bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of acute increases in bone matrix degradation on circulating levels of bone-gla protein
Authors: Dr. Riggs Lawrence B. , Tsai Kei-Sung , Mann Kenneth G.
Published: February 2010

Serum bone Gla-prolein (BGP), also called osteocalcin, is a specific and sensitive measure of bone turnover in a variety of metabolic bone disorders. Although some BGP diffuses into the circulation after synthesis by osteoblasts, most is incorporated into bone matrix where it remains until bone is resorbed. Thus, serum BGP could reflect bone formation, bone resorption, or a combination of both. The relationship of serum BGP to the components of bone turnover was evaluated in 18 normal women (mean age 48 yr; range 30-70) who received a continuous 24-h intravenous infusion of the 1-34 synthetic fragment of bovine parathyroid hormone. Mean ± SE for urinary hydroxyproline excretion, an index of bone resorption, increased (from 22.7 ± 2.2 to 38.5 ± 3.7 μg/100 ml glomerular filtrate [GF], p < .001), whereas levels of serum alkaline phosphatase, an index of bone formation, were unchanged (from 20 ± 1 to 20 ± 1 U/liter, NS). Despite the increase in bone resorption, levels of serum BGP decreased (from 8.8 ± 0.8 to 6.8 ng/dl, p < .001). The data suggest that circulating levels of BGP are a measure of bone formation but, at least in subjects with normal renal function, not a measure of bone resorption. Presumably BGP in bone matrix is degraded during osteoclastic resorption into fragments that either are not recognized by an antiserum raised against the native molecule or are rapidly cleared from the circulation.
Calcitonin receptor binding as a marker of osteoclast heterogeneity in osteopetrotic rodents
Authors: Rouleau Marie F. , Warshawsky Hershey , Marks Sandy C. , Goltzman David
Published: February 2010

We have employed a radioautographic technique to examine in vivo receptor binding of calcitonin to osteoclasts in four rodent mutants with osteopetrosis. 125I-Labeled calcitonin was injected intravenously alone or with excess unlabeled calcitonin to osteopetrotic (op/op), osteosclerotic (oc/oc), and microphthalmic (mi/mi) mice and to incisor absent (ia/ia) rats. Similar experiments were performed simultaneously in phenotypically normal littermates. Specific binding of calcitonin to receptors on osteoclasts and osteoclast morphology were then examined by light and electron microscope radioautography. Calcitonin binding was increased in mi/mi mice, where osteoclasts were abundant but reduced in size, and was also increased in op/op mice in association with an undulated and redundant osteoclast cell membrane. Binding of the hormone was markedly diminished on osteoclasts of oc/oc mice and ia/ia rats. Thus, in these rodent models of osteopetrosis all of which manifest reduced skeletal remodeling and share a recessive pattern of inheritance, considerable heterogeneity of osteoclast characteristics was demonstrable. Although calcitonin may play no primary pathogenetic role in most forms of this disease, calcitonin receptor binding is a morphological and functional marker of osteoclasts that can be used in assessing the pathophysiology of disorders of bone remodeling.
Attainment of therapeutic fluoride levels in serum without major side effects using a slow-release preparation of sodium fluoride in postmenopausal osteoporosis
Authors: Pak Charles Y.C. , Sakhaee Khashayar , Gallagher Christopher , Parcel Carol , Peterson Roy , Zerwekh Joseph E. , Lemke Martha , Britton Faye , Hsu Ming-Chue , Adams Beverley
Published: February 2010

The bioavailability, biochemical effects, and safety of a slow-release preparation of sodium fluoride were examined. In 8 normal volunteers, a single administration of slow-release sodium fluoride (25 mg) caused a slow rise and gradual decline in serum fluoride concentration, thus avoiding sharp peaks produced by a rapid-release preparation. In 37 patients with postmenopausal osteoporosis, serum fluoride concentration was kept within the “therapeutic window” (95-100 ng/ml) during long-term intermittent sodium fluoride (slow-release) therapy (25 mg twice/day, given for 3 months in each 5-month cycle over five cycles). Serum fluoride was also kept within the therapeutic window in 64 patients who took sodium fluoride (slow release) continuously over 12 months. Serum osteocalcin concentration increased progressively during fluoride treatment (correlation coefficient of 0.88, p < .001 for the relationship between serum osteocalcin and duration of therapy). Side effects to slow-release sodium fluoride therapy, assessed in 101 patients at two study sites, were minor and included diarrhea in 2 patients, nausea in 2 patients, abdominal pain and cramping in 2 patients, foot pain in 2 patients, and joint pain in 6 patients. Thus, slow-release sodium fluoride confers desired level of fluoride in serum, while providing safety of usage.
The effect of 1α-hydroxyvitamin D3 on the mineralization defect in disodium etidronate-treated paget's disease — a double-blind randomized clinical study
Authors: Ralston S.H. , Boyce B.F. , Cowan R.A. , Fogelman I. , Smith M.L. , Jenkins A. , Boyle I.T.
Published: February 2010

A double-blind randomized study of 29 patients with symptomatic Paget's disease was conducted comparing the clinical, biochemical, and histomorphometric responses to 3-month treatment with placebo (10 patients), low-dose disodium etidronate (EHDP) (5–7 mg/kg/day) (10 patients), and low-dose EHDP plus 1α-hydroxy-vitamin D3 (1αD3) 0.5 mcg daily (9 patients). In placebo-treated patients no significant changes were observed in symptoms, biochemistry, or bone histomorphometry. Histologically apparent mineralization defects developed after 3 months of therapy in 90% of patients in the EHDP group, compared with 45% of patients in the EHDP/1αD3 group. In 19% of the patients treated with active medication, the mineralization defects in pagetic bone were accompanied by histological evidence of continued osteoclastic resorption. The development of mineralization defects was not related to serum levels of vitamin D metabolites, alkaline phosphatase, or intestinal calcium absorption but did correlate with the occurrence of hyperphosphatemia during treatment, which was most marked in patients treated with EHDP alone. Although mineralization defects were less frequent in the EHDP/1αD3 group, these patients also responded less well symptomatically, thus limiting the potential usefulness of this drug combination in Paget's disease. It is suggested that the diminished response in the EHDP/1αD3 group and the lower frequency of mineralization defects may have been related to an alteration in the intestinal absorption of EHDP by the vitamin D metabolite.

