Mouse Bone Chart
Edilma Howard
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High-resolution computed tomography (CT) is widely used to assess bone structure under physiological and pathological conditions. Although the analytic protocols and parameters for micro-CT (μCT) analyses in mice are standardized for long bones, vertebrae, and the palms in aging mice, they have not yet been established for craniofacial bones. In this study, we conducted a morphometric assessment of craniofacial bones, in comparison with long bones, in aging mice. Although age-related changes were observed in the microarchitecture of the femur, tibia, vertebra, and basisphenoid bone, and were more pronounced in females than in males, the microarchitecture of both the interparietal bone and body of the mandible, which develop by intramembranous ossification, was less affected by age and sex. By contrast, the condyle of the mandible was more affected by aging in males compared to females. Taken together, our results indicate that mouse craniofacial bones are uniquely affected by age and sex.
Bone ossification starts at the embryo stage and continues until approximately age 25 years in humans2. Bone ossification is achieved through intramembranous and endochondral ossification. Whereas intramembranous ossification involves the direct conversion of the mesenchyme to bone and forms the flat bones of the skull, clavicle, and most of the cranial bones, endochondral ossification involves a cartilage template that is later replaced by bone and forms the long bones and cranial base2. Bones are continuously and actively remodeled throughout life; therefore, bone morphology and matrix quality change during development and with aging.
Disruption of the homeostasis between bone formation by osteoblasts and bone resorption by osteoclasts results in imbalanced bone remodeling. For instance, osteopenia, a condition of low bone mass or low bone density, occurs when bone resorption exceeds bone formation, and its progression is influenced by diet, environmental factors, hormonal changes, and aging. Individuals with osteopenia have a higher risk of suffering bone fractures and developing osteoporosis, in which bone mineral density (BMD) measured by dual X-ray absorptiometry, the gold standard for measuring age-related changes, is significantly reduced by more than two-fold3,4,5.
In osteoporotic bones, bone remodeling shifts toward bone resorption, resulting in increased net bone loss with aging6. In addition, this bone loss depends on the amount of surface available for bone remodeling7. Therefore, bone loss in early osteoporosis is mainly a loss of trabecular bone due to the larger surface available for bone remodeling8. However, cortical bone becomes more porous with aging, which leads to increased endocortical surface. As a result, bone loss in late osteoporosis is mainly a loss of cortical bone7. Previous studies indicate that cortical bone strength in the femur9,10, as well as cortical thickness in various bones, decrease with aging11,12. In addition, trabecular bone loss has been observed in the femoral head and vertebrae with aging, along with decreased trabecular bone volume and increased trabecular bone separation11,13,14.
Although both estrogen and androgen inhibit bone resorption and enhance bone formation, estrogen plays a more dominant role in the inhibition of bone resorption21. This is evident in women with estrogen deficiency due to menopause, which is associated with rapid trabecular bone loss6. The prevalence of osteoporosis is, therefore, higher in postmenopausal women than in older men. The National Osteoporosis Foundation has estimated that 9.1 million women are affected by osteoporosis, while only 2.8 million men develop the disease. Nonetheless, older men still suffer poor health outcomes resulting from osteoporosis.
Long bones and the vertebrae have been rigorously studied in both human and mice, as they are common sites of fracture due to aging and osteopenia/osteoporosis. By contrast, traumatic injuries and fractures in craniofacial bones are most commonly caused by falls and motor vehicle accidents22,23. Although recent efforts have been made to quantify the bone morphometry of the skull (e.g. cortical thickness, density, and porosity), the trabecular morphometry of the diploe has not been studied in detail24,25,26.
Micro-computed tomography (μCT) allows for the visualization of three-dimensional (3D) structures and is widely used to evaluate bone quality and morphology in small animals, under physiological and pathological conditions. μCT analyses are useful for evaluating the fine structure of bone/cartilage mineral components, particularly in the field of bone and cartilage research27,28,29,30. Qualifiable and quantifiable analyses of long bones and vertebrae, with a set of standardized parameters, are well established in the field; by contrast, a standardized protocol for craniofacial bones has not been yet defined. While some craniofacial bones are formed through endochondral ossification (e.g. the mandibular condyle and cranial base31,32), most are formed through intramembranous ossification. A few μCT studies have been conducted in young mice33, but a rigorous methodology for evaluating aging craniofacial bones has not been tested and validated. This study thus aims to evaluate the changes that occur in the structural properties of craniofacial bones in aging mice through μCT imaging analyses.
