Influence of a single loading episode on gene expression in healing rat Achilles tendons

Abstract

Mechanical loading stimulates tendon healing via mechanisms that are largely unknown. Genes will be differently regulated in loaded healing tendons, compared with unloaded, just because of the fact that healing processes have been changed. To avoid such secondary effects and study the effect of loading per se, we therefore studied the gene expression response shortly after a single loading episode in otherwise unloaded healing tendons. The Achilles tendon was transected in 30 tail-suspended rats. The animals were let down from the suspension to load their tendons on a treadmill for 30 min once, 5 days after tendon transection. Gene expression was studied by Affymetrix microarray before and 3, 12, 24, and 48 h after loading. The strongest response in gene expression was seen 3 h after loading, when 150 genes were up- or downregulated (fold change ≥2, P ≤ 0.05). Twelve hours after loading, only three genes were upregulated, whereas 38 were downregulated. Fewer than seven genes were regulated after 24 and 48 h. Genes involved in the inflammatory response were strongly regulated at 3 and 12 h after loading; this included upregulation of iNOS, PGE synthase, and IL-1β. Also genes involved in wound healing/coagulation, angiogenesis, and production of reactive oxygen species were strongly regulated by loading. Microarray results were confirmed for 16 selected genes in a repeat experiment (N = 30 rats) using real-time PCR. It was also confirmed that a single loading episode on day 5 increased the strength of the healing tendon on day 12. In conclusion, the fact that there were hardly any regulated genes 24 h after loading suggests that optimal stimulation of healing requires a mechanical loading stimulus every day.

Primary gene response to mechanical loading in healing rat Achilles tendons

“…Extraction of RNA was carried out by a combination of the Trizol method and RNeasy total mini kit (QIAGEN, Sollentuna, Sweden) (10,38). The tendons were kept frozen during homogenization and were pulverized one by one by a tungsten ball in liquid nitrogen-cooled vessels in a Retsch mixer mill MM 200 (Retsch, Haan, Germany).…”

“…Yet there is always a risk of re-rupture if too much loading is applied after injury. In rats, short daily episodes of loading of an otherwise unloaded healing Achilles tendon increases the strength of the healing tissue (1,9,10). In human patients, early weight bearing after surgical repair of Achilles tendon ruptures has been suggested to improve the clinical outcome (26,43).…”

“…The mechanisms behind the effect of mechanical stimulation are not fully understood, and the signaling pathways involved in tendon mechano-transduction in vivo are not well described. We previously studied the changes in gene expression that arise when an unloaded healing tendon is exposed to 30 min of loading (10). We used the Affymetrix microarray technique and studied the expression pattern at 3, 12, 24, and 48 h after one loading episode.…”

Inflammation and tendon healing

“…Although the outcome of loading at different phases of healing was the same, different mechanisms could be involved. In the same tendon healing model, it was shown that mechanical loading regulates inflammation-related genes during the inflammatory phase of tendon healing [2][3][4] . However, the possible influence of mechanical loading on magnitude and polarization of immune cells during the course of tendon healing needs to be further investigated.…”

“…Ruptured tendons heal slowly, and management of tendon injuries is challenging. Mechanical loading improves healing of tendons and has been shown to modulate inflammation-related genes, which points to the important role of inflammation during tendon healing [2][3][4] . There are other factors affecting tendon healing, like nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids.…”