Opposing effects of Sca-1(+) cell-based systemic FGF2 gene transfer strategy on lumbar versus caudal vertebrae in the mouse.

Our previous work showed that a Sca-1(+) cell-based FGF2 therapy was capable of promoting robust increases in trabecular bone formation and connectivity on the endosteum of long bones. Past work reported that administration of FGF2 protein promoted bone formation in red marrow but not in yellow marrow. The issue as to whether the Sca-1(+) cell-based FGF2 therapy is effective in yellow marrow is highly relevant to its clinical potential for osteoporosis, as most red marrows in a person of an advanced age are converted to yellow marrows. Accordingly, this study sought to compare the osteogenic effects of this stem cell-based FGF2 therapy on red marrow-filled lumbar vertebrae with those on yellow marrow-filled caudal vertebrae of young adult W(41)/W(41) mice. The Sca-1(+) cell-based FGF2 therapy drastically increased trabecular bone formation in lumbar vertebrae, but the therapy not only did not promote bone formation but instead caused substantial loss of trabecular bone in caudal vertebrae. The lack of an osteogenic response was not due to insufficient engraftment of FGF2-expressing Sca-1(+) cells or inadequate FGF2 expression in caudal vertebrae. Previous studies have demonstrated that recipient mice of this stem cell-based FGF2 therapy developed secondary hyperparathyroidism and increased bone resorption. Thus, the loss of bone mass in caudal vertebrae might in part be due to an increase in resorption without a corresponding increase in bone formation. In conclusion, the Sca-1(+) cell-based FGF2 therapy is osteogenic in red marrow but not in yellow marrow.

Fluoride directly stimulates proliferation and alkaline phosphatase activity of bone-forming cells.

Fluoride is one of the most potent but least well understood stimulators of bone formation in vivo. Bone formation was shown to arise from direct effects on bone cells. Treatment with sodium fluoride increased proliferation and alkaline phosphatase activity of bone cells in vitro and increased bone formation in embryonic calvaria at concentrations that stimulate bone formation in vivo.

Regulation of serum 1alpha,25-dihydroxyvitamin D3 by calcium and phosphate in the rat.

A new radioreceptor assay was used to quantify changes in serum concentration of 1alpha,25-dihydroxyvitamin D3 in rats with low calcium or low phosphate diets. Low availability of either ion elicits a fivefold increase in the circulating concentration of 1alpha,25-dihydroxyvitamin D3. The enhancement of 1alpha,25-dihydroxyvitamin D3 concentration in response to calcium deficiency is dependent on the presence of the parathyroid or thyroid glands (or both), suggesting that this effect is mediated by parathyroid hormone. In contrast, the response of phosphate deficiency is independent of these glands and may result from an action of low serum phosphate concentration or some factor associated with phosphate depletion on the renal synthesis of the 1alpha,25-dihydroxyvitamin D3 hormone.

Bone crystal maturation in renal osteodystrophy in humans.

Calcium, phosphorus, sodium, carbonate, magnesium, and hydroxyproline were measured in iliaccrest biopsies of 22 normal volunteers and 24 selected patients with renal osteodystrophy. Histologic classification revealed that 10 were mildly abnormal, 8 osteomalacic, and 6 osteofibrotic. Bone density measurements were performed on an additional 12 normal, 11 mildly abnormal, 6 osteomalacic, and 10 osteofibrotic subjects. The results revealed an increase in magnesium and adecrease in carbonate apparent in the minimal and osteomalacic lesions and a much greater change in osteofibrosis. The bone density was decreased in patients with osteofibrosis. These observations would appear to be explained by postulation of an impairment of the normal maturational process of bone whereby there is an increase in amorphous calcium phosphate and a decrease in apatite crystal. The data suggest that the maturational defect is present as soon as any abnormality can be identified histologically, is present to the same degree in osteomalacia, and is most severe in osteofibrosis. In comparison of two sets of six patients matched for age and duration of dialysis, neither acidosis nor vitamin D therapy appeared to influence the severity of the maturational defect.

Decrease in serum immunoreactive parathyroid hormone in rats and in parathyroid hormone secretion in vitro by 1,25-dihydroxycholecalciferol.

