bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Transforming Growth Factor β1 Genotype with Therapeutic Response to Active Vitamin D for Postmenopausal Osteoporosis
Authors: Yamada Yoshiji , Harada Atsushi , Hosoi Takayuki , Miyauchi Akimitsu , Ikeda Kyoji , Ohta Hiroaki , Shiraki Masataka
Published: April 2006

Transforming growth factor β (TGF-β) is an important regulator of bone metabolism, its effects being intertwined with those of estrogen and vitamin D. A T→C polymorphism in exon 1 of the TGF-β1 gene, which results in the substitution of proline for leucine, is associated with bone mineral density (BMD). However, it is not known whether this polymorphism affects the response to treatment with active vitamin D or to hormone replacement therapy (HRT) in individuals with osteoporosis. Changes in BMD at the lumbar spine (L2–L4 BMD) were compared among TGF-β1 genotypes in 363 postmenopausal Japanese women who were divided into three groups: an untreated, control group (n = 130), an active vitamin D treatment group (n = 117), and an HRT group (n = 116). TGF-β1 genotype was determined with an allele-specific polymerase chain reaction assay. In the control group, the rate of bone loss decreased according to the rank order of genotypes TT (homozygous for the T allele) > TC (heterozygous) > CC (homozygous for the C allele), with a significant difference detected between the CC and TT genotypes. The positive response of L2–L4 BMD to HRT increased according to the rank order of genotypes TT < TC < CC, although the differences among genotypes were not statistically significant. Individuals with the CC genotype responded to active vitamin D treatment with an annual increase in L2–L4 BMD of 1.6%, whereas those with the TT or TC genotypes similarly treated lost bone to a similar extent as did untreated subjects of the corresponding genotype. These results suggest that TGF-β1 genotype is associated with both the rate of bone loss and the response to active vitamin D treatment.
Genomic Organization of the Human Chondromodulin-1 Gene Containing a Promoter Region That Confers the Expression of Reporter Gene in Chondrogenic ATDC5 Cells
Authors: Yanagihara Itaru , Yamagata Masayo , Sakai Norio , Shukunami Chisa , Kurahashi Hiroki , Yamazaki Miwa , Michigami Toshimi , Hiraki Yuji , Ozono Keiichi
Published: April 2006

Chondromodulin-1 (ChM-1) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. To clarify the tissue-specific expression and the role of ChM-1 in pathophysiological conditions, we analyzed the structure of the human ChM-1 gene and its promoter. On the screening of a human genomic cosmid library using the human ChM-1 complimentary DNA (cDNA) as a probe, two clones were obtained that contained ChM-1 cDNA. The restriction enzyme map and nucleotide sequence revealed the human ChM-1 gene consisting of seven exons and exon-intron boundaries. The human ChM-1 gene was assigned to chromosome 13q14–21 by fluorescence in situ hybridization (FISH) using the clone as a probe. A primer extension analysis using total RNA extracted from human cartilage revealed a major transcription start site with the sequence CGCT+1GG. The region approximately 3-kilobase (kb) nucleotides upstream of the translation start site was then sequenced and analyzed in terms of promoter activity. We found that a region 446 base pairs (bp) upstream of the start site had promoter activity in COS7, HeLa, and ATDC5 cells. In structure the promoter is a TATA-less type without a GC-rich region. The transcription factors Sox9, Og12, and Cart-1 did not affect the promoter activity. The transcription factor Ying-Yang1 suppressed the promoter activity but GABP protein did not change the promoter activity. The construct containing −446/+87 fused to the SV40 enhancer and green fluorescent protein (GFP) exhibited expression of GFP corresponding to the differentiation of ATDC5 cells to mature chondrocytes. These results suggest that the element −446/+87 confers the cartilage-specific expression of this gene by some factor(s) other than Sox9, Og12, and Cart-1.
Cytoskeletal Abnormalities in Chondrocytes with EXT1 and EXT2 Mutations
Authors: Bernard Mark A. , Hogue Deborah A. , Cole William G. , Sanford Tiffany , Snuggs Mark B. , Montufar-Solis Dina , Duke P. Jackie , Carson Daniel D. , Scott Allison , Barry Van Winkle W. , Dr. Hecht Jacqueline T
Published: April 2006

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-μm repeat cross-bridges of α-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific α-actin, whereas β-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes.
Direct and Indirect Actions of Fibroblast Growth Factor 2 on Osteoclastic Bone Resorption in Cultures
Authors: Kawaguchi Hiroshi , Chikazu Daichi , Nakamura Kozo , Kumegawa Masayoshi , Hakeda Yoshiyuki
Published: April 2006

