Microdamage as a Bone Quality Component: Practical Guidelines for the Two-Dimensional Analysis of Linear Microcracks in Human Cortical Bone

Abstract

Microdamage is a component of bone quality believed to play an integral role in bone health. However, comparability between existing studies is fraught with issues due to highly variable methods of sample preparation and poorly defined quantification criteria. To address these issues, this article has two aims. First, detailed methods for preparation and analysis of linear microcracks in human ribs, specifically addressing troubleshooting issues cited in previous studies, are laid out. Second, new, partially validated criteria are proposed in an effort to reduce subjective differences in microcrack counts and measures, ensuring more comparable results between studies. Revised definitions based on current literature in conjunction with a digital atlas to reduce observer inaccuracy and bias are presented. The goal is to provide a practical methodology for bone biologists and biomechanists to collect and analyze linear microcracks for basic science research. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Keywords: AGING; BONE HISTOMORPHOMETRY; FRACTURE RISK ASSESSMENT; INJURY.

Figure 1

A stained linear microcrack highlighted by a white arrow. The image on the left is under bright field illumination, whereas the image on the right was photographed under FITC fluorescence. The dark pink color in the left image is the basic fuchsin permeating the open spaces, which appears red and in stark contrast to the unstained green portions of the bone in the right image.

Figure 2

Calculating cortical area. Example of a human rib cross‐section demonstrating how to calculate Ct.Ar. First, the Tt.Ar of the cross‐section is measured, including everything below the periosteal border. Second, the Es.Ar is measured, which is essentially the area of the medullary cavity. Finally, the Es.Ar is subtracted from the Tt.Ar, resulting in the total area of the cortex (Ct.Ar). Ct.Ar = cortical area; Tt.Ar = total area; Es.Ar = endosteal area.

Figure 3

Image of an overstained bone that is imaged under fluorescence. The excess of stain makes it difficult to distinguish fine features in the bone.