The present study determined the effects of 1,25-dihydroxycholecalciferol on serum immunoactive parathyroid hormone and on parathyroid hormone secretion in vitro. Rats injected i.p. with 1,25-dihydroxycholecalciferol, 130 pmol (2 U)/140 g body wt, which is probably a physiologic dose, had a significant 43% decrease in serum immunoreactive parathyroid hormone at 4 h. In addition, this dose of 1,25-dihydroxycholecalciferol inhibited the serum immunoreactive parathyroid hormone response to hypocalcemia induced by phosphate injection. Because the increment in serum immunoreactive parathyroid hormone was less but the decrement in serum calcium more in phosphate plus 1,25-dihydroxycholecalciferol-treated than in phosphate plus vehicle-treated rats, the impaired serum immunoreactive parathyroid hormone response to 1,25-dihydroxycholecalciferol could not be attributed to the change in serum calcium. In studies of parathyroid hormone secretion from bovine parathyroid tissue in vitro, the concentration of 1,25-dihydroxycholecalciferol used for most experiments was 1nM, which is in the range found in rat serum. 1,25-Dihydroxycholecalciferol at 1 or 100 nM significantly inhibited parathyroid hormone secretion when medium calcium concentration was normal (1.5 mM), high (3.0 mM), and low (1.0 mM). Maximum inhibition ranged from 19 to 74%; inhibition was generally seen after 2 h of incubation; and inhibition was sustained or progressive thereafter. Vitamin A, 0.1 muM, caused a marked stimulation of parathyroid hormone secretion. 1,25-Dihydroxycholecalciferol at 1 nM markedly reduced (44%) the effect of vitamin A to stimulate parathyroid hormone secretion. This effect of 1,25-dihydroxycholecalciferol was maximal at 1 h and persisted thereafter. Another steroid, hydrocortisone, 10 muM, did not inhibit parathyroid hormone secretion, suggesting that the 1,25-dihydroxycholecalciferol effect was not a nonspecific inhibitory effect on parathyroid cells. Because other workers have shown that parathyroid hormone directly stimulates 1,25-dihydroxycholecalciferol secretion, our results are consistent with the concept that there is a feedback loop where parathyroid hormone directly stimulates secretion of 1,25-dihydroxycholecalciferol, which in turn directly inhibits secretion of parathyroid hormone.

Chemical mutagenesis induced two high bone density mouse mutants map to a concordant distal chromosome 4 locus.

Phenotype-driven mutagenesis approach in the mouse holds much promise as a method for revealing gene function. Earlier, we have described an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to create genome-wide dominant mutations in the mouse model. Using this approach, we describe identification of two high bone density mutants in C57BL/6J (B6) background. The mutants, named as 12184 and 12137, have been bred more than five generations with wild-type B6 mice, each producing >200 backcross progeny. The average total body areal bone mineral density (aBMD) was 13-17% higher in backcrossed progeny from both mutant lines between 6 and 10 weeks of age, as compared to wild-type (WT) B6 mice (n=60-107). At 3 weeks of age the aBMD of mutant progeny was not significantly affected as compared to WT B6 mice. Data from 10- and 16-week old progeny show that increased aBMD was mainly related to a 14-20% higher bone mineral content, whereas bone size was marginally increased. In addition, the average volumetric BMD (vBMD) was 5-15% higher at the midshaft tibia or femur, as compared to WT mice. Histomorphometric analysis revealed that bone resorption was 23-34% reduced in both mutant mice. Consistent with histomorphometry data, the mRNA expression of genes that regulate osteoclast differentiation and survival were altered in the 12137 mutant mice. To determine the chromosomal location of the ENU mutation, we intercrossed both mutant lines with C3H/HeJ (C3H) mice to generate B6C3H F2 mice (n=164 for line 12137 and n=137 F2 for line 12184). Interval mapping using 60 microsatellite markers and aBMD phenotype revealed only one significant or suggestive linkage on chromosome 4. Since body weight was significantly higher in mutant lines, we also used body weight as additive and interactive covariate for interval mapping; both analyses showed higher LOD scores for both 12137 and 12184 mutants without affecting the chromosomal location. The large phenotype in the mutant mice compared to generally observed QTL effects (<5%) would increase the probability of identifying the mutant gene.

