Importance of bone mineral density measurements in evaluating fragility bone fracture risk in Asian Indian men.

UNLABELLED: We evaluated the effect of BMD on fracture risk prediction using FRAX® among Asian Indian men when used in conjunction with clinical risk factors. A majority of our subjects were either osteopenic or osteoporotic, and their fracture risk increased when FRAX® was used in conjunction with femur neck T-scores. INTRODUCTION: Asian Indian men living in the United States may represent a population that is at high and underappreciated risk for fragility bone fractures. PURPOSE: To evaluate the effect of BMD on fracture risk prediction using FRAX® among Asian Indian men when used in conjunction with clinical risk factors. METHODS: Forty four Asian Indian men (mean age 64.9 (±8.4) years) who had lived in the United States for an average of 33.6 (±10.6) years underwent BMD measurement at the proximal femur. Subjects were subjected to a general physical exam and history of fracture, hip fracture in a parent, current smoking and alcohol use, and diagnosis of inflammatory arthritis was obtained. Data from each subject were entered into the FRAX® algorithm and 10-year fracture probabilities were calculated using clinical risk factors (CRFs) alone and in combination with femur neck T-scores. RESULTS: Thirteen subjects (29.5%) had femur neck T-scores ≥ -1.0, 28 (63.6%) T-scores between -1.0 and -2.5, and three (6.8%) T-scores < -2.5. The 10-year probability of a major osteoporotic fracture based on a combination of clinical risk factors and femur neck T-scores was significantly higher than the fracture probability based on clinical risk factors alone (t(43) = 2.58, p = 0.01). CONCLUSIONS: Among Asian Indian men, the 10-year probability of a major osteoporotic fracture increases when femur neck T-scores are added to clinical risk factors in the FRAX® algorithm, and this population have a high fracture probability even in the absence of clinical risk factors.

Studies on congenital osteopetrosis in microphthalmic mice using organ cultures: impairment of bone resorption in response to physiologic stimulators.

The mechanism of congenital osteopetrosis in microphthalmic (mi) mice has been examined in bone organ cultures. Resorption was measured by the release of previously incorporated 45Ca in fetal long bones and newborn calvaria from mi mice and heterozygous or homozygous normal litter mates. Bones from mi mice showed a generalized resorption defect with decreased spontaneous or control resorption and failure to respond to parathyroid hormone (PTH), prostaglandin E2, 1,25 dihydroxy vitamin D3, vitamin A, or osteoclast activating factor (OAF) from human peripheral leukocytes or mouse spleen cells. Bones from heterozygotes showed a smaller response to PTH than bones from homozygous normals. Mutant bones failed to show an increase in lysosomal enzyme release in response to PTH or vitamin A, agents which increased release from bones of homozygous normals. Proline incorporation into collagenase-digestible protein was similar in cultures of normal and mutant bone and was inhibited by PTH and OAF. These results indicate that congenital osteopetrosis in mi mice is due to a generalized defect in the function and hormonal response of osteoclasts and suggests that this cell line is separate from the osteoblast cell line which shows no impairment of hormonal response.

The effects of parathyroid hormone, colchicine, and calcitonin on the ultrastructure and the activity of osteoclasts in organ culture.

The ultrastructure of osteoclasts was examined in fetal rat bones after stimulation or inhibition of resorption in culture. A central ruffled border area completely encircled by a clear zone was considered to represent the resorbing system of the cell. The proportion of ruffled border and clear zone in osteoclast cross sections was compared with changes in bone resorption as measured by the release of previously incorporated radioactive calcium ((45)Ca). In control cultures 55% of the osteoclast cross sections showed an area closely apposed to bone and this consisted mainly of clear zone; only 11% showed ruffled borders. Treatment with parathyroid hormone (PTH) increased (45)Ca release, increased the frequency of finding areas closely apposed to bone (79%), and markedly increased the frequency of the ruffled border area (64%). Colchicine given concurrently with PTH decreased the number of osteoclasts. Colchicine or calcitonin treatment after PTH stimulation decreased the proportion of ruffled border area significantly by 1 h; this was followed by a decrease in (45)Ca release. These inhibited osteoclasts resembled osteoclasts from control, unstimulated cultures, suggesting that the cells had returned to their inactive state. Colchicine-treated osteoclasts also showed a loss of microtubules and a massive accumulation of 100 A filaments, suggesting that synthesis of microtubular subunits had increased.

Divalent cation ionophores stimulate resorption and inhibit DNA synthesis in cultured fetal rat bone.

