bluuebunny/crossref_metadata_2025 huggingface dataset:
Potent and Selective Inhibition of Human Cathepsin K Leads to Inhibition of Bone Resorption In Vivo in a Nonhuman Primate
Authors: Stroup George B. , Lark Michael W. , Veber Daniel F. , Bhattacharyya Amit , Blake Simon , Dare Lauren C. , Erhard Karl F. , Hoffman Sandra J. , James Ian E. , Marquis Robert W. , Ru Yu , Vasko-Moser Janice A. , Smith Brian R. , Tomaszek Thadeus , Gowen Maxine
Published: April 2006

Cathepsin K is a cysteine protease that plays an essential role in osteoclast-mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB-357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB-357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin-releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB-357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis.
Acid Attack and Cathepsin K in Bone Resorption Around Total Hip Replacement Prosthesis
Authors: Konttinen Yrjö T. , Takagi Michiaki , Mandelin Jami , Lassus Jan , Salo Jari , Ainola Mari , Li Tian-Fang , Virtanen Ismo , Liljeström Mikko , Sakai Hideaki , Kobayashi Yasuhiro , Sorsa Timo , Lappalainen Reijo , Demulder Anne , Santavirta Seppo
Published: April 2006

Normal bone remodeling and pathological bone destruction have been considered to be osteoclast-driven. Osteoclasts are able to attach to bare bone surface and produce an acidic subcellular space. This leads to acid dissolution of hydroxyapatite, allowing cathepsin K to degrade the organic type I collagen-rich osteoid matrix under the acidic condition prevailing in Howship lacunae. Using a sting pH electrode, the interface membrane around a loosened total hip replacement prosthesis was found to be acidic. Confocal laser scanning disclosed irregular demineralization of the bone surface in contact with the acidic interface. Cathepsin K, an acidic collagenolytic enzyme, was found in interface tissue macrophages/giant cells and pseudosynovial fluid. Tissue extracts contained high levels of cathepsin K messenger RNA (mRNA) and protein. These observations suggest the presence of an acid- and cathepsin K-driven pathological mechanism of bone resorption, mediated not by osteoclasts in subosteoclastic space, but rather by the uncontrolled activity of macrophages in extracellular space.
Cooperative Effects of Exercise Training and Genistein Administration on Bone Mass in Ovariectomized Mice
Authors: Wu Jian , Wang Xin Xiang , Takasaki Misao , Ohta Atsutane , Higuchi Mitsuru , Ishimi Yoshiko
Published: April 2006

We reported that genistein, a soybean isoflavone, prevents bone loss caused by estrogen deficiency, without undesirable effects on the uterus. In this study, we examined cooperative effects of genistein administration and running exercise on bone mass in ovariectomized (OVX) mice. Female mice aged 7 weeks were either sham-operated or OVX and divided into six groups: (1) sham; (2) OVX; (3) OVX, treated with genistein at a submaximal dose (0.4 mg/day) subcutaneously (G); (4) OVX, exercised on a treadmill daily for 30 minutes/day at 12 m/minute on a 10° uphill slope (Ex); (5) OVX, given genistein and exercised (ExG); and (6) OVX, treated with 17β-estradiol (0.03 μg/day) in the same manner as genistein (E2). Four weeks after intervention, bone mass was estimated by dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). Bone mineral density (BMD) of the whole femur measured by DXA was higher in both the G and the Ex groups than in the OVX group. Furthermore, BMD in the ExG group was significantly higher than that in the groups receiving either intervention alone. Bone area in distal region of the femur was significantly higher in Ex and ExG groups as compared with those in the OVX and G groups. pQCT analysis showed that the cross-sectional areas (CSAs) and periosteum perimeter at midshaft of the femur did not differ in the sham and OVX groups but were significantly higher in Ex and ExG groups. Histomorphometric analysis showed that bone formation rate/bone surface (BFR/BS) was significantly higher in both Ex and ExG groups as compared with that in non-exercised groups. The bone volume (BV/TV) in the distal femoral cancellous bone was lower in the OVX than that in the sham group, and it was restored completely in the ExG group, as in the E2 group. Thickness of the trabecular bone (Tb.Th) was higher in Ex and ExG groups than that in the OVX and G groups. These results indicate that the combined intervention of moderate exercise and the submaximal dose of genistein administration show a cooperative effect in preventing bone loss in OVX mice.
Accelerated Up-Regulation of L-Sox5, Sox6, and Sox9 by BMP-2 Gene Transfer During Murine Fracture Healing
Authors: Uusitalo Hannele , Hiltunen Ari , Ahonen Matti , Gao Tie-Jun , Lefebvre Veronique , Harley Vincent , Kähäri Veli-Matti , Vuorio Eero
Published: April 2006

