bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Osteoblast Differentiation by Pasteurella Multocida Toxin (PMT): A Role for Rho GTPase in Bone Formation
Authors: Harmey Dympna , Stenbeck Gudrun , Nobes Catherine D , Lax Alistair J , Grigoriadis Agamemnon E
Published: April 2006

The role of the Rho-Rho kinase signaling pathway on osteoblast differentiation was investigated using primary mouse calvarial cells. The bacterial toxin PMT inhibited, whereas Rho-ROK inhibitors stimulated, osteoblast differentiation and bone nodule formation. These effects correlated with altered BMP-2 and −4 expression. These data show the importance of Rho-ROK signaling in osteoblast differentiation and bone formation.

Introduction: The signal transduction pathways controlling osteoblast differentiation are not well understood. In this study, we used Pasteurella multocida toxin (PMT), a unique bacterial toxin that activates the small GTPase Rho, and specific Rho inhibitors to investigate the role of Rho in osteoblast differentiation and bone formation in vitro.

Materials and Methods: Primary mouse calvarial osteoblast cultures were used to investigate the effects of recombinant PMT and Rho-Rho kinase (ROK) inhibitors on osteoblast differentiation and bone nodule formation. Osteoblast gene expression was analyzed using Northern blot and RT-PCR, and actin rearrangements were visualized after phalloidin staining and confocal microscopy.

Results: PMT stimulated the proliferation of primary mouse calvarial cells and markedly inhibited the differentiation of osteoblast precursors to bone nodules with a concomitant inhibition of osteoblastic marker gene expression. There was no apparent causal relationship between the stimulation of proliferation and inhibition of differentiation. PMT caused cytoskeletal rearrangements because of activation of Rho, and the inhibition of bone nodules was completely reversed by the Rho inhibitor C3 transferase and partly reversed by inhibitors of the Rho effector, ROK. Interestingly, Rho and ROK inhibitors alone potently stimulated osteoblast differentiation, gene expression, and bone nodule formation. Finally, PMT inhibited, whereas ROK inhibitors stimulated, bone morphogenetic protein (BMP)-2 and −4 mRNA expression, providing a possible mechanism for their effects on bone nodule formation.

Conclusions: These results show that PMT inhibits osteoblast differentiation through a mechanism involving the Rho-ROK pathway and that this pathway is an important negative regulator of osteoblast differentiation. Conversely, ROK inhibitors stimulate osteoblast differentiation and may be potentially useful as anabolic agents for bone.
Autosomal Dominant Hypocalcemia in Monozygotic Twins Caused by a De Novo Germline Mutation Near the Amino-Terminus of the Human Calcium Receptor
Authors: Hu Jianxin , Mora Stefano , Weber Giovanna , Zamproni Ilaria , Proverbio Maria Carla , Spiegel Allen M
Published: April 2006

To define the molecular pathogenesis of severe postnatal hypocalcemia in monozygotic twin sisters, we sequenced their CaR gene and identified a missense mutation, K29E. Expression of the mutant receptor in vitro showed a marked increase in Ca2+ sensitivity explaining the observed phenotype. Additional mutagenesis studies lead us to speculate concerning a novel mechanism whereby the K29E mutation may lead to receptor activation.

Introduction: Activating mutations of the Ca2+-sensing receptor (CaR) gene have been identified in subjects with autosomal dominant hypocalcemia. Study of such mutations has provided insight into the mechanism of activation of the CaR.

Materials and Methods: We performed biochemical and molecular genetic studies on monozygotic twin sisters who presented with early postnatal hypocalcemia and on their unaffected sister and parents. Functional characterization of mutant CaRs transfected in HEK-293 cells included immunoblots to monitor protein expression and Ca2+ stimulation of phosphoinositide hydrolysis to measure Ca2+ sensitivity.

Results: We identified a K29E missense mutation in the twin sisters but not in their parents or unaffected sister. The K29E mutant CaR showed a marked increase in Ca2+ sensitivity, including when it was co-transfected with wildtype CaR cDNA, consistent with a dominant effect. Substitution of K29 by aspartate equivalently increased CaR sensitivity, whereas conservative substitution by arginine did not.

Conclusions: Severe postnatal hypocalcemia in the twin sisters was caused by a de novo germline activating mutation. In a model of the Venus flytrap-like domain of the extracellular amino-terminus of the CaR, K29 is located close to a peptide loop, “loop 2,” that forms part of the dimer interface and is the site of 10 of the previously reported naturally occurring activating CaR mutations. We speculate that K29E increases Ca2+ sensitivity of the CaR by disrupting a salt bridge between K29 and an acidic residue in loop 2 and thereby changes the normal structure of loop 2 that maintains the CaR in its inactive conformation.
Blood Leptin and Adiponectin as Possible Mediators of the Relation Between Fat Mass and BMD in Perimenopausal Women
Authors: Kontogianni Meropi D , Dafni Urania G , Routsias John G , Skopouli Fotini N
Published: April 2006

Fat mass is a predictor of BMD; however, the mechanisms involved remain uncertain. Two adipokines, leptin and adiponectin, were examined as potential mediators of this relation in 80 perimenopausal women. Adiponectin did not exert any effect on BMD, whereas leptin exerted a negative one, with insulin acting as a confounder to this relation.