Two divalent cation ionophores, A23187 and Ionomycin, which are selective for calcium, stimulated the resorption of fetal rat long bones in organ culture at 0.1 to 1 micromolar but not at higher concentrations. Both agents inhibited DNA synthesis at concentrations that stimulated resorption. These results might explain the differences in ionophore effects on bone previously reported, and they imply that cell replication is not required for osteoclast formation in fetal rat long bone cultures.

Stimulation of DNA and collagen synthesis by autologous growth factor in cultured fetal rat calvaria.

Conditioned medium derived from organ or cell cultures prepared from 19- to 21-day fetal rat calvaria stimulated the incorporation of [3H]proline collagen and of [3H]thymidine into DNA in organ cultures of the same tissue. Addition of cortisol enhanced the effect on collagen but not on DNA synthesis. These effects appeared to be due to a nondialyzable and heat-stable growth factor.

Endotoxin: stimulation of bone resorption in tissue culture.

Bacterial endotoxins can stimulate the release of previously incorporated calcium-45 and tritiated proline from fetal rat bone in tissue culture. Endotoxin from Bacteroides melaninogenicus, an organism regularly found in the gingival crevice of man, produces a response similar to parathyroid hormone and is effective at doses as low as 0.1 microgram per milliliter. This response is inhibited by serum and dependent upon the presence of albumin. Endotoxins may play a role in the bone loss characteristic of human periodontal disease.

1,25-dihydroxycholecalciferol: a potent stimulator of bone resorption in tissue culture.

1,25-Dihydroxycholecalciferol (DHCC), isolated from kidney homogenates incubated with 25-hydroxycholecalciferol (HCC), stimulated the release of previously incorporated (45)45Ca from fetal rat bones in organ culture, at concentrations of 10(-10) to 10(-8)M. The dose response curves for 1,25-DHCC and 25-HCC, the parent compound, are parallel, but 1,25-DHCC is about 100 times as potent on a weight basis. Brief exposure to maximum doses of either agent leads to prolonged bone resorption.

25-Hydroxycholecalciferol: stimulation of bone resorption in tissue culture.

25-Hydroxycholecalciferol stimulates release of previously incorporated calcium-45 from fetal rat bones in doses of 0.9 to 27 units per milliliter. This effect cannot be produced by much larger doses of vitamin D(3). Comparison of stimulation of bone resorption by 25-hydroxycholecalciferol and parathyroid hormone reveals similarities with respect to time course, dose-response slope, and inhibition by calcitonin.

Complement-dependent stimulation of prostaglandin synthesis and bone resorption.

Complement-sufficient heterologous serum induced prostaglandin synthesis and resultant resorption in cultures of fetal rat long bones. Bone resorption was enhanced with unheated normal rabbit serum as compared to heated serum or serum from rabbits lacking the sixth component of complement (C6). Addition of functionally purified C6 restored resorptive activity in C6-deficient serum. Concentrations of prostaglandin E were increased in the culture media of bones incubated with complement-sufficient serum. The resorptive effects of active serum as well as the appearance of prostaglandin E in the media were inhibited by indomethacin.

Bone resorbing activity in supernatant fluid from cultured human peripheral blood leukocytes.

A new soluble mediator was found in supernatant fluid from cultures of human peripheral blood leukocytes that were stimulated by phytohemagglutinin, or by antigenic material present in human dental plaque deposits. This soluble Jactor produced bone resorption in organ cultures of fetal rat bones as measured by increased release of calcium-45, and also increased the number of active osteoclasts.

Restoration of parathyroid responsiveness in vitamin D-deficient rats by parenteral calcium or dietary lactose.

The effects of treatment with vitamin D, calcium, or lactose on the responsiveness of vitamin D-deficient rats to parathyroid hormone were compared. In the absence of vitamin D, parenteral calcium or dietary lactose administration resulted in increases in serum calcium concentration although not to the normal values obtained in animals given vitamin D. Dietary lactose also partially restored the low bone calcium content of vitamin D-deficient rats. Untreated vitamin D-deficient rats showed no significant changes in serum calcium concentration after parathyroidectomy or parathyroid extract administration. Vitamin D, lactose, and calcium all restored responsiveness to parathyroid hormone; serum calcium concentration decreased after parathyroidectomy and showed a dose-related increase in response to parathyroid extract. Hence, the unresponsiveness to parathyroid hormone in vitamin D deficiency may be due to a lack of calcium at a local site of action, presumably bone, rather than to the absence of vitamin D as a specific cofactor.

