bluuebunny/crossref_metadata_2025 huggingface dataset:
Do More Highly Organized Collagen Fibrils Increase Bone Mechanical Strength in Loss of Mineral Density After One-Year Running Training?
Authors: Puustjärvi Kaija , Nieminen Jyrki , Räsänen Tuomas , Hyttinen Mika , Helminen Heikki J. , Kröger Heikki , Huuskonen Jouni , Alhava Esko , Kovanen Vuokko
Published: April 2006

The aim of this study was to evaluate the effects of long-term running training on the structural properties of bone. Ten beagle dogs ran according to a strenuous progressive program (up to 40 km/day) for 1 year. At the end of the training program, there was a significant reduction in bone mineral density (up to 9.7%) in the vertebrae of the runner dogs as compared with 10 sedentary control dogs. Polarized light microscopy of the vertebral trabecular bone, however, displayed proportionally higher retardation values of the collagen network of the runner dogs than of the sedentary dogs, suggesting a reorganization in a more parallel manner in the collagen fibrils. The concentration and cross-linking of collagen in the bones remained similar in both groups. No differences were observed in the force to failure of bones of the two groups nor in the histomorphometric analysis of the bones. We suggest that the collagen network in the bones accounted for the maintenance of the strength properties in the bones of the runner dogs despite the loss of mineral density.
In Vitro Responses to 17β-Estradiol Throughout Pubertal Maturation in Female Human Bone Cells
Authors: Dr. Delaveyne-Bitbol Roselyne , Garabedian Michèle
Published: April 2006

To test the hypothesis that bone sensitivity to estrogens differ with the pubertal status, we cultured human osteoblasts (hOBs) from 14 girls (3–18 years) and examined the effects of repeated weekly doses of 17β-estradiol (E2, 10 pM–10 nM) on estradiol receptor (ER) and progesterone receptor (PR) expression, type I procollagen (PICP) and osteocalcin (BGP; bone Gla protein) production, and alkaline phosphatase (ALP) activity. The bone samples were divided into two equal groups according to the pubertal status and plasma E2 level of the donor. The two groups were significantly different for age (9 ± 1 and 15 ± 1 years), pubertal status (Tanner stages I–III and IV–V), and plasma E2 concentrations (17 ± 3 and 49 ± 4 pg/ml). ER and PR were expressed and not influenced by the sexual maturation in untreated cells. E2 increased ER in the two groups with nanomolar doses. Picomolar doses did not significantly increase ER expression but led to significant differences in the percentage of cells expressing ER in premenarchial (33%) and postmenarchial (7%) hOB cultures. In the two groups, E2 had no clear effect on PR expression, ALP activity, nor BGP production. But repeated weekly doses of E2 significantly influenced PICP production at picomolar doses. This effect depended upon the sexual maturation of the donor. E2 decreased PICP in premenarchial cultures and increased PICP in postmenarchial cultures. Thus, E2 modulates in vitro human bone cell metabolism and probably their phenotype and has different effects, depending on the pubertal status of the donor. Unlike what could have been expected, prepubertal and early pubertal hOBs appear to be specifically sensitive to picomolar doses of E2, suggesting that this hormone is a crucial regulator of bone metabolism even before puberty.
Tartrate-Resistant Bone Acid Phosphatase: Large-Scale Production and Purification of the Recombinant Enzyme, Characterization, and Crystallization
Authors: Kaija Helena , Jia Jia , Lindqvist Ylva , Andersson Göran , Prof. Vihko Pirkko
Published: April 2006

Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed in bone-resorbing osteoclasts and certain tissue macrophages in human tissues. The functions of TRAP in biological systems are not known. Elucidation of the three-dimensional structure of the active site could yield important information about the physiological substrate(s) of the enzyme. We have produced recombinant rat bone TRAP using a baculovirus expression vector system. The production was scaled up to a 30-l bioreactor, and a method of purification in large scale was developed. The enzyme is composed of one 34 kDa polypeptide chain. Trypsin digestion resulted in a preparation where two subunits of ∼23 kDa and ∼16 kDa appeared after disulfide reduction. Trypsin digestion activated the enzyme. We generated monoclonal antibodies against recombinant TRAP. One of the selected antibodies detected the 23 kDa subunit in Western blotting. The reduced and oxidized forms of the enzyme could be separated by Mono-S cation-exchange chromatography. Crystals of TRAP have been obtained with ammonium sulfate/polyethylene glycol as precipitant. They belong to space group P212121 or P21212 with unit cell dimensions a = 57.2 Å, b = 69.5 Å, and c = 87.2 Å and diffract to at least 2.2 Å resolution. A packing density value of 2.55 Å3/Da is consistent with one subunit in the asymmetric unit.
Demonstration of the Predominant Urine Osteocalcin Fragments Detectable by Two-Site Immunoassays
Authors: Matikainen Terhi , Käkönen Sanna-Maria , Pettersson Kim , Karp Matti , Lövgren Timo , Väänänen H. Kalervo , Hellman Jukka
Published: April 2006