Introduction: Fat mass is an important determinant of bone density, but the mechanism involved in this relation is uncertain. Leptin and adiponectin, as circulating peptides of adipocyte origin, are potential contributors to this relation. We investigated the role of leptin and adiponectin in mediating fat mass effects on the skeleton of perimenopausal women.

Materials and Methods: Twenty-five premenopausal and 55 postmenopausal, healthy women (42–68 years old) participated in our study. Lumbar spine BMD (BMDL2-L4) and total body BMC (TBBMC) were measured with DXA, leptin levels with ELISA, and adiponectin levels with radioimmunoassay (RIA). Additionally, body composition analysis was performed, as well as measurements of several hormones.

Results: It was shown that serum leptin levels were negatively correlated with BMD (β = −0.005, p = 0.027) and TBBMC (β = −14.32, p = 0.013). The above correlation was observed only when serum insulin levels were included, as an independent variable, in the regression analysis model. Adiponectin was not significantly correlated with BMDL2-L4 nor with TBBMC, either in the presence or absence of insulin.

Conclusion: Circulating adiponectin does not seem to exert any effect on bone mass. In contrast, circulating leptin showed a negative correlation with bone mass, dependent on serum insulin levels.
Brittle IV Mouse Model for Osteogenesis Imperfecta IV Demonstrates Postpubertal Adaptations to Improve Whole Bone Strength
Authors: Kozloff Kenneth M , Carden Angela , Bergwitz Clemens , Forlino Antonella , Uveges Thomas E , Morris Michael D , Marini Joan C , Goldstein Steven A
Published: April 2006

The Brtl mouse model for type IV osteogenesis imperfecta improves its whole bone strength and stiffness between 2 and 6 months of age. This adaptation is accomplished without a corresponding improvement in geometric resistance to bending, suggesting an improvement in matrix material properties.

Introduction: The Brittle IV (Brtl) mouse was developed as a knock-in model for osteogenesis imperfecta (OI) type IV. A Gly349Cys substitution was introduced into one col1a1 allele, resulting in a phenotype representative of the disease. In this study, we investigate the effect of the Brtl mutation on whole bone architecture, strength, and composition across a range of age groups.

Materials and Methods: One-, 2-, 6-, and 12-month-old Brtl and wildtype (WT) mice were analyzed. Femurs were assessed at the central diaphysis for cortical geometric parameters using μCT and were subsequently mechanically tested to failure by four-point bending. Matrix material properties were predicted using μCT data to normalize data from mechanical tests. Raman spectroscopy and DXA were used to assess matrix composition.

Results: Our findings show a postpubertal adaptation in which Brtl femoral strength and stiffness increase through a mechanism independent of changes in whole bone geometry. These findings suggest an improvement in the material properties of the bone matrix itself, rather than improvements in whole bone geometry, as seen in previous mouse models of OI. Raman spectroscopic results suggest these findings may be caused by changes in mineral/matrix balance rather than improvements in mineral crystallinity.

Conclusions: Our findings parallel the currently unexplained clinical observation of decreased fractures in human OI patients after puberty. The Brtl mouse remains an important tool for investigating therapeutic interventions for OI.
Shockwave Stimulates Oxygen Radical-Mediated Osteogenesis of the Mesenchymal Cells From Human Umbilical Cord Blood
Authors: Wang Feng-Sheng , Yang Kuender D , Wang Ching-Jen , Huang Hui-Cheng , Chio Chi-Chian , Hsu Te-Yao , Ou Chia-Yu
Published: April 2006

Human umbilical cord blood (HUCB) mesenchymal progenitor cells expressed stro-1 or CD44 or CD29, and subsequently, differentiated toward osteogenic lineage. Physical shockwave treatment increased osteogenic activity of HUCB mesenchymal progenitor cells through superoxide-mediated TGF-β1 induction. Transplantation of shockwave-treated HUCB mesenchymal progenitor cells enhanced healing of segmental femoral defect in severe combined immunodeficiency disease (SCID) mice.

Introduction: Mesenchymal progenitor cells (MPCs) in the bone marrow are precursors to bone development. It remains uncertain whether MPCs are present in human umbilical cord blood (HUCB) and are capable of differentiating into osteogenic cell lineage. Extending from a model of shockwave (SW) promotion of bone marrow stromal cell differentiation toward osteoprogenitors in rats, we further investigated how physical SW mediated biological responses in regulating osteogenic differentiation of HUCB MPCs.

Materials and Methods: HUCB was subjected to SW treatment at different energy flux densities and impulses. Colony-forming units-stroma (CFU-Stroma), osteogenic activities (Cbfa1/Runx2 expression, bone alkaline phosphatase activity, and bone nodule formation), and bone formation by heterologous transplantation into SCID mice were assessed.

