bluuebunny/crossref_metadata_2025 huggingface dataset:
Models of spinal trabecular bone loss as determined by quantitative computed tomography
Authors: Block Jon E. , Smith Rebecca , Glueer Claus-Christian , Steiger Peter , Ettinger Bruce , Genant Harry K.
Published: February 2010

Noninvasive bone densitometry techniques have significantly improved our understanding of the pattern and magnitude of bone loss over the life span. Quantitative computed tomography (QCT) is capable of selectively measuring highly labile trabecular bone in the central portion of the vertebrae. Trabecular bone mineral density (mg/ml) was determined in 538 healthy women ranging in age from 20 to 80 using GE CT/T scanners at 80 kVp. Various statistical regressions were performed for the entire population to describe the general pattern of bone loss from the spine; a cubic model (r = -0.69, SEE = 26.0 mg/ml) was found to be statistically superior (p < 0.01) to linear, quadratic, or exponential models. An average bone loss was approximated from these regression analyses with a yearly absolute rate of -2.02 ± 0.097 mg/ml per year (p < 0.0001). The average rate of change for premenopausal women was -0.45 mg/ml per year (p < 0.05), for perimenopausal women was -4.39 mg/ml per year (p < 0.0001) and for postmenopausal women was -1.99 mg/ml per year (p < 0.0001). QCT values were also stratified into 5 and 10 year age groups and analyzed seperately for pre- and postmenopausal women. The 5 and 10 year interval stratification revealed no identifiable bone density decrements prior to midlife using analysis of variance statistical methods; significant losses of bone mineral density were noted to correspond with the usual time of menopause and to continue into old age. Various two-phase regressions were employed using age and menstrual status to improve the description of age- and menopause-related bone loss. For all women, a linear-exponential pattern (r = -0.70, SEE = 25.44 mg/ml) with a decrement of 25 mg/ml across the early menopause, appeared most plausible, amounting to an average bone loss of 60% over the life span. We provide normative data here on spinal trabecular bone density appropriate for clinical comparison of female subjects encompassing the adult age range and menopausal status.
Parathyroid hormone-related protein: Immunohistochemical localization in cancers and in normal skin
Authors: Danks J. A. , Ebeling P. R. , Hayman J. , Chou S. T. , Moseley J. M. , Dunlop J. , Kemp B. E. , Martin T. J.
Published: February 2010

An immunoperoxidase method has been developed to detect parathyroid hormone-related protein (PTHrP) in histological specimens of tumors and of normal skin. A rabbit polyclonal antiserum against PTHrP-(1–16) was used that did not cross-react with PTH-(1–34) either under radioimmunoassay conditions or at the high antiserum concentrations used in neutralizing biologic activity. PTHrP antigen was detected in the keratinocyte layer of normal skin and in 100% of 34 samples of squamous cell cancers but in only one of six breast cancers, and none of 15 other adenocarcinomata. It was also detected in four of four samples of renal cortical carcinoma and two of two of melanoma, both of which can be associated with hypercalcemia, and three of three small cell carcinomata of the lung. Immunologic detection of PTHrP could be useful in the diagnosis of tumors of squamous cell origin, particularly in the cytological differentiation of lung cancers, where it may be of value in distinguishing between squamous cell and small cell carcinoma on the one hand and poorly differentiated adenocarcinoma on the other.
Skeletal disease in primary hyperparathyroidism
Authors: Silverberg Shonni J. , Shane Elizabeth , de la Cruz Luz , Dempster David W. , Feldman Frieda , Seldin David , Jacobs Thomas P. , Siris Ethel S. , Cafferty Maureen , Parisien May V. , Lindsay Robert , Clemens Thomas L. , Dr. Bilezikian John P.
Published: February 2010

Most patients with primary hyperparathyroidism in the 1980s do not have evidence of bone disease when they are evaluated by conventional radiography. We sought to determine whether skeletal involvement can be appreciated when more sensitive techniques, such as bone densitometry and bone biopsy, are utilized. We investigated 52 patients with primary hyperparathyroidism. They had mild hypercalcemia, 2.8 ± 0.03 mmol/liter (11.1 ± 0.1 mg/dl), low normal phosphorus, 0.9 ± 0.03 mmol/liter (2.8 ± 0.1 mg/dl), and no symptoms or specific radiological signs of skeletal involvement.

The greatest reduction in bone mineral density was found at the site of predominantly cortical bone, the radius (0.54 ± 0.1 g/cm; 79 ± 2% of expected), whereas the site of predominantly cancellous bone, the lumbar spine (1.07 ± 0.03 g/cm2), was normal (95 ± 3% of expected). The site of mixed composition, the femoral neck (0.78 ± 0.14 g/cm2), gave an intermediate value (89 ± 2% of expected). Preferential involvement of cortical bone with apparent preservation of cancellous bone in primary hyperparathyroidism was confirmed by percutaneous bone biopsy. Over 80% of patients had a mean cortical width below the expected mean, whereas cancellous bone volume in over 80% of patients was above the expected mean. The results indicate that the majority of patients with asymptomatic primary hyperparathyroidism have evidence by bone densitometry and bone biopsy for cortical bone disease. The results also indicate that the mild hyperparathyroid state may be protective of cancellous bone. The therapeutic implications of these observations await further longitudinal experience with this study population.
Normal vertebral dimensions and normal variation in serial measurements of vertebrae
Authors: Davies K. Michael , Recker Robert R. , Heaney Robert P.
Published: February 2010

A total of 568 lateral spine radiographs of 191 generally healthy white perimenopausal women were used to determine the limits of normal for shape and dimensions of the vertebral bodies most often involved in osteoporotic fracture. Anterior and posterior vertebral heights for T7–L4 were used to compute wedge shape, relative posterior height, and relative serial change. Wedge shape was defined as anterior height minus posterior height divided by posterior height. Relative posterior height was defined as the posterior height of a vertebra minus the posterior height of the vertebra superior divided by the posterior height of the vertebra superior.

