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Keywords:

  • bone density;
  • puberty;
  • insulin-like growth factor I;
  • osteocalcin;
  • C3H/HeJ and C57BL/6J mice

Abstract

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

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.


INTRODUCTION

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

The risk of developing senile osteoporosis in men and postmenopausal osteoporosis in women is in large measure determined by the amount of bone mass accumulated during the active growth phases early in life. This accumulation of bone mass determines the peak bone density attained during the third decade of life.(1,2) About 60–70% of variance in peak bone density is determined genetically,(3) and 40–50% of peak bone density is accumulated during puberty.(4) Elucidation of the mechanisms regulating this dramatic increase in skeletal mass during puberty is important in the effort to identify potential preventive or interventive measures that would lower the risk of developing osteoporosis.

In humans, peak bone density is attained between 20 and 30 years of age.(1,2) The most rapid accumulation in bone mass occurs in postnatal growth, from birth roughly to 3 years of age, and during puberty, from 12 to 18 years of age.(5) This increase in bone mass closely correlates with an increase in body weight and height. Puberty is initiated 2 years earlier in girls than in boys and during this period of pubertal maturation, a sexual dimorphism is established. The pubertal growth spurt lasts roughly 2 years longer in boys than in girls, such that boys are taller, stronger, and exhibit higher peak bone mass than do girls.(6)

The most beneficial period for osteoporosis prevention may be during the rapid growth phases that occur within the first 20 years of life.(7) The attainment of higher peak bone mass during this period should lead to reduced risk for fracture later in life by increasing peak bone mass.

In this study we are particularly interested in the different rates of bone remodeling occurring before, during, and after puberty. In studies comparing bone mass accumulation, Slemenda et al. found that higher peak bone mass in black girls is associated with lower bone turnover during puberty compared with that of white girls.(8) During puberty, a complex series of events occurs that impacts the changes in bone turnover and attainment of peak bone mass, such as periosteal expansion and endosteal contraction, longitudinal growth, modeling-dependent remodeling,(9) and sex hormone-dependent reduction in bone turnover or remodeling.(10) With the development of assays for serum/urine levels of bone formation and resorption markers, the measurement of bone turnover in different conditions of growth or disease becomes possible. During growth and in osteolytic diseases, a high bone turnover rate exists.(11–14)

Studies using mice as a model system have shown that mice, like humans, attain a peak bone density (at 4–6 months of age). Some strains experience age-related bone loss (1 year and older) leading to osteopenia.(15,16) Different strains of inbred mice have been shown to exhibit different peak bone densities(17) and responded differently to ovariectomy.(18) Similar to human studies using monozygotic and dizygotic twins, different strains of inbred mice provide genetically identical animals to dissect out the role of individual genes and their products in the development of a trait or disease. However, unlike the design restraints on sample size and genetic diversity in humans, mice provide a relatively unlimited number of genetically identical subjects. Two inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), which have the same body size and weight, previously have been shown to have very different peak bone densities.(17,19–21) Therefore, we chose to compare these two strains to study the following: (1) the relative contribution of postnatal (from birth until puberty), pubertal, and postpubertal growth to the final peak bone density of each strain; (2) the differences in bone remodeling between these two strains that may contribute to these different bone densities; (3) the differences in serum level of insulin-like growth factors (IGFs) between these two strains during postnatal, pubertal, and postpubertal growth; and (4) to determine if and when the sexual dimorphism in peak bone mass seen in humans after puberty also is seen in these mice.

MATERIALS AND METHODS

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

Recombinant human (rh) IGF-I (rhIGF-I) was a gift from Ciba-Geigy (Basel, Switzerland); rhIGF-II was purchased from BACHEM Chemicals (Torrance, CA, USA). Rabbit polyclonal antiserum against IGF-I (kindly provided by L.E. Underwood and J.J Van Wyk) was obtained form the National Hormone and Pituitary Program (Baltimore, MD, USA). Osteocalcin synthetic peptide was obtained from SynPep Corp. (Dublin, CA, USA). Paranitrophenol phosphate was purchased from Fluka (Buchs, Switzerland). All other chemicals were enzyme grade and purchased from Fisher Scientific (Tustin, CA, USA) or Sigma Chemical Co. (St. Louis, MO, USA).

In vivo work

Animals

Inbred C3H and B6 breeder mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). On receipt, the mice were allowed to acclimate to the new environment for 7 days and were then mated to produce the pups used in this study.