Results: Few CD34+ stem cells (1.3%) and stro-1+ cells (1.0%) were present in the freshly prepared mononuclear cells (MNCs) from HUCB. The number of stro-1+ cells, but not CD34+, increased to 72.4% in the adherent cell culture over 6 days. Stro-1+ cells co-expressed CD44 and CD29 markers and grew into CFU-Stroma that matured into bone nodules. We found that the SW treatment (0.16 mJ/mm2 energy flux density, 200 impulses) elicited superoxide production and promoted formation of CFU-Stroma, but not of hematopoietic CFU-Mix. SW also enhanced the production of transforming growth factor (TGF)-β1, but not of interleukin (IL)-3 or granulocyte monocyte-colony stimulating factor (GM-CSF). Neutralization of TGF-β1 significantly reduced SW-promoted CFU-Stroma formation. Superoxide scavenging by superoxide dismutase blocked SW enhancement of TGF-β1 production and formation of CFU-Stroma. Administration of SW-treated HUCB MPCs to SCID mice with femoral segmental defects facilitated dense, bridging callus and gap closure.

Conclusion: HUCB MPCs subjected to SW treatment is a potential source for stem cells useful in the treatment of orthopedic disorders. An optimal physical SW treatment enhanced osteogenesis through superoxide-mediated TGF-β1 production. Physical stimulation is an alternative method for extending mesenchymal stem cells of HUCB.
Pubertal Girls Only Partially Adapt to Low Dietary Calcium Intakes
Authors: Abrams Steven A , Griffin Ian J , Hicks Penni D , Gunn Sheila K
Published: April 2006

We evaluated the effects of low calcium in the diets of young adolescent girls. We measured calcium absorption and excretion using stable isotopes. We found partial adaptation to low intakes but a persistent large deficit relative to recommended intakes. Low calcium intakes pose a substantial risk of inadequate calcium retention.

Introduction: A substantial number of adolescent girls in the United States have habitual calcium intakes <500 mg/day (about 40% of the current recommended intake). The ability to adapt to these very low intakes by increasing calcium absorption and decreasing calcium excretion is not known. We sought to determine the effects of recommended (REC-Ca) versus very low (LO-Ca) calcium intakes on calcium absorption and excretion in white and black girls.

Materials and Methods: Pubertal, but premenarcheal girls, were adapted to low or recommended calcium intakes for at least 2 weeks before each study. Calcium absorption (n = 51) and endogenous fecal calcium excretion (n = 36 of the 51) were determined by dual-tracer stable isotope studies. Subjects were then switched to the other diet for at least 6 weeks, and the study was repeated.

Results: Calcium intake was 389 ± 10 mg/day on LO-Ca and 1259 ± 35 mg/day on REC-Ca diets. Fractional absorption increased from 44.9 ± 1.9% on REC-Ca to 63.4 ± 1.7% on LO-Ca (p < 0.01), but the net calcium absorption remained less than one-half the value on LO-Ca as on REC-Ca. Despite decreases in both endogenous fecal calcium excretion and urinary calcium excretion, net calcium balance was much lower on LO-Ca compared with REC-Ca1 (131 ± 14 versus 349 ± 32 mg/day, respectively; p < 0.001). We found significantly lower urinary calcium excretion but not calcium absorption in black girls compared with white girls.

Conclusions: Very low calcium intakes are only partially adapted to by increased absorption and decreased excretion. Very low calcium intakes place both white and black pubertal girls at substantial risk for inadequate calcium retention.
Terminal Differentiation of Osteoblasts to Osteocytes Is Accompanied by Dramatic Changes in the Distribution of Actin-Binding Proteins
Authors: Kamioka Hiroshi , Sugawara Yasuyo , Honjo Tadashi , Yamashiro Takashi , Takano-Yamamoto Teruko
Published: April 2006

Immunofluorescence staining of actin-binding proteins in osteoblasts and osteocytes was performed. α-Actinin, myosin, and tropomyosin showed similar organization in both osteoblastic stress fibers and osteocyte processes. However, fimbrin, villin, filamin, and spectrin showed dramatic differences in distribution between osteoblasts and osteocytes. This study suggested that terminal differentiation of osteoblasts to osteocytes is accompanied by highly dramatic changes in the distribution of actin-binding proteins.

Introduction: We previously reported that osteocyte shape is dependent on actin filaments. To analyze the terminal differentiation from osteoblasts to osteocytes, we investigated the actin-binding proteins, which are the control elements in the dynamic organization of the actin cytoskeleton.

Materials and Methods: We used primary chick osteocytes and osteoblasts, the phenotypes of which were confirmed by use of OB7.3, a chick osteocyte-specific monoclonal antibody and by detection of alkaline phosphatase activity, respectively. Immunofluorescence staining was performed for visualizing actin-binding proteins. Furthermore, we applied shear stress at 12 dyns/cm2 to the cells and compared the changes in fimbrin distribution.

Results: Immunofluorescence staining of fimbrin and α-actinin showed their presence in the processes of osteocytes, with especially strong signals of fimbrin at the sites of divarication of the processes. Anti-villin was reactive with the osteocyte cytoplasm but not with the processes. Interestingly, anti-villin immunoreactivity was much stronger in osteocytes than in osteoblasts. Filamin was localized along the stress fibers of osteoblasts but was seen only in those in the proximal base of osteocyte processes. Myosin and tropomyosin were found to have a similar pattern in both stress fibers of osteoblasts and osteocyte processes. The difference in the distribution of anti-spectrin staining was highly dramatic. Osteoblasts immunostained with anti-spectrin showed punctate signals on their cytoplasmic membranes, whereas anti-spectrin in osteocytes detected a filamentous organization; and the spectrin was totally colocalized with actin from the distal portion of the cytoplasmic processes to the cell center. In osteoblasts, shear stress induced recruitment of fimbrin to the end of stress fibers. However, fimbrin in the osteocyte processes did not change its localization.