The degree of normal wedging depended on position: wedge values progressed down the spine, from a mean of −0.106 at T7 to 0.048 at L4. The minimum normal wedge value ranged from −0.209 at T7 to −0.083 at L4.

Radiographs on a cohort of 28 osteoporotic women were evaluated both by ordinary clinical reading of the radiographs and by using the standards developed from the normal subjects. Initial agreement between the two modes of assessment was 85.4%, and in resolving the remaining disagreements the clinician agreed that he had initially misread the films in all but 3.2%. On the basis of this limited experience, we conclude that the use of such objective standards, in a computer-operated algorithm, is more accurate than routine radiographic assessment.

Serial x-rays spanning a 15–20 year period in these women permitted determination of both reproducibility and longitudinal change in vertebral dimensions in the perimenopause. The mean relative serial change was not significantly different from zero for all vertebrae. The normal lower bound for relative change in measured vertebral heights ranged between −10 and −16% anteriorly and −11 and −19% posteriorly.

Normal vertebral dimensions were found to vary from vertebra to vertebra; criteria for deformity should allow for this variation to avoid misdiagnosis.
Ultrastructural localization of tartrate-resistant, purple acid phosphatase in rat osteoclasts by histochemistry and immunocytochemistry
Authors: Clark Samuel A. , Ambrose Wallace W. , Anderson Thomas R. , Terrell Regina S. , Dr. Toverud Svein U.
Published: February 2010

The intracellular localization of the tartrate-resistant purple acid phosphatase in osteoclasts of developing rat bone has been determined immunocytochemically using an antiserum to the purified bone-derived purple acid phosphatase. The localization of the immunoreactivity was compared with the results of enzyme histochemistry using p-nitrophenylphosphate as substrate and 10 mM tartrate. Both methods revealed the presence of the enzyme in numerous vesicles of various sizes up to 2–3 μm in diameter and in granules. There was no immunoreactivity in the Golgi apparatus, and tartrate completely inhibited the histochemical activity of this organelle. No consistent extracellular activity could be detected, nor was any reaction product observed at the ruffled border. The localization of the tartrate-resistant purple acid phosphatase in osteoclasts is consistent with an intracellular function for this enzyme.
G protein-dependent activation of a phosphoinositide-specific phospholipase C in UMR-106 osteosarcoma cell membranes
Authors: Babich Michael , King Kathleen L. , Nissenson Robert A.
Published: February 2010

Recent evidence suggests that guanyl nucleotide binding (G) proteins are involved in receptor-mediated bone resorption and in osteoblastic function, but the nature of the G protein coupled to effectors that are involved in these skeletal effects is unknown. The purposes of this study were to determine (1) whether a G protein mediates activation of phosphoinositide-specific phospholipase C in UMR-106 rat osteosarcoma cells, and (2) whether parathyroid hormone (PTH) and a PTH-like protein (PLP) associated with humoral hypercalcemia of malignancy promote GTP-dependent PIP2 hydrolysis. Addition of GTP (10−4 M) or guanosine 5′-0-(3-thiotriphosphate, GTPγS, 10−5 M) to membranes prepared from UMR-106 cells labeled with [3H]myo-inositol increased both [3H]inositol trisphosphate (IP3) and [3H]inositol bisphosphate (IP2) formation. The increases in [3H]IP2 and [3H]IP3 produced by GTP were 8.6- and 4.3-fold, respectively. GTPγS produced a 17.6- and 11.9-fold increase in [3H]IP2 and [3H]IP3, respectively. The stimulatory effects of GTP and GTPγS were dose dependent (GTP ED50 = 3.9 × 10−6 M; GTPγS ED50 = 2.5 × 10−7 M) and progressive over 10 minutes and required the presence of Mg2+. GTP (10−4 M) and GTPγS (10−5 M) decreased membrane [3H]phosphoinositides concomitantly with increased [3H]IP2 and [3H]IP3. The GDP analog guanosine 5′-O-(2-thiodiphosphate, GDPβ) alone did not alter [3H]IP2 or [3H]IP3 production but at 10−4 M blocks the stimulatory effects of GTP and GTPγS. NaF (3 × 10−2 M) produced a 2.8- and 2.0-fold stimulation of [3H]IP2 and [3H]IP3, respectively. In the presence of 10−4 M GTP, bPTH-(1–34) (1 μg/ml) produced an increase in [3H]IP3 and [3H]IP2 of 23.5 + 3.0% (p < 0.001) and 14.1 + 2.5% (p < 0.01) within 2 minutes. hPLP-(1–34)amide (1 μg/ml) produced a 19.8 + 5.3% (p < 0.05) and 13.2 + 4.8% (p < 0.05) increase in [3H]IP3 and [3H]IP2. We conclude that UMR-106 membranes possess a G protein-sensitive phosphoinositide-specific phospholipase C. Conceivably, this signal transduction pathway contributes to the skeletal actions of PTH and PLP.
Equivalency of various methods for estimating osteoid seam width
Authors: Quarles L. Darryl , Lobaugh Bruce
Published: February 2010

We compared the true osteoid seam width (TOSW) as measured by a modification of the orthogonal intercept lengths with various methods of estimating seam widths, including (1) the commonly used length measurements at four equidistant points (O.Wi/4PT), (2) osteoid area divided by the osteoid perimeter (O.Ar/O/Pm) or the bone/osteoid interface (O.Ar/B.Bd), and (3) a novel method for estimating seam width defined as the osteoid area divided by the major axis of the seam (O.Ar/Axis). All methods for approximating osteoid seam width significantly exaggerated the true osteoid seam dimension by an amount that ranged from 16 to 23%. However, the relative accuracy of all methods of estimating osteoid seam width are equivalent as evidenced by the similar mean difference from the TOSW (3.4, 4.1, 5.1, and 3.8) demonstrated by O.Wi/5PT, O.Ar/Axis, O.Ar/O.Pm, O.Ar/B.Bd, respectively. Regression analysis of the various estimates of seam width on TOSW also demonstrated the equivalency of these methods. Moreover, all estimates could be employed to discriminate seams of normal dimensions from abnormally wide seams in bone specimens derived from patients with osteomalacia.