Genetic regulation of femoral bone mineral density: complexity of sex effect in chromosome 1 revealed by congenic sublines of mice.

The findings that sex-specific effects on femoral structure and peak bone mineral density (BMD) are linked to quantitative trait loci (QTL) provide evidence for the involvement of specific genes that contribute to gender variation in skeletal phenotype. Based on previous findings that the BMD QTL in chromosome 1 (Chr 1) exerts a sex-specific effect on femoral structure, we predicted that congenic sublines of mice that carry one or more of the Chr 1 BMD loci would exhibit gender difference in the volumetric BMD (vBMD) phenotype. To test this hypothesis, we compared skeletal parameters of male and female of five C57BL/6J (B6).CAST/EiJ (CAST)-1 congenic sublines of mice that carry overlapping CAST chromosomal segments from the vBMD loci in Chr 1. Femur vBMD measurements were performed by the peripheral quantitative computed tomography in male and female mice at 16 weeks of age. The skeletal phenotype of the C175-185 and C178-185 congenic sublines of mice provided evidence for the presence of the BMD1-4 locus at 178-180 Mb from the centromere. This QTL affects femur vBMD only in female mice. In contrast, CAST chromosomal region carrying BMD1-1 locus increased femur vBMD both in male and female mice. Furthermore, a gender specific effect on BMD of femur mid-shaft region (mid-BMD) was identified at 168-176 Mb in Chr 1 (F=16.49, P=0.0002), while no significant effect was found on total femur BMD (F=2.67, P=0.11). Moreover, this study allowed us to locate a body weight QTL at 168-172 Mb of Chr 1, the effect of this locus was altered in female mice that carry CAST chromosomal segment 168-176 Mb of Chr 1. Based on this study, we conclude that Chr 1 carries at least two vBMD gender-dependent loci; one genetic locus at 178-180 Mb (BMD1-4 locus) which affects both mid-shaft and total femur vBMD in female mice only, and another gender-dependent locus at 168-176 Mb (BMD1-2 locus) which affects femur mid-shaft vBMD in female but not male mice.

Skeletal growth factor is produced by human osteoblast-like cells in culture.

Human bone cells isolated from femoral heads were cultured in BGJb medium containing bovine serum albumin (100 micrograms/ml), insulin (1 microgram/ml) and epidermal growth factor (10 ng/ml), and the conditioned medium collected. The medium was concentrated, chromatographed using HPLC gel filtration (TSK 2000 SW), and assayed for mitogenic activity using [3H]thymidine incorporation into embryonic chick calvarial cells. The conditioned medium contained mitogenic activity which eluted with a different elution time than insulin or epidermal growth factor. Characterization of this activity suggests that it was due to human skeletal growth factor (SGF), a mitogen which had been previously isolated from human bone matrix. Common properties include: stimulation of DNA synthesis in cultured embryonic chick calvarial cells, competition with human SGF for binding to anti-SGF antibodies, elution from HPLC gel filtration as a large factor (Mr 100,000) under native conditions but as a small factor (Mr 10,000) under dissociative conditions (4 M guanidine HCl), elution time on HPLC reverse-phase chromatography (small SGF), inactivation by dithiothreitol, stability to heat, acidic or alkaline conditions and inactivation by trypsin and chymotrypsin. These observations provide evidence that human bone cells produce SGF. Conditioned medium from human skin cell cultures also contained mitogenic activity. However, the activity was less than that from bone cells and did not cross-react with the rat anti-SGF antibodies.

Skeletal growth factor and other growth factors known to be present in bone matrix stimulate proliferation and protein synthesis in human bone cells.

