Age- and Strain-Related Differences in Bone Microstructure and Body Composition During Development in Inbred Male Mouse Strains

Childhood and adolescence are critical periods for the accrual of peak bone mass and structure, defined as the maximal values of skeletal traits present at the end of skeletal maturation [1‐3]. Twin and family studies suggest that 60–80% of peak bone mass variability is attributable to genetic factors, whilst up to 40% of the remaining variability can be influenced by modifiable factors such as lifestyle and body composition [1, 3]. Given that peak bone characteristics may have lifelong influences on bone properties and affect osteoporotic fracture risk later in life [2, 3], a thorough understanding of their determinants is required for the development of novel strategies to combat osteoporosis and prevent fragility fractures.

Among other modifiable factors, the relationship between body composition (muscle and fat mass) and bone during growth in humans has gained considerable interest [1, 4]. Several pediatric studies suggest that muscle mass and bone mass are closely linked during development [4‐7]. Less clear is, however, whether muscle development is driving skeletal development (mechanostat theory) and therefore, whether the attainment of peak muscle mass precedes the acquisition of peak bone mass [8‐10]. Studies on the relationship between fat and bone parameters in children, adolescents and young adults have revealed positive, negative or no associations [4, 11, 12]; these discrepancies may reflect methodological differences and/or perplexing bone–fat interactions. Despite the growing evidence, pediatric studies in this area are challenged by uncontrolled genetic and environmental heterogeneity, undesirable radiation exposure during longitudinal bone assessments and bone measurements restricted to certain anatomical sites (e.g., assessment of the distal radius and tibia using high-resolution peripheral quantitative computed tomography or HR-pQCT) [13].

Conversely, animal models provide unique opportunities to study aspects of skeletal health and disease. In particular, inbred mouse strains have a homogeneous genetic background (similar to identical twins) and exhibit various bone phenotypes that can be studied under stringent environmental control. Importantly, bone mass and biomechanical properties in inbred mice appear to be strain dependent [14‐21]. For instance, Beamer et al. demonstrated large variations in bone mineral content (BMC) and density (BMD) among 11 inbred strains of mice [14]. Profound strain differences in BMD were found between the C57BL/6J and C3H/HeJ mice, which are extensively used in skeletal research and considered low and high bone mass strains, respectively. In contrast, normal skeletal phenotypes have been poorly characterized in young DBA/2JRj mice and rarely compared to those of other mouse strains [14, 16, 17]. Strain differences have also been reported in muscle [22, 23] and fat mass [24, 25], whereas investigations that have concomitantly explored bone, muscle and fat are scarce [26].

There are surprisingly limited data on normal patterns of development, especially around skeletal maturity. Significant controversies have been reported regarding the attainment of peak bone properties, even within a given mouse strain, and most available reports have focused on C57BL/6J mice [26‐29]. Thus, this period requires further investigation in C57BL/6J mice, but also in other commonly used strains.

As such, we explored age-related changes and strain differences in bone microstructure, body composition and selected biomarkers during development among 3 commonly used male and female inbred mouse strains, namely C57BL/6J, DBA/2JRj and C3H mice, and in this study we report the results for the male animals. Male mice remain understudied compared to female mice [14, 17, 19‐21, 30], despite the fact that optimization of skeletal acquisition is crucial for both sexes and skeletal diseases affect both women and men [1‐3].

This study presents baseline data that describe normal skeletal growth patterns and changes in body composition with age in 3 inbred mouse strains. We confirm and extend previous observations regarding strain specificity in bone microstructure. We also provide new evidence on inter- and intra-strain variability in the patterns (i.e., timing, magnitude, site) of cortical and trabecular bone development. Finally, this work advances our understanding regarding the relationships of skeletal microstructure, body composition, and relevant biomarkers (periostin and myostatin) and set the basis for delineating the contributions of these relationship in osteoporosis and sarcopenia.