Of patients treated with EHDP alone, 90% had symptomatic and biochemical improvement, suggesting that this agent is generally an effective and safe treatment in Paget's disease. However, in view of the high incidence of mineralization defects, we would continue to advise caution in the use of EHDP where there is significant deformity of, or lytic lesions, in weight-bearing bones.
Changes in the mineral density distribution in human bone with age: Image analysis using backscattered electrons in the SEM
Authors: Reid S.A. , Boyde A.
Published: February 2010

We report a study to test the feasibility of studying mineral density distributions in bone using the backscattered electron signal in scanning electron microscopy. Samples were human sixth ribs ranging in age from 8 weeks to 59 years, embedded in polymethylmethacrylate (PMMA), cut, polished, and carbon coated. The proportions of pixels falling in a uniform set of gray level slices of the BSE signal were determined using a microcomputer-based image analysis system interfaced directly to the SEM. The amount of high-density bone gradually increased with age at the expense of low-density bone, and there was an associated compression of the range of the mineral density distribution. Age-related differences were noted between the density distributions in the outer and inner rib cortices. The distribution in the inner cortex in neonates was influenced by the inclusion of densely mineralized endochondral bone and cartilage trabeculae formed at the growth cartilage zone. In adults it appeared that greater bone turnover occurred in the outer cortex, perhaps reflecting a differential mechanical loading across the rib. The technique enabled rapid, unbiased discrimination between the bone of neonates, children, and adults.
Rapid action of 1,25-dihydroxyvitamin D3 on calcium transport in perfused chick duodenum: Effect of inhibitors
Authors: Nemere Ilka , Dr. Norman Anthony W.
Published: February 2010

Transport of 45Ca from the lumen to the venous effluent was studied in duodena of normal, vitamin D3-replete (+D) chicks perfused through the celiac artery with 130 pM 1,25(OH)2D3 or vehicle. Administration of actinomycin D 3 h prior to perfusion did not alter the unstimulated transport rate or diminish the response to exogenous 1,25(OH)2D3: After 40 min exposure to the seco-steroid, 45Ca in the vascular effluent was 140% of control levels. The anti-microfilament agent cytochalasin b and the ionophore monensin, an inhibitor of Golgi function, similarly failed to suppress 1,25(OH)2D3-stimulated calcium transport. In pilot studies, Golgi and basal-lateral membrane fractions were prepared from duodenal epithelium of vitamin D-deficient (-D) chicks treated with vehicle or 650 pmol of 1,25(OH)2D3in vivo 2 h, 10 h, or 15 h before sacrifice, as well as from +D birds. Analyses of Golgi fractions for cathepsin B (CB) activity revealed a biphasic response with time, increasing to 200% of -D levels 2 h after 1,25(OH)2D3 administration and in equivalent preparations from +D birds. Less pronounced increases in acid phosphatase activity were observed in the same membrane fractions. In basal-lateral membranes, enhanced CB activity was detectable 10 h after 1,25(OH)2D3in vivo, rose to 155% of -D levels at 15 h, and to 245% of controls in fractions from +D birds, whereas acid phosphatase was 75%, 81%, and 125% of controls, respectively, at these times. Levels of CB in whole homogenates from which membranes were isolated rose to 200% of controls at 2 h and 10 h after 1,25(OH)2D3, but declined to 112% of controls in +D chicks. Acid phosphatase levels in the same homogenates were lower than or equivalent to control levels. These findings suggested additional perfusion studies in normal birds. Although vascular perfusion with iodoacetate and 1,25(OH)2D3, or leupeptin and 1,25(OH)2D3, failed to inhibit the effect of the vitamin D metabolite, 1,25(OH)2D3-enhanced calcium transport was abolished by addition of 1 mM leupeptin to the lumenal medium, but not by 1 mM pepstatin. The combined data suggest that lysosomes or their hydrolases may be involved in 1,25(OH)2D3-mediated transport of calcium across intestinal epithelial cells.
Microvascular organization at the epiphyseal-metaphyseal junction of growing rats
Authors: Arsenault A. Larry
Published: February 2010

The microarchitecture of the rat metaphyseal nutrient artery, the major blood supply to the calcifying epiphyseal growth plate, was studied by light microscopic serial sections, model reconstruction of serial sections, and scanning electron microscopy of plastic corrosion castings. These techniques illustrate how the metaphyseal artery undergoes extensive arborization and anastomosis upon route through the metaphysis. From this vascular network, closed capillary sprouts abut hypertrophic chondrocytes bordered by scaffolds of mineralized matrix.
Comparison of the effects of auranofin, gold sodium thiomalate, and penicillamine on resorption of cultured fetal rat long bones
Authors: Vargas Socorro J. , Jones Thomas G. , Hurley Marja M. , Raisz Lawrence G.
Published: February 2010