The purpose of the study was to investigate the effect of skeletal growth factor/insulinlike growth factor II and other growth factors known to be present in bone matrix on the proliferation and differentiation of human bone cells. Cells were isolated by collagenase digestion from femoral heads obtained during hip replacement operations. Cells were cultured in DMEM medium with 10% calf serum. Third to fifth passage cells were plated in multiwell plates and the medium changed to low serum (0.1%) for 2 days. The medium was changed to serum-free medium prior to addition of growth factors. Cell proliferation was measured by the incorporation of [3H]thymidine into DNA and by the percentage of cells that incorporate bromodeoxyuridine. Protein synthesis was measured by the incorporation of [3H]proline into trichloroacetic acid-precipitable material. Skeletal growth factor/insulinlike growth factor II and insulinlike growth factor I stimulated cell proliferation and protein synthesis in a dose-dependent manner. Alkaline phosphatase-specific activity was not increased by these factors. Transforming growth factor beta 1 did not affect cell proliferation but stimulated protein synthesis and increased the specific activity of alkaline phosphatase. Fibroblast growth factor did not affect any of the cell parameters. These studies suggest that skeletal growth factor/insulinlike growth factor II, insulinlike growth factor I, and transforming growth factor beta 1 may play a role in the local control of the proliferation and differentiation of human osteoblasts.

Specific activity of skeletal alkaline phosphatase in human osteoblast-line cells regulated by phosphate, phosphate esters, and phosphate analogs and release of alkaline phosphatase activity inversely regulated by calcium.

We assessed the significance of Ca and phosphate (P(i)) as determinants of (1) the amount of skeletal alkaline phosphatase (ALP) activity in SaOS-2 (human osteosarcoma) cells and normal human bone cells, and (2) the release of ALP activity from the cells into the culture medium. After 24 h in serum-free BGJb medium containing 0.25-2 mM P(i), the specific activity of ALP in SaOS-2 cells was proportional to P(i) concentration (r = 0.99, p < 0.001). The P(i)-dependent increase in ALP activity was time dependent (evident within 6 h) and could not be attributed to decreased ALP release, since P(i) also increased the amount of ALP activity released (r = 0.99, p < 0.001). Parallel studies with Ca (0.25-2.0 mM) showed that the amount of ALP activity released from SaOS-2 cells was inversely proportional to the concentration of Ca (r = -0.85, p < 0.01). This effect was rapid (i.e., observed within 1 h) and could not be attributed to a decrease in the amount of ALP activity in the cells. Phase distribution studies showed that the effect of low Ca to increase ALP release reflected increases in the release of both hydrophilic ALP (i.e., anchorless ALP, released by phosphatidylinositol-glycanase activity) and hydrophobic ALP (i.e., phosphatidylinositol-glycan-anchored ALP, released by membrane vesicle formation). The range of Ca-dependent changes in ALP-specific activity was much smaller than the range of P(i)-dependent changes. The observed correlation between skeletal ALP-specific activity and P(i) was not unique to osteosarcoma cells or to P(i). Similar effects were seen in normal human bone cells in response to P(i) (r = 0.99, p < 0.001) and in SaOS-2 cells in response to a variety of P(i) esters and analogs (e.g., beta-glycero-P(i) and molybdate). Further studies indicated that the effects of phosphoryl compounds on ALP-specific activity could not be correlated with effects on ALP reaction kinetics, cell proliferation, or acid phosphatase activity and that the beta-glycero-P(i)-dependent increase in ALP activity was blocked by cycloheximide but not actinomycin D. Together these data suggest that the function of skeletal ALP may be regulated by P(i) and that Ca may be involved in ALP release.

Insulinlike growth factor II and transforming growth factor beta regulate collagen expression in human osteoblastlike cells in vitro.

Insulinlike growth factor II (IGF-II) and transforming growth factor beta (TGF-beta) are the most abundant polypeptide growth factors found in human bone matrix and are produced by human bone cells in vitro. IGF-II and TGF-beta 1 increased total protein synthesis, collagenous protein synthesis, and the steady-state level of type I procollagen mRNA in a time-dependent manner in osteoblastlike cells isolated from human bone. Type III procollagen mRNA expression was low in untreated cultures and was not affected by IGF-II or TGF-beta. TGF-beta 1 elevated type I procollagen mRNA rapidly, with the maximal observed change at 10 h. In contrast, procollagen mRNA levels increased more slowly in response to IGF-II and reached a lower maximal level than with TGF-beta, but the response was sustained through 24 h. Collagenous protein synthesis in IGF-II- and TGF-beta-treated cells increased in parallel with increases in procollagen mRNA levels and was higher at 21 h for TGF-beta 1 and at 36 h for IGF-II. The difference in the time course and magnitude of change in type I procollagen mRNA levels in response to IGF-II and TGF-beta 1 suggests that these two growth factors work through distinct mechanisms that provide both a rapid transient response and a later sustained response in bone matrix biosynthetic activity.

