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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

Bone density is known to be determined by many factors, including age, calcium balance, exercise, and genetics.(1-9) Although the role of genetics in determining bone density is well established, specific genes that affect bone density have not been identified. Previous studies have shown significant differences in bone mineral content and bone size among different mouse strains.(10) To identify genes affecting bone density, a murine model has been developed for the purpose of investigating differences in bone density among inbred strains of mice.(11) Eleven strains of mice were screened using peripheral quantitative computerized tomography, and two were selected with widely different femoral bone densities. Mice of the strain C3H/HeJ have a mean femoral peak bone density of 0.69 ± 0.03 mg/mm3, while mice of the strain C57BL/6J have a mean femoral peak density of 0.45 ± 0.01 mg/mm3, p < 0.001 at 12 months of age. C3H/HeJ and C57BL/6J mice have significantly different femoral bone densities at all ages tested from 2–12 months. Bone densities of vertebrae and metacarpals were also higher in C3H/HeJ mice than C57BL/6J mice, indicating apparent differences between strains in both cortical and trabecular bone density. Although there were no differences in the external perimeter or length of the femur, the C3H/HeJ mouse femurs had greater cortical bone volume and smaller medullary cavity volume (0.82 ± 0.02 and 7.8 ± 0.09 mm3, respectively) compared with C57BL/6J (0.61 ± 0.01 and 14.1 ± 0.4 mm3, respectively, p < 0.001). These apparent differences in endosteal bone volume regulation might arise from different rates of bone resorption or bone formation or both.

Recently, we found that alkaline phosphatase activity in serum and bones of C3H/HeJ was higher than in C57BL/6J mice,(12) which is consistent with our observations that bone formation was increased in C3H/HeJ compared with C57BL/6J mice.(13) Furthermore, we found that the number of ALP-positive colonies produced in vitro per 106 bone marrow cells was greater in C3H/HeJ compared with C57BL/6J mice, which suggests that osteoblastic progenitor cells are more numerous in the bone marrow of C3H/HeJ mice. This observation leads to the question of whether osteoclastic progenitor cell numbers might also differ between C3H/HeJ and C57BL/6J mice. Preliminary observations that urine pyridinoline/creatinine excretion was lower in C3H/HeJ mice (451 ± 61 ng/mg) than in C57BL/6J mice (595 ± 167 ng/mg, p < 0.05, respectively),(14) suggest that the C57BL/6J mice have an increased rate of resorption compared to C3H/HeJ mice.

The current study was undertaken to test the specific hypotheses that in vivo osteoclast numbers and in vitro osteoclast formation from bone marrow cells are quantitatively different between C57BL/6J and C3H/HeJ mice; to assess bone resorption by the osteoclasts that develop in vitro; and to determine possible differences in capacities of osteoblasts from these two mouse strains to support osteoclast formation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

Materials

Alpha minimal essential medium (α-MEM) and fetal bovine serum for marrow cell osteoblast cocultures were purchased from GIBCO BRL (Gaithersburg, MD, U.S.A.). Dulbecco's minimal essential medium (DMEM) and Fe-supplemented bovine calf serum for osteoblast cultures were purchased from Mediatech (Herndon, VA, U.S.A.) and Hyclone (Logan, UT, U.S.A.), respectively. Penicillin and streptomycin were purchased from Gemini Bio-Products (Calabasas, CA, U.S.A.). Dexamethasone (Sigma, St. Louis, MO, U.S.A.) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (kindly provided by Hoffman LaRoche, Nutley, NJ, U.S.A.) were made as anhydrous ethanol stocks then diluted in marrow cell media. Final concentrations of ethanol were < 0.01%. Synthetic salmon calcitonin (Sigma) was used to inhibit osteoclast formation in vitro.

Animals

C57BL/6J and C3H/HeJ mice were obtained at different postweanling ages or as pregnant females from The Jackson Laboratory (Bar Harbor, ME, U.S.A.). Pregnant Swiss Webster mice were obtained from B & K Universal Ltd. (Freemont, CA, U.S.A.). All mice were housed at least 1 week before being used as sources of bone marrow or calvaria cells. All protocols were reviewed and approved by the Institutional Animal Care and Use Committees of the appropriate institutions.

