A possible link between loading, inflammation and healing: Immune cell populations during tendon healing in the rat

Abstract

Loading influences tendon healing, and so does inflammation. We hypothesized that the two are connected. 48 rats underwent Achilles tendon transection. Half of the rats received Botox injections into calf muscles to reduce mechanical loading. Cells from the regenerating tissue were analyzed by flow cytometry. In the loaded group, the regenerating tissue contained 83% leukocytes (CD45+) day 1, and 23% day 10. The M1/M2 macrophage ratio (CCR7/CD206) peaked at day 3, while T helper (CD3+CD4+) and Treg cells (CD25+ Foxp3+) increased over time. With Botox, markers associated with down-regulation of inflammation were more common day 5 (CD163, CD206, CD25, Foxp3), and M1 or M2 macrophages and Treg cells were virtually absent day 10, while still present with full loading. The primary variable, CCR7/CD206 ratio day 5, was higher with full loading (p = 0.001) and the Treg cell fraction was lower (p < 0.001). Free cage activity loading is known to increase size and strength of the tendon in this model compared to Botox. Loading now appeared to delay the switch to an M2 type of inflammation with more Treg cells. It seems a prolonged M1 phase due to loading might make the tendon regenerate bigger.

Cox-2 inhibition and the composition of inflammatory cell populations during early and mid-time tendon healing

“…In this model, mechanical loading or microtrauma by needling both regulate the expression of genes related to inflammation. The composition of leukocyte subpopulations, as studied by flow cytometry, was found to be remarkably similar between similarly treated animals, but highly variable over time and sensitive to degrees of mechanical loading 2 . This suggests that most factors that influence healing exert their effects primarily via an influence on inflammation.…”

“…Single cells were washed, and red blood cell was removed using ACK lysis buffer (155 mM NH3Cl, 10 mM KHCO3, and 88 μM EDTA). Trypan blue (Life technologies) was used to count live cells 2,7,8 .…”

“…In a rat Achilles tendon transection model, the inflammation resolves between 5 and 10 days, as long as the tissue is at least partially protected from mechanical loading. Full loading prolongs the inflammatory phase of healing, increases strength and leads to dramatic changes in the composition of the leukocyte population 2 . We have used a standardized rat Achilles tendon healing model in a series of previous studies related to inflammation [2][3][4] .…”

“…Full loading prolongs the inflammatory phase of healing, increases strength and leads to dramatic changes in the composition of the leukocyte population 2 . We have used a standardized rat Achilles tendon healing model in a series of previous studies related to inflammation [2][3][4] . In this model, mechanical loading or microtrauma by needling both regulate the expression of genes related to inflammation.…”

Inflammation and tendon healing

“…During early tendon healing, the newly formed tissue is composed of a great percentage of leukocytes (CD45) 67 . The leukocyte subpopulation composition was greatly influenced by mechanical loading in study I.…”