bluuebunny/crossref_metadata_2025 huggingface dataset:
A Dose-Finding Study of Zoledronate in Hypercalcemic Cancer Patients
Authors: Body J. J. , Lortholary A. , Romieu G. , Vigneron A. M. , Ford J.
Published: April 2006

Zoledronate is a new heterocyclic imidazole bisphosphonate that is the most potent bisphosphonate administered in humans because it is 100–850 times more potent than pamidronate, according to in vitro or animal models of bone resorption. We conducted an open-label, dose-finding, single-dose phase I study in tumor-induced hypercalcemia (TIH), which has been similarly used as a model to determine the active doses of other bisphosphonates. The primary objective was to determine, with a dose escalation schedule, two nontoxic dose levels of zoledronate able to induce normocalcemia in at least 80% of patients with TIH after rehydration (corrected Ca for albumin levels ≥2.75 mmol/l). Based on estimates of potency, the starting dose was 0.002 mg/kg, and further tested doses were 0.005, 0.01, 0.02, and 0.04 mg/kg. To obtain a more precise estimate of the response rate, we treated 10 more patients at the highest of the two effective dose levels. The median infusion time of zoledronate was 30 minutes. Thirty out of the 33 treated patients were evaluable for efficacy. Thirty percent of the patients had breast cancer and 54% had metastatic bone involvement. For all groups combined, mean Ca levels at baseline was 3.0 mmol/l. The two effective dose levels were 0.02 mg/kg and 0.04 mg/kg. Five out of five patients became normocalcemic after 0.02 mg of zoledronate/kg and 14 out of 15 after 0.04 mg of zoledronate/kg. The success rate of the latter dose was thus 93% (95% confidence interval [CI] 68–100%). At this dose, the first day of normocalcemia was day 2 or 3 for all but one patient. The duration of normocalcemia for the two effective doses could be assessed in nine patients; seven patients remained normocalcemic throughout the trial (32–39 days). The fall in serum Ca was accompanied by a marked fall in fasting urinary Ca excretion. Zoledronate was well tolerated: 7 out of 33 patients developed transient hypophosphatemia, and 3 developed transient hypocalcemia. The only clinically detectable side effect was an increase in body temperature occurring in 10 (30%) patients. In summary, very low doses of zoledronate (0.02 mg/kg and 0.04 mg/kg, i.e., 1.2 mg and 2.4 mg for a 60-kg individual, respectively) administered by a short-time infusion effectively treated patients with TIH. The fall in serum Ca was rapid, and normocalcemia was often maintained for several weeks. Zoledronate was well tolerated. Future trials will determine whether prolonged treatment with this potent compound can have greater effects on the skeletal morbidity rate in patients with tumor bone disease than can be achieved with currently available bisphosphonates.
Development and Characterization of a Human In Vitro Resorption Assay: Demonstration of Utility Using Novel Antiresorptive Agents
Authors: James Ian E. , Lark Michael W. , Zembryki Denise , Lee-Rykaczewski Elizabeth V. , Hwang Shing Mei , Tomaszek Thaddeus A. , Belfiore Pietro , Gowen Maxine
Published: April 2006

A human in vitro resorption assay has been developed using osteoclastoma-derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the αvβ3 integrin, and inhibitors of cathepsin K and the osteoclast ATPase. The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human αvβ3-mediated cell adhesion assay for the vitronectin receptor antagonists (r2 = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H+-ATPase inhibitors (r2 = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r2 = 0.80). Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue. These cells can be stored long-term in liquid nitrogen and upon thawing maintain their bone-resorbing phenotype. The cryopreserved cells can be cultured on bovine cortical bone for 24–48 h and resorption can be measured by either confocal microscopy or biochemical assays. The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds. In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C-telopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent assay. Resorption can be measured reproducibly using a 48-h incubation of osteoclasts on bone slices, or a 24-h incubation with bone particles. This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis.
Morphometric Dual-Energy X-Ray Absorptiometry of the Spine: Report of a Large Series and Correlation with Axial Bone Mineral Density
Authors: Bianco Antonio C. , Malvestiti Luiz F. , Gouveia Cecilia H. A. , Wehba Salim , Lewin Shlomo , Marone Marilia M. S.
Published: April 2006

We studied vertebral morphometry and its relation to bone mineral density (BMD) in normal Brazilian women (n = 605). All women (age 22–97 years) were ambulatory and healthy. A lateral spine scan was done for morphometric X-ray absorptiometry using an imaging densitometer. In 429 of these women, BMD of the spine and proximal femur also were measured using dual-energy X-ray absorptiometry. All women were white with mean (± 1 SD) age of 53.7 (± 9.5) years. About 21% of the women over 50 years had a T score for spine BMD lower than –2.5 SD, and 7% had a femoral neck BMD below this osteoporosis threshold. Vertebral heights (anterior, HA; middle, HM; and posterior, HP) and ratios (HA/HP and HM/HP) were assessed. There was no systematic difference between younger (20–49 years) and older (50+ years) women in heights or ratios. The vertebral heights were normalized for those observed in each individual case for the L2–L4 sequence. This normalization was adequate for all vertebral heights; the Z score averaged about +0.1. The average Z score for HA/HP was +0.01, but that for the HM/HP was −0.72, indicating that the latter ratio might differ from the reference population used (white American and European women). We observed a small positive correlation between vertebral heights and spine or femur BMD, but this was due entirely to the influence of body size on BMD. On a group basis, the HM/HP showed a significant association with axial BMD; the 1 SD difference between the lowest and highest quartile was associated with a difference of 8–15% (0.5–1.0 SD) in axial BMD.
A Cross-Sectional Study of Smoking and Bone Mineral Density in Premenopausal Parous Women: Effect of Body Mass Index, Breastfeeding, and Sports Participation
Authors: Jones Graeme , Scott Fiona Sara
Published: April 2006