We have isolated and characterized human osteocalcin (OC) fragments from pubertal urine. The fragments were isolated by immunoaffinity chromatography based on monoclonal antibody 6F9 and further purified by reverse phase chromatography. The major isolated forms, which were detectable with two-site immunofluorometric assays for serum OC, span residues 6–30 and 7–30 as determined by mass spectrometry and N-terminal amino acid sequencing. Full-length OC was not detectable in the supernatant fraction of urine but could be extracted with guanidinium hydrochloride from the sediment of urine samples. Urine samples from subjects with different menopausal status were measured by two different two-site assays. Urine OC (uOC) concentrations were 12- to 16-fold higher in the pubertal group than in the adult group. Also, the uOC concentration in a postmenopausal group was significantly higher than in a premenopausal group. The difference was 125% and 75% (values for p < 0.0001), respectively, when measured with the two assays. uOC concentrations in postmenopausal subjects on hormone replacement therapy were indistinguishable from the premenopausal subjects. The fact that uOC can be measured by a noncompetetive two-site assay design offers improved analytical sensitivity. Urine as the sample matrix is also especially interesting because the predominant markers of bone resorption, collagen type I peptides or cross-links, are performed on urine samples. Our results from the technical validation of two-site assays for uOC and from applying these to human pubertal and pre- and postmenopausal samples calls for more extensive clinical validation.
Parathyroid Hormone and Mechanical Usage Have a Synergistic Effect in Rat Tibial Diaphyseal Cortical Bone
Authors: Ma Yanfei , Jee Webster S. S. , Yuan Zhongzhi , Wei Wei , Chen Hongka , Pun Sunwah , Liang Haohai , Lin Chaohua
Published: April 2006

Previous reports showed that bone mass and architecture only partially recovered by remobilization (RM) after immobilization (IM)-induced osteopenia, and that parathyroid hormone (PTH) had an anabolic effect on the skeleton. The aim of this study was to determine whether low doses of PTH could restore IM-induced cortical bone loss and whether a combination of PTH plus loading (RM) treatment would be more effective than the PTH in unloaded (IM) limbs. One hundred and sixty 6-month-old rats were divided into aging and IM groups. The right hindlimb of the rat was immobilized by elastic bandage for 18 weeks, and then groups of rats were either kept IM or RM and treated with 30 μg or 80 μg of hPTH(1–38)/kg/day for 2, 10, and 20 weeks. Fluorescent-labeled, undecalcified cross-sections of right tibial shafts were studied. We found that RM for 20 weeks after 18 weeks of IM only partially recovered IM-induced muscle weight loss and PTH had no effect on muscle weight in either IM or RM limbs; that RM for 20 weeks after 18 weeks of IM partially restored some minimal cortical width by stimulating periosteal and endocortical bone formation and decreasing endocortical resorption; that PTH treatment of IM limbs completely restored IM-induced cortical bone loss and added extra bone by stimulating bone formation indices on all bone surfaces and depressing bone resorption on endocortical surface; that PTH treatment of RM limbs produced similar anabolic effects as in IM limbs with 30 μg/kg/day dose but the 80 μg/kg/day dose-treated limbs had a higher periosteal bone formation rate, which created a larger cross-sectional area, more cortical bone area, and a thicker cortex than the same dose treated IM limbs; and that PTH 80 μg/kg/day treatment produced more anabolic effect than the 30 μg/kg/day in both IM and RM limbs. We concluded that reloading the hindlimb by RM after long-term IM could not recover the cortical bone mass. PTH at employed doses was able to completely restore IM-induced cortical bone loss, and this effect was independent of mechanical stimulation. However, when PTH was combined with mechanical loading (RM), a synergistic anabolic effect on periosteal bone formation occurred which increased the cross sectional area that can increase bone strength.
Extension of Phenotype Associated with Structural Mutations in Type I Collagen: Siblings with Juvenile Osteoporosis Have an α2(I)Gly436 → Arg Substitution
Authors: Dawson Paul A. , Kelly Thaddeus E. , Marini Joan C.
Published: April 2006

Mutations in the type I collagen genes have been identified as the cause of all four types of osteogenesis imperfecta (OI). We now report a mutation that extends the phenotype associated with structural abnormalities in type I collagen. Two siblings presented with a history of back pain and were diagnosed with juvenile osteoporosis, based on clinical and radiological examination. Radiographs showed decreased lumbar bone density and multiple compression fractures throughout the thoracic and lumbar spines of both patients. One child has moderate short stature and mild neurosensory hearing loss. However, neither child has incurred the long bone fractures characteristic of OI. Protein studies demonstrated electrophoretically abnormal type I collagen in samples from both children. Enzymatic cleavage of RNA:RNA hybrids identified a mismatch in type I collagen α2 (COL1A2) mRNA. DNA sequencing of COL1A2 cDNA subclones defined the mismatch as a single-base mutation (1715G → A) in both children. This mutation predicts the substitution of arginine for glycine at position 436 (G436R) in the helical domain of the α2(I) chain. Analysis of genomic DNA identified the mutation in the asymptomatic father, who is presumably a germ-line mosaic carrier. The presence of the same heterozygous mutation in two siblings strongly suggests that the probands display the full phenotype. Taken together, the clinical, biochemical, and molecular findings of this study extend the phenotype associated with type I collagen mutations to cases with only spine manifestations and variable short stature into adolescence.
BMP-6 Is an Autocrine Stimulator of Chondrocyte Differentiation
Authors: Grimsrud Christopher D. , Romano Paul R. , D'souza Mary , Puzas J. Edward , Reynolds Paul R. , Rosier Randy N. , O'keefe Regis J.
Published: April 2006