Animal protocol

The female breeders were housed individually several days before expected parturition and fed a breeders' diet (10% fat content; Harlan Teklad, Placentia, CA, USA). The pups were weaned and weighed on postnatal day 21 using a digital scale (ACCULAB V-600) to the nearest 0.1 g. At weaning, the pups were fed regular rodent chow (4% fat content; Harlan Teklad) and housed 4–5 pups per cage with cages of females alternating with cages of males on each rack to eliminate uneven pheromone distribution. Pubertal maturation in B6 female mice begins when serum estradiol increases on day 26 and vaginal opening occurs by day 31.(22) In humans, puberty starts roughly 2 years later in males than in females and ends later in males than females.(23) We calculated that in mice, this difference would constitute a 2–3 day delay for males compared with females, so that puberty should be completed by day 35 in males. Based on this data, we chose to collect pups at days 7, 14, and 23 (before puberty starts); day 31 (at the end of puberty for the females); day 35 (at the end of puberty for the males); and on days 42, 49, and 56. Pups were killed by CO2 inhalation (dry ice) followed by decapitation. Whole blood was collected, allowed to clot, and then centrifuged. The serum was skimmed off and stored at −70°C until assayed. The left femur was dissected free of soft tissue keeping the condyles and femoral neck intact. This bone was used for the bone extract assay to determine bone-specific alkaline phosphatase (ALP) activity and bone size data. The right femur and proximal tibia were dissected free and stored in 70% ethanol for volumetric bone density and geometric parameter determination by peripheral quantitative computed tomography (pQCT) using an XCT Research M instrument (Norland Medical Systems, Inc., Fort Atkinson, WI, USA). The experimental procedures performed in this study comply with the National Institutes of Health (NIH) guide for the Care and Use of Laboratory Animals. All animals studies were reviewed and monitored by the Animal Studies Subcommittee at the Jerry L. Pettis Veterans Administration Medical Center (Loma Linda, CA, USA).

Bone densitometry

Volumetric bone density and geometric parameters were determined by pQCT. Previously, pQCT has been validated as a reliable method of determining volumetric bone density and geometric parameters in mice.(17) Routine calibration was performed daily with a defined standard (cone phantom) containing hydroxyapatite embedded in lucite (Norland Medical Systems, Inc.). The voxel size was set at 0.07 mm and a half-millimeter-thick slice was scanned at the middiaphysis. Analysis of the scans was performed using the manufacturer-supplied software program (STRATEC MEDIZINTECHNIK GMBH Bone Density Software, version 5.40C; Norland Medical Systems). Volumetric bone density and geometric parameters were estimated with Loop analysis. For cortical analysis, the threshold was set at 350 mg/cm3 for the 23- to 56-day-old mice. The 7- and 14-day-old mice had lower bone densities than did the 23- to 56-day-old mice and could not be measured at the 350 mg/cm3 threshold. Therefore, we used a threshold of 150 mg/cm3 for the 7- to 23-day-old mice in the cortical analysis. For cancellous analysis, the outer threshold was set at 250 mg/cm3 and the inner threshold was set at 300 mg/cm3 for 23- to 56-day-old mice, and 125 mg/cm3 and 150 mg/cm3 for 7-to 14-day-old mice, respectively. Values for 7- and 14-day-old mice may be less accurate than for the older mice because of the lower threshold used, but we have included them in this article because they show the rapid growth that occurs during the postnatal period (days 7–23).

Biochemical assays

Femoral bone extraction

The freshly dissected left femora were rinsed in 1× phosphate-buffered saline (PBS) for 24 h at 4°C and then transferred to 0.01% Triton X-100 (Sigma Chemicals) for 72 h at 4°C to extract the membrane bound ALP from the osteoblast cell surfaces. Bone-specific ALP activity and total protein content were determined for this extract. The femora were dehydrated in 75% ethanol and then allowed to dry out completely at 37°C. Once dry, the femoral length was measured to the nearest 0.01 mm using a caliper (Dial Caliper; Mitutoyo Corp., Japan) and the bones were weighed to the nearest 0.01 mg using a balance (Millibalance model 7500, Electrobalance DTL; Cahn Instruments, Inc., Cerritos, CA, USA).

ALP assay of bone extracts

ALP activity of the bone extract was determined using a paranitrophenol phosphate substrate as described previously.(24)

Protein assay

The total protein content of the bone extract was determined using Bradford assay (BioRad, Hercules, CA, USA)

Osteocalcin assay

Osteocalcin levels in serum were measured by specific radioimmunoassay (RIA) that was previously validated for mice. The mouse osteocalcin RIA had a CV of less than 8%.(25)

Separation of IGF binding proteins from IGFs

Because IGF binding proteins (IGFBPs) produce artifacts in IGF RIAs, complete separation of the IGFBPs from the IGFs is necessary in order for the IGF determinations to be valid. We have used previously validated BioSpin protocol to separate IGFs from IGFBPs.(26)

IGF-I RIA

IGF-I was measured by specific RIA as previously described.(27) The cross-reactivity of IGF-II in the IGF-I RIA was less than 0.5%. The sensitivity of the IGF-I RIA was less than 50 ng/liter; the intra- and interassay CVs were less than 10%.

IGF-II RIA

IGF-II was measured by specific RIA as previously described.(27) The cross-reactivity of IGF-I in the IGF-II RIA was less than 2%. The sensitivity of the IGF-II RIA was 0.1 μg/liter; the intra- and interassay CVs were less than 10%.

Statistics

Results are reported as mean ± SD for 7–9 animals per group and compared by two-tailed three-way analysis of variance (ANOVA) and Newman-Keuls post hoc test using commercially available statistical software (STATISTICA; StatSoft, Inc., Tulsa, OK, USA). Results were considered significantly different for p < 0.05.

RESULTS

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

The pattern of growth in body weight, femur weight, size, and mineral density have been determined for C3H and B6 mice for the postnatal, pubertal, and postpubertal growth periods. Although the general pattern of growth was similar for the two strains of mice, there were differences in femur bone growth. These differences were statistically significant as determined by three-way ANOVA (strain, sex, and day). The results of further analysis with Newman-Keuls post hoc testing are given in the tables and the figure legends.