Osteoclast numbers in vivo

Analysis of osteoclasts in demineralized bone sections is based on previously described procedures.(15) Mice were euthanized at 5.5 weeks of age by urethane injection. The right distal femur was fixed in neutral buffered 10% formalin for 24 h, demineralized, and prepared for parafin sectioning. Midcoronal sections (2 μm thick) of the distal femurs were stained for tartrate-resistant acid phosphatase (TRAP) as described(15) except that counterstaining was by Harris hematoxylin. Osteoclasts were identified as TRAP positive cells on bone surfaces. Bone surface length, number of osteoclasts, and fraction of bone surface occupied by osteoclasts were measured in the secondary spongiosa of the distal femur using a color video microscopy imaging system and Osteomeasure 2.31 program (OsteoMetrics, Inc., Atlanta, GA, U.S.A.). The sampling site was defined as the area between endosteal cortical bone surfaces extending from 0.3 mm to 0.78 mm proximal of the distal femur midepiphyseal plate. All contiguous optical fields within this area were analyzed at a magnification of ×1400.

Osteoclast formation in vitro

Mice for bone marrow and spleen cell preparations were euthanized by CO2 inhalation. Femurs were aseptically removed, placed on ice in α-MEM, and dissected free of soft tissues. The ends of the bones were removed and the marrow was flushed from the midshaft with a syringe and 25 guage needle using cold α-MEM.(14) Marrow cells from two to five female mice per strain were washed and resuspended in α-MEM containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. The washed marrow cells (5 × 105/well) were cocultured with neonatal calvaria cells (1 × 104/well) from outbred Swiss/Webster mice or from inbred (C3H/HeJ or C57BL/6J) mice in 24-well plates (Corning, Corning, NY, U.S.A.) in a final volume of 1 ml. Cultures were fed with media containing 10 nM 1, 25(OH)2D3 and 10 nM dexamethasone and were changed at day 3 by removing 0.5 ml of media and replacing it with fresh media. The marrow/osteoblast cultures were stopped at day 6. Groups of six replicate wells were used for each incubation condition. Spleen cells from 3.5-week-old mice were prepared as described previously(16) and were cocultured with calvaria cells as described above for bone marrow cells.

Osteoblast cells used for coculturing with marrow cells were isolated by collagenase digestion from calvariae of newborn mice. Mice 1–2 days of age were euthanized by CO2 inhalation, and calvariae were dissected free from adhering soft tissue. The calvariae were digested for 15 minutes at 37°C with 1 mg/ml collagenase A (Boehringer Mannheim, Indianapolis, IN, U.S.A.) in DMEM, then the supernatant was discarded and the calvaria were digested further in collagenase A for 90 minutes. The cells released were washed in DMEM + 10% calf serum and plated in the same media in 100 mm plates. Cells were grown until confluence and frozen in aliquots for subsequent bone marrow or spleen coculture studies. The calvarial cells were thawed 24 h prior to use, so all experiments were performed with cells at passage two.

Osteoclast identification in cocultures

Cultures were rinsed, fixed, and stained for TRAP following kit directions (Sigma, St. Louis, MO, U.S.A.). Cells were counted as osteoclasts if they were both TRAP positive and had three or more nuclei per cell. As reported previously in studies of similar mixed culture preparations,(17,18) osteoclasts that developed in the cocultures formed resorption pits on mineralized dentin slices. Other studies have demonstrated that similar cultures express calcitonin receptors.(19,20) We found that cultures maintained without 1,25(OH)2D3 had very few TRAP positive multinucleated cells, and that addition of calcitonin to cultures containing 1,25(OH)2D3 prevented formation of TRAP positive multinucleated cells and pit formation on dentin slices (see Results), supporting the conclusion that these cells are osteoclasts. There were no TRAP positive adherent cells 24 h after plating and some TRAP positive mononucleated cells but no multinucleated cells at 3 days. We observed TRAP positive multinucleated cells only at 6 days, suggesting that they developed between 4 and 6 days of culture and were not already present in the marrow cell population.

We analyzed six replicate wells per incubation condition unless otherwise noted, and, when osteoclast number was high, numbers of osteoclasts were estimated by counting cells in random fields comprising 15% of the well surface. Estimated cell counts were highly reproducible and compared closely with counts of entire wells. To minimize effects of interassay variation, statistical comparisons were limited, unless otherwise noted, to assessment of differences within each experiment. All observations were repeated at least twice. Statistical analysis was performed by one-ay analysis of variance (ANOVA) using the SYSTAT program (Systat, Inc., Evanston, IL, U.S.A.). Data are expressed as mean ± SEM.

