bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of fluoride on bone and implant histomorphometry in growing rats
Authors: Turner Russell T. , Francis Rita , Hannon Kathleen S. , Brown Daniel , Garand Joseph , Bell Norman H.
Published: February 2010

The effects of fluoride at concentrations of 2.0 and 4.5 mM in drinking water on growth rate, vitamin D, water and mineral metabolism, bone histomorphometry, and osteoinduction of demineralized allogenic bone matrix (DABM) were compared in the rat. Whereas fluoride did not influence fluid intake or growth rate at the lower concentration, it increased fluid intake and inhibited growth rate at the higher concentration. Fluoride produced dose-related increases in serum fluoride and alkaline phosphatase but did not alter serum 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D. Serum calcium and phosphate were reduced by fluoride at concentrations of 2.0 mM but not 4.5 mM. Cancellous bone fractional area was increased by fluoride at 2.0 mM and was reduced by fluoride at 4.5 mM. Fluoride had no effect on cancellous bone surface length or the percentage surface lined by osteoblasts and osteoclasts. Fluoride increased medullary area and decreased the endosteal bone formation rate. Fluoride increased periosteal bone formation and apposition rates at concentrations of 2.0 mM but not 4.5 mM. Fluoride inhibited mineralization in DABM implants, and at the higher concentration, fluoride increased the formation of new bone matrix. These results indicate that in the rat, fluoride increases cortical and trabecular bone at therapeutic doses and reduces trabecular bone at toxic doses. The serum concentration of flouride at therapeutic doses in the rat is similar to that in patients with osteoporosis who are on treatment with fluoride. In the rat, there is a narrow range between toxic and therapeutic doses.
Parathyroid hormone-induced ornithine decarboxylase activity in fetal rat osteoblasts
Authors: Dr. van Leeuwen J.P.T.M. , Bos M. P. , Herrmann-Erlee M.P.M.
Published: February 2010

Induction of ornithine decarboxylase (ODC, E.C. 4.1.1.17) activity by parathyroid hormone (PTH) in cultured fetal rat osteoblasts was studied. PTH induced ODC activity and stimulated cAMP production in a dose-dependent manner, the ED50 for cAMP being five times as high as that for ODC. Induction of ODC activity by PTH was partly inhibited by actinomycin D and cycloheximide, with 40 and 55% inhibition, respectively. PTH increased the intracellular ionized calcium concentration ([Ca2+]1), which was absent in a Ca2+-free medium. Blocking calcium influx, lowering the extracellular calcium concentration, and adding trifluoperazine inhibited both induction of ODC activity and stimulation of cAMP production by PTH. A23187 (100 nM and 1 μM), combined with a low dose of PTH (4 nM), resulted in a synergistic induction of ODC activity and an inhibition of cAMP production. A23187 inhibited induction of ODC activity as well as stimulation of cAMP production by the dose of PTH (20 nM) maximally effective in inducing ODC activity. Forskolin together with this maximal dose of PTH resulted in an additive effect on ODC activity and a synergistic stimulation of cAMP production. The current results show similarities and differences with respect to results obtained with osteoblasts from other species and osteoblast cell lines. The present data indicate that (1) PTH stimulates ODC activity and this is partly due to new enzyme synthesis; (2) calcium is involved in induction of ODC activity and stimulation of cAMP production by PTH; furthermore, it is suggestive that calmodulin and/or protein kinase C are involved; and (3) stimulation of cAMP production by PTH depends on an optimal intracellular calcium concentration range.
Hypercalcemia fails to suppress elevated serum parathyroid hormone concentrations during lactation in rats
Authors: Garner Sanford C. , Boass Agna , Dr. Toverud Svein U.
Published: February 2010

We have previously reported that increased serum immunoreactive parathyroid hormone (iPTH) in the lactating (L) rat is generally accompanied by hypocalcemia when diets containing 0.4% calcium (Ca) or less are fed. However, instances were also observed in which elevated iPTH levels did not coincide with a hypocalcemia signal. To test the hypothesis that iPTH levels can remain elevated even in the presence of hypercalcemia in lactation, a diet containing 1.2% Ca and 0.4% phosphorus (P) was fed to lactating rats in three experiments (A, B, and C) to achieve serum ionized calcium (ICa) levels approximately 10% above levels for nonmated (NM) controls. The serum ICa of NM controls fed the 1.2% Ca diet was slightly, but significantly, elevated, and serum iPTH (determined by an N-terminal specific assay) was significantly suppressed compared with NM controls fed a 0.4% Ca diet. In experiment A, L rats fed a 1.2% Ca diet had 81% higher serum iPTH levels than NM controls fed the same diet in spite of a mean (+SEM) ICa level of 1.77 + 0.05 mM for L rats versus 1.46 + 0.01 mM for NM controls; NM controls fed a 0.4% Ca diet had serum ICa of 1.37 + 0.01 mM. This novel finding of significantly higher iPTH and ICa in L compared with NM rats fed a 1.2% Ca and 0.4% P diet was confirmed in experiment B with eight rats in each group of L or NM rats fed either the 1.2% or the 0.4% Ca diet. In experiment C, Ca IP (3 mg per 100 g body weight), which raised the serum ICa level of 20 day L rats fed 1.2% Ca diet to 2.20 + 0.18 mM at 40 minutes postinjection, did not suppress the elevated serum iPTH level (14.5 + 1.2 pg/ml for L versus 11.3 + 0.6 for NM). In NM rats fed the 0.4% Ca diet, however, Ca IP caused a rise in serum ICa to 1.86 + 0.04 mM and suppressed serum iPTH from 11.9 + 0.7 to 9.5 + 0.4 pg/ml (p < 0.02) at 40 minutes. In experiment D injected iPTH-(1–34) disappeared from the circulation of L rats at a rate not significantly different from that in NM rats. It is suggested that during lactation PTH secretagogues other than hypocalcemia can stimulate PTH secretion so that even supraphysiological serum Ca levels do not suppress PTH secretion to the same degree as in NM rats.
Cyclooxygenase inhibitors enhance cell growth in an osteoblastic cell line, MC3T3-E1
Authors: Fujimori Akira , Tsutsumi Masaharu , Fukase Masaaki , Fujita Takuo
Published: February 2010