Physical measurements

Body weight

Weight gain in all the mice was increased roughly 3-fold over day 7 during postnatal growth (days 7–23). There was no difference in body weights between females of C3H and B6 mice at any time points. Male C3H mice were heavier than B6 male mice at day 42 and day 56 (Tables 1 and 2). Within each strain, males gained more weight than females during pubertal growth (in B6, 161% for males and 133% for females; in C3H, 158% for males and 143% for females). Males were heavier than females at day 31 and thereafter for both strains (Tables 1 and 2).

Table Table 1.. Age Changes in Measured Parameters for Male C3H and B6 Mice
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Table Table 2.. Age Changes in Measured Parameters for Female C3H and B6 Mice
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Femoral bone weight

The fastest rate of femoral growth was during the postnatal period (days 7–23) when dry femoral weight increased 8-fold in B6 and 7-fold in C3H mice. The C3H bones were significantly heavier than the B6 bones at day 42 and day 49 in male mice and at day 56 in female mice (Tables 1 and 2).

Femoral bone length

Femoral bone length increased more than 2-fold in both strains of mice during postnatal period. Although femora of B6 mice were shorter on day 7, the length increased more than in C3H mice during postnatal growth (days 7–23) so that there was no difference between strains from day 23 (Tables 1 and 2). Within strains, male femoral length was not different from that of females, either during or after puberty.

Middiaphysis geometric parameters

Area

Total area at the middiaphysis of the femur increased 2-fold during postnatal growth (days 7–23) in both B6 and C3H mice. There was no difference by strain or sex in total area on day 7 and day 14. Total area in B6 male mice was 9% (p = 0.028) greater than that of C3H males on day 49 and 17% (p < 0.001) greater on day 56. B6 female mice had 13% (p = 0.008) greater total area than did C3H females from day 23 onward (Tables 1 and 2). The total area at the middiaphysis in B6 males was 9% (p = 0.005) greater than that of B6 females on day 56 and the value for C3H males was 16% (p < 0.001) greater than that of C3H females on day 49.

Cortical area

Cortical area at the middiaphysis of the femur was not different by strain or sex during rapid postnatal growth (days 7–23) but cortical area increased by 82% in B6 mice and 63% in C3H mice (Tables 1 and 2). C3H male mice had greater cortical area than did B6 male mice on day 31 (17%, p = 0.02) through day 56 (13%, p = 0.005). C3H female mice had greater cortical area than that of B6 female mice on day 31 (20%, p = 0.007), day 42 (16%, p = 0.03), and day 56 (15%, p = 0.01). Within strains, cortical area was 13% (p = 0.03) greater in B6 male mice than in B6 female mice by day 56, while cortical area was 19% (p < 0.001) greater in C3H male mice than in C3H female mice by day 42.

Medullary area

Medullary area increased dramatically during rapid postnatal growth (days 7–23) in both strains of mice (Fig. 1). Medullary area at the middiaphysis of the femur was significantly greater in B6 mice than in C3H mice from day 14 (45% in B6 male mice vs. C3H male mice [p = 0.001] and 55% in B6 female mice vs. C3H female mice [p < 0.001]). B6 mice had greater medullary area than did C3H mice for the duration of the study. At day 56, B6 male and female mice exhibited 149% and 108% greater medullary area than C3H male and female mice, respectively. Within strain, there was no difference between sexes at any time during the study.

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Figure FIG. 1. Changes in medullary area in C3H and B6 mice from day 7 to day 56. Medullary area increased significantly between days 7 and 23 in both strains. Medullary area was greater by 45% or more in B6 mice compared with C3H mice for both sexes from day 14 onward. Medullary area was not significantly different in males versus females for either strain. Values expressed as mean of 7–9 animals ± SD; B6 male mice, closed triangles; B6 female mice, closed circles; C3H male mice, open triangles; C3H female mice, open circles.

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Periosteal circumference

Periosteal circumference at the middiaphysis of the femur did not differ across strain or sex during rapid postnatal growth (days 7–14) (Fig. 2). B6 female mice had 12% (p < 0.001) greater periosteal circumference than that of C3H female mice at day 56, and B6 male mice had 8% (p < 0.001) greater periosteal circumference than that of C3H male mice at day 56. Within strains, periosteal circumference in B6 male mice was 5% (p = 0.01) greater than that of B6 female mice by day 56 and periosteal circumference was 8% (p < 0.001) greater in C3H male mice than that of C3H female mice by day 42.

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Figure FIG. 2. Changes in periosteal circumference in C3H and B6 mice from day 7 to day 56. At day 23 periosteal circumference was 5% (p = 0.03) greater in B6 male mice and 7% (p < 0.01) in B6 female mice than in C3H male and female mice, respectively. On day 56, periosteal circumference was 8% (p < 0.001) greater in B6 male mice and 12% (p < 0.01) greater in B6 female mice than in C3H male and female mice, respectively. Within strain, B6 male mice had 5% (p = 0.01) greater periosteal circumference than B6 female mice on day 56 and C3H male mice had 8% (p < 0.001) greater periosteal circumference than C3H female mice on day 42. Values expressed as mean of 7–9 animals ± SD; B6 male mice, closed triangle; B6 female mice, closed circles; C3H male mice, open triangles; C3H female mice, open circles; SD = bars.

