Tie-fibre structure and organization in the knee menisci

Abstract

The collagenous structure of the knee menisci is integral to the mechanical integrity of the tissue and the knee joint. The tie-fibre structure of the tissue has largely been neglected, despite previous studies demonstrating its correlation with radial stiffness. This study has evaluated the structure of the tie-fibres of bovine menisci using 2D and 3D microscopy techniques. Standard collagen and proteoglycan (PG) staining and 2D light microscopy techniques were conducted. For the first time, the collagenous structure of the menisci was evaluated using 3D, second harmonic generation (SHG) microscopy. This technique facilitated the imaging of collagen structure in thick sections (50-100 μm). Imaging identified that tie-fibres of the menisci arborize from the outer margin of the meniscus toward the inner tip. This arborization is associated with the structural arrangement of the circumferential fibres. SHG microscopy has definitively demonstrated the 3D organization of tie-fibres in both sheets and bundles. The hierarchy of the structure is related to the organization of circumferential fascicles. Large tie-fibre sheets bifurcate into smaller sheets to surround circumferential fascicles of decreasing size. The tie-fibres emanate from the lamellar layer that appears to surround the entire meniscus. At the tibial and femoral surfaces these tie-fibre sheets branch perpendicularly into the meniscal body. The relationship between tie-fibres and blood vessels in the menisci was also observed in this study. Tie-fibre sheets surround the blood vessels and an associated PG-rich region. This subunit of the menisci has not previously been described. The size of tie-fibre sheets surrounding the vessels appeared to be associated with the size of blood vessel. These structural findings have implications in understanding the mechanics of the menisci. Further, refinement of the complex structure of the tie-fibres is important in understanding the consequences of injury and disease in the menisci. The framework of meniscus architecture also defines benchmarks for the development of tissue-engineered replacements in the future.

Keywords: blood vessels; meniscus; second harmonic generation microscopy; structure; tie-fibres.

Figure 1

Fast green and safranin-o staining of meniscal sections taken from: (a) anterior; (b) central (Andrews et al. 2013) with permissions; and (c) posterior portions of the medial meniscus of a bovine stifle. (d) A photo of a medial meniscus identifying the locations of the sections stained for histology. Tie-fibre architecture was variable between sections with increased prevalence in posterior sections. These patterns were comparable amongst all specimens that were evaluated. Scale bar: 15 mm.

Figure 2

Fast green and safranin-o staining of radial cross-sections. (Left) The radially oriented tie-fibres (arrows) emanating from the outer edge of the meniscus that appears to be an extension of the surface lamellar layer (dashed arrows). (Right) Many tie-fibres extend perpendicularly into the meniscus from the contact surfaces and are associated with increased PG staining (arrows). Scale bar: 5 mm.

Figure 3

SHG microscopy image demonstrating the arborization of tie-fibres as they divide into smaller fibres and subdivide the larger fascicles into smaller bundles (dashed ellipses). Arrows identify areas where bundles branch to subdivide the fascicles. Scale bar: 100 μm.

Figure 4

Histological images of meniscal sections stained with fast green and safranin-o. (a) Tie-fibres and a PG-rich regions (shown as pink colour) surrounding a blood vessel denoted by dashed ellipse (b) along a cut oblique to the blood vessel orientation (c) cut is perpendicular to the predominant blood vessel direction and shows several blood vessels in close proximity. (d) Schematic of the PG-rich perivascular regions in the menisci. Scale bar: ˜100 μm.

Figure 5

Top: SHG microscopy image identifying a tie-fibre sheet running diagonally from the bottom left to top right of the image. The sheets appears as groups of fibres running in parallel. Fibre bundles can be seen to arborize from the edge of the sheet to wrap around adjacent circumferential fascicles (arrows). Scale bar: 100 μm, both images are the same scale. (Bottom) Breakout image from the dashed rectangle (top). Solid arrows identify that fibres that appear crimped are actually following the contour of the circumferential fascicle. Dashed arrow identifies a small sheet of fibres wrapping over the top of another tie-fibre bundle.

Figure 6

(a,c) SHG of a blood vessel cut perpendicularly; (b,d) the blood vessel has been grazed along its length. Green colour identifies collagen, and red colour elastin as identified by IIF. Arrows identify the tie-fibres that surround the blood vessel and collagen-sparse region. (c,d) Three-dimensional images of sections. All section images are 250 μm square.