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Reliable markers of bone formation are essential to the investigation of metabolic bone disorders. In this regard, evidence indicates that circulating levels of human osteocalcin (OC) correlate with the skeletal isoenzyme of alkaline phosphatase and can be used as an index of bone formation. A disadvantage of using serum OC as a marker of formation is its diurnal variation. To address this problem we carried out our studies to determine the usefulness of urine in the assessment of bone turnover. Using a midmolecule specific human OC RIA, we were able to detect OC in urine of normal adults (42 mugeq/g creatinine), normal children (849 mu/geq/g creatinine), and Paget's disease patients (613 mugeq/g creatinine). Immunoreactive fragments of OC in human urine and human serum were separated by high pressure liquid chromatography. Multiple fragments were found in normal adult urine that were not detected in normal adult serum. Uremic and Paget's disease sera contain several immunoreactive forms of OC, other than the intact molecule, not found in normal adult serum. Additionally, both Paget's disease sera and urine contained a specific peak of immunoreactive material, eluting at 25% acetonitrile, that was not found in any other serum or urine tested. Urinary OC (uOC) correlated with both skeletal alkaline phosphatase (r = 0.91) and serum OC (r = 0.83), indices of skeletal formation. While uOC has a diurnal variation similar to that of serum OC, determinations of 24-h uOC give integrated values of daily bone turnover rates. Z-Score analysis indicates that uOC (z = 14.04) is better able to distinguish between normal children with high bone turnover and normal adults than either skeletal alkaline phosphatase (z = 8.87) or serum OC (z = 9.01).

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 D 3 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. (J Bone Miner Res 1999;14:39-46)

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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.

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Clinical studies on the use of sodium fluoride (NaF) in osteoporotic patients have demonstrated increased spinal bone mass without a reduction in vertebral fracture incidence, and a trend towards reduced appendicular bone mass with an increase in peripheral fracture incidence. As previous reports have suggested that NaF becomes incorporated into bone's crystal structure, possibly affecting bone strength, we sought to examine the relationship among bone fluoride content, bone mass, and skeletal fragility. Twenty-one-day-old female Sprague-Dawley rats were treated with four different doses of NaF. The tibiae were subjected to histomorphometric and biochemical analyses, and the femora were tested in torsion for the properties of strength, stiffness, energy storage capacity, and angular deformation. The results showed that over 50% of the skeleton in these rats was turned over in the presence of NaF. The four different doses resulted in a linear increase in bone F concentration and suggested excellent absorption and incorporation of this drug. No changes in histomorphometric indices of bone formation or turnover were found. Despite the large fraction of bone formed during NaF treatment, and the linear increase in bone fluoride content in relation to dose, there were no changes observed in any of the mechanical properties. These results suggest that, even extensive incorporation of fluoride into bone, in the absence of an effect on bone mass or remodeling, does not significantly alter its capacity to withstand mechanical loads.