal model in goats. Twenty-five Chinese mountain goats were used in which they were either ovariectomized (OVX) and fed with a low-calcium diet (n = 16) or sham-operated (SHAM; n = 9). Monthly photodensitometric analysis on proximal tibial metaphysis and calcaneus was performed. Two iliac crest biopsy specimens obtained before and 6 months after OVX were used for bone mineral density (BMD) measurement with peripheral quantitative computed tomography (pQCT). Lumbar vertebrae (L2 and L7), humeral heads, and calcanei were collected for BMD measurement after euthanasia. The humeral heads and calcanei were used in biomechanical indentation test. BMD measurement showed a significant 25.0% (p = 0.006) decrease in BMD of the iliac crest biopsy specimens 6 months after OVX. It also was statistically significant when compared with the SHAM (p = 0.028). BMD at L2, L7, calcaneus, and humeral head reduced by 24–33% (p ranged from 0.001 to 0.011) when compared with the SHAM. Photodensitometry showed a continuous decrease in bone density after OVX. There were significant decreases of 18.9% in proximal tibial metaphysis (p = 0.003) and 21.8% in calcaneus (p = 0.023) in the OVX group 6 months postoperatively. Indentation test on the humeral head and calcaneus showed a significant decrease 52% (p = 0.006) and 54% (p = 0.001), respectively, in energy required for displacement of 3 mm in the OVX group compared with the SHAM group. The decreases correlated significantly to the decrease in BMD of the corresponding specimens (r2 = 0.439 and 0.581; p < 0.001 for both). In conclusion, this study showed that OVX plus a low-calcium diet could induce significant osteopenia and deterioration of mechanical properties of the cancellous bone in goats.
Apparent Cure of a Newborn with Malignant Osteopetrosis Using Prednisone Therapy
Authors: Iacobini Metello , Migliaccio Silvia , Roggini Mario , Taranta Anna , Werner Beate , Panero Alessandra , Teti Anna
Published: April 2006

A newborn girl with hemorrhagic purpura, suspected neonatal sepsis, and pale and dry skin was lethargic with remarkable hepatosplenomegaly, convergent strabismus, severe anemia, and elevated alkaline phosphatase activity. Radiographs showed a generalized increase in bone density, small medullary cavities, sclerosis of the skull and vertebrae, transverse wavy stripes of sclerotic bone in the metaphyses, and bone-in-bone appearance in phalanges of hands and feet. On this basis, she was diagnosed with malignant infantile osteopetrosis. On the first day of life, the infant was given a blood transfusion and vitamin K (1 mg intravenously [iv]). Corticosteroid therapy was started with prednisone (2 mg/kg per day). She showed marked improvement of symptoms. On the 26th day and 42nd day of life, she received additional blood transfusions. On the 49th day, the patient was discharged and corticosteroid therapy was continued at a regimen of 5 mg/day. Subsequent blood sample analyses revealed normal values for age. At 1 year of life, a bone marrow sample showed normal white and red cell lineages. X-ray confirmed attenuation of the bone sclerosis; therefore, bone marrow transplantation (BMT) was not implemented. At the age of 1.5 years, prednisone therapy was discontinued gradually and withdrawn before the age of 2 years. Subsequent follow-up showed normalization of all radiological and hematologic parameters. At present, the patient is 3 years old and appears healthy with apparently complete regression of the disease.
Good Maintenance of Exercise-Induced Bone Gain with Decreased Training of Female Tennis and Squash Players: A Prospective 5-Year Follow-Up Study of Young and Old Starters and Controls
Authors: Kontulainen Saija , Kannus Pekka , Haapasalo Heidi , Sievänen Harri , Pasanen Matti , Heinonen Ari , Oja Pekka , Vuori Ilkka
Published: April 2006

