bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: Comparison with TGF-β1
Authors: Chen Theresa L. , Bates Rebecca L. , Dudley Andrew , Hammonds Glenn R. , Amento Edward P.
Published: February 2010

The biologic effects of recombinant human bone morphogenetic protein-2b (BMP-2b = BMP-4) were studied and compared with transforming growth factor-β1 (TGF-β1) in fetal rat osteoblast-like (ROB) cells. Similar to the effects of TGF-β1, BMP-2b stimulated DNA and collagen synthesis as well as protein accumulation. Unlike TGF-β1, which inhibited alkaline phosphatase activity, BMP-2b enhanced enzyme activity eight-to ninefold over the control level. The present study demonstrates direct actions of BMP-2b on bone-associated cells to stimulate osteogenic phenotypes in vitro and provides a cellular mechanism for the induction of bone formation by BMP-2b in vivo.
Journal of Bone and Mineral Research
Authors: Nussbaum Samuel R. , Potts John T.
Published: February 2010

The two most frequent causes for hypercalcemia are primary hyperparathyroidism and hypercalcemia associated with malignancy. Elevated or inappropriately high PTH serum levels are the hallmark of hyperparathyroidism. Sensitive immunometric assays for the secreted, biologically active, intact parathyroid hormone molecule, PTH-(l-84), employ two populations of region-specific antibodies, take advantage of saturation kinetics rather than competitive binding, and have many technical advantages over conventional radioimmunoassay. Approximately 90% of patients with primary hyperparathyroidism have elevated serum levels of PTH-(l-84) by immunometric assay; the remainder have inappropriately elevated values of PTH for the serum calcium concentration. Clinical correlation studies comparing measurements of PTH using antisera that recognize the carboxyl, midregion, or amino terminus of PTH with PTH levels determined by immunometric assays demonstrate elevated values in equivalent numbers of hyperparathyroid individuals. Immunometric assays for PTH-O-84) have their greatest value in separating patients with hyperparathyroidism from those with hypercalcemia of malignancy. In earlier studies using region-specific antisera, there was virtually always an overlap of serum PTH levels in hyperparathyroidism and hypercalcemia associated with malignancy. In contrast, analysis of results using PTH-(l-84) immunometric assays in several hundred reported patients shows a complete separation of PTH values. Clinical judgment, combined with measurement of PTH in the setting of hypercalcemia, can lead to the diagnosis of hyperparathyroidism with confidence in essentially all patients.
Journal of Bone and Mineral Research
Authors: Marcus Robert
Published: February 2010

One of the common dilemmas in clinical endocrine practice is the management of patients with hyperparathyroidism (HPT) who either cannot or will not undertake definitive surgical treatment. Therapeutic decisions would be simplified by the availability of a nonsurgical approach to control biochemical abnormalities and forestall the consequences of this condition. In the short term, oral phosphorus salts can be safe and effective. For chronic therapy, only estrogens and progestins have been examined in sufficient detail to merit discussion. Estrogens normalize serum and urinary calcium in the majority of older women with HPT. Biochemical control is generally maintained for as long as patients continue treatment. The data support the view that estrogen inhibits the actions of PTH, particularly on bone, but does not reduce the abnormal PTH secretion. Androgenic progestins may also lower serum and urinary calcium in HPT, but current data suggest that normalization of serum calcium levels is more likely to occur with estrogen. Although reduction in urinary calcium excretion should prove effective in preventing nephrolithiasis, it is unclear whether hormone therapy provides protection against fracture. Whether or not hormone replacement is prescribed, nonsurgical management requires a highly committed patient who is willing to undergo extensive and prolonged follow-up.
The osteocalcin propeptide is not secreted in vivo or in vitro
Authors: Gundberg Caren M. , Clough Mary E.
Published: February 2010

Osteocalcin is initially synthesized as an 11 kD molecule consisting of a 23-residue translocation signal peptide that is cleaved during translation, a 26-residue propeptide that targets the protein for gamma-carboxylation, and the 49-residue mature protein. Although the majority of newly synthesized osteocalcin is deposited into bone matrix, a small amount can be detected in blood, and it is this characteristic that has led to its current clinical use as a specific index of osteoblastic activity. Nothing is known, however, about the fate of the propeptide. If osteocalcin and the propeptide are cosecreted, then the concentration of the propeptide could also be useful as a marker of osteoblastic function and, further, may be superior to osteocalcin because it would be unaffected by binding to bone. To test this hypothesis, we synthesized a peptide corresponding to 21 residues of the osteocalcin propeptide from humans and produced a polyclonal antibody to this peptide. Human sera were screened for the presence of the propeptide, and the human osteosarcoma cell line MG-63 was tested for secretion of the propeptide. We could not detect any osteocalcin propeptide in sera from normal adults or individuals with renal failure or primary hyperparathyroidism or those on long-term coumadin therapy. Likewise there was no propeptide present in media from cells grown in the presence of vitamin K, l,25-(OH)2D3, warfarin, or warfarin plus 1,25-(OH)2D3. In contrast, the cell extract, characterized by high-performance liquid chromatography, contained mature osteocalcin, free propeptide, and the proosteocalcin precursor when cells were grown in the presence of 1,25-(OH)2D3 alone. When warfarin was added to the culture, only proosteocalcin precursor was detected in the cell extract. These data establish that the osteocalcin propeptide is not secreted in vitro or in vivo and verify that carboxylation is not required for the conversion of the precursor to osteocalcin.
Transforming growth factor β1 stimulates type II collagen expression in cultured periosteum-derived cells
Authors: Izumi Toshihiro , Scully Sean P. , Heydemann Ahlke , Bolander Mark E.
Published: February 2010

