bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and characterization of a conditionally transformed adult human osteoblastic cell line
Authors: Bodine Peter V.N. , Trailsmith Mark , Komm Barry S.
Published: February 2010

Many osteoblastic cell lines are currently in use, but these have limitations either in terms of their relevance to adult human biology and disease or in terms of their suitability for biochemical and molecular analyses. Consequently, we undertook the development of conditionally transformed adult human osteoblastic cell lines. Osteoblasts were obtained from a normal explant cancellous bone chip culture. These cells were infected with adenovirus-ori− SV40 tsA 209, which encodes a temperature-sensitive large T-antigen mutant. Cells immortalized with this virus express a transformed phenotype at the permissive temperature of 34°C but revert to a normal phenotype at the nonpermissive temperature of 40°C. Using this approach, we have isolated several cell clones and describe the characterization of one that was designated HOB-02-C1. Immunocytochemistry revealed that >95% of the cells express the large T-antigen at both temperatures. These cells exponentially proliferate at 34°C with a doubling time of ˜2 days but irreversibly stop dividing at 40°C. However, cell volume increases >2-fold when the cells are maintained for 6 days at the higher temperature. This clone expresses α1 type (I) procollagen mRNA and secretes type I procollagen C-peptide at both temperatures, although the levels were slightly elevated at 40°C. The cell line expresses alkaline phosphatase activity at 34°C, and the basal level of this enzyme increases 2- to 6-fold at 40°C. Alkaline phosphatase activity is induced 4- to 8-fold by 1α,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures, but transforming growth factor-β1 (TGF-β1) suppresses enzyme expression >90% at 40°C. Vitamin D3 also induces a 10-fold increase in osteocalcin secretion when the clone is maintained at 34°C, and this induction is enhanced > 8-fold at 40°C. Parathyroid hormone and forskolin stimulate a 4- to 6-fold increase in the production of intracellular cyclic AMP (cAMP) by the cells at 34°C, and this stimulation is enhanced 2- to 4-fold at 40°C. In contrast, prostaglandin E2 stimulates a 7- to 8-fold increase in cAMP only when the cells are maintained at 34°C. This cell line secretes TGF-β1 and interleukin-6 (IL-6) at 34°C, but only the basal secretion of IL-6 increases 70% at 40°C. Finally, alizarin red-S histochemical staining demonstrates that these cells produce mineralized nodules at both temperatures. In summary, the results of this study indicate that the HOB-02-C1 cells have a mature osteoblastic phenotype. Consequently, this new cell line and others obtained in a similar fashion should be valuable in vitro tools for cellular, biochemical, and molecular studies of adult human osteoblast biology.
Vitamin D receptor gene polymorphisms are not related to bone turnover, rate of bone loss, and bone mass in postmenopausal women: The OFELY study
Authors: Garnero Patrick , Borel Olivier , Sornay-Rendu Elisabeth , Arlot Monique E. , Delmas Pierre D.
Published: February 2010

Vitamin D receptor (VDR) gene polymorphisms have been reported to account for most of the well established genetic influence on bone mineral density (BMD). However, discordant studies have been published and it is still not clear whether VDR genotypes influence bone mass accretion and/or postmenopausal bone loss. In this study, we analyzed VDR gene polymorphisms, i.e., that of BsmI, ApaI, and TaqI restriction enzymes in 268 untreated postmenopausal women 1–26 years postmenopausal. There were 37 BBAA homozygote (absence of BsmI and ApaI restriction sites on both alleles), 55 bbaa homozygote (presence of restriction sites on both alleles), and 176 heterozygotes. At baseline, women between the three genotypes did not differ significantly in age, years since menopause, body mass index (BMI), nor dietary calcium intake. We found no relationship between VDR genotypes and bone turnover assessed by three serum markers of bone formation and three urinary bone resorption markers, nor with BMD measured at the spine, hip, forearm, and whole body by dual-energy X-ray absorptiometry (DXA). Rates of bone loss assessed by repeated DXA measurements over 2 years were highly significant (p = 0.02–0.0001) at all skeletal sites except for the lumbar spine but did not differ between genotypes at any sites either before or after adjustment for potential confounding factors such as years since menopause, BMI, calcium intake, serum 25 hydroxyvitamin D levels, and baseline BMD. When we restricted the analysis to early postmenopausal women, within 10 years of menopause (n = 128), lumbar spine bone loss became significant, but no significant difference between VDR genotypes in the rate of bone loss measured at any site was found. We conclude that VDR genotypes are not predictive of bone turnover, rate of postmenopausal bone loss, and bone mass in either early or late postmenopausal women. In a subgroup of women with a low calcium intake (below 600 mg/day), we also found no significant differences between genotypes in BMD and the rate of bone loss measured at any site, although the sample size (n = 64) may be too small to detect small differences. In conclusion, these data, along with the absence of relationships between VDR gene polymorphisms and peak bone mass that we recently reported, suggest that the determination of VDR genotypes is probably not a useful clinical test for the risk assessment of osteoporosis.
Evidence-based medicine: New priority for an old paradigm
Authors: Feussner John R.
Published: February 2010