Insulin-like growth factor (IGF)-I, -II, IGF binding proteins (IGFBP)-3, -4, and -5 levels in the conditioned media of normal human bone cells are skeletal site-dependent.

The skeleton in its function of affording strength and support to the body is subject to differential mechanical loading which has been implicated to mediate some of its effects on bone formation via the insulin-like growth factors (IGFs), which are important regulators of bone metabolism. We, therefore, sought to conduct the present study with the hypothesis that the skeletal site-dependent differences in mechanical loading and other variables including stage of osteoblast differentiation would be associated with site-specific differences in the production of the IGF system components. To test this hypothesis, conditioned media (CM) from normal human bone cells (control and IGF-II-treated 48-h cultures) from five different skeletal sites were obtained and assayed for IGF-I, IGF-II (following separation of IGF binding proteins [IGFBPs]), IGFBP-3, IGFBP-4, and IGFBP-5 protein levels employing specific radioimmunoassays for each protein. IGF-I levels were lower than any other IGF system component but were significantly different between the various sites tested. IGF-II levels were greatest in the CM from mandibular cells, followed by calvarial and rib cells, and least in the marrow stromal cells. IGFBP-3 levels were highest in the CM of vertebral cells and lowest in the CM of rib and mandibular cells. The relative abundance of IGFBP-4 in decreasing order was observed in mandibular, calvarial, vertebral, rib, and stromal cells' CM. IGFBP-5 was produced maximally by the calvarial cells, followed by the mandibular, vertebral, stromal, and rib cells. IGFBP-4 appeared to be the IGF system component most abundantly produced by all the cell types from the skeletal sites tested. On a molar basis, the IGFBPs in general were estimated to be produced at a higher magnitude than the IGFs. These findings indicate that there are skeletal site-dependent differences in the production of IGF system components and suggest that the regulation of bone metabolism may vary at the various skeletal sites.

The causes of bone scintigram hot spots in fluoride-treated osteoporotic patients.

We previously described new bone formation in fluoride-treated osteoporotic patients. Since then, several investigators have contended that fluoride-induced "hot spots," as seen on bone scintigrams, represent stress fractures. To further evaluate this issue we analyzed scintigrams, radiographs, and quantitative computer tomography (CT) scans of the spine and femoral condyles from 129 patients, obtained before and during therapy. Hot spots, new or of increased intensity, were more than twice as common in the weight-bearing peripheral skeleton than in nonweight-bearing sites (p less than 0.001). The hot spots were usually diffuse, multiple, bilateral, and mostly seen early in therapy, a pattern quite different from that expected of stress fractures. Previously, we postulated that mechanical stress and fluoride act synergistically to stimulate new bone formation. If this hypothesis were correct, we would expect to see a greater increase in femoral condyle bone density in patients with hot spots. Consistent with this hypothesis, patients who developed hot spots in the knees had a greater increase in condylar density (22 +/- 2.5 vs. 9 +/- 3.3 mg/cc) than those without hot spots and stress fractures were not seen in either group. Additionally, patients with more than 5 hot spots in the peripheral skeleton had greater increases in spinal and condylar density than those with fewer than 5 hot spots. Finally, stress fractures were found in less than 2% of our patients. While we do not exclude the possibility that some additional patients may have had stress fractures, our data support the hypothesis that the great majority of hot spots seen on the scintigrams are the sites of new bone formation.

Postnatal and pubertal skeletal changes contribute predominantly to the differences in peak bone density between C3H/HeJ and C57BL/6J mice.