Fracture repair is the best-characterized situation in which activation of chondrogenesis takes place in an adult organism. To better understand the mechanisms that regulate chondrogenic differentiation of mesenchymal progenitor cells during fracture repair, we have investigated the participation of transcription factors L-Sox5, Sox6, and Sox9 in this process. Marked up-regulation of L-Sox5 and Sox9 messenger RNA (mRNA) and smaller changes in Sox6 mRNA levels were observed in RNAse protection assays during early stages of callus formation, followed by up-regulation of type II collagen production. During cartilage expansion, the colocalization of L-Sox5, Sox6, and Sox9 by immunohistochemistry and type II collagen transcripts by in situ hybridization confirmed a close relationship of these transcription factors with the chondrocyte phenotype and cartilage production. On chondrocyte hypertrophy, production of L-Sox5, Sox9 and type II collagen were down-regulated markedly and that of type X collagen was up-regulated. Finally, using adenovirus mediated bone morphogenetic protein 2 (BMP-2) gene transfer into fracture site we showed accelerated up-regulation of the genes for all three Sox proteins and type II collagen in fractures treated with BMP-2 when compared with control fractures. These data suggest that L-Sox5, Sox6, and Sox9 are involved in the activation and maintenance of chondrogenesis during fracture healing and that enhancement of chondrogenesis by BMP-2 is mediated via an L-Sox5/Sox6/Sox9-dependent pathway.
Effects of Daily Treatment with Parathyroid Hormone on Bone Microarchitecture and Turnover in Patients with Osteoporosis: A Paired Biopsy Study
Authors: Dempster David W. , Cosman Felicia , Kurland Etah S. , Zhou Hua , Nieves Jeri , Woelfert Lillian , Shane Elizabeth , Plavetić Katarina , Müller Ralph , Bilezikian John , Lindsay Robert
Published: April 2006

We examined paired iliac crest bone biopsy specimens from patients with osteoporosis before and after treatment with daily injections of 400 U of recombinant, human parathyroid hormone 1–34 [PTH(1–34)]. Two groups of patients were studied. The first group was comprised of 8 men with an average age 49 years. They were treated with PTH for 18 months. The second group was comprised of 8 postmenopausal women with an average age 54 years. They were treated with PTH for 36 months. The women had been and were maintained on hormone replacement therapy for the duration of PTH treatment. Patients were supplemented to obtain an average daily intake of 1500 mg of elemental calcium and 100 IU of vitamin D. The biopsy specimens were subjected to routine histomorphometric analysis and microcomputed tomography (CT). Cancellous bone area was maintained in both groups. Cortical width was maintained in men and significantly increased in women. There was no increase in cortical porosity. There was a significant increase in the width of bone packets on the inner aspect of the cortex in both men and women. This was accompanied by a significant decrease in eroded perimeter on this surface in both groups. Micro-CT confirmed the foregoing changes and, in addition, revealed an increase in connectivity density, a three dimensional (3D) measure of trabecular connectivity in the majority of patients. These findings indicate that daily PTH treatment exerts anabolic action on cortical bone in patients with osteoporosis and also can improve cancellous bone microarchitecture. The results provide a structural basis for the recent demonstration that PTH treatment reduces the incidence of osteoporosis-related fractures.
Prevalence and Causes of Low Bone Density and Fractures in Kidney Transplant Patients
Authors: Patel Sanjeev , Kwan Jonathan T. C. , McCloskey Eugene , McGee Geraldine , Thomas Gill , Johnson Debbie , Wills Rachel , Ogunremi Lara , Barron Jeffrey
Published: April 2006

Osteoporosis is known to occur in patients with kidney transplants, but limited information is available about the prevalence and causes of this complication. We asked all 330 patients with kidney transplants in our unit to participate in this study of whom 165 (50%) agreed to do so. The characteristics of the participating patients were similar to the remaining 165 nonparticipants. Seventy of 165 (42%) of the participants were women of whom 40 were postmenopausal in contrast to the men of whom only one was hypogonadal. Bone mineral density (BMD) was significantly reduced at the radius (Z score, −1.5) and femoral neck (Z score, −0.7), but the lumbar spine was normal. BMD was lower in women than men at all skeletal sites. Osteoporosis was found in 10–44% and osteopenia was found in 35–50% of women depending on the site. BMD was related inversely to time since transplantation and cumulative prednisolone dose. Twenty-seven of the 165 (16%) patients had either vertebral deformities or a history of a low trauma fracture after transplantation. This fracture group consisted of 10/27 (37%) men and 17/27 (63%) women, of whom 14 were postmenopausal. Fracture patients tended to be older and have a longer duration of renal failure, dialysis, transplantation, greater cumulative steroid dose, and higher bone resorption markers than the nonfracture group. No differences were found for cumulative doses of cyclosporin or tacrolimus. Logistic regression showed that only duration of dialysis and time since transplantation significantly increased fracture risk, with odds ratio (OR) for each year of dialysis or transplantation being 1.21 (CI, 1.00–1.48) and 1.14 (CI, 1.05–1.23), respectively. These data show that low bone density and fractures are common in patients with kidney transplant and are determined by both pre- and posttransplant variables. Fracture risk was greatest in women, particularly if they were postmenopausal and we recommend that this subgroup is targeted for assessment and treatment.
Ibandronate: A Comparison of Oral Daily Dosing Versus Intermittent Dosing in Postmenopausal Osteoporosis
Authors: Riis B. J. , Ise J. , Von Stein T. , Bagger Y. , Christiansen C.
Published: April 2006