Differences between the methods, however, were observed that may have practical importance. In this regard, the direct procedure of determining distance demonstrated less variance than the indirect estimate of width. As a result, the direct measurement required fewer samples (n = 13) to detect a significant difference to normal and could discriminate smaller deviations in seam width (1.7 μm) at a given sample size compared with O.Ar/Axis (n = 28; 2.9 μm), O.Ar/O.Pm (n = 42; 3.4 μm), and O.Ar/B.Bd (n = 42; n = 3.2). However, in spite of the differences in the various methods, all width estimates can be successfully employed to discriminate small changes in seam widths provided that adequate numbers of seams are evaluated and mean values compared.
Noninvasive axial and peripheral assessment of bone mineral content: A comparison between osteoporotic women and normal subjects
Authors: van Berkum F.N.R. , Birkenhäger J.C. , Zeelenberg J. , Pols H.A.P. , Birkenhäger-Frenkel D.H. , van Veen L.C.P. , Trouerbach W.T. , Stijnen T.
Published: February 2010

We compared different methods of bone densitometry in women with spinal osteoporosis and normal subjects to assess their discriminatory capability. The methods used included: quantitative computed tomography of the spine (QCT) specified as to trabecular (QCTtrab) and cortical bone (QCTcort), dual-photon absorptiometry of the spine (DPAspine), single-photon absorptiometry of the distal and proximal forearm (SPAdist and SPAprox), and quantitative roentgen microdensitometry of the phalanx (QMD). A total of 25 postmenopausal osteoporotic women and 24 healthy comparison subjects matched for age and years since menopause were studied. In the osteoporotic group an average decrement of the axial bone mineral density of −50% (p < 0.001) and −20% (p < 0.001) were observed for QCTtrab and QCTcort, respectively. For DPAspine, SPAdist, SPAprox, and QMD the difference between normal and osteoporotic subjects was −20% (p < 0.001), −12% (p < 0.05), −7% (NS), and −6% (NS), respectively. With the peripheral measurements (SPA and QMD), alone or in combination, no adequate discrimination between women with or without vertebral compression fractures could be obtained. Although QCTtrab showd the highest diagnostic sensitivity (81%), it appears not to be superior to DPAspine. Combinations of the various axial and peripheral measurements did not result in an essentially better sensitivity.

In normal women as well as in osteoporotic individuals the trabecular and cortical QCT measurements showed two opposite trends, suggesting an increase in cortical and a decrease in trabecular density from L1 to L3.
Parathyroid cell number and size in hypercalcemic rats: A stereologic study employing modern unbiased estimators
Authors: Wernerson Annika , Reinholt Finn P. , Svensson Olle
Published: February 2010

Recently developed stereologic methods for unbiased estimations of particle number and size were employed to study parathyroid growth in normal and hypercalcemic young rats. Thus, the parathyroid cell number and size of parathyroid secretory cells were estimated by both the disector method and the volume-weighted mean volume method. The glandular volume was calculated from serial sections, and the volume density of secretory cells was estimated by conventional sterologic techniques. Three groups of animals were studied: normal rats at 3 weeks of age, hypercalcemic rats at 7 weeks of age, and age-matched controls. Hypercalcemia was induced by feeding the animals a purified diet that was nutritionally adequate except for low amounts of phosphate (0.02%) from 3 weeks of age. During the period from 3 to 7 weeks of age, the number of parathyroid secretory cells increased by 100%, whereas the mean cell volume increased by 20%. However, when calculated per gram body weight the volume and number of cells were larger in the younger animals. The phosphate-depleted animals grew slowly and developed severe hypercalcemia. Their parathyroid secretory cells were smaller, and each gland contained fewer cells than in age-matched controls. The lower cell number and cell volume, however, were proportional to the reduced body weight. Data from the 3-week-old animals indicate that the reduced cell number and size in hypercalcemic rats reflected growth arrest rather than atrophy.
Structure-activity relationships of parathyroid hormone analogs in the opossum kidney cell line
Authors: Carnes David L. , Cole Judith A. , Forte Leonard R. , Poelling Richard E. , Thorne Pamela K. , Eber Sammy
Published: February 2010

