Elastic fibres are broadly distributed in tendon and highly localized around tenocytes

Abstract

Elastic fibres have the unique ability to withstand large deformations and are found in numerous tissues, but their organization and structure have not been well defined in tendon. The objective of this study was to characterize the organization of elastic fibres in tendon to understand their function. Immunohistochemistry was used to visualize elastic fibres in bovine flexor tendon with fibrillin-1, fibrillin-2 and elastin antibodies. Elastic fibres were broadly distributed throughout tendon, and highly localized longitudinally around groups of cells and transversely between collagen fascicles. The close interaction of elastic fibres and cells suggests that elastic fibres are part of the pericellular matrix and therefore affect the mechanical environment of tenocytes. Fibres present between fascicles are likely part of the endotenon sheath, which enhances sliding between adjacent collagen bundles. These results demonstrate that elastic fibres are highly localized in tendon and may play an important role in cellular function and contribute to the tissue mechanics of the endotenon sheath.

Fig 1

Structure of tendon modified from (Kastelic et al. 1978) showing hierarchical organization of collagen bundles. Research presented in this document focuses on the fibre level of organization.

Fig 2

Dissection scheme for tendon organizational analysis. (a) Bovine flexor tendon with excised section marked by solid lines (X-X). Horizontal and vertical lines represent transverse and longitudinal cutting planes, respectively. (b) Transverse section clearly depicting the endotenon sheath (arrowhead) surrounding tendon fascicles. (c) Longitudinal section illustrating the collagen crimp structure aligned along the direction of loading.

Fig 3

Elastin, fibrillin-1 and fibrillin-2 dual antibody immunostaining depicting overall elastic fibre organization in untreated specimens with cell nuclei highlighted in blue. (a) Longitudinal elastin and fibrillin-1 fibres conform to wavy tendon structure and are densely distributed around tenocytes. Merged image highlights the lack of colocalization between fibrillin-1 and elastin (white arrowheads). (b) Longitudinal fibrillin-1 and fibrillin-2 fibres following wavy tendon structure with a high level of colocalization. (c) Transverse elastin and fibrillin-1 depicting elastic fibres as points surrounding tenocytes (white arrowhead) and a concentrated distribution between fascicles (yellow arrowhead). (d) Transverse fibrillin-1 and fibrillin-2 microfibrils distributed around tenocytes (white arrowhead) and between fascicles (yellow arrowhead) showing a high level of colocalization.

Fig 4

Detailed fibrillin-1 organization in tendon showing longitudinally oriented fibres present along arrays of cells and transversely oriented fibres between collagen fascicles. (a) Longitudinal 3D reconstruction from a 20-μm z-stack showing multiple fibres localized around tenocytes. (b) Transverse section highlighting areas of dense microfibril distribution around cells and between adjacent fascicles. (c) High magnification image showing interaction of elastic fibres and cells in transverse section. (d) Interfascicle 3D reconstruction of loose mesh-like elastic fibre structure found between collagen fascicles.

Fig 5

Three-dimensional reconstruction of elastic fibre organization (green) with outline of cell nuclei (blue) highlighted by dotted lines. Multiple elastic fibres surround groups of cells and have a branching structure. Fibres are also present between cells, but in fewer numbers.

Fig 6

Comparison of basic histology and pretreated immunohistochemistry for detecting elastic fibres. (a) Basic structure of tendon stained with H&E showing wavy collagen structure and cells arranged in rows. (b) Sparse elastic fibre organization (white arrowheads) as observed with Miller's stain. (c) Structure of tendon following hyaluronidase pretreatment with disrupted organization and lack of cell nuclei staining. (d) Enhanced immunodetection of elastic fibres following pretreatment with hyaluronidase.