The objective of this study was to compare efficacy and safety of continuous versus intermittent oral dosing of ibandronate. Two hundred forty women aged 55–75 years with postmenopausal osteoporosis were randomized to active treatment or placebo. Similar total doses of ibandronate were provided by treatment regimens with either continuous 2.5 mg of ibandronate daily (n = 81) or intermittent 20 mg of ibandronate every other day for the first 24 days, followed by 9 weeks without active drug (n = 78). The placebo group (total, n = 81) was crossed over after 12 months to receive either continuous (n = 37) or intermittent ibandronate (n = 35). By 24 months, bone mineral density (BMD) had increased significantly relative to baseline in both active treatment groups. The continuous and intermittent groups showed statistically equivalent increases in lumbar spine BMD of +5.64% (±0.53) and +5.54% (±0.53) and in total hip of +3.35% (±0.40) and +3.41% (±0.40), respectively (per protocol population). Biochemical markers of bone turnover decreased significantly in both treatment groups. The level of marker suppression was similar, although the intermittent group displayed, as expected, more fluctuation over the treatment period. The frequency of adverse events was similar in the treatment groups. In conclusion, the intermittent and continuous regimens showed equivalent changes in BMD and bone turnover. These results confirm previous preclinical findings indicating that the efficacy of ibandronate depends on the total oral dose given rather than on the dosing schedule. This supports development of new flexible dosing regimens targeted to minimize the frequency of dosing, which are expected to improve convenience and lead to enhanced long-term patient compliance.
Glucocorticoid Excess During Adolescence Leads to a Major Persistent Deficit in Bone Mass and an Increase in Central Body Fat
Authors: Abad Veronica , Chrousos George P. , Reynolds James C. , Nieman Lynnette K. , Hill Suvimol C. , Weinstein Robert S. , Leong Gary M.
Published: April 2006

Endogenous Cushing's syndrome (CS) in children causes growth retardation, decreased bone mass, and increased total body fat. No prospective controlled studies have been performed in children to determine the long-term sequelae of CS on peak bone mass and body composition. A 15-year-old girl with Cushing disease (CD), and her healthy identical co-twin, were followed for 6 years after the CD was cured. At the 6-year follow-up both twins had areal bone mineral density (BMD) and body composition determined by dual-energy X-ray absorptiometry (DXA) and three-dimensional quantitative computed tomography (3DQCT). Z scores for height, weight, and body mass index (BMI) were −2.3, −0.8 and 0.2, and 1.2, 0.2, and −0.6, in the twin with CD and her co-twin, respectively. In the twin with CD, areal BMD and bone mineral apparent density (BMAD) at different sites varied from 0.7 to 3 SD below her co-twin. Volumetric lumbar spine bone density Z score was −0.75 and 1.0, and total body, abdominal visceral, and subcutaneous fat (%) was 42, 10, and 41 versus 26, 4, and 17 in the twin with CD and her co-twin, respectively. The relationship between total body fat and L2-L4 BMAD was inverse in the twin with CD (p < 0.05), which by contrast in her co-twin was opposite and direct (p < 0.001). In the twin with CD, despite cure, there was a persistent deficit in bone mass and increase in total and visceral body fat. These observations suggest that hypercortisolism (exogenous or endogenous) during adolescence may have persistent adverse effects on bone and fat mass.
Quantitative Ultrasound and Trabecular Architecture in the Human Calcaneus
Authors: Nicholson P. H. F. , Müller R. , Cheng X. G. , Rüegsegger P. , Van Der Perre G. , Dequeker J. , Boonen S.
Published: April 2006

Relationships between quantitative ultrasound (QUS), density (bone volume density [BV/TV]), and trabecular architecture were investigated in 69 calcaneal cancellous bone cubes. Ultrasound signal velocity, phase velocity, attenuation, and broadband ultrasonic attenuation (BUA) measurements were made along the mediolateral axis. Density and architectural parameters were measured using microcomputed tomography (μCT). Density yielded the best correlations with QUS (r2 = 73–77%). Of the individual architectural parameters, correlations with QUS were highest for the Structure Model Index (SMI), a parameter quantifying the relative proportion of rods and plates (r2 = 57–63%). After adjustment for density, significant associations with QUS remained for SMI, trabecular spacing (Tb.Sp), and trabecular number (Tb.N), although the variance in QUS attributable uniquely to individual architectural parameters was at best 4%. In multivariate regression models, combinations of density and architectural parameters explained 76–82% of the variance in QUS, representing an r2 increase of, at most, 8% compared with using density alone. However, multivariate models using combinations of architectural parameters alone (i.e., density excluded) also had a good predictive ability for QUS (r2 = 73–81%). Thus, although these data show modest but significant density-independent relationships between QUS and trabecular architecture in the human calcaneus for the first time, the causal relationships behind the variation in acoustic properties remain obscure. Given the relative weakness and complexity of the emerging associations between QUS and architecture, it is prudent to regard QUS measurements in calcaneal bone primarily as an indicator of bone density.
Vitamin A Antagonizes Calcium Response to Vitamin D in Man
Authors: Johansson S. , Melhus H.
Published: April 2006