This prospective 5-year follow-up study of 64 adult female racquet sports players and 27 controls assessed the changes in the playing-to-nonplaying arm bone mineral content (BMC) differences to answer three questions: (1) Are training-induced bone gains lost with decreased training? (2) Is the bone response to decreased training different if the playing career has been started before or at puberty rather than after it? (3) Are the possible bone changes related to the changes in training? The players were divided into two groups according to the starting age of their tennis or squash playing. The mean starting age was 10.5 years (SD, 2.2) among the players who had started training before or at menarche (young starters; n = 36) while 26.4 years (SD, 8.0) among those players who had begun training a minimum of 1 year after menarche (old starters; n = 28). At baseline of the 5-year follow-up, the mean age of the young starters was 21.6 years (SD, 7.6) and that of old starters was 39.4 years (SD, 10.5). During the follow-up, the young starters had reduced the average training frequency from 4.7 times a week (2.7) to 1.4 times a week (1.3) and the old starters from 4.0 times a week (1.4) to 2.0 times a week (1.4), respectively. The 5-year follow-up revealed that despite reduced training the exercise-induced bone gain was well maintained in both groups of players regardless of their clearly different starting age of activity and different amount of exercise-induced bone gain. The gain was still 1.3–2.2 times greater in favor of the young starters (at the follow-up, the dominant-to-nondominant arm BMC difference was 22% [8.4] in the humeral shaft of the young starters versus 10% [3.8] in the old starters, and 3.5% [2.4] in controls). In the players, changes in training were only weakly related to changes in the side-to-side BMC difference (rs = 0.05–0.34, all NS), and this was true even among the players who had stopped training completely a minimum 1 year before the follow-up. In conclusion, if controlled interventions will confirm our findings that an exercise-induced bone gain can be well maintained with decreased activity and that the maintenance of the bone gain is independent of the starting age of activity, exercise can be recommended for preventing osteoporosis and related fractures.
The Dose-Response Effects of Ethanol on the Human Fetal Osteoblastic Cell Line
Authors: Maran A. , Zhang M. , Spelsberg T. C. , Turner R. T.
Published: April 2006

Alcohol is a risk factor for the development of osteoporosis, especially in men. Chronic alcohol abuse decreases bone mass, which contributes to the increased incidence of fractures. To better understand the mechanism of action of ethanol on bone metabolism, we have studied the dose-response effects of ethanol on conditionally immortalized human fetal osteoblasts (hFOB) in culture. Ethanol treatment had no significant effects on osteoblast number after 1 day or 7 days. Ethanol treatment did not reduce type I collagen protein levels at either time point at any dose but slightly reduced alkaline phosphatase activity after 7 days. The messenger RNA (mRNA) levels for alkaline phosphatase, type I collagen, and osteonectin were unaltered by 24 h of ethanol treatment but a high dose (200 mM) reduced mRNA levels for the two bone matrix proteins after 7 days. Ethanol treatment led to dose-dependent increases in transforming growth factor β1 (TGF-β1) mRNA levels and decreases in TGF-β2 mRNA levels. The concentration of ethanol in the medium decreased with time because of evaporation but there was little degradation caused by metabolism. These results, which show that cultured osteoblasts are less sensitive than osteoblasts in vivo, suggest that the pronounced inhibitory effects of ethanol on bone formation are not caused by direct cell toxicity.
Bone Mineralization and Osteoblast Differentiation Are Negatively Modulated by Integrin αvβ3
Authors: Cheng Su-Li , Lai Chung-Fang , Blystone Scott D. , Avioli Louis V.
Published: April 2006

