Cyclic loading of tendon fascicles using a novel fatigue loading system increases interleukin-6 expression by tenocytes

Abstract

Repetitive strain or 'overuse' is thought to be a major factor contributing to the development of tendinopathy. The aims of our study were to develop a novel cyclic loading system, and use it to investigate the effect of defined loading conditions on the mechanical properties and gene expression of isolated tendon fascicles. Tendon fascicles were dissected from bovine-foot extensors and subjected to cyclic tensile strain (1 Hz) at 30% or 60% of the strain at failure, for 0 h (control), 15 min, 30 min, 1 h, or 5 h. Post loading, a quasi-static test to failure assessed damage. Gene expression at a selected loading regime (1 h at 30% failure strain) was analyzed 6 h post loading by quantitative real-time polymerase chain reaction. Compared with unloaded controls, loading at 30% failure strain took 5 h to lead to a significant decrease in failure stress, whereas loading to 60% led to a significant reduction after 15 min. Loading for 1 h at 30% failure strain did not create significant structural damage, but increased Collagen-1-alpha-chain-1 and interleukin-6 (IL6) expression, suggesting a role of IL6 in tendon adaptation to exercise. Correlating failure properties with fatigue damage provides a method by which changes in gene expression can be associated with different degrees of fatigue damage.

Fig. 1

(a) Complete fascicle straining system; (b) close up of an individual loading chamber; (c) drawing of the loading chamber highlighting the components: To secure a fascicle between the upper and the bottom grip, the upper grip is removed from the chamber, the 1-mm thick plate and the screws are removed from the upper grip, and the fascicle is placed on the flat end of the upper grip. The 1-mm thick plate is then dropped back into place and secured with two screws, which are 2 mm apart. The upper grip is then inserted into the chamber, where it is prevented from rotating by a pin at the top of the chamber. The bottom grip is removed and the other end of the fascicle is placed between the two bottom screw holes. The bottom grip is then dropped back into place and secured with two screws. The chamber is filled with medium and sealed with a Plexiglas cylinder. O-rings prevent the chamber from leaking. The chamber is then secured in a rig, which holds up to 16 chambers, and the upper grip is connected to the actuator arm (see image a).

Fig. 2

Quasi-static failure stress of samples after previous fatigue loading. Control samples are presented as 0 h loading. Filled circles show samples subjected to 30% of the strain at failure. Triangles show samples loaded to 60% of the failure strain. *Significant difference between 30% and 60% loading groups (independent t-test). Data are presented as mean ± SE. (n = 6).

Fig. 3

Fold change in mRNA expression of the fatigue-loaded specimens relative to control specimens as represented by the 2^−ΔΔCt values normalized to 18S (a) or GAPDH (b). The median is represented by the line through the group of individual data points. The dashed line indicates a value of 1, representing the no-change level. *Significant difference between fatigue-loaded and control specimens (Wilcoxon test) (n = 7). 18S, 18S ribosomal RNA; COL1A1, collagen alpha chain 1; COL1A2, collagen alpha chain 2; CTGF, connective tissue growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGF1, insulin-like growth factor 1, IL6, interleukin 6; IL6R, interleukin 6 receptor; TGFb1, transforming growth factor beta 1; TGFb2, transforming growth factor beta 2; TGFb3, transforming growth factor beta 3.