Maturation of the Meniscal Collagen Structure Revealed by Polarization-Resolved and Directional Second Harmonic Generation Microscopy

Maxime Pinsard,Hélène Richard,Marie-Claire Schanne-Klein,François Légaré,Sheila Laverty,Julia Dubuc

doi:10.1038/s41598-019-54942-0

Abstract

We report Polarization-resolved Second Harmonic Generation (P-SHG) and directional SHG (forward and backward, F/B) measurements of equine foetal and adult collagen in meniscus, over large field-of-views using sample-scanning. Large differences of collagen structure and fibril orientation with maturation are revealed, validating the potential for this novel methodology to track such changes in meniscal structure. The foetal menisci had a non-organized and more random collagen fibrillar structure when compared with adult using P-SHG. For the latter, clusters of homogeneous fibril orientation (inter-fibrillar areas) were revealed, separated by thick fibers. F/B SHG showed numerous different features in adults notably, in thick fibers compared to interfibrillar areas, unlike foetal menisci that showed similar patterns for both directions. This work confirms previous studies and improves the understanding of meniscal collagen structure and its maturation, and makes F/B and P-SHG good candidates for future studies aiming at revealing structural modifications to meniscus due to pathologies.

Highlights

Second Harmonic Generation (SHG) microscopy is a recent and powerful technique to image the structure of biological specimens as it provides submicron spatial resolution, has low phototoxicity and a high depth selectivity and penetration
The forward over backward SHG signals (F/B) ratio increases with the level of homogeneity of noncentrosymmetric structures within the focal volume and has been related to the size of the collagen fibrils for collagen rich tissues[18,19]
We demonstrate the unique potential of SHG and Polarization-resolved SHG (P-SHG) to measure the collagen structure in the meniscus, and we use these two techniques together with histology to characterize the structural differences between equine foetal and adult meniscal ECM

Summary

Introduction

SHG microscopy is a recent and powerful technique to image the structure of biological specimens as it provides submicron spatial resolution, has low phototoxicity and a high depth selectivity and penetration. Up to now, the local orientation of the meniscal collagen structures has been mainly characterized by OPT, only to a resolution of ~15 μm permitting identification of the collagen fascicles but not of the individual fibers[4] These fibers were later imaged by SHG microscopy in some areas from bovine samples[5], but their orientation was discussed only in terms of the SHG intensity. Understanding and imaging the meniscal ECM could provide new knowledge to support studies on meniscal degradation events in joint degeneration, and potentially new insights towards meniscal regeneration in the future

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Conclusion