Numerous bone matrix proteins can interact with αv-containing integrins including αvβ3. To elucidate the net effects of the interaction between these proteins and αvβ3 on osteoblast function, we developed a murine osteoblastic cell line that overexpressed human αvβ3. Human αvβ3-integrin was expressed on cell membrane, in which its presence did not alter the surface level of endogenous mouse αvβ3. The expressed human αvβ3 was functional because cell adhesion to osteopontin was increased and this increment was abolished by antibody against human αvβ3. The proliferation rate of cells overexpressing αvβ3 (αvβ3-cells) was increased whereas matrix mineralization was decreased. To elucidate the mechanisms leading to inhibition of matrix mineralization, the expression of proteins important for mineralization was analyzed. Alkaline phosphatase activity and the expression of osteocalcin, type I collagen, and bone sialoprotein (BSP) were decreased whereas osteopontin was stimulated in αvβ3-cells. The regulation of osteopontin, osteocalcin, and BSP expression was mediated via transcriptional mechanism because their promoter activities were altered. Examination of molecules involved in integrin signaling indicated that activator protein-1 (AP-1) and extracellular signal-regulated kinase (Erk) activities were enhanced whereas c-jun N-terminal kinase (JNK) activity was decreased in αvβ3-cells. The activity of p38 and the levels of focal adhesion kinase (FAK) and vinculin were not altered. Moreover, the adhesions of αvβ3-cells to type I collagen and fibronectin were inhibited, which was attributed to decreased β1-integrin levels on cell surface. In conclusion, overexpressing αvβ3-integrin in osteoblasts stimulated cell proliferation but retarded differentiation, which were derived via altered integrin-matrix interactions, signal transduction, and matrix protein expression.
Strontium Ranelate Increases Cartilage Matrix Formation
Authors: Henrotin Y. , Labasse A. , Zheng S. X. , Galais Ph. , Tsouderos Y. , Crielaard J. M. , Reginster J. Y.
Published: April 2006

Based on previous studies showing that strontium ranelate (S12911) modulates bone loss in osteoporosis, it could be hypothesized that this drug also is effective on cartilage degradation in osteoarthritis (OA). This was investigated in vitro on normal and OA human chondrocytes treated or not treated with interleukin-1β (IL-1β). This model mimics, in vitro, the imbalance between chondroformation and chondroresorption processes observed in vivo in OA cartilage. Chondrocytes were isolated from cartilage by enzymatic digestion and cultured for 24–72 h with 10−4−10−3 M strontium ranelate, 10−3 M calcium ranelate, or 2 · 10−3 M SrCl2 with or without IL-1β or insulin-like growth factor I (IGF-I). Stromelysin activity and stromelysin quantitation were assayed by spectrofluorometry and enzyme amplified sensitivity immunoassay (EASIA), respectively. Proteoglycans (PG) were quantified using a radioimmunoassay. Newly synthesized glycosaminoglycans (GAGs) were quantified by labeled sulfate (Na235SO4) incorporation. This method allowed the PG size after exclusion chromatography to be determined. Strontium ranelate, calcium ranelate, and SrCl2 did not modify stromelysin synthesis even in the presence of IL-1β. Calcium ranelate induced stromelysin activation whereas strontium compounds were ineffective. Strontium ranelate and SrCl2 both strongly stimulated PG production suggesting an ionic effect of strontium independent of the organic moiety. Moreover, 10−3 M strontium ranelate increased the stimulatory effect of IGF-I (10−9 M) on PG synthesis but did not reverse the inhibitory effect of IL-1β. Strontium ranelate strongly stimulates human cartilage matrix formation in vitro by a direct ionic effect without stimulating the chondroresorption processes. This finding provides a preclinical basis for in vivo testing of strontium ranelate in OA.
The Effect of a Single Dose of Osteoprotegerin in Postmenopausal Women
Authors: Bekker Pirow J. , Holloway Donna , Nakanishi Arline , Arrighi Michael , Leese Philip T. , Dunstan Colin R.
Published: April 2006

Osteoprotegerin (OPG), a tumor necrosis factor (TNF) receptor family member, is a critical regulator of bone resorption. It is an important inhibitor of the terminal differentiation and activation of osteoclasts. This randomized, double-blind, placebo-controlled, sequential dose escalation study was conducted in postmenopausal women to determine the effect of a single subcutaneous (sc) dose of OPG on bone resorption as indicated by the biochemical markers, urinary N-telopeptide (NTX) and deoxypyridinoline (DPD), which are stable collagen degradation products. NTX levels decreased within 12 h after OPG administration. At the highest dose administered (3.0 mg/kg), a mean percent decrease in NTX of approximately 80% was observed 4 days after dosing. Six weeks after dosing a mean decrease of 14% in NTX was observed. The levels of bone-specific alkaline phosphatase (BSAP), a marker of bone formation, did not change for approximately 3 weeks after dosing. Thereafter, a modest decrease, reaching approximately 30% at 6 weeks, was observed in the 3.0-mg/kg dose group. The rapid decrease from baseline in NTX and delayed decrease in BSAP indicated that OPG acted primarily on osteoclasts to decrease bone resorption. OPG injections are well tolerated. This study, for the first time, indicates that a single sc injection of OPG is effective in rapidly and profoundly reducing bone turnover for a sustained period and that OPG therefore may be effective in treatment of bone diseases characterized by increased bone resorption such as osteoporosis.
Interaction Between the Vitamin D Receptor Gene and Collagen Type Iα1 Gene in Susceptibility for Fracture
Authors: Uitterlinden André G. , Weel Angelique E. A. M. , Burger Huib , Fang Yue , Van Duijn Cornelia M. , Hofman Albert , Van Leeuwen Johannes P. T. M , Pols Huibert A. P.
Published: April 2006

