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.

© 2012 The Authors. Journal of Anatomy © 2012 Anatomical Society.

Figures

Fig. 1

A schematic diagram of equine fetlock showing the metacarpophalangeal joint. The enlarged rectangle shows a cross-section of the distal end of the third metacarpal. The joint surface has two different radii of curvature and the junction between these is marked by a cortical ridge, which is a common site of early lesions. The loading of the joint varies across the surface. The palmar surface which articulates with the sesamoids is exposed primarily to compressive loads and the dorsal surface which articulates with the proximal phalanx is exposed to greater shear loads. The thickness of the cartilage also varies, with the thinnest cartilage on the palmar surface and the region with the thickest cartilage is close to the cortical ridge and marked with an arrow.

Fig. 2

Multi-modal imaging of the tidemark in a 6-year-old horse. (A) Second harmonic generation (SHG) image showing the collagen matrix. The individual collagen fibres are too fine to be resolved individually; however, their arrangement determines the texture of the image. (B) Two-photon fluorescence (TPF) image showing the distribution of endogenous fluorophores and (C) a coherent anti-Stokes Raman scattering (CARS) image taken at the CH2 resonance. (D) Merged image where blue = SHG, green = TPF and red = CARS. The tidemark is evident in all three imaging modalities. In the CARS image the chondrocytes are seen filling their lacunae above the tidemark but below the tidemark the lacunae are empty.

Fig. 3

Images in the plane of the tidemark. These images are taken from a z-stack of images taken in a cartilage plug from the cortical ridge of a 6-year-old horse. The stack of images started in the radial tissue and finished in the calcified tissue, with each image being separated by a 1-μm step. The images displayed here were identified as the plane of the tidemark due to a step change in fluorescence intensity between adjacent images as the field of view moved into the calcified tissue. Contrast in the second harmonic generation image (A) is from the collagen matrix and in the two-photon fluorescence image (B) from endogenous fluorophores. The collagen imaging in (A) shows an absence of fibres in the plane of the tidemark and both (A) and (B) show smooth lacuna boundaries.

Fig. 4

The cement line in transverse section. In all images the calcified cartilage is in the top half of the image and the subchondral bone in the bottom half. CC, calcified cartilage; CL, cement line; SB, subchondral bone. (A) SHG image showing differences in collagen organisation between the cartilage and the bone. (B) TPF image of the same field showing the accumulation of fluorescent debris in the lacunae and intense fluorescence at the cement line. (C–E) Higher resolution SHG imaging of collagen fibres at the cement line interface between the calcified cartilage and the subchondral bone. Insets show regions with fibres crossing the cement line at an increased magnification. In (D) there are so many fibres crossing the cement line that it appears obscured in places.

Fig. 5

An example of the histograms derived from directionality analysis on SHG images of two regions (size 25 × 25 μm) of radial zone cartilage, region 1 (from the palmar area) showing highly parallel collagen fibre arrangement and region 2 (from the dorsal area) showing a much less ordered collagen fibre arrangement. The values on the y axis are normalised so that the average value of the Fourier components is equal to 1. The analysis presented in the text was based on 90 regions of interest in each of the five horses used in the directionality study. The visibility (peak height – background) of the peak was used to quantify the variations in collagen fibre organisation.

Fig. 6

Regional variations in the degree of parallel organisation of the collagen fibres. SHG images are from (A) palmar region, (B) cortical ridge region and (C) dorsal region in the 14-year-old horse. In (A) there is a 16° change in the collagen fibre angle across the tidemark. The collagen fibre arrangement appears more ordered and parallel in (A) compared with (B) and (C), and this was confirmed by the Fourier transform analysis.

Fig. 7

Regional variations in cell organisation in a 14-year-old horse. (A) Typical images from the palmar region, (B) typical images from the cortical ridge region and (C) typical images from the dorsal region. In the merged images the auto-fluorescence is green and the SHG from collagen fibres is blue. In the cortical ridge and dorsal regions the width of the lacunae (dimension parallel to the tidemark) is greater than in the palmar region.

Fig. 8

Calcified cartilage underlying early focal lesions, from a 15-year-old horse(A–C), and from a 14-year-old horse (D,E). (A) and (B) are from the same region with (A) showing the collagen fibres and (B) showing the endogenous fluorophores. (C) Higher resolution image from the region highlighted in (B) where increased fluorescence can be seen filling the gaps in the collagen matrix (green arrows). In the merged image (D), blue represents collagen imaged through SHG and green represents endogenous fluorophores measured by the TPF. In this region the tidemark appeared irregular. The zone of calcified cartilage is very thick in places (+ 400 μm). Gaps in the collagen matrix were seen beneath the lesion, seen most clearly in (A) and (E).