Pit formation on sperm whale dentin slices was determined by modification of the procedure described by Takada et al.(21) Marrow cells from 4.5-week-old C3H/HeJ or C57BL/6J (5 × 105/well) were cocultured with neonatal calvaria cells (1 × 104/well) from Swiss Webster mice, on 1-mm-thick, 1.5 cm2 dentine slices in 1.9 cm2 wells and cultured as for TRAP positive osteoclast formation. At 6 days, the cells were removed from the dentin by brief sonication in 0.01 M NaOH. Pits were then stained with 1% toluidine blue in 1% sodium borate or in acid hematoxylin (Sigma Chemical Co.). Pits stained with acid hematoxylin had more well defined edges and were used for area measurements. Random optical fields observed by transmitted light microscopy were digitized using a color video camera, and pit areas were determined using the Sigma Scan/Image program (Jandel Scientific, San Rafael, CA, U.S.A.).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

In vivo characteristics of C3H/HeJ and C57BL/6J mice

Previous studies comparing inbred mouse strains have determined that bone length and animal weight did not differ between the C3H/HeJ and C57BL/6J strains of mice.(11) Since differences in total body weight could influence bone density based on weight bearing, in this study we confirmed that the average animal weight at each age examined was not significantly different between the two mouse strains. The mean weights for C3H/HeJ and C57BL/6J mice at 7 weeks of age, for example, were 15.80 ± 0.19 g and 15.75 ± 0.21 g, respectively (n = 8/group).

Osteoclast numbers in vivo in C3H/HeJ and C57BL/6J mice

Distal femur trabecular bone in the secondary spongiosa of 5.5-week-old female C57BL/6J mice contained approximately twice as many osteoclasts compared with C3H/HeJ mice (Table 1). C57BL/6J mice had a slightly higher bone surface length but this was not statistically significant. Numbers of osteoclasts were significantly greater (p < 0.001) in C57BL/6J than C3H/HeJ whether the numbers were expressed per bone surface length or per total area analyzed.

Table Table 1.. Osteoclast Numbers in Secondary Spongiosa of Distal Femur of 5.5-Week-Old Female C57BL/6J and C3H/HeJ Mice Were Determined as Described in the Materials and Methods
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Osteoclast formation in vitro

When marrow cells from C3H/HeJ and C57BL/6J mice were cocultured with Swiss Webster osteoblasts in the presence of 10−8 M 1,25(OH)2D3, multinucleated, TRAP positive osteoclasts formed by 6 days. In a representative experiment, C57BL/6J cultures had 907 ± 110 osteoclasts/well and C3H/HeJ cultures had 243 ± 36 osteoclasts/well. In the same experiment, addition of calcitonin (10 mU/ml) reduced osteoclast formation more than 95% to 38 ± 7 osteoclasts/well and 7 ± 4 osteoclasts/well, in C57BL/6J and C3H/HeJ cultures, respectively (p < 0.001 for each). Osteoclast formation was also dependent on 1,25(OH)2D3 (Fig. 1). These characteristics, together with our observations (below) that mixed bone marrow cultures formed pits in dentine slices, support the assumption that the TRAP positive multinucleated cells observed in the cocultures are osteoclast-like cells.

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Figure FIG. 1.. 1,25(OH)2D3 dose response for C3H/HeJ and C57BL/6J marrow osteoclast formation. Cocultures were established with Swiss Webster osteoblasts and femoral marrow cells as described in the Materials and Methods. Data points are mean ± SEM. The error bars are obscured by the symbols in all but one point. The C57BL/6J (circles) cultures had significantly more osteoclasts from 10−10 to 10−7 M 1,25(OH)2D3 (p < 0.001 at 10−10 to 10−8 M and p < 0.05 at 10−7 M) than the C3H/HeJ mice (squares). n = 6 replicates per dose.

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Comparison of osteoclast formation in vitro

There was a significantly greater number of osteoclasts generated per 106 marrow cells from C57BL/6J compared with C3H/HeJ femurs at all ages tested from 3–14 weeks of age, as summarized in Table 2. These experiments were repeated with marrow cells from female mice of different ages and with marrow cells from both male and female mice at 8 weeks of age. In 11 out of 12 cell preparations, the number of osteoclasts per 106 bone marrow cells was significantly higher for C57BL/6J mice than the C3H/HeJ mice. To assess the variation from animal to animal and verify that the differences between mouse strains were not simply due to errors in counting cells of the bone marrow preparations, an experiment was conducted comparing four replicate groups from each strain. Each group contained marrow cells pooled from the femurs of two mice. Cells of each group were counted separately and osteoclast formation was determined in three culture wells per group (Table 3). These data demonstrated that, in all mice tested, C57BL/6J marrow cells gave rise to more osteoclast formation in vitro than C3H/HeJ marrow cells.

