μCT-based, in vivo dynamic bone histomorphometry allows 3D evaluation of the early responses of bone resorption and formation to PTH and alendronate combination therapy

Abstract

Current osteoporosis treatments improve bone mass by increasing net bone formation: anti-resorptive drugs such as bisphosphonates block osteoclast activity, while anabolic agents such as parathyroid hormone (PTH) increase bone remodeling, with a greater effect on formation. Although these drugs are widely used, their role in modulating formation and resorption is not fully understood, due in part to technical limitations in the ability to longitudinally assess bone remodeling. Importantly, it is not known whether or not PTH-induced bone formation is independent of resorption, resulting in controversy over the effectiveness of combination therapies that use both PTH and an anti-resorptive. In this study, we developed a μCT-based, in vivo dynamic bone histomorphometry technique for rat tibiae, and applied this method to longitudinally track changes in bone resorption and formation as a result of treatment with alendronate (ALN), PTH, or combination therapy of both PTH and ALN (PTH+ALN). Correlations between our μCT-based measures of bone formation and measures of bone formation based on calcein-labeled histology (r = 0.72 - 0.83) confirm the accuracy of this method. Bone remodeling parameters measured through μCT-based in vivo dynamic bone histomorphometry indicate an increased rate of bone formation in rats treated with PTH and PTH+ALN, together with a decrease in bone resorption measures in rats treated with ALN and PTH+ALN. These results were further supported by traditional histology-based measurements, suggesting that PTH was able to induce bone formation while bone resorption was suppressed.

Minimizing Interpolation Bias and Precision Error in In Vivo µCT-Based Measurements of Bone Structure and Dynamics

“…To precisely align the trabeculae in each scan, a three-step image registration procedure was performed, as described previously 12 , using an open-source image registration toolkit 14 . Briefly, the cortical bone in the baseline ( b ) and follow-up ( f ) scans was aligned to result in an initial transformation matrix, T 1 .…”

“…Combining the resulting three transformation matrices resulted in an overall transformation T = T 3 T 2 T 1 , which describes the rotation and translation required to correctly align the individual trabeculae of the two scans. This registration technique has been previously shown to result in a precise alignment of the trabecular compartment, allowing for accurate measurements of bone formation and resorption 12 .…”

“…In addition to tracking changes in the trabecular microstructure, several recent studies have developed in vivo dynamic bone histomorphometry techniques that utilize registered μCT images to identify regions of bone formation and resorption, allowing for longitudinal assessment of bone remodeling and structural changes in the caudal vertebra 16, 21, 23 and at the proximal tibia 3, 12 . In these studies, animals are scanned at multiple time-points, and the resulting μCT images are registered, thresholded, and subtracted to identify, on a voxel-by-voxel basis, locations where bone has formed or resorbed 3, 12, 21 .…”

“…In these studies, animals are scanned at multiple time-points, and the resulting μCT images are registered, thresholded, and subtracted to identify, on a voxel-by-voxel basis, locations where bone has formed or resorbed 3, 12, 21 . However, this new analysis requires a direct voxel-by-voxel subtraction between image pairs.…”

“…Although application of MAT will not necessarily reduce the magnitude of the interpolation errors within a single image, by rotating the baseline and follow-up images by the same amount, this technique eliminates the biases that result from directly comparing an image that has been rotated with one that remains fixed. This new transformation method was then applied to two image analysis scenarios: (1) the evaluation of trabecular bone microstructure, and (2) the identification of regions of trabecular bone formation/resorption using in vivo dynamic bone histomorphometry 12 in repeated scans of rat tibiae. We hypothesized that the MAT scheme would result in reduced interpolation bias and improved reproducibility of these measurements as compared to the standard transformation (ST) scheme, where only the follow-up image is rotated.…”