Conclusion: We found that terminal differentiation of osteoblasts to osteocytes was accompanied by highly dramatic changes in the distribution of actin-binding proteins, changes of which may affect cellular function.
Effects of Simvastatin on Bone Turnover and BMD: A 1-Year Randomized Controlled Trial in Postmenopausal Osteopenic Women
Authors: Rejnmark Lars , Buus Henrik Niels , Vestergaard Peter , Heickendorff Lene , Andreasen Frederik , Larsen Lytken Mogens , Mosekilde Leif
Published: April 2006

To study effects of statins on human bone, 82 postmenopausal women were randomized to 1-year treatment with simvastatin 40 mg/day or placebo. The study showed no effect of simvastatin on biochemical bone markers or on BMD at the hip or spine. Thus, our results do not support a general beneficial effect of simvastatin on bone.

Introduction: Statins have been reported to cause bone anabolic as well as antiresorptive effects, and therefore statins have been suggested as potential agents in treatment of osteoporosis.

Materials and Methods: In a double-blinded design, 82 healthy postmenopausal women with osteopenia were randomized to 1-year simvastatin treatment 40 mg/day or placebo. BMD and plasma levels of cholesterol, parathyroid hormone (PTH), and biochemical bone markers were measured at baseline, after 1 year of treatment (week 52), and 26 weeks after withdrawal of treatment (week 78). Calcium supplements (400 mg/day) were administrated during the entire 1.5-year study period.

Results: Seventy-eight women completed the 1-year treatment. After 1 year, simvastatin but not placebo caused reduced plasma cholesterol (−27% versus +1%, p < 0.001) and low-density lipoprotein (LDL) levels (−43% versus +1%, p < 0.001). After withdrawal of treatment, cholesterol and LDL levels returned to baseline levels and no longer differed from the placebo group. However, plasma levels of PTH and biochemical bone markers did not differ between groups at week 52 or 78. Compared with placebo, simvastatin caused no changes in BMD at the lumbar spine, total hip, femoral neck, or whole body at week 52 or 78. However, a significant increase in BMD was found in response to simvastatin at the forearm. Within the simvastatin group, changes in cholesterol levels did not correlate to BMD changes at any site.

Conclusions: Our results do not support a general beneficial effect of simvastatin on bone.
Identification of Novel Regulators Associated With Early-Phase Osteoblast Differentiation
Authors: de Jong Diana S , Vaes Bart LT , Dechering Koen J , Feijen Alie , Hendriks Jose MA , Wehrens Ron , Mummery Christine L , van Zoelen Everardus JJ , Olijve Wiebe , Steegenga Wilma T
Published: April 2006

Key regulatory components of the BMP-induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2-induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP-induced osteoblast differentiation.

Introduction: Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2-induced osteoblast differentiation.

Materials and Methods: Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high-density microarrays. The regulated genes were grouped by means of model-based clustering, and protein functions were assigned. Real-time quantitative RT-PCR analysis was used to validate BMP2-induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP-induced osteoblast differentiation.

Results and Conclusions: A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represented the subset of immediate early genes. Regulation of expression of these genes was direct, independent of de novo protein synthesis and independent of the cell type studied. The intermediate early and late early genes consisted primarily of genes related to processes that modulate morphology, basement membrane formation, and synthesis of extracellular calcified matrix. The late early genes require de novo protein synthesis and show osteoblast specificity. In vivo and in vitro experiments showed that the transcription factors Hey1 and Tcf7 exhibited expression characteristics and cell type specificity very similar to those of the osteoblast specific transcription factor Cbfa1, and constitutive expression of Tcf7 in C2C12 cells differentially regulated osteoblast differentiation marker genes.
Cortical and Trabecular Bone Mineral Loss From the Spine and Hip in Long-Duration Spaceflight
Authors: Lang Thomas , LeBlanc Adrian , Evans Harlan , Lu Ying , Genant Harry , Yu Alice
Published: April 2006

We measured cortical and trabecular bone loss using QCT of the spine and hip in 14 crewmembers making 4- to 6-month flights on the International Space Station. There was no compartment-specific loss of bone in the spine. Cortical bone mineral loss in the hip occurred primarily by endocortical thinning.

Introduction: In an earlier study, areal BMD (aBMD) measurements by DXA showed that cosmonauts making flights of 4- to 12-month duration on the Soviet/Russian MIR spacecraft lost bone at an average rate of 1%/month from the spine and 1.5%/month from the hip. However, because DXA measurements represent the sum of the cortical and trabecular compartments, there is no direct information on how these bone envelopes are affected by spaceflight.

Materials and Methods: To address this, we performed a study of crewmembers (13 males and 1 female; age range, 40-55 years) on long-duration missions (4-6 months) on the International Space Station (ISS). We used DXA to obtain aBMD of the hip and spine and volumetric QCT (vQCT) to assess integral, cortical, and trabecular volumetric BMD (vBMD) in the hip and spine. In the heel, DXA was used to measure aBMD, and quantitative ultrasound (QUS) was used to measure speed of sound (SOS) and broadband ultrasound attenuation (BUA).