Fibroblast growth factor 2 (FGF-2 or basic FGF) is known to show variable actions on bone formation and bone resorption. This study was undertaken to elucidate the mechanisms whereby FGF-2 affects bone metabolism, especially bone resorption, using three different culture systems. FGF-2 at 10−9 M and higher concentrations induced osteoclastic cell formation in the coculture system of mouse osteoblastic cells and bone marrow cells, and this induction was abrogated by nonsteroidal anti-inflammatory drugs (NSAIDs). 45Ca release from prelabeled cultured mouse calvariae stimulated by FGF-2 (10−8 M) was also inhibited by NSAIDs, and the inhibition was stronger by NSAIDs, which are more selective for inhibition of cyclooxygenase 2 (COX-2) than COX-1, suggesting the mediation of COX-2 induction. COX-2 was highly expressed and its messenger RNA (mRNA) level was stimulated by FGF-2 in osteoblastic cells whereas it was undetectable or not stimulated by FGF-2 in cells of osteoclast lineage. To further investigate the direct actions of FGF-2 on osteoclasts, resorbed pit formation was compared between cultures of purified osteoclasts and unfractionated bone cells from rabbit long bones. FGF-2 (≥10−12 M) stimulated resorbed pit formation by purified osteoclasts with a maximum effect of 2.0-fold at 10−11 M, and no further stimulation was observed at higher concentrations. However, FGF-2 at 10−9 M − 10−8 M stimulated resorbed pit formation by unfractionated bone cells up to 9.7-fold. NS-398, a specific COX-2 inhibitor, did not affect the FGF-2 stimulation on purified osteoclasts but inhibited that on unfractionated bone cells. We conclude that FGF-2 at low concentrations (≥10−12 M) acts directly on mature osteoclasts to resorb bone moderately, whereas at high concentrations (≥10−9 M) it acts on osteoblastic cells to induce COX-2 and stimulates bone resorption potently.
Culture in Vector-Averaged Gravity Under Clinostat Rotation Results in Apoptosis of Osteoblastic ROS 17/2.8 Cells
Authors: Sarkar Devanand , Dr. Nagaya Takashi , Koga Kazuo , Nomura Yoshio , Gruener Raphael , Seo Hisao
Published: April 2006

Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clinostat to generate a vector-averaged gravity environment. We then evaluated the responses of osteoblast-like ROS 17/2.8 cells subsequent to rotation at 50 revolutions per minute (rpm) for 6–24 h. We found that the cells started to detach from the substrate between 12 h and 24 h of rotation in clinostat but not in stationary cultures or after horizontal rotation (the latter serving as a motion control for turbulence, shear forces, and vibrations). At 24 h, 35% of clinorotated cells had detached and the cells underwent apoptotic death as evidenced by DNA fragmentation analysis, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining, and flow cytometry with Annexin V staining. The apoptotic death was associated with perinuclear distribution of cell-surface integrin β1 and disorganization of actin cytoskeleton. These results suggest that vector-averaged gravity causes apoptosis of osteoblasts by altering the organization of the cytoskeleton. We hypothesize that apoptotic death of osteoblasts might play an important role in the pathogenesis of osteoporotic bone loss as observed in actual space flights.
Direct Action of Naturally Occurring Estrogen Metabolites on Human Osteoblastic Cells
Authors: Robinson John A. , Waters Katrina M. , Turner Russell T. , Spelsberg Thomas C.
Published: April 2006

This article describes experiments that were performed to examine the direct action of estrogen metabolites on cultured human osteoblast cells. The human fetal osteoblastic cell line, hFOB/ER9, which expresses high levels of the estrogen receptor (ER) alpha, was used to examine the direct effects of 16α-hydroxyestrone (16α-OHE1) and 2-hydroxyestrone (2-OHE1) on osteoblast differentiation. The 16α-OHE1 caused a decrease in osteocalcin (OC) secretion to a maximum of 40% of control values (vehicle-treated cells) at 10−7 M. Alkaline phosphatase (AP) activity was significantly induced at 10−7 M 16α-OHE1 with greater than 500% of control at 10−6 M 16α-OHE1. Finally, AP steady-state messenger RNA (mRNA) levels were increased within 24 h of 16α-OHE1 treatment. In contrast to 16α-OHE1, 2-OHE1 had no effects on the secretion of OC, AP activity, or AP gene expression. The 2-OHE1 also did not display any antiestrogen activity because treatment in combination with 17β-estradiol (E2) and 16α-OHE1 had no significant effect on the reduction in OC secretion or induction of AP activity. Similar to E2, 16α-OHE1 stimulated the expression of an early response gene, a TGF-β inducible early gene, designated TIEG, as early as 60 minutes after treatment, whereas treatment with 2-OHE1 displayed no effect. Support that the 16α-OHE1 regulation of these osteoblasts (OB) markers was mediated through the ER is shown by the fact that the estrogen antagonist ICI 182,780 abrogated these effects. These data suggest that 16α-OHE1 is a potent estrogen agonist on human osteoblastic hOB/ER9 cells. In contrast, 2-OHE1 displayed no estrogenic or antiestrogenic activity in this human osteoblast cell model.
Bone Has a Sexually Dimorphic Response to Aromatase Deficiency
Authors: Öz Orhan K. , Zerwekh Joseph E. , Fisher Carolyn , Graves Kathy , Nanu Lydia , Millsaps Rita , Simpson Evan R.
Published: April 2006