Osteoporosis is a common disease with a strong genetic component. Polymorphisms in the vitamin D receptor (VDR) gene have been implicated in osteoporosis but explain only a small part of the genetic effect on bone mineral density (BMD) while their effect on fractures is still uncertain. Recently, a G to T polymorphism in an Sp1 site in the collagen type Iα1 (COLIA1) gene was found to be associated with reduced BMD and with increased fracture risk. To analyze the combined influence of polymorphisms in the VDR gene and the COLIA1 gene in determining the susceptibility to osteoporotic fracture, we studied 1004 postmenopausal women. The “baT ” VDR haplotype, constructed from three adjacent restriction fragment length polymorphisms, was found to be overrepresented among fracture cases (p = 0.009). This corresponded to an odds ratio (OR) of 1.8 (95% CI, 1.0–3.3) for heterozygous carriers and 2.6 (95%CI, 1.4–5.0) for homozygous carriers of the risk haplotype. The effect was similar for vertebral and nonvertebral fractures and, most importantly, independent of BMD. We observed significant interaction (p = 0.03) between VDR and COLIA1 genotype effects. Fracture risk was not VDR genotype-dependent in the COLIA1 “reference” group (genotype GG) while in the COLIA1 “risk” group (genotypes GT and TT) the risk of fracture was 2.1 (95%CI, 1.0–4.4) for heterozygous and 4.4 (95%CI, 2.0–9.4) for homozygous carriers of the VDR risk haplotype. We conclude that both the VDR and the COLIA1 polymorphisms are genetic markers for osteoporotic fracture in women, independent of BMD. Our data indicate that interlocus interaction is likely to be an important component of osteoporotic fracture risk.
Postnatal and Pubertal Skeletal Changes Contribute Predominantly to the Differences in Peak Bone Density Between C3H/HeJ and C57BL/6J Mice
Authors: Richman C. , Kutilek S. , Miyakoshi N. , Srivastava A. K. , Beamer W. G. , Donahue L. R. , Rosen C. J. , Wergedal J. E. , Baylink D. J. , Mohan S.
Published: April 2006

Previous studies have shown that 60–70% of variance in peak bone density is determined genetically. The higher the peak bone density, the less likely an individual is to eventually develop osteoporosis. Therefore, the amount of bone accrued during postnatal and pubertal growth is an important determining factor in the development of osteoporosis. We evaluated the contribution of skeletal changes before, during, and after puberty to the development of peak bone density in C3H/HeJ (C3H) and C57BL/6J (B6) mice. Volumetric bone density and geometric parameters at the middiaphysis of femora were measured by peripheral quantitative computed tomography (pQCT) from days 7 to 56. Additionally, biochemical markers of bone remodeling in serum and bone extracts were quantified. Both B6 and C3H mice showed similar body and femoral weights. B6 mice had greater middiaphyseal total bone area and thinner cortices than did C3H mice. Within strains, males had thicker cortices than did females. C3H mice accumulated more minerals throughout the study, with the most rapid accumulation occurring postnatally (days 7–23) and during pubertal maturation (days 23–31). C3H mice had higher volumetric bone density as early as day 7, compared with B6 mice. Higher serum insulin-like growth factor I (IGF-I) was present in C3H mice postnatally at day 7 and day 14. Until day 31, B6 male and female mice had significantly higher serum osteocalcin than C3H male and female mice, respectively. Alkaline phosphatase (ALP) was found to be significantly higher in the bone extract of C3H mice compared with B6 mice at day 14. These data are consistent with and support the hypothesis that the greater amount of bone accrued during postnatal and pubertal growth in C3H mice compared with B6 mice may be caused by increased cortical thickness, increased endosteal bone formation, and decreased endosteal bone resorption.
Inaccuracies Inherent in Patient-Specific Dual-Energy X-Ray Absorptiometry Bone Mineral Density Measurements: Comprehensive Phantom-Based Evaluation
Authors: Bolotin H. H. , Sievänen H. , Grashuis J. L. , Kuiper J. W. , Järvinen T. L. N.
Published: April 2006

