Osteoclast formation in bone marrow cultures from two inbred strains of mice with different bone densities.

For the purpose of identifying genes that affect bone volume, we previously identified two inbred mouse strains (C57BL/6J and C3H/HeJ) with large differences in femoral bone density and medullary cavity volume. The lower density and larger medullary cavity volume in C57BL/6J mice could result from either decreased formation or increased resorption or both. We recently reported evidence suggesting that bone formation was increased in vivo and that osteoblast progenitor cells are more numerous in the bone marrow of C3H/HeJ compared with C57BL/6J mice. In the present study, we determined whether osteoclast numbers in vivo and osteoclast formation from bone marrow cells in vitro might also differ between the two mouse strains. We have found that the number of osteoclasts on bone surfaces of distal humerus secondary spongiosa was 2-fold higher in 5.5-week-old C57BL/6J mice than in C3H/HeJ mice of the same age (p < 0.001). Bone marrow cells of C57BL/6J mice cocultured with Swiss/Webster mouse osteoblasts consistently produced more osteoclasts than did C3H/HeJ bone marrow cells at all ages tested from 3.5-14 weeks of age (p < 0.001). Osteoclast formation was also greater from spleen cells of 3.5-week-old C57BL/6J mice than C3H/HeJ mice. The distribution of nuclei per osteoclast and the 1, 25-dihydroxyvitamin D3 dose dependence of osteoclast production from bone marrow cells were similar. Osteoclasts that developed from both C57BL/6J and C3H/HeJ marrow cells formed pits in dentin slices. Cultures from C57BL/6J marrow cells formed 2.5-fold more pits than cultures from C3H/HeJ marrow cells (p < 0.02). We compared the abilities of C57BL/6J and C3H/HeJ osteoblasts to support osteoclast formation. When bone marrow cells from either C57BL/6J or C3H/HeJ mice were cocultured with osteoblasts from either C57BL/6J or C3H/HeJ newborn calvaria, the strain from which osteoblasts were derived did not affect the number of osteoclasts formed from marrow cells of either strain. Together, these observations suggest that genes affecting the bone marrow osteoclast precursor population may contribute to the relative differences in bone density that occur between C3H/HeJ and C57BL/6J mouse strains.

Alkaline phosphatase levels and osteoprogenitor cell numbers suggest bone formation may contribute to peak bone density differences between two inbred strains of mice.

Previous studies have shown that C3H/HeJ (C3H) mice have higher peak bone density than C57BL/6J (B6) mice, at least in part because of differences in rates of bone resorption. The current studies were intended to examine the alternative, additional hypothesis that the greater bone density in C3H mice might also be a consequence of increased bone formation. To that end, we measured two presumptive, indirect indices of bone formation and osteoblast number in these inbred strains of mice: alkaline phosphatase (ALP) activity in serum, bones, and bone cells; and the number of ALP-positive colony-forming units (CFU) in bone marrow stromal cell cultures. We found that C3H mice had higher serum levels of ALP activity than B6 mice at 6 (118 vs. 100 U/L, p < 0.03) and 32 weeks of age (22.2 vs. 17.2 U/L, p < 0.001). Tibiae from C3H mice also contained higher levels of ALP activity than tibiae from B6 mice at 6 (417 vs. 254 mU/mg protein, p < 0.02) and 14 weeks of age (132 vs. 79 mU/mg protein, p < 0.001), as did monolayer cultures of bone-derived cells from explants of 7.5-week-old C3H calvariae and femora (8.2 times more, p < 0.02, and 4.6 times more, p < 0.001, respectively). Monolayer cell cultures prepared by collagenase digestion of calvariae from newborn and 6-week-old mice also showed similar strain-dependent differences in ALP-specific activity (p < 0.001 for each). Our studies also showed more ALP-positive CFU in bone marrow stromal cell cultures from 8-week-old C3H mice, compared with B6 mice (72.3 vs. 26.1 ALP-positive CFU/culture dish, p < 0.001). A similar result was seen for ALP-positive CFU production at 6 and 14 weeks of age, and the difference was greatest for the CFU that contained the greatest numbers of ALP-positive cells. Because skeletal ALP activity is a product of osteoblasts and has been shown to correlate with rates of bone formation, and because the number of ALP-positive CFU is believed to reflect the number of osteoprogenitor cells, the current data are consistent with the general hypothesis that bone formation may be greater in C3H than B6 mice because of a difference in osteoblast number. Our data further suggest that peak bone density may be greater in C3H mice than B6 mice due to a combination of decreased bone resorption and increased bone formation.