Chondrogenesis can occur during a bone repair process, which is related to several growth factors. Transforming growth factor β1, (TGF-β1,) downregulates the expression of type II collagen by chondrocytes in vitro, but injection of TGF-β1, into the periosteum in vivo increases type II collagen mRNA levels and initiates chondrogenesis.(1) We examined the effect of TGF-β1, on collagen gene expression in a bovine periosteum-derived cell culture system to evaluate its direct effect on the periosteum. Cultured cells expressed alkaline phosphatase and collagen proα1,(I) and proα1(II) mRNAs. A low level of type II collagen synthesis was demonstrated by immunoprecipitation. TGF-β1, had no effect on periosteal cell proliferation. Expression of collagen proα1(I) mRNA did not change with TGF-β1, treatment, but alkaline phosphatase mRNA showed a dose-dependent decrease. Expression of collagen proα1(II) mRNA was stimulated 2.7-fold by TGF-β1. TGF-β1, also caused a 2.6-fold increase in type II collagen synthesis by immunoprecipitation. These findings indicate that TGF-β1 is an enhancer of the expression of the chondrocyte phenotype of the periosteal cells and suggest that TGF-β1, is important in initiating and promoting cartilage formation in vivo.
Comparison of bone mineral content among Japanese, Koreans, and Taiwanese assessed by dual-photon absorptiometry
Authors: Sugimoto Toshitsugu , Tsutsumi Masaharu , Fujii Yoshio , Kawakatsu Mitsuru , Negishi Hirokuni , Lee Myung Chull , Tsai Keh-Sung , Fukase Masaaki , Fujita Takuo
Published: February 2010

Throughout the world the diagnosis and management of osteoporosis currently involves the measurement of bone mineral content. There are, however, no studies comparing bone mineral content among Asian people. This cross-sectional study was designed to quantify spine and femur bone mineral density (BMD) in Japanese and compare BMD among Asian people (Japanese, Koreans, and Taiwanese) using the same model dual-photon system (Norland Model 2600). Following a peak BMD in the third and fourth decades, the Japanese BMD values of the lumbar spine and femoral neck showed a clear decrease (annual loss of 0.99 and 0.74%, respectively) with age in females. On the other hand, Japanese BMD values were stable in males until the fifth decade. There was some decrease in BMD with age after the fifth decade, which was much less obvious than that in females. An age-dependent loss of BMD was clearly observed in Japanese and Korean but not in Taiwanese females. Korean males seemed to have a clearer age-dependent loss of BMD compared to Japanese males. Our findings indicate that differences may exist in the BMD of Asian people and that in addition to the quantitative determination of individual BMD, dual-photon absorptiometry may be useful for the comparison of BMD among different ethnic and cultural groups.
Defective bone formation by hyp mouse bone cells transplanted into normal mice: Evidence in favor of an intrinsic osteoblast defect
Authors: Ecarot Brigitte , Glorieux F.H. , Desbarats M. , Travers R. , Labelle L.
Published: February 2010

The hypophosphatemic (Hyp) mouse is an animal model for human hypophosphatemic vitamin D-resistant rickets. We have reported that bone cells isolated from Hyp mice born to homozygous mutant females produce abnormal bone when transplanted into normal mice. To test whether an environmentally acquired defect of the mutant cells contributed to the impaired bone formation observed in transplants, periostea and osteoblasts from normal and Hyp littermates were transplanted intramuscularly into normal animals. To test more specifically for an hypophosphatemia-induced cell alteration before transplantation, bone cells isolated from phosphate-depleted normal mice were transplanted into normal animals. The bone nodules formed in 2 week transplants were characterized by measuring their osteoid thickness and volume. Impaired bone formation was evidenced in Hyp transplants compared to normal littermate transplants by increased osteoid thickness and volume. In contrast to cells from mutant mice, cells isolated from normal mice with comparable hypophosphatemia produced normal bone. These results indicate that the inability of Hyp osteoblasts to produce normal bone when placed in a normal environment is not the consequence of prior exposure to an altered environment but likely of an intrinsic cellular abnormality. These observations add further support to the concept that the osteoblast is an important target for the Hyp mutation.
Physicochemical action of potassium-magnesium citrate in nephrolithiasis
Authors: Dr. Pak Charles Y. C. , Koenig Karl , Khan Rubina , Haynes Sharon , Padalino Paulette
Published: February 2010