To elucidate the significance of endogenous prostaglandin E2 (PGE2) in osteoblastic cell function, we studied the effects of cyclooxygenase inhibitors on cell growth and alkaline phosphatase (ALP) activity in MC3T3-E1 cells. UMR-106 cells were also used as references in our experiments. MC3T3-E1 cells, cultured in α-minimal essential medium containing 10% fetal bovine serum, were shown to produce PGE2, which was markedly suppressed in the presence of indomethacin. Addition of indomethacin resulted in an increase in DNA content and [3H]thymidine incorporation. A similar growth stimulatory effect was observed when structurally different cyclooxygenase inhibitors, that is, acetyl salicylic acid (ASA), flurbiprofen, and piroxicam, were added. These cyclooxygenase inhibitors, however, differed in their effects on ALP activity. Indomethacin and ASA enhanced ALP activity, whereas flurbiprofen and piroxicam suppressed it. We then examined the effects of exogenous addition of PGE2. Although exogenous PGE2 at 6 × 10−6 M slightly stimulated cell growth, it inhibited cell growth at 6 × 10−8 M and 6 × 10−7 M. ALP activity was reduced in a dose-dependent fashion by exogenous PGE2. These results suggest that PGE2 produced by MC3T3-E1 may be suppressing cell proliferation and that cyclooxygenase inhibitors, per se, may stimulate cell growth by inhibiting endogenous PGE2 production in MC3T3-E1 cells. UMR-106 cells also produced PGE2, although less than MC3T3-E1 cells. In UMR-106 cells, the cyclooxygenase inhibitors did not influence DNA content or ALP activity as distinctly as in MC3T3-E1 cells. DNA content in these cells was reduced only by a high concentration of exogenous PGE2 (6 × 10−6 M), and ALP activity was unaltered by exogenous PGE2. Thus, although endogenous prostaglandins had a growth inhibitory effect on UMR-106 cells similar to those on MC3T3-E1 cells, its role may be less important in UMR-106 cells.
Effects of transforming growth factor α and interleukin-1 on DNA synthesis, collagen synthesis, procollagen mRNA levels, and prostaglandin E2 production in cultured fetal rat calvaria
Authors: Dr. Hurley Marja M. , Fall Pamela , Harrison John R. , Petersen Donna N. , Kream Barbara E. , Raisz Lawrence G.
Published: February 2010

Transforming growth factor α (TGF-α) and interleukin-1 (IL-1) have been shown to affect bone metabolism in vitro by prostaglandin-dependent and PG-independent mechanisms. We assessed the effects of the combination of these two agents on [3H]thymidine (TdR) incorporation into DNA, DNA content, [3H]proline incorporation into collagenase-digestible (CDP), noncollagen protein (NCP), and PGE2 production in 21 day fetal rat calvaria cultured for 24–96 h. We also determined whether TGF-α plus IL-1 altered procollagen mRNA levels at 96 h. TGF-α, 1–30 ng/ml, produced a 41–59% increase in TdR incorporation into DNA, but the effect was partially blocked by human recombinant IL-1. At 96 h TGF-α alone or in combination with IL-1 significantly increased the DNA content of calvaria. At 96 h, TGF-α inhibited CDP labeling and the addition of IL-1 further enhanced this inhibitory effect. The enhanced inhibitory effect of TGF-α plus IL-1 on collagen synthesis was associated with a synergistic increase in prostaglandin accumulation in the medium. Addition of indomethacin blocked PGE2 accumulation and partially reversed the inhibitory effect of TGF-α alone or in combination with IL-1 on collagen synthesis. TGF-α decreased procollagen mRNA levels by 55%, but the combination of TGF-α plus IL-1 decreased procollagen mRNA levels by 82%. Our results show that TGF-α and IL-1, which are both produced by certain tumors as well as activated macrophages, appear to act synergistically to increase prostaglandin synthesis and inhibit collagen synthesis in vitro. Thus these agents may have a regulatory role on bone formation in vivo.
Modulation of responsiveness of the adenylate cyclase system in avian chondroprogenitor cells by pertussis toxin, PTH, and PGE2
Authors: Pines Mark , Yosif Bernard , Hurwitz Shmuel
Published: February 2010

Chondroprogenitor cells, derived from avian tibia epiphyseal growth plate, were cultured in vitro. Incubation of these cells with pertussis toxin augmented their cAMP response to parathyroid hormone (PTH), attenuated the response to forskolin, but did not modify the response to PGE2. Pertussis toxin modulation of the cAMP response was accompanied by ADP ribosylation of two proteins with molecular weights of 39 and 40 kD. Using specific antibodies, the 39 kD protein was identified as the inhibitory guanine nucleotide binding protein (Gi) of the adenylate cyclase system. The other ADP-ribosylated protein has not been identified.

Preincubation of the chondroprogenitor cells with PTH or PGE2 resulted in time-dependent heterologous desensitization of the cAMP response to a second challenge of either hormone. The cells did not recover from the densensitization for at least 18 h after removal of the hormones. PTH and PGE2 treatment did not affect the cAMP response to forskolin and cholera toxin. The PTH-dependent cAMP production was also not altered by forskolin treatment. PTH homologous desensitization was not affected by pertussis toxin treatment, but the heterologous desensitization due to PGE2 was significantly attenuated.