As outcomes research gains new importance in the practice of medicine, scientists publishing the results of clinical trials in JBMR must gain new appreciation for the elegant scientific methods and principles of analysis that make such investigations credible. Over the past year, during my tenure as Editor of the Journal, I have become progressively more interested in assuring that the clinical research reported in JBMR is of the highest quality. With this in mind, I asked John Feussner, M.D., a leading researcher in health outcomes investigations, to review recent manuscripts dealing with clinical trials that were published in JBMR and to search for areas in which we can make improvements. In the accompanying editorial, Dr. Feussner reports the result of his review. Not to my surprise, he was able to find errors of commission and omission that have affected several of the reported studies in the Journal. Although concern might be raised based upon reaching this conclusion from a review of a limited number of papers, it is more important to concentrate on the correctable factors that have been raised. Indeed, while many may consider this disquieting, I prefer to believe that the gauntlet has been thrown down and that we will respond to the challenges Dr. Feussner has provided us. In this regard, we must modify our clinical trials methodology and adhere to the principles that he has outlined in the accompanying editorial and allow that these principles guide our review of submitted manuscripts in the future. I believe that those of us in the editorial office are ready to refine the review process, paying newly directed attention to the methodological and analytical plans underlying clinical trials, and thereby move the quality of published clinical research in the Journal to even higher standards. The questions remains, however, Are you, the scientist/author and the scientist/reviewer, ready?
A novel gene expressed in rat ameloblasts codes for proteins with cell binding domains
Authors: Černý Radim , Slaby Ivan , Hammarström Lars , Wurtz Tilmann
Published: February 2010

Two variants of an mRNA sequence are identified that are expressed at high levels in rat ameloblasts during the formation of the enamel matrix. The sequences contain open reading frames for 407 and 324 amino acid residues, respectively. The encoded proteins, which we call amelins, are rich in proline, glycine, leucine, and alanine residues and contain the peptide domain DGEA, an integrin recognition sequence. The sequences coding for the C-terminal 305 amino acid residues, the 3′ nontranslated part, and a microsatellite repeat at the nontranslated 5′ region are identical in both mRNA variants. The remaining 5′ regions contain 338 nucleotides unique to the long variant, 54 common nucleotides, and 46 nucleotides present only in the short variant. Eleven nucleotides have the potential to code for 5 amino acids of both proteins in different reading frames. The reading frame of the longer variant includes codons for a typical N-terminal signal peptide. The amelins are likely to be constituents of the enamel matrix and the only proteins that have so far been implicated in binding interactions between the ameloblast surface and its extracellular matrix. (J Bone Miner Res 1996;11:883-891)
Herbimycin A, a tyrosine kinase inhibitor, impairs hypercalcemia associated with a human squamous cancer producing interleukin-6 in nude mice
Authors: Moriyama Keiji , Williams Paul J. , Niewolna Maria , Dallas Mark R. , Uehara Yoshimasa , Mundy Gregory R. , Yoneda Toshiyuki
Published: February 2010

Interleukin-6 (IL-6) is a multifunctional cytokine that is produced not only by a variety of normal cells but also by cancer cells. IL-6 produced by cancer cells stimulates the proliferation of these cancer cells in an autocrine/paracrine manner and causes paraneoplastic syndromes including hypercalcemia, cachexia, and leukocytosis. We have reported previously that a human oral squamous cancer associated with hypercalcemia produces large amounts of IL-6, that animals bearing this cancer exhibit elevated levels of plasma IL-6, and that neutralizing antibodies to human IL-6 reverse hypercalcemia in tumor-bearing animals, indicating an important role of IL-6 in the hypercalcemia in this model. Because these cancer cells overexpress epidermal growth factor receptors (EGFR) with intrinsic tyrosine kinase (TK) activity similar to many other squamous cancers, we examined the effects of herbimycin A, a tyrosine kinase inhibitor, on IL-6 production and hypercalcemia in animals bearing this cancer to develop a new approach to treat the hypercalcemia associated with malignancy. Intraperitoneal administration (once a day for 2 days) of herbimycin A to cancer-bearing hypercalcemic mice reduced the plasma levels of human IL-6 and impaired the hypercalcemia. During 2-day treatment with herbimycin A, no changes were observed in tumor size. Of interest, plasma levels of mouse, but not human, soluble IL-6 receptors were also elevated. However, herbimycin A showed no effects on plasma levels of mouse soluble IL-6 receptors. Herbimycin A suppressed the tyrosine autophosphorylation of EGFR and IL-6 mRNA expression and production, all of which were stimulated by EGF. The data raise the possibility that TK inhibitors may be potential mechanism-based therapeutic agents for the treatment of hypercalcemia associated with squamous cancers which overexpress EGFR. (J Bone Miner Res 1996;11:905-911)
High extracellular calcium stimulates osteoclast-like cell formation and bone-resorbing activity in the presence of osteoblastic cells
Authors: Kaji Hiroshi , Sugimoto Toshitsugu , Kanatani Masanori , Chihara Kazuo
Published: February 2010

The present study was performed to examine the effect of the high concentration of extracellular calcium ([Ca2+]e) on osteoclast-like cell formation and bone-resorbing activity in the presence or absence of osteoblasts. High [Ca2+]e (3 and 5 mM) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although high [Ca2+]e did not affect the formation of osteoclast-like cells from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in mouse spleen cell cultures. The osteoclast-like cells, newly formed by high [Ca2+]e in the presence of osteoblasts, possessed the ability to form pits on the dentine slices. The conditioned medium from osteoblastic MC3T3-E1 cells treated with high [Ca2+]e (5 mM) significantly increased the formation of osteoclast-like cells from hemopoietic blast cells, compared with the control medium. Dantrolene, an inhibitor of calcium mobilization from the intracellular calcium pool, and indomethacin significantly blocked high [Ca2+]e-stimulated osteoclast-like cell formation in the presence of osteoblasts, although voltage-dependent calcium channel blockers and anti-insulin-like growth factor I antibody did not affect it. High [Ca2+]e, however, significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures, although high [Ca2+]e inhibited bone-resorbing activity in isolated rabbit osteoclasts. An increase in the extracellular magnesium concentration (5 mM) affected neither osteoclast-like cell formation nor bone-resorbing activity. In conclusion, high [Ca2+]e stimulated osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts, presumably via osteoblasts. (J Bone Miner Res 1996;11:912-920)
Progesterone and dexamethasone stimulate proliferation and differentiation of osteoprogenitors and progenitors for adipocytes and macrophages in cell populations derived from adult rat vertebrae
Authors: Ishida Yoichiro , Tertinegg Inka , Heersche Johan N. M.
Published: February 2010