We compared three antirheumatic agents: auranofin (Aur), gold sodium thiomalate (GST), and penicillamine (Pen) for their effect on resorption in control unstimulated cultures of fetal rat long bones and in cultures stimulated by parathyroid hormone (PTH), prostaglandin E2 (PGE2), and murine interleukin-1 (mIL-1). Aur (3 × 10−6M) and GST (10−4M) inhibited PTH-stimulated bone resorption by 39 and 42%, respectively. The same concentrations of Aur and GST inhibited PGE2-stimulated bones by 72 and 44, respectively, and mIL-1-stimulated bones by 74 and 50%, respectively. Pen (10−4M) was not effective against any of the stimulators. Dose-response curves showed that Aur was at least 10 times more potent than GST. Inhibition by Aur was sustained after removal of the drug, while there was full recovery from GST. Aur inhibited 3H-thymidine and 3H-proline incorporation into bones, while GST had no effect. Aur and GST decreased β-glucuronidase activity to undetectable levels at five days of culture. Part of the therapeutic effectiveness of Aur and GST may reside in their ability to inhibit periarticular destruction by inhibiting PGE2- and IL-1-mediated osteoclastic bone resorption.
Evidence that calcium modulates circulating 25-hydroxyvitamin D in man
Authors: Dr. Bell Norman H. , Shaw Sheryl , Turner Russell T.
Published: February 2010

We previously demonstrated in normal subjects that 1,25-dihydroxyvitamin D3 (1,25(OH)2D) can prevent the increase in serum 25-hydroxyvitamin D (25-OHD) which occurs in response to vitamin D. An investigation was carried out in eight normal subjects, therefore, to determine whether increases in calcium intake would alter the response of serum 25-OHD to challenge with vitamin D. In control studies, vitamin D, 100,000 U/d for 4 d, significantly increased mean serum 25-OHD from 18 ± 3 to 42 ± 5 ng/ml (p < 0.001), an increment of 24 ng/ml (133%). Mean serum calcium, ionized calcium, phosphorus, creatinine, and 1,25(OH)2D did not change. In contrast, the same dose of vitamin D and calcium, 2,000 mg/d for 4 d, administered to the same eight subjects produced an increase in mean serum 25-OHD from 19 ± 3 to 31 ± 4 ng/ml (p < 0.001), an increment of only 12 ng/ml (63%) and significantly less than the control (p < 0.02). Mean serum calcium (8.8 ± 0.1 vs. 9.2 ± 0.1 mg/dl, p < 0.01) and ionized calcium (4.79 ± 0.07 vs. 4.85 ± 0.08 mg/dl, p < 0.05) increased significantly in response to vitamin D and calcium, mean serum phosphorus and creatinine did not change, and mean serum 1,25(OH)2D decreased significantly (37 ± 2 vs. 31 ±4 pg/ml, p < 0.02). In a postcontrol study in six of the normal subjects, vitamin D again significantly increased mean serum 25-OHD from 17 ± 3 to 39 ± 9 ng/ml (p < 0.02), an increment of 22 ng/ml (129%). Urinary calcium increased significantly on each of the four days of the administration of calcium as compared to the control study. The results provide evidence that changes in calcium intake can modulate the metabolism of 25-OHD in man.
Characterization of a human osteoblastic osteosarcoma cell line (SAOS-2) with high bone alkaline phosphatase activity
Authors: Murray Elsa , Provvedini Diego , Curran Deborah , Catherwood Bayard , Sussman Howard , Manolagas Stavros
Published: February 2010

We characterized the alkaline phosphatase activity of the human osteogenic sarcoma cell line, SAOS-2, and studied the regulation of this enzyme and 3',5'-cyclic adenosine monophosphate levels by 1,25-dihydroxyvitamin D3 and triamcinolone acetonide. We report that the basal alkaline phosphatase activity of SAOS-2 cells was 100–1000 times greater than that of other established human osteogenic sarcoma cell lines. The enzymatic activity was thermolabile, could be inhibited by levamisole and L-homoarginine, but not by L-phenylalanine, and was immunoprecipitable with anti-bone/liver/kidney, but not with anti-placental antibody, confirming that it is the tissue-unspecific or bone/liver/kidney isoenzyme. However, in contrast to other established human osteosarcoma cell lines (TE-85, SAOS-1), in which alkaline phosphatase activity is stimulated several-fold by the steroid hormones 1,25-dihydroxyvitamin D3 and hydrocortisone, the alkaline phosphatase activity of SAOS-2 cells was not affected by 1,25-dihydroxyvitamin D3 treatment despite the presence of classical receptors for this hormone. Furthermore, administration of the potent glucocorticoid analogue, triamcinolone acetonide, induced only a modest increase in activity. The SAOS-2 cell line expressed low basal cAMP levels (28 pmol/106 cells) which could be increased 25–40 times by pretreatment with parathyroid hormone. However, unlike other osteoblastic models, in which PTH-induced cAMP stimulation is modulated by 1,25-dihydroxy vitamin D3 and glucocorticoids, neither of these hormones had an effect on the PTH-stimulated cAMP levels in SAOS-2 cells. We conclude that the SAOS-2 cell line is an osteoblastic cell model which expresses high levels of tissue-unspecific alkaline phosphatase activity and exhibits limited responsiveness to two steroid hormones. These cells may prove useful in determining the structure and processing of alkaline phosphatase and in studying other features of the osteoblastic phenotype, such as cAMP production, and their hormonal regulation.
Influence of lactation and pregnancy + lactation on mechanical properties and mineral content of the rat femur
Authors: Peng Tai-Chan , Kusy Robert P. , Garner Sanford C. , Hirsch Philip F. , De Blanco Maria C.
Published: February 2010