These results suggest that exposure of chondroprogenitor cells to PTH and PGE2 results in heterologous desensitization of the cAMP response. The desensitization is not due to changes in the adenylate cyclase activity. The pertussis toxin-sensitive G proteins are involved in the PTH heterologous rather than homologous desensitization of the cAMP response.
Expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals
Authors: Taylor Linda M. , Tertinegg Inka , Okuda Ayako , Heersche Johannes N.M.
Published: February 2010

Metatarsal bones of 15-day-old mouse embryos contain proliferative tartrate-resistant acid phosphatase (TRAP) negative (-) osteoclast progenitors that progressively differentiate into multinucleated TRAP positive (+) osteoclasts. Using histochemical and autoradiographic techniques, we have examined the expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals. Fresh mouse metatarsals from embryos aged 14–17 days and metatarsals from 15-day-old embryos cultured for 1, 2, 3, and 6 days were stained for TRAP. Calcitonin binding to osteoclasts and their precursors was studied by incubating metatarsals with [125I]salmon calcitonin (sCT) and quantitating grain counts from autoradiographs of tissue sections. Calcitonin receptors first appear on nonproliferating osteoclast precursors, most often just after or simultaneously with the development of TRAP activity. The effect of sCT on the development of TRAP+ mononuclear preosteoclasts was examined by culturing 15-day-old metatarsals in the continuous presence of 5 mU sCT for periods of up to 3 days and quantitating the number of TRAP+ mononuclear preosteoclasts that develop. Calcitonin did not affect the differentiation of osteoclasts up to the stage of the TRAP+ mononuclear preosteoclast.
Role of bone and kidney in parathyroid hormone-related peptide-induced hypercalcemia in rats
Authors: Rizzoli René , Caverzasio J. , Chapuy M.C. , Martin T.J. , Bonjour J.P.
Published: February 2010

A protein responsible for the biochemical syndrome similar to primary hyperparathyroidism associated with certain tumors has been recently characterized and its effects at the level of bone and kidney reported. However, the relative role of tubular reabsorption of calcium (Ca) and bone resorption in the pathogenesis of hypercalcemia induced by this factor is still debated. We investigated the effects of a synthetic amino-terminal fragment of parathyroid hormone-related protein [PTHrP-(1–34)] administered chronically by intraperitoneal osmotic minipumps in thyroparathyroidectomized (TPTX) rats. Clearance studies performed on day 6 of treatment after a 24 h fast revealed an increase in renal tubular reabsorption of Ca and a decrease in renal tubular reabsorption of phosphate (Pi), accompanied by an increase in cAMP excretion. PTHrP-(1–34) (90 pmol/h) stimulated bone resorption as evaluated by an increment in fasting urinary Ca excretion. Although the bone resorption inhibitor aminopropylidene diphosphonate fully corrected urinary Ca excretion and reduced plasma Ca from 3.04 ± 0.07 to 2.44 ± 0.21 mM (p < 0.05), this latter value remained considerably higher than in TPTX control rats (1.54 ± 0.12 mM, p < 0.01). In contrast, when the agent WR-2721, which is known to decrease the renal tubular reabsorption of Ca by a PTH-independent mechanism, was given, a further drop in plasma Ca and an increase in urinary Ca excretion were observed. These findings are similar to those found in animals implanted with the hypercalcemic Leydig cell tumor. In conclusion, this study indicates that stimulation of renal tubular reabsorption of Ca is an important determinant of the increase in plasma Ca induced by the chronic infusion of PTHrP-(1–34) in TPTX rats.
Protein kinase C activity in UMR-106-01 cells: Effects of parathyroid hormone and insulin
Authors: Iida-Klein Akiko , Varlotta Vicky , Hahn Theodore J.
Published: February 2010

The calcium and phospholipid-dependent protein kinase C (PKC) system appears to play an important role in mediating hormonal effects in various tissues including bone. Accordingly, we characterized PKC activity in the UMR-106–01 rat osteosarcoma osteoblastlike cell line and examined its hormonal regulation. UMR- 106–01 cells were found to possess a classic, phorbol ester-activated PKC system, which was highly calcium and phospholipid dependent. A 30 s exposure to 10 nM bovine parathyroid hormone (PTH) (1–34) increased cytosolic and membrane-bound PKC activity by 12 and 157%, respectively, resulting in a 2.2-fold increase in the membrane-bound to cytosolic (MB/C) activity ratio (all p < 0.01). The MB/C activity ratio was highest at 20 min, exhibiting a 2.8-fold increase over the control values (p < 0.01). In contrast, 10 nM insulin increased cytosolic PKC activity but decreased membrane-bound activity, resulting in a 61% decrease in the MB/C activity ratio at 20 min (p < 0.02). Moreover, insulin reduced PTH stimulation of the PKC activity ratio by 42 and 62% at 30 s and 20 min, respectively (p < 0.02). Thus, PTH and insulin have opposing effects on the PKC activity ratio in UMR-106–01 cells.
Influence of body habitus and race on bone mineral density of the midradius, hip, and spine in aging women
Authors: Desimone Daniel P. , Stevens June , Edwards Jeanne , Shary Judith , Gordon Leonie , Dr. Bell Norman H.
Published: February 2010