We investigated the effects of the sex hormone progesterone (Prog) and the synthetic glucocorticoid dexamethasone (Dex) on proliferation and differentiation of progenitor cells of osteogenic, adipocytic, and hemopoietic lineages in cell populations derived from explants of adult female rat lumbar vertebrae. The cell populations were obtained by culturing bone explants in plasma clots immersed in α-minimum essential medium plus 10% fetal calf serum (standard medium) and then subculturing the outgrowth cells in standard medium plus 50 μg/ml of ascorbic acid, 5 mM β-glycerophosphate, and with or without Prog or Dex. On day 6 of culture, these populations were analyzed for cAMP responses to parathyroid hormone (PTH), prostaglandin E2 (PGE2), and isoproterenol (IPT). Increases in intracellular cAMP were seen in response to PTH, PGE2, and IPT, and culturing in medium containing Prog increased these responses. At various time periods between days 4–27 of culture, the cultures were evaluated for the presence of bone nodules, alkaline phosphatase (AP)-positive colonies, adipocytes, monocytes, and macrophages. Prog and Dex increased the number of bone nodules and AP-positive colonies. The effect of Prog on bone nodule formation was smaller than that of Dex. In addition, the effect of Dex on bone nodule formation was evident after 10 days of culture, while the Prog-induced effects became significant at days 16–20 of culture. Both hormones also increased the number of Sudan IV-positive colonies (adipocytes), certain types of α-naphthyl butyrate esterase (α-NBE)-positive colonies (monocytes, macrophages, and T-lymphocytes), and ED2-positive colonies (macrophages). Prog-treated cultures contained more colonies of small spindle-shaped α-NBE-positive cells and fewer colonies of small round α-NBE-positive cells when compared with Dex-treated cultures. These data indicate that cell populations derived from adult rat lumbar vertebrae contain, among others, osteoprogenitors and progenitors for adipocytes and macrophages that are stimulated to proliferate and differentiate by Prog and Dex. The data also suggest that the effects of Prog and Dex differ qualitatively and quantitatively. (J Bone Miner Res 1996;11:921–930)
Role of nonenzymatic glycosylation of type I collagen in diabetic osteopenia
Authors: Dr. Katayama Yasuyuki , Akatsu Takuhiko , Yamamoto Michiko , Kugai Nobuo , Nagata Naokazu
Published: February 2010

Formation of advanced glycation end products (AGEs) in extracellular matrix (ECM) is implicated in the development of chronic diabetic complications. However, the involvement of AGEs in diabetic bone disease has not been well established. We have examined whether AGEs are increased in the bone collagen of streptozotocin-induced diabetic rats in vivo and whether glycation of type I collagen affects the functions of osteoblastic cells in vitro. During 12 weeks of observation, AGEs in collagen extracted from the tibiae of diabetic rats increased in a time-dependent manner and were significantly higher than controls at every time point. In vitro, the incubation of collagen with glucose-6-phosphate resulted in a time-dependent increase of AGEs. When osteoblastic cells isolated from fetal rat calvaria were cultured on AGE-modified type I collagen, it dose-dependently inhibited phenotypic expressions of osteoblasts. Among osteoblastic parameters, nodule formation was the most sensitive, being inhibited by ∼70% by the glycation of collagen for only 1 week. Alkaline phosphatase activity and osteocalcin secretion were inhibited by 20–30% and 15–70%, respectively, by the glycation of collagen for 1–5 weeks. These results indicate that AGE-modified collagen affects osteoblastic cell differentiation and function in vitro and suggest that similar changes occurring in vivo may contribute to diabetic osteopenia. (J Bone Miner Res 1996; 11:931–937)
Limited chondro-osteogenesis by recombinant human transforming growth factor-β1 in calvarial defects of adult baboons (papio ursinus)
Authors: Ripamonti U. , Bosch C. , Van Den Heever B. , Duneas N. , Melsen B. , Ebner R.
Published: February 2010

The therapeutic utility of a single application of recombinant human transforming growth factor-β (hTGF-β) has not been previously tested in large osseous wounds in primates. Sixteen calvarial defects, 25 mm in diameter, were prepared in four adult male baboons (Papio ursinus). In each animal, three defects were treated with increasing doses of hTGF-β1 in conjunction with baboon insoluble collagenous bone matrix as carrier (5, 30, and 100 μg of hTGF-β1/g of matrix). The fourth defect was implanted with collagenous matrix without hTGF-β1 as control. Serial undecalcified sections were prepared from the specimens harvested on day 30. Islands of cartilage and endochondral osteogenesis were found in hTGF-β1-treated defects, irrespective of the doses used. Histomorphometry of the defect site showed no significant differences between control and hTGF-β1-treated specimens with regard to bone and osteoid volumes. However, analysis of the regenerated tissue in proximity to the defect margins only showed that, on average, greater amounts of bone formed in specimens that were treated with 5 and 30 μg of hTGF-β1 when compared with controls. This suggests a possible effect on osteoblastic cells originating from the periosteal and endosteal spaces of the severed calvaria. Overall, however, this difference has no therapeutic implications for the healing of large cranial wounds in primates. The present findings indicate that a single application of hTGF-β1, in conjunction with collagenous matrix, results in limited chondro-osteogenesis in defects of membranous bone of adult baboons. (J Bone Miner Res 1996;11:938-945)
Assessment of cancellous bone structure: Comparison of strut analysis, trabecular bone pattern factor, and marrow space star volume
Authors: Croucher Peter I. , Garrahan Nigel J. , Compston Juliet E.
Published: February 2010

