bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of prostaglandins E1, E2, and F2α on osteoclast formation in mouse bone marrow cultures
Authors: Collins David A. , Chambers Timothy J.
Published: February 2010

Prostaglandins (PG) act as direct inhibitors of mature osteoclasts, but although resorption-inhibition is also observed initially PG increase bone resorption in organ culture. This suggests that PG influence bone resorption in organ culture through actions on cell types other than mature osteoclasts. We have therefore tested the effects of PG E1, E2, and F2α on the differentiation of osteoclastic phenotype in mouse bone marrow cultures using bone resorption and calcitonin receptors (CTR) as markers of osteoclastic differentiation. We found that PGE2 (10−6–10−9 M) and PGE1 (10−6–10−7 M) induced a significant increase in CTR-positive cell numbers, to levels five to eight times those seen in controls and similar to the number induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone resorption was increased (10−7 M PGE2 and 10−6 M PGE1) in association with the increased CTR-positive cell numbers, suggesting that the PG also induced resorptive function. 1,25-(OH)2D3 increased both the number of CTR-positive cells and the extent of resorption per cell; the additional presence of PG did not affect the number of CTR-positive cells but did reduce bone resorption compared with 1,25-(OH)2D3 alone. PGF2α had no significant effect on CTR-positive cell induction or bone resorption. The results suggest that PGE1 and E2 induce osteoclastic differentiation in mouse bone marrow cultures and inhibit the function of the osteoclasts thus formed. The explanation for this apparently paradoxical behavior may be that osteoclast generation occurs in a different microenvironment from that in which osteoclast function is regulated and that PG production may be inversely modulated at the two sites, in response to a given stimulus, to effect a change in bone resorption.
Amino-terminal sequence homology of two chick kidney mitochondrial proteins immunoisolated with monoclonal antibodies to the cytochrome P450 of 25-hydroxyvitamin D3-1α-hydroxylase
Authors: Moorthy Bhagavatula , Mandel Mark L. , Ghazarian Jacob G.
Published: February 2010

We demonstrate the unique capability of monoclonal antibodies for the specific immunodetection and characterization of two antigenic proteins occurring in normal chick kidney mitochondrial extracts. The antigens were adsorbed to cyanogen bromide-activated Sepharose gel coupled to monoclonal antibodies (MAbs) of the IgM class raised against cytochrome P4501α, which inhibit equally the 25-hydroxyvitamin D3 1α- and 24-hydroxylase catalytic activities (Mandel et al. 1990 J Clin Lab Immunol, in press). The two identified antigenic proteins are polypeptides with apparent molecular weights of 57,000 and 55,000 daltons. The 1α-hydroxylase cytochrome P450 has been shown to have a molecular weight of 57,000 daltons (Mandel et al. 1990 Biochim Biophys Acta 1034:239–246). The optimal antigen: Gel ratio for maximal antigen binding as cytochrome P450 heme, which was determined spectrally, was found to be 1.3 nmol cytochrome P450 per g MAb- coupled Sepharose. At this ratio the total binding capacity of the gel was 1 nmol cytochrome P450 per g Sepharose. The two polypeptides were desorbed with 0.1% Emulgen 911 in 25% glycerol at pH 3.0 and separated by SDS-gel electrophoresis. The amino-terminal sequences of the two antigens were determined by automated Edman degradation with an on-line analyzer of PTH derivatives. The sequences in both antigens were 100% homologous. Complete amino acid composition analysis also revealed that their amino acid compositions were highly similar. These findings suggest that the smaller protein may be a proteolytic cleavage product of the 1α-hydroxylase P450 cytochrome and may represent a putative 24-hydroxylase antigen.
Classification of vertebral fractures
Authors: Eastell Richard , Cedel Sandra L. , Wahner Heinz W. , Riggs B. Lawrence , Dr. Melton L. Joseph
Published: February 2010

Although it is a cardinal feature of involutional osteoporosis, there is often disagreement on what constitutes a vertebral fracture. We measured vertebrae T4-L5 in 52 healthy women to develop a normal range (mean ± 3 SD) for vertebral shape and used these data to assess the prevalence of vertebral fractures. We classified vertebral fractures by type of deformity (wedge, biconcavity, or compression) and further by the degree of deformity (grades 1 and 2). In 195 postmenopausal women who were an age-stratified random sample of the Rochester population (ages 47–94), 40 (21%) had vertebral fractures (mean, 2 per person). There was a similar number of compression and wedge fractures, and grade 2 fractures were as common as grade 1. In a referral sample of 74 women with suspected osteoporosis, 62 (84%) had vertebral fractures (mean, 3.3 per person). Wedge fractures were most common, and grade 2 fractures were more common than grade 1. The distribution of type and grade of fractures differed between the two patient groups (P < 0.01). Bone mineral density of the lumbar spine was related to mean fracture grade (r = −0.33, P < 0.05) and to fracture number (r = −0.57, P < 0.001) but not to fracture type. We conclude that a comprehensive approach is required in describing vertebral fractures. Using this approach we found distortion in the fracture characteristics of women referred to an osteoporosis clinic compared to women in the community.
Enzymatic isolation of chondrocytes from immature rabbit articular cartilage and maintenance of phenotypic expression in culture
Authors: Glade Michael J. , Kanwar Yasphal S. , Hefley Thomas J.
Published: February 2010