Effect of potassium-magnesium citrate on urinary biochemistry and crystallization of stone-forming salts was compared with that of potassium citrate at same dose of potassium in five normal subjects and five patients with calcium nephrolithiasis. Compared to the placebo phase, urinary pH rose significantly from 6.06 ± 0.27 to 6.48 ± 0.36 (mean ± SD, p < 0.0167) during treatment with potassium citrate (50 mEq/day for 7 days) and to 6.68 ± 0.31 during therapy with potassium-magnesium citrate (containing 49 mEq K, 24.5 mEq Mg, and 73.5 mEq citrate per day). Urinary pH was significantly higher during potassium-magnesium citrate than during potassium citrate therapy. Thus, the amount of undissociated uric acid declined from 118 ± 61 mg/day during the placebo plase to 68 ± 54 mg/day during potassium citrate treatment and, more prominently, to 41 ± 46 mg/day during potassium-magnesium citrate therapy. Urinary magnesium rose significantly from 102 ± 25 to 146 ± 37 mg/day during potassium-magnesium citrate therapy but not during potassium citrate therapy. Urinary citrate rose more prominently during potassium-magnesium citrate therapy (to 1027 ± 478 mg/day from 638 ± 252 mg/day) than during potassium citrate treatment (to 932 ± 297 mg/day). Consequently, urinary saturation (activity product) of calcium oxalate declined significantly (from 1.49 × 10−8 to 1.03 × 10−8 M2) during potassium-magnesium citrate therapy and marginally (to 1.14 × 10−8 M2) during potassium citrate therapy. Moreover, the formation product of calcium phosphate (brushite), indicative of inhibition against spontaneous nucleation, rose more prominently during potassium-magnesium citrate treatment (from 4.62 × 10−7 to 8.78 × 10−7 M2) than during potassium citrate therapy (to 6.08 × 10−7 M2). The inhibition against agglomeration of calcium oxalate increased marginally (p = 0.02) during potassium-magnesium citrate therapy, whereas it did not change significantly during potassium citrate treatment. Thus, potassium-magnesium citrate (at same dose of potassium) is more effective than potassium citrate in inhibiting the crystallization of uric acid and calcium oxalate in urine.
Bisphosphonate Space Measurement in Paget's Disease of Bone Treated with APD
Authors: Stone M. D. , Marshall D. H. , Hosking D. J. , Perkins A. C. , Evans A. J. , Wastie M. L.
Published: February 2010

The bisphosphonate space (BPS) is a quantitative measurement of skeletal uptake of 99mTc-HMDP. We measured BPS in 36 patients with Paget's disease of bone, both before and 6 months after treatment with intravenous APD (disodium pamidronate) infusions. BPS fell after treatment, but proportionally less than serum alkaline phosphatase (ALP) and fasting urinary hydroxyproline/creatinine (HYPRO). There was no dose-response relationship between the dose of APD given and the percentage reduction in ALP and HYPRO at 6 months. Log dose of APD/pretreatment BPS, however, predicted the percentage reduction in ALP and HYPRO very well, and from the respective regression equations it was possible to predict the dose of APD needed to achieve normal values of ALP and HYPRO. In the 10 patients who achieved a normal ALP and 9 patients a normal HYPRO after more than 6 months treatment with APD (range 7–18 months), the predicted dose of APD agreed closely with the actual dose. In conclusion, our data support the idea that log dose APD/pretreatment BPS is a valid predictor of biochemical response in Paget's disease.
Crush fracture syndrome in senile osteoporosis: A nutritional consequence?
Authors: Prof. Dr. Rico H. , Revilla M. , Villa L. F. , Hernandez E. R. , Fernandez J. P.
Published: February 2010

Osteoporosis is a very important age-related health problem. The body's composition changes with age, and these changes are a true reflection of aging and of the individuals's nutritional status. Mineral content changes have been reported in vertebral osteoporosis. Interestingly, enough, there have not been reports on concomitant water, fat, and fat-free mass changes associated with this condition. In this report, changes in the latter parameters are compared between patients with osteoporosis and controls. The four components (water, mineral, fat, and fat-free mass) were found significantly reduced (p < 0.001) in osteoporosis. Serum albumin and protein mass were also reduced (p < 0.001).
An assay system utilizing devitalized bone for assessment of differentiation of osteoclast progenitors
Authors: Amano Shigeru , Hanazawa Shigemasa , Kawata Yasuhiro , Ohta Kazuzo , Kitami Hidekazu , Kitano Shigeo
Published: February 2010

The present study provides a novel assay system to examine the differentiation of osteoclast progenitors on devitalized bone slices. We used the population of bone cells liberated enzymatically from 14-day-old mouse embryonal calvariae as a source of osteoclast progenitors. The analysis of differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts was assessed in terms of the formation of TRAP-positive cells and pits or resorption lacunae, respectively, on devitalized bone slices. Osteoclasts having bone-resorbing activity appeared when the calvarial cell population was cultured in the presence of 1α,25-(OH)2D3 on devitalized bone slices. The resorbing activity increased in a 1α,25-(OH)2D3 dose-related manner. However, calcitonin, a potent inhibitor of differentiation and activation of osteoclast lineage cells, reduced the area of the resorption lacunae in a dose-dependent fashion. The bone-resorbing cells on the bone slices expressed an obvious ruffled border and clear zone, structures specific to mature osteoclasts. These results suggest that osteoclast progenitors in the mouse calvarial population examined differentiated into mature osteoclasts in the presence of 1α,25-(OH)2D3 on devitalized bone slices. Further, using this assay system we assessed the effect of some other osteotropic factors on the differentiation of osteoclast progenitors to mature osteoclasts. IL-1, IL-6, and PTH increased the formation of TRAP-positive cells and pits and the area of resorption lacunae in a dose-dependent fashion. However, prostaglandin E2 was unable to induce the formation of resorption lacunae, although a significant appearance of TRAP-positive cells was observed at a concentration of 200 ng/ml. In contrast, the stimulating effect of these osteotropic factors on the formation of resorption lacunae was inhibited significantly by indomethacin treatment (200 ng/ml). This novel assay system is thus useful for study of the differentiation of osteoclast progenitors to preosteoclasts and mature osteoclasts on devitalized bone slices and also for assessment of the effect of cytokines and hormones on the differentiation of osteoclast progenitors.
Influence of prostaglandins on DNA and matrix synthesis in growth plate chondrocytes
Authors: O'Keefe Regis J. , Crabb Ian D. , Puzas J. Edward , Rosier Randy N.
Published: February 2010