The quality of bone was assessed from femurs of rats both during lactation and after pregnancy + lactation. Mechanical properties of stiffness, strength, toughness, and ductility were measured, along with standard measurements of dry weight, ash weight, and total bone mineral. No changes occurred during the first week of lactation. During the second and third weeks of lactation all bone parameters except ductility decreased significantly. These data are consistent with bone losing mineral in order to supplement the dietary calcium intake necessary for milk production. In other experiments, femurs were collected from nulliparous rats and from rats that had previously undergone 1–3 pregnancy + lactations. The largest changes in bone mineral and mechanical properties occurred after a single pregnancy + lactation period, although significant further decreases in stiffness and strength occurred after the second pregnancy + lactation. No additional losses occurred following the third pregnancy + lactation. Even 5 months after only one pregnancy + lactation period, the bone quality was still impaired as all bone properties were lower than in nulliparous controls. Because the changes, especially stiffness and strength, were relatively larger than the changes in dry and ash weights of bone, measurements of these mechanical properties provide a more sensitive method to evaluate the quality of bone.
Mechanism of fibroblast attachment to bone extracellular matrix: Role of a 44 kilodalton bone phosphoprotein
Authors: Dr. Somerman Martha J. , Prince Charles W. , Sauk John J. , Foster Ruth A. , Butler William T.
Published: February 2010

While the exact mechanisms regulating bone homeostasis are unknown, it is generally accepted that factors with the capacity to regulate cell attachment and spreading play a role in osteogenesis. A 44 kDa bone phosphoprotein (44K BPP), isolated from rat bone and synthesized by osteoblasts, was evaluated for its role in attachment and spreading of fibroblasts. In uncoated plates, enhanced cell attachment and spreading were observed when fibroblasts were exposed to the 44K BPP. The attachment properties of the bone phosphoprotein are different from those of fibronectin, in that the 44K BPP did not promote cell attachment in type I collagen wells, as was seen with fibronectin. Also, 44K BPP continued to enhance cell attachment up to 24 h, whereas cell attachment declined in time with cells exposed to fibronectin. Cycloheximide did not alter 44K BPP promotion of cell attachment, indicating that de novo protein synthesis was not required. These studies suggest that the 44K BPP is important in the regulation of cell attachment and spreading at sites of mineralization.
Effect of epidermal growth factor infusion on serum and urine calcium in mice
Authors: Dr. Shevrin Daniel H. , Lad Thomas E. , Kukreja Subhash C.
Published: February 2010

Transforming growth factors (TGFs) have been implicated in the pathogenesis of the hypercalcemia in malignancy (HM). In order to evaluate the role of these growth factors (epidermal growth factor (EGF) and TGF-α acting via the EGF receptor) in the development of HM, we studied the effect of 2 doses of EGF (0.1 and 0.3 μg/g/day) given for 7 days as a continuous infusion on serum and urine calcium in athymic mice. These infusions had no effect on serum and urine Ca values in this study. In order to assess the biological activity of the infused EGF, other known effects on gastric and pancreatic weights were evaluated. EGF-infused animals had significantly greater gastric and pancreatic weights than controls. Thus, EGF infusion into mice in doses which elicited known biological effects failed to have an effect on serum and urine Ca. An infusion of bovine parathyroid hormone 1–34 at the dose of 0.1 μg/g/day resulted in significant hypercalcemia.
The effect of low calcium diet, mithramycin, and dichlorodimethylene bisphosphonate on humoral hypercalcemia of malignancy in nude mice transplanted with the canine adenocarcinoma tumor line (CAC-8)
Authors: Dr. Rosol T.J. , Capen C.C.
Published: February 2010

The effect of a low calcium diet, mithramycin, or dichlorodimethylene bisphosphonate were evaluated in nude mice with humoral hypercalcemia of malignancy associated with the transplanted canine adenocarcinoma (CAC-8). Low calcium (0.01%) diet significantly reduced serum calcium levels in hypercalcemic nude mice and reduced urine calcium excretion to control levels. Mithramycin (8 mg/kg) decreased serum calcium concentration and urine calcium excretion to the range of control non-tumor-bearing nude mice at day 5 after a single injection, but there was no change in the number of tartrate-resistant acid phosphatase-positive osteoclasts in lumbar vertebrae. Osteoclasts from CAC 8-bearing nude mice after mithramycin administration were decreased in size, had small ruffled borders, and increased relative size of clear zones. Dichlorodimethylene bisphosphonate (Cl2MDP) (45 mg/kg) partially reduced serum calcium concentration of hypercalcemic tumor-bearing nude mice, decreased urine calcium excretion to control levels, and markedly reduced the numbers of tartrate-resistant acid phosphatase-positive osteoclasts in lumbar vertebrae. Osteoclasts from Cl2MDP-treated nude mice were smaller and had a reduced frequency of ruffled borders than saline-treated hypercalcemic nude mice. In vitro bone resorption induced by CAC-8 extract was significantly reduced by C12MDP and mithramycin. The results of these investigations suggest that the hypercalcemia and hypercalciuria associated with HHM in nude mice with CAC-8 are the combined result of altered calcium homeostasis in the bone, kidney, and intestine. Chemotherapeutic agents that specifically affect only bone or feeding a low calcium diet alone may not completely ameliorate the hypercalcemia of HHM.
Procollagen type I carboxy-terminal extension peptide in serum as a marker of collagen biosynthesis in bone. Correlation with iliac bone formation rates and comparison with total alkaline phosphatase
Authors: Parfitt A.M. , Simon L.S. , Villanueva A.R. , Krane S.M.
Published: February 2010