Aromatase synthesizes estrogen from androgen precursors. To better understand the role of estrogen in skeletal metabolism and growth, we have assessed long bone growth and histomorphometry in aromatase-deficient (ArKO) mice. The age range for the animals was 5–7 months. At this age mice have already achieved peak bone density but continue slow bone growth. Femur length, an index of long bone growth, showed decreased growth in ArKO males compared with wild-type (wt) littermates but no significant difference in females. Radiographically, compared with age- and sex- matched littermates both ArKO males and females showed osteopenia in the lumbar spine. Histologically, both ArKO males and females showed an osteoporotic-type picture, characterized by significant decreases in trabecular bone volume and trabecular thickness. However, compared with wt littermates female ArKO animals showed a bone remodeling picture consistent with increased bone turnover, much like early postmenopausal osteoporosis in humans. On the other hand, male ArKO animals showed decreases in both osteoblastic and osteoclastic surfaces compared with wt littermates, similar to age-related osteopenia. These findings suggest that osteoporosis seen in aromatase-deficient mice may arise from different bone remodeling activities between males and females. These results also show that the ArKO model exhibits the expected results of estrogen deficiency and may be a good model for investigating sex-specific responses to estrogen deficiency. Furthermore, they imply that estrogen is important for attaining peak bone mass in male as well as in female mice.
Vitamin K2 (Menatetrenone) Effectively Prevents Fractures and Sustains Lumbar Bone Mineral Density in Osteoporosis
Authors: Shiraki Masataka , Shiraki Yumiko , Aoki Choju , Miura Masakazu
Published: April 2006

We attempted to investigate whether vitamin K2 (menatetrenone) treatment effectively prevents the incidence of new fractures in osteoporosis. A total of 241 osteoporotic patients were enrolled in a 24-month randomized open label study. The control group (without treatment; n = 121) and the vitamin K2–treated group (n = 120), which received 45 mg/day orally vitamin K2, were followed for lumbar bone mineral density (LBMD; measured by dual-energy X-ray absorptiometry [DXA]) and occurrence of new clinical fractures. Serum level of Glu-osteocalcin (Glu-OC) and menaquinone-4 levels were measured at the end of the follow-up period. Serum level of OC and urinary excretion of deoxypyridinoline (DPD) were measured before and after the treatment. The background data of these two groups were identical. The incidence of clinical fractures during the 2 years of treatment in the control was higher than the vitamin K2–treated group (χ2 = 10.935; p = 0.0273). The percentages of change from the initial value of LBMD at 6, 12, and 24 months after the initiation of the study were −1.8 ± 0.6%, −2.4 ± 0.7%, and −3.3 ± 0.8% for the control group, and 1.4 ± 0.7%, −0.1 ± 0.6%, and −0.5 ± 1.0% for the vitamin K2–treated group, respectively. The changes in LBMD at each time point were significantly different between the control and the treated group (p = 0.0010 for 6 months, p = 0.0153 for 12 months, and p = 0.0339 for 24 months). The serum levels of Glu-OC at the end of the observation period in the control and the treated group were 3.0 ± 0.3 ng/ml and 1.6 ± 0.1 ng/ml, respectively (p < 0.0001), while the serum level of OC measured by the conventional radioimmunoassay (RIA) showed a significant rise (42.4 ± 6.9% from the basal value) in the treated group at 24 months (18.2 ± 6.1% for the controls; p = 0.0081). There was no significant change in urinary DPD excretion in the treated group. These findings suggest that vitamin K2 treatment effectively prevents the occurrence of new fractures, although the vitamin K2–treated group failed to increase in LBMD. Furthermore, vitamin K2 treatment enhances γ-carboxylation of the OC molecule.
Transient Chondrogenic Phase in the Intramembranous Pathway During Normal Skeletal Development
Authors: Nah Hyun-Duck , Pacifici Maurizio , Gerstenfeld Louis C. , Adams Sherrill L. , Kirsch Thorsten
Published: April 2006

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.
Comparative Effects of Estrogen and Raloxifene on B Lymphopoiesis and Bone Loss Induced by Sex Steroid Deficiency in Mice
Authors: Onoe Yoshiko , Miyaura Chisato , Ito Masako , Ohta Hiroaki , Nozawa Shiro , Suda Tatsuo
Published: April 2006

Estrogen deficiency caused by ovariectomy (OVX) results in a marked bone loss because of stimulated bone resorption. We have reported that OVX selectively stimulates B lymphopoiesis in mouse bone marrow, which is somehow related to bone resorption. Estrogen prevents both the increased B lymphopoiesis and the bone resorption caused by estrogen deficiency. Raloxifene also has a potent estrogenic activity for bone with minimal estrogenic activity for the uterus. To examine the effects of raloxifene on B lymphopoiesis and bone resorption, OVX mice were given either estrogen or raloxifene subcutaneously for 2–4 weeks using a miniosmotic pump. Reduced uterine weight in OVX mice was restored completely by 17β-estradiol (E2). Some 300-fold higher doses of raloxifene increased uterine weight of OVX mice, but only slightly. The number of B220- positive pre-B cells was increased markedly in bone marrow after OVX. The increased B lymphopoiesis was prevented not only by E2 but by raloxifene. In OVX mice, the trabecular bone volume (BV) of the femoral distal metaphysis was reduced markedly, when measured by microcomputed tomography (μCT) scanning and dual-energy X-ray absorptiometry. Both E2 and raloxifene similarly restored it. Like estrogen deficiency, androgen deficiency induced by orchidectomy (ORX) also resulted in a marked bone loss and increased B lymphopoiesis. Both E2 and raloxifene prevented the changes in ORX mice. These results indicate that both estrogen deficiency and androgen deficiency similarly stimulate B lymphopoiesis in mouse bone marrow, which accompany bone loss. Raloxifene exhibits estrogenic actions in bone and bone marrow to prevent bone loss and regulate B lymphopoiesis without inducing estrogenic action in the uterus.
Direct Measurement of Hormone-Induced Acidification in Intact Bone
Authors: Belinsky Glenn S. , Tashjian Armen H.
Published: April 2006

Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6-2′-0-dibutyryladenosine-cAMP (db-cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively.
Morphometric X-Ray Absorptiometry and Morphometric Radiography of the Spine: A Comparison of Prevalent Vertebral Deformity Identification
Authors: Dr. Rea J. A. , Chen M. B. , Li J. , Blake G. M. , Steiger P. , Genant H. K. , Fogelman I.
Published: April 2006

Prevalent vertebral deformities are associated with a substantially increased risk of subsequent vertebral and nonvertebral fractures. Knowledge of vertebral fracture status is an important component in the prediction of further fractures in patients with osteoporosis. This study reports a comparison of the quantitative identification of vertebral deformities on morphometric X-ray absorptiometry (MXA) scans and conventional radiographs (MRX) in 161 postmenopausal women (mean age ± SD, 64 ± 7.1 years) recruited from patients referred by their family doctor for bone density measurement (n = 119) and osteoporotic subjects with known vertebral deformities attending an osteoporosis clinic (n = 42). Each subject had MXA scans and MRXs of the thoracolumbar spine, to image the vertebrae from T4–L4, at a single visit. The scans and radiographs were analyzed by two trained observers using six points to quantify the shape of each vertebral body. From these points, three vertebral heights were measured: anterior, middle, and posterior. Vertebral deformities were identified using the algorithms proposed by Eastell and by McCloskey. Generally good to excellent agreement (per vertebra, κ = 0.87–0.93; per subject, κ = 0.81–0.91) was observed between the two algorithms used for quantitative vertebral deformity identification using MXA or MRX More moderate agreement (per vertebra, κ = 0.70–0.79; per subject, κ = 0.67–0.75) was seen when comparing the same algorithm between MXA and MRX Agreement between MXA and MRX for the McCloskey algorithm was better than for the Eastell algorithm, largely because of the lower number of false positives produced by the McCloskey methodology. Deformity identification by MXA was limited because of poor image quality, primarily in the upper thoracic spine. One in six MRX deformities were missed by MXA as they occurred in vertebrae not visualized sufficiently for analysis on the MXA scans. Deformity identification was poorer in the upper thoracic spine in analyzable vertebrae with a sensitivity of 50.0% for MXA in terms of MRX using the Eastell algorithm for the vertebral levels T4–T7, compared with 80.6% for L1–L4. MXA proved to be more effective at identifying moderate to severe MRX deformities producing a sensitivity of 22.0% for MXA in terms of identifying MRX grade 1 deformities using the Eastell algorithm, compared with 81.6% for grade 2 deformities. Although MXA image quality is inferior to that of conventional radiographs, MXA has distinct advantages such as a substantially reduced effective dose to the patient and acquisition of a single image of the spine. MXA is a potentially useful, relatively fast, low-radiation technique to identify prevalent vertebral deformities, particularly moderate to severe deformities in the middle/lower thoracic and lumbar spine, in conjunction with morphometric radiography in some patients.
Identification of Vertebral Deformities in Women: Comparison of Radiological Assessment and Quantitative Morphometry Using Morphometric Radiography and Morphometric X-Ray Absorptiometry
Authors: Ferrar L. , Jiang G. , Barrington N. A. , Professor Eastell R.
Published: April 2006

Our aim was to compare normal vertebral reference values for morphometric radiography (MRX) and morphometric X-ray absorptiometry (MXA) and to compare these methods for the identification of vertebral deformities. We calculated MXA reference values (Hologic QDR 4500 A) for 327 women (ages 22–88 years) randomly selected from local General Practice lists in Sheffield, U.K. MRX reference values were calculated from spinal radiographs for 123 of these subjects (ages 56–88 years). We used these reference values to identify deformities in the MRX and MXA reference populations and in 83 women with osteoporosis (ages 49–87 years). We observed differences in mean deformity of vertebral height ratios measured by MRX and MXA, especially for the mid-to-posterior ratio. We compared agreement between quantitative methods (MRX and MXA) and qualitative radiological assessment. Severity of deformity was defined by semiquantitative (SQ) assessment. Agreement was moderate for MRX (k = 0.59; 95% CI = 0.43–0.77) and for MXA (k = 0.47; 95% CI = 0.29–0.66) in the reference population. Agreement was good for MRX (k = 0.86; 95% CI = 0.82–0.89) and MXA (k = 0.71; 95% CI = 0.66–0.75) in the osteoporotic population. MRX and MXA correctly identified a greater proportion of moderate or severe deformities compared with mild deformities. Sensitivity, specificity, predictive values, and accuracy were slightly better for MRX than for MXA. Although MXA agrees well with qualitative radiological assessment, the large proportion of vertebrae excluded from analysis because of poor image quality limits the diagnostic value of the technique. Reference intervals should be technique specific.
Chronic Intravenous Aminobisphosphonate Therapy Increases High-Density Lipoprotein Cholesterol and Decreases Low-Density Lipoprotein Cholesterol
Authors: Adami Silvano , Braga Vania , Guidi Giancesare , Gatti Davide , Gerardi Daniela , Fracassi Elena
Published: April 2006

