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

Abstract

In order to investigate the three-dimensional structure of the collagen fibrils in articular cartilage, full-thickness canine humeral cartilage was microtomed into perpendicular sections that included both the articular surface and the subchondral bone and approximately 100 successive parallel sections that were each 6 μm thick and from a different cartilage depth. Each section was imaged using polarized light microscopy with a 5× objective (2.0 μm pixel size), generating two quantitative images (angle and retardation). Selected sections were also imaged using a 40× objective (0.25 μm pixel size). At an increased depth from the articular surface, the angle and retardation results in the perpendicular sections showed the well-known 90° change in fibril orientation between the surface and the deep cartilage. In contrast, the retardation results of the parallel sections decreased from the articular surface and remained approximately 0 through most of the radial zones, while the angle results of the parallel sections only changed about 30°. The territorial matrix morphology surrounding 61 chondrocyte clusters was quantified by its length, aspect ratio, and orientation. The cellular clusters in the surface cartilage were ellipsoidal in both parallel and perpendicular sections. In the radial zone, the cellular clusters were oriented in vertical columns in the perpendicular sections and as circular groupings in the parallel sections. This orthogonal imaging technique could provide a better understanding of the three-dimensional territorial and interterritorial fibrils in articular cartilage, the disturbance of which could signify the onset of degenerative cartilage diseases such as osteoarthritis.

Keywords:

• articular cartilage
• PLM
• birefringence
• parallel sections
• collagen network
• chondrocyte cluster

ACKNOWLEDGMENTS

Y. Xia is supported by the R01 grants (AR 45172, AR 52353) from the National Institutes of Health. The authors are grateful to Drs. C. Les and H. Sabbah (Henry Ford Hospital, Detroit) for providing the canine joints; Mr. Ilco Aksovski (Department of Physics, Oakland University) for an initial exploratory study of parallel sections by PLM; Dr. Nagarajan Ramakrishnan (current address: Defence Laboratory, Ratanada Palace, Rajasthan, India) for help with PLM 5× imaging; Prof. Ekrem Cicek (Mehmet Akif Ersoy University, Burdur, Turkey) for exploring PLM cell cluster imaging; Dr. Matthew Szarko (current address: St. George’s University of London, UK) and Mr. Farid Badar (Department of Physics, Oakland University) for helpful discussions; Mr. David Kahn (Department of Physics, Oakland University) and Miss Aimee Xia (University of Michigan, Ann Arbor) for editorial comments.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.