Age-related and sex-specific effects on architectural properties and biomechanical response of the C57BL/6N mouse femur, tibia and ulna

Abstract

Aging is known to reduce bone quality and bone strength. We sought to determine how aging affects the biomechanical and architectural properties of various long bones, and if sex influences age related differences/changes. While researchers have extensively studied these changes in individual bones of mice, there is no comprehensive study of the changes in the bones from the same mice to study the changes with aging. We performed three point bending tests and microcomputed tomography (microCT) analysis on femurs, tibiae and ulnae. Three point bending tests were utilized to calculate biomechanical parameters and imaging was also performed using high resolution microCT to reveal both cortical and trabecular microarchitecture C57BL/6N mice were divided into three age groups: 6, 12 and 22 months. Each age and sex group consisted of 6-7 mice. The ultimate load to failure (UL), elastic stiffness (ES), modulus of elasticity (E) and the moment of inertia about bending axis (MOI) for each bone was calculated using three point bending test. MicroCT scans of all the bones were analyzed to determine cortical bone volume per tissue volume (C.BV/TV), trabecular bone volume per tissue volume (Tb.BV/TV), cortical bone area (B.Ar) using CTAn's microCT analysis and tested for correlation with the biomechanical parameters. Mean (standard error) values of UL in femur decreased from 19.8(0.6) N to 12.8(1.1) N (p < .01) and 17.9(0.6) N to 14.6(1.0) N (p = .02) from 6 to 22 months groups in males and females respectively. Similarly, UL in tibia decreased from 19.8(0.5) N to 14.3(0.2) N (p < .01) and 14.4(0.6) N to 9.5(1.0) N (p < .01) from 6 to 22 months group in males and females respectively. ES in femur decreased from 113.2(7) N/mm to 69.6(6.7) N/mm (p < .01) from 6 to 22 months in males only. ES in tibia decreased from 78.6(3.2) N/mm to 65.0(2.3) N/mm (p = .01) and 53.1(2.9) N/mm to 44.0(1.7) N/mm (p = .02) from 6 to 22 months in males and females respectively. Interestingly, ES in ulna increased from 8.2(0.8) N/mm to 10.9(1.0) N/mm (p = .051) from 6 to 22 months of age in females only. E in femur decreased from 4.0(0.4) GPa to 2.8(0.2) GPa (p = .01) and 6.7(0.5) GPa to 4.5(0.4) GPa (p = .01) from 6 to 22 months of age in males and females respectively while tibia showed no change. However, E in ulna increased from 7.0(0.8) GPa to 11.0(1.1) GPa (p = .01) from 6 to 22 months of age in females only. Changes in age and sex-related bone properties were more pronounced in the femur and tibia, while the ulna showed fewer overall differences. Most of the changes were observed in biomechanical compared to architectural properties and female bones are more severely affected by aging. In conclusion, our data demonstrate that care must be taken to describe bone site and sex-specific, rather than making broad generalizations when describing age-related changes on the biomechanical and architectural properties of the skeleton.

Keywords: Aging; Biomechanical parameters; C57BL/6N; Femur; Gender; Three-point bending; Tibia; Ulna.

Fig. 1

Femur (left), Tibia (middle) and Ulna (right) loaded in three point bending test.

Fig. 2

Calculation of (A) elastic and plastic work to failure (B) displacement in elastic and plastic regions and cross section of (C) Femur, (D) Tibia & (E) Ulna showing actual loading orientation for the calculation of bending strength and moment of inertia using BoneJ.

Fig. 3

Biomechanical parameters males: Ultimate load (UL), Elastic stiffness (ES), Modulus of elasticity (E), and Moment of inertia (MOI) in Femurs (F), Tibiae (T), and Ulnae (U). Values that are too close are staggered apart to help with differentiation. (Data are individual value and mean ± standard error).

Fig. 4

Biomechanical parameters females: Ultimate load (UL), Elastic stiffness (ES), Modulus of elasticity (E), and Moment of inertia (MOI) in Femurs (F), Tibiae (T), and Ulnae (U). (Values that are too close are staggered apart to help with differentiation).

Fig. 5

MicroCT parameters males: Cortical bone area (B.Ar), Cortical BV/TV (C.BV/TV), and Trabecular BV/TV (Tb.BV/TV) in Femurs (F), Tibiae (T), and Ulnae (U). (Values that are too close are staggered apart to help with differentiation.)

Fig. 6

MicroCT parameters females: Cortical bone area (B.Ar), Cortical BV/TV (C.BV/TV), and Trabecular BV/TV (Tb.BV/TV) in Femurs (F), Tibiae (T), and Ulnae (U). (Values that are too close are staggered apart to help with differentiation).

Fig. 7

Summary of biomechanical parameters grouped as structural, material and geometric properties (Both males and females) and the significant changes at 12 months & 22 months of age while p value was set to 0.05.

Fig. 8

Graphs of biomechanical parameters showing significant correlation with microCT parameters (males) while p was set to 0.05. Pearson correlation value is shown as “r” in each graph.

Fig. 9

Graphs of biomechanical parameters showing significant correlation with microCT parameters (females) while p was set to 0.05. Pearson correlation value is shown as “r” in each graph.

Figure S1

Biomechanical parameters males: Ultimate load (UL), Elastic stiffness (ES), and Moment of inertia (MOI) in Ulnae (U). (Data are mean ± standard error)

Figure S2

Biomechanical parameters females: Ultimate load (UL), Elastic stiffness (ES), and Moment of inertia (MOI) in Ulnae (U). (Data are mean ± standard error)