While parathyroid hormone-related protein (PTHrP) has been characterized as an important negative regulator of chondrocyte maturation in the growth plate, the autocrine or paracrine factors that stimulate chondrocyte maturation are not well characterized. Cephalic sternal chondrocytes were isolated from 13-day embryos, and the role of bone morphogenetic protein-6 (BMP-6) as a positive regulator of chondrocyte maturation was examined in monolayer cultures. Progressive maturation, which was accelerated in the presence of ascorbate, occurred in the cultures. During maturation, the cultures expressed high levels of BMP-6 mRNA which preceded the induction of type X collagen mRNA. Treatment of the cultures with PTHrP (10−7 M) at the time of plating completely abolished BMP-6 and type X collagen mRNA expression. Removal of PTHrP after 6 days was followed by the rapid (within 24 h) expression of BMP-6 and type X collagen mRNA, with BMP-6 again preceding type X collagen expression. The addition of exogenous BMP-6 (100 ng/ml) to the cultures accelerated the maturation process both in the presence and absence of ascorbate and resulted in the highest levels of type X collagen. When exogenous BMP-6 was added to PTHrP containing cultures, maturation occurred with the expression of high levels of type X collagen, despite the presence of PTHrP in the cultures. Furthermore, BMP-6 did not stimulate expression of its own mRNA in the PTHrP treated cultures, but it did stimulate the expression of Indian hedgehog (Ihh) mRNA. These latter findings suggest that while PTHrP directly inhibits BMP-6, it indirectly regulates Ihh expression through BMP-6. Other phenotypic changes associated with chondrocyte differentiation were also stimulated by BMP-6, including increased alkaline phosphatase activity and decreased proliferation. The results suggest that BMP-6 is an autocrine factor that initiates chondrocyte maturation and that PTHrP may prevent maturation by inhibiting the expression of BMP-6.
Characterization of Mouse Cathepsin K Gene, the Gene Promoter, and the Gene Expression
Authors: Li Yi-Ping , Chen Wei
Published: April 2006

Cathepsin K, a lysosomal cysteine protease, is abundantly and selectively expressed in osteoclasts and has a specialized role in osteoclast-mediated bone resorption. In contrast to function studies, transcription regulation of cathepsin K remains largely unknown. In this study, the gene encoding mouse cathepsin K and the promoter have been isolated and completely sequenced. In addition, the temporal and spatial expressions of cathepsin K have been characterized. Intrachromosomal mapping studies revealed that the gene contains eight exons and seven introns spanning ∼10.6 kb of genomic DNA, a genomic organization that was highly conserved with respect to its human homology. Analysis of the 9 kb 5′ flanking region indicates that this gene lacks canonical TATA and CAAT boxes and contains multiple putative transcription regulatory elements which are also present in the comparable position of 5′ flanking region of human cathepsin K gene. Mouse cathepsin K was found to be a single-copy gene. Northern blot analysis of RNAs from a number of mouse tissues revealed that cathepsin K mRNA is selectively expressed in osteoclast. The selective expression of cathepsin K was confirmed by anticathepsin K immunohistochemical staining. The sequence of cathepsin K expression was linked to osteoclast differentiation in vivo and in vitro by a tartrate-resistant acid phosphatase–anticathepsin K dual immunostaining technique. Cathepsin K is initially expressed at the preosteoclast stage and throughout the mature osteoclast stage. The primer extension assay indicated a major transcription start site 58 bp upstream of the initiator Met codon. The characterization of the cathepsin K gene, its promoter, and the temporal and spatial expression may provide valuable insights into its osteoclast-specific expression and the molecular mechanisms responsible for osteoclast activation.
Differential Effects of 20-Epi Vitamin D Analogs on the Vitamin D Receptor Homodimer
Authors: Dr. Koszewski Nicholas J. , Reinhardt Timothy A. , Horst Ronald L.
Published: April 2006

Vitamin D analogs have received increased attention because of their possible therapeutic benefits in treating osteoporosis and various proliferative disorders. Several analogs were examined for their effects on DNA binding of the vitamin D receptor (VDR) homodimer complex with the murine osteopontin vitamin D response element. All of the tested analogs increased complex binding by recombinant human VDR in the electrophoretic mobility shift assay and notable differences in mobility of these complexes were observed. A panel of C-terminal anti-VDR antisera were screened for their ability to interact with analog-bound VDR homodimer complexes or as a heterodimer complex with recombinant human retinoid X receptor α (rhRXRα). Like calcitriol, analog-bound heterodimer complexes were largely resistant to interaction with these antisera; however, striking differences were observed with the various antisera in an analogous homodimer binding experiment. KH1060 and CB1093, analogs with 20-epi conformations, produced homodimer complexes that were 3- to 6-fold more resistant to supershifting with Ab180 compared with the hormone or EB1089. Chymotrypsin digestion in combination with Western blotting using a C-terminal anti-VDR antiserum revealed similar digestion patterns for all ligands. However, KH1060- and CB1093-bound VDR complexes were more resistant to digestion than either calcitriol or EB1089. Finally, the ability of these compounds to yield stable homodimer complexes was assessed by challenging preformed homodimer with the exogenous addition of rhRXRα extracts. Although new heterodimer complexes appeared in a time-dependent fashion, the preformed homodimer complexes exhibited stable binding throughout the time course of the experiment. The results indicate that VDR homodimers are targets of vitamin D analogs with differential effects on C-terminal protein conformation that may partially explain the varied biological responses of these compounds.
Osteogenic Phenotype Expression of Allogeneic Rat Marrow Cells in Porous Hydroxyapatite Ceramics
Authors: Akahane M. , Ohgushi H. , Yoshikawa T. , Sempuku T. , Tamai S. , Tabata S. , Dohi Y.
Published: April 2006