The objective of this cross-sectional study was to describe the relationship between cigarette smoking, effect modifiers, and bone density in premenopausal parous women. We studied a sample of 276 women (mean age 33 years) from Southern Tasmania. The study factors were cigarette smoking, body mass index (BMI), sports participation, and breastfeeding history. Bone mineral density was measured utilizing an Hologic QDR 2000 densitometer and converted to Z scores using the group mean and variance. There were 118 current smokers and 158 nonsmokers. Smokers had lower bone mass at all sites (femoral neck, −0.32 SD, 95% confidence interval [CI] −0.60 to −0.04; lumbar spine, −0.49 SD, 95% CI −0.76 to −0.22; total body, −0.40 SD, 95% CI −0.66 to −0.14). Stratifying by BMI revealed that this association was only present, but greater in magnitude, for those with a BMI <25 kg/m2. Smokers who had breastfed at least one child had an additional deficit in bone mass (femoral neck, −0.48 SD, 95% CI −0.89 to −0.07; lumbar spine, −0.39 SD, 95% CI −0.80 to 0.02; total body, −0.37 SD, 95% CI −0.77 to 0.06) while smokers who took part in competitive sport had significant increments in bone mass (femoral neck, 0.74 SD, 95% CI 0.31 to 1.17; lumbar spine, 0.48 SD, 95% CI 0.03 to 0.93; total body, 0.42 SD, 95% CI 0.00 to 0.84). Neither of these two associations were present in nonsmokers. In conclusion, current smoking was associated with substantial deficits in bone mass in our sample of women, particularly those with a BMI <25 kg/m2. In addition, smoking may prevent the usual postweaning recovery phase of bone after breastfeeding while sports participation may offset the negative effect of smoking on bone mass. These observations need to be confirmed in longitudinal studies but they imply that past studies of smoking in this age group may have missed important associations as they did not consider possible effect modifiers.
Altered Postnatal Expression of Insulin-Like Growth Factor-I (IGF-I) and Type X Collagen Preceding the Perthes' Disease–Like Lesion of a Rat Model
Authors: Kikkawa Masanori , Imai Shinji , Hukuda Sinsuke
Published: April 2006

The spontaneously hypertensive rat (SHR) is a widely used animal model for the study of hypertension. It also exhibits an osteonecrosis of the femoral epiphysis that resembles the clinical features of Perthes' disease in humans. In this rat model, occlusion of the epiphyseal vessels occurs as a result of a breakdown of the mechanically vulnerable epiphysis. The postnatal development of the epiphysis recapitulates the serial events of the endochondral ossification (i.e., cartilage formation), chondrocyte hypertrophy, cartilage mineralization, vascularization, and introduction of osteoblasts that form the secondary ossification center within the epiphysis. In the present study, a detailed radiographic and histological analysis demonstrates that the osteonecrosis is preceded by a disturbance of the cartilage mineralization and a disturbance of the ossification, despite a normal hypertrophy of the epiphyseal cartilage. These observations suggest that abnormal development of the femoral epiphysis occurs much earlier than manifestation of the osteonecrosis. They lead us to a hypothesis that yet-unclarified transitional events between the cartilage hypertrophy and the cartilage mineralization may be affected in SHRs. Type X collagen is a developmentally regulated matrix molecule that is implicated in the mineralization of the hypertrophied chondrocytes. We show that the expression of type X collagen during epiphyseal ossification is delayed in SHRs (vs. normal controls), suggesting disturbed growth and/or differentiation of the epiphyseal chondrocytes. Postnatal growth and differentiation of the chondrocytes at least partly depend on insulin-like growth factor-I (IGF-I), which is produced by the chondrocytes in response to the pituitary growth hormone and stimulates cartilage growth in situ. The present study demonstrates an altered IGF-I expression during early postnatal life in SHRs and suggests that the altered IGF-I expression as well as the following delay in upregulation of type X collagen may cause the mechanical vulnerability of the femoral epiphysis in SHRs.
Mutations of the GNAS1 Gene, Stromal Cell Dysfunction, and Osteomalacic Changes in Non–McCune–Albright Fibrous Dysplasia of Bone
Authors: Bianco P. , Riminucci M. , Majolagbe A. , Kuznetsov S. A. , Collins M. T. , Mankani M. H. , Corsi A. , Bone H. G. , Wientroub S. , Spiegel A. M. , Fisher L. W. , Gehron ROBEY P.
Published: April 2006