Calcium deficiency in fluoride-treated osteoporotic patients despite calcium supplementation.

To test the hypothesis that the osteogenic response to fluoride can increase the skeletal requirement for calcium, resulting in a general state of calcium deficiency and secondary hyperparathyroidism, we assessed calcium deficiency, spinal bone density, by quantitative computed tomography, and serum PTH in three groups of osteoporotic subjects. Two of the three groups had been treated with fluoride and calcium (at least 1500 mg/day) for 32 +/- 19 months. Group 1 consisted of 16 fluoride-treated subjects who had shown rapid increases in spinal bone density (+ 3.8 +/- 2.6 mg/cm2 month), group II consisted of 10 fluoride-treated subjects who had shown decreases or only slow increases in spinal bone density (-0.05 +/- 0.6 mg/cm3 month), and group III consisted of 10 age-matched untreated osteoporotic controls. Calcium deficiency was assessed by measurement of calcium retention after calcium infusion. The results of our studies showed that 1) 94% of the subjects in Group I were calcium deficient compared with only 30% in groups II and III (P < 0.01 for each); 2) the subjects in group I retained more calcium (79%) than the subjects in group II (60%, P < 0.001) or the subjects in group III (64%, P < 0.005); 3) calcium retention was proportional to serum PTH (r = 0.37, n = 36, P < 0.03); and 4) calcium retention was proportional to the (previous) fluoride-dependent increase in quantitative computed tomography spinal bone density (in groups I and II, r = 0.48, n = 26, P < 0.02). To test the hypothesis that the calcium deficiency and the secondary hyperparathyroidism that were associated with the positive response to fluoride would respond to concomitant calcitriol treatment, a subgroup of 7 calcium-deficient subjects were selected from group I and treated with calcitriol (plus fluoride and calcium) for an average of 7 months. The calcitriol therapy reduced the calcium deficit in all 7 subjects, decreasing calcium retention from 80% to 62% (P < 0.02), and decreasing PTH from 50 to 28 pg/mL (P < 0.02). Together, these data indicate that fluoride-treated osteoporotic subjects may develop calcium deficiency in proportion to the effect of fluoride to increase bone formation, and this calcium deficit is responsive to calcitriol therapy.

Synthetic peptide-based immunoassay for amino-terminal propeptide of type I procollagen: application for evaluation of bone formation.

Serum biochemical markers are powerful tools for the evaluation of bone turnover. In this study, we developed a radioimmunoassay, using a synthetic peptide for the N-terminal fragment of human type I [alpha 1(I)] procollagen (N-PCP). A 14-amino acid peptide was synthesized from the amino terminus and used to generate antibodies in rabbits. The synthetic peptide was used as standard and tracer in the assay. Both native type I amino procollagen (PINP), which was purified from skin fibroblasts, and human serum displaced tracer binding in parallel with the synthetic peptide. The range for measurement of N-PCP in serum was 0.7 to 30 micrograms/L (0.21-9.18 nmol/L). In a sample of 17 normal adults and 13 children (ages 9-16 years) there was a strong correlation between serum N-PCP determined by this assay and both skeletal alkaline phosphatase isoenzyme and osteocalcin, markers of bone formation. Serum concentrations of N-PCP in a group of normal children were eightfold higher than concentrations in normal adults, with no overlap between the two groups. N-PCP also correlated with C-terminal type I procollagen determined with a commercially available kit (r = 0.92).

Interleukin-6 messenger RNA expression and interleukin-6 protein secretion in cells isolated from normal human bone: regulation by interleukin-1.

Recent evidence suggests that cytokines, in addition to regulating hematopoiesis and immune functions, may be important paracrine regulators of bone turnover. Interleukin-1 (IL-1) and IL-6 are cytokines that are produced by and affect both hematopoietic and nonhematopoietic cell types. IL-1 stimulates bone resorption and inhibits osteoblast proliferation and collagen production. Previous reports that IL-6 was secreted in murine osteoblast and bone organ cultures in response to IL-1 and PTH suggested that IL-6 has paracrine effects on bone resorption or formation. To determine whether IL-6 has a paracrine function in human bone, IL-6 expression in cells isolated from normal human bone was investigated. IL-6 mRNA levels in untreated cultures were low and variable, and IL-6 secretion was undetectable. PTH had no effect on IL-6 mRNA levels or IL-6 secretion. IL-1 beta increased IL-6 mRNA levels, maximally 40-fold at 12 h. IL-1 beta increased IL-6 secretion to 0.13 nM, more than 80-fold that of untreated controls at 12 h. IL-1 beta also increased IL-1 beta mRNA levels, maximally 9-fold at 12 h, but did not increase cellular levels or secretion of IL-1 beta protein. Recombinant human IL-6 at 0.5-5 nM stimulated resorption in neonatal mouse calvarial organ cultures but had no effect on human bone-derived cell DNA synthesis or type I procollagen mRNA levels. The results suggest that IL-6 production by human osteoblasts may function to enhance osteolytic activity of IL-1 but does not affect proliferative and matrix biosynthetic aspects of bone formation that were tested. Because osteoblasts and bone marrow cells are in close proximity, IL-6 produced by osteoblasts may also function to amplify IL-1 stimulation of immune responses and hematopoiesis in bone marrow.

