Distribution of proteins within different compartments of tendon varies according to tendon type

Abstract

Although the predominant function of all tendons is to transfer force from muscle to bone and position the limbs, some tendons additionally function as energy stores, reducing the energetic cost of locomotion. To maximise energy storage and return, energy-storing tendons need to be more extensible and elastic than tendons with a purely positional function. These properties are conferred in part by a specialisation of a specific compartment of the tendon, the interfascicular matrix, which enables sliding and recoil between adjacent fascicles. However, the composition of the interfascicular matrix is poorly characterised and we therefore tested the hypothesis that the distribution of elastin and proteoglycans differs between energy-storing and positional tendons, and that protein distribution varies between the fascicular matrix and the interfascicular matrix, with localisation of elastin and lubricin to the interfascicular matrix. Protein distribution in the energy-storing equine superficial digital flexor tendon and positional common digital extensor tendon was assessed using histology and immunohistochemistry. The results support the hypothesis, demonstrating enrichment of lubricin in the interfascicular matrix in both tendon types, where it is likely to facilitate interfascicular sliding. Elastin was also localised to the interfascicular matrix, specifically in the energy-storing superficial digital flexor tendon, which may account for the greater elasticity of the interfascicular matrix in this tendon. A differential distribution of proteoglycans was identified between tendon types and regions, which may indicate a distinct role for each of these proteins in tendon. These data provide important advances into fully characterising structure-function relationships within tendon.

Keywords: endotenon; histology; immunohistochemistry; interfascicular matrix; proteoglycans.

Figure 1

Schematic showing the hierarchical structure of tendon in which collagen aggregates to form subunits of increasing diameter, the largest of which is the fascicle. Fascicles are interspersed by the interfascicular matrix (IFM, also known as the endotenon). Reproduced from Thorpe et al., 2015a,b with kind permission from Wiley publications.

Figure 2

Representative images showing AB/PAS staining for proteoglycans in the SDFT (A) and CDET (B). Scale bar: 100 μm.

Figure 3

Representative images showing immunohistochemical staining of decorin in the SDFT (A) and CDET (B). Scale bar: 100 μm. There were no significant differences in decorin staining intensity between tendon types or regions (C,D). Individual data points are shown, with lines representing IFM and FM regions in the same image (C). In (D), data are displayed as mean ± SD.

Figure 4

Representative images showing immunohistochemical staining of lumican in the SDFT (A) and CDET (B). Scale bar: 100 μm. Staining intensity was significantly greater in the CDET IFM than in the SDFT IFM and in the CDET FM (C,D). Individual data points are shown, with lines representing IFM and FM regions in the same image (C). In D, data are displayed as mean ± SD. **P < 0.01; ***P < 0.001.

Figure 5

Representative images showing immunohistochemical staining of biglycan in the SDFT (A) and CDET (B). Scale bar: 100 μm. Staining intensity was significantly greater in the SDFT IFM than in the FM (C,D). Individual data points are shown, with lines representing IFM and FM regions in the same image (C). In D, data are displayed as mean ± SD. *P < 0.05.

Figure 6

Representative images showing immunohistochemical staining of fibromodulin in the SDFT (A) and CDET (B). Scale bar: 100 μm. Staining intensity was significantly greater in the CDET than in the SDFT FM (C,D). Individual data points are shown, with lines representing IFM and FM regions in the same image (C). In D, data are displayed as mean ± SD. **P < 0.01.

Figure 7

Representative images showing immunohistochemical staining of lubricin in the SDFT (A) and CDET (B). Scale bar: 100 μm. Staining intensity was significantly greater in the SDFT IFM than in the FM (C,D). Individual data points are shown, with lines representing IFM and FM regions in the same image (C). In D, data are displayed as mean ± SD. **P < 0.01.

Figure 8

Representative images showing Elastic von Gieson's staining of the SDFT (A) and CDET (B). Scale bar: 50 μm. Elastic fibres are visible as blue/black lines. In the SDFT, elastin staining was predominantly localised to the IFM (arrow) with a small number of elastic fibres evident within the FM (dashed arrows). In the CDET, a small number of elastic fibres were identified in the IFM and in the FM. Quantification of the percent staining of elastin demonstrated significantly greater elastin in the SDFT IFM than in the FM (C). It was not possible to perform statistical analyses comparing the SDFT and CDET due to low numbers of stained elastic fibres in the CDET groups. ***P < 0.001.