Nowadays, bisphosphonates are considered the drugs of choice for the treatment of several bone disorders. Their exact mechanism of action is not clear but recently it has been reported that the aminobisphosphonates inhibit cholesterol biosynthesis and that this might be relevant for their actions on bone osteoclasts. The study includes 87 postmenopausal women with moderate to severe osteoporosis. The patients were randomly assigned to intravenous (iv) infusion of 50 mg of the aminobisphosphonate Neridronate dissolved in 100 ml of saline solution every 2 months for a year (44 patients). The remaining 43 served as controls. At the time of each infusion blood samples were obtained for the evaluation of total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), apolipoprotein A-I (Apo A-I), apolipoprotein B (Apo B), and total and bone alkaline phosphatase (AP). Free deoxypyridinoline (f-DPD) was measured in fasting urine specimens. In the control group no significant changes were observed throughout the study period for any of the biochemical variables. In the Neridronate-treated patients both bone AP and f-DPD excretion fell significantly by 15–20%. In these patients serum total cholesterol and serum triglycerides showed marginal decreases, which were occasionally significant. LDL-C and Apo B fell by 5–6% and these changes were statistically significant at most time points. Apo A-I and HDL-C rose progressively with time. At the 12th month, HDL-C rose 17–18% (p < 0.0001) above the baseline values. Similar findings were obtained in four postmenopausal women given high iv doses of Pamidronate or Alendronate. In conclusion aminobisphophonates, at least when given iv, induce remarkable and unexpected effects on lipid metabolism with a final profile that might be clinically relevant.
Suppressed Bone Turnover by Bisphosphonates Increases Microdamage Accumulation and Reduces Some Biomechanical Properties in Dog Rib
Authors: Mashiba Tasuku , Hirano Toru , Turner Charles H. , Forwood Mark R. , Johnston C. Conrad , Burr David B.
Published: April 2006

It has been hypothesized that suppression of bone remodeling allows microdamage to accumulate, leading to increased bone fragility. This study evaluated the effects of reduced bone turnover produced by bisphosphonates on microdamage accumulation and biomechanical properties of cortical bone in the dog rib. Thirty-six female beagles, 1–2 years old, were divided into three groups. The control group (CNT) was treated daily for 12 months with saline vehicle. The remaining two groups were treated daily with risedronate (RIS) at a dose of 0.5 mg/kg per day or alendronate (ALN) at 1.0 mg/kg per day orally. After sacrifice, the right ninth rib was assigned to cortical histomorphometry or microdamage analysis. The left ninth rib was tested to failure in three-point bending. Total cross-sectional bone area was significantly increased in both RIS and ALN compared with CNT, whereas cortical area did not differ significantly among groups. One-year treatment with RIS or ALN significantly suppressed intracortical remodeling (RIS, 53%; ALN, 68%) without impairment of mineralization and significantly increased microdamage accumulation in both RIS (155%) and ALN (322%) compared with CNT. Although bone strength and stiffness were not significantly affected by the treatments, bone toughness declined significantly in ALN (20%). Regression analysis showed a significant nonlinear relationship between suppressed intracortical bone remodeling and microdamage accumulation as well as a significant linear relationship between microdamage accumulation and reduced toughness. This study showed that suppression of bone turnover by high doses of bisphosphonates is associated with microdamage accumulation and reduced some mechanical properties of bone.
Chromosomal Mapping of Osteopenia-Associated Quantitative Trait Loci Using Closely Related Mouse Strains
Authors: Beneš Helen , Weinstein Robert S. , Zheng Wenhui , Thaden John J. , Jilka Robert L. , Manolagas Stavros C. , Reis Robert J. Shmookler
Published: April 2006

