This study investigated in more detail the growth of male and female C57Bl/6 mice using laboratory measurements of density, modulus and composition of cortical bone from the tibiae, as well as geometrical properties using pQCT scanning. It was designed to form the baseline for future studies comparing wild-type animals with those in which the genetic alterations have been introduced.

A colony of C57Bl/6 mice were kept in standard conditions in accordance with UK Home Office guidelines. Animals were euthanized at the ages of 1,2,3,6,9 and 12 months. After measuring the total length each tibia was divided using a mineralogical saw. First the proximal metaphysis was removed, as judged visually by where the shaft became more uniform. An adjacent slice, approximately 2 mm thick, was then cut from the proximal diaphysis for ultrasonic measurement. The distal metaphysis was then removed leaving the main part of the diaphysis for density and composition measurements. A syringe filled with PBS was used to wash out the marrow from within the diaphysis. The density of the diaphyseal cortical bone was measured using Archimedes’ principle. To determine the composition of the bone, the specimens were dehydrated in pre-dried crucibles at 105 °C for 24 hours, then ashed at 600 °C for 24 hours. An ultrasonic method was used to determine the elastic modulus of the bone in the 2 mm slice cut from the end of the diaphysis. Left tibial bones were scanned in a Stratec XCT Research M pQCT densitometer and from this image the total cross-sectional area, the cross-sectional area of the cortical bone and the polar cross-sectional moment of inertia (CSMI) were calculated. The volumetric bone mineral density (‘cortical density’) was determined using a bone threshold of 570 mg cm-3 to identify the cortex. The same threshold was used for all samples to ensure that the same density of bone would be detected in all samples.

Growth of C57Bl/6

Male C57Bl/6 mice were heavier and had greater tibial lengths than the corresponding females (Fig. 1(a,b)). The females grew relatively more slowly than the males in the early months, but both mass and tibial length appeared to reach their maximum by about 6 months of age for both sexes. Similar trends were seen in the mineral and organic mass fractions (Figs. 1(c) (d)); mineral content increased and organic content decreased and both appeared to have reached a steady value by about 3-4 months of age. This pattern was not fully reflected in the density or modulus (Figs. 1(e) and (f)). The density of the bone showed a significant difference with age and sex. Despite the increase in mineral content, expressed as a fraction of the dry mass, density and mineral content were poorly correlated. The bone modulus in the males appeared to follow a growth-like increase with age but the females did not show such a clear pattern.

Linear correlations with similar gradients were found between modulus and density for both males and females but with the females having a greater modulus than the males for a given density (Fig. 2). The gradients and correlation coefficients were: 13.8 GPa cm3 g-1 and R2adj = 0.37 for the males and 14.1 GPa cm3 g-1 and R2adj = 0.44 for the females and the residuals were normally distributed in both cases showing a linear fit is adequate.

pQCT measurements

Measurements were made from a single slice at the centre of the diaphysis. There was an increase in volumetric BMD that was similar to the growth pattern described above; the peak being reached before the age of 6 months (Fig. 3(a)), and there was no difference between males and females. Sex differences were found, though, for total and cortical cross-sectional areas (Figs. 3(b), and (c)) and for the cross-sectional moment of inertia (Fig. 3(d)) with males always having greater values than females. For both sexes, peak values were all achieved by 3 months of age or soon after.

The objective of this study was to describe in more detail the age-related maturation of the mechanical and material properties of cortical bone in C57Bl/6 mice, which have become one of the standard laboratory models for studies of bone. Nearly all the parameters measured from the bones reached their maximum value at between 3 and 6 months of age, probably earlier in this time span rather than later but to be more precise is not possible given the study design. The body weight of the mouse followed the same pattern. These data would support other studies suggesting skeletal maturity is reached at about 4 months.

The torsional rigidity of the bone is given by the product of the modulus and the cross-sectional moment of inertia, EI. Using the modulus values measured above and the corresponding CSMI obtained from the mid-diaphysis from the pQCT measurements enables this to be estimated. Interestingly, though the individual measurements change only slowly once maturity is reached it can be seen from Fig. 4 that this continues to increase throughout the whole study period, though having only 3 male animals at the 12-month time point indicate a degree of caution must be exercised at this point..

In summary we have shown that skeletal maturity for most factors in C57Bl/6 mice has occurred before the age of 6 months. The females grew more slowly than the males and were smaller, but there were no differences between modulus or volumetric BMD, though the modulus did not appear to follow the usual growth curve in the females. Bone mineral mass fraction and density however, behaved differently in males and females. These differences, both in magnitude and in change with age, between the sexes would suggest using only one sex in experiments to avoid complications arising from this source.

Figure 1

Growth (mean and standard deviation) of male and female C57Bl/6 mice results in an increase in (a) the mass of the animal and (b) the length of the tibia. Within the tibia there were changes in (c) the mass fraction of mineral, (d) the mass fraction of organic material (both expressed as fractions of the wet mass), (e) the density and (f) the modulus of the bone matrix which were different in males and females.

Figure 2

The relationship between modulus and density was not strong and similar in males and females. The regression lines show that, on average, for a given density the females have a higher modulus. (Mean and standard deviation shown)

Figure 3

Peripheral QCT measurements (mean and standard deviation) from the mid-shaft of the tibia. (a) Volumetric BMD, (b) total cross-sectional area, (c) cortical cross-sectional area and (d) the cross-sectional moment of inertia.

Figure 4

Estimation of the torsional rigidity (mean and standard deviation), EI, of the tibiae derived from the product of the modulus, E, and the CSMI, I, suggests that it continues to increase throughout the study period, in contrast to other measures which indicate that maturity has been reached.