Osteoporosis and fractures of the hip are less common in black women and in obese white women than in nonobese white women. To determine the effects of race, age, and body weight on bone mineral density (BMD), BMD of the lumbar spine, trochanter, and femoral neck were determined by dual-photon absorptiometry in 131 nonobese white women and 34 nonobese black women, all of whom were within 30% of their ideal body weight, and in 24 obese white women and 27 obese black women, all of whom weighed more than 30% of their ideal body weight and were in the same age range as the nonobese women. All of them were 51 years of age or older, and most of them were postmenopausal. BMD of the midradius was measured by single-photon absorptiometry. Whereas body weight was significantly higher in the black as compared to the white women, the ages of the two groups were not different from each other. BMD declined with age and increased with body weight in both the black and white women at each of the four sites measured. After adjusting for age and weight by covariate analyses, black women had greater BMDs than white women at the midradius, lumbar spine, and femoral neck (p < 0.001), but not at the trochanter (p = 0.18). The increases in BMD observed in the obese and black women in the present study are consistent with the previous findings that osteoporosis and fractures of the hip are less common in black and in obese white women than in nonobese white women.
The partial degradation of osteonectin by a bone-derived metalloprotease enhances binding to type I collagen
Authors: Dr. Tyree Bernadette
Published: February 2010

Cultured neonatal rat calvaria produce latent metalloproteases capable of degrading collagen, gelatin, and osteonectin. The osteonectin-degrading activity was further characterized and found to be optimally active between pH 6 and 8 and inhibited with EDTA and 1,10-phenanthroline but not phenylmethylsulfonyl fluoride. Analysis of the degradation products of osteonectin by SDS-PAGE in the presence of dithiothreitol showed the generation of a somewhat stable 32,000 mw cleavage product. Comparison of the binding properties of this cleavage product with intact osteonectin indicated that the fragment retained its ability to bind hydroxyapatite in the presence of high salt (2 M NaCl). Importantly, the binding of osteonectin to type I collagen fibrils was enhanced by limited proteolysis.
Osteotropic agents induce the differential secretion of granulocyte-macrophage colony-stimulating factor by the osteoblast cell line MC3T3-E1
Authors: Dr. Horowitz Mark C. , Coleman David L. , Ryaby James T. , Einhorn Thomas A.
Published: February 2010

Osteoblasts play a central role in the regulation of bone remodeling. Not only are they responsible for the formation of new bone, but they also regulate bone resorption. These cells also exert regulatory influences outside the bone in that they are able to regulate hematopoiesis. However, obtaining pure populations of osteoblasts devoid of contaminating cell types remains problematic. One approach to this problem is the use of cloned osteoblastic cell lines. To this end we have used MC3T3-E1, a cloned murine osteoblast cell line of C57BL/6 origin. We report that MC3T3-E1 cells respond to lipopolysaccharide (LPS) and, to a lesser extent, parathyroid hormone (PTH) by the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). However, 1,25-(OH)2D3, a potent activator of osteoblasts, fails to induce these cells to secrete GM-CSF. These results suggest that MC3T3-E1 cells respond to osteotropic agents in a hierarchical fashion. Secretion of GM-CSF is not constitutive but rather requires active induction of the cells. MC3T3 cells fail to secrete detectable levels of interleukin-2 (IL-2), IL-3, or IL-4, regardless of whether or not the cells are activated. The data indicate that MC3T3-E1 cells secrete cytokines in response to osteotropic agents in a way similar to that of normal primary osteoblasts. Therefore, MC3T3-E1 cells may serve as a good in vitro model for primary osteoblasts.
Characterization of a Ca2+-ATPase in osteoclast plasma membrane
Authors: Bekker Petrus J. , Gay Carol V.
Published: February 2010

The plasma membrane fraction of chicken osteoclasts was purified utilizing 20% continuous Percoll gradients. Biochemical marker enzyme analysis (ouabain-sensitive Na+, K+-ATPase and 5′-nucleotidase) indicated that plasma membrane enrichment was 11.87-fold and 7.25-fold, respectively, and contamination with mitochondria, endoplasmic reticulum, and lysosomes was low as determined by succinic dehydrogenase, NADH dehydrogenase, and N-acetylglucosaminidase activities, respectively. SDS latency of Na+K+-ATPase and 5′-nucleotidase activities of the isolated plasma membranes revealed that 43-50% of vesicles were sealed, with 10-16% in the inside-out orientation, depending on the membrane fraction used. Electron microscopy confirmed the vesicular nature of the plasma membrane fraction. The plasma membrane Ca2+-ATPase had a high-affinity (KCa = 0.22 μM; Kmax = 0.16 μmol/mg per min) and a low-affinity (KCa = 148 μM; Vmax = 0.37 μtmol/mg per min) component. Calmodulin (0.12 μM) had no effect on Ca2+-ATPase activity. However, trifluoperazine (0.1 mM), a calmodulin antagonist, strongly inhibited especially the high-affinity component of the enzyme. Vanadate and lanthanum also caused inhibition. In the presence of CDTA, a potent Ca2+ and Mg2+ chelating agent, high-affinity Ca2+-ATPase activity was abolished, indicating that trace Mg2+ was essential for activity. The Ca2+-ATPase substrate curve using ATP showed a high-affinity (Km = 12.3 μM; Kmax= 0.022 μmol/mg per min) and a low-affinity (Km = 43.8 μM; Vmax = 0.278 μmol/mg per min) component. These results demonstrate that osteoclasts have a plasma membrane Ca2+-ATPase with characteristics similar to the enzyme responsible for active calcium extrusion in other cells.
The diltiazem analog TA-3090 mimics the actions of high extracellular Ca2+ on parathyroid function in dispersed bovine parathyroid cells
Authors: Dr. Chen Chu J. , Brown Edward M.
Published: February 2010