Cancellous bone architecture is an important determinant of bone strength. Recently, several approaches to the assessment of bone structure in histological sections have been described; however, no comparative studies of these different methods have been reported. We have developed computerized methods for the simultaneous assessment of several indices of bone structure, including trabecular bone pattern factor, marrow space star volume, node-to-terminus ratio, trabecular number, and trabecular separation. The relationships between these variables has been examined in iliac crest cancellous bone obtained from 41 healthy subjects, 17 male and 24 female, aged 20–80 years (mean 47.9 years). All structural indices assessed showed significant correlations with cancellous bone area (p < 0.0001). Values for trabecular bone pattern factor and marrow space volume were highly correlated (r = 0.789; p < 0.0001). A comparison of indices obtained by strut analysis with trabecular bone pattern factor and marrow space star volume also revealed significant relationships, especially for the terminus-to-terminus strut length (r = 0.704 and r = 0.634, respectively; p < 0.0001) and node to terminus ratio (r = −0.947 and r = −0.788, respectively; p < 0.0001). The node-to-terminus ratio and trabecular bone pattern factor showed significant relationships with age which were independent of sex, cancellous bone area and trabecular width (p < 0.01 and p < 0.005, respectively). Our results demonstrate strong correlations between the different two-dimensional indices of bone structure in cancellous bone from healthy subjects. Trabecular penetration is likely to be an important determinant of all these variables, which may therefore reflect connectivity; however, direct comparison of these methods with three-dimensional techniques is required to establish their true relationship to bone structure. (J Bone Miner Res 1996;11:955-961)
The impact of total enteral nutrition on distraction osteogenesis in a rat model
Authors: Dr. Lumpkin C. K. , Aronson J. , Shen X. C. , Gao G. G. , Skinner R. A. , Badger T. M.
Published: February 2010

Limb lengthening by gradual mechanical distraction, termed distraction osteogenesis (DO), results in new bone formation. We have developed a rat tibial model for DO and have proceeded to study the effects of nutrition on this process. We have combined the intragastric diet delivery system of total enteral nutrition (TEN) with DO in the rat model. The first study was designed to address the weight loss associated with DO in dogs and patients. Rats in the chow + DO group lost 10% body weight over the 20-day distraction period but gradually gained weight back to the preoperative level by the end of the 5th week of the bone consolidation period. In contrast, in the TEN + DO group, a weight gain was recorded during the 20-day distraction phase. A second study was conducted to determine the effects of TEN on the rate and histology of regenerate bone formation. The weight changes replicated those seen in the first study. Standardized radiographs, taken on day 20, revealed increases (p > 0.003) in regenerate bone formation in the TEN group when compared with the chow group. Increased numbers of osteoclasts in the TEN group may indicate an accelerated entry into the remodeling phase of consolidation. Serum IGF-I values, taken at day 20, did not differ between the groups. These results demonstrate that the nutritional support dramatically increased the mineralized bone formed over the 20-day distraction period. (J Bone Miner Res 1996;11:962–969)
Effects of hysterectomy on bone in intact rats, ovariectomized rats, and ovariectomized rats treated with estrogen
Authors: Dr. Goulding Ailsa , Gold E. , Lewis-Barned N. J.
Published: February 2010

To determine whether the uterus plays any role in mediating the ability of estrogen to conserve bone in the rat, eight groups of animals (n = 8) with their skeletons labeled with 45Ca were studied. Rats were ovariectomized (OVX), hysterectomized (Hyst), or given sham operations (Sham) and then pair-fed a low-hydroxyproline casein diet for 4 weeks. The groups were treated orally with 17β-estradiol (E2) or vehicle, and serial measurements of biochemical markers of bone breakdown were made in weeks 1, 2, and 4. The femur density was measured by dual-energy X-ray absorptiometry (DXA), and skeletal calcium and 45Ca content were determined chemically. Final total body calcium values (mg) in the eight treatment groups were (means ± SD): Sham, 2573 ± 179; Sham + E2, 2635 ± 159; Hyst, 2537 ± 151; Hyst + E2, 2410 ± 151; OVX, 2189 ± 146; OVX + E2, 2559 ± 172; OVX/Hyst, 2138 ± 132; and OVX/Hyst + E2, 2460 ± 140. Ovariectomy raised biochemical markers of bone resorption (urinary 45Ca, hydroxyproline, and deoxypyridinoline), lowered DXA bone mineral density, and reduced total body calcium and 45Ca content in both Hyst and Sham-Hyst animals (p < 0.001), whereas E2 treatment prevented these changes. Hysterectomy did not impair the ability of E2 to conserve bone in OVX rats. Thus, we conclude that estrogen-mediated induction of growth factors from uterine tissue does not play an essential role in mediating the bone-conserving actions of estrogen in the rat. (J Bone Miner Res 1996;11:977-983)
Influence of Ca2+ concentration on the clearance and circulating levels of intact and carboxy-terminal iPTH in pentobarbital-anesthetized dogs
Authors: D'Amour P. , Rousseau L. , Rocheleau B. , Pomier-Layrargues G. , Huet P.M.
Published: February 2010