Porous hydroxyapatite (HA) ceramics were combined with either allogeneic (ACI) or isogeneic (Fischer 344) rat marrow cells and implanted in subcutaneous sites of Fischer rats. FK506 as an immunosuppressant or saline was administered to the recipient rats. The implanted marrow/HA composites were harvested on day 28 and analyzed for bone-forming capability by determining osteoblastic phenotype expression levels of protein synthesis and gene expression. The alkaline phosphatase (ALP) activity and osteocalcin (OC) contents were very low and mRNAs (Northern blot analysis) were not detected in the allografts without FK506. However, high activity of ALP and high content of OC were found and mRNAs were detected in the allografts with FK506 and in the isografts (with and without FK506). This analysis indicates the osteogenic potential of allogeneic marrow cells in the presence of FK506. The histologic sections revealed that allografts without FK506 did not show bone formation but did show the infiltration of many small cells in the ceramics indicating an immunologic reaction, however, the allografts with FK506 and the isografts (with and without FK506) showed consistent de novo bone formation on the HA pore surface. These results indicate that FK506 can suppress the immunologic reaction in the allografts and induce a favorable conditions to support osteoblastic differentiation of allogeneic rat marrow stromal stem cells on the surface of HA ceramics. Therefore, our study suggests the feasibility of clinical transplantation of allogeneic bone marrow for a selected bone graft in applications using adjuvant systemic immunosuppression.
An Automated Analysis of Intracortical Porosity in Human Femoral Bone Across Age
Authors: Dr. Stein M. S. , Feik S. A. , Thomas C. D. L. , Clement J. G. , Wark J. D.
Published: April 2006

The matrix of human cortical bone is arranged around a network of vascular spaces (hereafter referred to as “pores”). Our aim was to investigate age-related differences in human cortical porosity (total pore area divided by cortical bone area), pore size and number, and surface to volume ratios, while adjusting for sex, height, and weight. Ninety-six specimens of entire transverse sections of human femoral diaphysis, from subjects aged 21–92 years, were examined. We used our established automated image acquisition and analysis system which measures pores from entire sections of multiple specimens of bone. Over 400,000 pores were recorded. Results showed a greater porosity in older bone (p < 0.01) but marked variation in porosity for any given age. The cohort median, of the specimen medians, of pore cross-sectional area was 2050 μm2. Older specimens did not have more pores than younger specimens but had a greater proportion of larger pores (p < 0.05) and greater intraspecimen variation in pore size (p < 0.001). The pore surface to bone matrix volume ratio was a median 2.3 mm2/mm3. This varied more than 4-fold between individuals but did not relate to age. No simple relationships were found between any of the measured parameters and either sex, height, or weight, even after adjustment for age. We conclude that the greater porosity in older specimens is due to greater pore size rather than a larger number of pores. Age, however, explains little of the inter-individual variation in the parameters studied.
Bone Loss in Celiac Disease Is Related to Secondary Hyperparathyroidism
Authors: Dr. Selby Peter L. , Davies Michael , Adams Judith E. , Mawer E. Barbara
Published: April 2006

Celiac disease is a major cause of intestinal malabsorption. Previous studies have demonstrated that celiac disease is associated with significant osteoporotic bone loss. These studies have suggested that successful treatment of the malabsorption is associated with amelioration of the bone loss. Such studies have failed to examine bone mass at peripheral skeletal sites which is more likely to be responsive to changes in parathyroid hormone (PTH) in response to calcium malabsorption. We have examined bone density in the lumbar spine, femoral neck, and distal forearm in 35 patients with celiac disease who had been established on gluten-free diet. In addition, the concentrations of PTH and 1,25-dihydroxyvitamin D (1,25(OH)2D) were measured. Bone density was below that expected for the subject's age and gender at all sites. This was most marked in the distal forearm where the bone density was 1.40 SD below expected (p < 0.0001). In the forearm, there was a negative relationship between bone density and PTH concentration (r = −0.49, p = 0.009). In the forearm and lumbar spine, there was a negative relationship between 1,25(OH)2D concentration and bone density. Bone mass was not related to the concentration of 25-hydroxyvitamin D at any of the skeletal sites measured. Bone density is reduced in the peripheral skeleton in celiac disease and this deficit persists despite treatment with apparent normalization at axial skeletal sites. This reduction in bone mass is related to the presence of secondary hyperparathyroidism which should be sought in all patients with treated celiac disease.
Expression and Localization of Estrogen Receptor-β in Murine and Human Bone
Authors: Vidal Olle , Kindblom Lars-Gunnar , Ohlsson Claes
Published: April 2006

Estrogens have profound effects on bone metabolism. Cellular responses to estrogens are mediated by estrogen receptors (ERs) which belong to the nuclear receptor superfamily. Two estrogen receptors, ERα and ERβ, have been cloned. Previously expression of ERα has been shown in osteoblasts. Here we demonstrate that the transcript for ERβ can be detected in the human osteosarcoma cell lines (MG-63 and SaOS-2) and in cultured human osteoblast-like cells. We also show that ERβ protein is present in nuclear extracts from these cells. Furthermore, ERβ immunoreactivity is found in sections of murine and human bone. Murine and human osteoblast and osteocyte nuclei are immunoreactive for ERβ. Osteoclasts are also ERβ immunoreactive but the staining is mainly cytoplasmic. The present study demonstrates that ERβ is present in all the cellular compartments involved in bone formation and bone resorption, both in human and in murine bone tissue.
Effect of Bisphosphonate (Incadronate) on Fracture Healing of Long Bones in Rats
Authors: Li Jiliang , Mori Satoshi , Kaji Yoshio , Mashiba Tasuku , Kawanishi Jun , Norimatsu Hiromichi
Published: April 2006