Table Table 2.. Osteoclast Formation in Cocultures of Newborn Swiss Webster Osteoblasts and Either C57BL/6J or C3H/HeJ Marrow Cells or Spleen Cells
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Table Table 3.. Variation in Osteoclast Formation Between Animals of the Same Strain
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To ascertain whether the differences in osteoclast formation observed in female mice also occurred in male mice, an experiment was done with marrow cells from male C3H/HeJ and C57BL/6J mice. Within the same strain, male and female femur marrow cells produced comparable numbers of osteoclasts. Osteoclast formation from male C57BL/6J mice was significantly higher than from male C3H/HeJ mice (Table 2).

In one experiment with the youngest mice (3.5-week-old), we isolated cells from the spleen as well as bone marrow. In cocultures with Swiss Webster osteoblasts, spleen and bone marrow cells from C57BL/6J mice produced more osteoclasts compared with C3H/HeJ mice (Table 2).

Previous studies of mouse bone marrow and osteoblast cocultures normally treated the cultures with 1,25(OH)2D3 at 10−8 M. One possible explanation for the difference in osteoclast formation between C3H/HeJ and C57BL/6J mice is that osteoprogenitor cells in the two strains are differentially dependent on 1, 25(OH)2D3 concentration. To test this possibility, we treated cocultures with 1,25(OH)2D3 doses from 10−7 to 10−11 M. The maximal number of osteoclasts formed at 10−9 M 1,25(OH)2 D3 in cultures from bone marrow cells of both mouse strains (Fig. 1). At all doses tested, the C57BL/6J bone marrow cell population produced more osteoclasts than the C3H/HeJ bone marrow cell population.

Not only were there more osteoclasts formed in vitro per 106 marrow cells in the C57BL/6J mice, but at all ages examined, from 3.5 to 14 weeks, the number of nucleated marrow cells per femur was higher in the C57BL/6J mice, probably as a result of the larger medullary cavity. In the experiment described in Table 2, for example, 6.5- to 7-week-old C3H/HeJ mice had 4.25 ± 0.34 × 106 nucleated marrow cells per femur, while C57BL/6J mice had 7.08 ± 0.30 × 106 cells per femur. Thus, with 83 ± 2 and 498 ± 79 osteoclasts formed per 106 nucleated marrow cells, the C3H/HeJ and C57BL/6J mice formed 353 ± 9 and 3530 ± 43 osteoclasts per femur, respectively.

To determine whether osteoclast size might vary between the two mouse strains, TRAP positive cells formed in vitro were divided into three groups based on the number of nuclei per cell (Fig. 2). In all three size classes, osteoclasts from C57BL/6J mice were more numerous than osteoclasts from C3H/HeJ mice (p < 0.001). There was no significant difference between mouse strains in the number of osteoclast nuclei per cell. C3H/HeJ marrow-derived osteoclasts had, on average, 5.55 ± 0.13 nuclei, while C57BL/6J marrow derived osteoclasts had 5.46 ± 0.05 nuclei per cell (n = 6). Because there were more osteoclasts per well in C57BL/6J marrow derived cultures, the total number of osteoclast nuclei per culture well was greater in C57BL/6J than in C3H/HeJ cultures.

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Figure FIG. 2.. Distribution of nuclei per osteoclast. Cocultures were established with Swiss Webster osteoblasts as described in the Materials and Methods. TRAP positive cells with one or two nuclei were not counted. The numbers of TRAP positive osteoclasts (mean ± SEM) with 3–5, 6–10, and 11–20 nuclei/cell were significantly greater in C57BL/6J (hatched bars) than in C3H/HeJ (solid bars) cultures, p < 0.001 for each. The average number of nuclei per osteoclast was 5.55 ± 0.13 for C3H/HeJ cultures and 5.46 ± 0.05 for C57BL/6J cultures (NS, n = 6 replicates per group).