The role of hormone secretion and hormone clearance in the differential control of circulating levels of intact (I-) and carboxy-terminal (C-) immunoreactive parathyroid hormone (iPTH) was evaluated in 18 pentobarbital-anesthetized dogs. Catheters were installed in the aorta, left renal, and hepatic veins for sampling. Hepatic and renal blood flows were calculated from sulfobromophtalein (BSP) and p-aminohippuric acid (PAH) extraction and clearance. I- and C-iPTH were measured during a 1 h of infusion of CaCl2 or Na2EDTA. High-performance liquid chromatography (HPLC) profiles of I- and C-iPTH in and out of the liver and kidney were also obtained. Data on two dogs (one CaCl2 and one Na2EDTA infusion) were pooled for the analysis of one parathyroid function using a four-parameter mathematical model. Results obtained in the basal state and during analysis of the parathyroid function were also compared with those of 24 awakened dogs. Results are means ± SD. Anesthetized dogs had lower levels of Ca2+ (1.29 ± 0.03 vs. 1.34 ± 0.04 mmol/l; p < 0.001) and higher levels of I- (11.5 ± 5.7 vs. 3.0 ± 1.9 pmol/l, p < 0.001) and C-iPTH (52 ± 20.9 vs. 22.8 ± 10.5 pmol/l; p < 0.001) than awakened dogs. Their stimulated (S) and nonsuppressible (NS) I-iPTH levels were increased 2- and 4-fold, respectively, while similar C-iPTH levels rose only 1.35- and 1.75-fold; this caused their S (4.4 ± 0.7 vs. 6.8 ± 1.9; p < 0.001) and NS (24.6 ± 11.8 vs. 49.8 ± 27.5; p < 0.05) C-iPTH/I-iPTH ratios to decrease. This was not explained by different renal clearance rates of I- and C-iPTH since both were similar at ∼10 ml/kg/minute and unaffected by Ca2+ concentration. Clearance of all I- and C-iPTH HPLC molecular forms by the kidney appeared equal. A 50% decrease in the hepatic clearance of I-iPTH to ∼12 ml/kg/minute in pentobarbital-anesthetized dogs, related to a lower hepatic blood flow, explained the higher levels of S and NS I-iPTH in these animals. I-iPTH hepatic clearance was unaffected by Ca2+ concentration. C-iPTH hepatic clearance was much lower at ∼5 ml/kg/minute, abolished by hypercalcemia, and reduced by the influence of anesthesia on hepatic blood flow. This also explained the higher S C-iPTH levels in anesthetized animals. I-PTH(1–84) detected by the C-iPTH assay explained only 37.6% of the hepatic C-iPTH clearance in hypocalcemia and 73.3% in hypercalcemia. Overall, our results indicate that total C-iPTH clearance is about 40.2% that of I-iPTH in hypocalcemia and 41.3% in hypercalcemia. This would only explain a 2.4- to 2.5-fold difference in circulating levels of I- and C-iPTH if secretion rates were equal; the larger difference observed in S and NS C-iPTH/ I-iPTH ratio values is thus mainly explained by different production rates.
Effect of parathyroid hormone on renal calbindin-D28k
Authors: Hemmingsen Claus , Staun Michael , Lewin Ewa , Nielsen Palle K. , Olgaard Klaus
Published: February 2010

The present investigation was conducted to examine the effects of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) on renal calbindin-D28k in rats. Four groups of studies were performed: (1) parathyroidectomy (PTX) or a sham operation followed by infusion of 1,25-dihydroxyvitamin D (1,25[OH]2D) or vehicle; (2) infusions of PTH(1-34), PTH(1-84), 1,25(OH)2D, or vehicle; (3) infusion of PTHrP(1-34), PTHrP (1-86), PTH(1-34), or vehicle; and (4) injections of calcium or vehicle. PTX reduced renal calbindin-D28k levels even when plasma concentrations of 1,25(OH)2D were kept constant by infusion of 1,25(OH)2D. Infusions of PTH(1-34), PTH(1-84), and 1,25(OH)2D all increased renal calbindin-D28k and plasma calcium, whereas PTHrP(1-34) and PTHrP(1-86) increased renal calbindin-D28k before an increase of plasma calcium took place. Hypercalcemia induced by the injection of calcium did not affect the levels of renal calbindin-D28k. The present data suggest that PTH and PTHrP exert a direct effect on renal calbindin-D28k, which is not mediated by changes of 1,25(OH)2D or calcium.
The influence of growth hormone on cancellous and cortical bone of the vertebral body in aged rats
Authors: Andreassen Troels T. , Melsen Flemming , Oxlund Hans
Published: February 2010

The influence of growth hormone administration on cancellous and cortical bone of the vertebral body in 2-year-old male rats has been investigated. All rats were injected for 80 days, then killed. Controls were given saline, and three recombinant human growth hormone (rhGH) injected groups were given either rhGH (2.7 mg/kg/day) for the first 20 or 40 days, followed by saline injection, or rhGH for all 80 days. Tetracycline labeling was performed on days 41 and 69. In all groups given rhGH, an increase in the cortical bone volume was found. In the rhGH 40-day group, single labeling corresponding to injection on day 41 was seen all around the anterior surface of the vertebral body wall (toward the abdominal cavity). In the rhGH 80-day group, double labeling was seen all around the anterior surface of the vertebral body, and a substantial increase in the mineralizing surface/total surface, mineral apposition rate, and mineralized bone formation rate was found. In the cortical bone of the anterior wall, cavities had developed in the rhGH 40- and 80-day groups. In the cancellous bone, no differences in bone volume, bone volume/total volume, or bone surface/bone volume were seen, but in the middle part of the vertebral body a decrease in the mineralizing surface/total surface was found in the rhGH 80-day group. The height of the vertebral body was not influenced by rhGH administration, whereas the transversal and midsaggital diameters were increased in the rhGH 80-day group. The compressive mechanical strength of the vertebral body specimens was increased in the rhGH 80-day group, and this increase most likely could be explained by formation and deposition of cortical bone.
Evidence that interleukin-1, but not interleukin-6, affects costochondral chondrocyte proliferation, differentiation, and matrix synthesis through an autocrine pathway
Authors: Horan J. , Dean D.D. , Kieswetter K. , Schwartz Z. , Boyan B.D.
Published: February 2010

