Glucocorticoid-treated mice have localized changes in trabecular bone material properties and osteocyte lacunar size that are not observed in placebo-treated or estrogen-deficient mice

Abstract

This study compares changes in bone microstructure in 6-month-old male GC-treated and female ovariectomized mice to their respective controls. In addition to a reduction in trabecular bone volume, GC treatment reduced bone mineral and elastic modulus of bone adjacent to osteocytes that was not observed in control mice nor estrogen-deficient mice. These microstructural changes in combination with the macrostructural changes could amplify the bone fragility in this metabolic bone disease.

Introduction: Patients with glucocorticoid (GC)-induced secondary osteoporosis tend to fracture at higher bone mineral densities than patients with postmenopausal osteoporosis. This suggests that GCs may alter bone material properties in addition to BMD and bone macrostructure.

Materials and methods: Changes in trabecular bone structure, elastic modulus, and mineral to matrix ratio of the fifth lumbar vertebrae was assessed in prednisolone-treated mice and placebo-treated controls for comparison with estrogen-deficient mice and sham-operated controls. Compression testing of the third lumbar vertebrae was performed to assess whole bone strength.

Results: Significant reductions in trabecular bone volume and whole bone strength occurred in both prednisolone-treated and estrogen-deficient mice compared with controls after 21 days (p < 0.05). The average elastic modulus over the entire surface of each trabecula was similar in all the experimental groups. However, localized changes within the trabeculae in areas surrounding the osteocyte lacunae were observed only in the prednisolone-treated mice. The size of the osteocyte lacunae was increased, reduced elastic modulus around the lacunae was observed, and a "halo" of hypomineralized bone surrounding the lacunae was observed. This was associated with reduced (nearly 40%) mineral to matrix ratio determined by Raman microspectroscopy. These localized changes in elastic modulus and bone mineral to matrix ratio were not observed in the other three experimental groups.

Conclusions: Based on these results, it seems that GCs may have direct effects on osteocytes, resulting in a modification of their microenvironment. These changes, including an enlargement of their lacunar space and the generation of a surrounding sphere of hypomineralized bone, seem to produce highly localized changes in bone material properties that may influence fracture risk.

FIG. 1

A representative sample of an elastic modulus map of an individual trabecula from four treatment groups is shown. Each figure represents a mean of >8000 points of data from each trabecula. (A) A trabecula from a prednisolone-treated mouse with a mean E of 24.2 ± 1.9 × 10⁹ N/m² (GPa). A significant reduction in E (30% below the mean values) was observed at both the remodeling surface (shown within white dotted closed-loop lines) and around the osteocyte lacunae within the trabeculae (shown with black dotted closed-loop lines). (B) A trabecula from a placebo-treated male mouse showing a mean elastic modulus (E) of 23.8 ± 3.1 GPa with the expected reduction only over the osteocyte lacunae. (C) A trabecula from an ovariectomized mouse (after 21 days of estrogen deficiency) with a mean E of 24.8 ± 3.2 GPa, which was similar to the other study groups. E was reduced at the remodeling surface and over the osteocyte lacunae but not surrounding the osteocyte lacunae. (D) A sham-operated control with a mean E of 23.4 ± 3.0 GPa.

FIG. 1

A representative sample of an elastic modulus map of an individual trabecula from four treatment groups is shown. Each figure represents a mean of >8000 points of data from each trabecula. (A) A trabecula from a prednisolone-treated mouse with a mean E of 24.2 ± 1.9 × 10⁹ N/m² (GPa). A significant reduction in E (30% below the mean values) was observed at both the remodeling surface (shown within white dotted closed-loop lines) and around the osteocyte lacunae within the trabeculae (shown with black dotted closed-loop lines). (B) A trabecula from a placebo-treated male mouse showing a mean elastic modulus (E) of 23.8 ± 3.1 GPa with the expected reduction only over the osteocyte lacunae. (C) A trabecula from an ovariectomized mouse (after 21 days of estrogen deficiency) with a mean E of 24.8 ± 3.2 GPa, which was similar to the other study groups. E was reduced at the remodeling surface and over the osteocyte lacunae but not surrounding the osteocyte lacunae. (D) A sham-operated control with a mean E of 23.4 ± 3.0 GPa.

FIG. 2

Raman microspectroscopic imaging of prednisolone-treated (GC) and placebo-treated (PL) mouse trabecula. (A) A comparison of the Raman spectrum of a typical bone embedded in methylmethacrylate and methylmethacrylate alone. (B) GC treatment reduces the mineralized tissue around the osteocyte lacunae and this is not seen in the PL-treated trabecula. (C) Quantitative mineral to organic band area ratios along the lines shown in B. There is a decrease in mineralized tissue (inorganic) to matrix (organic) peaks in mice treated with prednisolone. This change in the mineral to matrix ratio is not seen in the placebo-treated, sham-operated, or estrogen-deficient (OVX) mice (C).

FIG. 3

Biomechanical tests of the third lumbar vertebral body of mice from each treatment group (n = 8/group). Animals treated with prednisolone and estrogen-deficient mice have significant reductions in all measured mechanical property parameters compared with the control groups. P_max represents the maximum load measured in Newtons. *p < 0.05 from prednisolone-treated mice and **p < 0.05 from sham-operated mice.