Elastase treatment of tendon specifically impacts the mechanical properties of the interfascicular matrix

Abstract

The tendon interfascicular matrix (IFM) binds tendon fascicles together. As a result of its low stiffness behaviour under small loads, it enables non-uniform loading and increased overall extensibility of tendon by facilitating fascicle sliding. This function is particularly important in energy storing tendons, with previous studies demonstrating enhanced extensibility, recovery and fatigue resistance in the IFM of energy storing compared to positional tendons. However, the compositional specialisations within the IFM that confer this behaviour remain to be elucidated. It is well established that the IFM is rich in elastin, therefore we sought to test the hypothesis that elastin depletion (following elastase treatment) will significantly impact IFM, but not fascicle, mechanical properties, reducing IFM resilience in all samples, but to a greater extent in younger tendons, which have a higher elastin content. Using a combination of quasi-static and fatigue testing, and optical imaging, we confirmed our hypothesis, demonstrating that elastin depletion resulted in significant decreases in IFM viscoelasticity, fatigue resistance and recoverability compared to untreated samples, with no significant changes to fascicle mechanics. Ageing had little effect on fascicle or IFM response to elastase treatment. This study offers a first insight into the functional importance of elastin in regional specific tendon mechanics. It highlights the important contribution of elastin to IFM mechanical properties, demonstrating that maintenance of a functional elastin network within the IFM is essential to maintain IFM and thus tendon integrity. STATEMENT OF SIGNIFICANCE: Developing effective treatments or preventative measures for musculoskeletal tissue injuries necessitates the understanding of healthy tissue function and mechanics. By establishing the contribution of specific proteins to tissue mechanical behaviour, key targets for therapeutics can be identified. Tendon injury is increasingly prevalent and chronically debilitating, with no effective treatments available. Here, we investigate how elastin modulates tendon mechanical behaviour, using enzymatic digestion combined with local mechanical characterisation, and demonstrate for the first time that removing elastin from tendon affects the mechanical properties of the interfascicular matrix specifically, resulting in decreased recoverability and fatigue resistance. These findings provide a new level of insight into tendon hierarchical mechanics, important for directing development of novel therapeutics for tendon injury.

Keywords: Elastase; Elastin; Fatigue; Interfascicular matrix; Tendon.

Graphical abstract

Fig. 1

Schematic showing IFM recovery testing protocol. To test the ability of the IFM to recover, the opposite ends of each fascicle were removed, so that only 10 mm of intact IFM was left connecting the fascicles. Four lines were drawn across the two fascicles in the central test region to track local displacements; samples shown schematically and pictorially, where samples are pictured on a blue cutting board (a). A time-displacement graph pictorially represents the test protocol and times at which images were analysed. Four specific test points were identified; A: initial point, B: maximum displacement, C: immediately after removal of load and D: after a 10 second recovery period (b). Still frames of the sample were extracted from the video at each selected time point (c). Tracking algorithms were adopted to investigate the displacement of the lines, and thus fascicles, during each test (d).

Fig. 2

Validation of elastase treatment. (a-d) Representative confocal images showing tendon explants immunolabelled for elastin (red) and cell nuclei (blue): fresh (a), after incubation in control buffer (b), in 0.2 U/ml elastase solution (c) and 2 U/ml elastase solution (d). Scale bar: 20 μm. Visible elastin fibres are noted with arrows. Quantitative investigation of tendon matrix composition compares elastin (e), GAG (f) and collagen (g) content for fresh, control and elastase treated samples. Significant differences between treatments are identified by: *** p<0.001 (normally distributed data – ANOVA). Data are displayed as mean ± standard deviation.

Fig. 3

Creep curves of young SDFT IFM (a) and old SDFT IFM (b), highlighting the variability between samples by plotting the maximum (Max) and minimum (Min) curves in each group, alongside the average (Av). Fresh samples are shown in black, control samples in grey, elastase treated with a dotted line. Labelling is not used for elastase treated samples as it evident all elastase treated samples failed at less than 100 cycles.

Fig. 4

Effect of elastin depletion on IFM fatigue properties in young and old samples. Data compares cycles to failure (a), creep between cycles 1 and 10 (b) and secondary creep rate of maximum displacement (c) for young and old SDFT IFM samples from fresh, control and elastase treated groups (n = 5/age group; total samples tested: 15/treatment/tendon). Significant differences are flagged with: **p<0.01 and ***p<0.001 (Cycles to Failure: normally distributed – ANOVA, all remaining IFM fatigue data not normally distributed – Mann-Whitney test).

Fig. 5

Effect of elastin depletion on IFM loading response and recovery. Loading and recovery were visually monitored by tracking markers across the IFM samples. Angle deviation (degrees) of lines was first determined under the application of 75% of the predicted failure extension (a). Load was removed, and then recovery of lines relative to their start point was measured immediately after loading (b) and 10 s after the removal of load (c). Graphs compare young and old SDFT IFM samples in the fresh, control and elastase treated groups (n = 5/age group; total samples tested: 2/treatment/tendon). Significant differences are flagged with: *p<0.05 and **p<0.01 (data non-normally distributed – Mann-Whitney test). For more details regarding loading protocol refer to Fig. 1.