Altered mechanical environment of bone cells in an animal model of short- and long-term osteoporosis

Abstract

Alterations in bone tissue composition during osteoporosis likely disrupt the mechanical environment of bone cells and may thereby initiate a mechanobiological response. It has proved challenging to characterize the mechanical environment of bone cells in vivo, and the mechanical environment of osteoporotic bone cells is not known. The objective of this research is to characterize the local mechanical environment of osteocytes and osteoblasts from healthy and osteoporotic bone in a rat model of osteoporosis. Using a custom-designed micromechanical loading device, we apply strains representative of a range of physical activity (up to 3000 με) to fluorescently stained femur samples from normal and ovariectomized rats. Confocal imaging was simultaneously performed, and digital image correlation techniques were applied to characterize cellular strains. In healthy bone tissue, osteocytes experience higher maximum strains (31,028 ± 4213 με) than osteoblasts (24,921 ± 3,832 με), whereas a larger proportion of the osteoblast experiences strains >10,000 με. Most interestingly, we show that osteoporotic bone cells experience similar or higher maximum strains than healthy bone cells after short durations of estrogen deficiency (5 weeks), and exceeded the osteogenic strain threshold (10,000 με) in a similar or significantly larger proportion of the cell (osteoblast, 12.68% vs. 13.68%; osteocyte, 15.74% vs. 5.37%). However, in long-term estrogen deficiency (34 weeks), there was no significant difference between bone cells in healthy and osteoporotic bone. These results suggest that the mechanical environment of bone cells is altered during early-stage osteoporosis, and that mechanobiological responses act to restore the mechanical environment of the bone tissue after it has been perturbed by ovariectomy.

Figure 1

Diagram of custom-designed micro-loading device in position under the confocal microscope (A) and close-up (B). Relationship among bone sample, loading platens, and microscope objective shown in (C) and (D).

Figure 2

Confocal image of PMMA-embedded fluorescent microsphere (A), with the contour plot of strain within it under 3000 με loading (B). Diagram of analytical solution for spherical inclusion in a homogenous material (C), adapted from Bilgen and Insana (42). Comparison of experimental and analytical results over a range of applied loads is shown in (D). To see this figure in color, go online.

Figure 3

Diagram of removal of proximal and distal ends of femur, followed by longitudinal sectioning of the sample (A–C). Imaging was performed at the middiaphysis, ∼50 μm below the cut surface (dotted line in A and box in (C). Confocal scans were performed from cut face through depth of bone (D), allowing visualization of the lacunar-canalicular network (E) and osteoblast pericellular space (green in G, and the osteocytes (red in F) and osteoblasts (red in H)). To see this figure in color, go online.

Figure 4

Confocal scans of the same location in a femur sample at (A) 0 μm and (D) 50 μm from the cut surface. (Green staining) Cell viability (B and E); (red staining) cytotoxicity (C and F) (with scale bar, 100 μm). Thresholding of (D) for quantification of cell viability is shown in (G). To see this figure in color, go online.

Figure 5

Confocal images of (A) a sample osteocyte and (B) osteoblast at 0 με. Digital image correlation (DIC) is applied to characterize the maximum principal strain distribution in (C) the osteocyte, (D) the osteoblast at 3000 με (scale bar, 10 μm) and (E) strain amplification in an osteocyte cell process. To see this figure in color, go online.

Figure 6

Average maximum principal strain distributions observed after 5 and 34 weeks in osteoporotic (OVX) and healthy (SHAM) osteoblasts as a percentage of cell area. n = 4 for 5-week groups, n = 2 for the 34-week groups, (a) p < 0.05 versus SHAM-5 at corresponding strain level, and (b) p < 0.05 versus OVX-5 at the corresponding strain level.

Figure 7

Average maximum principal strain distributions observed after 5 and 34 weeks in osteoporotic (OVX) and healthy (SHAM) osteocytes as a percentage of cell area. n = 2 for 5-week groups, n = 2 for 34-week groups, (a) p < 0.05 versus SHAM-5 at corresponding strain level, and (b) p < 0.05 versus OVX-5 at the corresponding strain level.