Activating missense mutations of the GNAS1 gene, encoding the α subunit of the stimulatory G protein (Gs), have been identified in patients with the McCune–Albright syndrome (MAS; characterized by polyostotic fibrous dysplasia, café au lait skin pigmentation, and endocrine disorders). Because fibrous dysplasia (FD) of bone also commonly occurs outside of the context of typical MAS, we asked whether the same mutations could be identified routinely in non-MAS FD lesions. We analyzed a series of 8 randomly obtained, consecutive cases of non-MAS FD and identified R201 mutations in the GNAS1 gene in all of them by sequencing cDNA generated by amplification of genomic DNA using a standard primer set and by using a novel, highly sensitive method that uses a protein nucleic acid (PNA) primer to block amplification of the normal allele. Histologic findings were not distinguishable from those observed in MAS-related FD and included subtle changes in cell shape and collagen texture putatively ascribed to excess endogenous cyclic adenosine monophosphate (cAMP). Osteomalacic changes (unmineralized osteoid) were prominent in lesional FD bone. In an in vivo transplantation assay, stromal cells isolated from FD failed to recapitulate a normal ossicle; instead, they generated a miniature replica of fibrous dysplasia. These data provide evidence that occurrence of GNAS1 mutations, previously noted in individual cases of FD, is a common and perhaps constant finding in non-MAS FD. These findings support the view that FD, MAS, and nonskeletal isolated endocrine lesions associated with GNAS1 mutations represent a spectrum of phenotypic expressions (likely reflecting different patterns of somatic mosaicism) of the same basic disorder. We conclude that mechanisms underlying the development of the FD lesions, and hopefully mechanism-targeted therapeutic approaches to be developed, must also be the same in MAS and non-MAS FD.
Phenotypic Stability of Articular Chondrocytes In Vitro: The Effects of Culture Models, Bone Morphogenetic Protein 2, and Serum Supplementation
Authors: Stewart Matthew C. , Saunders Kathryn M. , Burton-Wurster Nancy , Macleod James N.
Published: April 2006

Numerous in vitro culture models have been developed for the investigation of chondrocyte and cartilage biology. In this study, we investigated the stability of the chondrocytic phenotype in monolayer, aggregate, pellet, and explant culture models and assessed the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) and serum supplementation on the phenotype in each model. Phenotypic effects were assessed by analyses of procollagen type II, aggrecan, (V+C)− fibronectin, and procollagen type I messenger RNA expression. In monolayer cultures, we noted a characteristic loss of procollagen type II and induction of procollagen type I expression. The aggregate and pellet culture models supported matrix protein gene expression profiles more reflective of in vivo levels. In explant cultures, expression of matrix protein genes was consistently depressed. Treatment with rhBMP-2 significantly increased the expression of procollagen type II and aggrecan in monolayer cultures; however, other models showed comparatively little response. Similarly, serum supplementation significantly down-regulated procollagen type II and aggrecan expression in monolayer cultures but had less effect on gene expression in the other models. Serum supplementation increased procollagen type I expression in monolayer and aggregate cultures. These results suggest that the influence of exogenous BMP-2 and serum on expression of chondrocyte-specific matrix protein genes is influenced by aspects of substrate attachments, cellular morphology, and/or cytoskeletal organization. Finally, the analyses of fibronectin expression suggest that V and C region alternative splicing in chondrocytes is linked to the establishment of a three-dimensional multicellular complex.
Variations in Three-Dimensional Cancellous Bone Architecture of the Proximal Femur in Female Hip Fractures and in Controls
Authors: Ciarelli T. E. , Fyhrie D. P. , Schaffler M. B. , Goldstein S. A.
Published: April 2006

Cubes of cancellous bone were obtained from proximal femora of women with hip fractures (n = 26) and from female cadaveric controls (n = 32) to compare architecture and mechanics between groups. Specimens were scanned on a microcomputed tomography system. Stereologic algorithms and model-based estimates were applied to the data to characterize the three-dimensional cancellous microstructure. Cubes were mechanically tested to failure to obtain mechanical properties. Specimens from control subjects had significantly higher bone volume fraction, trabecular number, and connectivity than specimens from patients with hip fractures; no difference in trabecular thickness was observed between groups. Both maximum modulus and ultimate stress were significantly higher in the control than in the fracture group, consistent with the higher bone volume found in the control group. No statistical differences in any of these architectural or mechanical variables were found when groups were matched for bone volume. Specimens from both patients with hip fractures and controls demonstrated strong relationships between trabecular number and bone volume fraction that were statistically equivalent, suggesting that for a given bone mass, both groups have the same overall number of trabeculae. However, there was an architectural difference between fracture and control groups in terms of the three-dimensional spatial arrangement of trabeculae. Fracture specimens had a significantly more anisotropic (oriented) structure than control specimens, with proportionately fewer trabecular elements transverse to the primary load axis, even when matched for bone volume. Relationships between mechanical and architectural parameters were significantly different between groups, suggesting that fracture and control groups have different structure-mechanics relationships, which we hypothesize may be a consequence of the altered three-dimensional structure between groups.
Human Myeloma Cells Promote the Recruitment of Osteoblast Precursors: Mediation by Interleukin-6 and Soluble Interleukin-6 Receptor
Authors: Karadag Abdullah , Scutt Andrew M. , Croucher Peter I.
Published: April 2006