We measured iliac bone formation rates on all surfaces after double tetracycline labeling, serum levels of type 1 procollagen carboxy-terminal extension peptide (pColl-I-C), and serum levels of total alkaline phosphatase activity (TAP) in four normal subjects and in 44 patients with various forms of metabolic bone disease. In three patients with enzymatic evidence of liver disease both biochemical serum markers were disproportionately raised. In a patient with idiopathic axial osteosclerosis serum pColl-I-C was selectively increased by more than ten-fold. In the remaining 44 subjects pColl-I-C and TAP levels correlated significantly with each other (r = 0.70) and both showed the same directional changes and broadly similar correlations with iliac bone formation rate expressed in different ways. In general, pColl-I-C levels correlated better with cancellous bone formation rates and TAP levels with cortical bone formation rates. There was a modest improvement in prediction of bone formation rate with multiple regression using both markers. In 15 patients with typical uncomplicated postmenopausal osteoporosis, neither biochemical marker, singly or jointly, correlated significantly with any expression of bone formation rate. Disadvantages to the use of pColl-I-C as a marker include a significant contribution to the serum level from type 1 collagen biosynthesis in tissues other than bone, and (probably) variable metabolic clearance. For both biochemical markers the most consistently high correlations (r = 0.77–0.79) were found with total bone formation rate for the entire biopsy core volume, which is the best estimate available from a biopsy of formation rate at the bone organ level of organization in vivo. The core volume as a referent also allows the amount of bone formed on cortical, endocortical, and cancellous surfaces to be compared. Measurement of serum pColl-I-C levels merits further study as a noninvasive index of bone metabolism. Differences between normal and abnormal subjects in the relationships between a variety of biochemical markers and a variety of histologic indices have the potential for providing insight into the pathogenesis of osteoporosis.
Tamoxifen prevents the skeletal effects of ovarian hormone deficiency in rats
Authors: Turner Russell T. , Wakley Glenn K. , Hannon Kathleen S. , Bell Norman H.
Published: February 2010

To determine whether the nonsteroidal antiestrogen tamoxifen behaves as either an agonist or antagonist of estrogen on bone, the effects of ovariectomy, 17β-estradiol, and tamoxifen were compared on radial growth at the tibial diaphysis in young adult female rats. Ovariectomy and 17β-estradiol did not alter serum calcium, phosphate, or 25-hydroxyvitamin D. Ovariectomy increased serum 1,25-dihydroxyvitamin D in one experiment but not in the other. Tamoxifen increased the serum calcium and phosphate by itself and did not change serum 1,25-dihydroxy vitamin D in ovariectomized rats. Ovariectomy produced significant increases in medullary area, periosteal bone formation rate, and periosteal bone apposition rate compared to values in sham-operated animals and did not change endosteal bone formation rate. The increase in medullary area resulted from an increase in osteoclast number and resorbing surface length. Although endosteal forming surface length decreased, this was compensated for by an increase in the apposition rate. 17β-estradiol and tamoxifen each prevented the increases in bone formation rate and medullary area in ovariectomized rats. Tamoxifen reduced the length of the resorbing surface and osteoclast number to values observed in sham-operated animals. The findings demonstrate that in the rat, tamoxifen acts as an estrogen agonist by preventing the skeletal alterations that result from ovarian hormone deficiency.
J774A.1 macrophage cell line produces PDGF-like and non-PDGF-like growth factors for bone cells
Authors: Cheng S. -L. , Rifas L. , Shen V. , Tong B. , Pierce G. , Deuel T. , Peck William A.
Published: February 2010

In light of evidence that macrophages participate in the local regulation of bone remodeling, we have examined the production of peptide stimulators of bone cell growth and specialization by the J774A.1 macrophage cell line. Cultured J774A.1 cells secrete growth-promoting activities which have an affinity for heparin. The first partially purified materia), termed HEP I, appears to contain platelet-derived growth factor (PDGF)-like activity. It has a molecular weight of about 30,000 daltons, inhibits the binding of labeled PDGF to its receptors, reacts with polyclonal anti-human PDGF antibody, and exhibits mitogenic activity for osteoblasts, which is partially blocked by anti-PDGF antisera. Like PDGF, HEP I is active in a wide variety of mesenchyme-derived cells, including osteoblasts, chondrocytes, smooth muscle cells, fibroblasts, 3T3 cells and NRK cells. The J774A.1 cells contain mRNA, which hybridizes to a v-sis DNA probe, suggesting that they express the c-sis gene, which contains the code for a PDGF-like protein. The second factor, HEP II, has an approximate molecular weight of 20,000 daltons and possesses substantial mitogenic activity for osteoblasts, chondrocytes, and smooth muscle cells, but is not mitogenic for fibroblasts, 3T3 cells, and NRK cells. HEP II appears to be a unique bone cell mitogen, which is distinct from the growth factors presently known. Neither HEP I nor HEP II contained interleukin 1, a macrophage product known to promote bone resorption and perhaps the growth and activity of osteoblasts.
Total bone calcium in normal women: Effect of age and menopause status
Authors: Gallagher J.C. , Goldgar D. , Moy Alan
Published: February 2010

Bone density in different regions of the skeleton was measured in 392 normal women aged 20–80 years by dual photon absorpiometry. In premenopausal women, aged 25–50 years, multiple regression analysis of regional bone density on age, height, and weight showed a small significant decrease in total bone density (<0.01) but no significant change in other regions of the skeleton. In postmenopausal women there were highly significant decreases in all regions of the skeleton (p < 0.001), and bone density in these areas decreased as a logarithmic function of years since menopause.

