Osteoclast Formation in Bone Marrow Cultures from Two Inbred Strains of Mice with Different Bone Densities

Abstract

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)

Postnatal and Pubertal Skeletal Changes Contribute Predominantly to the Differences in Peak Bone Density Between C3H/HeJ and C57BL/6J Mice

“…The difference in cortical thickness between C3H and B6 mice appeared to be caused by significantly smaller endosteal circumference in C3H mice (approximately 20% less) compared with B6 mice. The increase in cortical thickness of approximately 25% during puberty (days [23][24][25][26][27][28][29][30][31][32][33][34][35] in both strains of mice appeared mainly to be caused by increased periosteal circumference, because the endosteal circumference did not show significant change during this period (Fig. 3).…”

“…Consistent with this idea were the findings that (1) C3H mice exhibited significantly higher ALP activity in the femoral bone extract than did B6 mice at the time when maximal differences in bone density between the two strains occur, (2) C3H mice exhibited greater endosteal bone formation rate than B6 mice by histomorphometric analysis, (21) and (3) there were fewer osteoclast precursors in the marrow of C3H mice than in B6 mice. (28) Further studies are needed to evaluate the relative contribution of endosteal bone formation versus endosteal bone resorption in contributing to differences in cortical thickness between the two strains of mice during postnatal growth.…”

“…In addition, serum levels of IGF-I and osteocalcin provided statistically significant contributions to the prediction of various bone parameters (Table 3). To further evaluate the relative contribution of the previously mentioned five independent variables to predict the gender differences in bone size, we performed multiple regression analyses using data from prepubertal (days 7-23), pubertal (days [23][24][25][26][27][28][29][30][31][32][33][34][35], and postpubertal (days 35-56) periods. We found that the relative contribution of gender to differences in periosteal circumference was much greater during the postpubertal period than the prepubertal period (␤-coefficient of 0.36 vs. 0.11).…”

Role of genetic background in determining phenotypic severity throughout postnatal development and at peak bone mass in Col1a2 deficient mice (oim)

“…By one month of age, C3 mice have a greater cortical bone width coupled with a smaller marrow cavity diameter than B6 femurs and these differences persist and become more exaggerated until one year of age [38,40]. Additionally, by 12 weeks of age, C3 femurs have thicker trabeculi and smaller trabecular separation compared to B6 femurs [41]. These findings suggest that differences in adult bone shape and strength may be due to allelic variability that influences in utero bone development and that these differences persist throughout life.…”

Evidence that IGF-binding protein-5 functions as a growth factor

“…Osteoblast cells used were isolated by collagenase digestion from calvariae of newborn C3H/HeJ mice as described previously (28). The cells released were washed in DMEM + 10% calf serum and plated in the same media in 10-cm plates.…”