Structural alterations in the parathyroid hormone (PTH) molecule produce marked changes in biologic activity. We examined the relative sensitivity of PTH-stimulated cAMP formation and PTH-inhibitable Na+- dependent phosphate transport responses to bovine PTH analogs [bPTH-(1–34), bPTH-(1–84), 8,18-norleucine-34-tyrosinamide bPTH-(1-34), bPTH-(7–34)-amide, 8,18-norleucine-34-tyrosinamide bPTH-(3–34), transaminated bPTH-(1–34)] and the human PTH-related peptide of malignancy (1–34) in cultured opossum kidney cells. The rank order of potency for stimulation of cAMP formation was bPTH-(1–34) = hPTHrP-(1–34) > nle bPTH-(1–34) > bPTH-(1–84) ≥ TAbPTH-(1–34). Nle bPTH-(3–34) and bPTH-(7–34) did not affect cAMP formation in intact cells at concentrations up to 10 μM. The rank order of potency for the inhibition of phosphate transport was bPTH-(1–34) = hPTHrP-(1–34) > nle bPTH-(1–34) > bPTH-(1–84) = TAbPTH-(1–34) > nle bPTH-(3–34). TAbPTH-(1–34) was a full agonist and inhibited phosphate transport at concentrations that did not increase cAMP formation, but nle bPTH-(3–34) was a partial agonist in spite of its inability to stimulate cAMP formation. Bovine PTH-(7–34) had no effect on phosphate transport. This study indicates that changes in the PTH molecule produce analogs that apparently discriminate between the cAMP-stimulating activity and phosphate transport-inhibiting activities of the native hormone. These analogs may prove useful in defining PTH receptor subtypes based on differences in their pharmacologic properties in the regulation of physiologic responses in PTH target cells.
Early strain-related changes in enzyme activity in osteocytes following bone loading in vivo
Authors: Skerry Timothy M. , Lanyon Lance E. , Bitensky Lucille , Chayen Jo
Published: February 2010

The skeleton's architecture is matched to the changing loads to which it is subjected because mechanical loading directly or indirectly influences the activity of cell populations to deposit, maintain, or remove bone tissue as appropriate. This responsiveness to load bearing presupposes that certain cells are sensitive to load itself or to its consequences within the tissue. The nature of this effect and the cells concerned have not yet been determined.

In this series of experiments, bones were exposed in vivo to a single short period of dynamic loading, which if repeated daily had been shown to result in increased new bone formation. There was an increase in the activity of glucose 6-phosphate dehydrogenase (G6PD) in the periosteal cells adjacent to the bone surface 6 min after this single period of loading. This increase was proportional to the strain magnitude in the bone tissue in the immediate vicinity of the cells. In osteocytes, although the G6PD activity in each individual cell was unchanged by loading, the number of cells displaying activity was increased. This increase was also directly proportional to the applied strain in that area of the cortex (52% compared with 26% active osteocytes at a strain of 0.002). Activation of G6PD was unaccompanied by any equivalent changes in the activities of either glyceraldehyde 3-phosphate dehydrogenase (GA3PD) or lactate dehydrogenase (LDH). This finding is consistent with loading increasing the activity of the oxidative part of the pentose monophosphate shunt pathway. It is also consistent with stimulation of a synthetic process, such as the production of RNA from ribose 5-phosphate.

These results show that intermittent loading of bone tissue produces rapid strain-related effects on the metabolism of both osteocytes and periosteal cells. This change is consistent with an increase in their synthetic activity.
The characterization, regulation, and function of insulin receptors on osteoblast-like clonal osteosarcoma cell line
Authors: Dr. Pun K. K. , Lau P. , Ho P. W. M.
Published: February 2010

The properties and regulation of insulin receptors on monolayers of cultured clonal osteoblastic rat osteosarcoma UMR-106 cells and human osteosarcoma U20S cells were studied. Confluent cultures of UMR-106 cells bound lactoperoxidase-labeled, HPLC-purified [125I]A-14-monoiodinated insulin in a reversible, saturable, and specific manner. Binding was related inversely to the incubation temperature. Prolonged period of steady-state binding was achieved at all temperatures studied. Competition curves demonstrated half-maximal inhibition of [125I]insulin binding at an unlabeled insulin concentration of about 1 nM. Scatchard analysis of the binding data was curvilinear, suggesting negative cooperativity, and revealed that UMR-106 osteoblasts contained about 87,000 receptor sites per cell according to a two-site model. Bound [125I]insulin dissociated from osteoblasts with a t1/2 of about 15 minutes at 22°C. The dissociation curve was multiexponential, and the addition of native insulin accelerated the dissociation of intact but not degraded [125I]insulin. Preincubation with 125 nM insulin for 1 h induced 70% loss of binding sites and reduced the total insulin bound by 30%. When monolayers were treated with the lysosomotropic agent chloroquine, a 40% increase in cell-associated radioactivity that could not be dissociable in fresh buffer was observed. The use of an energy depleter, sodium fluoride, completely inhibited the effects of chloroquine. Similar results were obtained for human osteosarcoma U20S cells except that the number of receptor sites was far less than that of UMR-106 cells. Insulin increased collagen synthesis at a half-maximal concentration of 1 nM. To conclude, cultured rat and human osteoblasts possess insulin receptors that exhibit kinetic properties and specificity similar to those of other insulin target cells. Receptor-bound insulin is internalized and degraded by a chloroquine-sensitive, energy-requiring reaction. Insulin receptor on bone cells modulates the synthesis of collagen and this role may be important in bone homeostasis.
Regulation of 1,25-dihydroxyvitamin d3 by calcium in the parathyroidectomized, parathyroid hormone-replete rat
Authors: Weisinger Jose R. , Favus Murray J. , Langman Craig B. , Dr. Bushinsky David A.
Published: February 2010