Peak bone mineral density (BMD) is a highly heritable trait in humans and is currently the best predictor of skeletal fragility underlying osteoporosis. The SAMP6 mouse strain displays unusually low BMD at maturity, and age-dependent osteopenia associated with defective osteoblastogenesis. To identify quantitative trait loci (QTLs) influencing bone density, we constructed crosses between SAMP6 and either AKR/J or SAMP6, two related mouse strains of higher peak BMD. Due to common ancestry of these strains, intercross parents differed at only 39–40% of 227 highly-polymorphic genotyping markers, thus restricting our search to this informative portion of the genome and reducing the number of mice required for QTL significance. Using dual energy X-ray absorptiometry (DEXA), we measured spinal BMD in F2 cross progeny at 4 months of age, and selectively genotyped those in the highest and lowest quartiles for BMD. Based on linear regression of bone density on genotype, including Composite Interval Mapping to enhance mapping precision while adjusting for effects of distal markers, we identified multiple QTLs significantly affecting spinal BMD; these were mapped to regions of chromosomes 2 (two sites, one confirmed in both crosses), 7, 11, 13 and 16. One of these loci had been previously identified as a significant bone-density QTL, while 3 substantiate QTLs suggested by a low-power study of 24 recombinant-inbred mouse lines. Such recurrent appearance of QTLs, especially in crosses involving distantly-related strains, implies that polymorphism at these loci may be favored by evolution and might underlie variation in peak bone density among humans.
Transforming Growth Factor-β1 Gene Polymorphism, Bone Turnover, and Bone Mass in Italian Postmenopausal Women
Authors: Bertoldo Francesco , D'Agruma Leonardo , Furlan Federico , Colapietro Francesca , Lorenzi Maria Tiziana , Maiorano Nunzia , Iolascon Achille , Zelante Leopoldo , Locascio Vincenzo , Gasparini Paolo
Published: April 2006

Transforming growth factor β1 (TGF-β1) is abundant in bone and is an important regulator of the osteoclastic-osteoblastic interaction (coupling). The sequence variation, 713–8delC in the TGF-β1 gene has previously been found to be associated with very low bone mass in osteoporotic women and with increased bone turnover in both osteoporotic and normal women. The possible association of this polymorphism with bone mass and bone turnover has now been investigated in 256 postmenopausal Italian women. A significant association of TGF-β1 with bone mass was detected in the populations. Subjects carrying the sequence variation 713–8delC (Tt) genotype showed a significantly lower bone mineral density (BMD) at the hip than those without sequence variation in the genotype (TT). Individuals carrying the tt genotype have a more severe osteoporosis (P = 0.0001 vs. TT and Tt genotypes). The frequency of the fragility fractures was significantly lower in individuals with TT genotype than in those with the Tt and tt genotypes (χ2 = 21.9; P = 0.006). Furthermore a significant association was found between 713–8delC and bone turnover. The results suggest a strong evidence for an association among the 713–8delC allele of the TGF-β1 gene and the femoral BMD, the prevalence of osteoporotic fractures, and finally a high bone turnover in a sample of Italian postmenopausal women.
Expression of Osteoclast Differentiation Signals by Stromal Elements of Giant Cell Tumors
Authors: Atkins Gerald J. , Haynes David R. , Graves Stephen E. , Evdokiou Andreas , Hay Shelley , Bouralexis Stelios , Findlay David M.
Published: April 2006

The mechanisms by which primary tumors of the bone cause bone destruction have not been elucidated. Unlike most other lytic bone tumors, osteoclastomas, otherwise known as giant cell tumors (GCT), contain osteoclast-like cells within the tumor stroma. A new member of the TNF-ligand superfamily member, osteoclast differentiation factor (ODF/OPGL/RANKL/TRANCE), was recently identified. ODF was shown to directly stimulate osteoclastogenesis, in the presence of M-CSF. In this study, the expression of ODF was examined in a number of tumor samples associated with bone lysis in vivo. In addition, we investigated expression of the ODF receptor on osteoclast precursors, RANK, as well as the ODF inhibitor osteoprotegerin (OPG), and another TNF-ligand superfamily member, TRAIL, previously shown to abrogate the inhibitory effects of OPG. We report here the novel finding that GCT stromal cells contain abundant ODF mRNA, whereas the giant cell population exclusively expresses RANK mRNA. These results are consistent with the osteoclast-mediated bone destruction by these tumors. We also report the expression of OPG and TRAIL mRNA in GCT samples. A comparison with other lytic and nonlytic tumors of bone showed that GCT express more ODF and TRAIL mRNA relative to OPG mRNA. In addition, GCT were found to express a number of cytokines previously reported to play central roles in osteoclastogenesis, namely, IL-1, −6, −11, −17, as well as TNF-α. Importantly, GCT were also found to express high levels of M-CSF mRNA, a cytokine shown to be an essential cofactor of ODF, and a survival factor for mature and developing osteoclasts. Furthermore, expression of these molecules by stromal cells isolated from GCT continued in vitro. Thus GCT constitutively express all of the signals that are currently understood to be necessary for the differentiation of osteoclasts from precursor cells.
A Novel Synthetic Triazolotriazepine Derivative JTT-606 Inhibits Bone Resorption by Down-Regulation of Action and Production of Bone Resorptive Factors
Authors: Chikazu Daichi , Shindo Masanori , Iwasaka Toshiki , Katagiri Mika , Manabe Noriyo , Takato Tsuyoshi , Nakamura Kozo , Kawaguchi Hiroshi
Published: April 2006