The studies included here identify factors affecting cartilage digestion by crude bacterial collagenase (cCGN) and describe a cartilage digestion medium that maximizes both tissue digestion rate and viable cell yield. The basal digestion medium contained 100 mM NaCl, 3 mM K2HPO4, 1 mM CaCl2, 1 mM MgSO4, 10 mM NaHCO3, 60 mM sorbitol, 5 mg/ml of dextrose, 1 mg/ml of albumin, and 2 mg/ml of cCGN in 25 mM HEPES at pH 7.2. Approximately 45% of articular cartilage tissue was digested in this basal medium in 6 h at 37°C, yielding 6.8 × 106 viable cells per g tissue digested. The addition of 30 μM tosyllysylchloromethane (TLCM) increased the fraction of tissue digested in 6 h to 68% (p < 0.05) and doubled viable cell yields to 13.6 × 106 per g tissue digested (p < 0.05). Withholding Mg, decreasing NaCl to 70 mM, and adding 30 mM KCl increased fractional tissue digestion to 81% (p < 0.01) and doubled viable cell yield yet again (to 29.9 × 106 viable cells per g tissue digested). Supplementation with TLCM increased the rate of cartilage digestion and the yield of viable cells regardless of cCGN source or lot. Additional trypsin (0.25%) inhibited tissue digestion and decreased cell yield; this effect was reversible with the addition of TLCM. The cartilage digestion medium developed in these studies (low Mg with added K and TLCM) was very effective in digesting articular, scapular, rib, and growth plate cartilage, as well as in yielding a large number of viable chondrocytes. These cells grew well in culture and maintained their chondrocytic characteristics, secreting predominantly type II collagen and large macromolecular forms of chondroitin sulfate-rich proteoglycans.
Spine deformity index (SDI) versus other objective procedures of vertebral fracture identification in patients with osteoporosis: A comparative study
Authors: Sauer Peter , Leidig Gudrun , Minne Helmut W. , Duckeck Günter , Schwarz Wolfgang , Siromachkostov Lubomir , Ziegler Reinhard
Published: February 2010

Radiologic identification of vertebral fractures is most important in the diagnosis and monitoring of patients with spinal osteoporosis. Different methods, using vertebral height measurements for fracture identification, have therefore been developed. We compared four methods for fracture identification in spinal x-rays of 62 female patients with primary osteoporosis. The methods of Hedlund and Gallagher,(1) Melton et al.,(2) and Davies et al.(3) are based on the ratio of heights within one vertebra or of the height ratios of adjacent vertebrae; all three methods result in counting the number of vertebral fractures. The fourth method of Minne et al.(4) relates anterior, middle and posterior heights of the vertebrae between T5 and L5 to the respective heights of T4. The relative vertebral heights of patients with osteoporosis are compared to the respective relative heights (anterior, middle, and posterior) of normal subjects (T5-L5). This allows the identification of fractured vertebrae, as well as a quantification of the extent of deformation due to these fractures (spine deformity index, SDI).

The same measurement data of 62 spinal x-rays of anterior, middle, and posterior heights between T4 and L5 were used to detect vertebral fractures by the four different methods. Correlation between the number of identified fractures by the different methods ranged between r = 0.56 and 0.83. On the other hand, we found a remarkable difference in the mean number of identified fractures and a discrepancy in the identification of single vertebrae as fractured or not. All four methods revealed an accumulation of fractures in the midthoracic area and in the region of transition from thoracic to lumbar spine. Vertebral fractures as identified by SDI were not detected by the other three methods in 12–29% of the cases, even if vertebral height reduction was more than 6 mm. The reliability of each method was examined by the determination of “decreasing” number of fractures during follow-up. A decrease in the number of fractures was found in about 25% patients, if using the three methods(1–3) that count only the number of fractures. We obtained a 3.6% decrease in the number of fractures using the fourth method.(4) Furthermore, the decrease in SDI values in follow-up was within the range of variance.

We therefore believe that SDI and related procedures are reliable in quantifying spinal osteoporosis and monitoring during follow-up.
Assessment by reflection ultrasound method of the effect of intermittent slow-release sodium fluoride-calcium citrate therapy on material strength of bone
Authors: Dr. Zerwekh Joseph E. , Antich Peter P. , Sakhaee Khashayar , Gonzales Jerome , Gottschalk Frank , Pak Charles Y. C.
Published: February 2010

It has been suggested that fluoride therapy, while increasing bone mass, produces bone with inferior mechanical properties. In the present report this hypothesis was tested using a novel reflection ultrasound technique. Transiliac crest bone biopsies were obtained from 16 patients with osteoporosis and vertebral compression fractures (12 women and 4 men, mean age 56 years) before and after approximately 2 years of intermittent slow-release sodium fluoride therapy (25 mg twice a day) combined with continuous calcium citrate supplementation. Samples were analyzed by a reflection ultrasound method, which analyzes ultrasound velocity with a sample site resolution of 200 μm and thus provides a measure of the mechanical property of single trabeculae (material). For the group, mean fractional change in velocity increased 6.1 ± 2.3% (SEM) from a mean value of 3303 ± 80 to 3484 ± 55 m/s (p = 0.028). A total of 13 patients (81%) demonstrated higher velocities after treatment. Thus reflection ultrasound analysis of bone appears to provide a sensitive means of assessing changes in the material property of bone. Furthermore, these results suggest that the treatment regimen utilized in these patients improves strength of bone at the material or trabecular level largely independently of change in bone mass. The combination therapy also increased spinal (L2-L4) bone density for the group as assessed by dual-photon absorptiometry (5.3 ± 2.0%). There was no significant correlation between the change in ultrasound velocity and bone density (r = 0.0026, p = 0.996). This finding supports the suggestion that the strength of whole vertebrae (structure) could also have been increased following treatment as a result of a rise in bone mass, possibly from an increased number of trabeculae. Taken together the results support the possibility that intermittent slow-release sodium fluoride treatment in combination with calcium citrate can enhance the strength of whole bone (spine) by improving the microscopic or material property of bone, as well as by increasing bone mass.
Iron as a possible aggravating factor for osteopathy in itai-itai disease, a disease associated with chronic cadmium intoxication
Authors: Noda Makoto , Yasuda Masanori , Kitagawa Masanobu
Published: February 2010