For unknown reasons, the highest incidence of osteoporosis is found in northern Europe. In these populations, the sunlight exposure is limited and the vitamin A intake is high. The interaction between vitamin A and D has been the subject of several in vitro and animal studies. We have studied the acute effects of vitamin A and D on calcium homeostasis in 9 healthy human subjects. We compared the effect of (i) 15 mg of retinyl palmitate, (ii) 2 μg of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], (iii) 15 mg of retinyl palmitate plus 2 μg of 1,25(OH)2D3, and (iv) placebo in a double-blind crossover study. The subjects took vitamin preparations at 10:00 p.m. and the following day blood samples were collected five times from 8:00 a.m. to 4:00 p.m. Serum levels of 1,25(OH)2D3 and retinyl esters increased (1.7-fold and 8.3-fold, respectively; p < 0.01). As expected, serum calcium (S-calcium) increased (2.3%; p < 0.01) and S-parathyroid hormone (PTH) decreased (−32%,;p < 0.05) after 1,25(OH)2D3 intake. In contrast, retinyl palmitate intake resulted in a significant decrease in S-calcium when taken alone (−1.0%; p < 0.05) and diminished the calcium response to 1,25(OH)2D3 after the combined intake (1.4%; p < 0.01). S-PTH was unaffected by retinyl palmitate. No significant changes in serum levels of the degradation product of C-telopeptide of type I collagen (CrossLaps), or U-calcium/creatinine levels were found. In conclusion, an intake of vitamin A corresponding to about one serving of liver antagonizes the rapid intestinal calcium response to physiological levels of vitamin D in man.
Acidic Fibroblast Growth Factor Signaling Inhibits Peroxynitrite-Induced Death of Osteoblasts and Osteoblast Precursors
Authors: Kelpke S. S. , Reiff D. , Prince C. W. , Thompson J. A.
Published: April 2006

After trauma injury to the musculoskeletal system, conditions such as ischemia and inflammation involve excess production of superoxide (), nitric oxide (·NO), and their reaction product, peroxynitrite (ONOO−). Exposure of murine osteoblasts and rat-derived primary osteoblast precursors to ONOO− resulted in a dose- and time-dependent delayed cell death that was more characteristic of apoptosis than necrosis. Exposure of both cell populations to ONOO− immediately enhanced phosphorylation and nitration of tyrosine residues within several polypeptides. Treatment of osteoblasts and osteoblast precursors with exogenous acidic fibroblast growth factor (FGF-1) enhanced cellular growth, increased endogenous levels of tyrosine phosphorylation, and significantly induced expression of both osteopontin and osteocalcin messenger RNA (mRNA) as well as osteopontin protein. Pretreatment of both cell populations with exogenous FGF-1 prevented ONOO−-mediated death. Cell signaling induced by FGF-1 pretreatment had no major effect of total levels of tyrosine nitration after ONOO− treatment. Collectively, these in vitro efforts show that FGF-1 signaling renders osteoblasts and osteoblast precursors resistant to the cytotoxic effects of ONOO−. Consequently, results presented here predict the therapeutic use of this growth factor for promoting the progression of bone repair mechanisms after fracture trauma.
Hypoparathyroidism Associated with Aneurysm of the Left Subclavian Artery (Kommerell's Diverticulum) in an Adult Patient with a Chromosome 22q11.2 Deletion
Authors: Trombetti Andrea , Bottani Armand , George François , Rizzoli René
Published: April 2006

Hypoparathyroidism may either be acquired or of congenital origin. From the latter group, which represents a minority of cases, agenesis or hypoplasia of the parathyroid glands resulting in symptomatic hypocalcemia in the newborn or infant frequently is caused by a microdeletion of chromosome 22q11.2. We describe a man in whom hypoparathyroidism was first diagnosed at the age of 59 years. The endocrine disorder was found to be associated with this chromosome imbalance and also with an aneurysm of the left subclavian artery (Kommerell's diverticulum) compressing the esophagus and trachea. Given the potential implication for genetic counseling, a 22q11.2 deletion should be considered in the differential diagnosis of adult patients with hypoparathyroidism of unknown origin and should be searched for by appropriate molecular cytogenetic technique.
Developmental Distribution of Collagen Type XII in Cartilage: Association with Articular Cartilage and the Growth Plate
Authors: Gregory Kate E. , Keene Douglas R. , Tufa Sara F. , Lunstrum Gregory P. , Morris Nicholas P.
Published: April 2006

Collagen type XII is a member of the fibril-associated collagens and is characterized by a short triple-helical domain with three extended noncollagenous NC3 domains. Previous studies suggested that collagen XII is a component of cartilage but little is known about its spatial-temporal distribution. This study uses a polyclonal antibody to the purified NC3 domain to investigate its developmental distribution in rat forelimb. Collagen XII was present at the joint interzone on embryonic day 16 (E16d) and restricted to the presumptive articular cartilage by E18d. Labeling of the articular surface intensified as development progressed postnatally (day 1 [1d] to 28d) and extended approximately six cell diameters deep. In juvenile rats, collagen XII antibodies also labeled the longitudinal and transverse septa of stacked chondrocytes in the growth plate. However, collagen XII was not associated at any developmental stage with the cartilaginous secondary ossification center and was only weakly expressed in epiphyseal cartilage. Ultrastructural localization of the NC3 domain epitope showed labeling of the surface of collagen II fibrils both in tissue and in isolated fibrils. The results presented provide further evidence that articular cartilage differs substantially from the underlying epiphyseal cartilage and that different chondrocytic developmental fates are reflected in the composition of their extracellular matrix starting early in development. In addition, collagen XII was distributed in areas of cartilage with more organized fibril orientation and may have a role in promoting alignment or stabilizing such an organization, thereby creating a matrix capable of withstanding load-bearing forces.
Dentin Matrix Protein 1 Is Predominantly Expressed in Chicken and Rat Osteocytes But Not in Osteoblasts
Authors: Toyosawa S. , Shintani S. , Fujiwara T. , Ooshima T. , Sato A. , Ijuhin N. , Komori T.
Published: April 2006

