Hyperlipidemia affects multiscale structure and strength of murine femur

Abstract

To improve bone strength prediction beyond limitations of assessment founded solely on the bone mineral component, we investigated the effect of hyperlipidemia, present in more than 40% of osteoporotic patients, on multiscale structure of murine bone. Our overarching purpose is to estimate bone strength accurately, to facilitate mitigating fracture morbidity and mortality in patients. Because (i) orientation of collagen type I affects, independently of degree of mineralization, cortical bone׳s micro-structural strength; and, (ii) hyperlipidemia affects collagen orientation and μCT volumetric tissue mineral density (vTMD) in murine cortical bone, we have constructed the first multiscale finite element (mFE), mouse-specific femoral model to study the effect of collagen orientation and vTMD on strength in Ldlr(-/-), a mouse model of hyperlipidemia, and its control wild type, on either high fat diet or normal diet. Each µCT scan-based mFE model included either element-specific elastic orthotropic properties calculated from collagen orientation and vTMD (collagen-density model) by experimentally validated formulation, or usual element-specific elastic isotropic material properties dependent on vTMD-only (density-only model). We found that collagen orientation, assessed by circularly polarized light and confocal microscopies, and vTMD, differed among groups and that microindentation results strongly correlate with elastic modulus of collagen-density models (r(2)=0.85, p=10(-5)). Collagen-density models yielded (1) larger strains, and therefore lower strength, in simulations of 3-point bending and physiological loading; and (2) higher correlation between mFE-predicted strength and 3-point bending experimental strength, than density-only models. This novel method supports ongoing translational research to achieve the as yet elusive goal of accurate bone strength prediction.

Keywords: Collagen type I; High fat diet; Hyperlipidemia; Mouse bone; Multiscale finite element.

Figure 1

mFE model of murine femur (a) under 3-point bending and (b) one-legged stance. 1mm bins 1 to 7 are indicated from mid-shaft to proximal femur in (b) and used for collection of data from microscopy investigations.

Figure 2

% of bright area on longitudinal sections of murine femora. The percentages are shown per quadrant (A, anterior; L, lateral; P, posterior; M, medial) and per bin (numbered as 1 to 7, moving up from mid-shaft towards the proximal femur), per mouse group, averaged over the femora of the 4 mice in the group. The data of individual femora follow the trend of the averaged data.

Figure 3

Birefringent signal by CPL and fluorescent signal by confocal microscopy. We show examples of regions with specific percentages of bright areas: (a) 70% of bright area, occurring e.g. at the anterior quadrant of WT; and (b) 40% of bright area, e.g. at anterior mid-shaft of ND MUT and at anterior of HFD MUT. Confocal microscopy shows matrix and osteocyte lacunae of (c) ND WT and (d) HFD MUT. In enlargements, different collagen orientation patterns are emphasized with white markings.

Figure 4

Correlation of microindentation parameter Avg US (1st-L) with elastic modulus in anterior-posterior direction. The correlation was stronger for (a) collagen-density (CD) models than (b) density-only (D) models of 3 femora per group. The p-value of the r² were significant, p<0.01.

Figure 5

Comparison of distributions of ε_zz at cortical mid-shaft between collagen-density (CD) and density-only (D) models. For 3-point bending, ε_zz was larger for CD than D at the anterior quadrant of ND WT (488±58 µε vs. 301±26 µε; Table I).

Figure 6

Comparisons of distributions of ε_zz at cortical mid-shaft among collagen-density models. For one-legged stance, the absolute value of ε_zz was larger for (a) HFD WT than (b) HFD MUT at the anterior (336±56 µε vs. 44±18 µε; Table II, #2) and the posterior quadrant (977±72 µε vs. 393±30 µε; Table II, #4). The absolute value of ε_zz was larger for (a) HFD WT than (c) ND MUT at the posterior quadrant (977±72 µε vs. 585±51 µε; Table II, #4).

Figure 7

Correlations between experimental bone strength and mFE-predicted strength for 3-point bending. The correlations were computed for ND WT, HFD WT and ND MUT at the anterior quadrant, for which ε_zz significantly differed between collagen-density (CD) models and density-only (D) models (Table I). The p-values of the r² were significant, p<10⁻³.