Itai-itai disease is thought to be the result of chronic cadmium (Cd) intoxication. We examined 23 autopsy cases of itai-itai disease and 18 cases of sudden death as controls. Urine and blood samples from 10 patients were collected before they died and revealed the presence of severe anemia and renal tubular injuries. Undecalcified sections of iliac bone were stained with Aluminon reagent, an ammonium salt of aurintricarboxylic acid, and Prussian blue reagent in all cases of itai-itai disease. These two reagents reacted at the same mineralization fronts. X-ray microanalysis revealed the presence of iron at mineralization fronts in itai-itai disease. Five patients showed evidence of hemosiderosis in the liver, spleen, and pancreas, probably as a result of post transfusion iron overload. Renal calculi and calcified aortic walls were also stained with Prussian blue reagent in several patients. Neither ferritin nor transferrin were visualized at mineralization fronts in itai-itai disease by immunohistochemical staining. These results suggest that iron is bound to calcium or to calcium phosphate by a physicochemical reaction. A marked osteomalacia was observed in 10 cases of itai-itai disease by histomorphometry. Regression analyses of data from cases of itai-itai disease suggested that an Aluminon-positive metal inhibited mineralization and that renal tubules were injured. Since bone Cd levels were increased in itai-itai disease, it is likely that renal tubules were injured by exposure to Cd. Therefore, stainable bone iron is another possible aggravating factor for osteopathy in itai-itai disease, and a synergistic effect between iron and Cd on mineralization is proposed.
Coexistence of reduced function of natural killer cells and osteoclasts in two distinct osteopetrotic mutations in the rat
Authors: Dr. Popoff Steven N. , Jackson Mary E. , Koevary Steven B. , Marks Sandy C.
Published: February 2010

Recent evidence suggesting that immune cells and their products (cytokines) play an important role in the regulation of skeletal development and function, particularly of the osteoclast, implies that immune cell dysfunction may be involved in the pathogenesis of certain skeletal disorders. The mammalian osteopetroses are a pathogenetically heterogeneous group of skeletal disorders characterized by skeletal sclerosis resulting from reduced osteoclast-mediated bone resorption. Using a 51Cr-release microcytotoxicity assay we demonstrated that splenic natural killer (NK) cell activity was significantly reduced in two distinctly different osteopetrotic mutations in the rat, osteopetrosis (op) and toothless (tl). To determine whether this reduction in NK cell-mediated cytotoxicity is caused by decreased cell number and/or function in these osteopetrotic mutants, we quantitated NK cells by analyzing mononuclear cell suspensions labeled for two-color fluorescence with OX8 and OX 19 monoclonal antibodies in a fluorescence-activated cell sorter. Flow cytometry of these double-labeled cells revealed that the percentage of NK cells (OX8+/OX19− subset) in op and tl spleens was not significantly different from that of normal spleens. These results suggest that NK cells in these osteopetrotic mutants are functionally defective. Thus aberrations in osteoclast and NK cell function coexist in these mutations, and their developmental relationships deserve further study.
Interspecies comparison of renal cortical receptors for parathyroid hormone and parathyroid hormone-related protein
Authors: Orloff John J. , Goumas Douglas , Wu Terence L. , Stewart Andrew F.
Published: February 2010

Parathyroid hormone (PTH) and PTH-related proteins (PTHrP) interact with a common receptor in rat bone cells and in canine renal membranes with similar affinity, but PTHrP are substantially less potent than PTH in stimulating adenylate cyclase in canine renal membranes; in contrast, PTH and PTHrP are equipotent in stimulating adenylate cyclase in rat bone cells. This discrepancy has been largely viewed as reflecting differences in the relative efficiency of signal transduction for PTHrP between bone and kidney assay systems. To test the alternative (but not mutually exclusive) hypothesis that these differences could reflect interspecies differences in PTH receptors, we have characterized the bioactivity of amino-terminal PTHrP and PTH in rat and human renal cortical membranes (RCM) and compared them to results we previously reported in canine RCM.

The stability of PTH and PTHrP peptides under binding and adenylate cyclase assay conditions was greater than 80% for each species. Competitive inhibition of [125I](Tyr36)hPTHrP-(1–36)NH2 binding to rat RCM by bPTH-(1–34) and (Tyr36)hPTHrP-(1–36)NH2 yielded nearly identical binding dissociation constants (3.7 and 3.6 nM, respectively), and binding to human RCM demonstrated slightly greater potency for PTHrP (0.5 nM) than for PTH (0.9 nM). Similarly, adenylate cyclase stimulating activity was equivalent for the two peptides in rat RCM, but PTHrP was twofold more potent than PTH in human RCM. Covalent photoaffinity labeling of protease-protected rat RCM yielded an apparent 80 kD receptor protein, and crosslinking of human RCM labeled an 85 kD receptor, indistinguishable in size from the canine renal PTH receptor.