We previously showed that the calcium channel blocker diltiazem raises cytosolic Ca2+ and inhibits PTH release in bovine parathyroid cells. To investigate further possible mechanisms underlying these effects, we examined the effects of the more potent diltiazem analog TA-3090, which is a Ca2+ channel antagonist in vascular smooth muscle, on several aspects of the function of dispersed bovine parathyroid cells. Like diltiazem, TA-3090 (10-6-10-4) produced a dose-dependent inhibition of immunoreactive PTH release at 0.5 mM Ca2+ and raised the cytosolic Ca2+ concentration by 25-50% in fura-2-loaded parathyroid cells in the presence but not in the absence of extracellular Ca2, suggesting that it activated rather than inhibited Ca2+ channels. To determine whether this compound affects other aspects of parathyroid function, we examined its effects on the inhibition of cAMP accumulation by Ca2+, a process we recently found to involve inhibition of cAMP generation by G1 through a receptorlike mechanism, which is independent of changes in cytosolic Ca2+, TA-3090 (10−4 M) inhibited dopamine-stimulated cAMP accumulation by up to 75% (from 663 to 166 fmol per 105 cells), with a higher apparent potency at greater extracellular Ca2+ concentrations. Moreover, the addition of 10−4 M TA-3090 potentiated the inhibitory effects of both Ca2+ and Mg2+, decreasing the concentration of the divalent cation necessary to produce half-maximal inhibition of cAMP accumulation by about twofold. In the absence of extracellular Ca2+, however, TA-3090 had no effect on the stimulation of cAMP by dopamine or on the inhibition of dopamine-stimulated cAMP accumulation by PGF2a, which also regulates cAMP via G1. Finally, the effects of TA-3090 on the inhibition of cAMP by Ca2+ were totally abolished following preincubation with pertussis toxin for 20-24 h. These data suggest that TA-3090 not only modulates the function of bovine parathyroid cells at the level of Ca2+ channels per se but also may affect cAMP metabolism by potentiating the effects of high extracellular Ca2+ concentrations at or near the putative Ca2+ “receptor” or “sensor.”
Recombinant murine interferon-γ inhibits the fusion of mouse alveolar macrophages in vitro but stimulates the formation of osteoclastlike cells on implanted syngeneic bone particles in mice in vivo
Authors: Vignery Agnès , Niven-Fairchild Tracy , Shepard Michael H.
Published: February 2010

Osteoclasts are multinucleated cells that originate from the fusion of mononuclear precursors and are responsible for bone resorption. Indirect evidence from in vitro studies suggests that IFN-γ and TNF-α inhibit and stimulate bone resorption, respectively, but contradictory results have emerged from the literature regarding the effects of IFN-γ on macrophage multinucleation. Using highly sensitive model systems, the present work demonstrates that, in mice, rMuIFN-γ inhibits the fusion of alveolar macrophages in vitro but augments the number of osteoclastlike cells on implanted syngeneic bone particles in vivo. Although rMuTNF-α fails to stimulate macrophage multinucleation in either system, treatment of implanted animals with rMuIFN-γ appears to limit the inflammatory reaction and favor tissue repair.
Prostaglandin E2 inhibits formation of osteoclastlike cells in long-term human marrow cultures but is not a mediator of the inhibitory effects of transforming growth factor β
Authors: Chenu C. , Kurihara N. , Mundy G.R. , Roodman G. David
Published: February 2010

Prostaglandins are important local regulators of bone cell function and have been shown to have multiple effects on osteoclasts. Using a human bone marrow culture system in which multinucleated cells with osteoclast characteristics form, we have recently shown that TGF-β is a potent inhibitor of osteoclastlike cell formation and appears to act at several stages of their development. Because it has been suggested that the effects of TGF-β are mediated via a prostaglandin-dependent mechanism, we determined the effects of prostaglandin E2 (PGE2) on total and osteoclastlike cell formation (detected by reactivity with the 23c6 monoclonal antibody, which identifies osteoclasts) in human marrow cultures and tested whether prostaglandin synthesis was responsible for the inhibitory effects of TGF-β on multinucleated cell formation. These studies show that PGE2 is a potent inhibitor of both 23c6-positive and negative multinucleated cell formation in human marrow cultures and that the effects of TGF-β on multinucleated cell formation are not mediated by PGE2.
Mechanisms underlying the stimulation of PTH release by GppNHp in permeabilized bovine parathyroid cells
Authors: Leboff Meryl S. , Oetting Marguerite , Brown Edward M.
Published: February 2010

We examined changes in cAMP and inositol phosphate metabolism to assess the contribution of the guanine nucleotide regulatory (G) protein(s) regulating adenylate cyclase and phospholipase C in mediating the stimulatory effects of GppNHp on PTH release from permeabilized bovine parathyroid cells. To examine the role of Gs, the G protein stimulating adenylate cyclase, and cAMP on PTH release, permeabilized cells were incubated with either GppNHp or isoproterenol, and the effects of these agents on PTH release and cellular cAMP content were determined by RIA. To study the effects of GppNHp on inositol phosphate accumulation, permeabilized cells prelabeled with [3H]inositol were exposed to GppNHp, and inositol phosphates were measured using ion-exchange chromatography. These studies revealed that isoproterenol produced a dose-dependent increment in cAMP content in permeabilized cells with no significant effect on PTH release. Conversely, GppNHp rapidly and markedly elevated PTH release with a smaller and delayed rise in cAMP content. GppNHp- also promoted a dose-dependent increase in inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) accumulation, suggesting activation of phosphoinositide hydrolysis. Addition of dioctanoylglycerol, however, a synthetic diacylglycerol (DG) that activates protein kinase C, produced a much smaller increment in PTH release than GppNHp. Moreover, reducing the free calcium concentration to < 10−9 M by adding 10 mM EGTA to the permeabilization medium dissociated the effects of GppNHp and DG on secretion, increasing GppNHp-stimulated PTH release while reducing PTH secretion evoked by DG. In summary, (1) changes in cAMP content cannot account for the effects of GppNHp on PTH release; and (2) GppNHp increases the hydrolysis of phosphoinositides, but the addition of exogenous diacylglycerol does not mimic quantitatively the effects of GppNHp on hormone release. Thus, a G protein may be involved in the control of PTH release in permeabilized cells, perhaps by directly stimulating secretion at a locus distal to the regulation of cAMP or phosphoinositide metabolism.
Effects of parathyroid hormone, calcitonin, and dibutyryl-cyclic AMP on osteoclast area in cultured chick tibia
Authors: Pandalai Sudha , Dr. Gay Carol V.
Published: February 2010