Prostaglandins are locally produced in a number of tissues in response to a variety of stimuli, including local growth factors and systemic hormones. The present investigation characterizes prostaglandin effects on growth plate chondrocytes. Since cyclic adenosine monophosphate (cAMP) may act as a prostaglandin-stimulated second messenger, the effects of prostaglandins A1, D2, E1, E2, F2α, and I2 (10−10-10−6 M) on cAMP levels and thymidine incorporation were evaluated. The stimulation of cAMP and thymidine incorporation by the various prostaglandin metabolites were dose dependent and highly correlated (r = 0.99, p < 0.001). The magnitude of the effect varied but was maximal at 10−6 M for each of the prostaglandins. Prostaglandins of the E series (E1 and E2) were the most potent, causing significant effects at 10−10 M and with maximal 12- and 13-fold increases in DNA synthesis after a 24 h exposure. Prostaglandins D2 and A1 maximally stimulated thymidine incorporation by 4.7- and 3.1-fold but caused significant increases only at 10−8 M. Prostaglandins F2α and I2 were the least stimulatory, producing small but significant increases in thymidine incorporation at 10−6 M (30 and 100% stimulations). A causal relationship between cAMP and thymidine incorporation was further verified by the ability of dibutyryl-cAMP to increase DNA synthesis. Long-term chondrocyte cultures treated continuously with PGE2 demonstrated an increase in cell number, confirming the proliferative effect. Indomethacin did not alter the potent dose-dependent stimulations of chondrocyte DNA synthesis by TGF-β1, basic FGF, or PTH, indicating that these known mitogens act independently of prostaglandin metabolism. PGE2 was further examined for its effects of matrix synthesis. PGE2 inhibited collagen synthesis with a maximal 42% decrease but did not alter noncollagen protein synthesis. In contrast, PGE2 maximally increased sulfate incorporation by 35% and caused a small dose-dependent inhibition in alkaline phosphatase activity. Thus, prostaglandins alter DNA and matrix synthesis in growth plate chondrocytes and may have an important role in chondrocyte metabolism in the growth plate, fracture callus, and other areas of endochondral ossification.
Contribution of vertebral deformities to chronic back pain and disability
Authors: Ettinger Bruce , Black Dennis M. , Nevitt Michael C. , Rundle Amy Chen , Cauley Jane A. , Cummings Steven R. , Genant Harry K.
Published: February 2010

Among 2992 white women aged 65–70 years recruited from population-based listings, we measured radiographic vertebral dimensions of T5–L4 and calculated ratios of heights: anterior/posterior, mid/posterior, and posterior/posterior of either adjacent vertebra. The degree of deformity for each vertebra was analyzed in terms of the number of standard deviations (SD) that ratio differed from the mean ratio calculated for the same vertebral level in this population. We correlated the severity of each woman's worst vertebral deformity with back pain, back disability in six activities of daily living, and height loss since age 25. Only 39.4% of the cohort had no vertebral deformity; 10.2% had a deformity ≥ 4 SD. Vertebral deformities < 4 SD below the mean were not associated with increased back pain, disability, or loss of height. In contrast, women whose deformity was ≥ 4 SD had a 1.9 (95% CI, 1.5–2.4) times higher risk of moderate to severe back pain and a 2.6 (95% CI, 1.7–3.9) times higher risk of disability involving the back; they were also 2.5 (95% CI, 2.0–3.2) times more likely to have lost ≥ 4 cm in height. All three types of vertebral deformity (wedge, end plate, and crush) were equally associated with these outcomes. Multiple deformities < 4 SD did not increase the likelihood of these three outcomes, but multiple deformities >≥ 4 SD tended to be associated with increased back pain, disability, and height loss. This large cross-sectional study suggests that vertebral deformities cause substantial pain, disability, or loss of height only if vertebral height ratios fall 4 SD below the normal mean. Much back pain could not be attributed to vertebral deformities, suggesting other causes.
Intermittent administration of bovine PTH-(1–34) increases serum 1,25-dihydroxyvitamin D concentrations and spinal bone density in senile (23 month) rats
Authors: Mitlak Bruce H. , Williams Daniel C. , Bryant Henry U. , Paul Donald C. , Neer Robert M.
Published: February 2010