Parathyroid hormone (PTH) is a major stimulus for the renal production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Elevated arterial blood ionized calcium ([Ca2+]) depresses serum 1,25-(OH)2D3 in nonparathyroidectomized rats even when serum PTH is maintained at high levels by infusion. However, suppression by [Ca2+] of endogenous PTH, causing the fall in 1,25-(OH)2D3, cannot be excluded. To determine whether [Ca2+] regulates 1,25-(OH)2D3 in the absence of a variation in PTH, we parathyroidectomized (PTX) rats (post-PTX calcium levels <7.0 mg/dl), inserted arterial and venous catheters, and then replaced PTH using an osmotic pump. We varied [Ca2+] by infusing either 75 mM sodium chloride (control), 0.61 μmol/min of EGTA (EGTA), or calcium chloride at 0.61 μmol/min (low calcium) or 1.22 μmol/min (high calcium) for 24 h 5 days after surgery. Blood was then drawn from the rat through the arterial catheter. Compared with the control, [Ca2+] fell with EGTA, remained constant with the low-calcium infusion, and rose with the high-calcium infusion. 1,25-(OH)2D3 was correlated inversely with [Ca2+] in all four groups together (r = −0.635, n = 34, p < 0.001), within the control group alone (r = −0.769, n = 11, p < 0.002), and within the EGTA group alone (r = −0.774, n = 10, p < 0.003). Serum phosphorus, PTH, and arterial blood pH were not different in any group, and none correlated with serum 1,25-(OH)2D3. We conclude that 1,25-(OH)2D3 levels are regulated by [Ca2+] independently of serum PTH, phosphorus, and acid-base status, all of which support the hypothesis that [Ca2+] is a principal regulator of serum 1,25-(OH)2D3 in the rat.
Trabecular bone remodeling and bone mineral density in the adult cat during chronic dietary acidification with ammonium chloride
Authors: Ching Shelley V. , Norrdin Robert W. , Dr. Fettman Martin J. , LeCouteur Richard A.
Published: February 2010

Ammonium chloride (NH4CI) is used as a urinary acidifier in the treatment and prevention of feline urologic syndrome. It is reported to cause alterations in calcium and bone metabolism in humans, dogs, and rats. Adult cats with normal renal function were fed 1.5% NH4CI for 6 months to study the effects of chronic dietary acidification on trabecular bone remodeling of the iliac crest and bone mineral density (BMD) of lumbar vertebral trabecular bone and femoral cortex. Histomorphometric analyses of iliac crest biopsies were performed before and after treatment. Static and dynamic parameters of bone resorption and formation were determined. Single-energy quantitative computed tomography (SEQCT) was used to measure lumbar trabecular and femoral cortical BMD. There were no significant treatment effects in iliac crest trabecular bone remodeling or BMD of the vertebrae and femora. Bone remodeling activity decreased with time in both acidotic and control cats. Vertebral BMD increased with time in both groups of cats, whereas no change was seen in the femora. Thus, chronic dietary acidification for 6 months with therapeutic levels of NH4CI produced no significant changes in trabecular bone remodeling or bone mineral density in adult cats.
Mechanism of amphotericin B stimulation of net calcium efflux from neonatal mouse calvariae
Authors: Blair Neil F. , Krieger Nancy S. , Bushinsky David A.
Published: February 2010

Amphotericin B is a polyene antifungal agent that binds to membrane sterols, creating aqueous pores that permit ion fluxes sufficient to cause cell lysis. It has also been shown to alter ion transport in mammalian cells, including proton secretion from renal tubular cells. The latter effect can lead to distal renal tubular acidosis in patients treated for systemic fungal infections. Based on the understanding that osteoclast-mediated bone resorption is dependent on proton secretion, we examined the effect of amphotericin B on calcium efflux from neonatal mouse calvariae in organ culture. Amphotericin B (5 μg/ml) stimulated net calcium efflux from calvariae within 24 h to a level almost as great as that produced by a maximally effective concentration of parathyroid hormone. The stimulated calcium efflux was completely inhibited by both 10 ng/ml salmon calcitonin, a physiologic inhibitor of osteoclast activity, and 4 × 10−4 M acetazolamide, a specific inhibitor of carbonic anhydrase, the enzyme necessary for substantial proton generation by osteoclasts. These results indicate a direct effect of amphotericin B on bone in vitro to stimulate osteoclast-mediated calcium efflux.
Rapid publication: Impairment of macrophage colony-stimulating factor production and lack of resident bone marrow macrophages in the osteopetrotic op/op Mouse
Authors: Dr. Felix R. , Cecchini M.G. , Hofstetter W. , Elford P.R. , Stutzer A. , Fleisch H.
Published: February 2010

Mouse calvaria-derived osteoblastlike cells have been shown to produce macrophage colony-stimulating factor (M-CSF). This factor may be involved in osteoclastogenesis and thus in bone resorption. In the present study we investigated whether the production of M-CSF was altered in the osteopetrotic mouse mutant strain op/op, characterized by a decrease in osteoclast number and an impairment of bone resorption. Whole calvariae and cells, as well as skin and lung fibroblasts, of the op/op mouse were found to produce no measurable M-CSF, in contrast to tissue and cells derived from normal littermates. M-CSF was identified by colony assay in semisolid media and by inhibition of the biologic activity with antiserum against M-CSF. Furthermore, the number of resident macrophages, identified by F4/80 antigen (F4/80 Ag) immunohistochemistry, was drastically decreased in bone and bone marrow of the op/op mouse, but in skin these cells were normal in number and morphology. These findings suggest that both M-CSF and resident macrophages play a role in the mechanism of bone resorption. The op/op mouse appears to be a valuable model to further investigate such a hypothesis.
Chemotactic response of osteoblastlike cells to transforming growth factorβ
Authors: Pfeilschifter Johannes , Wolf Oliver , Naumann Antje , Minne Helmut W. , Mundy Gregory R. , Ziegler Reinhard
Published: February 2010