Development of a new radioimmunoassay for human osteocalcin: evidence for a midmolecule epitope.

Osteocalcin is a vitamin K-dependent bone-specific protein that can be found circulating in the serum. The circulating levels of osteocalcin have been shown to be an index of bone turnover. Existing radioimmunoassays for osteocalcin have been shown to be specific for C-terminal epitopes, a region that is identical in the human and bovine osteocalcin. There are, however, five amino acids different in the N-terminal region of the molecule. We describe here an immunoassay for a midmolecule epitope of osteocalcin using osteocalcin purified from human femoral head bone powder. Antibody specificity was determined using tryptic digests and a synthetic fragment of human osteocalcin. This assay has only a partial crossreactivity with bovine osteocalcin. This is the first report of an assay against a midmolecular epitope of osteocalcin involving a region in which the human and bovine osteocalcins differ. Osteocalcin levels determined by this assay have a significant correlation with both the total serum alkaline phosphatase and the serum skeletal alkaline phosphatase levels in normal adult human serum and, to a greater degree, in sera of patients with conditions associated with increased bone turnover (Paget's disease, hyperparathyroidism, and newborn sera). These correlations are greater than those previously reported for C-terminal assays, suggesting the possibility that different regions of the molecule may elicit different information concerning bone turnover.

Acetylcholinesterase in abnormal human muscle.

Fifty human muscle biopsies were examined for histochemical localization of acetylcholinesterase (AChE) activity. Six normal muscle samples had AChE at the myoneural junctions and around the periphery of many fibers. The AChE within the sarcoplasm itself was found in only a few atrophied fibers. However, 21 of 44 biopsies of abnormal muscles had sarcoplasmic AChE in more than 10% of their fibers. Such cases included Duchenne, limb-girdle and facio-scapulo-humeral dystrophy, neurogenic and spinal muscle atrophy, spinal cord injury, peripheral nerve injury, Schwartz-Jampel syndrome and myasthenic syndrome. Sarcoplasmic AChE is found in embryo muscle and usually declines after birth. It appears after denervation in the chicken but not the rat and remains in muscles of chickens with an inherited muscular dystrophy. The results of the human muscle study support the idea that in the human, as in the chicken, interruption of a neurally-mediated regulation of AChE results in the reappearance of high AChE activity in the sarcoplasm of the muscle fibers.

Incidence of acetylcholinesterase in the sarcoplasm of human and chicken muscles.

Fifty-nine biopsies of human muscle, 53 of them abnormal, 6 normal, were studied for the histochemical localization of acetylcholinesterase (AChE) using frozen sections and light microscopy. In addition to AChE which was found at the myoneural and myotendon junction, specific staining was found around the periphery of many fibers from normal and abnormal muscles. Moreover, AChE activity was found to be high in the sarcoplasm of more than 10% of the fibers from 28 biopsies of abnormal muscle including cases of hemiplegia, spinal cord injury, denervation and neuropathy, infantile spinal muscle atrophy, Duchenne, limb-girdle and facioscapulohumeral dystrophies, Schwartz-Jampel syndrome and a myasthenic syndrome. Of the muscles from experimental animals examined, only the Rhesus monkey exhibited AChE around the periphery of the fibers, and only the dystrophic chicken and not the dystrophic mouse or hamster, showed extensive sarcoplasmic AChE. Histograms of muscle fiber diameters indicated that AChE in the sarcoplasm was associated with fibers of all sizes, depending on the nature of the disorder examined. Fibers containing AChE were smaller than unstained fibers in dystrophic chicken muscle. The results suggest that in the human, sarcoplasmic AChE is reversibly repressed during muscle maturation and that its mode of regulation by motor neurons is similar to that found in the chicken.