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Endosteal circumference

Endosteal circumference of the middiaphysis increased 65–90% during rapid postnatal growth (days 7–23) and a total of 7–10% during pubertal (days 23–31) and postpubertal growth (days 31–56) in B6 mice (Fig. 3). In C3H mice, endosteal circumference increased by 60–70% during rapid postnatal growth (days 7–23) but did not change significantly throughout the rest of the study. Endosteal circumference was significantly greater in B6 mice than in C3H mice for the entire study. Within strains, endosteal circumference did not differ across sex for the entire study (Fig. 3).

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Figure FIG. 3. Changes in endosteal circumference in C3H and B6 mice from day 7 to day 56. Endosteal circumference was significantly greater in B6 mice compared with C3H mice at all age groups except at day 7. Endosteal circumference was not significantly different in male versus female mice for either strain. Endosteal circumference increased significantly between days 7 and 23 after which there was no significant difference in endosteal circumference. Values expressed as mean of 7–9 animals ± SD; B6 male mice, closed triangles; B6 female mice, closed circles; C3H male mice, open triangles; C3H female mice, open circles; SD = bars.

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Cortical thickness

Cortical thickness at the middiaphysis of the femur was roughly 30% (p < 0.01) greater in C3H mice than in B6 mice throughout the study. Within strains, cortical thickness was 9% (p < 0.05) greater in C3H male mice than in C3H female mice from day 42. But no significant differences across sex were evident in B6 mice (Fig. 4). The greatest increase in cortical thickness occurred during rapid postnatal (days 7–23) and pubertal (days 23–31) growth, while the rate of change in cortical thickness decreased dramatically after puberty.

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Figure FIG. 4. Changes in cortical thickness in C3H and B6 mice from day 23 to day 56. Cortical thickness was significantly greater in C3H mice compared with B6 mice at all time points studied. Cortical thickness was significantly greater in male C3H mice compared with female C3H mice at days 42, 49, and 56. In B6 mice, the cortical thickness was significantly different in male mice compared with female mice. Cortical thickness increased significantly during puberty in both strains of mice. Values expressed as mean of 7–9 animals ± SD; B6 males mice, closed triangles; B6 female mice, closed circles; C3H male mice, open triangles; C3H female mice, open circles.

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Bone densitometry

Mineral content

Total mineral content of the femoral middiaphysis increased 4-fold in both B6 mice and C3H mice during rapid postnatal growth (days 7–23; Fig. 5). Total mineral content in C3H mice was roughly 40% (p < 0.01) greater than that of B6 mice throughout the study (44% on day 7, 54% on day 31, and 38% on day 56). Within strain, total mineral content of B6 male mice was 25% (p = 0.01) greater than that of B6 female mice and 23% (p < 0.001) greater in C3H male mice than in C3H female mice on day 42.

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Figure FIG. 5. Changes in middiaphyseal total mineral content in C3H and B6 mice from day 7 to day 56. On day 23, C3H male mice had 56% (p < 0.001) and C3H female mice had 51% (p < 0.01) greater total mineral content than B6 male and female mice, respectively. On day 35, C3H male mice had 42% (p < 0.001) and C3H female mice had 39% (p < 0.001) greater total mineral content than B6 male and female mice, respectively. On day 56, C3H males had 36% (p < 0.001) and C3H females had 40% (p < 0.001) greater total mineral content than B6 male and female mice, respectively. Within strain, B6 male mice had 25% (p = 0.01) greater mineral content than B6 female mice by day 42 and C3H male mice had 23% greater mineral content (p < 0.001) than C3H female mice on day 42. Values expressed as mean of 7–9 animals ± SD; B6 male mice, closed triangle; B6 female mice, closed circles; C3H male mice, open triangles; C3H female mice, open circles; SD = bars.

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Density

Total volumetric bone density of the middiaphysis increased more in C3H mice (females, 81% and males, 83%) than in B6 mice (females, 37% and males, 51%) during rapid postnatal growth (days 7–23). Greater volumetric bone density was present in C3H mice on day 7 (C3H vs. B6 female mice, 27%; C3H vs. B6 male mice, 41%; p < 0.05), and by day 56, this difference was even greater (C3H vs. B6 female mice, 60%; C3H vs. B6 male mice 62%; p < 0.001). Within strain, there was no significant difference in volumetric bone density between sexes (Tables 1 and 2). The greatest difference in rate of total density accretion occurred during rapid postnatal (days 7–23) and pubertal (days 23–31) growth (Figs. 6 and 7). The rate of bone density gain in C3H mice during postnatal and pubertal growth was twice that of B6 mice, while it was not different after puberty.

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Figure FIG. 6. Changes in bone density in C3H and B6 mice from day 7 to day 56. Because bone density was not different between male and female mice of either strain, the data for both sexes were pooled (p < 0.01). Total volumetric bone density of the middiaphysis was about 30% greater in C3H mice than in B6 mice at day 7. C3H mice exhibited 70% more bone density than did B6 mice after puberty (day 31). Bone density was significantly higher in C3H mice than B6 mice at all time points (p < 0.01). Bone density more than doubled during puberty in both strains of mice. Values expressed as mean of 7–9 animals ± SD; B6 mice, closed triangle; C3H mice, open triangles; SD = bars.

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Figure FIG. 7. Changes in the rate of volumetric bone density accretion per day in C3H and B6 mice from day 7 to day 56. C3H mice had a 2.8-fold and a 1.6-fold higher rate of bone accretion from day 7 to day 14 and from day 23 to day 31, respectively, compared with B6 mice. The gain in bone density for each time period was calculated from the mean value for each time period and was divided by the duration in days to obtain the rate. Male and female mice were calculated separately and used as replicate values for the calculation of significance. B6 mice, closed triangles; C3H mice, open triangles.