This study was designed to test whether bisphosphonates disturb the process of fracture healing. Female Sprague-Dawley rats were injected with either two doses of bisphosphonate (incadronate) (10 μg/kg and 100 μg/kg) or vehicle three times a week for 2 weeks. Right femora were then fractured and fixed with intramedullary wires. Incadronate treatment was stopped in pretreatment groups (P-10 and P-100 groups), while the treatment was continued in continuous treatment groups (C-10 and C-100 groups). Animals were sacrificed at 6 and 16 weeks after surgery. Soft X-ray of all fractured femora was taken. After mechanical testing, fractured femora were stained in Villanueva bone stain and embedded in methyl methacrylate. Cross-sections near fracture line were analyzed by microradiography and histomorphometry. Radiographic study showed that bony callus was present in all the fractures and incadronate treatment led to a larger callus, especially in C-100 group at both 6 and 16 weeks. Histologic study showed that the process of fracture healing in pretreatment groups was delayed at 6 weeks, but reached control level thereafter and showed same characteristics as in control at 16 weeks. Woven bony callus could still be seen in continuous treatment groups at 16 weeks. Mechanical study indicated that the ultimate load of C-100 group was slightly higher than the other treatment groups and control. The results suggest that pretreatment with incadronate did not affect fracture healing at 16 weeks after fracture. However, continuous incadronate treatment could lead to larger callus, but it delayed remodeling process during fracture healing, especially with high-dose treatment.
Bone's Early Responses to Mechanical Loading Differ in Distinct Genetic Strains of Chick: Selection for Enhanced Growth Reduces Skeletal Adaptability
Authors: Dr. Pitsillides Andrew A. , Rawlinson Simon C. F. , Mosley John R. , Lanyon Lance E.
Published: April 2006

Bone's functional competence is established and maintained, at least partly, by mechanisms involving appropriate adaptation to mechanical loading. These appear to fail in chickens selectively bred either for maximum egg (Egg-type) or meat (Meat-type) production, which show high rates of fracture and skeletal abnormality, respectively. By measuring several early strain-induced responses in cultured embryonic tibiotarsi from commercially bred (Egg-type and Meat-type) and wild-type (Wild-type) chicks, we have investigated the possibility that these skeletal failures are the product of a compromised ability to respond appropriately to loading-induced mechanical strain. Axial loads engendering peak dynamic (1 Hz) longitudinal strains of between −1300 με and −1500 με (for 10 minutes) in vitro in tibiotarsi from the three types of 18-day-old chicks increased periosteal osteoblast glucose 6-phosphate dehydrogenase (G6PD) activity in both Wild-type (26%, p < 0.01) and Egg-type (49%, p < 0.001) chicks in situ, while Meat-type chicks did not show any significant changes (11%). Load-induced increases in medium nitrite accumulation (stable nitric oxide [NO] metabolite) were produced in Egg-type and Wild-type tibiotarsi (82 ± 12%, p < 0.01; 39 ± 8%, p < 0.01), respectively. In contrast, loading produced no change in NO release from Meat-type chick tibiotarsi. These changes in NO release correlated with load-related increases in G6PD activity (R2 = 0.98, p < 0.05) in the different chick types. Wild-type and Meat-type tibiotarsal periosteal osteoblasts responded in a biphasic manner to exogenous prostacyclin (PGI2), with maximal stimulation of G6PD activity at 10−7 M and 10−6 M PGI2. However, Egg-type chick osteoblasts showed smaller, progressive increases up to 10−5 M PGI2. These results indicate that early phases of the adaptive response to loading differ in different genetic strains of embryonic chick; that skeletal abnormalities which develop in genetically selected, high growth rate chicks may reflect a compromised ability to respond to load; and that load-induced increases in osteoblastic G6PD activity appear to be closely associated with increased rates of NO release. It is probable that similar genetically related differences in bones' responsiveness to mechanical loading occur in other species.
Longitudinal Bone Mineral Density Changes in Female Child Artistic Gymnasts
Authors: Nickols-Richardson S. M. , O'Connor P. J. , Shapses S. A. , Lewis R. D.
Published: April 2006

Changes in bone mineral density (BMD), and related factors, in female child artistic gymnasts (n = 9) and their age- (±0.3 years), height- (±2.8 cm), and weight- (±1.7 kg) matched controls (n = 9) were prospectively examined. It was hypothesized that gymnasts would possess higher BMD at baseline, 6, and 12 months later and have greater gains in BMD over 1 year compared with controls. BMD (g/cm2) of the total proximal femur (TPF), Ward's triangle (WT), trochanter (Troch), femoral neck (FN), lumbar spine (LS, L1–L4), and total body (TB) were measured by dual-energy X-ray absorptiometry. Physical activity was measured by a 7-day recall; daily dietary intakes of energy and nutrients were estimated from 3-day records. Serum osteocalcin and urinary pyridinium cross-links were measured by radioimmunoassay and high performance liquid chromatography, respectively. Gymnasts versus controls possessed significantly higher BMD at all sites measured. Although not significantly different (p > 0.05), gymnasts compared with controls had moderately larger percentage changes in Troch (% Δ = 8.6 ± 3.0 vs. 3.8 ± 5.1%, d = 0.41), FN (% Δ = 6.1 ± 1.2 vs. 3.9 ± 1.6%, d = 0.55), LS (% Δ = 7.8 ± 1.1 vs. 6.8 ± 1.6%, d = 0.26), and TB BMD (% Δ = 5.6 ± 0.8 vs. 3.4 ± 0.7%, d = 0.98) as evidenced by the magnitude of the effect sizes (d). Gymnasts versus controls possessed a lower percentage body fat (p < 0.01) and engaged in more hours of very hard activity (p < 0.0001). Calcium, as a percentage of adequate intake, decreased over 12 months (p < 0.01), and urinary cross-links significantly decreased over 6 months in both groups. Female child gymnasts possess higher BMD at the TPF and related sites, LS, and TB compared with nongymnast controls, and 1 year of gymnastics training moderately increases Troch, FN, LS, and TB BMD for gymnasts compared with controls. These findings lend support to the idea that gymnastics training in childhood helps maximize peak BMD.
Targeted Expression of Calcitonin Gene-Related Peptide to Osteoblasts Increases Bone Density in Mice
Authors: Ballica Rabia , Valentijn Karine , Khachatryan Armen , Guerder Sylvie , Kapadia Shanta , Gundberg Caren , Gilligan James , Flavell Richard A. , Vignery Agnès
Published: April 2006