Results and Conclusions: aBMD was lost at rates of 0.9%/month at the spine (p < 0.001) and 1.4-1.5%/month at the hip (p < 0.001). Spinal integral vBMD was lost at a rate of 0.9%/month (p < 0.001), and trabecular vBMD was lost at 0.7%/month (p < 0.05). In contrast to earlier reports, these changes were generalized across the vertebrae and not focused in the posterior elements. In the hip, integral, cortical, and trabecular vBMD was lost at rates of 1.2-1.5%/month (p < 0.0001), 0.4-0.5%/month (p < 0.01), and 2.2-2.7%/month (p < 0.001), respectively. The cortical bone loss in the hip occurred primarily by cortical thinning. Calcaneal aBMD measurements by DXA showed smaller mean losses (0.4%/month) than hip or spine measurements, with SOS and BUA showing no change. In summary, our results show that ISS crewmembers, on average, experience substantial loss of both trabecular and cortical bone in the hip and somewhat smaller losses in the spine. These results do not support the use of calcaneal aBMD or QUS measurements as surrogate measures to estimate changes in the central skeleton.
Longitudinal In Vivo Effects of Growth Hormone Overexpression on Bone in Transgenic Mice
Authors: Eckstein Felix , Weusten Axel , Schmidt Corina , Wehr Ulrich , Wanke Rüdiger , Rambeck Walter , Wolf Eckhard , Mohan Subburaman
Published: April 2006

In this study we examined the effect of systemic overexpression of GH on bone in transgenic mice longitudinally in vivo over a period of 9 months. We observed substantially increased BMC in GH transgenic mice and a significant reduction in serum osteocalcin. GH effects on bone were strongly dependent on gender and developmental stage.

Introduction: State-of-the-art bone marker and microimaging technology was applied in this longitudinal study to examine bone metabolism, BMC, bone density, and cortical bone structure over the life span of growth hormone (GH) transgenic (tg) mice.

Materials and Methods: Thirty-eight mice from four genetic groups (male, female, tg, and controls) were examined with DXA, and their femur and tibia were examined with peripheral QCT (pQCT). Osteocalcin (formation) and collagen cross-links (resorption) from serum and urine were also measured at postnatal weeks 3, 6, 9, 12, 18, 26, and 38.

Results: GH tg mice displayed a significant increase in body weight (up to 50%) and BMC (up to 90%), but serum osteocalcin was significantly reduced compared with controls. GH tg females (but not males) displayed increased trabecular density over controls up to week 12. In contrast, male (but not female) GH tg mice displayed a higher cortical cross-sectional area than controls. Cortical density was significantly lower in both male and female GH tg mice compared with control mice.

Conclusions: The increase in BMC in GH tg mice is associated with reduced serum osteocalcin levels, indicating that bone turnover may be lower than in the control mice. On a structural level, bone responds to GH excess in a gender-specific manner, with alterations varying substantially between different developmental stages.
Chondrosarcoma in Association With Primary Hyperparathyroidism
Authors: Bhatia Ajay , Cannon Stephen , Briggs Tim , Keen Richard W
Published: April 2006

We describe two female patients, 66 and 36 years of age, with both primary hyperparathyroidism and chondrosarcoma. Case 1 had a chondrosarcoma of the right scapula, and case 2 had chondrosarcoma of the left proximal tibia. Both patients underwent surgical resection of their chondrosarcoma and subsequent parathyroid surgery. Histological analysis of the excised parathyroid in case 1 showed a parathyroid carcinoma and in case 2 showed a parathyroid adenoma. Including these two patients, there is now a total of six cases that have been reported in the literature describing the association between hyperparathyroidism and bone malignancy. We believe that this small number makes it unlikely that there is an association between these two conditions, although we speculate that there may be an underlying genetic basis.
Imbalance of Osteoclastogenesis-Regulating Factors in Patients With Celiac Disease
Authors: Taranta Anna , Fortunati Dario , Longo Maurizio , Rucci Nadia , Iacomino Enzo , Aliberti Ferdinando , Facciuto Enzo , Migliaccio Silvia , Bardella Maria Teresa , Dubini Antonella , Borghi Maria Orietta , Saraifoger Silvia , Teti Anna , Bianchi Maria Luisa
Published: April 2006

Celiac disease is an autoimmune disorder characterized by atrophy of the intestine villi triggered by ingestion of gluten in genetically susceptible individuals. The association between celiac disease and low BMD has been recognized, but the mechanisms of disturbance are poorly understood. We show imbalance of cytokines relevant to bone metabolism in celiac patients' sera and the direct effect of these sera on in vitro bone cell activity.

Introduction: Celiac disease is associated with mineral metabolism derangement and low BMD. We investigated whether imbalance of serum factors in celiac patients could affect human bone cell activity in vitro.

Materials and Methods: We studied two groups of celiac patients—one on a gluten-free diet and another before the diet—both with decreased bone mass. Patients were investigated for bone turnover markers, and their sera were used for culturing bone cells from healthy donors and evaluate changes in cell activity.