Although osteocytes are the most abundant cells in bone, little is known about their function, and no specific marker protein for osteocytes has been described. Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein expressed in tooth organ and bone. Our previous work showed that in the chicken, which is not capable of forming tooth, DMP1 messenger RNA (mRNA) is highly expressed in bone by Northern blot analysis. To clarify the significance of DMP1 expression in bone, the expression of DMP1 mRNA and its protein was examined in the chicken and rat. In the chicken, DMP1 mRNA was detected only in bone tissues and was localized in osteocytes and preosteocytes but not in osteoblasts. Similarly, in the rat, DMP1 mRNA was predominantly expressed in osteocytes and preosteocytes in bone matrix but not in osteoblasts located at the bone surface. Antiserum was raised against the peptide from rat DMP1, and the localization of DMP1 was examined by immunohistochemistry. In the development of bone, DMP1 was first detected in newly formed bone matrix after osteoblastic cells had been embedded within it. After the appearance of typical osteocytes, DMP1 was localized in the pericellular bone matrix of osteocytes, including their processes. These data show that DMP1 is a bone matrix protein specifically expressed in osteocytes and preosteocytes and suggest that DMP1 plays a role in bone homeostasis because of its high calcium ion-binding capacity.
Regulation of Osteoclast Differentiation by Fibroblast Growth Factor 2: Stimulation of Receptor Activator of Nuclear Factor κB Ligand/Osteoclast Differentiation Factor Expression in Osteoblasts and Inhibition of Macrophage Colony-Stimulating Factor Function in Osteoclast Precursors
Authors: Chikazu Daichi , Katagiri Mika , Ogasawara Toru , Ogata Naoshi , Shimoaka Takashi , Takato Tsuyoshi , Nakamura Kozo , Kawaguchi Hiroshi
Published: April 2006

This study investigated the mechanism of direct and indirect actions of fibroblast growth factor 2 (FGF-2) on osteoclast differentiation using two mouse cell culture systems. In the coculture system of osteoblasts and bone marrow cells, FGF-2 stimulated osteoclast formation. This effect was decreased markedly by osteoprotegerin (OPG) or NS-398, a selective cyclo-oxygenase 2 (COX-2) inhibitor. FGF-2 (≥10−9 M) stimulated receptor activator of nuclear factor κB ligand/osteoclast differentiation factor (RANKL/ODF) messenger RNA (mRNA) expression from 2 h to 7 days in cultured osteoblasts. NS-398 did not affect the early induction but decreased the later one, indicating that the later effect is mediated by COX-2 induction in osteoblasts. To study the direct action of FGF-2 on osteoclast precursors, we used mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of soluble RANKL/ODF (sRANKL/ODF) and macrophage colony-stimulating factor (M-CSF). Although osteoblasts expressed all FGF receptors (FGFR-1 to −4), only FGFR-1 was detected in C7 cells at various differentiation stages. FGF-2 alone or in combination with sRANKL/ODF did not induce osteoclastogenesis from C7 cells; however, FGF-2 from lower concentrations (≥10−11 M) significantly decreased osteoclast formation induced by M-CSF in the presence of sRANKL/ODF. FGF-2 did not alter mRNA levels of M-CSF receptor (Fms) or RANK in C7 cells. Immunoprecipitation/immunoblotting analyses revealed that tyrosine phosphorylation of several cellular proteins including Fms in C7 cells induced by M-CSF was inhibited by FGF-2 in the presence of sRANKL/ODF. We conclude that FGF-2 regulates osteoclast differentiation through two different mechanisms: (1) an indirect stimulatory action via osteoblasts to induce RANKL/ODF partly through COX-2 induction and prostaglandin production and (2) a direct inhibitory action on osteoclast precursors by counteracting M-CSF signaling.
Adherent Endotoxin on Orthopedic Wear Particles Stimulates Cytokine Production and Osteoclast Differentiation
Authors: Bi Yanming , Seabold Joscelyn M. , Kaar Scott G. , Ragab Ashraf A. , Goldberg Victor M. , Anderson James M. , Greenfield Edward M.
Published: April 2006

