The orientation of collagen molecules is an important determinant of their functionality in connective tissues. The objective of the current study is to establish a method to determine the alignment of collagen molecules in histological sections of cartilage by polarized Fourier transform infrared imaging spectroscopy (FT-IRIS), a method based on molecular vibrations. Polarized FT-IRIS data obtained from highly oriented tendon collagen were utilized to calibrate the derived spectral parameters. The ratio of the integrated areas of the collagen amide I/II absorbances was used as an indicator of collagen orientation. These data were then applied to FT-IRIS analysis of the orientation of collagen molecules in equine articular cartilage, in equine repair cartilage after microfracture treatment, and in human osteoarthritic cartilage. Polarized light microscopy (PLM), the most frequently utilized technique to evaluate collagen fibril orientation in histological sections, was performed on picrosirius red-stained sections for comparison. Thicknesses of each zone of normal equine cartilage (calculated based on differences in collagen orientation) were equivalent as determined by PLM and FT-IRIS. Comparable outcomes were obtained from the PLM and FT-IRIS analyses of repair and osteoarthritis tissues, whereby similar zonal variations in collagen orientation were apparent for the two methods. However, the PLM images of human osteoarthritic cartilage showed less obvious zonal discrimination and orientation compared to the FT-IRIS images, possibly attributable to the FT-IRIS method detecting molecular orientation changes prior to their manifestation at the microscopic level.