We examined the effect of intermittent administration of bovine parathyroid hormone (1–34) (bPTH) on spinal bone mineral content (BMC) and bone mineral density (BMD), serum 1,25-dihydroxyvitamin D concentrations, and serum markers of osteoblast function in senile male and female rats (23 and 24 months of age, respectively). Sexually mature young (3 month) male rats were similarly treated for comparison. bPTH administration increased serum osteocalcin concentrations without changing serum inorganic phosphate or calcium concentrations in either group of old animals. In young animals, PTH administration increased the serum calcium and inorganic phosphate concentrations significantly (p < 0.05), although values remained within the normal range. In the vehicle-treated male rats, serum 1,25-dihydroxyvitamin D concentrations were lower in the senile than in the young animals (18 ± 5 versus 47 ± 6 pg/ml, p < 0.05). PTH administration resulted in significantly increased serum 1,25-dihydroxyvitamin D concentrations in the senile and young male animals (both, p < 0.05) and the final mean serum 1,25-dihydroxy vitamin D concentrations were not statistically different (68 ± 9 versus 85 ± 6 pg/ml respectively; p = NS). Serum 1,25-dihydroxyvitamin D concentrations were significantly (p < 0.05) higher in the PTH-treated senile female rats than the sex-matched, vehicle-treated controls. The pretreatment spinal BMC and BMD as assessed by dual-energy x-ray absorptiometry (DEXA) were significantly higher in the senile male animals than in the young animals. Spinal BMC and BMD decreased in the vehicle-treated senile male rats (p < 0.05) over the 3 weeks of the study despite a gain in weight. bPTH administration prevented this fall in spinal BMC and increased spinal BMD (p < 0.05). Spinal BMC and BMD increased significantly (p < 0.05) in the young vehicle-treated rats, and PTH administration caused a further significant increase in both parameters of bone mass. Spinal BMD was significantly (p < 0.05) higher in the PTH-treated senile female rats than in vehicle-treated controls. These findings demonstrate that intermittent PTH administration increases serum 1,25-dihydroxyvitamin D concentrations of senile animals to the levels of PTH-treated, young, sexually mature animals. In addition, PTH administration arrests bone loss and increases spinal BMD of senile rats.
Effect of dietary phosphate deprivation and supplementation of recipient mice on bone formation by transplanted cells from normal and X-linked hypophosphatemic mice
Authors: Ecarot B. , Glorieux F.H. , Desbarats M. , Travers R. , Labelle L.
Published: February 2010

The hypophosphatemic (Hyp) mouse is the murine homolog for human hypophosphatemic vitamin D-resistant rickets. We previously reported that bone cells isolated from normal and Hyp mice produced abnormal bone when transplanted intramuscularly into mutant mice. To assess the role of hypophosphatemia on bone formation in transplants, normal and Hyp mouse periostea were pair transplanted into control or phosphate (P)-supplemented Hyp mice and into control or P-deprived normal mice. The bone nodules formed in transplants after 2 weeks were characterized by measuring the thickness of the surrounding osteoid seams and the relative osteoid volume. P restriction in normal recipient mice impaired bone formation by transplanted normal cells and aggravated the defective bone formation by Hyp cells. The osteoid thickness and volume remained significantly higher in Hyp transplants than in normal cotransplants, however. P supplementation of Hyp recipient mice normalized bone formation by transplanted normal cells but not by Hyp cells. However, a marked decrease in osteoid thickness and volume was observed in Hyp transplants down to values observed in normal recipient mice. These results indicate that hypophosphatemia is not the only cause of abnormal bone formation in the Hyp mouse but that an osteoblast dysfunction contributes to the bone disease. These observations further support the concept that the osteoblast may be an important target for the Hyp mutation.
Effect of transforming growth factor β on parathyroid hormone receptor binding and cAMP formation in rat osteosarcoma cells
Authors: Seitz Patricia K. , Zhu Bao-Ting , Cooper Cary W.
Published: February 2010

Transforming growth factor β (TGF-β) is now recognized as an important growth regulator and modulator in bone, where it apparently acts in an autocrine or paracrine fashion. In an effort to help elucidate how TGF-β may interact with parathyroid hormone (PTH) to influence bone turnover, we examined the idea that TGF-β might alter the number or affinity of PTH receptors in osteoblastic bone cells, PTH receptor binding was assessed in cultured ROS 17/2.8 cells using [125I]PTHrP-(1–34) as labeled ligand. Specific binding to intact cells was measured in the presence of up to 1 μM unlabeled rPTH-(1–34), and cAMP in cell extracts was determined by RIA. Incubation of ROS cells with 2 ng/ml of TGF-β for the maximally effective time of 3 days increased the number of PTH binding sites (Bmax) by 47 ± 13%, with no change in the Kp (3 nM). TGF-β also increased the intracellular cAMP response to 0.3 nM rPTH-(1–34) (ED50) by 53 ± 22%. Both effects were dose dependent, with 1–4 ng/ml of TGF-β producing maximal effects, and both effects were blocked by the protein synthesis inhibitor cycloheximide (2–5 μM). Since TGF-β induced comparable increases in both PTH binding and cAMP formation, the findings suggest that TGF-β can increase the number of functional PTH receptors in cultured ROS 17/2.8 cells. This effect may reflect an action of TGF-β to slow replication and promote differentiated functions in these cells.
Bone mineral density in elderly men and women: Results from the framingham osteoporosis study
Authors: Hannan Marian T. , Felson David T. , Anderson Jennifer J.
Published: February 2010