Aseptic loosening of orthopedic implants is thought to be caused primarily by osteoclast differentiation induced by bone resorptive cytokines produced in response to phagocytosis of implant-derived wear particles. This study examined whether adherent endotoxin on the wear particles is responsible for inducing osteoclast differentiation as well as production of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α). Removal of adherent endotoxin almost completely inhibited the responses to titanium (Ti) particles by both murine marrow cells and human peripheral blood monocytes. In vivo experiments showed that endotoxin removal reduced particle-induced osteolysis by 50–70%. Addition of lipopolysaccharide (LPS) to the “endotoxin-free” particles restored their ability to induce cytokine production and osteoclast differentiation in vitro. Moreover, marrow cells from mice that are hyporesponsive to endotoxin because of mutation of Toll-like receptor 4 induced significantly less cytokine production and osteoclast differentiation in response to Ti particles with adherent endotoxin than did marrow cells from normoresponsive mice. This mutation also resulted in significantly less particle-induced osteolysis in vivo. Taken together, these results show that adherent endotoxin is involved in many of the biological responses induced by orthopedic wear particles and should stimulate development of new approaches designed to reduce the activity of adherent endotoxin in patients with orthopedic implants.
Scanning Electrochemical Microscopy at the Surface of Bone-Resorbing Osteoclasts: Evidence for Steady-State Disposal and Intracellular Functional Compartmentalization of Calcium
Authors: Berger Christine E. M. , Rathod Hersha , Gillespie James I. , Horrocks Benjamin R. , Datta Harish K.
Published: April 2006

Osteoclast resorptive activity occurs despite the presence of extremely high levels of ionized calcium ([Ca2+]) within the osteoclast hemivacuole, which is generated as a by-product of its resorptive activity. Previous in vitro observations have shown that increases in extracellular [Ca2+] ([Ca2+]e) in the surrounding medium can inhibit the osteoclast resorptive activity. Therefore, it has been suggested that the osteoclast acts as a “sensor” for [Ca2+]e, and that high [Ca2+]e leads to an increase in intracellular [Ca2+] ([Ca2+]i), thereby inhibiting osteoclasts in a negative feedback manner. In this report we have carried out an experimental and theoretical analysis of calcium disposal during osteoclast activity to evaluate how in vitro models relate to in vivo osteoclast activity, where it is possible that high [Ca2+]e may be present in the hemivacuole but not over the nonresorbing surface of the cell. Scanning electrochemical microscopy (SECM) studies of [Ca2+] and superoxide anion () generation by bone-resorbing osteoclasts on the surface of a bovine cortical bone slice were compared with microspectofluorometric measurements of the levels of [Ca2+]i in single osteoclasts and the effect of [Ca2+]i on various aspects of osteoclast function. The generation of by the osteoclasts has been shown to be positively correlated with osteoclast resorptive function and can therefore serve as an index of acute changes in osteoclast activity. The SECM of bone-resorbing osteoclasts at the surface of a bone slice revealed a continuous steady-state release of Ca2+. Even after prolonged incubation lasting 3 h the near-surface [Ca2+]e in the solution above the cell remained <2 mM. The SECM real-time measurement data were consistent with the osteoclast acting as a conduit for continuous Ca2+ disposal from the osteoclast-bone interface. We conclude that the osteoclast distinguishes [Ca2+]e in the hemivacuole and in the extracellular fluid above the cell which we denote [Ca2+]e′. We found that an increase in [Ca2+]i may be associated with activation; inhibition; or be without effect on generation, bone-matrix, or bone resorption. Similarly, osteoclast adhesion and bone-resorbing activity was affected by [Ca2+]e′ but showed no correlation with [Ca2+]i. The data suggest the existence of functional compartmentalization of [Ca2+]i within the osteoclast, where elevated calcium may have an inhibitory, excitatory, or no effect on the overall osteoclast activity while exerting a selective effect on different functional modalities. These observations lead to the conclusion that far from being inhibited by Ca2+ generated, the osteoclast by virtue of the observed functional compartmentalization is highly adapted at carrying out its activity even when the level of [Ca2+] in resorptive lacunae is elevated.
Effects of Raloxifene, a Selective Estrogen Receptor Modulator, on Bone Turnover Markers and Serum Sex Steroid and Lipid Levels in Elderly Men
Authors: Doran Patrick M. , Riggs B. Lawrence , Atkinson Elizabeth J. , Khosla Sundeep
Published: April 2006

Several lines of evidence implicate estrogen deficiency as a cause of bone loss in elderly men. Thus, in 50 elderly men (mean age ± SD, 69.1 ± 6.0 years), we performed a randomized blinded study to assess the effect of 6 months of treatment with 60 mg/day of raloxifene (a selective estrogen receptor modulator [SERM] that has an agonist effect on bone but is not feminizing) versus placebo on bone turnover markers. The mean changes in bone turnover markers, serum sex steroid, or lipid levels with treatment did not differ between groups. However, changes in urinary cross-linked N-telopeptide of type I collagen (NTX) excretion were related directly to the baseline serum estradiol level in the raloxifene (r = 0.57; p = 0.004) but not in the placebo-treated (r = 0.15; p = 0.485) men (p = 0.015 for the difference between groups). Moreover, the men in whom NTX excretion decreased after raloxifene treatment had significantly lower baseline estradiol levels (mean ± SEM, 22 ± 2 pg/ml) than the men in whom urinary NTX excretion didn't change or increased after raloxifene therapy (30± 3 pg/ml; p = 0.03), and no such difference was found in the placebo group. Thus, raloxifene has no significant effect on bone turnover markers or lipid levels in elderly men. However, the association noted between baseline estradiol levels and the change in urine NTX excretion in the raloxifene-treated men suggests that a subset of men with low estradiol levels may respond to raloxifene or other SERMs, and further studies are needed to directly test this possibility.
Determinants of Bone Mineral Density in Older Men and Women: Body Composition as Mediator
Authors: Pluijm S. M. F. , Visser M. , Smit J. H. , Popp-Snijders C. , Roos J. C. , Lips P.
Published: April 2006