We conclude that rat, canine, and human renal cortical PTH receptors exhibit species specificity. The previously observed differences between rat bone cells and canine renal membranes in the efficiency of signal transduction by PTHrP may be explained, at least in part, by these species differences.
Effects of voluntary exercise on bone mineral content in rats
Authors: Newhall Katherine M. , Rodnick Kenneth J. , van der Meulen Marjoleine C. , Carter Dennis R. , Marcus Robert
Published: February 2010

We used a voluntary running model to explore the relationship between average daily running distance and bone mineral status of rats. A total of 60 male Sprague-Dawley rats were randomly assigned at 6 weeks of age to a sedentary control group (n = 22) or to a group with unlimited access to a running wheel (n = 38). The running distance of exercising rats was monitored daily, and steady-state running levels ranged from 3.2 to 18.1 km/day. At the end of the experimental period, femora and tibiae were dissected and bone mineral content (BMC, g/cm) and bone mineral density (BMD, g/cm2) were measured by single-photon absorptiometry. Cross-sectional morphometry was examined by taking a transverse section of the femoral middiaphysis. Hindlimb percentage fat was significantly higher in controls than in runners (20.0 ± 1.2 versus 11.1 ± 0.6, p < 0.001), and soleus mass was greater in runners than in controls (371 ± 8.1 versus 320 ± 0.8 mg, p < 0.001). Femoral and tibial lengths, weights, and volumes were significantly higher in runners than in controls (p < 0.005). BMC and BMD were higher in runners than in controls at all sites apart from the distal femur. Cross-sectional areas at the femoral midshaft were greater in running rats than in sedentary controls (6.26 ± 0.1 versus 5.45 ± 0.3 mm2, p < 0.02), as was the polar moment of inertia (15.6 ± 0.6 versus 12.7 ± 0.2 mm4, p < 0.05). No positive correlation was found between distance run and BMC, BMD, cross-sectional area, or polar moment of inertia. Conversely, there was a highly significant positive correlation between body weight and most bone measurements and a negative correlation between distance run and body weight and femoral and tibial wet weight and volume. Partial correlations correcting for differences in body weight suggested that exercise level was not an independent predictor of BMC. In summary, voluntary running activity markedly increased femoral and tibial BMC in young male rats compared to weight-matched sedentary controls. However, within the exercising group substantially higher running distances produced no further increase in BMC.
Lack of a direct effect of estrogen on proliferation and differentiation of normal human osteoblast-like cells
Authors: Keeting Philip E. , Scott Robert E. , Colvard Douglas S. , Han In K. , Spelsberg Thomas C. , Riggs B. Lawrence
Published: February 2010

Although osteoblasts contain estrogen receptors, it is unclear whether estrogen has direct effects on osteoblast proliferation and differentiation. We evaluated the effects of 17β-estradiol treatment (1 pM to 10 nM) on the proliferation and differentiation of cultured normal adult human cells that expressed many of the phenotypic characteristics and hormonal sensitivities of mature osteoblasts (hOB cells). Treatment of hOB cells with estradiol for as long as 144 h did not affect the rate of DNA synthesis and had minimal, if any, effects on differentiated function. Whereas alkaline phosphatase activity was increased by nearly twofold (P < 0.01) when the hOB cells were treated with 1 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], treatment with estradiol had no effect when given alone and did not affect the cells' response to 1,25-(OH)2D3. Similarly, the release of bone gla protein (BGP, osteocalcin) was induced by treatment with 1,25-(OH)2D3 (P < 0.05), but estradiol treatment did not affect this response. Cellular levels of mRNA for alkaline phosphatase and BGP were not altered by estradiol treatment. We conclude that estradiol treatment does not have major effects on the growth or differentiation of cultured hOB cells. These results are consistent with previous observations in vivo that indicate estrogen acts principally to decrease bone resorption, not to modulate its formation.
Immunoaffinity purification of parathyroid hormone-related protein from bovine milk and human keratinocyte-conditioned medium
Authors: Stewart Andrew F. , Wu Terrence L. , Insogna Karl L. , Milstone Leonard M. , Burtis William J.
Published: February 2010

Parathyroid hormone-related proteins (PRHrP) are a novel family of proteins that appear to be responsible for humoral hypercalcemia of malignancy. Although PTHrP derived from human tumors have been purified and their N-terminal amino acid sequence determined, and although the structure of the PTHrP gene and its alternatively spliced mRNA transcripts have been defined, the secretory and circulating form(s) of the protein are unknown. Purification of PTHrP in the past has been difficult, requiring multiple chromatographic steps and months or years to complete.

To define naturally occurring PTHrP species we have developed a rapid and efficient immunoaffinity purification method. Bovine milk (250 ml) and human keratinocyte-conditioned medium (3000 ml) were affinity purified using a 300 μl affinity-purified polyclonal anti-PTHrP-(1–36) antibody column and a single RP-HPLC step. Purification required only 7–10 days and yielded a 3–4% recovery. Quantities of PTHrP sufficient for silver-stained SDS-PAGE, Western analysis, and N-terminal amino acid sequence were obtained.