Previous studies have shown that 60-70% of variance in peak bone density is determined genetically. The higher the peak bone density, the less likely an individual is to eventually develop osteoporosis. Therefore, the amount of bone accrued during postnatal and pubertal growth is an important determining factor in the development of osteoporosis. We evaluated the contribution of skeletal changes before, during, and after puberty to the development of peak bone density in C3H/HeJ (C3H) and C57BL/6J (B6) mice. Volumetric bone density and geometric parameters at the middiaphysis of femora were measured by peripheral quantitative computed tomography (pQCT) from days 7 to 56. Additionally, biochemical markers of bone remodeling in serum and bone extracts were quantified. Both B6 and C3H mice showed similar body and femoral weights. B6 mice had greater middiaphyseal total bone area and thinner cortices than did C3H mice. Within strains, males had thicker cortices than did females. C3H mice accumulated more minerals throughout the study, with the most rapid accumulation occurring postnatally (days 7-23) and during pubertal maturation (days 23-31). C3H mice had higher volumetric bone density as early as day 7, compared with B6 mice. Higher serum insulin-like growth factor I (IGF-I) was present in C3H mice postnatally at day 7 and day 14. Until day 31, B6 male and female mice had significantly higher serum osteocalcin than C3H male and female mice, respectively. Alkaline phosphatase (ALP) was found to be significantly higher in the bone extract of C3H mice compared with B6 mice at day 14. These data are consistent with and support the hypothesis that the greater amount of bone accrued during postnatal and pubertal growth in C3H mice compared with B6 mice may be caused by increased cortical thickness, increased endosteal bone formation, and decreased endosteal bone resorption.

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.

Acute inhibition of mineralization and stimulation of bone resorption mediated by hypophosphatemia.

Using rats previously labeled with 45Ca, the effects of a severely phosphate deficient diet on calcium mobilization from bone into serum were examined in both intact and thyroparathyroidectomized (TPTX) RATS. With the TPTX animals, increased calcium mobilization from bone was evident 12 hr after the rats had been placed on the low phosphorus diet. At that time period, both TPTX and intact rats had become severely hypophosphatemic. However, in intact rats, calcium mobilization was not observed until 48 hr had elapsed. Both intact and TPTX hypophosphatemic rats developed hypercalcemia. To determine if inhibition of calcium deposition into bone contributed to this change, the course of 45Ca movement from blood into bone was followed in an experiment where rats received a single injection of the isotope at the time the low phosphorus diet was given. The animals on the low phosphorus diet showed a significantly lower bone specific activity and a higher serum specific activity compared to the control group, indicating calcium deposition into bone was inhibited. We conclude that the acute response to hypophosphatemia, resulting from the low phosphorus dietary regimen, was an increase in bone resorption and an inhibition of bone mineralization. The increase in bone resorption occurred more rapidly in TPTX rats than in the intact animals.

Increases in number and size of osteoclasts in response to calcium or phosphorus deficiency in the rat.

In this study, the two determinants of the rate of osteoclastic bone resorption, cell number and cell activity, were evaluated to characterize the response to two chronic resorptive stimuli, a calcium-deficient diet and a phosphorus-deficient diet, fed to rats for 8 days. The number of osteoclast nuclei was determined directly and the resorptive activity of osteoclasts indirectly from cell size. In our sampling site in the tibial diaphysis, there were marked increases in osteoclast layer width (i.e., cell size) and in the number of osteoclasts and osteoclast nuclei in both test groups. Also, in both test groups the increase in the number of osteoclast nuclei was greater than the increase in osteoclast width, emphasizing the importance of osteoclast stem cell activity in the resorptive response. When the data on the control and test animals were pooled, there was a strong correlation between the width of the osteoclast layer and the number of osteoclast nuclei (r = 0.87, P less than 0.001). Thus, these two resoprtive stimuli produced proportional changes in osteoclast cell number and probably activity, raising the possibility that the resorptive response is sterotyped. These results also suggest the existence of a control mechanism which coupled the proliferative activity of osteoclast stem cells and the resorptive activity of osteoclasts. Despite the fact that the rats were subjected to chronic resorptive stimuli, forming surface and the total number of osteoblasts were also increased. Moreover, in data pooled from control and test rats, there was a strong correlation between the number of osteoclast nuclei and the number of osteoblasts (r = 0.92, P less than 0.001). This implies that chronic resorptive stimuli directly or indirectly stimulate osteogenic stem cells to increase production of osteoblasts as well as osteoclasts. The observed coupling of differentiated cell number probably contributes to the established phenomenon of coupling between the rates of bone accretion and resorption.

Androgens directly stimulate proliferation of bone cells in vitro.