Although the effects of interleukin-1 (IL-1) and interleukin-6 (IL-6) on articular cartilage chondrocytes have been reported, little is known concerning the effects of these cytokines on growth plate chondrocytes. In this study, we examined the effect of IL-1α, IL-1β, and IL-6 on growth plate chondrocyte proliferation, differentiation, and matrix production as a function of cell maturation and examined the ability of these cells to produce IL-1α and IL-1β. Confluent fourth passage cultures of rat costochondral resting zone and growth zone chondrocytes were treated with 0–100 ng/ml of IL-1α, IL-1β, or IL-6 for 24 h and then assayed for [3H]-thymidine incorporation, alkaline phosphatase specific activity, [35S]-sulfate incorporation, and percent collagen production. Neutralizing polyclonal antibodies were used to confirm the specificity of response to each cytokine. Treatment of resting zone cells with IL-1α produced a significant, dose-dependent decrease in [3H]-thymidine incorporation, while similarly treated growth zone cells were unaffected by treatment with this cytokine. IL-1α also stimulated alkaline phosphatase specific activity and inhibited [35S]-sulfate incorporation by resting zone chondrocytes, but had no affect on growth zone chondrocytes. When collagen production was examined, it was observed that IL-1α had a stimulatory affect on growth zone cells but no affect on resting zone cells. When the effect of IL-1β was examined, it was observed that this cytokine inhibited [3H]-thymidine incorporation by resting zone cells and stimulated isotope incorporation in growth zone cells. IL-1β also stimulated alkaline phosphatase specific activity and inhibited [35S]-sulfate incorporation by resting zone chondrocytes but had no affect on growth zone chondrocytes. In contrast to IL-1α, IL-1β stimulated collagen production by resting zone cells but not growth zone cells. IL-6 had no affect on any of the parameters measured in either cell type. When cytokine production was measured, it was found that IL-1α was produced by both cell types, while IL-1β was produced only by resting zone cells. Resting zone cells secreted both IL-1α and IL-1β into the media, but 75% of the total cytokine produced by these cells was retained in the cell layer. In contrast, growth zone cells did not secrete measurable IL-1α into the media. These results suggest that IL-1α and IL-1β target resting zone cells, inducing them to differentiate and acquire a phenotype characteristic of the more mature growth zone cells. Moreover, resting zone chondrocytes produce both IL-1α and IL-1β, suggesting the possibility of an autocrine effect of these cytokines on the cells.
Surface adhesion-mediated regulation of chondrocyte-specific gene expression in the nontransformed RCJ 3.1c5.18 rat chondrocyte cell line
Authors: Mcdougall Skye , Fu Y-Hsien , Lowe Gina N. , Williams Alexia , Polendo Ruben , Benya Paul D. , Iida-Klein Akiko , Fang Meika A. , Hahn Theodore J.
Published: February 2010

Recent evidence suggests that decreased chondrocyte function in osteoarthritis and other articular disorders may be due to chondrocyte dedifferentiation produced by altered regulatory signals from the cartilage extracellular matrix (ECM). However, there are currently no mammalian chondrocytic cell line systems adapted to the study of this process. We therefore examined the effects of ECM growth conditions on markers of differentiated chondrocytic phenotype expression in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line, including type II collagen expression, aggrecan production, link protein gene expression, and parathyroid hormone (PTH) receptor number. RCJ cells grown in monolayer on plastic exhibited a dedifferentiated phenotype characterized by flattened cell morphology, with >80% type I collagen and <5% type II collagen production, as determined by two-dimensional gel mapping electrophoresis of collagen cyanogen bromide peptides. In addition, aggrecan production was low, and link protein mRNA was not expressed at detectable levels. After transfer to growth under minimal attachment conditions on the surface of a composite type I collagen/agarose (0.15%–0.8%) gel (CAG) for 7 days, RCJ cells developed a rounded, chondrocytic morphology and a pattern of differentiated, chondrocytic gene expression, with 79% type II and 8% type I collagen production. Steady-state type I and type II procollagen mRNA levels were altered in parallel with collagen protein expression. In cells grown on CAG, aggrecan production increased 6-fold, and there was a marked increase in both aggrecan core protein and link protein mRNA levels. In addition, maximal PTH-stimulated cAMP generation increased 15-fold in association with an increased PTH receptor number. Therefore, the RCJ chondrocyte cell line is highly sensitive to ECM regulation of chondrocyte-specific gene expression.
Raloxifene analog (LY117018 HCL) ameliorates cyclosporin A—Induced osteopenia in oophorectomized rats
Authors: Bowman A.R. , Sass D.A. , Marshall I. , Ma Y.F. , Liang H. , Jee W.S.S. , Epstein S.
Published: February 2010