Transforming growth factor β (TGF-β) has multiple effects on bone cell metabolism in vitro but its exact role in bone remodeling still needs to be defined. Here we demonstrate that TGF-β is chemotactic for osteoblastlike cells from fetal rat calvariae and osteoblastlike ROS 17/2.8 osteosarcoma cells. Maximal chemotaxis occurred at 5-15 pg/ml of TGF-β and was observed with TGF-β1 and TGF-β2 at equivalent concentrations. Conditioned medium from osteoblastlike cells containing latent TGF-β failed to stimulate chemotactic migration. However, chemotactic activity was observed in conditioned medium that had been transiently acidified. Since acidification is known to activate TGF-β, these results suggest that only active TGF-β is capable of inducing a chemotactic response. Preincubation of osteoblastlike cells with TGF-β in concentrations from 10 pg/ml to 1 ng/ml for 48 h abolished a subsequent chemotactic response of these cells to TGF-β, indicating that TGF-β-induced chemotaxis is a transient phenomenon. Since TGF-β may be released from the bone matrix and/or activated during bone resorption, the chemotactic activity of TGF-β for osteoblastlike cells may be important for the recruitment of osteoblastlike cells to sites of bone remodeling.
Fluoride bioavailability from slow-release sodium fluoride given with calcium citrate
Authors: Dr. Pak Charles Y. C. , Sakhaee Khashayar , Parcel Carol , Poindexter John , Adams Beverley , Bahar Abdillahi , Beckley Robert
Published: February 2010

Clinical pharmacology of slow-release sodium fluoride given with calcium citrate was examined in acute and long-term studies. Following a single oral administration of 50 mg slow-release sodium fluoride, a peak serum fluoride concentration (Cmax) of 184 ng/ml was reached in 2 h; thereafter, serum fluoride concentration declined with a T1/2 of 5.9 h. The concurrent administration of calcium citrate (400 mg calcium) gave an equivalent Tmax (time required to attain Cmax) and T1/2, but a lower Cmax of 135 ng/ml. The coadministration of a meal with fluoride also reduced Cmax but increased Tmax. The area under the serum concentration curve of slow-release sodium fluoride was reduced 17-27% by a meal or calcium citrate. Thus, calcium citrate reduced fluoride absorption and peak fluoride concentration in serum of slow-release sodium fluoride but did not affect the time required to reach peak concentration or the rate of subsequent decline. The effect of a meal was similar, except for a longer period required to reach peak serum concentration. During long-term administration of 25 mg slow-release sodium fluoride coadministered with 400 mg calcium as calcium citrate on a twice daily schedule, the trough level of serum fluoride could be kept between 95 and 190 ng/ml, believed to be the therapeutic window.
Osteoporosis as the sole presentation of bone marrow mastocytosis
Authors: Lidor Cobi , Frisch Bertha , Gazit Dan , Gepstein Reuven , Hallel Tom , Mekori Yoseph A.
Published: February 2010

Three (young) adults with severe generalized osteopenia and vertebral compression fractures were studied. Extensive clinical and laboratory investigations were not contributory. Undecalcified bone biopsies demonstrated multiple mast cell granulomas in the marrow in two patients and numerous mast cells diffusely distributed throughout the bone marrow in the third patient. Mast cells may serve as a pathogenic agent in osteoporosis. Therefore, we conclude that isolated skeletal mastocytosis without clinical evidence of mast cell mediator release should be sought in the evaluation of a patient with unexplained severe bone loss.
Heterogeneity of human bone
Authors: Ninomiya James T. , Tracy Russell P. , Calore James D. , Gendreau Mark A. , Kelm Robert J. , Mann Kenneth G.
Published: February 2010

Matched samples of bone from the lumbar spine and tibia were obtained at autopsy from three adult males who had no known evidence of metabolic bone disease at the time of their demise. The soluble noncollagenous bone proteins were quantitatively extracted from these samples and assayed for the relative content of two bone-associated proteins, osteocalcin and osteonectin. When compared to trabecular bone, cortical bone had higher levels of osteocalcin and much lower levels of osteonectin. When concentration is expressed per gram of dried bone, the osteocalcin excess in cortical bone ranged from 30- to 32-fold, and the osteonectin excess in trabecular bone ranged from 21- to 47-fold. These differences were significant (P < 0.01) using analysis of variance. We conclude that the human skeleton is not homogeneous with regard to these biochemical markers and that cortical and trabecular bone are biochemically quite distinct. This implies that these two types of bone may be subject to distinct regulatory mechanisms and that global assessments of skeletal function and bone quality based upon soluble markers should be applied with caution. The data also imply that a differential assessment of skeletal performance may be possible using biochemical serum markers.
Phorbol esters modulate the high Ca2+-stimulated accumulation of inositol phosphates in bovine parathyroid cells
Authors: Dr. Kifor Olga , Congo Dale , Brown Edward M.
Published: February 2010