An extensive series of dual-energy X-ray absorptiometry (DXA) scans and dual polyenergetic X-ray simulation studies of 150 different phantom arrays were carried out to evaluate quantitatively the extent of systematic inaccuracies inherent in DXA in vivo bone mineral density (BMD). These measurements are particularly relevant to lumbar vertebral and proximal femoral sites. The phantoms were specially fabricated near perfect absorptiometric representations of bone material, red marrow (RM) and yellow marrow (YM), and extraosseous mixtures of fat (F) and lean muscle that spanned the full range of soft tissue anthropometrics encountered clinically. In each case, the DXA-measured BMD values obtained using Hologic, Lunar, and Norland densitometers were found to be virtually the same and to be in excellent agreement with the corresponding quantitative simulation study BMD results. Comparisons of the known phantom BMD values and DXA-measured BMD in each case allowed the BMD inaccuracies to be evaluated. These present findings show that these ubiquitous inaccuracies in DXA BMD methodology are of in vivo soft tissue anthropometric genesis. It is found that patient-specific DXA-measured in vivo BMD inaccuracies as high as 20% or more can be readily anticipated clinically, particularly in cases of osteopenic, osteoporotic, and elderly patients. As these inaccuracies exceed considerably DXA precision errors, they may compromise patient-specific evaluations of fracture risk and, in prospective studies, mask or exaggerate clinically significant true changes in BMD. It is concluded that the magnitudes and variability of inherent inaccuracies in DXA-measured in vivo BMD underscore the need for prudence and circumspection in interpretations and assessments of DXA-based clinical studies.
Parathyroid Hormone/Parathyroid Hormone-Related Peptide Type 1 Receptor in Human Bone
Authors: Langub M. Chris , Monier-Faugere Marie-Claude , Qi Quanle , Geng Z. , Koszewski Nicholas J. , Malluche Hartmut H.
Published: April 2006

The parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor (denoted as PTH-1R) is a key signaling factor through which calcium-regulating hormones PTH and PTHrP exert their effects on bone. There are contradictory reports regarding the capability of osteoclasts to express PTH-1R. To address this issue in humans, bone biopsy specimen samples from 9 normal controls and 16 patients with moderate to severe secondary renal hyperparathyroid bone disease (2°HPT) with elevated PTH levels were studied to determine whether osteoclasts in the bone microenvironment express PTH-1R messenger RNA (mRNA) and protein. We report that osteoclasts express the PTH-1R mRNA but the protein is detected only in patients with 2°HPT. The PTH-1R mRNA and protein also were found in osteoblasts, osteocytes, and bone marrow cells. Receptor expression was higher in osteoclasts and osteoblasts of patients with 2°HPT than normal controls (98.0 ± 1.1% vs. 65.7 ± 14.3% and 65.8 ± 3.4% vs. 39.1 ± 6.2%; p < 0.01, respectively). Approximately half of osteoclasts found in bone of patients with 2°HPT have the PTH-1R protein. In patients with 2°HPT, a positive relationship exists between erosion depth, a parameter of osteoclastic activity, and the percentage of osteoclasts with PTH-1R protein (r = 0.58; p < 0.05). In normal controls, an inverse relationship exists between the percentage of osteoblasts with receptor mRNA, mRNA signals/cell, and serum PTH levels (r = −0.82 and p < 0.05 and r = −0.78 and p < 0.01, respectively). The results provide the novel evidence of PTH-1R in human osteoclasts and suggest a functional role for the receptors in 2°HPT.
Human Osteoclast Cathepsin K Is Processed Intracellularly Prior to Attachment and Bone Resorption
Authors: Dodds Robert A. , James Ian E. , Rieman David , Ahern Reina , Hwang Shing Mei , Connor Janice R. , Thompson Scott D. , Veber Daniel F. , Drake Fred H. , Holmes Stephen , Lark Michael W. , Gowen Maxine
Published: April 2006