In the search for a new class of bone-sparing agents, we have conducted random screening of the domestic chemical library using 45Ca release assay from prelabeled cultured neonatal mouse calvariae and identified a novel synthetic triazolotriazepine JTT-606 as a candidate for a potent inhibitor of bone resorption. JTT-606 inhibited 45Ca release dose dependently not only in the control calvarial culture but also in the stimulated cultures by interleukin-1α (IL-1α), fibroblast growth factor 2 (FGF-2), and parathyroid hormone (PTH). JTT-606 also inhibited both basal and stimulated osteoclast-like (OCL) cell formation in the coculture of mouse osteoblastic cells and bone marrow cells dose dependently, indicating its inhibitory effect on osteoclast differentiation. Ex vivo OCL cell formation by cultured bone marrow cells collected from ovariectomized (OVX) mice also was decreased dose dependently by in vivo application of JTT-606 to a level similar to that from sham-operated mice. Furthermore, JTT-606 inhibited resorbed pit formation by isolated mature osteoclasts as well as by unfractionated bone cells derived from rabbit long bones in the control and FGF-2–stimulated cultures dose dependently, indicating both the direct and the indirect actions of JTT-606 on mature osteoclast function. In addition, JTT-606 reduced production of IL-1α, tumor necrosis factor α (TNF-α), IL-6, and granulocyte-macrophage colony–stimulating factor (GM-CSF) in the human peripheral blood mononuclear cell culture. In vivo analyses of mature OVX rats revealed that the application of JTT-606 for 12 weeks increased the BMD of the lumbar spine and decreased the levels of serum osteocalcin and urine deoxypyridinoline to levels similar to those of 17β-estradiol–treated OVX rats. We propose that JTT-606 may inhibit both osteoclast differentiation and function by down-regulating both the action and the production of bone resorptive factors. It is speculated that JTT-606 could be a potent agent for the treatment of osteopenic disorders with elevated osteoclastic bone resorption.
Changes in Cortical Bone Mineralization in the Developing Mandible: A Three-Dimensional Quantitative Computed Tomography Study
Authors: Maki K. , Miller A. , Okano T. , Shibasaki Y.
Published: April 2006

Quantitative computed tomography (QCT) was completed in 34 subjects between the ages of 9 and 33 years with symmetrical mandibles in order to investigate the three-dimensional cortical bone mineral density (BMD) distribution in the mandible. The number and distribution of the pixels were determined at three levels: (1) representing the entire mandibular bone; (2) the cortical bone at 60% above the baseline defined as the segmentation level (around 1050 mg/cm3) and representative of only cortical bone; and (3) the highest mineralized cortical bone (>1250 mg/cm3). The geometrical distribution of the highest mineralized areas was evaluated by three-dimensional reconstruction of the images. The total number of pixels for the entire mandible increased significantly at each time point represented at four increasing ages groups (9–11 years of age, 12–14 years of age, 15–17 years of age, and >18 years of age). The male and female subjects had a similar total number of pixels for the entire mandible before the age of 11, but the male subjects showed a significantly larger total number of mandibular pixels after that age. Comparison of the number of pixels for pure cortical bone (60% segmentation level) and the highest mineralized cortical bone indicated a significant increase with maturation with the greatest change occurring between the 13-year and 16-year age groups. However, the ratio of cortical bone/total bone increased at a more rapid rate in the male subjects and reached a plateau by the 16-year age group, showing distinct differences in mineralization of the mandible between the sexes.
Patients with Prior Fractures Have an Increased Risk of Future Fractures: A Summary of the Literature and Statistical Synthesis
Authors: Klotzbuecher Carolyn M. , Ross Philip D. , Landsman Pamela B. , Abbott Thomas A. , Berger Marc
Published: April 2006

Numerous studies have reported increased risks of hip, spine, and other fractures among people who had previous clinically diagnosed fractures, or who have radiographic evidence of vertebral fractures. However, there is some variability in the magnitudes of associations among studies. We summarized the literature and performed a statistical synthesis of the risk of future fracture, given a history of prior fracture. The strongest associations were observed between prior and subsequent vertebral fractures; women with preexisting vertebral fractures (identified at baseline by vertebral morphometry) had approximately 4 times greater risk of subsequent vertebral fractures than those without prior fractures. This risk increases with the number of prior vertebral fractures. Most studies reported relative risks of approximately 2 for other combinations of prior and future fracture sites (hip, spine, wrist, or any site). The confidence profile method was used to derive a single pooled estimate from the studies that provided sufficient data for other combinations of prior and subsequent fracture sites. Studies of peri- and postmenopausal women with prior fractures had 2.0 (95% CI = 1.8, 2.1) times the risk of subsequent fracture compared with women without prior fractures. For other studies (including men and women of all ages), the risk was increased by 2.2 (1.9, 2.6) times. We conclude that history of prior fracture at any site is an important risk factor for future fractures. Patients with a history of prior fracture, therefore, should receive further evaluation for osteoporosis and fracture risk
Differences in Skeletal Kinetics Between Vertebral and Humeral Bone Measured by 18F-Fluoride Positron Emission Tomography in Postmenopausal Women
Authors: Dr. Cook Gary J. R. , Lodge Martin A. , Blake Glen M. , Marsden Paul K. , Fogelman Ignac
Published: April 2006