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Biochemical assays

Serum IGF-I

C3H female mice had significantly higher serum levels of IGF-I than did B6 mice on day 7 and day 14, whereas C3H male mice exhibited higher serum IGF-I levels at day 7 (Tables 1 and 2). In the pooled data from male and female mice, C3H mice had greater serum IGF-I levels than B6 mice at days 7, 14, 35, and 42 (data not shown).

Serum IGF-II

On day 7 and day 14, IGF-II levels were not different by strain or sex. By day 23, serum IGF-II was nearly undetectable and remained at low levels for the duration of the study (data not shown).

Serum osteocalcin

Serum osteocalcin was significantly higher in B6 mice compared with that of C3H mice from day 7 (77%, p < 0.01) to 42 (33%, p < 0.01) in males and from day 7 (94%, p < 0.01) to 31 (42%, p < 0.01) in females. Serum osteocalcin increased roughly 2-fold during postnatal growth (days 7–23) in male mice of both strains and then decreased steadily until day 56. However, in female mice, serum osteocalcin levels doubled from days 7–14 and then decreased steadily until day 56 (Tables 1 and 2). Serum osteocalcin levels in female mice of both strains were 32% higher than those in male mice on day 14 (p < 0.01), but were not different at other time points.

Bone extract ALP

Bone extract ALP normalized for total extractable protein showed a 4-fold increase in activity during the rapid-bone modeling associated with increased bone length and size during postnatal growth (days 7–23) in both strains, although bone extract from C3H mice contained 2-fold greater ALP activity on day 14 than that of B6 mice (Fig. 8). A decrease of 30% in ALP activity occurred during puberty (days 23–31). Within strains, there was no difference in ALP activity between sexes at any time.

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Figure FIG. 8. Changes in bone-specific ALP activity in bone extracts for C3H and B6 mice from day 7 to day 56. On day 14, bone-specific ALP activity in C3H male mice was 75% (p < 0.001) and in C3H female mice was 95% (p < 0.001) greater than B6 male and female mice, respectively. On day 23, C3H male mice had 21% (p < 0.01) greater bone-specific ALP activity than B6 male mice, but C3H female mice were not different from B6 female mice. Values expressed as mean of 7–9 animals ± SD; B6 male mice, closed triangle; B6 female mice, closed circles; C3H male mice, open triangles; C3H female mice, open circles; SD = bars.

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Correlations

In both strains, body weight was highly correlated with all other physical, geometric, and densitometry measurements throughout the study (data not shown). Serum IGF-I levels were correlated positively with physical, geometric, and densitometry measurements in both strains, especially during postnatal growth but less so during pubertal and postpubertal growth (data not shown). In the pooled data from C3H and B6 strains of mice belonging to both sexes, multiple regression analyses were carried out using strain, sex, age, IGF-I, and osteocalcin as independent variables (Table 3). The β-coefficients, which represent the relative contribution of each independent variable in the prediction of the dependent variable, reveal that age is the most important predictor of various bone parameters, as expected. Furthermore, strain contributed predominantly to the differences in density, endosteal circumference, and cortical thickness. 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 23–35), 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 (β-coefficient of 0.36 vs. 0.11). To determine if gender differences in periosteal circumference can be explained based on IGF-I, we determined the relative contribution of strain, sex, and age to differences in IGF-I using data from prepubertal (days 7–23) and postpubertal periods (days 35–56). We found that strain, sex, and age nearly contributed equally (β-coefficient of 0.39, 0.3, and 0.26, respectively) during the postpubertal period. However, during the prepubertal period sex had no effect on serum IGF-I as expected (β-coefficient = 0.01).

Table Table 3.. Multiple Regression Analysis in the Pooled Data from Males and Females of C3H and B6 Strains of Mice
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DISCUSSION

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

In previous studies, two inbred strains of mice, C3H and B6, exhibited similar body weights but different volumetric peak bone densities.(17,19–21) To identify the growth period(s) in which this divergence in bone density becomes apparent, we evaluated temporal skeletal changes in C3H and B6 mice belonging to both sexes before, during, and after puberty. Our data show that although C3H mice exhibit slightly higher total bone density compared with B6 mice at 7 days of age (approximately 25%), total bone density is 69% greater in C3H mice compared with B6 mice at 31 days of age, despite similar body size, body weight, and bone length. Accordingly, the gain in bone density in C3H mice during postnatal (days 7–23) and pubertal (days 23–31) growth is more than twice that of B6 mice. In addition, the findings that the gain in bone density increases by more than 70% during puberty (days 23–31) attest that rapid skeletal changes occur during this period. Therefore, the postnatal (days 7–23) and pubertal (days 23–31) growth periods appear to be critical in the development of the difference in bone density between C3H and B6 mice. The difference in total bone density seen at day 35 between C3H and B6 mice was maintained during the rest of the study period.

