A multi‐modal multiphoton investigation of microstructure in the deep zone and calcified cartilage

Abstract

Multi-modal multiphoton microscopy was used to investigate tissue microstructure in the zone of calcified cartilage, focussing on the collagen fibre organisation at the tidemark and cement line. Thick, unstained and unfixed sagittal sections were prepared from the equine metacarpophalangeal joint. Second harmonic generation (SHG) provided contrast for collagen, two-photon fluorescence (TPF) for endogenous fluorophores, and coherent anti-Stokes Raman scattering (CARS) allowed the cells to be visualised. The structure of radial and calcified cartilage was found to vary with location across the joint, with the palma regions showing a more ordered parallel arrangement of collagen fibres than the cortical ridge and dorsal regions. These patterns may be associated with regional variations in joint loading. In addition, the cell lacunae had a greater diameter in the dorsal region than in the palmar region. At the cement line some collagen fibres were observed crossing between the calcified cartilage and the subchondral bone. At the tidemark the fibres were parallel and continuous between the radial and calcified cartilage. Beneath early superficial lesions the structure of the tidemark and calcified cartilage was disrupted with discontinuities and gaps in the fibrillar organisation. Cartilage microstructure varies in the deep zones between regions of different loading. The variations in collagen structure observed may be significant to the local mechanical properties of the cartilage and therefore may be important to its mechanical interactions with the subchondral bone. The calcified cartilage is altered even below early superficial lesions and therefore is important in the understanding of the aetiology of osteoarthritis.

Fibril deformation under load of the rabbit Achilles tendon and medial collateral ligament femoral entheses

“…The relative smoothness and abruptness of this boundary, as well as the shape of cell lacunae in its vicinity, are indicators of the commonly referred to tidemark, identified histologically . Similarities can be observed between these images and those of others that reference a tidemark or mineralization line and employ multiphoton microscopy (e.g., enamel–dentin interface of the tooth, calcified‐uncalcified cartilage interface at the equine metacarpophalangeal joint). The focus of these researchers was not on ligament/tendon interfaces; however, histological study has shown the tidemark of articular cartilage to be continuous with that of fibrocartilaginous entheses, provided they are in close proximity .…”

Chemically specific imaging and in-situ chemical analysis of articular cartilage with stimulated Raman scattering

“…Second harmonic (SHG) imaging of collagen is well established [5,6]. Its application to cartilage is complicated by the fact that the individual fibres of type II collagen are finer than those of type I and below the limit of resolution, but valuable information on its organisation in cartilage has been derived by image processing [7,8] and polarization senstive measurements [9]. Elastin shows strong two photon fluorescence (TPF) and this technique has revealed the somewhat unexpected presence of an extensive network of elastin fibres in the surface zone of articular cartilage [10,11].…”

Quantitative SHG imaging in osteoarthritis model mice, implying a diagnostic application

“…The acquired signal reflects the localization and amount of fibrous structures of the collagen molecule. Therefore, it is proposed that SHG imaging enables the clinician to detect both quantitative and qualitative changes in the cartilage matrix at a microscopic resolution [6][7][8][9][10][11][12][13]. Several animal OA models, including histopathological assessments of their osteoarthritic processes, are well established [14][15][16][17].…”