The neuropeptide calcitonin gene-related peptide (CGRP) is concentrated in fine sensory nerve endings innervating all tissues, including bone. CGRP inhibits osteoclasts, stimulates insulin-like growth factor I and inhibits tumor necrosis factor alpha production by osteoblasts in vitro. To investigate the role of CGRP in bone in vivo, mice were engineered to express CGRP in osteoblasts by placing the human CGRP gene under the control of the rat osteocalcin promoter (Ost-CGRP tg+ mice). Calvaria cultures from transgene positive (tg+), but not tg− mice, produced bioactive CGRP. Trabecular bone density and bone volume, determined by peripheral quantitative computed tomography and bone histomorphometry, respectively, were higher in tg+ than tg− littermates. This increase in bone volume was associated with an increased bone formation rate. Trabecular bone density decreased in tg+ mice as a result of ovariectomy, but remained higher than in sham tg− mice. Targeting CGRP to osteoblasts appears to favor the establishment of a higher trabecular bone mass in mice.
Mechanical Tension-Stress Induces Expression of Bone Morphogenetic Protein (BMP)-2 and BMP-4, but Not BMP-6, BMP-7, and GDF-5 mRNA, During Distraction Osteogenesis
Authors: Sato Motohiko , Ochi Takahiro , Nakase Takanobu , Hirota Seiichi , Kitamura Yukihiko , Nomura Shintaro , Yasui Natsuo
Published: April 2006

Bone lengthening with osteotomy and gradual distraction was achieved in 57 rats, and the effect of mechanical tension-stress on gene expression of bone morphogenetic proteins (BMPs) was investigated by in situ hybridization and Northern blot analysis using probes of BMP-2, BMP-4, BMP-6, BMP-7, and growth/differentiation factor (GDF)-5. There was a lag phase for 7 days after femoral osteotomy until gradual distraction was carried out for 21 days at a rate of 0.25 mm/12 h using a small external fixator. The signals of the above BMPs mRNA were not detected in the intact rat bone but they were induced after osteotomy except those for BMP-7. By 4 days after osteotomy, BMP-2 and BMP-4 mRNAs were detected in chondrogenic precursor cells in the subperiosteal immature callus. BMP-6 and GDF-5 mRNA were detected in more differentiated cells in chondroid bone. By 7 days after osteotomy, cartilaginous external callus and bony endosteal callus were formed. Meanwhile, the signals of BMP-2 and BMP-4 mRNAs declined to preoperative levels, whereas the signals of BMP-6 and GDF-5 mRNAs were rather elevated. As distraction was started, the callus elongated and eventually separated into proximal and distal segments forming a fibrous interzone in the middle. Expression of BMP-2 and BMP-4 mRNAs was markedly induced at this stage. Their signals were detected widely among chondrogenic and osteogenic cells and their precursor cells sustaining mechanical tension-stress at the fibrous interzone. BMP-6 and GDF-5 mRNAs were detected exclusively in chondrogenic cells at both ends of the fibrous interzone, where endochondral ossification occurred. But neither mRNA was detected in terminally differentiated hypertrophic chondrocytes. As distraction advanced, the cartilage was progressively resorbed from both ends and new bone was formed directly by intramembranous ossification. There was no new cartilage formation in the advanced stage of distraction. The signals of BMP-6 and GDF-5 mRNA declined by this stage, while those of BMP-2 and BMP-4 were maintained at high level for as long as distraction was continued. After completion of distraction, the fibrous interzone fused and the lengthened segment was consolidated. BMP-2, BMP-4, BMP-6, nor GDF-5 was expressed at this stage. The signals of BMP-7 were not detected throughout the experiment. The present results suggest that excellent and uninterrupted bone formation during distraction osteogenesis owes to enhanced expression of BMP-2 and BMP-4 genes by mechanical tension-stress. Abundant gene products of BMP-2 and BMP-4 could induce in situ bone formation by paracrine and autocrine mechanisms.
A Novel T Cell Cytokine Stimulates Interleukin-6 in Human Osteoblastic Cells
Authors: Rifas Leonard , Avioli Louis V.
Published: April 2006

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a heavy lymphocytic infiltration into the synovial cavity, resulting in the secretion of a variety of cytokines which ultimately leads to destruction of joint tissue. Among the infiltrating cells are activated T cells which produce specific cytokines capable of osteoclast progenitor cell expansion, fusion, and activation. Cultures of activated human T cells and human osteoblasts (hOBs) were used to study the possibility that lymphokines may act on osteoblasts to produce the osteoclastogenic factor interleukin-6 (IL-6). Purified T cells were activated with a combination of anti-CD3 and anti-CD28 antibodies, cocultured with hOBs in direct physical contact or separated by a transwell system, and conditioned media (CM) were assayed for IL-6 production. After a 72 h incubation period, activated T cell–hOB interaction resulted in a 100-fold increase of IL-6 production over basal levels. The immunosuppressant cyclosporine A (CsA) inhibited T cell tumor necrosis factor alpha and IL-6 production but did not inhibit the T cell induction of IL-6 from hOB. Assay of activated T-cell CM on hOB revealed that a soluble factor, not cell-cell contact, was the major inducer of IL-6. The induction of IL-6 mRNA by both activated T cell CM and CsA-treated activated T cell CM was confirmed by Northern blot analysis. Neutralizing antibodies to IL-13 and IL-17 did not affect IL-6 production. These findings suggest that activated T cells produce a novel, potent, IL-6 inducing factor that may be responsible for the bone loss observed in RA patients.
Glucocorticoids Decrease Interleukin-6 Levels and Induce Mineralization of Cultured Osteogenic Cells from Children with Fibrous Dysplasia
Authors: Stanton Robert P. , Hobson Grace M. , Montgomery Ben E. , Moses Priscilla A. , Smith-Kirwin Susan M. , Funanage Vicky L.
Published: April 2006