The differences in cortical thickness between the two strains of mice reflect the greater accumulation of bone mineral in C3H mice compared with B6 mice during postnatal growth. This difference in cortical thickness between C3H and B6 mice developed during the rapid growth of the postnatal (days 7–23) and pubertal (days 23–31) periods. 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 23–35) 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). Although neither the periosteal nor the endosteal circumference showed significant change during postpubertal growth (days 35–56) in C3H mice, both the periosteal and the endosteal circumference increased in B6 mice during this period, resulting in B6 mice having significantly greater periosteal circumference than C3H mice at 56 days of age. Consistent with these data, Sheng et al.(21) recently have shown, by histomorphometric analysis, that the total cross-sectional area of the femur at the middiaphysis is significantly greater in the B6 mice at 6 weeks of age compared with C3H mice.

The cellular mechanisms that contribute to the greater cortical thickness in C3H mice compared with B6 mice can only be speculated at this time. In this regard, the findings that endosteal but not periosteal circumference is significantly different between the two strains of mice during postnatal growth suggested that differences in endosteal bone formation and resorption could, in part, contribute to the observed differences in cortical thickness. 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.

The divergence in total volumetric bone density at the femoral middiaphysis between C3H and B6 strains during postnatal growth may, in part, be caused by greater bone area in the C3H mice than in the B6 mice because of differences in cortical thickness. However, the possibility that the bones of C3H mice during early postnatal growth may be more mineralized or less porous compared with B6 mice cannot be ruled out because C3H mice exhibit approximately 30% greater bone density than B6 mice despite similar bone area on day 7. Although the finding that C3H mice exhibit greater ash weight and calcium content compared with the B6 mice at 9 weeks of age(19) are consistent with this idea, further studies are needed to evaluate whether or not C3H bones contain less porosity or more mineralization of the bone matrix compared with B6 mice during postnatal growth when the greatest difference in density was seen despite similarities in bone area.

The rapid increase in total mineral content during postnatal growth (days 7–23) appeared mainly to be caused by increased bone size, which increased by more than 50% during this period (Fig. 9). This increase in bone size appeared to be caused by increased periosteal bone formation rate because both osteocalcin levels in the serum and ALP activity in bone extract increased during this period. In terms of the individual factors that contribute to the increased periosteal bone formation, we postulate that IGF-I may be a potential candidate because: (1) serum levels of IGF-I increased dramatically during this period (Tables 1 and 2) and showed significant correlation with changes in periosteal circumference and biochemical measurements of bone formation during this period (Table 3); and (2) mice lacking IGF-I exhibit dramatic reduction in postnatal skeletal growth.(29) Consistent with a role for IGF-I, our findings also show significantly greater serum IGF-I levels in C3H mice than in B6 mice on days 7, 14, 35, and 42 in the pooled data from male and female mice. Consistent with these data, we have previously shown that the IGF-I differences are statistically significant up to 10 months of age.(20) Further studies are needed to evaluate the cause and effect relationship between changes in serum levels of IGF-I and skeletal changes in C3H and B6 mice during postnatal growth.

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Figure FIG. 9. A model of the changes in bone size during postnatal, pubertal, and postpubertal growth in C3H and B6 mice. On day 7, the periosteal circumference of C3H and B6 femora was similar; as illustrated by the cross-sectional diagram of the femur at the middiaphysis, B6 mice had a 20% greater endosteal circumference than did C3H mice, and C3H mice had 34% greater volumetric bone density than did B6 mice. Between days 7 and 23 periosteal circumference increased 50% in C3H mice and 58% in B6 mice, while endosteal circumference increased by 90% in both strains. On day 23, the periosteal circumference of C3H and B6 femora was similar; B6 mice had 16% greater endosteal circumference than did C3H mice, and C3H mice had 69% greater volumetric bone density than did B6 mice. On day 35, B6 mice had about 4% greater periosteal circumference than did C3H mice, B6 had a 22% greater endosteal circumference than did C3H mice, and C3H mice had 56% greater volumetric bone density than did B6 mice. On day 56, B6 mice had 8–12% greater periosteal circumference than did C3H mice, B6 had a 35% greater endosteal circumference than did C3H mice, and C3H mice had 61% greater volumetric bone density than did B6 mice.

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In contrast to postnatal growth, the mineral accumulation during puberty appears to be predominantly because of decreased bone resorption caused by increased male and female sex steroid hormones. There are a number of findings that support this idea. First, the increase in total bone density during puberty in both C3H and B6 mice was associated with a decrease in biochemical markers of bone remodeling in both serum and bone extracts. Second, we and others have shown that the increase in mineral accumulation during sexual development in girls is associated with decreased bone turnover.(8,10,30) Third, Slemenda et al.(8) have shown that black children accumulated 10% greater bone mass and had significantly reduced bone turnover, as measured by serum levels of osteocalcin and tartrate-resistant acid phosphatase, compared with white children during pubertal growth. Although sex steroid hormone-induced changes in cytokine production(31–33) have been implicated in the reduced bone turnover that occurs during puberty, the cause and effect relationship between cytokine production and the reduction in bone turnover during puberty remains to be established.