Slices of osteoclast-enriched endosteal surfaces of 3 week chick tibia were cultured for 1-3 days. Osteoclasts on the bone surface were made visible by acridine orange fluorescence. Osteoclast area was measured by image analysis. Parathyroid hormone (PTH) caused osteoclasts to increase in area about 40%, and calcitonin (CT) caused a decrease in area also of about 40%. Subsequent addition of dibutyryl cyclic AMP to PTH- or to CT-treated cells resulted in a further change of 40 and 30%, respectively. Application of the cyclic AMP analog alone had no effect. All responses were rapid, occurring in 2-4 minutes.
Osteoclasts isolated from membranous bone in children exhibit nuclear estrogen and progesterone receptors
Authors: Pensler Jay M. , Radosevich James A. , Higbee Russell , Langman Craig B.
Published: February 2010

Osteoclasts were isolated from membranous bone from four children without metabolic bone disease who were undergoing craniotomy for either tumor or trauma. Both freshly isolated osteoclasts and those cultured for 4–7 days exhibited the following characteristics: production of tartrate-resistant acid phosphatase (9.5–14.8 units), contraction in response to application of 100 mg/ml of human calcitonin, and formation of resorption lacunae on devitalized bone wafers. Nuclear estrogen and progesterone receptors were demonstrated by immunohistochemical techniques and quantitated in two of the patients by radioimmunoassay (estrogen receptor RIA, 23.6 and 23.8 cpm/μg protein; progesterone receptor RIA, 36.7 and 74.2 cpm/μg protein). The demonstration of sex steroid hormone receptors in the nucleus of osteoclasts derived from children with normal membranous bone has estabished a potential mechanism whereby direct modulation of bone resorption by the sex steroid estrogen and progesterone may occur.
Bone mineral loss and recovery after 17 weeks of bed rest
Authors: Leblanc Adrian D. , Schneider Victor S. , Evans Harlan J. , Engelbretson David A. , Krebs Jean M.
Published: February 2010

The purpose of this work was to determine the rate and extent of bone loss and recovery from long-term disuse and in particular from disuse after exposure to weightlessness. For this purpose, bed rest is used to simulate the reduced stress and strain on the skeleton. This study reports on the bone loss and recovery after 17 weeks of continuous bed rest and 6 months of reambulation in six normal male volunteers. Bone regions measured were the lumbar spine, hip, tibia, forearm, calcaneus, total body, and segmental regions from the total-body scan. The total body, lumbar spine, femoral neck, trochanter, tibia, and calcaneus demonstrated significant loss, p < 0.05. Expressed as the percentage change from baseline, these were 1.4, 3.9, 3.6, 4.6, 2.2, and 10.4, respectively. Although several areas showed positive slopes during reambulation, only the calcaneus was significant (p < 0.05), with nearly 100% recovery. Segmental analysis of the total-body scans showed significant loss (p < 0.05) in the lumbar spine, total spine, pelvis, trunk, and legs. During reambulation, the majority of the regions demonstrated positive slopes, although only the pelvis and trunk were significant (p < 0.05). Potential redistribution of bone mineral was observed: during bed rest the bone mineral increased in the skull of all subjects. The change in total BMD and calcium from calcium balance were significantly (p < 0.05) correlated, R = 0.88.
T lymphocyte surface antigen markers in osteoporosis
Authors: Rosen Clifford J. , Usiskin Keith , Owens Marilyn , Barlascini Cornelius O. , Belsky Martin , Adler Robert A.
Published: February 2010

Two distinct syndromes of osteoporosis have been postulated: type I, which is characterized by accelerated bone loss occurring in women during the early postmenopausal period; and type II, an age-related process of bone loss affecting both men and women in and after the seventh decade. Recently there has been indirect evidence linking local products of the immune system with bone remodeling. We therefore studied peripheral blood profiles of specific lymphocyte phenotypes in women with type I osteoporosis and in older women and men with type II osteoporosis. The ratio of CD4-bearing (T helper) cells to CD8-bearing (T cytotoxic-suppressor) cells (CD4/CD8 ratio) was elevated in women with symptomatic type I disease. In addition there was a significant negative correlation (r = -0.62, p < 0.001) between the CD4/CD8 ratio and the spinal bone mineral density measured by dual-photon absorptiometry. In contrast, older men with a history of fracture (hip or spine) had CD4/CD8 ratios similar to control men. The number of T cells bearing IL-2R or VLA-1 was not different between osteoporotic subjects and controls in either men or women. This study supports the concept that local products of the immune system may be directly or indirectly involved in the pathogenesis of type I osteoporosis.
Effects of estrogen on daylong circulating calcium, phosphorus, 1,25-dihydroxyvitamin d, and parathyroid hormone in postmenopausal women
Authors: Packer Elizabeth , Holloway Leah , Newhall Katherine , Kanwar Gurinderjit , Butterfield Gail , Marcus Robert
Published: February 2010

