Collagen orientation in compact bone: I. A new method for the determination of the proportion of collagen parallel to the plane of compact bone sections.

Previous work has shown that different types of osteons that have different mechanical properties with regard to tension and compression can be identified and studied by polarized light microscopy. This study reports the development of an automated method for the analysis of that proportion of collagen in transverse sections of human femurs that lies parallel with the plane of section. The method is based upon the use of circularly polarized light to detect collagen with a major component lying in the plane of the section, correcting the measurement for bright collagen under these circumstances with a corresponding darkfield measurement to determine the area of bone within each field of the section analyzed. The method has been validated in several different ways. The measured value of bright collagen under circularly polarized light depends upon the section thickness, since a linear relationship was found between section thickness and bright areas for a large number of microscopic fields. Plane parallel sections are therefore recommended for this study. The pattern of distribution of bright areas was the same for different observers and different set detection levels in any one section. The pattern found in adjacent sections from the same region of the shaft was identical.