Different mechanisms activated by mild versus strong loading in rat Achilles tendon healing

<div><p>Background</p><p>Mechanical loading stimulates Achilles tendon healing. However, various degrees of loading appear to have different effects on the mechanical properties of the healing tendon, and strong loading might create microdamage in the tissue. This suggests that different mechanisms might be activated depending on the magnitude of loading. The aim of this study was to investigate these mechanisms further.</p><p>Methods</p><p>Female rats had their right Achilles tendon cut transversely and divided into three groups: 1) unloading (calf muscle paralysis by Botox injections, combined with joint fixation by a steel-orthosis), 2) mild loading (Botox only), 3) strong loading (free cage activity). Gene expression was analyzed by PCR, 5 days post-injury, and mechanical testing 8 days post-injury. The occurrence of microdamage was analyzed 3, 5, or 14 days post-injury, by measuring leakage of injected fluorescence-labelled albumin in the healing tendon tissue.</p><p>Results</p><p>Peak force, peak stress, and elastic modulus of the healing tendons gradually improved with increased loading as well as the expression of extracellular matrix genes. In contrast, only strong loading increased transverse area and affected inflammation genes. Strong loading led to higher fluorescence (as a sign of microdamage) compared to mild loading at 3 and 5 days post-injury, but not at 14 days.</p><p>Discussion</p><p>Our results show that strong loading improves both the quality and quantity of the healing tendon, while mild loading only improves the quality. Strong loading also induces microdamage and alters the inflammatory response. This suggests that mild loading exert its effect via mechanotransduction mechanisms, while strong loading exert its effect both via mechanotransduction and the creation of microdamage.</p><p>Conclusion</p><p>In conclusion, mild loading is enough to increase the quality of the healing tendon without inducing microdamage and alter the inflammation in the tissue. This supports the general conception that early mobilization of a ruptured tendon in patients is advantageous.</p></div

Primers used for qRT-PCR (purchased from applied Biosystems).

<p>Primers used for qRT-PCR (purchased from applied Biosystems).</p

Results from gene expression and microdamage analyses.

<p><b>A)</b> Gene expression for extracellular matrix genes, 5 days after tendon injury. The fold change of genes for <i>Collagen I</i> (<i>COL1A1</i>), <i>collagen III</i> (<i>COL3A1</i>), <i>collagen V</i> (<i>COL5A1</i>), and <i>lysyl oxidase</i> (<i>LOX</i>) are shown. Three different loading conditions were tested; unloading (Botox and steel-orthosis), mild loading (Botox), and strong loading (free cage activity), N = 11–12. <b>B)</b> Leakage of fluorescent protein (BSA-FITC) as a sign of bleeding and microdamage: 3, 5, or 14 days after tendon injury. The result describes the fraction of the fluorescence in the tendon tissue compared to the fluorescence in the blood plasma (ratio of (counts per second / mg specimen) / (counts per second / mg blood plasma)). Two different loading conditions were tested: mild (Botox) and strong loading (N = 12). The rats were intravenously injected with BSA-FITC 1 hour before euthanasia. The fluorescence detected in the tendon tissue was normalized to the tissue weight and the fluorescence detected in the blood plasma. * p < 0.001.</p

Gene expression, 5 days post-injury.

<p>Gene expression, 5 days post-injury.</p

Raw data from fluorescence measurements.

<p>Raw data from fluorescence measurements.</p

Overview of the fluorescence value measured.

<p>Overview of the fluorescence value measured.</p

Mechanical properties of rat Achilles tendons, 8 days post-injury.

<p>Mechanical properties of rat Achilles tendons, 8 days post-injury.</p

Unloading device.

<p>Steel-orthosis, used in the unloaded group.</p

Mechanical testing results.

<p>Mechanical testing results for peak force, transverse area, peak stress, and elastic modulus, 8 days after tendon injury. Three different loading conditions were tested: unloading (Botox and steel-orthosis), mild loading (Botox), and strong loading (free cage activity). N = 10.</p