Cathepsin K is a member of the papain superfamily of cysteine proteases and has been proposed to play a pivotal role in osteoclast-mediated bone resorption. We have developed a sensitive cytochemical assay to localize and quantify osteoclast cathepsin K activity in sections of osteoclastoma and human bone. In tissue sections, osteoclasts that are distant from bone express high levels of cathepsin K messenger RNA (mRNA) and protein. However, the majority of the cathepsin K in these cells is in an inactive zymogen form, as assessed using both the cytochemical assay and specific immunostaining. In contrast, osteoclasts that are closer to bone contain high levels of immunoreactive mature cathepsin K that codistributes with enzyme activity in a polarized fashion toward the bone surface. Polarization of active enzyme was clearly evident in osteoclasts in the vicinity of bone. The osteoclasts apposed to the bone surface were almost exclusively expressing the mature form of cathepsin K. These cells showed intense enzyme activity, which was polarized at the ruffled border. These results suggest that the in vivo activation of cathepsin K occurs intracellularly, before secretion into the resorption lacunae and the onset of bone resorption. The processing of procathepsin K to mature cathepsin K occurs as the osteoclast approaches bone, suggesting that local factors may regulate this process.
The Bone Morphogenetic Proteins Antagonist Noggin Inhibits Membranous Ossification
Authors: Aspenberg Per , Jeppsson Charlotte , Economides Aris N.
Published: April 2006

Bone morphogenetic proteins (BMPs) are expressed and secreted during fracture repair. Although they are likely to be required for this process, little is known about their physiological role in bone regeneration. Noggin is a protein that specifically binds and inactivates several BMPs. It plays fundamental roles during early embryonal development and limb morphogenesis by this BMP-inactivating activity. This study shows that Noggin can modify bone formation in vivo in the adult animal and, thus, indirectly, that BMP signaling is indispensable in this process. A noggin mutein (hNgΔB2-Fc) engineered so as to display increased bioavailability was used. Bilateral titanium bone chambers were inserted in 70 rats, and side comparisons for bone formation in the chambers were done. The hNgΔB2-Fc had no effect on total amount of tissue formed in the chamber but decreased the amount of bone compared with both buffer controls and a control made up of an Fc-tagged IL-6Rα protein, which had no effects of its own. Also, wild-type noggin inhibited bone formation. Thus, endogenous BMP signaling is necessary for normal bone regeneration.
Collagen Metabolism Is Markedly Altered in the Hypertrophic Cartilage of Growth Plates from Rats with Growth Impairment Secondary to Chronic Renal Failure
Authors: Álvarez Jesús , Balbín Milagros , Fernández Marta , López José M.
Published: April 2006