Surprisingly, we found that serum osteocalcin levels were significantly lower in C3H mice compared with B6 mice throughout the study. Because ALP activity in bone extract was significantly higher in the C3H mice than in the B6 mice during postnatal growth, we anticipated serum osteocalcin levels also to be higher during this period in C3H mice. There are a number of potential explanations for this discrepancy. It is possible that osteocalcin expression in osteoblasts is higher in C3H mice compared with B6 mice; however, a smaller proportion of synthesized osteocalcin was released into the extracellular fluid compared with the matrix compartment in C3H mice compared with B6 mice, resulting in reduced levels in serum. Metabolic differences in serum osteocalcin (e.g., half-life and clearance rate) between C3H and B6 mice also may contribute to the lower levels of serum osteocalcin in C3H mice compared with B6 mice. Alternatively, the higher level of osteocalcin produced in the B6 mice may act as an inhibitor of bone formation during postnatal growth because bone formation was increased at the endosteum in mice lacking osteo-calcin.(34)

Our findings also show that sexual dimorphic skeletal changes occurred in mice during puberty as in the case of humans.(4) This sexual dimorphism may be attributed to greater bone size and cortical thickness in men than in women,(35) which in turn may be caused by a later start and a longer pubertal growth period in boys than girls.(5) However, volumetric bone mineral density (BMD) does not appear to be different between the two sexes in humans.(36–38) Within strains, we found that 8-week-old male mice had a greater total mineral content and a larger periosteal circumference than did female mice, despite similar endosteal circumference. Thus, male mice of both strains exhibited increased bone size and cortical thickness resulting in greater total mineral content compared with female mice. The molecular mechanisms that are responsible for the increased periosteal bone formation and/or reduction in periosteal bone resorption in the male mice compared with female mice have yet to be established.

In conclusion, the difference in bone density between C3H and B6 strains of mice is established early and maintained throughout the period of growth. Two growth periods, postnatal and pubertal, are particularly critical to the development of peak BMD. Although the difference in bone density is maintained throughout, there are differences in the pattern of growth between strains that suggest that the genes that contribute to the difference in BMD during each period of growth may be different. This is consistent with the evidence from quantitative trait loci studies(39) that multiple genes contribute to the difference in bone density. The genes that are expressed differentially in the skeleton of C3H and B6 mice during postnatal and pubertal growth may provide clues regarding the genes that contribute to the BMD differences between these two strains.