We have sought to investigate regional differences in skeletal kinetics between lumbar vertebrae and the humerus of postmenopausal women with 18F-fluoride positron emission tomography (PET). Twenty-six women, mean age 62 years, had dynamic PET scans of the lumbar spine and lower humerus after the injection of 180 MBq 18F-fluoride ion. Plasma arterial input functions (IFs) were calculated from a mean IF measured arterially from 10 women and scaled according to late individual venous activity. Vertebral and humeral time activity curves were measured by placing regions of interest (ROI) over lumbar vertebrae and the humeral shaft. Using a three-compartmental model and nonlinear regression analysis the macroconstant Ki, representing plasma clearance of fluoride to bone mineral, and the individual rate constants K1 (related to regional skeletal blood flow) and k2 to k4 describing transport between plasma, an extracellular fluid compartment and a bone mineral compartment, were measured. Mean vertebral Ki (3.47 × 10−2 ml · min−1 · ml−1) and K1 (1.08 × 10−1 ml · min−1 · ml−1) were found to be significantly greater than humeral Ki (1.64 × 10 2 ml min−1 ml−1; P < 0.0001) and K1 (3.90 × 10−2 ml · min−1 · ml−1; P < 0.0001) but no significant differences were found in k2, k3, and k4. These findings confirm differences in regional skeletal kinetics between lumbar vertebrae and the lower humerus. These observations may help increase our understanding of the regional differences in pathophysiology and response to treatment that have been observed in sites consisting predominantly of either trabecular or cortical bone. 18F-fluoride PET may prove to be a valuable technique in the noninvasive measurement of regional skeletal metabolism.
Increased Expression of Bone Sialoprotein in Bone Metastases Compared with Visceral Metastases in Human Breast and Prostate Cancers
Authors: Waltregny David , Bellahcène Akeila , De Leval Xavier , Florkin Benoǐt , Weidle Ulrich , Castronovo Vincent
Published: April 2006

The recent demonstration that bone sialoprotein (BSP) is expressed in osteotropic cancers suggests that this bone matrix protein might be implicated in the preferential seed and growth of metastatic cells in bone. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. The exact mechanisms by which BSP may favor bone metastases formation are not clearly established yet. Although BSP expression has been detected in breast, prostate, lung, thyroid, and neuroblastoma primary tumors, no information regarding its expression in metastases is available to date. In this study, we have examined BSP expression in 15 bone and 39 visceral metastatic lesions harvested from 8 breast cancer patients and 7 prostate cancer patients who died of disseminated disease. We were able to retrieve the primary lesions from 5 of the 8 breast cancer patients as well as from all 7 prostate cancer patients. All the primary breast tumor patients and 5 of the 7 primary prostate cancer patients expressed a detectable level of BSP. Bone metastases from all 8 breast cancer patients and from 5 out of 7 prostate cancer patients exhibited detectable levels of the protein. Metastatic cells in close contact with bone trabeculae usually were highly positive for BSP. BSP also was detected in secondary lesions developed at visceral sites including liver, thyroid, lung, and adrenal glands. However, BSP expression was significantly lower in visceral metastases than in skeletal ones (Mann-Whitney test, p < 0.05). Our data represent the first demonstration of an increased expression of BSP in bone metastases compared with nonskeletal metastases in human breast and prostate cancers and add weight to the body of evidence attributing a significant role to this protein in the genesis of bone metastases. (J Bone Miner Res 2000;15:834–843)
Increased Marrow-Derived Osteoprogenitor Cells and Endosteal Bone Formation in Mice Lacking Thrombospondin 2
Authors: Hankenson Kurt D. , Bain Steven D. , Kyriakides Themis R. , Smith Erica A. , Goldstein Steven A. , Bornstein Paul
Published: April 2006

The phenotype of thrombospondin 2 (TSP2)–null mice includes abnormalities in collagen fibrils and increases in ligamentous laxity, vascular density, and bleeding time. In this study, analyses by computerized tomography (CT) revealed that cortical density was increased in long bones of TSP2-null mice. Histomorphometric analysis showed that the mid-diaphyseal endosteal bone formation rate (BFR) of TSP2-null mice was increased in comparison with that of wild-type (WT) animals. Although microgeometric analysis showed that periosteal and endosteal radii were reduced, the mechanical properties of femurs from TSP2-null mice were not significantly different from those of controls, presumably because of the concomitant increase in endosteal bone mass. Bone loss in ovariectomized mice was equivalent for WT and mutant mice, a finding that indicates that TSP2-null animals are capable of normal bone resorption. To further explore the cellular basis for the increased endosteal BFR in TSP2-null mice, marrow stromal cells (MSCs) were isolated and examined in vitro. These cells were found to be present in increased numbers in a colony forming unit (CFU) assay and showed an increased rate of proliferation in vitro. We conclude that TSP2 regulates the proliferation of osteoblast progenitors, directly or indirectly, and that in its absence endosteal bone formation is increased. (J Bone Miner Res 2000;15:851–862)