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Osteoclasts that developed from C57BL/6J and C3H/HeJ bone marrow cells in cocultures with Swiss/Webster osteoblasts resorbed pits in dentin slices. Pits stained by toluidine blue or by acid hematoxylin exhibited characteristic morphologies described previously. C57BL/6J bone marrow cell cultures from 4.5-week-old mice produced 2.5-fold as many pits per dentin slice as were produced by C3H/HeJ cultures (p < 0.01; Table 4). In the same experiment, the number of TRAP positive multinucleated osteoclasts that formed in plastic wells was also greater for C57BL/6J than C3H/HeJ cultures. Addition of 10 mU/ml calcitonin decreased the number of pits formed by osteoclasts to < 3% (Table 4). Areas of the pits formed in cultures from both strains of mice had a broad range. The mean pit area in C3H/HeJ cultures was 16% higher than the mean area in C57BL/6J cultures but was not significantly different. When pit area values were log transformed, however, the transformed values exhibited a normal distribution (Fig. 3) and were statistically different (p < 0.05).

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Figure FIG. 3.. Areas of pits formed in dentin slices by C57BL/6J and C3H/HeJ osteoclasts. Areas were determined in a random sample of pits (473 from C57BL/6J, 392 from C3H/HeJ) formed by mixed cultures of 4.5-week-old C57BL/6J (hatched bars) or C3H/HeJ (solid bars) marrow cells and newborn Swiss/Webster osteoblasts. Histogram classes are powers of two. The mean area of C3H/HeJ pits, shown in Table 3, was significantly different (p < 0.05) from the mean area of C57BL/6J pits when areas were log transformed.

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Table Table 4.. Pit Formation in Marrow Cell Osteoblast CoCultures Plated on Dentine Slices
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Since there were virtually no osteoclasts formed in the absence of osteoblasts in our experiments, and previous studies have demonstrated the importance of osteoblasts in inducing osteoclast formation, we hypothesized that osteoblasts in the marrow or on the endosteum of C57BL/6J and C3H/HeJ mice may influence the relative amount of osteoclast formation. Although our experiments using osteoblasts from a third unrelated mouse strain (Swiss Webster) suggested that differences in osteoclast formation between C57BL/6J and C3H/HeJ marrow arise from differences in the marrow cells, these experiments do not rule out possible effects of osteoblast genotype on the induction of osteoclast formation. To assess whether the number of osteoclasts generated in vitro was determined by the strain from which the osteoblasts were derived, experiments were performed using neonatal osteoblastic cells from either C57BL/6J or C3H/HeJ calvariae, with marrow cells from either the same strain or from the heterologous strain. Surprisingly, there was no effect of the osteoblast cells to alter numbers of osteoclasts formed from either mouse strain (Fig. 4). Whether Swiss Webster, C57BL/6J, or C3H/HeJ cells were used as the osteoblast source, there were always more osteoclasts formed from C57BL/6J marrow than from C3H/HeJ marrow.

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Figure FIG. 4.. Osteoclast formation in cocultures with C57BL/6J and C3H/HeJ osteoblasts. Marrow cells from 9-week-old mice were cocultured with osteoblasts from either newborn C57BL/6J or C3H/HeJ calvariae. The C57BL/6J marrow cells (hatched bars) produced more osteoclasts compared with C3H/HeJ marrow cells (solid bars), whether they were plated with C57BL/6J or C3H/HeJ osteoblasts, p < 0.001 for each. Values are mean ± SEM, n = 6 per group.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

C57BL/6J mice had more osteoclasts on endosteal bone surfaces compared with C3H/HeJ mice. The site in the humerus that we investigated is an area of active bone remodeling in which relative rates of bone formation and resorption determine the amount of trabecular bone present. We speculate that the increased number of osteoclasts in C57BL/6J bone results in increased resorption compared with C3H/HeJ mice. This supports previous evidence for increased resorption in C57BL/6J mice that was based on increased pyridinoline excretion.(14)