Cyclosporin A (CsA) administered to the oophorectomized (Ox) rat exacerbates the high turnover osteopenia associated with estrogen deficiency. 17β-estradiol replacement therapy prevents this bone loss. The aim of this study was to see whether an estrogen-like compound, Raloxifene analog (LY117018 HCL, Ral) could likewise ameliorate CsA-induced osteopenia in the Ox rat. Sixty 6-month-old Sprague-Dawley rats, divided into five groups, underwent oophorectomy. One group acted as a basal group and the others received either vehicle (group B), CsA 15 mg/kg/day (group C), Ral 3 mg/kg/day (group D), or CsA 15 mg/kg/day and Ral 3 mg/kg/day (group E) for 28 days by gavage. A sixth sham operated group of 12 rats received vehicle only (group A). Rats were weighed and bled on days 0, 14, and 28 for measurement of ionized calcium, glucose, osteocalcin (BGP), 17β-estradiol, and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). Tibiae were removed on day 28 for bone histomorphometry after double tetracycline and calcein labeling. Oophorectomy caused a significant gain in weight in groups B and C which was prevented by Ral in groups D and E. Randomized blood glucose levels and 1,25(OH)2D3 levels were elevated in both CsA-treated groups. Blood ionized calcium levels were lower in vehicle (group B) compared with sham (group A) on day 28. Ox (group B) had significantly higher serum BGP levels compared with sham-operated rats. Serum BGP levels were further elevated in group C compared with vehicle and were lowered in both Ral-treated groups to vehicle levels by day 28. Bone histomorphometry revealed a high turnover osteopenia with increased parameters of bone formation and resorption and loss of cancellous bone volume postoophorectomy (group B). CsA (group C) exacerbated the effects of oophorectomy. Ral (group D) completely prevented the high turnover osteopenia caused by oophorectomy and was able to attenuate substantially the effects of CsA in the Ox rat (group E). Ral therapy ameliorated CsA-induced osteopenia in the Ox rat and might prove a useful agent in preventing bone loss in postmenopausal women receiving CsA.
Down-regulation of the receptor for parathyroid hormone (PTH) and PTH-related peptide by PTH in primary fetal rat osteoblasts
Authors: Jongen J. W. J. M. , van Hove E. C. Willemstein , van der Meer J. M. , Bos M. P. , Juppner H. , Segre G. V. , Abou-Samra A. B. , Feyen J. H. M. , Herrmann-Erlee M. P. M.
Published: February 2010

We studied the effects of parathyroid hormone (PTH) on PTH parathyroid hormone related peptide (PTHrP) receptor mRNA level, PTHrP binding, and PTH-stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblasts, derived from fetal rat calvariae (ROB). Cells isolated during 10–70 minutes of collagenase treatment were seeded at a density of 25,000 cells/cm2 and cultured for 4 days. These cells show a fast increase in cAMP production after stimulation for 5 minutes with 20 nM bovine parathyroid hormone(1-34) (bPTH(1-34)). When ROB are incubated with bPTH(1-34) (0.04–40 nM) for 24 h, a dose-dependent decrease of the PTH/PTHrP receptor mRNA level, PTHrP binding, and PTH-stimulated cAMP accumulation can be observed. Pretreatment of ROB with a high concentration of bPTH(1-34) (40 nM) leads within 15 minutes to a decrease in PTH-stimulated cAMP accumulation. However, it takes ≥3 h before a significant decrease in PTH/PTHrP receptor mRNA level can be observed. Also a significant decrease in PTHrP binding is observed after only 4 h of incubation with bPTH(1-34). Compared with bPTH(1-34), pretreatment of ROB with bPTH(3-34) (40 and 100 nM) for 24 h causes smaller decreases in PTH-stimulated cAMP accumulation, PTHrP binding, and in the PTH/PTHrP receptor mRNA level. We investigated the possible involvement of the protein kinase A signaling pathway in the regulation of the PTH/PTHrP receptor mRNA expression. Both forskolin and (Bu)2AMP decreased PTHrP binding and PTH/PTHrP mRNA levels. These observations suggest that chronic activation of the PKA signaling pathway may down-regulate PTH/PTHrP receptor expression and thus hormone responsiveness in “normal” osteoblasts. In short, we found that the decrease of the PTH-stimulated cAMP accumulation after long-term pretreatment with bPTH(1-34) is correlated with both PTH/PTHrP receptor mRNA level and PTHrP binding. These data also suggest that the initial desensitization (<30 minutes) of PTH-stimulated cAMP responsiveness by pretreatment with a high concentration of bPTH(1-34) (40 nM) is not dependent on the number of available PTH/PTHrP receptors. The protein kinase A signaling pathway is involved in the regulation of the PTH/PTHrP receptor, but, regarding the effect of bPTH(3-34), other signaling systems are also involved.
Signal transduction by basic fibroblast growth factor in rat osteoblastic py1a cells
Authors: Hurley Marja M. , Marcello Kristina , Abreu Christine , Kessler Michael
Published: February 2010

Basic fibroblast growth factor (bFGF) is a potent mitogen for bone. In this study, we utilized the clonal rat osteoblastic cell line, Py1a, to examine signal transduction by bFGF and to determine the role of mitogen activated protein kinases (MAPK) and induction of c-fos mRNA in the mitogenic response to bFGF. Stimulation of [3H]thymidine incorporation (TDR) into DNA by bFGF was determined in the presence of phorbol myristate acetate of (PMA) to down-regulate the protein kinase C (PKC) pathway, genistein, an inhibitor of tyrosine kinase and H-7, a PKC inhibitor, bFGF 10−8 M and PMA 10−7 M increased TDR by 242 and 245%, respectively. Treatment with bFGF or PMA for 5 or 30 minutes increased tyrosine phosphorylation of multiple proteins, and immunoblotting with MAPK-specific antibody revealed that two of these bands were the 42 and 44 kD isoforms of MAPK. PMA and bFGF induced c-fos mRNA expression at 30 minutes. Genistein at 10 μg/ml blocked the mitogenic effect of bFGF and partially inhibited the mitogenic effect of PMA. Genistein at 100 μg/ml also blocked both bFGF- and PMA-induced increases in c-fos mRNA. A 24 h pretreatment with PMA at 10−7 M inhibited the mitogenic response, tyrosine phosphorylation of MAPK, and induction of c-fos mRNA subsequent to the addition of PMA, but not bFGF. H-7 at 50 μM blocked bFGF-induced mitogenesis and c-fos induction, but did not inhibit bFGF-induced tyrosine phosphorylation of MAPK. In this study, we show that the signaling pathway of bFGF and PMA are similar in that they both induce tyrosine phosphorylation of MAP kinases and activate c-fos. However, the signaling pathways ultimately diverge in that once the PKC pathway is down-regulated by PMA pretreatment or blocked by the PKC inhibitor H-7, tyrosine phosphorylation of MAP kinase, c-fos induction, and the mitogenic effect of PMA is blocked. In contrast, down-regulation of the PKC pathway inhibits c-fos and the mitogenic response to bFGF, but not bFGF's effects on tyrosine phosphorylation of MAP kinase.
Ketoconazole and phorbol myristate acetate regulate osteoclast precursor fusion in primary murine marrow culture
Authors: Fan Xian , Biskobing Diane M. , Bain Steven , Dr. Rubin Janet
Published: February 2010