Our study investigated bone mineral density of the proximal femur and ultradistal and proximal radius in a population of elderly men and women. The Framingham study started in 1948, following a population-based sample for evaluation of cardiovascular risk factors and events. During the 20th biennial Framingham examination (1988–89) we conducted the Framingham osteoporosis study, measuring bone mineral density in the proximal femur and distal and proximal radius for 1154 study participants. Ages ranged from 68 to 98 years, with a mean age of 76 years. Bone mineral density was measured using Lunar SP2 and DP3 densitometers. This cross-sectional study evaluates mean bone mineral density measurements at each site by 5 year age intervals for men and women, testing for trends in bone density with age. Analyses were repeated adjusting for weight and height. Among the 446 and 708 women, bone mineral density of the femur and bone mineral content of the proximal radius were inversely and significantly related to age in both sexes and were considerably higher in men than women at all sites. The linear decline with age group in our cross-sectional study remained after multivariate adjustment for height and weight. The ultradistal radius showed no significant correlation with age for either sex. There were significant correlations between the bone measurements made at different sites for both men and women (range in r = 0.27–0.89). Cross-sectional curves of bone mineral density with age showed no significant differences in slope between males and females. In this cross-sectional study, we found that increased age was significantly associated with decreased bone mass in a linear and equivalent fashion for both men and women through the elderly years in the proximal femur and proximal radius.
Appendicular bone mineral and a woman's lifetime risk of hip fracture
Authors: Black Dennis M. , Cummings Steven R. , Melton L. Joseph
Published: February 2010

Appendicular bone mass is inversely related to the risk of hip fracture in short-term prospective studies, but hip fractures typically occur about 30 years after menopause. We developed a model that estimates a woman's lifetime risk of hip fracture based on measurement of radial bone mass at age 50 using short-term prospective data relating bone mass to hip fracture, the correlation between bone mass at age 50 and later years, the age-specific incidence of hip fracture and mortality, and prospective data about bone mass and mortality. We estimate that a 50-year-old white woman has a 19% lifetime risk of hip fracture if her radial bone mass is at the 10th percentile for her age and an 11% lifetime risk if her bone mass is at the 90th percentile. Improved measurement techniques that have a higher predictive value for hip fracture in short-term studies could substantially increase this gradient of lifetime risk and therefore be more clinically useful.
Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: An in vitro model of osteoblast development
Authors: Quarles L. Darryl , Yohay Daniel A. , Lever Laura W. , Caton Rashmi , Wenstrup Richard J.
Published: February 2010

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1–9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and β-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. β-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of β-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.
Maturation-associated changes in the cellular composition of mouse calvariae and in the biochemical characteristics of calvarial cells separated into subclasses on percoll density gradients
Authors: Wong G.L. , Ng M.C.
Published: February 2010

The effects of tissue maturation on the cellular composition and biochemical characteristics of bone were studied in neonatal, young adult, and aging mice. Osteoblast subclasses were isolated on Percoll density gradients. Neonatal calvariae consisted almost exclusively of cells banding at low and intermediate buoyant density. High buoyant density cells constituted 5–10% of total cells at 10 days of age but increased to 50–60% by 5 weeks of age. These latter cells were released late during collagenase digestion. This indicates that they arise from the deeper layer of bone. For this reason, we consider them putative osteocytes. We established that constitutive secretion of IGF-I and TGF-β and activities of cellular alkaline phosphatase paralleled those of the tissue of origin in all cell groups and was highest in cells of intermediate buoyant density. These activities declined rapidly after cessation of growth at 5 weeks of age in both bone and isolated cells. Between 5 and 8 weeks of age, the hormonal response to PTH also declined dramatically. The maximum cAMP induced by PTH declined by about 70% in highly responsive cells of intermediate buoyant density and fell to insignificant levels in cells of high buoyant density. We found that a cyclic AMP response to PTH was positively correlated with stimulated secretion of IGF-I by this hormone in cells from animals of all ages. Despite their inability to respond to PTH with increases in cAMP and IGF-I, adult bone cells of high buoyant density continued to respond to PTH with increases in the secretion of TGF-β. The stimulatory effect of PTH on the secretion of TGF-β could not be mimicked by dibutyryl-cAMP or forskolin in any subclass of osteoblasts, in contrast to IGF-I, which was increased in the presence of these compounds. In summary, we show that PTH increases secretion of TGF-β by bone cells. In addition, separation of bone cells from mature mice on the basis of buoyant density permits separation of specific osteoblastic subclasses whose numbers and biochemistry change with maturity. Furthermore, these data suggest that osteocytic cells (1) accumulate in mature bone, (2) undergo loss of selective PTH responses, including cAMP and IGF-I, and (3) retain cAMP-independent PTH responsiveness as shown by increased secretion of TGF-β.
Effects of resistance and endurance exercise on bone mineral status of young women: A randomized exercise intervention trial
Authors: Snow-Harter Christine , Bouxsein Mary L. , Lewis Barbara T. , Carter Dennis R. , Marcus Robert
Published: February 2010