DNA synthesis is not necessary for osteoclastic responses to parathyroid hormone in cultured fetal rat long bones.

Osteoclasts, the principal cells mediating bone resorption, are believed to increase their size, number, and resorbing activity in response to parathyroid hormone (PTH) through mechanisms dependent upon the fusion of specific mononuclear precursor cells into either new or existing multinucleated osteoclasts. To address the question of whether these actions of PTH are dependent on the replication of osteoclast precursor cells, we examined the ability of an inhibitor of DNA synthesis, hydroxyurea (HU), to alter bone resorption, osteoclast formation, and DNA synthesis in cultured fetal rat bones treated with PTH. We found that HU significantly reduced [3H]thymidine incorporation into the bones and labeling of osteoclast nuclei by greater than 90%, but did not prevent PTH from stimulating bone resorption, measured as the release of 45Ca, or from increasing the number of osteoclasts in the bones. In bones cultured without PTH, HU decreased the rate of bone resorption, but not the number of osteoclasts per bone. We conclude that in fetal rat bone cultures, PTH can increase osteoclast number and stimulate bone resorption by affecting existing osteoclasts and osteoclast precursors, and that replication of osteoclast precursor cells is not necessary for PTH to stimulate a resorptive response. In unstimulated cultures it appears that HU inhibits bone resorption by affecting mechanisms that are independent of changes in osteoclast number and that may be influenced by cell replication or other unknown factors.

Partial purification of osteoclast-activating factor from phytohemagglutinin-stimulated human leukocytes.

Osteoclast-activating factor (OAF) is a soluble mediator found in supernates of human peripheral leukocytes which have been cultured with antigens or phytomitogens. OAF is a potent stimulator of osteoclastic resorption of fetal bone in organ culture. The present studies were designed to characterize OAF chemically. Bone resorbing activity from supernates of leukocytes cultured without added plasma was not lost on dialysis using a membrane with a molecular weight cutoff of 3,500, but was lost when heated to 60 degrees C for 30 min. The activity was lost after treatment with trypsin or pronase but not after treatment with ribonuclease or neuraminidase. Papain, which inactivated parathyroid hormone at a concentration of 25 mug/ml, did not inactivate OAF at 250 mug/ml. OAF did not react with an antibody to bovine parathyroid hormone which cross-reacts with human parathyroid hormone. OAF was also distinguished from active metabolites of vitamin D and from prostaglandin by extraction procedures and immunoassay for prostaglandin E(2). When the medium from activated leukocytes cultured with autologous plasma was fractionated by gel filtration on Sephadex, bone resorbing activity eluated both with plasma proteins and in lower molecular weight fractions. However, when medium from leukocytes cultured without added plasma was chromatographed, all the OAF activity was eluted in a sharp low molecular weight peak located between chymotrypsinogen (25,000 molecular weight) and ribonuclease A (13,700 molecular weight). This peak contained about 4% of the total protein originally present in the supernate. Its activity was destroyed by overnight incubation at 37 degrees C at pH 6 or 8, but not at pH 7.2. After incubation at 4 degrees C, the activity was lost at pH 3 or 10, but not at pH 4-9. The active fraction from Sephadex G-100 was therefore chromatographed at pH 7.2 on DEAE cellulose and carboxymethyl cellulose. The active material was not adsorbed; however, about sevenfold further purification was achieved by removal of contaminants. The material obtained after sequential Sephadex, DEAE and, carboxymethyl cellulose chromatography stimulated resorption of fetal rat bone in culture at concentrations of 0.75-3 mug protein/ml, indicating that this preparation of OAF was nearly as potent as bovine parathyroid hormone in this system.

Effects of DNA and prostaglandin synthesis inhibitors on the stimulation of bone resorption by epidermal growth factor in fetal rat long-bone cultures.

We examined two inhibitors of DNA synthesis, hydroxyurea (HU) and aphidicholin (APC), and two inhibitors of prostaglandin cyclooxygenase, indomethacin and flufenamic acid, for their effects on the resorptive responses of fetal rat long-bone cultures to epidermal growth factor (EGF) and parathyroid hormone (PTH). As we have previously found, HU decreased unstimulated 45Ca release but had little effect on the resorptive response to PTH. HU also did not block resorption stimulated by EGF. Addition of the cyclooxygenase inhibitor, indomethacin, did not alter the resorptive responses of unstimulated or PTH-treated cultures in either the presence or absence of HU or the resorptive response of bones cultured with EGF alone. However, indomethacin completely blocked the resorptive response to EGF of bones that were cultured with HU. The effects of indomethacin on EGF-mediated resorption in HU-treated cultures appeared to be related to an inhibition of prostaglandin synthesis since flufenamic acid had similar effects. However, the effects of HU on the resorptive response to EGF may not have resulted solely from its inhibitory action on DNA synthesis since APC, in the absence of cyclooxygenase inhibitors, completely blocked EGF-mediated resorption without significantly affecting the response to PTH. These results demonstrate that the mechanisms regulating PTH- and EGF-mediated resorption in fetal rat long-bone cultures differ, and imply that a component of EGF-mediated resorption in these cultures is dependent on sustained DNA synthesis.

