Different gene response to mechanical loading during early and late phases of rat Achilles tendon healing

Abstract

Mechanical loading stimulates tendon healing both when applied in the inflammatory phase and in the early remodeling phase of the process, although not necessarily via the same mechanisms. We investigated the gene response to mechanical loading in these two phases of tendon healing. The right Achilles tendon in rats was transected, and the hindlimbs were unloaded by tail suspension. The rats were exposed to 5 min of treadmill running 3 or 14 days after tendon transection. Thereafter, they were resuspended for 15 min or 3 h until euthanasia. The controls were suspended continuously. Gene analysis was first performed by microarray analysis followed by quantitative RT-PCR on selected genes, focusing on inflammation. Fifteen minutes after loading, the most important genes seemed to be the transcription factors EGR1 and C-FOS, regardless of healing phase. These transcription factors might promote tendon cell proliferation and differentiation, stimulate collagen production, and regulate inflammation. Three hours after loading on , inflammation was strongly affected. Seven inflammation-related genes were upregulated according to PCR: CCL20, CCL7, IL-6, NFIL3, PTX3, SOCS1, and TLR2. These genes can be connected to macrophages, T cells, and recruitment of leukocytes. According to Ingenuity Pathway Analysis, the recruitment of leukocytes was increased by loading on, which also was confirmed by histology. This inflammation-related gene response was not seen on Our results suggest that the immediate gene response after mechanical loading is similar in the early and late phases of healing but the late gene response is different. This study investigates the direct effect of mechanical loading on gene expression during different healing phases in tendon healing. One isolated episode of mechanical loading was studied in otherwise unloaded healing tendons. This enabled us to study a time sequence, i.e., which genes were the first ones to be regulated after the loading episode.

Response to mechanical loading in rat Achilles tendon healing is influenced by the microbiome

“…This might explain the tendency to stronger tendons in fully loaded contaminated rats compared to the clean (p = 0.056). The early callus is dominated by different leukocytes, and the composition of these cells undergoes dramatic change during the early healing phases in our model [8][9][10]. Full loading, in contrast to partial unloading, can result in tissue microdamage followed by a pro-inflammatory response [7].…”

“…The magnitude of loading appears to activate different mechanisms which will ultimately lead to different effects on the mechanical properties [7]. The Achilles tendon heals through callus formation and the initial tissue contains weak matrix with primarily leukocytes infiltrating this matrix [8]. Full loading, when applied to this matrix, has been shown to induce microdamage and alter the immune cell composition and ultimately promote a pro-inflammatory response [7,9].…”

“…Full loading, when applied to this matrix, has been shown to induce microdamage and alter the immune cell composition and ultimately promote a pro-inflammatory response [7,9]. Hence, in part, the response to mechanical loading seems to be closely connected to immune cell reactions and thereby possibly to changes in the microbiome [8][9][10]. We therefore hypothesized that changes in the microbiome, would also influence the response to mechanical loading.…”

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.…”