Results: The N-terminal telopeptide of procollagen type I and interleukin (IL)-6 were higher than normal in patients not on the gluten-free diet. IL-1β and TNF-α/β were normal in all patients. IL-12 was reduced in all patients, whereas IL-18 was reduced only in patients on the diet. The RANKL/Osteoprotegerin (OPG) ratio was increased in patients not on the gluten-free diet. Persistently increased osteoclast numbers were obtained from peripheral blood mononuclear cells of healthy donors on incubation with sera of patients not on the gluten-free diet versus control sera and sera from patients on the diet. In human osteoblasts from healthy individuals, IL-18 was reduced on incubation with sera from all patients, whereas OPG expression was lower when sera from patients not on the diet were used. Proliferation, alkaline phosphatase, and nodule mineralization were increased in osteoblast cultures containing sera from all celiac patients, either on or not on the gluten-free diet.

Conclusions: We conclude that bone loss in celiac disease might also be caused by a cytokine imbalance directly affecting osteoclastogenesis and osteoblast activity.
Regeneration of Defects in Articular Cartilage in Rat Knee Joints by CCN2 (Connective Tissue Growth Factor)
Authors: Nishida Takashi , Kubota Satoshi , Kojima Shunji , Kuboki Takuo , Nakao Kyouji , Kushibiki Toshihiro , Tabata Yasuhiko , Takigawa Masaharu
Published: April 2006

CTGF/CCN2, a hypertrophic chondrocyte-specific gene product, possessed the ability to repair damaged articular cartilage in two animal models, which were experimental osteoarthritis and full-thickness defects of articular cartilage. These findings suggest that CTGF/CCN2 may be useful in regeneration of articular cartilage.

Introduction: Connective tissue growth factor (CTGF)/CCN2 is a unique growth factor that stimulates the proliferation and differentiation, but not hypertrophy, of articular chondrocytes in vitro. The objective of this study was to investigate the therapeutic use of CTGF/CCN2.

Materials and Methods: The effects of recombinant CTGF/CCN2 (rCTGF/CCN2) on repair of damaged cartilage were evaluated by using both the monoiodoacetic acid (MIA)-induced experimental rat osteoarthritis (OA) model and full-thickness defects of rat articular cartilage in vivo.

Results: In the MIA-induced OA model, quantitative real-time RT-PCR assays showed a significant increase in the level of CTGF/CCN2 mRNA, and immunohistochemical analysis and in situ hybridization revealed that the clustered chondrocytes, in which clustering indicates an attempt to repair the damaged cartilage, produced CTGF/CCN2. Therefore, CTGF/CCN2 was suspected to play critical roles in cartilage repair. In fact, a single injection of rCTGF/CCN2 incorporated in gelatin hydrogel (rCTGF/CCN2-hydrogel) into the joint cavity of MIA-induced OA model rats repaired their articular cartilage to the extent that it became histologically similar to normal articular cartilage. Next, to examine the effect of rCTGF/CCN2 on the repair of articular cartilage, we created defects (2 mm in diameter) on the surface of articular cartilage in situ and implanted rCTGF/CCN2-hydrogel or PBS-hydrogel therein with collagen sponge. In the group implanted with rCTGF/CCN2-hydrogel collagen, new cartilage filled the defect 4 weeks postoperatively. In contrast, only soft tissue repair occurred when the PBS-hydrogel collagen was implanted. Consistent with these in vivo effects, rCTGF/CCN2 enhanced type II collagen and aggrecan mRNA expression in mouse bone marrow-derived stromal cells and induced chondrogenesis in vitro.

Conclusion: These findings suggest the utility of CTGF/CCN2 in the regeneration of articular cartilage.
In Vitro Differentiation of CD14 Cells From Osteopetrotic Subjects: Contrasting Phenotypes With TCIRG1, CLCN7, and Attachment Defects
Authors: Blair Harry C , Borysenko Christopher W , Villa Anna , Schlesinger Paul H , Kalla Sara E , Yaroslavskiy Beatrice B , García-Palacios Verónica , Oakley Jennifer I , Orchard Paul J
Published: April 2006

We studied osteoclastic differentiation from normal and osteopetrotic human CD14 cells in vitro. Defects in acid transport, organic matrix removal, and cell fusion with deficient attachment were found. Analysis of genotypes showed that TCIRG1 anomalies correlated with acid transport defects, but surprisingly, organic matrix removal failure correlated with CLCN7 defects; an attachment defect had normal TCIRG1 and CLCN7.

Introduction: Osteopetrotic subjects usually have normal macrophage activity, and despite identification of genetic defects associated with osteopetrosis, the specific developmental and biochemical defects in most cases are unclear. Indeed, patients with identical genotypes often have different clinical courses. We classified defects in osteoclast differentiation in vitro using four osteopetrotic subjects without immune or platelet defects, three of them severe infantile cases, compared with normals.

Materials and Methods: Osteoclast differentiation used isolated CD14 cells; results were correlated with independent analysis of two key genes, CLCN7 and TCIRG1. CD14 cell attachment and cell surface markers and extent of differentiation in RANKL and colony-stimulating factor (CSF)-1 were studied using acid secretion, bone pitting, enzyme, and attachment proteins assays.

