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. 2020 Jan:130:115126.
doi: 10.1016/j.bone.2019.115126. Epub 2019 Oct 31.

The age-related decrease in material properties of BALB/c mouse long bones involves alterations to the extracellular matrix

Amy Creecy  1 , Sasidhar Uppuganti  2 , Madeline R Girard  3 , Siegfried G Schlunk  3 , Chidi Amah  4 , Mathilde Granke  2 , Mustafa Unal  5 , Mark D Does  3 , Jeffry S Nyman  6
Affiliations

The age-related decrease in material properties of BALB/c mouse long bones involves alterations to the extracellular matrix

Amy Creecy et al. Bone. 2020 Jan.

Abstract

One possibility for the disproportionate increase in fracture risk with aging relative to the decrease in bone mass is an accumulation of changes to the bone matrix which deleteriously affect fracture resistance. In order to effectively develop new targets for osteoporosis, a preclinical model of the age-related loss in fracture resistance needs to be established beyond known age-related decreases in bone mineral density and bone volume fraction. To that end, we examined long bones of male and female BALB/c mice at 6-mo. and 20-mo. of age and assessed whether material and matrix properties of cortical bone significantly differed between the age groups. The second moment of area of the diaphysis (minimum and maximum principals for femur and radius, respectively) as measured by ex vivo micro-computed tomography (μCT) was higher at 20-mo. than at 6-mo. for both males and females, but ultimate moment as measured by three-point bending tests did not decrease with age. Cortical thickness was lower with age for males, but higher for old females. Partially accounting for differences in structure, material estimates of yield, ultimate stress, and toughness (left femur) were 12.6%, 11.1%, and 40.9% lower, respectively, with age for both sexes. The ability of the cortical bone to resist crack growth (right femur) was also 18.1% less for the old than for the young adult mice. These decreases in material properties were not due to changes in intracortical porosity as pore number decreased with age. Rather, age-related alterations in the matrix were observed for both sexes: enzymatic and non-enzymatic crosslinks by high performance liquid chromatography increased (femur), volume fraction of bound water by 1H-nuclear magnetic resonance relaxometry decreased (femur), cortical tissue mineral density by μCT increased (femur and radius), and an Amide I sub-peak ratio I1670/I1640 by Raman spectroscopy increased (tibia). Overall, there are multiple matrix changes to potentially target that could prevent the age-related decrease in fracture resistance observed in BALB/c mouse.

Keywords: Advanced glycation end-products; Bone quality; Bound water; Collagen; Raman spectroscopy; Toughness.

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Figures

Figure 1:
Figure 1:
Structural characteristics of the mid-diaphysis with representative μCT images of male femurs (A) and radii (B). The minimum principal second moment of area (Imin) of the femur (C) and the maximum principal second moment of area (Imax) of the radius (D) was higher for old mice than for young adult mice, irrespective of sex. Cortical thickness (Ct.Th) of the femur (E) and radius (F) however was higher for old females but lower for old males compared to young adult mice. **p<0.01 and ****p<0.0001 within male. ###p<0.001 and ####p<0.0001 within female.
Figure 2:
Figure 2:
Cortical porosity from μCT evaluations of the left and right femurs. Representative three-dimensional renderings from the intact (A) and notched femurs (B) show that the higher resolution of the latter partially resolved what appear to be vascular channels. By 12 μm (C) or 6 μm (D) voxel size, porosity either did not vary between the age groups or was lower with age. Pore number for both the left (E) and right (F) imaging was lower in the diaphysis from old mice than from young adult mice. . *p<0.05 and ****p<0.0001 within male. #p<0.05 and ####p<0.0001 within female.
Figure 3:
Figure 3:
Estimated material properties of cortical bone. Drawings of the three-point bending tests are shown for both the femur (A) and radius (B). Yield stress of the cortical bone was lower for old mice than for the young adult mice when the femur (C), but not the radius (D), was tested. While toughness was lower for both the old male and female mice, compared to respective young adult mice, when the femur was tested (E), it was only lower for old than for young adult males when the radius was tested (F). **p<0.01, ***p<0.001 and ****p<0.0001 within male. #p<0.05 and ####p<0.0001 within female.
Figure 4:
Figure 4:
Crack resistance of the femoral cortex. In fracture toughness tests, a crack is propagated by loading a femur mid-shaft with a micro-notch on the tension side of bending (A). The stress intensity to initiate the crack was estimated from the yield moment, and the work during crack growth (Wcrack) per bone cross-sectional area (Ct.Ar) was estimated from the area under the moment vs. span-adjusted displacement curve (B). Kc,init (C) and Wcrack/Ct.Ar (D) were lower for old than young adult mice in both sexes. ***p<0.001 and ****p<0.0001 within male. ##p<0.01 and ####p<0.0001 within female.
Figure 5:
Figure 5:
Age-related changes in bone matrix characteristics of BALB/c femoral diaphysis. Ct.TMD of the intact femoral mid-shaft (A) and notched femoral mid-shaft (B) was higher at 20-mo. than at 6-mo. Mature enzymatic (PYD) collagen crosslink was higher (C), while bound water was lower (D) with advanced age. A non-enzymatic collagen crosslink (PE) was also higher for aged mice than young adult mice (E). Cross-sectional area of the mid-shaft was only higher with age for female mice (F). 6-mo mice are represented by circles and 20-mo. mice are represented by squares. **p<0.01, ***p<0.001, ****p<0.0001 within male. #p<0.05, ##p<0.01, ###p<0.001 and ####p<0.0001 within female.
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