Depth‐dependent anisotropies of amides and sugar in perpendicular and parallel sections of articular cartilage by Fourier transform infrared imaging

Abstract

Full thickness blocks of canine humeral cartilage were microtomed into both perpendicular sections and a series of 100 parallel sections, each 6 μm thick. Fourier Transform Infrared Imaging (FTIRI) was used to image each tissue section eleven times under different infrared polarizations (from 0° to 180° polarization states in 20° increments and with an additional 90° polarization), at a spatial resolution of 6.25 μm and a wavenumber step of 8 cm−1. With increasing depth from the articular surface, amide anisotropies increased in the perpendicular sections and decreased in the parallel sections. Both types of tissue sectioning identified a 90° difference between amide I and amide II in the superficial zone of cartilage. The fibrillar distribution in the parallel sections from the superficial zone was shown to not be random. Sugar had the greatest anisotropy in the upper part of the radial zone in the perpendicular sections. The depth-dependent anisotropic data were fitted with a theoretical equation that contained three signature parameters, which illustrate the arcade structure of collagens with the aid of a fibril model. Infrared imaging of both perpendicular and parallel sections provides the possibility of determining the three-dimensional macromolecular structures in articular cartilage. Being sensitive to the orientation of the macromolecular structure in healthy articular cartilage aids the prospect of detecting the early onset of the tissue degradation that may lead to pathological conditions such as osteoarthritis.

Quantitative Determination of Morphological and Territorial Structures of Articular Cartilage from Both Perpendicular and Parallel Sections by Polarized Light Microscopy

“…This represented a near random organization of the fibrils and was consistent with the FTIRI results of the same cartilage sections [38]. Because of this low retardation, any angle measurement was likely to contain large errors, indicated by the large error bars in the angle profiles of parallel sections (Fig.…”

“…In the SZ cartilage, the collagen fibrils in parallel planes were found to have a finite structural anisotropy, established by the motional anisotropy of water molecules in μMRI [44] and the amide anisotropy in Fourier-transform infrared imaging (FTIRI) [36, 38]. These previous assessments of the surface fibril structure by different physical mechanisms are consistent with the birefringent results in this PLM report.…”

“…To facilitate the imaging of the same section by both visible light in PLM and infrared light in Fourier-transform infrared imaging (the Fourier-transform infrared imaging results of this project are reported elsewhere [38]), the cartilage sections were placed directly on the MirrIR slides (a specially coated microscope slide from Kevley Technologies, Chesterland, Ohio) without a cover glass. A number of additional cartilage sections from several neighboring blocks were covered with the usual mounting media and cover glass, and were also imaged in PLM as quantitative comparisons [19].…”

“…This sectioning and imaging approach allows all structural zones to be imaged together in one experiment. Alternatively, articular cartilage can also be studied using thin sections cut parallel to the articular surface (termed as the parallel sections in this report), i.e., each parallel section is cut from a particular depth in cartilage [30–38]. The goal of this investigation is to map the cartilage anisotropies and cellular morphology by imaging cartilage cut from two orthogonal planes – perpendicular sections and a series of sequentially numbered parallel sections through the entire cartilage depth.…”

Vibrational spectroscopy of articular cartilage

“…In AC, the polarized FTIR studies have revealed that the intensities of the amide I, the amide II, and the amide III regions vary strongly when polarization plane of infrared light is altered (19,(57)(58)(59)(60), whereas the carbohydrate region shows only weak anisotropy in the radial zone of AC (58,61). It is known that the transition moments of the amide I and II bonds are qualitatively perpendicular to each other (60).…”