Fibrous dysplasia (FD) is a progressive bone disease in which abnormal fibroblast proliferation results in the replacement of normal cancellous bone with an immature fibrous tissue that is poorly mineralized. The disease manifests itself in the monostotic form in which only one bone is involved and the polyostotic form in which multiple bones at different sites are affected. The McCune–Albright syndrome is a variation of the polyostotic form in which patients demonstrate a greater extent of bone involvement and a variety of endocrinopathies. Somatic activating mutations in the GNAS gene have been demonstrated in the fibrotic lesions of patients affected with either monostotic or polyostotic FD. The increased cAMP levels caused by the G-protein mutations lead to increased interleukin-6 (IL-6) levels in the affected tissues, resulting in abnormal osteoblast differentiation and increased osteoclastic activity. Utilizing cell culture techniques that have been developed for mammalian bone marrow stromal cells, we have successfully cultured osteogenic stem cells from the affected stroma of 11 FD patients. Cells cultured from patients with polyostotic FD showed a high frequency of the Gsα mutation, whereas cells from monostotic FD patients showed a low frequency of the mutation. Both the normal and FD cells displayed the osteogenic phenotype when exposed to medium containing glucocorticoids. Glucocorticoids also caused a dramatic inhibition of IL-6 mRNA and protein levels in osteogenic cells cultured from the FD patients. These findings suggest that chemical alteration of cellular function may lead to new treatment options for patients with FD.
Age-Related Osteogenic Potential of Mesenchymal Stromal Stem Cells from Human Vertebral Bone Marrow
Authors: Dr. D'Ippolito Gianluca , Schiller Paul C. , Ricordi Camillo , Roos Bernard A. , Howard Guy A.
Published: April 2006

Mesenchymal stem cells (MSCs) residing in bone marrow (BM) are the progenitors for osteoblasts and for several other cell types. In humans, the age-related decrease in bone mass could reflect decreased osteoblasts secondary to an age-related loss of osteoprogenitors. To test this hypothesis, BM cells were isolated from vertebral bodies of thoracic and lumbar spine (T1–L5) from 41 donors (16 women and 25 men) of various ages (3–70 years old) after death from traumatic injury. Primary cultures were grown in alpha modified essential medium with fetal bovine serum for 13 days until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP+) were considered to have osteogenic potential. BM nucleated cells were plated (0.5, 1, 2.5, 5, or 10 × 106 cells/10-cm dish) and grown in dexamethasone (Dex), which promotes osteoblastic differentiation. The optimal plating efficiency using BM-derived cells from donors of various ages was 5 × 106 cells/10-cm dish. BM-derived cells were also grown in the absence of Dex at this plating density. At the optimal plating density, in the presence of Dex, the number of CFU-F/ALP+ present in the BM of the younger donors (3–36 years old) was 66.2 ± 9.6 per 106 cells (mean ± SEM), but only 14.7 ± 2.6 per 106 cells in the older donors (41–70 years old). With longer-term culture (4–5 weeks) of these BM cells in medium containing 10 mM β-glycerophosphate and 100 μg/ml ascorbic acid, the extracellular matrix mineralized, a result consistent with mature osteoblastic function. These results demonstrate that the number of MSCs with osteogenic potential (CFU-F/ALP+) decreases early during aging in humans and may be responsible for the age-related reduction in osteoblast number. Our results are particularly important in that the vertebrae are a site of high turnover osteoporosis and, possibly, the earliest site of bone loss in age-related osteoporosis.
Localization of Smads, the TGF-β Family Intracellular Signaling Components During Endochondral Ossification
Authors: Prof. Sakou Takashi , Onishi Toshiyuki , Yamamoto Takuya , Nagamine Tomonori , Sampath T. Kuber , ten Dijke Peter
Published: April 2006

Members of the transforming growth factor-β (TGF-β) family transduce signals from the cell membrane to the nucleus via specific type I and type II receptors and Smad proteins. Smad1 and Smad5 mediate intracellular signaling of bone morphogenetic protein (BMP), whereas Smad2 and Smad3 transduce TGF-β signaling. Smad4 is a common mediator required for both pathways. Smad6 and Smad7 inhibit signaling by members of the TGF-β superfamily. Here, we examined the expression of Smad1 to Smad7 proteins during endochondral ossification of epiphyseal plate of growing rats using immunohistochemical techniques. The expression of Smad proteins was correlated with the expression of TGF-β1 and its receptors, and BMP-2/4 and BMP receptors. The results show that TGF-β1 and BMP-2/4 were actively expressed in chondrocytes that are undergoing proliferation and maturation, which overlaps with expression of their corresponding type I and type II receptors. The Smads, however, exhibited a distinct expression pattern, respectively. For example, Smad1 and Smad5 were highly expressed in proliferating chondrocytes and in those chondrocytes that are undergoing maturation. The TGF-β/activin-restricted Smads were also expressed in a nearly complementary fashion; Smad2 was strongly expressed in proliferating chondrocytes, whereas Smad3 was strongly observed in maturing chondrocytes. Smad4 was broadly expressed in all zones of epiphyseal plate. Inhibitory Smads, Smad6 and Smad7, were strongly expressed in the zone of cartilage that contained mature chondrocytes. Our findings show a colocalization of the pathway-restricted and inhibitory Smads with activating ligands or ligands whose action they antagonize and their receptors in various zones of epiphyseal growth plate, suggesting that TGF-β superfamily Smad signaling pathways plays a morphogenic role during endochondral bone formation.
Hormone Replacement Therapy Prevents Osteoclastic Hyperactivity: A Histomorphometric Study in Early Postmenopausal Women
Authors: Dr. Eriksen Erik Fink , Langdahl Bente , Vesterby Annie , Rungby Jørgen , Kassem Mustapha
Published: April 2006