Multiple myeloma is associated with the development of osteolytic bone disease characterized by a disruption to normal bone resorption and bone formation. Although studies have shown that myeloma cells produce factors that promote bone resorption little data are available examining the mechanism of decreased bone formation or the factors that mediate this effect. In the present study we describe a novel in vitro coculture system in which to investigate the effect of myeloma cells on osteoblast recruitment and differentiation. Under appropriate conditions mesenchymal stem cells were shown to differentiate into colonies of cells, a proportion of which show characteristics of osteoblasts, in that they express alkaline phosphatase activity and stain positively for collagen and calcium. The addition of the human myeloma cells JJN-3, RPMI-8226, or NCI-H929 to these cultures stimulated a significant increase in the total number of colonies (p < 0.005) and the proportion of osteoblastic colonies (p < 0.005). Media conditioned by these cells also were able to promote the formation of both total and osteoblastic colonies (p < 0.005). The addition of an antibody against the interleukin-6 receptor (IL-6R) blocked myeloma cell and myeloma cell-conditioned media induced osteoblast recruitment (p < 0.01). Furthermore, media conditioned by myeloma cells incubated with phorbol ester, which promotes IL-6R shedding, or a metalloproteinase inhibitor, which inhibits IL-6R shedding, were able to stimulate (p < 0.005) and inhibit osteoblast recruitment (p < 0.005), respectively. In addition, soluble IL-6R (sIL-6R) and IL-6 together, but not alone, were able to promote osteoblastic colony formation (p < 0.01). Taken together these data show that myeloma cells promote osteoblast recruitment by release of sIL-6R from myeloma cells.
Interleukin-6 Gene Polymorphism Is Related to Bone Mineral Density During and After Puberty in Healthy White Males: A Cross-Sectional and Longitudinal Study
Authors: Lorentzon Mattias , Lorentzon Ronny , Nordström Peter
Published: April 2006

Bone mineral density (BMD) is under strong genetic control and is the major determinant of fracture risk. The cytokine interleukin-6 (IL-6) is an important regulator of bone metabolism and is involved in mediating the effects of androgens and estrogens on bone. Recently, a G/C polymorphism in position −174 of the IL-6 gene promoter was found. We investigated this genetic polymorphism in relation to BMD during late puberty and to peak bone mass, in healthy white males. We identified the IL-6 genotypes (GG, GC, and CC) in 90 boys, age 16.9 ± 0.3 years (mean ± SD), using polymerase chain reaction (PCR). BMD (g/cm2) at the femoral neck, lumbar spine, and total body was measured using dual energy X-ray absorptiometry. The volumetric BMD (vBMD; mg/cm3) of the lumbar spine was estimated. Differences in BMD in relation to the genotypes were calculated using analysis of variance (ANOVA). Subjects with the CC genotype had 7.9% higher BMD of the femoral neck (p = 0.03), 7.0% higher BMD of the lumbar spine (p < 0.05), and 7.6% higher vBMD of the lumbar spine (p = 0.04), compared with their GG counterparts. Using multiple regression, the IL-6 genotypes were independently related to total body BMD (CC > GG; p = 0.03), humerus BMD (CC > GG; p < 0.05), neck BMD (CC > GG; p = 0.01), spine BMD (CC > GG; p = 0.01), and spine vBMD (CC > GG; p = 0.008). At age 19.3 ± 0.7 years (mean ± SD; 88 men) the IL-6 genotypes were still independent predictors for total body BMD (CC > GG; p = 0.03), humerus BMD (CC > GG; p = 0.03), spine BMD (CC > GG; p = 0.02), and spine vBMD (CC > GG; p = 0.003), while the IL-6 genotypes were not related to the increase in bone density seen after 2 years. We have shown that polymorphism of the IL-6 gene is an independent predictor of BMD during late puberty and of peak bone mass in healthy white men.
Calvariae from Fetal Mice with a Disrupted Igf1 Gene Have Reduced Rates of Collagen Synthesis but Maintain Responsiveness to Glucocorticoids
Authors: Woitge Henning W. , Kream Barbara E.
Published: April 2006

The goals of this study were to examine the role of insulin-like growth factor I (IGF-I) on bone formation and to test the hypothesis that the inhibitory effects of glucocorticoids on bone formation are independent of the IGF-I pathway. In serum-free organ cultures of 18-day fetal mouse calvariae derived from Igf1 null mice (Igf1−/−) and their wild-type (Igf1+/+) and heterozygous (Igf1+/−) littermates, we measured the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), percent collagen synthesis (PCS), the incorporation of [3H]thymidine into DNA, and messenger RNA (mRNA) levels of osteoblast markers in the presence or absence of dexamethasone. After 24 h of culture, calvariae of all genotypes had similar levels of PCS. However, after 48–96 h of culture, PCS was significantly lower in Igf1−/− calvariae compared with Igf1+/+ calvariae. Treatment of calvariae with 100 nM of dexamethasone for 48–96 h decreased PCS in all genotypes. After 72 h of culture, [3H]thymidine incorporation was similar in all genotypes and 100 nM dexamethasone caused a significant reduction in [3H]thymidine incorporation in all genotypes. Dexamethasone at 100 nM decreased α1(I)-collagen (Col1a1) mRNA and increased alkaline phosphatase, bone sialoprotein, and osteopontin mRNA in all genotypes after 72 h of culture. Type I IGF receptor mRNA levels were highest in Igf1−/− calvarial cultures. Dexamethasone at 100 nM increased Igf2 and type I IGF receptor mRNA levels in all genotypes. We conclude that one intact allele for Igf1 is sufficient to maintain normal rates of collagen synthesis in fetal mouse calvarial cultures. Moreover, the inhibitory effects of glucocorticoids on collagen synthesis and cell replication are at least partially independent of the IGF-I pathway in this model.
Spontaneous, But Not Induced, Puberty Permits Adequate Bone Mass Acquisition in Adolescent Turner Syndrome Patients
Authors: Carrascosa A. , Gussinyé M. , Terradas P. , Yeste D. , Audí L. , Vicens-Calvet E.
Published: April 2006