We studied the effects of estrogen on daylong circulating levels of calcium, inorganic phosphorus, parathyroid hormone (PTH), and 1,25-(OH)2D3 (calcitriol) in a group of 10 postmenopausal women (68.5 ± 1.4 years, mean ± SEM). The study was conducted under strict dietary control, with mean calcium and phosphorus intakes of 845 and 970 mg. After treatment with conjugated equine estrogens, 1.25 mg/day, for 1 month, significant decreases in fasting (0800 h) serum levels were observed for calcium (9.09 ± 0.08 versus 9.46 ± 0.10 mg/dl, p < 0.01) and phosphorus (3.38 ± 0.10 versus 3.73 ± 0.08 mg/dl, p < 0.01). On the 0800 h fasting specimen, midmolecule PTH concentrations were higher (44.0 ± 7.9 versus 34 ± 8.2 pg/ml, p < 0.05), but intact PTH was unchanged (28.6 ± 2.7 versus 29.1 ± 1.7 pg/ml) and a rise in circulating calcitriol (39.8 ± 4.3 versus 31.6 ± 2.1 pg/ml) was marginally significant (p = 0.07). When data represented multiple samples averaged over 7 and 15 h, significant estrogen-related reductions in serum calcium and phosphorus concentrations were observed. In addition, estrogen was associated with a significant rise in the daylong (15 h) level of calcitriol (39.4 ± 4 versus 30.5 ± 2.4 pg/ml, p < 0.01). Daylong mid- and intact PTH concentrations were unchanged on estrogen compared to baseline values. No significant correlations were observed between changes in fasting calcitriol level and changes in fasting concentrations of calcium, phosphorus, or PTH. Further, the rise in daylong calcitriol concentration did not correlate significantly with changes in fasting or integrated values of calcium or PTH. However, the rise in calcitriol did correlate significantly with the decrements in 3 and 7 h serum phosphorus levels (Δcalcitriol versus Δ 7 h phosphorus, r = 0.65, p < 0.05; calcitriol fold-increase versus Δ 7 h phosphorus, r = 0.78, p < 0.01). We conclude that the rise in circulating calcitriol on estrogen appears to be related primarily to changes in blood levels of inorganic phosphorus, not to changes in calcium and PTH, the other primary regulators of calcitriol production.
Cellular responses to mechanical loading in vitro
Authors: Dr. Haj Alicia J. El , Minter Sarah L. , Rawlinson Simon C. F. , Suswillo Rosemary , Lanyon Lance E.
Published: February 2010

A technique has been established in which cancellous bone biopsies may be simultaneously perfused and subjected to mechanical load bearing. Assessments of cell viability over a period of 24 h were based on the cAMP response to parathyroid hormone, intracellular lactate dehydrogenase activity, and electron micrograph morphology. Two cellular responses to mechanical loading were demonstrated similar to those that follow “osteogenic” loading in vivo, as reported previously. These were (1) a rise in intracellular G6PD in lining cells immediately after loading, and (2) an increase in RNA synthesis measured in osteocytes 6 h after loading. In vivo the osteogenic response to loading was modulated by indomethacin. In these in vitro experiments, addition of indomethacin inhibited both the loading-related G6PD and the RNA responses.
Calcium metabolism in postmenopausal osteoporosis: The influence of dietary calcium and net absorbed calcium
Authors: Hasling C. , Charles P. , Jensen F. TaagehøJ , Mosekilde L.
Published: February 2010

A group of 85 females aged 48-77 years with postmenopausal crush fracture osteoporosis were investigated using a 7 day combined calcium balance and calcium47 tracer kinetic turnover study to assess the influence of dietary calcium and net absorbed calcium on bone metabolism. During the study, patients were on their habitual diet, as determined by a prestudy registration. Dietary calcium was measured after double serving of all the meals. All urine and feces were collected and analyzed for calcium content. Bone mineralization rate and bone resorption rate were determined by applying the continuously expanding calcium pool model to the tracer kinetic data.

Urine calcium excretion and net absorbed calcium were correlated (r = 0.64, p < 0.0001) with the following equation: urinary excreted calcium (mmol/day) = 2.4 + 0.4 x net absorbed calcium (mmol/day). Dermal calcium loss was not correlated with net absorbed calcium or urinary calcium. The net amount of absorbed calcium necessary to balance urinary and dermal losses was calculated to be 4.2 mmol calcium per day. The daily calcium intake necessary for obtaining a net absorbed calcium in excess of the urinary and dermal calcium losses and thereby ensure skeletal integrity was estimated to be 34.2 mmol calcium per day compared to an average intake of 27.9 + 7.6 (mean + SD) mmol/day.

Net absorbed calcium correlated negatively to bone resorption rate (r = -0.31, p < 0.005) and positively to bone mineralization rate (r = 0.29, p < 0.01) and to calcium balance (r = 0.66, p < 0.0001). Dietary calcium intake and calcium balance correlated positively (r = 0.38, p < 0.001). The minimum dietary calcium intake to ensure calcium balance was 34.5 mmol calcium per day when using this correlation.