Skeletal growth depends on growth plate cartilage activity, in which matrix synthesis by chondrocytes is one of the major processes contributing to the final length of a bone. On this basis, the present work was undertaken to ascertain if growth impairment secondary to chronic renal insufficiency is associated with disturbances of the extracellular matrix (ECM) of the growth plate. By combining stereological and in situ hybridization techniques, we examined the expression patterns of types II and X collagens and collagenase-3 in tibial growth plates of rats made uremic by subtotal nephrectomy (NX) in comparison with those of sham-operated rats fed ad libitum (SAL) and sham-operated rats pair-fed with NX (SPF). NX rats were severely uremic, as shown by markedly elevated serum concentrations of urea nitrogen, and growth retarded, as shown by significantly decreased longitudinal bone growth rates. NX rats showed disturbances in the normal pattern of chondrocyte differentiation and in the rates and degree of substitution of hypertrophic cartilage with bone, which resulted in accumulation of cartilage at the hypertrophic zone. These changes were associated with an overall decrease in the expression of types II and X collagens, which was especially marked in the abnormally extended zone of the hypertrophic cartilage. Unlike collagen, the expression of collagenase-3 was not disturbed severely. Electron microscopic analysis proved that changes in gene expression were coupled to alterations in the mineralization as well as in the collagen fibril architecture at the hypertrophic cartilage. Because the composition and structure of the ECM have a critical role in regulating the behavior of the growth plate chondrocytes, results obtained are consistent with the hypothesis that alteration of collagen metabolism in these cells could be a key process underlying growth retardation in uremia.
The Prospects of Estimating Trabecular Bone Tissue Properties from the Combination of Ultrasound, Dual-Energy X-Ray Absorptiometry, Microcomputed Tomography, and Microfinite Element Analysis
Authors: Van Lenthe G. H. , Van Den Bergh J. P. W. , Hermus A. R. M. M. , Huiskes R.
Published: April 2006

Osteoporosis commonly is assessed by bone quantity, using bone mineral density (BMD) measurements from dual-energy X-ray absorptiometry (DXA). However, such a measure gives neither information about the integrity of the trabecular architecture nor about the mechanical properties of the constituting trabeculae. We investigated the feasibility of deriving the elastic modulus of the trabeculae (the tissue modulus) from computer simulation of mechanical testing by microfinite element analysis (μFEA) in combination with measurements of ultrasound speed of sound (SOS) and BMD measurements. This approach was tested on 15 postmortem bovine bone cubes. The apparent elastic modulus of the specimens was estimated from SOS measurements in combination with BMD. Then the trabecular morphology was reconstructed using microcomputed tomography (μCT). From the reconstruction a mesh for μFEA was derived, used to simulate mechanical testing. The tissue modulus was found by correlating the apparent moduli of the specimens as assessed by ultrasound with the ones as determined with μFEA. A mean tissue modulus of 4.5 GPa (SD, 0.69) was found. When adjusting the μFEA-determined elastic moduli of the entire specimens with their calculated tissue modulus, an overall correlation of R2 = 96% with ultrasound-predicted values was obtained. We conclude that the apparent elastic stiffness characteristics as determined from ultrasound correlate linearly with those from μFEA. From both methods in combination, the elastic stiffness of the mineralized tissue can be determined as an estimator for mechanical tissue quality. This method can already be used for biopsy specimens, and potentially could be applicable in vivo as well, when clinical CT or magnetic resonance imaging (MRI) tools with adequate resolution reach the market. In this way, mechanical bone quality could be estimated more accurately in clinical practice.
Protective Effect of Short-Term Calcitriol or Cyclical Etidronate on Bone Loss After Cardiac or Lung Transplantation
Authors: Henderson Kathy , Eisman John , Keogh Anne , MaCdonald Peter , Glanville Allan , Spratt Phillip , Sambrook Philip
Published: April 2006

Bone loss is most rapid in the immediate period after cardiac or lung transplantation. This randomized study compared the efficacy of 6 months of treatment with either calcitriol (1,25-dihydroxyvitamin D3; 0.5 μg/day) or two cycles of etidronate plus calcium in preventing bone loss in 41 patients undergoing cardiac or lung transplantation. Patients were followed for 18 months after cessation of treatment. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA). There were no significant differences between groups with respect to age or cumulative dose of prednis(ol)one or cyclosporin over the 2 years. Bone loss did not differ between groups after 6 months and, despite 6 months prophylaxis with either agent, bone loss was significant in both groups at 6 months and 12 months. However, compared with an untreated reference group, both therapies offered significant protection at 6 months and etidronate provided significant protective carryover after therapy had been discontinued. These data suggest short-term prophylaxis with calcitriol or cyclical etidronate is partially effective in reducing bone loss after cardiac or lung transplantation but treatment needs to be continued for a longer term.