Results and Conclusions: CD14 cells from all subjects had similar lysosomal and nonspecific esterase activity. With the exception of cells from one osteopetrotic subject, CD14 cells from osteopetrotic and control monocytes attached similarly to bone or tissue culture substrate. Cells from one osteopetrotic subject, with normal CLCN7 and TCIRG1, did not attach to bone, did not multinucleate, and formed no podosomes or actin rings in RANKL and CSF-1. Attachment defects are described in osteopetrosis, most commonly mild osteopetrosis with Glantzman's thrombasthenia. However, this case, with abnormal integrin αvβ3 aggregates and no osteoclasts, seems to be unique. Two subjects were compound heterozygotes for TCIRG1 defects; both had CD14 cells that attached to bone but did not acidify attachments; cell fusion and attachment occurred, however, in RANKL and CSF-1. This is consistent with TCIRG1, essential for H+-ATPase assembly at the ruffled border. A compound heterozygote for CLCN7 defects had CD14 cells that fused in vitro, attached to bone, and secreted acid, TRACP, and cathepsin K. However, lacunae were shallow and retained demineralized matrix. This suggests that CLCN7 may not limit H+-ATPase activity as hypothesized, but may be involved in control of organic matrix degradation or removal.
Follistatin Restricts Bone Morphogenetic Protein (BMP)-2 Action on the Differentiation of Osteoblasts in Fetal Rat Mandibular Cells
Authors: Abe Yukiko , Abe Tatsuya , Aida Yoshitomi , Hara Yoshitaka , Maeda Katsumasa
Published: April 2006

We tested whether FS secretion might modulate BMP-2 actions by measuring FS levels and counting bone numbers of rat mandibular cells. In the presence of Dex, BMP-2 stimulated FS secretion at the early phase and augmented bone nodule by neutralizing with FS antibody. We concluded that BMP-2 facilitates FS secretion, and the FS restricts BMP-2 action on osteoblastogenesis.

Introduction: Bone morphogenetic proteins (BMPs) promote the differentiation of osteoprogenitor cells into osteoblasts. Activin A is involved in the regulation of bone formation. Follistatin (FS) antagonizes the bioactivities of BMP and activin A extracellularly.

Materials and Methods: In this study, we tested whether the induction of FS secretion might modulate the effects of BMP-2 on osteoblast development, using the bone nodule-forming cultures of fetal rat mandibular cells.

Results and Conclusions: In the presence of dexamethasone (Dex), BMP-2 stimulated the secretion of FS at the early phase (days 3-9) of the culture. Dex alone had no effect, and BMP-2 alone was less effective than the combination of the two. BMP-4 and -6 had little effect on FS secretion. Activin A inhibited the early upregulation of FS secretion when added with BMP-2 and Dex. In the presence of Dex, BMP-2 increased bone nodule numbers when added to early cultures. The addition of anti-FS antibody to cultures with BMP-2 and Dex augmented bone nodule formation. These results show that BMP-2 facilitates the secretion of FS in the presence of Dex, and the increased FS secretion restricts the action of BMP-2 on osteoblast differentiation.
An In Vitro Study of the Ultrasonic Axial Transmission Technique at the Radius: 1-MHz Velocity Measurements Are Sensitive to Both Mineralization and Intracortical Porosity
Authors: Bossy Emmanuel , Talmant Maryline , Peyrin Françoise , Akrout Leïla , Cloetens Peter , Laugier Pascal
Published: April 2006

The ultrasonic axial transmission technique allows for investigating skeletal sites such as the cortical layer of long bones (radius, tibia, phalanges). Using synchrotron radiation μCT, we investigated, in vitro, the relationships between 1-MHz axial transmission SOS measurements at the radius and site-matched measurements of C.Th, POR, MIN, and vBMD.

Introduction: The ultrasonic axial transmission technique allows for investigating skeletal sites such as the cortical layer of long bones (radius, tibia, phalanges).

Materials and Methods:Using synchrotron radiation μCT, we investigated, in vitro, the relationships between 1-MHz axial transmission speed of sound (SOS) measurements at the radius and site-matched measurements of cortical thickness (C.Th), intracortical porosity (POR), tissue mineralization (MIN), and volumetric BMD (vBMD). SOS measurements were based on bidirectional axial transmission and were performed with a 1-MHz proprietary probe on 39 excised human radii.

Results: The highest correlations between SOS values and bone parameters (R2SOS/POR = 0.28, p < 10−3; R2SOS/MIN = 0.38, p < 10−4; R2SOS/vBMD = 0.57, p < 10−3) were found for bone parameters assessed in a 1-mm-thick periosteal region of the cortex rather than throughout the whole cortex. The observed moderate correlation between SOS and C.Th values (R2SOS/C.Th = 0.20, p < 10−2) disappeared when controlled for other variables. The two best multilinear predictive models, including either BMD alone or the pair of dependent variables MIN and POR (all assessed in the periosteal cortex), were equally accurate in predicting SOS values (R2SOS/(POR,MIN) = 0.59, p < 10−5; R2SOS/vBMD = 0.57, p < 10−5).