In contrast to conventional purification schemes, affinity purification of PTHrP is rapid and efficient and can be applied to biologic samples that contain PTHrP in low abundance. These methods can be applied to the purification and characterization of the as yet undefined secretory and circulating forms of PTHrP.
Effect of chronic carbonic anhydrase inhibitor therapy on bone mineral density in white women
Authors: Dr. Pierce William M. , Nardin George F. , Fuqua Margaret F. , Sabah-Maren Emily , Stern Steven H.
Published: February 2010

A limited, dual-photon absorptiometry, single-center study of bone mineral density (BMD) was conducted on white female glaucoma subjects who were chronic users of the carbonic anhydrase (CA) inhibitors acetazolamide (Az) or methazolamide (Mz). In postmenopausal subjects long-term (>4 years) CA inhibitor use was associated with a bone-sparing effect as judged by spinal BMD in comparison to controls matched for age, sex, weight, and ethnic group or in comparison to a national normative data base. Short-term (0-2 years) postmenopausal CA inhibitor users and premenopausal subjects using CA inhibitors showed no sparing of spinal BMD. Femoral neck BMD was not affected by CA inhibitor therapy in any of the groups. This study supports a proposed role for carbonic anhydrase in human bone resorption and suggests a possible future utility for carbonic anhydrase inhibitors in prophylaxis or management of primary involutional osteoporosis. Future studies are necessary to verify and expand these findings, assess the effects of CA inhibitors on bone mechanical competence, and further develop CA inhibitors with some specificity for bone.
Isolation of avian osteoclasts: Improved techniques to preferentially purify viable cells
Authors: Oursler Merry Jo , Collin-Osdoby Patricia , Anderson Fred , Li Ling , Webber David , Dr. Osdoby Philip
Published: February 2010

Among the many different methods that have been used to obtain and study isolated osteoclasts from a variety of species, the egg-laying hen maintained on a low-calcium diet has proven to be one of the richest sources of relatively large numbers of osteoclasts. However, recent reports and our own observations indicate that only a very small proportion of the osteoclasts harvested by such methods are viable.(1) The difficulty in obtaining large numbers of viable osteoclasts has restricted studies of osteoclast function and regulation, and so new isolation methods were sought. This report describes an osteoclast isolation procedure designed to substantially enrich for large numbers of viable authentic osteoclasts. Size and cell density differences between osteoclasts and contaminating mononuclear cells have been exploited in developing the methods for osteoclast enrichment. Sequential nonenzymatic and enzymatic procedures, followed by cell density separations, have yielded three populations of osteoclasts derived from chick hatchlings maintained on a low-calcium diet. A corresponding decrease in bone-associated osteoclasts during the sequential isolation scheme has been monitored using an osteoclast-directed monoclonal antibody, 121F. The first two populations contain 40% osteoclasts, which are predominantly (>99%) nonviable, but the third population contains 8-fold more viable osteoclasts, effectively increasing the proportion of viable osteoclasts more than 25-fold in comparison with the first two populations. The osteoclast-like nature of the isolated viable population 3 cells was established by demonstrating ruffled border formation, possession of the 121F monoclonal antibody-reactive osteoclast antigen, bone particle resorption activity, and resorption pit formation on cortical bone slices revealed by transmission and scanning electron microscopy.
Estrogen and diphosphonate treatment provide long-term protection against osteopenia in ovariectomized rats
Authors: Dr. Wronski T.J. , Yen C.-F. , Scott K.S.
Published: February 2010

The goal of this study is to determine whether the previously observed, short-term protective effect of estrogen and diphosphonate compounds against osteopenia in ovariectomized (OVX) rats can be maintained for an entire year. Sham-operated control and OVX rats were treated intermittently with vehicle alone, estrogen, or the diphosphonate compounds etidronate disodium (EHDP) and risedronate (NE-58095) for 360 days after surgery. Their proximal tibiae and first lumbar vertebrae were processed undecalcified for quantitative bone histomorphometry. Both skeletal sites in vehicle-treated OVX rats were characterized by decreased cancellous bone volume and increases in most cellular and fluorochrome-based indices of bone formation and resorption. Treatment of OVX rats with estrogen or diphosphonate compounds depressed bone turnover and provided nearly complete protection against cancellous bone loss. Long-term EHDP treatment induced a moderate mineralization defect, as indicated by increased absolute osteoid volume and a high proportion of osteoid surfaces devoid of adjacent osteoblasts. In contrast, NE-58095 had minimal effects on bone mineralization. These findings indicate that diphosphonate compounds and estrogen provide long-term protection against tibial and vertebral osteopenia in OVX rats. They further indicate that diphosphonate compounds merit consideration as an alternative to estrogen for the prevention of postmenopausal bone loss.
Inhibition of bone resorption by inorganic phosphate is mediated by both reduced osteoclast formation and decreased activity of mature osteoclasts
Authors: Yates A. John , Oreffo Richard O.C. , Mayor Kevin , Mundy Gregory R.
Published: February 2010

High concentrations of inorganic phosphate (Pi) are known to inhibit bone resorption, although the mechanism(s) underlying this effect is unclear. To investigate whether Pi can inhibit the formation of osteoclasts we studied the effects of changes in Pi concentration between 1 and 4 mM on osteoclast-like cell formation in 1 week cultures of mouse bone marrow. Osteoclast-like cells were identified by multinuclearity, positive staining for tartrate-resistant acid phosphatase (TRAP), and contraction in response to calcitonin. Increasing concentrations of Pi inhibited formation of these cells in a dose-dependent manner. To study effects of Pi on the bone-resorbing activity of mature osteoclasts we isolated osteoclasts from calcium-deficient egg-laying hens or rat pups and incubated them on sperm whale dentine slices. High Pi concentrations markedly reduced both the number of resorption pits formed per dentine slice and the mean area of each pit in both avian and mammalian systems. These data indicate that high concentrations of Pi act on bone directly, both to inhibit generation of new osteoclasts from their precursor cells and to inhibit bone resorption by mature osteoclasts. These effects of extracellular Pi concentration may play an important modulatory role on bone turnover in vivo and have potential importance in several disease states in which Pi metabolism is perturbed.
Inhibiting and stimulating effects of TGF-β1 on osteoclastic bone resorption in fetal mouse bone organ cultures
Authors: Dieudonné S.C. , Foo P. , Van Zoelen E.J.J. , Burger E.H.
Published: February 2010

