Multiscale characterization of ovariectomized rat femur

Abstract

Estrogen deficiency activates bone resorbing cells (osteoclasts) and to a lesser extent bone forming cells (osteoblasts), resulting in a gap between resorption and formation that leads to a net loss of bone. These cell activities alter bone architecture and tissue composition. Thus, the objective of this study is to examine whether multiscale (10^-2 to 10^-7 m) characterization can provide more integrated information to understand the effects of estrogen deficiency on the fracture risk of bone. This is the first study to examine the effects of estrogen deficiency on multiscale characteristics of the same bone specimen. Sprague-Dawley female rats (6 months old) were obtained for a bilateral ovariectomy (OVX) or a sham operation (sham). Micro-computed tomography of rat femurs provided bone volumetric, mineral density, and morphological parameters. Dynamic mechanical analysis, static elastic and fracture mechanical testing, and nanoindentation were also performed using the same femur. As expected, the current findings indicate that OVX reduces bone quantity (mass and bone mineral density) and quality (morphology, and fracture displacement). Additionally, they demonstrated reductions in amount and heterogeneity of tissue mineral density (TMD) and viscoelastic properties. The current results validate that multiscale characterization for the same bone specimen can provide more comprehensive insights to understand how the bone components contributed to mechanical behavior at different scales.

Keywords: Bone micro-CT; Estrogen; Matrix mineralization; Nanoindentation; Osteoporosis.

Fig. 1.

(a) Steps of compartmentation for the 3D micro-CT image of a femoral bone. Masking was performed to identify the bone marrow cavity including trabecular bone (TB). CB₅₅ (55% of length) was digitally isolated. The total volume was determined, including CB, TB, and masked voxels. The TMD parameters were determined in individual TMD histograms of typical (b) whole bone, (c) CB, and (d) TB for sham and OVX groups.

Fig. 2.

Steps of segmentation for (a) cortical bone (CB₅₅, 55% of length) layers from periosteum to endosteum to obtain TMD (b) mean, (c) SD, (d) Low₅, and (e) High₅. The OVX group had significantly lower values of all TMD parameters at each distance compared to the sham group (p<0.046), except SD between 350 μm and 540 μm, and Low₅ between 675 μm and 810 μm (p>0.051).

Fig. 3.

(a) 3-point bending at 55% of femur length from the femoral head, (b) hysteresis (W) computed using a non-destructive load-displacement up to 0.01 mm, (c) non-destructive oscillatory bending displacement using a mean level of 0.01 mm and amplitude of 0.005 mm at the range of 0.5 to 3 Hz of DMA, and (d) static fracture tests for typical femurs of sham and OVX groups. Red dashed lines: nanoindentation locations

Fig. 4.

(a) Nanoindentation sites in a femur. Although this figure shows a bigger size with 1000 nm depth of indentation to clearly illustrate the locations, the real size of indentation is smaller (5 to 6 μm width) with 500 nm depth for 3×3 array sites for periosteal, core, and endosteal regions of the cortical bone. (b) Elastic modulus (E_b), plastic hardness (H), static viscoelastic normalized creep (Creep/P_max) and viscosity (η), and dynamic viscoelastic tangent delta (tan δ) are provided using a cycle of nanoindentation at the same site of bone matrix.