Second harmonic generation imaging reveals a distinct organization of collagen fibrils in locations associated with cartilage growth

ABSTRACT

Purpose: The articular-epiphyseal cartilage complex (AECC) is responsible for the expansion of the bone ends and serves the function of the articular cartilage in juvenile mammals. Bundles of collagen fibrils surrounding cells were in the literature observed more frequently near the articular surface of the AECC. The articular surface, the perichondrium, and cartilage canals are interfaces where appositional growth of the AECC has been demonstrated. The current study aimed to evaluate the potential of second harmonic generation (SHG) to locate the collagen fibril bundles near the articular surface and to examine whether a comparable collagen fibril organization could be observed near the other interfaces of the AECC. Materials and methods: The study included the femoral condyle of four piglets aged 82–141 days. The forward and backward scattered SHG, and their ratio, was analyzed across the AECC using objectives with different numerical aperture. Two-photon-excited fluorescence was used to visualize cells. Results: A similar pattern of collagen fibril organization was observed near the articular surface, around cartilage canals, and adjacent to the perichondrium. The pattern consisted of a higher ratio of forward to backward scattered SHG that increased relative to the surrounding matrix at lower numerical aperture. This was interpreted to reflect collagen fibril bundles in the territorial matrix of cells in these areas. Conclusions: The observed arrangement of collagen fibrils was suggested to be related to the presumed different growth activity in these areas and indicated that SHG may be used as an indirect and label-free marker for cartilage matrix growth.

KEYWORDS:

• Articular cartilage
• cartilage canal
• cartilage growth
• collagen fibrils
• second harmonic generation

Acknowledgments

We would like to thank Astrid Bjørkøy for technical support with the imaging system and Rajesh Kumar and Elisabeth Inge Romijn for assistance with the sample preparation. The HES stained sample was prepared at the Cellular and Molecular Imaging Core Facility (CMIC), Norwegian University of Science and Technology. We also thank Kristin Sæterbø for obtaining a fresh piglet from a local pig farm. The other piglets were received from Norwegian Pig Breeders Association, Norsvin, and funded by grant number 199598 and grant number 244212 from the Research Council of Norway.

Declaration of interest

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