We examined the effects of TPA on the high Ca2+-stimulated accumulation of inositol phosphates in bovine parathyoid cells to determine whether protein kinase C modulates phosphoinositide turnover in a fashion similar to that observed in other cell types stimulated by more classic Ca2+ mobilizing hormones. Following exposure of parathyroid cells to TPA (10−6 M) for 10 or 30 minutes, there was a time- and dose-dependent inhibition of the accumulation of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) stimulated by 3 mM Ca2+. Half the maximal observed inhibition took place at 1–10 nM TPA, with 50–60% inhibition of high Ca2+-stimulated accumulation of inositol phosphates at 10−6 M TPA. The active phorbol ester, 4β-phorbol didecanoate, produced similar effects; the inactive derivative, 4α-phorbol didecanoate, was without effect. When parathyroid cells were exposed to TPA (10−6 M) for varying times and were then incubated with high (3 mM) Ca2+, inhibition of inositol phosphate accumulation was observed with 10 or 30 minutes preincubation. In contrast, preincubation of cells with TPA for 3 or 18 h markedly enhanced the high (3 mM) Ca2+-induced increase in inositol phosphates. In cells preincubated with TPA for 18 h, binding sites for [3H]phorbol dibutyrate and total protein kinase C (PKC) activity were reduced by greater than 95% and by 71%, respectively, consistent with downregulation of the enzyme. These results suggest that the high extracellular Ca2+-stimulated increase in accumulation of inositol phosphates in parathyroid cells, which has been postulated to result from a receptorlike process, can be modulated by agonists of protein kinase C in a fashion similar to that observed with more classic Ca2+ mobilizing hormones. Activators of kinase C initially inhibit the generation of inositol phosphates, presumably as a result of reduced turnover of phosphoinositides, but subsequently enhance inositol phosphate accumulation, probably because of down-regulation of protein kinase C.
Bone-resorbing activity and prostaglandin E produced by human periodontal ligament cells in vitro
Authors: Saito Shigeru , Dr. Rosol Thomas J. , Saito Mayumi , Ngan Peter W. , Shanfeld Joseph , Davidovitch Zeev
Published: February 2010

Human periodontal ligament (PDL) cells were derived from healthy premolars extracted for orthodontic treatment and were utilized for in vitro experiments in passages 4–6. Human PDL cells were seeded in tissue culture tubes and incubated with interleukin-1α (Il-1α), IL-1β, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), indomethacin, parathyroid hormone (PTH), or their combinations, for 1 h. The medium was then replaced with serum-free BGJb medium and incubated for 24 h without further additions. Prostaglandin E (PGE) concentrations in the conditioned media (CM) were measured by radioimmunoassay, and bone-resorbing activity was measured using 45Ca-labeled neonatal mouse calvariae. The results of this study indicated that (1) unstimulated cultured PDL cells produced PGE, and PDL CM stimulated bone resorption; (2) cytokine-treated (IL-1α, IL-1β, and TNF-α) PDL cells had increased production of PGE and bone-resorbing activity compared to unstimulated PDL cells; (3) indomethacin completely inhibited PGE production from unstimulated PDL cells but only partially inhibited bone-resorbing activity, indicating that PDL cells produced nonprostaglandin bone-resorbing factor(s); (4) IFN-γ did not change PGE or bone-resorbing activity production by cytokine-stimulated PDL cells; and (5) PTH treatment of PDL cells in addition to cytokines (IL-1α, IL-1β, and TNF-α) had additive effects on the production of bone-resorbing activity and synergistic effects on PGE production compared to cytokine treatment alone.
Bisphosphonates in vitro specifically inhibit, among the hematopoietic series, the development of the mouse mononuclear phagocyte lineage
Authors: Dr. Cecchini Marco G. , Fleisch Herbert
Published: February 2010

Bisphosphonates (BP) are powerful inhibitors of bone resorption. We have previously shown that 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP), 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP), and dichloromethylenebisphosphonate (Cl2MBP) inhibit the proliferation of macrophages in vitro at concentrations that do not affect the viability of nonproliferating cells. In this study we further investigated whether the antiproliferative effect of these three BP is, among the hematopoietic series, preferential to the mononuclear phagocyte lineage. BP were unable to inhibit more than 30–40% of the [3H]thymidine (3H-TdR) incorporation into bone marrow cells stimulated to proliferate by multilineage colony-stimulating activity containing conditioned medium (multi-CSA). From the analysis of the colonies induced in semisolid medium by multi-CSA and recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF), a dose-dependent disappearance specific to the macrophage-containing colonies emerged. In contrast, the number and composition of colonies other than macrophage and, in particular, the granulocyte colonies were not affected by these compounds, even at high concentrations (100 μM) previously also shown to be toxic for nonproliferating macrophages. Since the macrophages, differently from polymorphonuclear phagocytes, are known to be highly pinocytotic, it is possible that by this means they selectively concentrate BP intracellularly, leading to toxic concentrations. We postulate tht BP may also act in vivo in addition to their effect on osteoclast activity, by a similar mechanism on osteoclast precursors and on bone resident macrophages, a source of cytokines stimulating bone resorption and leading to impaired osteoclast recruitment and activity. Furthermore, their specific effect on the mononuclear phagocyte lineage may also be relevant in other pathologic situations in which these cells are involved.
Effect of naproxen on cancellous bone in ovariectomized rats
Authors: Dr. Lane Nancy , Coble Toni , Kimmel Donald B.
Published: February 2010

Nonsteroidal anti-inflammatory drugs (NSAIDs) affect bone metabolism in vitro and in vivo. They delay but do not alter the outcome of healing processes in bone. In some bone loss models, they block bone resorption and slow the rate of loss. We studied the effect of naproxen, a potent NSAID, on cancellous bone of the proximal tibial metaphysis of 6-month-old adult female ovariectomized rats.

Animals were ovariectomized, divided into groups, and fed standard diets differing only in naproxen content for 42 days. The rats of the groups ate 2.0, 5.5, 12.7, and 32 mg naproxen per kg body weight per day, respectively. Serum levels of naproxen were determined. Bone volume, mineralizing surface, osteoblast activity, osteoclast surface, and bone resorption rate were determined by bone histomorphometric techniques.