The effects of TGF-β1 on osteoclastic resorption of fetal mouse calvaria and long bones at various stages of development was studied in organ culture. In resorbing calvariae and long bones with an established marrow cavity TGF-β1 (4–10 ng/ml) had a stimulating effect on 45Ca release that was partially inhibited by indomethacin. In primitive long bones, however, which were explanted before osteoclast invasion and excavation of a marrow cavity had started, TGF-β1 (1–4 ng/ml) inhibited 45Ca release by an indomethacin-insensitive mechanism.

Histomorphometry of long bones after staining for tartrate-resistant acid phosphatase (TRAP) revealed that TGF-β1 treatment inhibited the migration of TRAP-positive cells from periosteum to developing marrow cavity and inhibited cell fusion. However, the formation of (mononuclear) TRAP-positive cells in the periosteum-perichondrium was strongly enhanced.

These data suggest that TGF-β1 modulates various steps in the cascade of osteoclast development, recruitment, and activation in different ways, involving both prostaglandin-mediated and prostaglandin-independent pathways. Therefore the net effect of exogenous TGF-β1 on osteoclastic resorption in bone organ cultures depends on the relative prevalence of osteoclast progenitors, precursors, and mature osteoclasts in the tissue under study.
Spinal bone mineral density in 335 normal and obese children and adolescents: Evidence for ethnic and sex differences
Authors: Mccormick David P. , Ponder Stephen W. , Fawcett H. Daniel , Palmer Judy L.
Published: February 2010

Spinal bone mineral density (BMD) and anthropometric measures were studied in 312 nonobese and 23 obese black, white, and Hispanic children and adolescents age 5.00–18.99 years. In adolescents BMD correlated with age, weight, height, fat-free density, body mass index, and midarm circumference.

Utilizing the entire group of 312 nonobese subjects, mean Z scores were calculated for comparison versus reference subgroups for bone mineral density index (BMDI, BMD/weight). BMDI was greater for black than for white and Hispanic children and adolescents across all ages studied. Female adolescents accumulated spinal mineral more rapidly than male adolescents. Black males had greater mineral than white and Hispanic males. Differences in BMDI between subgroups could not be explained by differences in body weight or spinal vertebral size. BMDI proved a more sensitive measure for comparing subgroups than did BMD.

The study provides normative data and a quantitative methodology for analyzing differences in spinal mineral between groups of children and adolescents.
Structural studies of the mineral phase of calcifying cartilage
Authors: Rey C. , Beshah K. , Griffin R. , Glimcher M.J.
Published: February 2010

The calcified cartilage of the epiphyseal growth plate of young calves has been studied by x-ray diffraction, Fourier transform infrared spectroscopy, magic angle 31P nuclear magnetic resonance spectroscopy, and chemical composition. The powdered tissue was separated by density centrifugation as a function of mineral content and thus qualitatively of the age of the calcium-phosphorus mineral phase. The individual density centrifugation fractions were examined separately. X-ray diffraction of the samples, especially of the lowest density fractions, revealed very poorly crystalline apatite. Fourier transform infrared spectroscopy and 31P nuclear magnetic resonance spectroscopy revealed the presence of significant amounts of nonapatitic phosphate ions. The concentration of such nonapatitic phosphates increases during the early stages of mineralization but then decreases as the mineral content steadily rises until full mineralization is achieved.

The total concentration of carbonate ions was found to be much lower in calcified cartilage than in bone from the same organ (scapula). The carbonate ions are located in both A sites (OH−) and B sites (PO43-), with a distribution similar to that found in bone mineral. However, discrepancies between infrared resolution factors of phosphate and carbonate bands are consistent with a heterogeneous distribution of carbonate ions in poorly organized domains of the solid phase of calcium phosphate. These initial studies permit one to characterize the calcium phosphate mineral phase as a very poorly crystalline, immature calcium phosphate apatite, rich in labile nonapatitic phosphate ions, with a low concentration of carbonate ions compared with bone mineral of the same animal, indeed from the bone of the same organ (scapula).
In vitro studies on bone resorption in neonatal mouse calvariae using a modified dissection technique giving four samples of bone from each calvaria
Authors: Ljunggren Östen , Ransjö Maria , Lerner Ulf H.
Published: February 2010