Lumbar L2-L4 bone mineral density (BMD) values were measured in 37 adolescent and young adult Turner syndrome patients. Nine had developed spontaneous puberty and had had regular menses since menarche (12.55 years ± 1.17 years) to the time of BMD evaluation (14.96 years ± 1.26 years). In the other 28, puberty was induced with increasing doses of oral ethinyl estradiol (2.5-10.0 μg/day, for 2 years) and later administration of estrogen/gestagen therapy up to the time of BMD evaluation. In 18, the adolescent group, menarche appeared at 14.68 years ± 0.63 years and BMD was evaluated at 17.77 years ± 0.70 years, and in the other 10, the young adult group, menarche appeared at 14.47 years ± 0.53 years and BMD was evaluated at 20.90 years ± 0.68 year. BMD values were in the normal range in those who had developed spontaneous puberty (Z score values, −0.24 ± 0.22) and in the osteopenia range in those in whom puberty was induced (Z score values, −2.09 ± 0.79 and −2.18 ± 0.32 for the adolescent and young adult groups, respectively) p < 0.0001. Height Z score values were similar in all three groups (−3.45 ± 0.77, −3.15 ± 0.83, and −3.08 ± 0.33, respectively). No significant differences in calcium intake or physical activity were found among groups. Neither the karyotype distribution nor growth hormone (GH) therapy (five in the spontaneous puberty and six in the induced puberty groups had been treated for a 3.5- to 4.4-year period) explained the differences in BMD values. Because the main difference between groups was the availability of estrogens to bone tissue from infancy to menarche and of estrogens/gestagens from then on up to the time of BMD evaluation, our results suggest that normal gonadal function from infancy to adulthood may be required for adequate bone mass peaking. Early detection of osteopenia and improvement in general measures for adequate bone mass peaking (calcium intake and physical activity) should be considered mandatory in the health care of these patients.
More Broken Bones: A 4-Year Double Cohort Study of Young Girls With and Without Distal Forearm Fractures
Authors: Goulding A. , Jones I. E. , Taylor R. W. , Manning P. J. , Williams S. M.
Published: April 2006

Predictors of childhood fractures have not been investigated previously. This study was undertaken to determine whether a previous history of forearm fracture, low bone mineral density (BMD; both areal bone mineral density [aBMD, g/cm2] and volumetric bone mineral apparent density [BMAD, g/cm3]), or anthropometry, influence fracture risk in young girls. At baseline, two cohorts of girls, aged 3–15 years, were evaluated: 100 had recently broken a forearm (group 1) and 100 were fracture free (group 2). Four years later we restudied 170 of these girls (82 from group l and 88 from group 2). We now report the relationships of previous fracture history, baseline BMD (measured by dual-energy X-ray absorptiometry), baseline weight, and height to risk of new fracture. More new fractures occurred in group l (37 fractures in 24 girls) than in group 2 (8 fractures in 7 girls; p = 0.0007). The independent predictors for occurrence of a new fracture at any skeletal site in a multivariate model adjusting for age, weight, total body aBMD, and fracture history were previous fracture (hazard ratio [HR], 3.28; 95% CI, 1.41-7.64); age (HR per l-year increase, 0.91; 95% CI, 0.84-0.99); total body aBMD (HR per l SD decrease, 1.92; 95% CI, 1.31-2.81); and body weight (HR per l SD increase, 1.49; 95% CI, 1.06-2.08). Girls with two risk factors together had substantially greater fracture risk: previous fracture and low spinal BMAD (HR, 9.4; 95% CI, 2.8-32.0), previous fracture and high body weight (HR, 10.2; 95% CI, 2.8-37.6), or previous fracture and low total body aBMD (HR, 13.0; 95% CI, 3.9-43.1). We conclude that previous forearm fracture, low total body aBMD, low spinal BMAD, and high body weight each increase risk of new fractures within 4 years in young girls. Interventions to reduce the risk of fractures, particularly forearm fractures, in girls warrant further study.
Deficiency of the α-Subunit of the Stimulatory G Protein and Severe Extraskeletal Ossification
Authors: Eddy Mark C. , Jan de beur Suzanne M. , Yandow Suzanne M. , McAlister William H. , Shore Eileen M. , Kaplan Frederick S. , Whyte Michael P. , Levine Michael A.
Published: April 2006

Progressive osseous heteroplasia (POH) is a rare disorder characterized by dermal ossification beginning in infancy followed by increasing and extensive bone formation in deep muscle and fascia. We describe two unrelated girls with typical clinical, radiographic, and histological features of POH who also have findings of another uncommon heritable disorder, Albright hereditary osteodystrophy (AHO). One patient has mild brachydactyly but no endocrinopathy, whereas the other manifests brachydactyly, obesity, and target tissue resistance to thyrotropin and parathyroid hormone (PTH). Levels of the α-subunit of the G protein (Gsα) were reduced in erythrocyte membranes from both girls and a nonsense mutation (Q12X) in exon 1 of the GNAS1 gene was identified in genomic DNA from the mildly affected patient. Features of POH and AHO in two individuals suggest that these conditions share a similar molecular basis and pathogenesis and that isolated severe extraskeletal ossification may be another manifestation of Gsα deficiency.
A Skeletal Gene Database
Authors: Ho Nicola C. , Jia Libin , Driscoll Catherine C. , Gutter Emily M. , Francomano Clair A. , Drezner Marc K.
Published: April 2006