This study aimed to assess the relative importance of several determinants of bone mineral density (BMD) and to examine to what extent these potential determinants influence total hip BMD through body composition. The study population consisted of 522 participants (264 women and 258 men) of the Longitudinal Aging Study Amsterdam (LASA), aged 65 years and over, and living in Amsterdam and its vicinity. BMD of the total hip was measured using dual-energy X-ray absorptiometry (DXA). Potential determinants of BMD were age, weight change since age 25 years, lifestyle factors, chronic diseases, medication use, and hormonal factors. Potential mediators between the possible determinants and BMD were two measures of body composition: fat mass (FM) and appendicular muscle mass (AMM). Multiple regression analyses including all potential determinants in one model without body composition identified age, weight change, walking activity, and sex hormone-binding globulin (SHBG) as independent determinants for total hip BMD in women. In men, current smoking, participation in sports, and parathyroid hormone (PTH) concentration were independently associated with total hip BMD. When total hip BMD was regressed on the potential determinants and each measure of body composition, it appeared that FM, and to a lesser extent, muscle mass (MM), were independently related to BMD. In women, adjustment for FM reduced the strength of the associations of weight change, walking activity, and SHBG with total hip BMD. Adjustments for MM did not influence the associations between the determinants and BMD. In men, neither FM nor MM appeared to play a mediating role between the determinants and BMD. It can be concluded that (1) FM and MM are strong independent determinants of total hip BMD and that (2) FM possibly plays a mediating role in the association of weight change, walking activity, and SHBG with total hip BMD in women.
Assessment of Gene Regulation by Bone Morphogenetic Protein 2 in Human Marrow Stromal Cells Using Gene Array Technology
Authors: Locklin R. M. , Riggs B. L. , Hicok K. C. , Horton H. F. , Byrne M. C. , Khosla S.
Published: April 2006

Marrow stromal cells can differentiate into osteoblasts, adipocytes, myoblasts, and chondrocytes. Bone morphogenetic protein 2 (BMP-2) is a potent stimulator of osteoblastic differentiation, and identification of the genes regulated by BMP-2 in these cells should provide insight into the mechanism(s) of osteoblastic differentiation. Thus, we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (10 genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface proteins (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID−1, ID−2, ID−3, and ID−4. These encode dominant negative helix-loop-helix (dnHLH) proteins that lack the nuclear binding domain of the basic HLH proteins and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1, STAT1, and JunB. The changes in these nuclear binding factor mRNA levels were confirmed by real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). A further three transcription factors, core binding factor β (CBFβ), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment. In summary, we have used a gene chip microarray to identify a number of BMP-2 responsive genes in hMS cells. Thus, these studies provide potential candidate genes that may induce osteoblastic differentiation or, in the case of the ID proteins, block differentiation along alternate pathways.
Tissue Inhibitor of Metalloproteinases 1 and 2 Directly Stimulate the Bone-Resorbing Activity of Isolated Mature Osteoclasts
Authors: Sobue T. , Hakeda Y. , Kobayashi Y. , Hayakawa H. , Yamashita K. , Aoki T. , Kumegawa M. , Noguchi T. , Hayakawa T.
Published: April 2006

Tissue inhibitor metalloproteinases 1 (TIMP-1) and 2 have been reported to inhibit bone resorption. However, here, we report the direct action of both TIMP-1 and TIMP-2 on isolated rabbit mature osteoclasts to stimulate their bone-resorbing activity at significantly lower concentrations (∼ng/ml) than those (∼μg/ml) required for the inhibition of bone resorption. The cell population used in this study consisted of a mature osteoclast population with >95% purity. TIMP-1 (∼50 ng/ml) and TIMP-2 (∼8–10 ng/ml) increased the pit area excavated by the isolated mature osteoclasts. The stimulatory effects of TIMPs were abolished by simultaneous addition of anti-TIMP antibodies. At higher concentrations, the stimulation of bone resorption decreased reversely to the control level. The magnitude of the stimulatory effect of TIMP-2 was more than that of TIMP-1. Metalloproteinase inhibitors such as BE16627B and R94138 could not replace TIMPs with respect to the bone-resorbing activity, suggesting that the osteoclast-stimulating activity of TIMPs was independent of the inhibitory activity on matrix metalloproteinases (MMPs). TIMPs stimulated tyrosine phosphorylation of cellular proteins in the isolated mature osteoclasts. Both herbimycin A, an inhibitor of tyrosine kinases, and PD98059 and U0126, inhibitors of mitogen-activated protein kinase (MAPK), completely blocked the TIMP-induced stimulation of osteoclastic bone-resorbing activity. On the plasma membrane of osteoclasts, some TIMP-2-binding proteins were detected by a cross-linking experiment. These findings show that TIMPs directly stimulate the bone-resorbing activity of isolated mature osteoclasts at their physiological concentrations and that the stimulatory action of TIMPs is likely to be independent of their activities as inhibitors of MMPs.
Sexual Dimorphism in Vertebral Fragility Is More the Result of Gender Differences in Age-Related Bone Gain Than Bone Loss
Authors: Duan Yunbo , Turner Charles H. , Kim Bom-Taeck , Seeman Ego
Published: April 2006