A substantial body of cross-sectional data and a smaller number of intervention trials generally justify optimism that regular physical activity benefits the skeleton. We conducted an 8 month controlled exercise trial in a group of healthy college women (mean age = 19.9 years) who were randomly assigned to a control group or to progressive training in jogging or weight lifting. We measured the following variables: bone mineral density (BMD) of the spine (L2–4) and right proximal femur using dual-energy x-ray absorptiometry, dynamic muscle strength using the 1-RM method, and endurance performance using the 1.5 mile walk/run field test. A total of 31 women completed the 8 month study. For women completing the study, compliance, defined as the percentage of workout sessions attended, was 97% for the runners (range 90–1009/0) and 92% (range 88–100%) for the weight trainers. Body weight increased by approximately 2 kg in all groups (p < 0.05). Weight training was associated with significant increases (p < 0.01) in muscle strength in all muscle groups. Improvement ranged from 10% for the deep back to 54% for the leg. No significant changes in strength scores were observed in the control or running groups. Aerobic performance improved only in the running group (16%, p < 0.01). Lumbar BMD increased (p < 0.05) in both runners (1.3 ± 1.6%) and weight trainers (1.2 ± 1.8%). These results did not differ from each other but were both significantly greater than results in control subjects, in whom bone mineral did not change. No measure of bone mineral at the proximal femur changed significantly in any group. These results demonstrate that 8 months of supervised progressive training in either running or resistance exercise modestly increases lumbar spine mineral in young women.
Expression of mutant α(I)-procollagen in osteoblast and fibroblast cultures from a proband with osteogenesis imperfecta type IV
Authors: Chipman Stewart D. , Shapiro Jay R. , McKinstry Monique B. , Stover Mary Louise , Branson Philip , Rowe David W.
Published: February 2010

This study compares the synthesis of mutant type I collagen in cultured dermal fibroblasts and trabecular osteoblasts that were isolated from a patient with moderately severe osteogenesis imperfecta (type IV). Previous study of this patient's dermal fibroblasts revealed a 2000 dalton deletion located in cyanogen bromide peptide 4 of α2(D-collagen. The phenotype of the bone cell cultures was defined by a 3–4 day logarithmic phase doubling time, predominantly type I collagen production over type III and alkaline phosphatase activity 13.5 times dermal fibroblast levels. The current study revealed that both fibroblasts and osteoblasts synthesized a normal and a shortened α2(I) chain, each as the product of separate alleles. Following pepsin treatment of the procollagens, a shortened α1(I) chain was also seen in both cell types. Cyanogen bromide peptide mapping of osteoblast α-chains demonstrated the same deletions in the cyanogen bromide peptide 4 as observed in the fibroblast cyanogen bromide maps. PAGE analysis of oligonucleotide-specific cDNA that was reverse transcribed from RNA isolated from fibroblasts and osteoblasts also demonstrated the presence of two bands, one the normal size of α2(I) cDNA and a second species that was smaller by 54 base pairs. Sequencing of polymerase chain reaction-amplified cDNA fragments revealed an in-frame deletion of exon 12. This finding was confirmed by the RNase protection method. Genomic DNA sequencing detected a T→G point mutation in the second position of the 5′ splice donor site of intron 12. Therefore, in this patient with osteogenesis imperfecta there was no qualitative alteration in the osteoblast-specific expression of this mutant α2(I)-collagen allele compared to dermal fibroblasts.
Suppression of PTH and decreased action on bone are partially responsible for the low calcemic activity of 22-oxacalcitriol relative to 1,25-(OH)2D3
Authors: Finch Jane L. , Brown Alex J. , Mori Takashi , Nishii Yasuho , Slatopolsky Eduardo
Published: February 2010

We previously showed that OCT, an analog of 1,25-(OH)2D3 with little calcemic activity, can decrease PTH mRNA levels in normal rats and inhibit PTH secretion in cultured bovine parathyroid cells with the same potency as 1,25-(OH)2D3 and that in normal rats fed a normal calcium diet, administration of OCT (500 ng) for 5 days did not increase plasma Ca. Thus, to determine if PTH suppression by OCT contributes to its lack of calcemic activity and to further characterize the effects of OCT on Ca metabolism, we performed several studies in parathyroidectomized (PTX) rats. PTX rats, maintained on a normal diet (0.9% Ca), received daily injections of vehicle, 1,25-(OH)2D3 (200 ng/day), or OCT (200 ng/day) for 6 days. Plasma Ca was measured daily. Plasma Ca in control rats stayed between 6.60 and 7.40 mg/dl, whereas Ca increased to 12.9 ± 0.42 mg/dl in 1,25-(OH)2D3-treated rats and to 9.53 ± 0.35 mg/dl in OCT-treated rats after 6 days. With a Ca-deficient diet, control rats maintained a plasma Ca between 4.25 and 4.60 mg/dl, but Ca increased to 13.7 ± 0.24 mg/dl with 1,25-(OH)2D3 and to 7.29 ± 0.17 mg/dl with OCT. Since the elevation in Ca by OCT was similar with both diets, OCT appears to act primarily on bone. PTX rats were infused with PTH (1.84 μg/kg/day) via an Alzet pump to achieve normal plasma Ca and then treated daily with either vehicle or OCT (200 ng/day). After 6 days, OCT increased serum Ca to 10.7 ± 0.21 mg/dl over a control value of 8.58 ± 0.29 mg/dl. Thus, even when normocalcemic conditions were maintained with a nonsuppressible source of PTH, OCT had calcemic activity. These data demonstrate that OCT has calcemic activity, mainly in the bone, that is masked by its suppressive action on PTH. When compared to 1,25-(OH)2D3, however, the intrinsic calcemic activity of OCT is much less but its effects on PTH secretion are similar. Thus, OCT may be ideal for the treatment of the secondary hyperparathyroidism of chronic renal failure.
Maintenance of cancellous bone connectivity in primary hyperparathyroidism: Trabecular strut analysis
Authors: Parisien May , Mellish Robert W. E. , Silverberg Shonni J. , Shane Elizabeth , Lindsay Robert , Bilezikian John P. , Dempster David W.
Published: February 2010