The rats' dose-related serum naproxen levels ranged from 4 to 28 μg/ml. Naproxen inhibited up to 70% of the bone loss occurring after ovariectomy at a serum level of 4 μg/ml. We deduced that naproxen blocked bone resorption in ovariectomized rats by slowing osteoclast activity at all doses. In contrast, naproxen slowed bone formation only at serum levels > 20 μg/ml in ovariectomized rats. These findings may have clinical relevance in helping to prevent postmenopausal bone loss in women.
Treatment of humoral hypercalcemia of malignancy in rats with inhibitors of carbonic anhydrase
Authors: Brown Gregory M. , Morris Carol A. , Mitnick Mary Ann , Insogna Karl L.
Published: February 2010

The enzyme carbonic anhydrase has been suggested as a critical participant in osteoclast-mediated bone resorption. In humoral hypercalcemia of malignancy (HHM) intense osteoclastic bone resoprtion is principally responsible for the observed hypercalcemia. We therefore undertook to examine the effect of the carbonic anhydrase inhibitor acetazolamide on the hypercalcemia induced by the H500 Leydig cell tumor in Fisher rats, a well-described model of HHM. Acetazolamide treatment for 10 h at 10 mg/h resulted in a significant fall in serum calcium in the five drug-treated animals (14.2 ± 0.9 to 11.5 ± 0.1 mg/dl, p < 0.05). Conversely, the six animals infused with vehicle alone showed a significant rise in serum calcium (12.5 ± 0.5 to 13.8 ± 0.1 mg/dl, p < 0.05). At the end of the infusion, the acetazolamide-treated animals had a significantly lower mean serum calcium than those receiving vehicle alone (11.5 ± 0.1 versus 13.8 ± 0.1, p < 0.05). There was no significant change in serum phosphorus, urine calcium, urine phosphorus, or nephrogenous cyclic AMP excretion between the two groups. Acetazaolamide and HTS 5-(3-hydroxybenzoyl)-2-thiophenesulfonamide, another carbonic anhydrase inhibitor, both significantly inhibited in vitro bone resorption induced by 5 × 10−9 M 36Tyr(1–36)-PTHrP-amide (PTHrP, parathyroid hormone-related protein). Acetazolamide also inhibited the resorption induced by 10−8 M (1–141)-PTHrP and 2.5 × 10−9 M (1–74)-PTHrP. We conclude that acetazolamide is effective in lowering the serum calcium in animals with humoral hypercalcemia of malignancy. The data are consistent with the hypothesis that the mechanism of action for this effect is direct inhibition of osteoclast-mediated bone resorption.
Structural determinants of the capacity of heparin to inhibit collagen synthesis in 21-day fetal rat calvariae
Authors: Hurley Marja M. , Kream Barbara E. , Raisz Lawrence G.
Published: February 2010

Earlier work from our laboratory demonstrated that heparin inhibits type I collagen and DNA synthesis in fetal rat calvariae in vitro. In this paper we have analyzed the structural features of heparin that determine its inhibitory effect on collagen synthesis. These experiments were performed using unmodified heparins and low-molecular-weight heparins from different manufacturers, nonheparin glycosaminoglycans, desulfated heparins, anticoagulant and nonanticoagulant heparin, and chemically defined heparin oligosaccharides. Low-molecular-weight heparin (Mr 3700–5100) inhibited collagen synthesis, but oligosaccharides (disaccharides to decasaccharide, Mr 665–3000) did not. The glycosaminoglycans chondroitin sulfate B, heparan sulfate, and hyaluronic acid did not alter collagen synthesis but dextran sulfate was as inhibitory as unmodified heparin. Nonanticoagulant as well as anticoagulant low-molecular-weight heparin fractions inhibited collagen synthesis. Modification of heparin by total desulfation, O-desulfation, or N-desulfation and re-N-acetylation resulted in the loss of inhibitory property, suggesting that the degree of sulfation contributed to heparin's inhibitory effect. Low-molecular-weight heparins from different manufacturers were just as inhibitory as native heparin on collagen synthesis. We therefore conclude that low-molecular-weight heparin compounds offer no protection against heparin-induced osteoporosis. Our findings also suggest that the size and sulfation of a heparin-derived oligosaccharide contribute to its ability to inhibit collagen synthesis in bone.
Regulation of alkaline phosphatase by 1,25-dihydroxyvitamin D3 and ascorbic acid in bone-derived cells
Authors: Franceschi Renny T. , Young Jock
Published: February 2010

The bone, liver, and kidney isozyme of alkaline phosphatase (ALP) has been measured in MG-63 human osteosarcoma cells after treatment with ascorbic acid (AA) and/or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Both compounds were required to achieve maximum ALP activity. When grown in the absence of 1,25-(OH)2D3 cells had low basal ALP activity regardless of whether media contained AA. In AA-free medium, 1,25-(OH)2D3 (10 nM) increased ALP activity fourfold. Addition of AA further increased levels of ALP activity induced by 1,25-(OH)2D3 to 10–15 times those found in -AA controls. The earliest effects of 1,25-(OH)2D3 were seen after 24–48 h, and ALP activity continued to increase for 6–8 days. AA and 1,25-(OH)2D3 had similar effects on ALP activity in ROS 17/2.8 rat osteosarcoma cells. In MG-63 cells the effects of AA and 1,25-(OH)2D3 could not be simply explained by the ability of these compounds to inhibit cell growth because another mitotic inhibitor, hydroxyurea, had a minimal effect on ALP activity. 1,25-(OH)2D3-specific induction of ALP ± AA was totally blocked by inhibitors of protein and RNA synthesis. Maximal ALP induction was obtained when cells were plated at low density. Consistent with our previous report (Franceschi et al. 1988 J Biol Chem 263:18938–18945), 1,25-(OH)2D3 rapidly stimulated type 1 collagen synthesis and acid-precipitable hydroxyproline production in MG-63 cells and this stimulation was further increased by AA. These results suggest that induction of the osteoblast marker, ALP, is directly or indirectly coupled to collagen matrix synthesis and/or accumulation.