Systematic organization of documented data coupled with ready accessibility is of great value to research. Catalogs and databases are created specifically to meet this purpose. The Skeletal Gene Database evolves as part of the Skeletal Genome Anatomy Project (SGAP), an ongoing multi-institute collaborative effort, to study the functional genome of bone and other skeletal tissues. The primary objective of the Skeletal Gene Database is to create a contemporary list of skeletal-related genes, offering the following information for each gene: gene name, protein name, cellular function, disease(s) caused by mutation of the corresponding gene, chromosomal location, LocusLink number, gene size, exon/intron numbers, messenger RNA (mRNA) coding region size, protein size/molecular weight, Online Mendelian Inheritance in Man (OMIM) number of the gene, UniGene assignment, and PubMed reference. The database includes genes already known and published in the literature as well as novel genes not yet characterized but known to be expressed in skeletal tissue. It will be posted on the web for easy access and swift referencing. The data will be updated in tempo with current and future research, thereby providing an invaluable service to the scientific community interested in obtaining information on bone-related genes.
The Expression of Metalloproteinase-2, −9, and −14 and of Tissue Inhibitors-1 and −2 Is Developmentally Modulated During Osteogenesis In Vitro, the Mature Osteoblastic Phenotype Expressing Metalloproteinase-14
Authors: Filanti C. , Dickson G. R. , Di Martino D. , Ulivi V. , Sanguineti C. , Romano P. , Palermo C. , Manduca P.
Published: April 2006

During osteogenesis, in vitro, of tibial-derived rat osteoblasts (ROB) and derived clones, changes occur in the interactions of mature osteoblasts with the endogenous extracellular matrix (ECM) and these culminate in the formation of tridimensional nodules, which become sites of mineral deposition. We investigated if these changes might be mediated by remodeling of ECM, and we focused our study on the neutral metalloproteinases (MMPs), known agents of matrix remodeling, and on their tissue inhibitors (TIMPs). We report that during in vitro differentiation, osteoblasts express the secreted MMP-2 and −9 and the membrane gelatinase MMP-14. These, along with the tissue inhibitors TIMP-1 and −2, are developmentally regulated according to the maturation stage of osteoblasts. Their levels change in a similar association with osteoblast phenotypic maturation in different populations of ROB, which take different times to complete osteogenesis in vitro. MMP-14 expression coincides in both cell populations with the mature osteoblastic phenotype and is localized in the cells forming nodules. MMP-2 and −9 are expressed diffusely in the osteoblast population. Developmentally associated changes in the activation of MMP-2 are detected, associated in their timing with the expression of MMP-14 in both populations of ROB, and MMP-14 activates pro-MMP-2 in vitro. Expression of messenger RNAs (mRNAs) for the three MMPs increases up to the time of nodule formation. At this stage, TIMP-1 mRNA levels are lowest. TIMP-2 mRNA decreases throughout osteogenesis. In situ hybridization in 7-day-old rat tibias shows the strongest expression of MMP-14 among osteogenic cells, in lining osteoblasts on the newly formed trabeculae under the growth plate, and on the endosteal surface of cortical bone. Our data support the concept that the developmentally regulated expression of MMP-14 triggers localized proteolysis within the osteogenic population, concomitant in vitro to nodule formation.
Bisphosphonates Induce Breast Cancer Cell Death In Vitro
Authors: Fromigue Olivia , Lagneaux Laurence , Body Jean-Jacques
Published: April 2006

Breast cancer frequently spreads to bone and is almost always associated with osteolysis. This tumor-induced osteolysis is caused by increased osteoclastic bone resorption. Bisphosphonates are used successfully to inhibit bone resorption in tumor bone disease and may prevent development of new osteolytic lesions. The classical view is that bisphosphonates only act on bone cells. We investigated their effects on breast cancer cells using three human cell lines, namely, MCF-7, T47D, and MDA.MB.231, and we tested four structurally different bisphosphonates: clodronate, pamidronate, ibandronate, and zoledronate. We performed time course studies for each bisphosphonate at various concentrations and found that all four compounds induced a nonreversible growth inhibition in both MCF-7 and T47D cell lines in a time- and dose-dependent manner. The MDA.MB.231 cell line was less responsive. Bisphosphonates induced apoptosis in MCF-7 and cell necrosis in T47D cells. The inhibition of MCF-7 cell proliferation could be reverted almost completely by the benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk) inhibitor of caspases, suggesting that the apoptotic process observed in the MCF-7 cell line is mediated, at least partly, by the caspase system. Caspase activity was little changed by bisphosphonates in T47D cells and the inhibitor of caspase did not modify bisphosphonates effects. In summary, we found that bisphosphonates inhibit breast cancer cell growth by inducing cell death in vitro. Such effects could contribute to the beneficial role of bisphosphonates in the treatment and the prevention of tumor-induced osteolysis.
Structural Trends in the Aging Femoral Neck and Proximal Shaft: Analysis of the Third National Health and Nutrition Examination Survey Dual-Energy X-Ray Absorptiometry Data
Authors: Beck Thomas J. , Looker Anne C. , Ruff Christopher B. , Sievanen Harri , Wahner Heinz W.
Published: April 2006