Based on multiple regression analyses, the decrease in spine density and total bone calcium was 2.5–3.0 times greater in the 25 years after menopause than the 25 years before menopause. The largest change, however, occurred in the first five years after menopause. During this time the estimated annual change in spine density and total bone calcium was about 10 times greater than that in the premenopausal period. These results demonstrate the important effect of the menopause in determining bone mass in later life.
Bone turnover in spinal osteoporosis
Authors: Pødenphant Jan , Johansen Julia Sidenius , Thomsen Karsten , Riis Bente Juel , Leth Arne , Christiansen Claus
Published: February 2010

We have investigated biochemical indices of bone formation and bone resorption: serum alkaline phosphatase (sAP) plasma bone Gla protein (pBGP), fasting urinary hydroxyproline corrected for creatinine (FuHP/Cr), and fasting urinary calcium corrected for creatinine (FuCa/Cr) in 43 postmenopausal women with spinal fractures. Furthermore, histomorphometric indices of bone resorption and bone formation, as well as whole body retention (WBR) of 99m-technetium-diphosphonate (99mTc-DP), were determined. The results are compared to pre- and postmenopausal normal subjects. The results showed that indices of bone formation were mutually correlated except for sAP vs. WBR. sAP, WBR, and pBGP increased with age. sAP and WBR were not different between osteoporotics and age-matched controls, while pBGP and probably histological indices of bone formation were lower in osteoporotics than in age-matched controls. pBGP—and to a lesser extent sAP—were significantly correlated with all histological parameters reflecting bone formation. Finally, biochemical indices of bone resorption were high in osteoporotic patients and poorly correlated with histological bone resorption. The discrepancy between biochemical markers of bone formation may be related to the low sensitivity of sAP and WBR. Conversely, pBGP, sAP, and WBR may reflect different aspects of osteoblastic activity and bone mineralization. Finally, our data suggest that bone turnover increases with aging and that osteoporotic patients have higher bone resorption and probably lower bone formation than age-matched controls.
Evidence that stimulation of 1,25(OH)2D3 production in primary cultures of mouse kidney cells by cyclic AMP requires new protein synthesis
Authors: Korkor Adel B. , Gray Richard W. , Henry Helen L. , Kleinman Jack G. , Blumenthal Samuel S. , Garancis John C.
Published: February 2010

When primary culture of C75BL6 mouse cortical kidney cells in serum-free medium were incubated with unlabeled 25(OH)D3, they produced a metabolite which co-migrated with authentic 1,25(OH)2D3 and which could be measured by competitive receptor assay. A metabolite co-migrating with authentic 10-oxo-19-nor-25-OH-D3 was also produced. However, when cultures were incubated with 25(OH)D3 for 1 hour or longer, 10-oxo-19-nor-25-OH-D accounted for less than 15% of the total 3H-1,25(OH)2D3 displacement activity. Production of 1,25(OH)2D3 increased with increasing protein content of the cultures, with time of incubation, and with substrate concentration. The apparent Km was 1.4 ± 0.6 μM and Vmax 2.6 ± 0.4 pM/mg protein/hr. These cultures possessed a very high level of phosphodiesterase activity, as indicated by their high cyclic AMP (cAMP) response to IBMX. This high phosphodiesterase activity may have been responsible for the lack of stimulation of 1,25(OH)2D3 production by physiologic or near physiologic concentrations of parathyroid hormone (PTH) in the absence of IBMX. However, when IBMX 10−6M was present, bPTH 10−9M significantly increased production of both cAMP and 1,25(OH)2D3. There was a close correlation between 1,25(OH)2D3 production and cAMP content of the cultures (basal or stimulated). An incubation time of at least 4 hours was required for cAMP to increase 1,25(OH)2D3 production and was inhibited in the presence of cycloheximide and actinomycin D. This study further documents the regulation of renal 1,25(OH)2D3 synthesis by PTH in mammalian kidney and provides evidence for cAMP as a possibly important second messenger in this effect. The synthesis of 1,25(OH)2D3 in response to cAMP appears to depend upon the ability of the cell to synthesize new protein.
Interleukin-1β is a potent inhibitor of bone formation in vitro
Authors: Dr. Stashenko Philip , Dewhirst Floyd E. , Rooney Monique L. , Desjardins Laurie A. , Heeley John D.
Published: February 2010

The effect of interleukin-1β, the major component of osteoclast-activating factor (OAF), on bone formation by fetal rat osteoblast-rich cells was investigated. An in vitro culture system developed by Ecarot-Charrier et al. (1983) and Bellows et al. (1986) was utilized in which osteoblasts form mineralized nodules which closely resemble woven bone. Continuous exposure of cultures to homogenous IL-1β resulted in potent inhibition of mineralized nodule formation, which was half maximal at 0.1 U/ml (7.5 × 10−13M). Bone formation may thus be considerably more sensitive to IL-1β than is bone resorption (half maximal at 3.8 × 10−11M). Inhibition of bone formation occurred when cultures were exposed to IL-1β at both early and late time periods and was unaffected by the presence of indomethacin or by the osteoclast inhibitors calcitonin and γ-interferon. Instead, IL-1β exerted multiple inhibitory effects on osteoblast functions, including inhibition of collagen and noncollagen protein synthesis, alkaline phosphatase expression, and cell replication. On a dose response basis, the inhibition of protein synthesis correlated most closely with inhibition of bone formation. IL-1β is clearly inhibitory rather than stimulatory for bone formation as assessed in this system and is therefore unlikely to function as a coupling factor linking the processes of bone resorption and bone formation.
Production of hemopoietic growth factors by bone tissue and bone cells in culture
Authors: Dr. Felix R. , Elford P.R. , Stoercklé C. , Cecchini M. , Wetterwald A. , Trechsel U. , Fleisch H. , Stadler B.M.
Published: February 2010