Osteoclast formation in vitro was significantly greater from C57BL/6J than C3H/HeJ marrow cells at all ages tested. This suggests that the difference in osteoclast formation was not caused simply by a developmental delay of osteoclast precursor populations in C3H/HeJ mice which would eventually “catch up” to C57BL/6J mice. Osteoclast formation from spleen cells was also greater in C57BL/6J mice compared with C3H/HeJ mice, suggesting that possible differences in osteoclast precursor populations are not restricted to the bone marrow microenvironment but might be influenced by systemic factors that differ quantitatively between the mouse strains. One possible factor is circulating 1,25(OH)2D3. However, in a preliminary report,(22) circulating 1,25(OH)2D3 levels were lower in C57BL/6J than in C3H/HeJ mice, suggesting either that the sensitivity of C57BL/6J osteoclast precursor cells to 1,25(OH)2D3 might be greater in C57BL/6J mice or that different 1,25(OH)2D3 levels in vivo cannot account for different osteoclast formation in vitro. Our data from 1,25(OH)2D3 dose-response experiments suggest that the difference in osteoclast formation in vitro between C3H/HeJ mice and C57BL/6J mice is not due to differential sensitivity to 1,25(OH)2D3. Another possible systemic difference between the mouse strains is circulating estrogen or androgen levels. However, osteoclast production was not dependent on the sex of the mice from which the bone marrow cells were isolated. This result suggests that potential differences in sex steroid levels or cell sensitivity to sex steroids (or other sex-related differences) cannot account for the differences in osteoclast formation that occur between the two mouse strains. Last, circulating growth hormone and insulin-like growth factor (IGF) levels, which are positively associated with bone resorption and osteoclast formation,(23) might differ between C3H/HeJ and C57BL/6J mice. C3H/HeJ mice have higher circulating levels of growth hormone and IGF-I,(24) which may contribute to the higher bone formation that occurs in C3H/HeJ mice.(13) Higher IGF-I levels, however, would give rise to higher numbers of osteoclasts, so IGFs cannot account for the lower osteoclast numbers in C3H/HeJ mice.

Osteoclasts from C3H/HeJ and C57BL/6J mice resorbed pits in dentin slices, as described previously in studies using the same mixed culture model with different mouse strains.(17,18) The number of pits was greater in cultures from C57BL/6J than C3H/HeJ mice and was roughly proportional to the number of osteoclasts formed in the same experiment. The size of resorption pits was nearly the same in C3H/HeJ and C57BL/6J cultures and was statistically different only when pit areas were log transformed. Whether meaningful differences exist in the abilities of osteoclasts from the two strains to resorb bone cannot be determined from the present data. The data do indicate, however, that the reduced number of pits formed in C3H/H3J cultures does not result from defective pit formation.

Since it is well established that osteoblastic stromal cells are important regulators of osteoclastogenesis from bone marrow precursor cells, we initially considered the corollary hypothesis that differences in osteoclast formation from C3H/HeJ compared with C57BL/6J mouse marrow cells result from differences in the capacities of the stromal cells from the respective mouse strains to stimulate or support osteoclast formation. The stromal cells not only secrete soluble factors such as macrophage colony-stimulating factor (M-CSF), which stimulate osteoclast differentiation, but cell contact between stromal cells and osteoclast progenitor cells may also be necessary for osteoclast formation.(25) In the SAMP6 mouse genetic model for accelerated aging, decreased osteoclast formation in SAMP6 bone marrow culture was attributed to defective stromal cells rather than defective osteoclasts.(26) Results of the present study, however, do not support a difference in the ability of osteoblasts to support osteoclast formation in vitro. The results are consistent with the hypothesis that C3H/HeJ and C57BL/6J mouse strains differ in the number of bone marrow osteoclast progenitor cells, or in the ability of osteoblastic cells to differentiate into mature osteoclasts in the coculture system, or in both characteristics.

We found that C57BL/6J osteoblasts produced higher levels of IL-6 and granulocyte M-CSF compared with C3H/HeJ osteoblasts.(27) Although these differences in osteoblast cytokine production cannot account for the differences in osteoclast formation observed between the mouse strains (because in the present study C57BL/6J osteoblasts did not support increased osteoclast formation from marrow cells of either strain compared with C3H/HeJ osteoblasts) it is possible that differential cytokine signaling in the bone marrow in vivo results in increased numbers of osteoclast progenitor cells in C57BL/6J mice compared with C3H/HeJ mice.