Bone resorption in a modified bone culture system, based on incubation of small fragments from neonatal mouse calvarial bones, has been studied. Four bone fragments were dissected out from each mouse calvaria and were thereafter cultured in CMRL 1066 medium in plastic multiwell dishes. Bone resorption was assessed by 45Ca release from prelabeled bones. The rate of bone resorption in response to parathyroid hormone (PTH) was less in the anterior part of the calvaria compared to the posterior part. After removing the anterior region, four parietal bone fragments that showed identical basal and PTH-stimulated release of 45Ca could be dissected out from each mouse. Excretion of lactate dehydrogenase and β-glucuronidase was the same in bones cultured submerged or on grids. Uptake of [3H]thymidine in bones cultured submerged was 54% of [3H]thymidine uptake in bones cultured on grids. Dose-response curves, established by using parietal bone fragments, showed that the sensitivity and the magnitude of the increase in 45Ca release seen after stimulation with PTH, prostaglandin E2, and 1α-hydroxyvitamin D3 were the same for bones cultured submerged or on grids. The 45Ca release in response to stimulation with PTH, prostaglandin E2, and 1α-OHD3 was the same in calvarial fragments cultured submerged and those previously obtained with calvarial halves cultured on grids. Thus, even though the rate of DNA synthesis was slower in bones cultured submerged, the rate and the magnitude of resorption were the same in bones cultured on grids or submerged. These data show that it is possible to perform studies on bone resorption with small fragments of neonatal mouse parietal bones.
On the role of cyclic AMP as a mediator of bone resorption: γ-Interferon completely inhibits cholera toxin- and forskolin-induced but only partially inhibits parathyroid hormone-stimulated 45Ca release from mouse calvarial bones
Authors: Dr. Lerner Ulf H. , Ransjö Maria , Ljunggren Östen , Klaushofer Klaus , Hoffmann Oskar , Peterlik Meinrad
Published: February 2010

The effects of γ-interferon (γ-IFN) on bone resorption and cyclic AMP formation stimulated by parathyroid hormone (PTH), forskolin, and cholera toxin have been studied in cultured neonatal mouse calvarial bones. Bone resorption was assessed by the release of 45Ca from prelabeled mouse calvarial bone fragments. Cyclic AMP formation was quantified by analyzing the amount of the nucleotide in calvarial bone tissue. γ-IFN completely blocked the 45Ca release response to forskolin and cholera toxin in 96 h cultures. In contrast, the 45Ca release response to PTH was only partially inhibited, an effect that was seen over a wide range of PTH concentrations. The inhibitory effect of γ-IFN was dose dependent, with a threshold for action at 10 U/ml. Forskolin-stimulated 45Ca release could only be inhibited when γ-IFN was added simultaneously with forskolin; γ-IFN added to bones prestimulated with forskolin had no effect. The inhibitory effect of γ-IFN on PTH-stimulated 45Ca release was seen first after a time lag of 48 h. In contrast, calcitonin caused an inhibition after only 3 h. PTH and cholera toxin stimulation of radioactive calcium release was also inhibited by γ-IFN in bones treated with indomethacin. γ-IFN inhibited forskolin-induced 45Ca release in bones treated with the mitotic inhibitor hydroxyurea. No effect of γ-IFN on cyclic AMP formation induced by PTH, cholera toxin, or forskolin could be seen. These data show that γ-IFN inhibits forskolin- and cholera toxin-induced bone resorption by a mechanism unrelated to prostaglandin production or mitotic activity. We suggest that γ-IFN causes a delayed inhibition of bone resorption in mouse calvariae by inhibiting the recruitment of new osteoclasts and thereby totally blocks forskolin- and cholera toxin-induced bone resorption. The bone-resorptive effect of PTH is only partially blocked since the initial stages in the mechanism of action of PTH involve rapid activation of existing osteoclasts, insensitive to inhibition by γ-IFN. These data lend further support to the view that PTH stimulation of bone resorption is not solely mediated by cyclic AMP.
Superoxide dismutase and catalase activities in the growth cartilage: Relationship between oxidoreductase activity and chondrocyte maturation
Authors: Matsumoto H. , Silverton S.F. , Debolt K. , Dr. Shapiro I.M.
Published: February 2010

Superoxide dismutase (SOD) and catalase are enzymes that protect cells from radical attack. Catalase disproportionates hydrogen peroxide, and SOD is an oxidoreductase that serves to dismutate the superoxide anion. The objective of this communication was to measure the activity of these disproportionating enzymes in the chick tibial growth cartilage and to relate enzyme activity to chondrocyte maturation and tissue calcification. Analytic techniques were optimized for the measurement of both enzymes; particular care was taken to ensure that the values obtained were due to SOD and catalase, not to the presence of other oxidases or contaminants. Catalase and SOD had similar profiles of activity in cartilage. For both enzymes, the highest levels of activity were observed in premineralized cartilage; as chondrocytes matured there was a progressive decrease in the activity of SOD and catalase. Comparison of chondrocyte SOD activity with nonmineralizing tissues indicated that the activity of cultured cartilage cells was low. We also measured the SOD activity of avascular chondrodystrophic cartilage and found it to be less than that of proliferating cartilage. When cartilage was electrofocused, three SOD isozymes were detected. The pI of the major isozyme corresponded to the copper-zinc isoform. We suggest that the observed changes in enzymatic activity are dependent on a number of cartilage-specific factors that include the vascular supply, the local production of oxygen radicals by chondrocytes, and the oxidative state of the tissue.
Automated comparison of dual-photon absorptiometric studies of the lumbar spine
Authors: Verheij Luis F. , Blokland Jacobus A. K. , Papapoulos Socrates E. , Bijvoet Olav L. M. , Pauwels Ernest K. J.
Published: February 2010