This report describes the first observation of a direct mitogenic effect of androgens on isolated osteoblastic cells in serum-free culture. [3H]thymidine incorporation into DNA and cell counts were used as measures of cell proliferation. The percentage of cells that stained for alkaline phosphatase was used as a measure of differentiation. Dihydrotestosterone (DHT) enhanced mouse osteoblastic cell proliferation in a dose dependent manner over a wide range of doses (10(-8) to 10(-11) molar), and was maximally active at 10(-9) M. DHT also stimulated proliferation in human osteoblast cell cultures and in cultures of the human osteosarcoma cell line, TE89. Testosterone, fluoxymesterone (a synthetic androgenic steroid) and methenolone (an anabolic steroid) were also mitogenic in the mouse bone cell system. The mitogenic effect of DHT on bone cells was inhibited by antiandrogens (hydroxyflutamide and cyproterone acetate) which compete for binding to the androgen receptor. In addition to effects on cell proliferation, DHT increased the percentage of alkaline phosphatase (ALP) positive cells in all three bone cell systems tested, and this effect was inhibited by antiandrogens. We conclude that androgens can stimulate human and murine osteoblastic cell proliferation in vitro, and induce expression of the osteoblast-line differentiation marker ALP, presumably by an androgen receptor mediated mechanism.

Serum immunoreactive parathyroid hormone and 25-hydroxyvitamin D in patients with uremic bone diseases.

Bone histologic parameters and serum iPTH and 25-OHD were measured in 20 patients with end-stage renal failure treated with hemodialysis. By bone histologic criteria, the patients were divided into three groups: mild, osteomalacic, and fibrotic. The increase in serum iPTH was much greater in the fibrotic group than in the mild or osteomalacic groups. In the uremic patients as a group, there were significant correlations between serum iPTH and both percent marrow fibrosis and percent resorbing surface. In the mild and fibrotic groups together, serum iPTH was also correlated with percent forming surface. This and other findings suggested that most of the bone changes in the mild and fibrotic groups could be explained by excess PTH. The difference in bone changes and in serum iPTH between the mild and fibrotic groups could be related to our eariler findings that duration of renal disease was much greater in the fibrotic than in the mild group. The lowest increment in serum iPTH was found in the osteomalacic group. In this group, percent resorbing surface was not increased and there was only a slight increase in marrow fibrosis. Thus in all three groups, serum iPTH appeared to reflect parathyroid status. The cause of the elevated serum iPTH and for the intergroup differences was not apparent inasmuch as serum calcium was normal in all three groups. Serum 25-OHD was significantly elevated in the osteomalacic and fibrotic groups. Because none of our patients had received preparations containing vitamin D, the elevated serum 25-OHD in the osteomalacic and fibrotic groups is consistent with altered vitamin D metabolism in these two groups. There was a direct relationship between percent osteroid area and serum 25-OHD. However, whether or not altered vitamin D metabolism contributed to the mineralization defect in uremic bone disease could not be established.

Lack of a high prevalence of the BB vitamin D receptor genotype in severely osteoporotic women.

Studies of twins strongly suggest that more than 50% of the peak spinal bone density is determined by genetics. It was reported recently that this genetic effect is primarily determined by vitamin D receptor (VDR) alleles; specifically, a VDR genotype termed BB has been highly associated with low peak bone density. Homozygotes for the second VDR allele, bb, are associated with high peak bone density. If peak bone density is an important determinant of osteoporosis and if the VDR genotype is an important determinant of peak bone density, then patients with severe osteoporosis should have a high prevalence of the BB VDR genotype compared with that of control subjects. To test this hypothesis, we used Southern blot analysis to determine the VDR genotype of 41 Caucasian patients (72 +/- 14 yr) with severe osteoporosis (27 women with spinal bone densities below 50 mg/cm3 as determined by quantitative computed tomography; 14 women with spinal bone densities below 0.75 g/cm2 as determined by dual energy x-ray absorptiometry) and 23 Caucasian control subjects (68 +/- 7 yr) without osteoporosis (quantitative computed tomography values at or above the fracture threshold of 100 mg/cm3). Only 6 of the 41 individuals in the group with severe osteoporosis had the BB genotype, whereas 16 had the bb genotype. In the control group comprising 23 individuals, 7 had the BB genotype and only 6 had the bb genotype. We conclude that the BB VDR genotype is not a good predictor of risk for developing severe osteoporosis in our population.