This study was carried out to determine whether bone might be a source of hemopoietic growth factors. Both neonatal murine calvaria and primary cultures of cells isolated from calvaria released, upon stimulation with lipopolysaccharide, an activity that stimulated the growth of the interleukin (IL) 3-dependent cell lines, 32D cl, 123, and NSF 60. Upon gel filtration, this activity eluted with a molecular weight of 30,000 kDa. Further characterization, however, revealed that the major activity in conditioned medium was not IL 3. Activity was absorbed by DEAE-Sephacel at low salt concentration, whereas IL 3 does not adhere. Furthermore, an IL 3-specific antiserum did not neutralize the activity from cells and only partly neutralized the activity generated by whole calvaria. After gel filtration, the 30-kDa activity stimulated the growth of very large colonies in semisolid medium consisting mainly of granulocytes with the remainder being macrophages. No colony types belonging to other hemopoietic lineages were found, indicating, again, that the activity was not identical to IL 3. Subsequently, conditioned medium was fractionated by hydrophobic chromatography on Phenyl-Sepharose CL-4B, yielding two peaks of activity. Neutralization of activity with antisera to granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL 3 and use of colony assays showed that medium conditioned by whole calvaria contained GM-CSF and granulocyte CSF (G-CSF) in similar amounts together with a little IL 3, and medium conditioned with calvaria cells contained GM-CSF and little G-CSF. We conclude that bone releases hemopoietic growth factors that could contribute both to hemopoiesis and to the recruitment of osteoclasts from progenitors resident in the adjacent marrow.
Bone status of senescent male rats: Chemical, morphometric, and mechanical analysis
Authors: Kiebzak Gary M. , Smith Ronald , Gundberg Caren C. , Howe Juliette C. , Sacktor Bertram
Published: February 2010

The bone status of male rats 6, 12, and 24 months of age (n = 10) was examined. Femur calcium (Ca), phosphorus (P), and osteocalcin contents; serum chemistry; and mechanical properties of the bone were measured and correlated. Diaphyseal Ca, P, and osteocalcin contents were not different in animals 6 and 12 months of age but decreased significantly at 24 months: -7.4% for Ca, -4.2% for P, and -24% for osteocalcin compared to 12 months. Femurs from 24-month-old (senescent) rats were characterized by a scalloped appearance of the midfemoral endosteal surface and by cortical porosities. These age-associated changes coincided with nearly two-fold increases in serum immunoreactive parathyroid hormone (PTH) and osteocalcin. Serum Ca did not change with age, whereas serum P decreased (-14.8%) from 6 to 24 months. Maximum breaking force required to fracture femurs at midshaft did not change with age. Hence, the strength of the femur as an intact organ was not compromised with age despite the loss of diaphyseal Ca and P in the senescent animal. However, ultimate stress, a parameter that normalizes for differences in bone geometry and size, decreased 35% in femurs from 12- compared with 24-month-old animals. These mechanical results might be explained by the morphometric finding that, in contrast to the small but progressive age-associated increases in femur weight and length, the cortical and medullary areas increased at least two-fold. Therefore, the strength of the intact femur was maintained by architectural compensations, even though normalized tissue strength decreased with age. These findings suggest that bone status was compromised in the aged male rat.
1,25-dihydroxyvitamin D3 stimulates avian and mammalian cartilage growth in vitro
Authors: Burch Warner M. , Lopez-Claros Marcelo , Uskokovic Milan R. , Drezner Marc K.
Published: February 2010

We addressed the question of whether 1,25-dihydroxyvitamin D3 (1,25-(OH)2D) could directly stimulate cartilage growth in vitro. Pelvic leaflets from chick embryos and scapular growth plates from fetal pigs were organ cultured in serum-free medium in the presence and absence of 1,25-(OH)2D. After 3 days of incubation, 1,25-(OH)2D had increased the pelvic cartilage wet weight 42% and the dry weight 32% above the weight of cartilages incubated in medium alone. 1,25-(OH)2D (10−9M-10−12M) caused a dose-dependent increase in weight, with maximal increases at 10−9M. Furthermore, two deuterized derivatives of 1,25-(OH)2D, 26,27-D6-1,25-(OH)2D3 and 24,26,27-D8-1,25-(OH)2D3, stimulated pelvic cartilage growth in vitro. 26,27-D6-1,25-(OH)2D3 increased cartilage growth at concentrations as low as 10−13M. Growth plates from fetal pig scapulas were weighed then organ-cultured for 2 weeks in the presence and absence of 1,25-(OH)2D. 1,25-(OH)2D stimulated increases in growth plate weight above growth plates incubated in medium alone. 26,27-D6-1,25-(OH)2D3 appeared to be potent at lower concentrations than 1,25-(OH)2D on growth plate cartilage. Thus, 1,25-(OH)2D stimulated in vitro growth in two growing cartilage models, the avian pelvic cartilage and the mammalian scapular growth plate cartilage.
Effect of short-term glucocorticoids on serum osteocalcin in healthy young men
Authors: Godschalk Michael F. , Downs Robert W.
Published: February 2010

Young healthy men were studied during brief treatment with prednisone to determine the rapidity of the effects of glucocorticoids on serum osteocalcin. Seven subjects were given 60 mg of prednisone orally at 8 a.m. on 5 consecutive days. Serum osteocalcin fell to 68% of the pretreatment level within 24 hours after the first dose was administered (p < 0.01) and reached a nadir of 37% of baseline between 48 and 96 hours after treatment was begun (p < 0.005). When prednisone was discontinued, serum osteocalcin returned promptly to pretreatment levels. Similar, though less marked, effects were found with lower doses of prednisone. Serum osteocalcin was not different from baseline after 5 mg of prednisone in five subjects, but after treatment of five subjects each with 10, 15, or 20 mg of prednisone, osteocalcin levels were 83%, 78%, and 74% of baseline, respectively (p < 0.05). Serum osteocalcin levels fell rapidly with glucocorticoid administration, indicating that the effects of glucocorticoids on bone cells may be demonstrated long before clinical evidence of osteoporosis becomes apparent.
Bone mineral and glycosaminoglycans in newborn and mature rabbits
Authors: Grynpas Marc D. , Hunter Graeme K.
Published: February 2010