Hip scans of U.S. adults aged 20–99 years acquired in the Third National Health and Nutrition Examination Survey (NHANES III) using dual-energy X-ray absorptiometry (DXA) were analyzed with a structural analysis program. The program analyzes narrow (3 mm wide) regions at specific locations across the proximal femur to measure bone mineral density (BMD) as well as cross-sectional areas (CSAs), cross-sectional moments of inertia (CSMI), section moduli, subperiosteal widths, and estimated mean cortical thickness. Measurements are reported here on a non-Hispanic white subgroup of 2719 men and 2904 women for a cortical region across the proximal shaft 2 cm distal to the lesser trochanter and a mixed cortical/trabecular region across the narrowest point of the femoral neck. Apparent age trends in BMD and section modulus were studied for both regions by sex after correction for body weight. The BMD decline with age in the narrow neck was similar to that seen in the Hologic neck region; BMD in the shaft also declined, although at a slower rate. A different pattern was seen for section modulus; furthermore, this pattern depended on sex. Specifically, the section modulus at both the narrow neck and the shaft regions remains nearly constant until the fifth decade in females and then declined at a slower rate than BMD. In males, the narrow neck section modulus declined modestly until the fifth decade and then remained nearly constant whereas the shaft section modulus was static until the fifth decade and then increased steadily. The apparent mechanism for the discord between BMD and section modulus is a linear expansion in subperiosteal diameter in both sexes and in both regions, which tends to mechanically offset net loss of medullary bone mass. These results suggest that aging loss of bone mass in the hip does not necessarily mean reduced mechanical strength. Femoral neck section moduli in the elderly are on the average within 14% of young values in females and within 6% in males.
Role of Activator Protein 1 Transcriptional Activity in the Regulation of Gene Expression by Transforming Growth Factor β1 and Bone Morphogenetic Protein 2 in ROS 17/2.8 Osteoblast-like Cells
Authors: Palcy Sandrine , Bolivar Isabel , Goltzman David
Published: April 2006

In osteoblastic cells, transforming growth factor β1 (TGF-β1) has been found to regulate the expression of a variety of proto-oncogenes including c-fos, c-jun, and junB. The c-fos in particular has been implicated in the mitogenic effect of TGF-β1. Here, we examined the role of these early response genes in the regulation of osteoblast (OB) gene expression by two members of the TGF-β superfamily, TGF-β1 and bone morphogenetic protein 2 (BMP-2). In ROS 17/2.8 cells, TGF-β1 as well as BMP-2 up-regulated the expression of junB and c-fos messenger RNAs (mRNAs), and this increase was correlated in both cases with an increase in activator protein 1 (AP-1) DNA-binding activity involving JunB and c-Fos proteins. Protein kinase C (PKC)- and protein tyrosine kinase (PTK)-dependent pathways have been implicated in both TGF-β1 signaling and AP-1 gene regulation. Therefore, using the kinase inhibitors chelerythrine chloride and genistein, we showed that PKC and PTK activities, respectively, participated in TGF-β1- and BMP-2-induced increases in junB mRNA levels. Similarly, these kinase activities were involved in the stimulatory effect of BMP-2 on c-fos mRNA expression. Using a natural dominant negative for AP-1 transcriptional activity in ROS 17/2.8 cells, we then showed that AP-1 transcription factors mediated TGF-β1- and BMP-2-regulated expression of the (α1) collagen I gene as well as TGF-β1-regulated expression of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor. Our data emphasize the role of the AP-1 transcription factor in TGF-β1 and BMP-2 signaling and highlight the importance of this transcription factor family in the expression of OB genes.
Influence of Bone Tissue Density and Elasticity on Ultrasound Propagation: An In Vitro Study
Authors: de Terlizzi Francesca , Battista Stefano , Cavani Francesco , Canè Valerio , Cadossi Ruggero
Published: April 2006

Ultrasound (US) waves are mechanical vibrations that are applied to a material—bone tissue—in order to study its properties, that is, density, elasticity, and structure. In this study we evaluated in which way density and elasticity of the spongy bone influenced the transmission of 1.25 MHz US pulses. Twelve cylindrical specimens (diameter, 8 mm; height, 5 mm) excised from phalanxes of pig were decalcified with 0.5 M EDTA for different times (0, 2, and 5 days). During these periods, the samples underwent the following investigations: US transmission, density, and elasticity measurements. To assess the homogeneity of decalcification, the cross-sections of some samples were microradiographed. A detailed analysis of the US signal received was performed using velocity, Fourier analysis, and some parameters typical of signal processing technique. A good correlation was found between US velocity and density (r2 = 0.70); a lower correlation was found between velocity and elasticity (r2 = 0.59). If density and elasticity are considered simultaneously, the correlation with the US velocity improves significantly (r2 = 0.84). Fourier analysis enabled us to observe a shift of the main frequency toward lower values as the decalcification process advanced. We also observed that in the regressions weighted for density, US velocity correlated poorly with elasticity (r2 = 0.16), whereas signal processing parameters maintain a good correlation with elasticity (ultrasound peak amplitude [UPA], r2 = 0.48; slope, r2 = 0.62). In this study, it has been observed that when using a signal processing technique to analyze US pulses, it is possible to identify some parameters that are related in different ways to density and to elastic properties of bone. Our results show the potentiality of US technique to separate information on bone density and elasticity that X-ray-based densitometric methods do not provide.
Quantitative Ultrasound Does Not Reflect Mechanically Induced Damage in Human Cancellous Bone
Authors: Nicholson P. H. F. , Bouxsein M. L.
Published: April 2006