We conclude that excess net absorbed calcium in postmenopausal osteoporosis is not quantitatively excreted in the urine but seems to induce a more positive calcium balance by reducing bone resorption rate and increasing bone mineralization rate. The recommended calcium allowance in Western countries does not meet the minimum dietary calcium requirement to ensure calcium balance in patients with postmenopausal osteoporosis when dermal calcium loss is also taken into account
Effects of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate on mouse osteoclasts
Authors: Dr. Marshall Michael John , Wilson Andrew Sean , Davie Michael William John
Published: February 2010

A group of 5-day-old mice were injected intraperitoneally with (3-amino-1-hydroxypropylidine)-1,1-bisphosphonate (APD). Morphologic changes were observed in vitally stained osteoclasts on parietal bones 3 days later, and these were judged to be degenerative. At this time significantly increased numbers of nuclei per osteoclast and total numbers of osteoclast nuclei were observed. However, at 4 days after the injection of APD, the total numbers of osteoclasts were significantly reduced relative to controls. When parietal bones were maintained in culture, APD reduced osteoclast numbers and inhibited cell-mediated 45Ca2+ release. Exposure of bones to parathyroid hormone increased the number of osteoclasts counted 1 day later. This effect was not blocked by APD. Calcitonin prevented the reduction in osteoclast numbers due to APD in vitro. We conclude that APD has a direct effect on resorbing mouse osteoclasts
Estrogen inhibits release of tumor necrosis factor from peripheral blood mononuclear cells in postmenopausal women
Authors: Ralston Stuart H. , Russell R. Graham G. , Gowen Maxine
Published: February 2010

Cytokines, such as interleukin-1 (IL-1) and tumor necrosis factors (TNF), produced by cells of the monocyte-macrophage lineage in the local bone microenvironment, are potentially important local regulators of bone turnover. To investigate whether the protective effects of estrogen against postmenopausal bone loss may be mediated by inhibition of cytokine release, we studied the effects of 17β-estradiol, dihydrotestosterone, and hydrocortisone on TNF release from human peripheral blood mononuclear cells (PBMC) in vitro. In unstimulated cells derived from eight postmenopausal women, seven of whom had osteoporotic vertebral fractures, 17β-estradiol inhibited TNF release in a dose-dependent manner between 10−6 and 10−12 M but had no consistent effect on cells derived from men or premenopausal women. Dihydrotestosterone in concentrations of up to 10−6 M had no effect on TNF release in any patient group, whereas hydrocortisone at 10−6 M was a potent inhibitor of TNF release in all groups.

Since TNF is a potent stimulator of bone resorption, the inhibitory effect of estrogen on TNF release may be part of the mechanism by which it exerts a protective effect on the skeleton in postmenopausal women. These observations may also be relevant in other inflammatory diseases of connective tissue, such as rheumatoid arthritis, in which disease activity may fluctuate as estrogen levels change — during the menstrual cycle, in pregnancy, and after the menopause.
Frequency distributions of tetracycline-based measurements: Implications for the interpretation of bone formation indices in the absence of double-labeled surfaces
Authors: Foldes Joseph , Shih Mei-Shu , Parfitt A. Michael
Published: February 2010

The frequency distributions of mineral apposition rate (MAR) and mineralizing surface (MS), measured separately on the intracortical, endocortical, and cancellous surfaces in 46 normal subjects and 79 patients with postmenopausal osteoporosis, indicated that MAR has a finite lower limit of 0.3 μ/day (uncorrected for section obliquity) but that MS has no finite lower limit. We conclude that in the absence of labels MAR, and indices derived from it, must be treated as missing values, but that MS and indices with MS in the numerator should be allowed to take values of zero. To avoid infinite values for indices with MS in the denominator, we propose that osteoid mineralization rate (the reciprocal of mineralization lag time) and osteoblast vigor (the reciprocal of formation period) be used instead. For surfaces with genuine single labels (SL) but no double labels, we propose that MS is calculated as SL/2 and that for MAR either the lower limit of 0.3 or the mean measured value from other surfaces be used for calculating derived indices.
Calcium absorptive consistency
Authors: Heaney Robert P. , Weaver Connie M. , Lee Fitzsimmons Mary , Recker Robert R.
Published: February 2010

Calcium absorption efficiency was measured two or three times each in 74 premenopausal and 142 postmenopausal women under conditions predicted to alter absorptive performance. A woman's absorptive consistency was evaluated across differing loads, differing intervals, and substances of differing intrinsic absorbability. In all these circumstances there was a statistically significant correlation between a woman's absorption under differing test situations accounting for up to 60% of the variance typically found in cross-sectional studies. For example, when the same substance but at differing load levels was tested three times over an 8 week period, various coefficients of correlation ranged from + 0.773 to + 0.849 (P < 0.001). Even over intervals as long as 5 years correlation of absorption fraction within individuals remained significant (r = +0.487, P < 0.001).
Effects of progesterone on postovariectomy bone loss in aged rats
Authors: Barengolts E.I. , Gajardo H.F. , Rosol T.J. , D'Anza J.J. , Pena M. , Botsis J. , Kukreja S.C.
Published: February 2010

The effects of progesterone on oophorectomy-induced bone loss in aged rats were evaluated. Female rats aged 12 months were divided into three groups: (1) sham-operated controls (SHAM); (2) oophorectomized (OVX); (3) OVX rats treated with progesterone (OVX + PROG). After 20 weeks the dry weight, bone ash, and calcium content of femur, tibia, and fourth lumbar vertebra were significantly lower in OVX than in sham rats. These reductions did not occur in OVX rats treated with PROG. There was no difference in the bone composition between the control and progesterone-treated rats. Vertebral bone histomorphometry showed increased bone resorption as well as increased bone formation parameters in OVX rats. Progesterone treatment inhibited the increased resorption indices, but the bone formation remained elevated. The results indicate that progesterone therapy prevents the postovariectomy bone loss in aged rats. The protective effect of progesterone is mediated by inhibition of bone resorption while maintaining the increased bone formation. These findings suggest that progesterone alone may be a valuable agent for management of postmenopausal osteoporosis.