The relation between bone mineralization and glycosaminoglycans (GAG) was investigated in newborn and mature rabbit diaphyseal bone. Using the density fractionation technique, the bone was separated into fractions of increasing density from 1.4 to 2.3 grams/ml. Each fraction was analyzed by X-ray diffraction to determine the average crystal size. The GAG content of each fraction and of the unfractionated bone was determined by direct extraction and on a few fractions by sequential extraction in guanidine hydrochloride and guanidine/EDTA. There was a decrease in the GAG content with animal age and with increasing fraction density in the newborn rabbit. In one overlapping fraction (2.0–2.1 grams/ml), the GAG content was twice as high in the newborn as in the mature animal. Finally, the crystal size substantially increased from newborn to mature rabbits. Therefore, calcification and maturation of bone is associated with a decrease in the proteoglycan content of the organic matrix.
Effects of fluoride on parathyroid hormone secretion and intracellular second messengers in bovine parathyroid cells
Authors: Chenl Chu J. , Anast Constantine S. , Brown Edward M.
Published: February 2010

Fluoride ion (F−) alone or in conjunction with aluminum (Al3+) has been shown to stimulate the activity of guanine nucleotide-binding proteins (G proteins) in cell membrane preparations from a variety of cell types and in intact hepatic cells. Several studies have indicated that G proteins are involved in the regulation of parathyroid hormone (PTH) secretion. Intracellular second messengers which modulate PTH secretion (e.g., cAMP) have also been found to be regulated by G proteins. We have, therefore, employed F− as a probe to investigate the possible role of G proteins in the modulation of PTH release and the intracellular second messengers that have been implicated in the control of PTH secretion. F− produces a dose-dependent inhibition of PTH release with a maximal inhibitory effect (67%) at 5 mM. F− exerts its inhibitory effect within 5 min and the degree of suppression of PTH secretion gradually increases over 1 hr. F− (5 mM) inhibits PTH secretion at 0.5 mM Ca2+ to the level observed with 2 mM Ca2+ alone; moreover, the effects of F− and high Ca2+ are not additive. While 1 mM F− suppresses PTH secretion by only 21%, and 10 μM Al3+ has virtually no effect at all, together they inhibit PTH release approximately to the level (63% inhibition) observed with 5 mM NaF alone. In the presence of 10−5M dopamine, F− produces a concentration-dependent inhibition of cAMP accumulation (0.684 ± 0.033 pmoles/105 cells at 0 mM F−vs. 0.256 ± 0.048 at 5 mM F−). However, the F−-induced decrease in cAMP cannot account for the inhibition of PTH release by this agent, since addition of methylisobutylxanthine (10−4M) by F− -treated cells raises intracellular cAMP content above that of control cells but fails to reverse the inhibition of PTH release. The cytosolic calcium concentration in Fura-2-loaded cells increases from 210 ± 20 nM to 340 ± 44 nM after 5 mM F− was added to incubation media. Prior removal of extracellular Ca2+ by EGTA totally blocks the F−-induced rise in cytosolic Ca2+ without preventing the inhibition of PTH release by NaF. F− also produces a time- and dose-dependent increase in the accumulation of IP, IP2, and IP3 in cells prelabeled with [3H]inositol and incubated with 10 mM Li+, consistent with activation of phospholipase C. We conclude that F− is a potent inhibitor of PTH secretion. Its action is potentiated by Al3+, consistent with mediation via G protein(s). F− also mimics the effects of high Ca2+ on several intracellular second messengers in parathyroid cells (e.g., cAMP, cytosolic Ca2+, and IP3), presumably through activating G proteins, although the changes in these mediators may not be directly linked to inhibition of secretion. The tumor promoter TPA, an activator of protein kinase C, reverses the inhibition of PTH release by 2 mM Ca2+ but has no effect on the inhibition of secretion by F−, suggesting that F− may also affect G proteins related to secretion at a locus distal to the protein kinase C system.
Characterization of isolated and cultured chick osteoclasts: The effects of acetazolamide, calcitonin, and parathyroid hormone on acid production
Authors: Hunter Susan J. , Schraer Harald , Dr. Gay Carol V.
Published: February 2010

The effects of acetazolamide, calcitonin (CT), and parathyroid hormone (PTH) on acid production in isolated osteoclasts has been investigated. Osteoclasts were isolated from the endosteum of 3-week chick tibias and were maintained under culture conditions for 5 days. The cells were treated with acetazolamide (10×4M and 10−7M), CT (1 mU/ml and 0.31 mU/ml) and PTH (6.5 U/ml and 0.40 U/ml) for 1, 3, 6, and 18 hr. The cells were stained with acridine orange and the intensity of fluorescence measured by a light microscope photometer. Acetazolamide treatment resulted in a steady decline in intracellular acidity, suggesting that carbonic anhydrase plays a major role in acid production in isolated osteoclasts. Treatment with PTH produced a decline in acidity at 1 hr, followed by a peak at 3 hr and then a decline at 6 and 18 hr. The transient increase in acidity may be due to activation of carbonic anhydrase by PTH. Calcitonin treatment also resulted in a decline in cell acidity which was similar, but less pronounced than that resulting from acetazolamide treatment. These results indicate that calcitonin may mediate osteoclast activity by alterations in intracellular acid production.