We previously demonstrated an increase in cancellous bone volume and trabecular number in patients with mild primary hyperparathyroidism (PHPT). To test the hypothesis that this increase is due to preservation of cancellous bone architecture, we conducted a trabecular strut analysis using a new method that measures trabecular connectivity. Iliac crest biopsies from 37 patients with PHPT, 14 men (28–68 years) and 23 women (26–68 years), were examined histomorphometrically and compared to cadaveric samples from 24 age-matched subjects, 17 men and 7 women. Two-dimensional indices of cancellous structure–node number (N.Nd), terminus number (N.Tm), node to node (Nd.Nd), node to terminus (Nd.Tm), and terminus to terminus (Tm.Tm) strut lengths, and total strut length (TSL)–were measured and the ratio of node number to terminus number (N.Nd/N.Tm) calculated. TSL, N.Nd, and Nd.Nd were significantly higher in patients than in controls. TSL and Nd.Nd, but not N.Nd or Nd/Tm, decreased significantly with age in PHPT, indicating that age-related bone loss in PHPT occurs without significant loss of trabecular connectivity. Two-dimensional indices reflecting connectivity or the amount of bone, that is, N.Nd, Nd.Nd, N.Nd/N.Tm, and TSL, correlated positively with cancellous bone volume (BV/TV) and trabecular number (Tb.N) and negatively with trabecular spacing (Tb.Sp) in both PHPT and controls. Trabecular thickness (Tb.Th) correlated positively with Nd.Nd and Tb.N and negatively with Tm.Tm in PHPT but not in controls. The present data show that in PHPT there is not only greater cancellous bone volume and trabecular number but preserved trabecular connectivity as well. The data further support the hypothesis that in PHPT cancellous bone architecture is maintained.
Nitroblue tetrazolium reduction and bone resorption by osteoclasts in vitro inhibited by a manganese-based superoxide dismutase mimic
Authors: Ries William L. , Key Lyndon L. , Rodriguiz Ramona M.
Published: February 2010

Oxygen-derived free radicals are produced by osteoclasts. Oxygen radical formation occurs at the osteoclast/bone surface interface. This location next to bone implies that oxygen radicals, including but not limited to superoxide, are needed for bone resorption. Compounds that scavenge superoxide are being developed as pharmaceutical agents to inhibit the damaging effects of oxygen radical formation on tissues. One such scavenger is the Desferal-manganese complex (DMnC). DMnC reduced the amount of formazan staining produced by the interaction of oxygen radicals with nitroblue tetrazolium (NBT) in both individual mouse calvarial osteoclasts in tissue explants and isolated osteoclasts. As a result of the reduced concentrations of oxygen radicals, DMnC inhibited bone resorption by calvarial explants and isolated osteoclasts. Superoxide dismutase (SOD) inhibited NBT reduction and bone resorption by isolated osteoclasts but to a lesser degree than DMnC. Inhibition of bone resorption in the isolated osteoclast system increased in parallel to the concentration of DMnC in cultures. Desferal without Mn had no effect on bone resorption by isolated osteoclasts. These results support the hypothesis that osteoclasts produce oxygen radicals as part of the process of bone resorption.
Localization of parathyroid hormone-related protein mrna expression in breast cancer and metastatic lesions by in situ hybridization
Authors: Vargas Socorro J. , Gillespie Matthew T. , Powell Gerard J. , Southby Justine , Danks Janine A. , Moseley Jane M. , Martin John T.
Published: February 2010

Parathyroid hormone-related protein (PTHrP) has been identified immunohistochemically in 60% of breast carcinoma(1) and in 92% of breast cancer metastases in bone.(2) To establish whether the localization of the PTHrP antigen reflects protein synthesis and also to investigate the role of PTHrP in metastatic disease, as part of an ongoing study, we used in situ hybridization to study the localization of PTHrP mRNA in a retrospective series of primary breast tumors and their metastatic lesions. Paraffin sections of 17 primary and 26 metastatic lesions, 11 of which were in bone, were available for the study: 10 of the 17 (59%) primary lesions, 8 of 11 (73%) breast cancer metastases to bone, and 3 of 15 (20%) metastases to non-bone sites showed specific localization of PTHrP mRNA. These findings establish that PTHrP is commonly synthesized by primary breast cancers and support previous immunohistochemical studies reporting a higher incidence of PTHrP-positive tumor cells in skeletal metastases than in nonskeletal metastases.