We showed clear strain-specific phenotypic differences in cortical and trabecular bone microstructure. C3H/J mice had the thickest cortices at all skeletal sites followed by DBA/2JRj and C57BL/6J mice. Conversely, overall, C57BL/6J mice had smaller cortical bone area fraction and tissue mineral density than DBA/2JRj and C3H/J mice, whilst small differences were found between DBA/2JRj and C3H/J mice. These results confirm previous studies that compared C3H/J and C57BL/6J female [14, 17, 19‐21, 38] and male mice [15, 16, 18] and further indicate that male DBA/2JRj mice display intermediate values in cortical bone parameters.

The inferior cortical bone characteristics in C57BL/6J mice were counterbalanced by longer-term gains in the trabecular compartment. This is supported by the higher trabecular BV/TV and trabecular number, and lower trabecular separation seen in C57BL/6J mice compared to DBA/2JRj and C3H/J mice at least at 24 weeks of age. Our data are consistent with some [18, 21], but not all previous comparisons between C3H/J and C57BL/6J mice [14]. These discrepancies may be partially explained by differences in the methodologies used to assess bone structure (µCT vs. pQCT) and ages between ours and their experimental animals. The most pronounced characteristic of trabecular microstructure in DBA/2JRj mice was their consistently thinner trabeculae (vs. C57Bl/6J and C3H/J mice). Collectively, our work clarifies that strain differences are compartment specific and indicates that labelling a strain according to a sole complex trait such as bone mass may mask pronounced differences in cortical and trabecular bone properties.

We found that cortical and trabecular compartments may be differentially affected by age and that growth patterns differ among strains. In C57BL/6J mice, cortical and trabecular bone development peaked at the same timeframe (24 weeks of age). The same developmental pattern was seen for both cortical sites assessed. The vertebra and the proximal tibia shared similarities in trabecular bone development; however, in the distal femur we did not statistically identify age-related effects. The main mechanism of trabecular microstructure augmentation at the lumbar spine in C57BL/6J mice was thickening of the existing trabeculae, whereas trabecular number declined after 8 weeks of age. This decrease in trabecular number may be associated with altered remodeling processes, which aim to reorganize trabecular bone in response to prevalent loading conditions. In line with previous observations [29], the continuous increase in mean trabecular thickness may reflect the increased stress imposed to a reduced number of trabeculae. An alternative explanation why mean trabecular thickness increases, is that, as thinner trabeculae are resorbed, there is an increase in the average thickness of residual trabeculae. Previous observations have also shown that C57Bl/6J mice achieve peak structural, material and mechanical bone properties after 20 weeks of age [20, 28, 39]. In contrast, Glatt et al. studied age-related changes in bone morphology in male C57BL/6J mice and showed earlier increases (peak reached by 8 weeks) and subsequent maintenance (up to 24 weeks) in lumbar BV/TV, which were accompanied by elevations in trabecular number by 8 weeks and increases in trabecular thickness up to 16 weeks of age [29]. Small differences in growth patterns were seen in the distal femur and proximal tibia [29]. These discrepant findings can be in part attributed to differences in sampling frequency, intergroup variability and site evaluation (L4 vs. L5).

Normal growth patterns in DBA/2JRj and C3H/J mice remain poorly investigated. In DBA/2JRj mice, cortical bone maturity was reached at 24 weeks of age. Highest values of trabecular bone properties were, however, achieved earlier, between 8 and 16 weeks of age, depending on anatomical site, and these gains were associated with a higher number of trabeculae and favorable organization. In C3H/J mice, different growth patterns were observed at different anatomical sites. For example, maturity of cortical bone at the tibia was achieved at 24 weeks, whereas we did not statistically detect maximal values for most tibial trabecular parameters. This observation indicates that either there were no overt trabecular bone changes over time or that trabecular characteristics at this site peaked beyond the timeframe of our study. In contrast, at the femur, cortical thickness was greatest at 24 weeks, however, cortical bone area fraction decreased markedly after 16 weeks, suggesting some age-related thinning of the cortex. Skeletal maturity at the femoral trabecular sites was also achieved early, as indicated by peak femoral trabecular BV/TV at 8 weeks; this peak resulted primarily from increases in trabecular number rather than thickening. Trabecular bone properties in the lumbar spine reached maximal levels at 16 weeks and declined after this time point. Previously published data suggest that C3H/J mice display maximal skeletal morphological and biomechanical properties before 16 weeks of age [20]. We speculate that differences in load distribution characteristics in the different skeletal sites may contribute to the different patterns of age-related trabecular bone changes in C3H/J mice.