Conclusion: For the first time, the respective adjusted contributions of POR (−24 m/s%−1) and tissue mineralization (+3.5 m/s/mg/cm−3) to SOS values were assessed. These results suggest potential sensitivity of axial transmission SOS values to changes in cortical bone status under different pathological conditions or treatments affecting POR and/or tissue mineralization.
Osteoclast Deficiency Results in Disorganized Matrix, Reduced Mineralization, and Abnormal Osteoblast Behavior in Developing Bone
Authors: Dai Xu-Ming , Zong Xiao-Hua , Akhter Mohammed P , Stanley E Richard
Published: April 2006

Studies of the influence of the osteoclast on bone development, in particular on mineralization and the formation of the highly organized lamellar architecture of cortical bone by osteoblasts, have not been reported. We therefore examined the micro- and ultrastructure of the developing bones of osteoclast-deficient CSF-1R-nullizygous mice (Csf1r−/− mice).

Introduction: Colony-stimulating factor-1 receptor (CSF-1R)-mediated signaling is critical for osteoclastogenesis. Consequently, the primary defect in osteopetrotic Csf1r−/− mice is severe osteoclast deficiency. Csf1r−/− mice therefore represent an ideal model system in which to investigate regulation by the osteoclast of osteoblast-mediated bone formation during development.

Materials and Methods: Bones of developing Csf1r−/− mice and their littermate controls were subjected to X-ray analysis, histological examination by light microscopy and transmission electron microscopy, and a three-point bending assay to test their biomechanical strength. Bone mineralization in embryonic and postnatal bones was visualized by double staining with alcian blue and alizarin red. Bone formation by osteoblasts in these mice was also examined by double-calcein labeling and in femoral anlagen transplantation experiments.

Results and Conclusions: Frequent spontaneous fractures and decreased strength parameters (ultimate load, yield load, and stiffness) in a three-point bending assay showed the biomechanical weakness of long bones in Csf1r−/− mice. Histologically, these bones have an expanded epiphyseal chondrocyte region, a poorly formed cortex with disorganized collagen fibrils, and a severely disturbed matrix structure. The mineralization of their bone matrix at secondary sites of ossification is significantly reduced. While individual osteoblasts in Csf1r−/− mice have preserved their typical ultrastructure and matrix depositing activity, the layered organization of osteoblasts on the bone-forming surface and the direction of their matrix deposition toward the bone surface have been lost, resulting in their abnormal entrapment by matrix. Moreover, we also found that (1) osteoblasts do not express CSF-1R, (2) the bone defects in Csf1r−/− embryos develop later than the development of osteoclasts in normal embryos, and (3) the transplanted Csf1r−/− femoral anlagen develop normally in the presence of wildtype osteoclasts. These results suggest that the dramatic bone defects in Csf1r−/− mice are caused by a deficiency of the osteoclast-mediated regulation of osteoblasts and that the osteoclast plays an important role in regulating osteoblastic bone formation during development, in particular, in the formation of lamellar bone.
Targeted Expression of SHH Affects Chondrocyte Differentiation, Growth Plate Organization, and Sox9 Expression
Authors: Tavella Sara , Biticchi Roberta , Schito Anna , Minina Eleonora , Di Martino Davide , Pagano Aldo , Vortkamp Andrea , Horton William A , Cancedda Ranieri , Garofalo Silvio
Published: April 2006

The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation.

Introduction: Hedgehogs (Hhs) are a family of secreted polypeptides that play important roles in vertebrate development, controlling many critical steps of cell differentiation and patterning. Skeletal development is affected in many different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways cause severe abnormalities in the appendicular, axial, and cranial skeleton in man and other vertebrates.

Materials and Methods: Genetic manipulation of mouse embryos was used to study in vivo the function of SHH in skeletal development. By DNA microinjection into pronuclei of fertilized oocytes, we have generated transgenic mice that express SHH specifically in chondrocytes using the cartilage-specific collagen II promoter/enhancer. Transgenic skeletal development was studied at different embryonic stages by histology. The expression pattern of specific chondrocyte molecules was studied by immunohistochemistry and in situ hybridization.

Results: Transgenic mice died at birth with severe craniorachischisis and other skeletal defects in ribs, sternum, and long bones. Detailed analysis of long bones showed that chondrocyte differentiation was blocked at prehypertrophic stages, hindering endochondral ossification and trabecular bone formation, with specific defects in different limb segments. The growth plate was highly disorganized in the tibia and was completely absent in the femur and humerus, leading to skeletal elements entirely made of cartilage surrounded by a thin layer of bone. In this cartilage, chondrocytes maintained a columnar organization that was perpendicular to the bone longitudinal axis and directed toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was greatly increased in all cartilage, as well as the expression of parathyroid hormone-related protein (PTHrP) at the articular surface; while the expression of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of chondrocyte maturation, was greatly reduced and restricted to the displaced chondrocyte columns. Transgenic mice also revealed the ability of SHH to upregulate the expression of Sox9, a major transcription factor implicated in chondrocyte-specific gene expression, in vivo and in vitro, acting through the proximal 6.8-kb-long Sox9 promoter.

Conclusion: Transgenic mice show that continuous expression of SHH in chondrocytes interferes with cell differentiation and growth plate organization and induces high levels and diffuse expression of Sox9 in cartilaginous bones.