Samples were placed in a foam mold for stability during the μCT scans, which were performed at 15 m resolution for the head, third lumbar vertebra, tibia, and femur using a SCANCO vivaCT-40 system (SCANCO Medical AG, Fabrikweg, Switzerland; 70 peak kilovoltage and 145-A X-ray source). Air was used as the scan medium to provide the highest contrast between the sample and the background. 3D reconstruction and analysis of the μCT images were performed with the Dragonfly software [Version 2021.1 for Windows; Object Research Systems (ORS) Inc., Montreal, Canada] with DICOM files.
The difference in bone size according to age was taken into consideration when determining the volume of interest (VOI) for all bones. For the femur, trabecular bone measurements were taken 0.5 mm proximally from the distal epiphyseal growth plate, with 1 mm in height, and cortical bone parameters were measured at mid-diaphysis (which was located by taking the midpoint between the greater trochanter and intercondylar notch), extending to a length of 0.5 mm. For the tibia, trabecular bone measurements were taken 0.5 mm distally from the tibial metaphysis, with 1 mm in height, and cortical bone parameters were measured at the midpoint between the distal and proximal growth plates, extending to a length of 0.5 mm. For the vertebrae, the VOI for the body of the third lumbar vertebrae (L3) was determined relative to the height of the vertebra; measurements were taken from a VOI of 50% in height and 45% in width from the center of the body, and cortical thickness was calculated from the ventral vertebral cortex.
For craniofacial bones, several candidate bones were selected based on the clinical relevance and clear/ rich trabecular architecture. For the basisphenoid bone, VOI was taken from 200 m anteriorly to the spheno-occipital synchondrosis (SOS), with 1 mm in width and 500 m in length. For the interparietal bones, VOI was taken from the largest cross-sectional area, with 1.5 mm in width and 1 mm in length. For the mandibular trabecular bone, VOI was located at the first molar (M1) bifurcation, extending 30 slices between the M1 proximal and distal roots; trabecular bone below the M1 roots was outlined excluding the molars, lower incisors, and the mandibular cortical bone. For the mandibular condyle, the most anterior and posterior points of the mandibular condyle were located, and the VOI of the condyle was selected above the artificial line drawn from the anterior to the posterior points.
Our observations of age-related changes in the trabecular bone of the femur, tibia, and lumbar vertebra were consistent with previous findings, showing an increase in trabecular separation and decrease in trabecular BV/TV with aging in both male and female mice. For femoral and tibial Tb.Th measurements, there was an initial increase and a subsequent decrease as mice approached the end of their lives. Femoral and tibial cortical thickness decreased slightly, whereas vertebral cortical thickness increased slightly with aging, a similar pattern observed by Glatt et al.39 and Halloran et al.40. Cortical and trabecular measurements for female mice showed a higher degree and faster rate of bone loss compared to male mice.
Our results from the μCT analyses were validated with histological staining (Fig. S1), which showed that the thickness and length of trabecular bones in the femur, tibiae, and vertebrae were decreased with aging in both male and female mice. Previous studies showed that the bone marrow in the femur and tibiae changes to yellow marrow, which is composed of adipocytes, with aging more drastically in female mice44,45. In agreement with these findings, the femoral and tibial bone marrow changed to yellow marrow in the femurs of female mice older than 18 months and in the tibia of female mice older than 12 months, whereas in male mice it remained as red marrow in these bones until the age of 22 months. In addition, as seen in the μCT images, there was increased invasion of blood vessels into the subchondral bone of the mandibular condyles, while the thickness of fibrocartilage on the condylar surface was intact. Thus, the change seen in the mandibular condyle differed from that observed in other bones.
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