Hormone synthesis and secretion by rat parathyroid glands in tissue culture.

Rat parathyroid glands maintained in organ culture secrete biologically active parathyroid hormone (PTH) and synthesize and secrete labeled proteins from (3)H- or (14)C-labeled amino acids added to the medium. The amounts of biological activity and labeled protein in the medium are both inversely proportional to the calcium concentration. Some of the labeled low molecular weight protein was identified as PTH which had been synthesized and secreted in culture by preliminary isolation on Sephadex G-100 columns and further purification using an antibody to bovine PTH which cross-reacted with rat PTH. The cross-reacting antibody inhibited the biological effects of rat PTH and caused hypocalcemia in intact rats. The antibody bound some of the labeled low molecular weight protein of the medium at neutral pH so that it migrated as a large molecular weight complex on Sephadex. Biologically active, labeled PTH was recovered by dissociation of this complex in acid and rechromatography.

Prostaglandin G/H synthase-2 is required for maximal formation of osteoclast-like cells in culture.

We examined the effect on osteoclast formation of disrupting the prostaglandin G/H synthase genes PGHS-1 and-2. Prostaglandin E(2) (PGE(2)) production was significantly reduced in marrow cultures from mice lacking PGHS-2 (PGHS-2(-/-)) compared with wild-type (PGHS-2(+/+)) cultures. Osteoclast formation, whether stimulated by 1,25-dihydroxyvitamin D(3) (1,25-D) or by parathyroid hormone (PTH), was reduced by 60-70% in PGHS-2(-/-) cultures relative to wild-type cultures, an effect that could be reversed by providing exogenous PGE(2). Cultures from heterozygous mice showed an intermediate response. PGHS inhibitors caused a similar drop in osteoclast formation in wild-type cultures. Co-culture experiments showed that supporting osteoblasts, rather than osteoclast precursors, accounted for the blunted response to 1,25-D and PTH. This lack of response appeared to result from reduced expression of RANK ligand (RANKL) in osteoblasts. We cultured spleen cells with exogenous RANKL and found that osteoclast formation was 50% lower in PGHS-2(-/-) than in wild-type cultures, apparently because the former cells expressed high levels of GM-CSF. Injection of PTH above the calvaria caused hypercalcemia in wild-type but not PGHS-2(-/-) mice. Histological examination of bone from 5-week-old PGHS-2(-/-) mice revealed no abnormalities. Mice lacking PGHS-1 were similar to wild-type mice in all of these parameters. These data suggest that PGHS-2 is not necessary for wild-type bone development but plays a critical role in bone resorption stimulated by 1,25-D and PTH.

Effect of osteoclast activating factor from human leukocytes on bone metabolism.

The effects of osteoclast activating factor (OAF) released by normal human peripheral blood leukocytes cultured with phytohemagglutinin have been examined in organ culture. Like parathyroid hormone (PTH), OAF causes a rapid increased in the release of previously incorporated 45Ca from fetal rat bone after brief or continuous exposure; the bones also lose stable calcium and collagen content. The resorption response to OAF also resembles that of PTH in having a steep dose response curve and being only transiently inhibited by calcitonin and partially inhibited by increasing medium phosphate concentration. OAF-stimulated resorption was inhibited more effectively by cortisol than was PTH stimulation. The response to maximally effective doses of OAF was not enhanced by PTH or prostaglandin E2, but submaximal doses gave additive effects. Both OAF and PTH inhibit collagen synthesis in fetal rat calvaria at the concentrations that stimulate bone resorption.

Direct stimulation of bone resorption by thyroid hormones.