Acknowledgements

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

The authors acknowledge the expert technical assistance provided by Daniel Bruch and Joe Rung-Aroon in this study. Support for this work was received from the NIH grant AR31062, the National Medical Technology Test Bed, and the U.S. Department of the Army.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Lloyd T, Rollings N, Andon MB, Demers LM, Eggli DF, Kieselhorst K, Kulin H, Landis JR, Martel JK, Orr G, Smith P 1992 Determinants of bone density in young women. I. Relationships among pubertal development, total body bone mass, and total body bone density in premenarchal females. J Clin Endocrinol Metab 75:383387.
  • 2
    Theintz G, Buchs B, Rizzoli R, Slosman D, Clavien H, Sizonenko PC, Bonjour JP 1992 Longitudinal monitoring of bone mass accumulation in healthy adolescents: Evidence for a marked reduction after 16 years of age at the levels of lumbar spine and femoral neck in female subjects. J Clin Endocrinol Metab 75:10601065.
  • 3
    Eisman JA, Kelly PJ, Morrison NA, Pocock NA, Yeoman R, Birmingham J, Sambrook PN 1993 Peak bone mass and osteoporosis prevention. Osteoporos Int 3(Suppl 1):5660.
  • 4
    van der Meulen MCH, Carter DR 1999 Mechanical determinants of peak bone mass. In: RosenCJ, GlowackiJ, BilezikianJP (eds.) The Aging Skeleton. Academic Press, San Diego, CA, USA, pp. 105114.
  • 5
    Gertner JM 1999 Childhood and adolescence. In: FavusMJ (ed.) Primer on Metabolic Bone Diseases and Disorders of Mineral Metabolism, 4th Ed. Lippincott Williams & Wilkins, Philadelphia, PA, USA, pp. 4549.
  • 6
    Abbassi V 1998 Growth and normal puberty. Pediatrics 102(2 Pt 3):507511.
  • 7
    Seeman E, Tsalamandris C, Formica C 1993 Peak bone mass, a growing problem? Int J Fertil Menopausal Stud 38(Suppl 2):7782.
  • 8
    Slemenda CW, Peacock M, Hui S, Zhou L, Johnston CC 1997 Reduced rates of skeletal remodeling are associated with increased bone mineral density during the development of peak skeletal mass. J Bone Miner Res 12:676682.
  • 9
    Schiessl H, Frost HM, Jee WS 1998 Estrogen and bone-muscle strength and mass relationships. Bone 22:16.
  • 10
    Cadogan J, Blumsohn A, Barker ME, Eastell R 1998 A longitudinal study of bone gain in pubertal girls: Anthropometric and biochemical correlates. J Bone Miner Res 13:16021612.
  • 11
    Garnero P, Delmas PD 1998 Biochemical markers of bone turnover. Applications for osteoporosis. Endocrinol Metab Clin North Am 27:303323.
  • 12
    Ross PD, Knowlton W 1998 Rapid bone loss is associated with increased levels of biochemical markers. J Bone Miner Res 13:297302.
  • 13
    Weaver CM, Peacock M, Martin BR, McCabe GP, Zhao J, Smith DL, Wastney ME 1997 Quantification of biochemical markers of bone turnover by kinetic measures of bone formation and resorption in young healthy females. J Bone Miner Res 12:17141720.
  • 14
    Garnero P, Delmas PD 1997 Bone markers. Baillieres Clin Rheumatol 11:517537.
  • 15
    Kalu DN 1999 Animal models of the aging skeleton. In: RosenCJ, GlowackiJ, BilezikianJP (eds.) The Aging Skeleton. Academic Press, San Diego, CA, USA, pp. 3750.
  • 16
    Ornoy A, Katzburg S 1995 Osteoporosis: Animal models for the human disease. In: OrnoyA (ed.) Animal Models of Human Related Calcium Metabolic Disorders. CRC Press, New York, NY, USA, pp. 105126.
  • 17
    Beamer WG, Donahue LR, Rosen CJ, Baylink DJ 1996 Genetic variability in adult bone density among inbred strains of mice. Bone 18:397403.
  • 18
    Bain SD, Bailey MC, Celino DL, Lantry MM, Edwards MW 1993 High-dose estrogen inhibits bone resorption and stimulates bone formation in the ovariectomized mouse. J Bone Miner Res 8:435442.
  • 19
    Chen C, Kalu DN 1999 Strain differences in bone density and calcium metabolism between C3H/HeJ and C57BL/6J mice. Bone 25:413420.
  • 20
    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.
  • 21
    Sheng MH, Baylink DJ, Beamer WG, Donahue LR, Rosen CJ, Lau KH, Wergedal JE 1999 Histomorphometric studies show that bone formation and bone mineral apposition rates are greater in C3H/HeJ (high-density) than C57BL/6J (low-density) mice during growth. Bone 25:421429.
  • 22
    Ahima RS, Dushay J, Flier SN, Prabakaran D, Flier JS 1997 Leptin accelerates the onset of puberty in normal female mice. J Clin Invest 99:391395.
  • 23
    Bonjour JP, Theintz G, Buchs B, Slosman D, Rizzoli R 1991 Critical years and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab 73:555563.
  • 24
    Farley JR, Hall SL, Herring S, Tarbaux NM 1992 Two biochemical indices of mouse bone formation are increased, in vivo, in response to calcitonin. Calcif Tissue Int 50:6773.
  • 25
    Srivastava AK, Catillo G, Wergedal JE, Mohan S, Baylink DJ 2000 Development and application of a synthetic peptide based osteocalcin assay for the measurement of bone formation in mouse serum. Calcif Tissue Int 67:255259.
  • 26
    Mohan S, Libanati C, Dony C, Lang K, Srinivasan N, Baylink DJ 1995 Development, validation, and application of a radioimmunoassay for insulin-like growth factor binding protein-5 in human serum and other biological fluids. J Clin Endocrinol Metab 80:26382645.
  • 27
    Mohan S, Bautista CM, Herring SJ, Linkhart TA, Baylink DJ 1990 Development of valid methods to measure insulin-like growth factors-I and -II in bone cell-conditioned medium. Endocrinology 126:25342542.
  • 28
    Linkhart TA, Linkhart SG, Kodama Y, Farley JR, Dimai HP, Wright KR, Wergedal JE, Sheng M, Beamer WG, Donahue LR, Rosen CJ, Baylink DJ 1999 Osteoclast formation in bone marrow cultures from two inbred strains of mice with different bone densities. J Bone Miner Res 14:3946.
  • 29
    Baker J, Liu JP, Robertson EJ, Efstratiadis A 1993 Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:7382.
  • 30
    Mauras N, Rogol AD, Haymond MW, Veldhuis JD 1996 Sex steroids, growth hormone, insulin-like growth factor-1: Neuroendocrine and metabolic regulation in puberty. Horm Res 45:7480.
  • 31
    Manolagas SC, Bellido T, Jilka RL 1995 Sex steroids, cytokines and the bone marrow: New concepts on the pathogenesis of osteoporosis. Ciba Found Symp 191:187196.
  • 32
    Kimble RB, Srivastava S, Ross FP, Matayoshi A, Pacifici R 1996 Estrogen deficiency increases the ability of stromal cells to support murine osteoclastogenesis via an interleukin-1 and tumor necrosis factor-mediated stimulation of macrophage colony-stimulating factor production. J Biol Chem 271:2889028897.
  • 33
    Hofbauer LC, Gori F, Riggs BL, Lacey DL, Dunstan CR, Spelsberg TC, Khosla S 1999 Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: Potential paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinology 140:43824389.
  • 34
    Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, Karsenty G 1996 Increased bone formation in osteocalcin-deficient mice. Nature 382:448452.
  • 35
    Bonjour J-P, Rizzoli R 1995 Bone acquisition in adolescence. In: MarcusR, FeldmanD, KelseyJ (eds.) Osteoporosis. Academic Press, San Diego, CA, USA, pp. 465476.
  • 36
    Gilsanz V, Boechat MI, Roe TF, Loro ML, Sayre JW, Goodman WG 1994 Gender differences in vertebral body sizes in children and adolescents. Radiology 190:673677.
  • 37
    Kalender WA, Felsenberg D, Louis O, Lopez P, Klotz E, Osteaux M, Fraga J 1989 Reference values for trabecular and cortical vertebral bone density in single and dual-energy quantitative computed tomography. Eur J Radiol 9:7580.
  • 38
    Gilsanz V, Gibbens DT, Roe TF, Carlson M, Senac MO, Boechat MI, Huang HK, Schulz EE, Libanati CR, Cann CC 1988 Vertebral bone density in children: Effect of puberty. Radiology 166:847850.
  • 39
    Beamer WG, Rosen CJ, Shultz KL, Pung AE, Churchill GA, Donahue LR, Baylink DJ 1999 Mice congenic for bone density QTLs exhibit corresponding bone density changes. J Bone Miner Res 14(Suppl 1):S174.