Interestingly, we have observed differences between C3H/HeJ and C57BL/6J mice in osteoblast properties, osteoblast formation from marrow cell precursors in vitro,(12) and bone formation in vivo.(13) Osteoblasts isolated from femurs and calvariae of C3H/HeJ mice had higher ALP activities compared with osteoblasts from C57BL/6J mice, and the number of ALP positive stromal cell colonies formed per 106 marrow cells was greater in C3H/HeJ mice compared with C57BL/6J mice. In vivo studies found that endosteal bone mineral apposition rate and relative bone formation rate were significantly higher in C3H/HeJ mice than C57BL/6J mice. Thus, different genes affecting resorption and formation could contribute to the difference in bone density between C3H/HeJ and C57BL/6J mice. Furthermore, our findings indicate that bone marrow populations from C3H/HeJ and C57BL/6J mice have an inverse relationship between osteoblast and osteoclast progenitor cell numbers. Although these observations do not indicate a causal relationship, one could speculate that the increased osteoblast progenitor cell population and increased bone formation that are apparent in C3H/HeJ bone may create an environment that is inhibitory to osteoclast progenitor cell formation. However, how this mechanism could account for differences in osteoclast formation from spleen cells of the two mouse strains is currently unclear.

In summary, our data suggest that there are differences in osteoclast precursor populations of the bone marrow between C3H/HeJ and C57BL/6J mice. These differences could contribute to the differences in bone density observed between these two inbred mouse strains. The ultimate goal of our studies is to identify genes that can account for the difference in bone density between the C3H/HeJ and C57BL/6J mice. The results of the previous and the present study together suggest that genes that regulate osteoclast lineage cells, as well as genes that regulate osteoblast lineage cells, may contribute to the difference in bone densities which exists between the two inbred mouse strains.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference

The authors thank Ms. Angela Eason and Ms. Sarán Wilkins for expert technical assistance. We thank Dr. Helen Gruber of Carolinas Medical Center for consultation on histologic and histochemical methods. Funding for this work was supplied by grants from the National Institutes of Health #AR-43618 and #CA-34196 (W.G.B.), by a subcontract from U.S. Army Medical Research Acquisition Activity Grant DAMD17-96-1-6306 (W.G.B. and D.J.B.), by Medical Research grants from the Department of Veterans Affairs (T.A.L., J.R.F., D.J. B.), and by the Loma Linda University School of Medicine Research Support Fund.