Although hypercalcemia, osteoporosis, and increased bone turnover are associated with thyrotoxicosis, no direct effects of thyroid hormones on bone metabolism have been reported previously in organ culture. We have now demonstrated that prolonged treatment with thyroxine (T4) or triiodothyronine (T3) can directly increase bone resorption in cultured fetal rat long bones as measured by the release of previously incorporated 45Ca. T4 and T3 at 1 muM to 10 nM increased 45Ca release by 10-60% of total bone 45Ca during 5 days of culture. The medium contained 4 mg/ml of bovine serum albumin to which 90% of T4 and T3 were bound, so that free concentrations were less than 0.1 muM. The response to T4 and T3 was inhibited by cortisol (1 muM) and calcitonin (100 mU/ml). Indomethacin did not inhibit T4 response suggesting that T4 stimulation of bone resorption was not mediated by increased prostaglandin synthesis by the cultured bone. Matrix resorption was demonstrated by a decrease in extracted dry weight and hydroxyproline concentration of treated bones and by histologic examination which also showed increased osteoclast activity. The effects of thyroid hormones were not only slower than those of other potent stimulators of osteoclastic bone resorption (parathyroid hormone, vitamin D metabolites, osteoclast activating factor, and prostaglandins), but the maximum response was not as great. We conclude that T4 and T3 can directly stimulate bone resorption in vitro at concentrations approaching those which occur in thyrotoxicosis. This effect may explain the disturbances of calcium metabolism seen in hyperthyroidism.

Big and little forms of osteoclast activating factor.

We have further characterized osteoclast activating factor (OAF) using a bioassay for bone resorption which utilizes the release of previously incorporated (45)Ca from fetal rat long bones in organ culture. When supernatant media from activated leukocyte cultures were concentrated on Amicon PM10 membranes (assigned molecular weight cutoff 10,000 daltons) and chromatographed on Sephadex G-50 columns, the bone-resorbing activity eluted between the molecular weight markers chymotrypsinogen (25,000 daltons) and cytochrome c (12,500 daltons). This peak of biological activity has been called big OAF. When filtrates from the PM10 membranes were concentrated on Amicon UM2 membranes (assigned molecular weight cutoff 1,000 daltons) and chromatographed on Sephadex G-50 columns, some of the biological activity eluted between the molecular weight markers chymotrypsinogen and cytochrome c (big OAF), but there was a separate peak of biological activity which eluted with [(3)H]proline (140 daltons). This second peak has been called little OAF. Little OAF was eluted from Bio-Gel P6 columns between the molecular weight markers calcitonin (approximately 3,500 daltons) and vitamin B(12) (1,330 daltons), but was retained by Spectrapor dialysis tubing (nominal molecular weight cutoff 3,500 daltons). Big OAF was converted to little OAF by equilibration in 1 M NaCl or 2 M urea. Little OAF was self-associated back to big OAF by equilibration in buffers of low ionic strength (Tris-HCl 10-50 mM). Little OAF was extracted into the organic phase in ethyl acetate after acidification of the sample to pH 3.5. The biological activity remained in the aqueous phase after ethyl acetate extraction at pH 7.5-8.4. Little OAF has been purified more than 6,000-fold compared with the original material so that bone-resorbing activity is maximal in a sample with a protein concentration of 80 ng/ml.

Ovariectomy enhances and estrogen replacement inhibits the activity of bone marrow factors that stimulate prostaglandin production in cultured mouse calvariae.

To examine PG production in estrogen deficiency, we studied effects on cultured neonatal mouse calvariae of bone marrow supernatants (MSup) from sham-operated (SHAM), ovariectomized (OVX), or 17 beta-estradiol (OVX+E)-treated mice. MSups were obtained 3 wk after OVX when bone density had decreased significantly. 10-60% MSup increased medium PGE2 and levels of mRNA for inducible and constitutive prostaglandin G/H synthase (PGHS-2 and PGHS-1) and cytosolic phospholipase A2 in calvarial cultures. OVX MSups had twofold greater effects on PGHS-2 and medium PGE2 than other MSups. IL-1 receptor antagonist and anti-IL-1 alpha neutralizing antibody decreased MSup-stimulated PGHS-2 mRNA and PGE2 levels and diminished differences among OVX, sham-operated, and OVX+E groups. In contrast, antibodies to IL-1 beta, IL-6, IL-11, and TNF alpha had little effect. There were no significant differences in IL-1 alpha concentrations or IL-1 alpha mRNA levels in MSups or marrow cells. PGHS-2 mRNA in freshly isolated tibiae from OVX mice was slightly greater than from sham-operated. We conclude that bone marrow factors can increase PG production through stimulation of PGHS-2; that OVX increases and estrogen decreases activity of these factors; and that IL-1 alpha activity, together with additional unknown factors, mediates the differential MSup effects.