Osteoclast formation requires both precursor proliferation and then fusion into a multinuclear cell. These processes can be separated in primary murine marrow culture where osteoclastogenesis is stimulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Here we investigate the regulation of precursor fusion. Ketoconazole, an agent known to inhibit cell fusion, added during the fusion period (days 5–6), dose-dependently inhibited formation of tartrate-resistant acid phosphatase+ (TRAP+) multinucleated cells (TRAP+MNCs), maximally at 62 ± 4% (n = 10). TRAP+MNCs in cultures exposed to 48 h of ketoconazole (1 μM) during fusion had fewer nuclei compared with control (11.7 ± 0.6 vs. 15.1 ± 0.9). This inhibitory effect was completely reversed 24 h after removal of ketoconazole from culture. Phorbol myristate acetate (PMA) stimulated TRAP+MNC formation when given during the last 12 h of culture (2.3 ± 0.2 fold compared with control). This increased formation was unaffected by the addition of hydroxyurea and accompanied by an increase in nuclei per TRAP+MNC (15.5 ± 0.9 vs. 13.1 ± 0.6). Finally, staurosporine decreased TRAP+MNC formation in the presence or absence of PMA, implying that protein kinase C is involved in fusogenic processes. Regulation of fusion appears to be another mechanism by which bone remodeling can be modulated in vivo.
Dietary (n-3) and (n-6) polyunsaturates and acetylsalicylic acid alter ex vivo PGE2 biosynthesis, tissue IGF-I levels, and bone morphometry in chicks
Authors: Watkins Bruce A. , Shen Chwan-L. , Allen Kenneth G. D. , Seifert Mark F.
Published: February 2010

This study examined the effects of dietary (n-6) and (n-3) polyunsaturated fatty acids (PUFA) and acetylsalicylic acid (ASA) on bone ash content, morphometry, fatty acid composition, ex vivo PGE2 biosynthesis, tissue IGF-I concentration, and serum alkaline phosphatase (ALPase) activity in chicks. Newly hatched chicks were fed a semipurified diet containing soybean oil (S) or menhaden oil + safflower oil (M) at 90 g/kg. At 4 days of age, chicks were divided into four equal treatment groups receiving 0 mg (⊖) or 500 mg (⊕) of ASA/kg of diet: S⊖ASA, M⊖ASA, S⊕ASA, and M⊕ASA. Lipid and ASA treatments did not affect bone length, bone ash, or bone mineral content in chicks. Chicks fed M had increased fractional labeled trabecular surface and tissue level bone formation rates, independent of ASA treatment, compared with those given S. A significant fat × ASA interaction effect was found for trabecular bone volume, thickness, separation, and number. Chicks fed S had higher 20:4(n-6) but lower 20:5(n-3) concentrations in liver and bone compared with those given M. Ex vivo PGE2 biosynthesis was higher in liver homogenates and bone organ cultures of chicks fed S compared with the values for those given M at 17 days. ASA treatment decreased ex vivo PGE2 production in liver homogenates and bone organ cultures of chicks, independent of the dietary lipids. Chicks fed ASA had a lower concentration of IGF-I in tibiotarsal bone compared with those not given ASA at 19 days. Serum ALPase activity was higher in chicks given M compared with those fed S, but the values were reversed with ASA feeding. This study demonstrated that both dietary fat and ASA modulated bone PGE2 biosynthesis, and that (n-3) PUFA and fat × ASA interactions altered bone morphometry.
Normal ranges for vertebral height ratios and prevalence of vertebral fracture in hong kong chinese: A comparison with American caucasians
Authors: Lau E. M. C. , Chan H. H. L. , Woo J. , Lin F. , Black D. , Nevitt M. , Leung P. C.
Published: February 2010

We have established the normal ranges for vertebral height ratios and studied the prevalence of vertebral fracture in 481 Chinese women aged 70–79. Our results were compared with those of an American study with a similar methodology. Lateral spine radiographs centered at T8 and L3 were taken, and the anterior, middle, and posterior vertebral heights of each vertebra from T5 to L4 were measured. The means and standard deviations (SDs) of the anterior to posterior, middle to posterior, and posterior to posterior vertebral height ratios were derived after trimming the extreme values from the distribution. The Chinese had smaller vertebral size than the American Caucasians at all levels. Moreover, the anterior to posterior vertebral height ratios were consistently smaller in the Chinese than in the Americans from T10 onward. The means of other vertebral height ratios in the Chinese were similar to the American Caucasians, but the SDs were greater in the Chinese. The prevalence of vertebral fracture was 29% in the Chinese and 25% in the American Caucasians if a cut-off of 3 SD below the mean for vertebral height ratios was used. We conclude that population-specific means and SD should be used for defining vertebral fracture and that the prevalence of vertebral fracture is similar in Hong Kong Chinese and American Caucasians.