This study investigated the ability of quantitative ultrasound (QUS) to detect reductions in the elastic modulus of cancellous bone caused by mechanical damage. Ultrasonic velocity and attenuation were measured using an in-house parametric imaging system in 46 cancellous bone cores from the human calcaneus. Each core was subjected to a mechanical testing regime to (a) determine the predamage elastic modulus, (b) induce damage by applying specified strains in excess of the yield strain, and (c) measure the postdamage elastic modulus. The specimens were divided into four groups: a control group subjected to a nominally nondestructive 0.7% maximum strain (ϵm) and three damage groups subjected to increasing strain levels (ϵm = 1.5, 3.0, and 4.5%). QUS measurements before and after the mechanical testing showed no significant differences between the control group and damage groups, despite highly significant (p < 0.001) reductions in the elastic modulus of up to 72%. These results indicate that current QUS techniques do not intrinsically reflect the elastic properties of cancellous bone. This is consistent with ultrasonic properties being determined by other factors (apparent density and/or architecture), which normally are associated strongly with elastic properties, but only when bone is mechanically intact. Clinically, this implies that ultrasound cannot be expected to detect bone fragility in the absence of major changes in bone density and/or trabecular architecture.
Strong Prediction of Fractures Among Older Adults by the Ratio of Carboxylated to Total Serum Osteocalcin
Authors: Luukinen H. , Käkönen S.-M. , Pettersson K. , Koski K. , Laippala P. , Lövgren T. , Kivelä S.-L. , Väänänen H. K.
Published: April 2006

We examined serum total osteocalcin (TOC), carboxylated osteocalcin (COC), and their ratio (COC/TOC) by one-step two-site immunofluorescent assays in 87% (n = 792) of all home-dwelling persons of 70 years or older living in a defined area in northern Finland. Other baseline subject-related risk factors of fractures were assessed by postal questionnaires, interviews, clinical examinations, and tests. During a 5-year follow-up period, all falls and fractures (n = 106) were recorded by regular phone calls and by examining all the medical records yearly. Serum TOC and COC concentrations increased with advancing age and were higher in women than in men, but corresponding differences were not found in the case of COC/TOC. The adjusted relative risk of fracture was elevated in association with low (≤−1 SD from the mean) COC; hazard ratio (HR, 95% CI) 2.00 (1.20-3.36) and low COC/TOC; HR 5.32 (3.26-8.68), the relative risk being highest in the population older than 80 years; and HR 7.02 (2.42-20.39). The predictive value of low COC/TOC lasted 3 years. The multivariable-adjusted relative risk of hip fracture (n = 26) in regard to low COC/TOC ratio was 3.49 (1.12-10.86), as compared with the persons who did not suffer hip fractures. Our results suggest that serum COC concentrations and, more strongly, COC/TOC, predict the occurrence of fractures in older community-dwelling adults. The risk of fracture associated with low COC/TOC equals the hip fracture risk previously verified for concomitant high serum undercarboxylated OC concentrations and low bone mineral density.
“Café-Au-Lait Spots” Caused by Vitiligo in McCune-Albright Syndrome
Authors: Whyte Michael P. , Podgornik Michelle N. , Zerega Jan , Reinus William R.
Published: April 2006

Café-au-lait spots, fibrous dysplasia of bone, and endocrine gland hyperactivity are the principal features of McCune-Albright syndrome (MAS). Café-au-lait spots appear at, or soon after, birth. We illustrate “café-au-lait spots” acquired during middle age in a patient with MAS that are an illusion caused by vitiligo. This 64-year-old woman is the oldest patient reported with this disorder.
The Mechanical Properties of Cancellous Bone in the Proximal Tibia of Ovariectomized Rats
Authors: Hogan Harry A. , Ruhmann Sean P. , Sampson H. Wayne
Published: April 2006

The “mature rat model” is an effective and often-used surrogate for studying mechanisms and characteristics of estrogen-deficient osteopenia. The purpose of this study was to extend our understanding of this animal model to include the mechanical properties of cancellous bone in the proximal tibia. Female Sprague–Dawley rats were divided into two groups (n = 13 each) at 14 weeks of age: an ovariectomized group (OVX) and a sham-operated control group (sham). The study terminated after a duration of 5 weeks. Specimens 2 mm long were cut from the proximal tibial metaphysis just below the growth plate and tested using two methods: (1) “whole-slice” compression, in which the entire specimen is loaded between two larger flat platens and (2) “reduced-platen” compression (RPC), which uses platens sized and aligned to load only the cancellous bone in the center of the sample. Three-point bending tests also were conducted on the femur. The short duration of estrogen deficiency yielded only minimal differences (< 10%) in femoral cortical bone but dramatic reductions (∼60%) in cancellous bone properties as determined by the RPC method. Ultimate stress was 7.23 MPa ± 1.97 MPa for OVX versus 18.1 MPa ± 5.21 MPa for sham; and elastic modulus was 252 MPa ± 104 MPa for OVX versus 603 MPa ± 180 MPa for sham. These changes in mechanical properties are similar in many respects to the dramatic effects reported in histomorphometric studies. For the whole-slice method, differences in mechanical properties between the two groups were not as large because the test directly loads both cancellous and cortical bone, and the latter is not affected as severely by estrogen deficiency. In this case, ultimate stress and elastic modulus were only 30% (or less) lower for the OVX group. (J Bone Miner Res 2000;15:284–292)