Reference

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Reference
  • 1
    Takeshita T, Yamagata Z, Iijima S, Nakamura T, Ouchi Y, Orimo H, Asaka A 1992 Genetic and environmental factors of bone mineral density indicated in Japanese twins. Gerontology 38:4349.
  • 2
    Kelly PJ, Harris M 1995 Genetic regulation of peak bone mass. Acta Paediatr 411:2429.
  • 3
    Alekal L, Clasey JL, Fehling PD, Weigel RM, Boileau RA, Erdman JW, Stillman R 1995 Contributions of exercise, body composition, and age to bone mineral density in premenopausal women. Med Sci Sports Exerc 27:14771485.
  • 4
    Jouanny P, Guillemin F, Kuntz C, Jeandel C, Pourel J 1995 Environmental and genetic factors affecting bone mass. Similarity of bone density among members of healthy families. Arthritis Rheum 38:6167.
  • 5
    Livshits G, Vainder M, Pavlovsky O, Kobyliansky E 1996 Population biology of human aging: Ethnic and climatic variation of bone age scores. Hum Biol 68:293314.
  • 6
    Matkovic V 1996 Nutrition, genetics and skeletal development. J Am Coll Nutr 15:556569.
  • 7
    Ralston SH 1997 The genetics of osteoporosis. QJM 90:247251.
  • 8
    Ferrari S, Rizzoli R, Slosman D, Bonjour JP 1998 Familial resemblance for bone mineral mass is expressed before puberty. J Clin Endocrinol Metab 83:358361.
  • 9
    Hopper JL, Green RM, Nowson CA, Young D, Sherwin AJ, Kaymakci B, Larkins RG, Wark JD 1998 Genetic, common environment, and individual specific components of variance for bone mineral density in 10- to 26-year-old females: A twin study. Am J Epidemiol 147:1729.
  • 10
    Murray EJ, Song MK, Laird EC, Murray SS 1993 Strain-dependent differences in vertebral bone mass, serum osteocalcin, and calcitonin in calcium-replete and -deficient mice. Proc Soc Exp Biol Med 203:6473.
  • 11
    Beamer WG, Donahue LR, Rosen CJ, Baylink DJ 1996 Genetic variability in adult bone density among inbred strains of mice. Bone 18:397403.
  • 12
    Dimai HP, Linkhart TA, Linkhart SG, Donahue LR, Beamer WG, Rosen CJ, Farley JR, Baylink DJ 1998 Alkaline phosphatase levels and osteoprogenitor cell numbers suggest that bone formation may contribute to peak bone density differences between two inbred strains of mice. Bone 22:211216.
  • 13
    Sheng MH-C, Beamer WG, Donahue LR, Rosen CJ, Baylink DJ, Wergedal JE 1998 Phenotype studies show that endosteal bone formation is greater in C3 (high density) than B6 (low density) mice during growth. FASEB J 12:A508.
  • 14
    Baylink D, Farley J, Donahue L, Rosen CJ, Barr B, Lee JES, Beamer W 1995 Evidence that high peak bone density is due to decreased resorption not increased formation in a murine model. J Bone Miner Res 10:338.
  • 15
    Boyce BF, Wright K, Reddy SV, Koop BA, Story B, Devlin R, Leach RJ, Roodman GD, Windle JJ 1995 Targeting simian virus 40 T antigen to the osteoclast in transgenic mice causes osteoclast tumors and transformation and apoptosis of osteoclasts. Endocrinology 136:57515759.
  • 16
    Takahashi N, Akatsu T, Udagawa N, Sasaki T, Yamaguchi A, Moseley JM, Martin TJ, Suda T 1988 Osteoblastic cells are involved in osteoclast formation. Endocrinology 123:26002602.
  • 17
    Jimi E, Nakamura I, Amano H, Taguchi Y, Tsurakai T, Tamura M, Takahashi N, Suda T 1996 Osteoclast functions is activated by osteoblastic cells through a mechanism involving cell-to-cell contact. Endocrinology 137:21872190.
  • 18
    Girasole G, Passeri B, Jilka RL, Manolagas SC 1994 Interleukin-11: A new cytokine critical for osteoclast development. J Clin Invest 93:15161524.
  • 19
    Udagawa N, Takahashi N, Katagiri T, Tamura T, Wada S, Findlay DM, Martin TJ, Hirota H, Taga T, Kishimoto T, Suda T 1995 Interleukin (IL)-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors. J Exp Med 182:14611468.
  • 20
    Takahashi N, Udagawa N, Akatsu T, Tanaka H, Shionome M, Suda T 1991 Role of colony-stimulating factors in osteoclast development. J Bone Miner Res 6:977985.
  • 21
    Takada Y, Kusuda M, Hiura K, Sato T, Mochizuki H, Nagao Y, Tomura M, Yahiro M, Hakeda Y, Kawashima H, Kumegawa M 1992 A simple method to assess osteoclast-mediated bone resorption using unfractionated bone cells. Bone Miner 17:347359.
  • 22
    Chen C, Beamer WG, Donahue LR, Kalu DN 1996 Strain differences in serum 1,25(OH)2 levels and in intestinal vitamin D receptor occupancy between C3H/HeJ and C57BL/6Jmice. J Bone Miner Res 11 (Suppl 1):S316.
  • 23
    Mochizuki H, Hakeda Y, Wakatsuki N, Usui N, Akashi S, Sato T, Tanaka K, Kumegawa M 1992 Insulin-like growth factor-I supports formation and activation of osteoclasts. Endocrinology 131:10751080.
  • 24
    Rosen CJ, Dimai HP, Vereault D, Donahue LR, Beamer WG, Farley J, Linkhart S, Linkhart T, Mohan S, Baylink DJ 1997 Circulating and skeletal insulin-like growth factor-I (IGF-I) concentrations in two inbred strains of mice with different bone mineral densities. Bone 21:217223.
  • 25
    Suda T, Udagawa N, Nakamura I, Miyaura C, Takahashi N 1995 Modulation of osteoclast differentiation by local factors. Bone 17:87S91S.
  • 26
    Jilka RL, Weinstein RS, Takahashi K, Parfitt AM, Manolagas SC 1996 Linkage of decreased bone mass with impaired osteoblastogenesis in a muring model of accelerated senescence. J Clin Invest 97:17321740.
  • 27
    Linkhart T, Linkhart S, Farley J, Dimai HP, Quian H-Y, Horowitz M, Beamer W, Donahue LR, Rosen C, Baylink DJ 1996 Osteoblast production of osteolytic cytokines: Differences between low peak bone density C57BL/6J mice and high peak bone density C3H/HeJ mice. J Bone Miner Res 11 (Suppl 1):S167.