An automated image comparison procedure was developed to optimize the precision of bone mineral density measurements by dual-photon absorptiometry. Changed acquisition conditions cause differences between two images to be compared. Alignment of one image with respect to the other is performed by a transformation that involves a rotation, a horizontal or vertical shift, and a correction for the soft tissue level. The best possible transformation is found in a stepwise search, guided by initial estimations of its parameters. After optimum transformation of one image, the region of interest of the other image is applied to both of them. Duplicate measurements of 9 patients and 15 normal subjects were performed; automated analysis yielded improved precision with respect to manual analysis. The coefficient of variation (CV) was also computed. The CV for automated analysis was 2.00% for patients and 1.04% for normal subjects compared to 3.55 and 1.93%, respectively, for manual analysis. For phantoms, the precision was 2.67% for manual analysis and 0.49% for automated analysis.
Cortical osteoclasts are less sensitive to etidronate than trabecular osteoclasts
Authors: Chappard Daniel , Petitjean Muriel , Alexandre Christian , Vico Laurence , Minaire Pierre , Riffat Georges
Published: February 2010

Acute osteoporosis after spinal cord injury is related to an early increase in osteoclastic resorption. Healthy subjects subjected to bed rest similarly increase their osteoclast number in trabecular bone. Bisphosphonates possess a highly antiosteoclastic activity. The effects of a 120 day bed rest period, with or without etidronate therapy on cortical bone were measured in 15 subjects. Cortical thickness and cortical porosity were measured on transiliac bone biopsies taken before and after the bed rest period. Osteoclasts were detected histo-chemically and were counted with a semiautomatic image analyzer. Cortical thickness, cortical porosity, and cortical osteoclast number were not significantly modified in subjects submitted to bed rest alone. In the etidronate-treated patients, cortical bone mass parameters were also found to be unaffected, but the most striking feature was that the osteoclast number was unchanged. Trabecular osteoclasts, on the contrary, were increased in the untreated subjects (+95.2%) but decreased in the treated subjects (-78%). Bone cells may have heterogeneous responses according to their trabecular or cortical location. Cortical osteoclasts seem to be unaffected by etidronate therapy.
Comparison of total-body measurements by dual-energy x-ray absorptiometry and dual-photon absorptiometry
Authors: Prof. Nuti Ranuccio , Martini G. , Righi G. , Frediani B. , Turchetti V.
Published: February 2010

Both dual-photon absorptiometry (DPA) using 153Gd and dual-energy x-ray absorptiometry (DEXA) can be used for measurement of bone mineral content (BMC) and bone mineral density (BMD) of the total skeleton and its seven major regions. The short-term precision (coefficient of variation, CV) of DEXA for total-body BMD using the medium (20 minute) and fast (10 minute) speeds was 0.34 and 0.68% in 5 normal subjects; the corresponding CV in 5 osteoporotic females were 0.70 and 1.04%. The CV for BMD using DPA was 0.82% in 8 normal subjects and 0.70% in 12 osteoporotic patients. The CV for regional BMD using DPA was similar to fast-speed DEXA, without significant differences (p NS); precision with medium-speed DEXA was superior to DPA, and the differences were statistically significant (p < 0.05) for head, spine, trunk, ribs, and pelvis. Total-body measurements using both DPA and DEXA were done on 99 subjects (84 females and 15 males). Significant correlations (r = 0.98; p < 0.001) were found between DEXA and DPA measurements of both BMC and BMD. There were also significant correlations (r = 0.94–0.98; p < 0.001) between DEXA and DPA measurements of anatomic regions (head, trunk, spine, pelvis, ribs, arms, and legs). DPA and DEXA results for BMD of total skeleton, ribs, pelvis, and legs were similar (p NS), and statistically significant differences were found in head, spine, and arm measurements (p < 0.01, p < 0.01, and p < 0.05, respectively); regression equations allowed adjustment of DEXA values in patients already measured with the earlier DPA method.
Purinergic regulation of cytosolic calcium and phosphoinositide metabolism in rat osteoblast-like osteosarcoma cells
Authors: Kumagai Hiromichi , Sacktor Bertram , Filburn Charles R.
Published: February 2010

We have shown that ATP increases cytosolic Ca2+ in UMR-106 cells through P2-purinergic receptor stimulation (Calcif Tissue Int 45:251-254). This response was further characterized using cells loaded with indo-1/AM or prelabeled with [3H]inositol. ATP elicited a rapid transient increase in Ca2+ from 148 to 540 nM, followed by a biphasic decline (first rapid and then slower) to basal within 1 minute and then a late slow rise to 200 nM by 4 minutes. ADP also elicited a rapid transient increase, but this was followed by a second smaller transient and a later, slow increase above basal Ca2+. These transient increases in Ca2+ induced by ATP and ADP were dose dependent, detected at 10−6 M ATP and 10−7 M ADP, and saturated at 10−4 M with both nucleotides. The maximum increase in Ca2+ was 20% greater with ATP than ADP. EGTA chelation of extracellular Ca2+ abolished the biphasicity of the ATP-induced Ca2+ transient, the second ADP-induced transient, and all late slower increases in Ca2+. Desmethoxyverapamil pretreatment attenuated the biphasicity of the ATP-induced transient and the second peak elicited by ADP. Elevated extracellular Ca2+ (5 mM) prevented the return to the basal level that normally follows the ATP-induced Ca2+ transient and amplified the sustained increase in Ca2+ but had little effect on the response to ADP. IP3 and IP4 increased rapidly after addition of ATP, with I(1,4,5)P3 increasing before I(1,3,4)P3. These data indicate that P2-purinergic stimulation of UMR-106 cells causes three consecutive responses in cytosolic Ca2+: (1) a transient increase due to IP3-mediated mobilization of intracellular Ca2+; (2) a transient increase due in part to influx, probably associated with a Ca2+ channel; and (3) a later sustained increase that requires extracellular calcium.