In addition to age- and strain- related differences in skeletal morphology, we explored how changes in body composition (muscle, fat) and relevant biochemical markers relate to skeletal characteristics during development. In human studies, there is consensus that lean mass is positively associated with bone parameters [4‐7, 12]. Furthermore, some [8, 10], albeit not all [9], studies, have shown that peak muscle mass precedes peak bone mass, further supporting the notion that muscle accrual impacts bone acquisition. These positive association between lean mass and BMD have been largely attributed to the direct mechanical impact (muscle contractions, weight of muscle) of muscle on bone as described in the mechanostat theory [40].

We extend our understanding on the bone–muscle relationship during growth and propose that the relationship may be compartment dependent. We showed that the highest muscle mass and cortical bone properties occurred at 24 weeks of age in C3H/J and DBA/2JRj mice, whereas more variable cortical skeletal development patterns were seen in C3H/J mice. These results may reflect the observations that muscle mass increased progressively in C57BL/6J and DBA/2JRj mice, whereas it did not change over time in C3H/J mice. Due to our sampling frequency, it remains uncertain whether cortical bone acquisition follows peak muscle mass; nevertheless, our findings suggest that maximal values in both tissues may occur at the same timeframe. Our data could also be explained by available reports suggesting that mouse strains respond differently to mechanical stress. Indeed, C3H mice have been shown to be less sensitive to mechanical loading and unloading conditions than C57BL/6J mice [41, 42]. Alternatively, our finding may reflect collinear growth of muscle and bone tissues, regulated by genetic, endocrine and environmental factors [2, 32, 43].

Our findings do not support the notion that changes in muscle mass drive adaptations of trabecular bone during growth. For instance, overall, trabecular bone parameters reached maximum at 24 weeks in C57BL/6J, but at 8–16 weeks for DBA/2JRj mice, despite continuous increases in muscle mass by 24 weeks of age in both strains. These results may indicate that genetics, environmental and intrinsic factors and their complex interactions may override the effects of muscle mass on trabecular bone. It has been proposed that C3H/J mice possess sets of genes that lead to enhanced cortical bone properties and impaired trabecular bone [14, 16, 21]. Another possibility is that trabecular bone adaptations are driven by intrinsic bone factors. Indeed, the superior cortical bone structure in C3H/J mice is likely to carry a major part of mechanical load, leading to stress protective responses and resorption of trabecular bone [17, 21].

In addition to the mechanical interactions between bone and muscle, these tissues are also linked through secreted factors (i.e. myokines, osteokines, systemic hormones) [32, 33]. Myostatin, a potentially negative regulator of muscle growth and regeneration expressed predominantly in skeletal muscle, is a candidate of muscle–bone interactions. Age- and strain-related differences in myostatin levels were not reflected in the rather small muscle mass changes over time in either mouse strain. A potential explanation for these results is that myostatin abundance in serum may not reflect its activity, which is inhibited by several proteins (e.g., follistatin) [44]. Nevertheless, strain differences in myostatin levels might have contributed to the observed skeletal phenotypes during growth. C3H/J mice had the lowest myostatin levels and favorable cortical bone parameters, whereas C57BL/6J had the highest myostatin levels and the worst cortical microstructure. These observations are consistent with animal studies showing that myostatin knock out mice have increased periosteal circumference and tissue mineral density [33, 34].