Spine fractures usually occur less commonly in men than in women. To identify the structural basis for this gender difference in vertebral fragility, we studied 1013 healthy subjects (327 men and 686 women) and 76 patients with spine fractures (26 men and 50 women). Bone mineral content (BMC), cross-sectional area (CSA), and volumetric bone mineral density (vBMD) of the third lumbar vertebral body (L3) were measured by posteroanterior (PA) and lateral scanning using dual-energy X-ray absorptiometry (DXA). In this cross-sectional study, the diminution in peak vertebral body BMC from young adulthood to old age was less in men than in women (6% vs. 27%). This diminution was the net result of two opposing changes occurring concurrently throughout adult life: the removal of bone adjacent to marrow on the inner (endosteal) surface by bone resorption and the deposition of bone on the outer (periosteal) surface by bone formation. For L3, we estimated that men resorbed 3.7 g and deposited 3.1 g, producing a net loss of 0.6 g from young adulthood to old age and women resorbed 3.1 g and deposited only 1.2 g, producing a net loss of 1.9 g. Thus, based on our indirect estimates of periosteal gain and endosteal loss across life, the observed net diminution in BMC during aging was less in men than women because absolute periosteal bone formation was greater in men than women (3.1 g vs. 1.2 g) not because absolute bone resorption was less in men. On the contrary, the absolute amount of bone resorbed was greater in men than women (3.7 g vs. 3.1 g). Periosteal bone formation also increased vertebral body CSA 3-fold more in men than in women, distributing loads onto a larger CSA, so that the load imposed per unit CSA decreased twice as much in men than in women (13% vs. 5%). In men and women with spine fractures, CSA and vBMD were reduced relative to age-matched controls. However, vBMD was no different to the adjusted vBMD in age-matched controls derived assuming controls had no periosteal bone formation during aging. Thus, large amounts of bone are resorbed in men as well as in women, accounting for the age-related increase in spine fractures in both genders. Periosteal bone formation increases CSA and offsets bone loss in both genders but more greatly in men, accounting for the lower incidence of spine fractures in men than in women. We speculate that reduced periosteal bone formation, during growth or aging, may be in part responsible for both reduced vertebral size and reduced vBMD in men and women with spine fractures. Sexual dimorphism in vertebral fragility is more the result of gender differences in age-related bone gain than age-related bone loss.
The Biomechanical Basis of Vertebral Body Fragility in Men and Women
Authors: Duan Yunbo , Seeman Ego , Turner Charles H.
Published: April 2006

The aim of this study was to quantify the biomechanical basis for vertebral fracture risk in elderly men and women. A bone is likely to fracture when the loads imposed are similar to or greater than its strength. To quantify this risk, we developed a fracture risk index (FRI) based on the ratio of the vertebral body compressive load and strength. Loads were determined by upper body weight, height, and the muscle moment arm, and strength was estimated from cross-sectional area (CSA) and volumetric bone mineral density (vBMD). With loads less than the strength of the bone, the FRI remains <1. For any given load, once bone strength diminishes due to a falling vBMD, the FRI will increase. Should FRI approach or exceed unity, structural failure of the vertebra is likely. We measured vertebral body CSA vBMD of the middle zone of third lumbar vertebra by lateral and posteroanterior (PA) scanning using dual-energy X-ray absorptiometry (DXA) and calculated vertebral compressive stress (load per unit area) in 327 healthy men and 686 healthy women and 26 men and 55 postmenopausal women with vertebral fractures. Activities that require forward bending of the upper body caused ∼10-fold more compressive stress on the vertebra compared with standing upright. Men and women had similar peak vBMD in young adulthood. Because men have greater stature than women, the loads imposed on the vertebral body are higher (3754 ± 65 N vs. 3051 ± 31 N; p < 0.001). However, because CSA also was higher in men than women, peak load per unit CSA (stress) did not differ by gender (317.4 ± 4.7 N/cm2 vs. 321.9 ± 3.3 N/cm2, NS). The FRI was similar in young men and women and well below unity (0.42 ± 0.02 vs. 0.43 ± 0.01; NS). Gender differences emerged during aging; CSA increased in both men and women but more so in men, so load per unit area (stress) diminished but more so in men than in women. vBMD decreased in both genders but less so in men. These changes were captured in the FRI, which increased by only 21% in men and by 102% in women so that only 9% of elderly men but 26% of elderly women had an FRI ≥ 1. Men and women with vertebral fractures had an FRI that was greater than or equal to unity (1.03 ± 0.13 vs. 1.35 ± 0.13; p < 0.05) and was 2.04 SD and 2.26 SD higher than age-matched men and women, respectively. In summary and conclusion, young men and women have a similar vBMD, vertebral stress, and FRI. During aging, CSA increases more, and vBMD decreases less in men than in women. Thus, fewer men than women are at risk for fracture because fewer men than women have these structural determinants of bone strength below a level at which the loads exceed the bone's ability to tolerate them. Men and women with vertebral fractures have FRIs that are equal to or exceed unity. The results show that a fracture threshold for vertebrae can be defined using established biomechanical principles; whether this approach has greater sensitivity and specificity than the current BMD T score of −2.5 SD is unknown.