The effects of estrogen deficiency on cortical bone microporosity and mineralization

Abstract

Recent studies have demonstrated matrix-mineral alterations in bone tissue surrounding osteocytes in estrogen-deficient animals. While cortical bone porosity has been shown to be a contributor to the mechanical properties of bone tissue, little analysis has been done to investigate the effects of estrogen deficiency on bone's microporosities, including the vascular and osteocyte lacunar porosities. In this study we examined alterations in cortical bone microporosity, mineralization, and cancellous bone architecture due to estrogen deficiency in the ovariectomized rat model of postmenopausal osteoporosis. Twenty-week-old female Sprague-Dawley rats were subjected to either ovariectomy or sham surgery. Six weeks post-surgery tibiae were analyzed using high-resolution micro-CT, backscattered electron imaging, nanoindentation, and dynamic histomorphometry. Estrogen deficiency caused an increase in cortical bone vascular porosity, with enlarged vascular pores and little change in tissue mineral density in the proximal tibial metaphysis. Measurements of cancellous architecture corresponded to previous studies reporting a decrease in bone volume fraction, an increase in trabecular separation, and a decrease in trabecular number in the proximal tibia due to estrogen deficiency. Nanoindentation results showed no differences in matrix stiffness in osteocyte-rich areas of the proximal tibia of estrogen-deficient rats, and bone labeling and backscattered electron imaging showed no significant changes in mineralization around the vascular pores. The findings demonstrate local surface alterations of vascular pores due to estrogen deficiency. An increase in cortical vascular porosity may diminish bone strength as well as alter bone mechanotransduction via interstitial fluid flow, both of which could contribute to bone fragility during postmenopausal osteoporosis.

Keywords: Bone mechanotransduction; Cortical porosity; Osteocyte lacunar porosity; Osteoporosis; Vascular porosity.

Figure 1.

Top left: Micro-CT image of the rat proximal tibia demonstrating region of analysis; longitudinal height of region = 2 mm. Top right: Cross-sectional image from region analyzed for an OVX rat; cortical vascular and lacunar porosities were assessed in the anterior (A) and posterior (P) regions. Middle row: Micro-CT cross-sectional images of the anterior proximal tibial metaphysis of a SHAM and OVX rat (1-μm voxel size). Bottom row: 3D renderings of cortical vascular canals in the anterior region for a SHAM and OVX rat (osteocyte lacunae not shown); longitudinal height of section = 2 mm.

Figure 2.

Representative SHAM and OVX volumes of interests (VOIs) from the anterior proximal tibial metaphysis illustrating the segmentation of the cortical vascular canals (red) and osteocyte lacunae (yellow). The entire (unzoomed) cylindrical VOIs are 250 μm in diameter and 2 mm in height.

Figure 3.

Top row: Micro-CT cross-sectional images of the proximal tibial metaphysis of a SHAM and OVX rat (4-μm voxel size), taken from the same proximal tibia region analyzed for the cortical microporosities shown in Figure 1. Bottom row: Segmented volumes of interest (VOIs) used to assess the cancellous bone microarchitecture in SHAM and OVX; VOI height = 2 mm.

Figure 4.

Typical SEM images of a region of interest surrounding a vascular pore from a SHAM rat (left) and OVX rat (right). Both images taken from the posterior proximal tibia; scale bars = 20 μm.

Figure 5.

A schematic representation of the rat tibia showing the regions analyzed using nanoindentation. A matrix of 25 nanoindents, 5 μm apart, was made in the anterior (A) and posterior (P) of the cortical tibial metaphysis in an osteocyte-rich area.

Figure 6.

Micro-CT microarchitectural measures (mean ± standard deviation) for cancellous bone from the proximal tibia metaphysis for SHAM and OVX groups. (a) Cancellous bone volume fraction, BV/TV (%). (b) Trabecular thickness, Tb.Th (μm). (c) Trabecular separation, Tb.Sp (μm). (d) Trabecular number, Tb.N (1/mm), (e) Structure model index, SMI. *p < 0.05.

Figure 7.

Micro-CT microarchitectural measures (mean ± standard deviation) for cortical bone from the anterior and posterior regions of the proximal tibia metaphysis for SHAM and OVX groups. (a) Vascular canal porosity, Ca.V/TV (%). (b) Average vascular canal diameter, Ca.Dm (μm). (c) Vascular canal separation, Ca.Sp (μm). (d) Osteocyte lacunar porosity, Lc.V/TV (%). (e) Osteocyte lacunar density, Lc.N/TV (# per mm³). (f) Tissue mineral density, TMD (g/cm³). *p < 0.05.

Figure 8.

SEM assessment of mineralization (calibrated grayscale, mean ± standard deviation) around vascular pores from the anterior (top) and posterior (bottom) regions of the proximal tibia cortex. For both SHAM and OVX, mineralization level was lowest at the vascular pore surface; but there were no mineralization differences between SHAM and OVX. ^a different from 1–5 μm region, p < 0.05; ^b different from 6–10 μm region, p < 0.05; ^c different from 11–15 μm region, p < 0.05.