Periostin is expressed in several tissues including bone and skeletal muscle [35]. In bone, periostin is a structural component of bone matrix, but also acts as a signaling molecule, which, through different pathways (i.e., sclerostin/Wnt-b catenin pathway), enhances osteoblast function, and hence, bone formation [35]. Deletion of the periostin gene in mice results in low bone mass, less favorable cortical bone structure and low bone strength in young adult mice [45, 46]. Periostin is also expressed by skeletal myofibers and has been shown to contribute to muscle development, regeneration and differentiation [36]. In our work, in all mouse strains, periostin levels were maximal at 8 weeks of age and declined thereafter, a finding consistent with the age-related decreases in periostin reported previously [35]. Theoretically, these results may reflect rapid stimulation of bone formation and/or muscle development at earlier time points. Nevertheless, given that periostin is non-specific to bone or muscle and serum periostin levels are reflective of the metabolism of other tissues as well; future studies are needed to elucidate the contribution of periostin to musculoskeletal phenotypes during development.

The relationship between fat mass and BMD in pediatric and young adult population is controversial, as available studies have demonstrated a positive, negative or no association [4, 6, 12]. It is plausible that these discrepancies somewhat reflect the complex interactions between bone and fat. Although the mechanical contribution of fat mass is limited to its weight, biochemical links between bone and fat are suggested to play a major role in the interplay of these tissues and are mediated by adipokines, osteokines, hormones and inflammatory factors [47]. Low body fat content and adiposity during growth may also impact the timing of maturation by affecting the secretion of molecules that exert positive (IGF-1), dual (leptin) or negative effects (tumor necrosis factor or TNF-a, interleukin 6 or IL-6) on bone [47, 48]. We speculate that this may be one of the reasons why DBA/2JRj mice and C3H/J mice (higher fat mass at early time points) experience earlier maturation than C57BL/6J mice. We also showed that fat mass increased progressively in all strains and paralleled the highest values in cortical thickness, despite variable developmental patterns in trabecular bone parameters. Further studies are needed to disentangle the effects of normal body fat levels on bone, together with the mechanisms that mediate these effects.

This study is strengthened by the powerful μCT imaging technique, which is non-destructive, accurate and widely used for the evaluation of three-dimensional bone microstructure [31]. Indeed, μCT measurements are highly correlated with measurements acquired by (static) histomorphometry, dual-energy X-ray absorptiometry (DXA) and pQCT [31, 49]. The simultaneous assessments of bone microstructure, body composition and bone/muscle-related biomarkers allowed us to provide novel insights into the relationship between bone, muscle and fat. Another strength of our work is that all mouse strains were studied at the same time under the same experimental conditions; therefore, confounding variations in diet composition, housing and handing conditions of the animals were reduced.

This study has some shortcomings. Given that our data are cross-sectional, rather than longitudinal; this work provides insights into normal growth patterns by inference. As a proxy of body composition, we calculated muscle and fat sum by summing the masses of individual muscles and fat depots. Assessment of body composition using validated methodologies would have been more accurate in determining age-related changes in muscle and fat mass [26, 50]. Mechanical testing was not directly performed to assess bone strength; nevertheless, bone microstructure has been shown to be a major determinant of bone stiffness and failure load during growth [51]. This work focused on skeletal development; therefore, we included mice at the age of rapid bone accrual (8–24 weeks). Although we were able to statistically detect maximal values in several bone microstructural parameters, we may have missed those that occurred earlier (< 8 weeks) or later (> 24 weeks) than the time of observation or small time differences, which occurred between 8 and 24 weeks of age, but did not reach statistical significance.

This study provides a comprehensive characterization of normal skeletal development and body composition changes in three male inbred mouse strains. Our data confirm previous observations showing that C3H/J mice display the best cortical bone parameters, clarify earlier inconsistencies by demonstrating that C57BL/6J have favorable trabecular bone parameters, and suggest that the DBA/2JRj mice present with intermediate values. We further support strain differences in skeletal growth patterns; these differences affect the timing of development (i.e., C57BL/6J mice mature later than DBA/2JRj and C3H/J mice) and are compartment and site specific. Strain differences in body composition were observed early in the study and, albeit they were less pronounced than strain effects on skeletal phenotypes, they appeared to influence skeletal maturity. We anticipate that this work will aid in the informed selection of background strains and ages in genetic manipulations, lifestyle, pharmacological, and surgical interventions, will contribute to the interpretations of existing studies and will guide future research on the molecular and cellular regulation of peak bone mass.