In a randomized, double blind, clinical prospective trial comprising 35 women treated with either hormone replacement therapy (HRT) (cyclic estradiol/norethisterone acetate) or placebo we performed histomorphometric studies on paired bone biopsies obtained before and after 2 years of treatment. Untreated women developed a progressively more negative balance at individual bone multicellular units (BMUs) (i.e., wall thickness-erosion depth) (2.2 ± 1.7 μm vs. −5.7 ± 1.4 μm; p < 0.01), while women on HRT displayed preservation of bone balance (2.4 ± 2.4 μm vs. 2.5 ± 2.5 μm; NS). No significant differences in wall thickness between the two groups were demonstrable, but the untreated women developed a pronounced increase in erosion depth over 2 years (46.9 ± 1.8 μm vs. 52.0 ± 1.9 μm; p < 0.05), while the HRT group revealed no change (47.8 ± 2.7 μm vs. 44.6 ± 1.7 μm; NS). Furthermore, the placebo group displayed an increased osteoclastic erosion depth (17.8 ± 1.6 μm vs. 25.0 ± 1.7 μm; p < 0.001), compared with unchanged values in the HRT group (20.0 ± 1.6 μm vs. 16.9 ± 1.4 μm/day; NS). While the placebo group revealed a slight increase in volume referent resorption rate (35 ± 8% vs. 38 ± 8%; NS) the HRT group revealed a pronounced decrease (46 ± 8% vs. 28 ± 5%; p < 0.05). No significant changes in marrow star volume (an index of trabecular perforations) were demonstrable in either group. Our results demonstrate that bone remodeling in early postmenopausal women is characterized by progressive osteoclastic hyperactivity, which is reduced by cyclic HRT. This reduction of resorptive activity at the BMU level after HRT seems to precede the reduction in activation frequency demonstrated in previous studies on older postmenopausal women.
Identification and Characterization of a Novel Protein, Periostin, with Restricted Expression to Periosteum and Periodontal Ligament and Increased Expression by Transforming Growth Factor β
Authors: Horiuchi Keisuke , Amizuka Norio , Takeshita Sunao , Takamatsu Hiroyuki , Katsuura Mieko , Ozawa Hidehiro , Toyama Yoshiaki , Bonewald Lynda F. , Kudo Akira
Published: April 2006

We had previously identified the cDNA for a novel protein called osteoblast-specific factor 2 (OSF-2) from an MC3T3-E1 cDNA library using subtraction hybridization and differential screening techniques. Here we describe the localization, regulation, and potential function of this protein. Immunohistochemistry using specific antiserum revealed that in adult mice, the protein is preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure. Based on this observation and the fact that other proteins have been called OSF-2, the protein was renamed “periostin.” Western blot analysis showed that periostin is a disulfide linked 90 kDa protein secreted by osteoblasts and osteoblast-like cell lines. Nucleotide sequence revealed four periostin transcripts that differ in the length of the C-terminal domain, possibly caused by alternative splicing events. Reverse transcription- polymerase chain reaction analysis revealed that these isoforms are not expressed uniformly but are differentially expressed in various cell lines. Both purified periostin protein and the periostin-Fc recombinant protein supported attachment and spreading of MC3T3-E1 cells, and this effect was impaired by antiperiostin antiserum, suggesting that periostin is involved in cell adhesion. The protein is highly homologous to βig-h3, a molecule induced by transforming growth factor β (TGF-β) that promotes the adhesion and spreading of fibroblasts. Because TGF-β has dramatic effects on periosteal expansion and the recruitment of osteoblast precursors, this factor was tested for its effects on periostin expression. By Western blot analysis, TGF-β increased periostin expression in primary osteoblast cells. Together, these data suggest that periostin may play a role in the recruitment and attachment of osteoblast precursors in the periosteum.
On the Estrogen–Bone Relationship and Postmenopausal Bone Loss: A New Model
Authors: Frost Harold M.
Published: April 2006

In this model of estrogen effects on bone, a postulated mediator mechanism in marrow would affect modeling and remodeling only of bone next to or close to it. That mediator mechanism could sense estrogen. In response to that hormone, it would let remodeling of bone next to marrow proceed in its conservation mode. This would minimize losses of that bone and tend to prevent an osteopenia. But acute estrogen deficiency would make that mechanism switch remodeling of bone next to marrow to its disuse mode. Meanwhile, conservation-mode remodeling would continue for haversian and subperiosteal bone. The resulting losses of bone next to marrow would expand marrow cavities, thin cortices, and reduce trabecular bone “mass,” but would not reduce outside bone diameters. That scheme could explain the osteopenia that follows natural or experimental estrogen deficiency in mammalian females. If so, as estrogen secretion rises in girls at puberty they should begin accumulating more bone next to marrow. They do. Also if so, at menopause women should begin to lose that bone. They do. Those effects would exist in addition to known effects of estrogen on existing osteoclasts and osteoblasts.