Nanoscale Structure and Property Changes in Collagen Fibrils on the Extracorporeal Shockwave Therapy Applications of the Rat Achilles Tendon Injury

摘要

Extracorporeal Shockwave therapy (ESWT) is popular in the treatment of musculo-skeletal injuries. However, the mechanism by which such therapy might have an effect on tendon injury is unknown. The Achilles tendon consists mainly of collagen Type I fibers that contain collagen fibrils. When the Achilles tendon is injured, it is inflamed. The collagenase-induced Achilles tendinitis (CIAT) model has been widely used to study tendinitis. The major advantages of atomic force microscopy (AFM) over conventional optical and electron microscopy for bioimaging include its non-requirement of a special coating and vacuum, and its capability to perform imaging in all environments. Therefore, the changes in the ultrastructure and adhesion force of the collagen fibrils on the Achilles tendons of rats with CIAT model were observed using AFM. The changes in the structure of the Achilles tendons were evaluated based on the diameter and D-banding of the collagen fibrils. CIAT was induced with the injection of 30 μl of crude collagenase into male Sprague-Dawley rats (7-week old). The animals were each sacrificed on the each at the 2th, 3th, 7th, 12th, 19th, 26th, 33th, 40th, 47th, and 63th day after the collagenase injection. The normal and injured Achilles tendons were fixed in 4% buffered formalin and dehydrated with increasing concentrations of ethanol. AFM was performed using the non-contact mode at the resolution of 512 × 512 pixels with a scan speed of 0.8 line per second. The adhesion force was measured via the force-distance curve, resulted from the interactions between the AFM tip and the collagen fibril sample using the contact mode. In intact Achilles tendon, the mean diameter, D-periodicity and adhesion force of collagen fibrils were 130.38 ± 17.40 nm, 67.81 ± 2.37 nm, and 6.48 ± 0.81 nN. CIAT rats reached 128.68 ± 15.75 nm, 72.55 ± 12.60 nm, and 5.03 ± 0.88 nN at the end of the experiment. Then, fibril diameter (138.17%) and adhesion force (187.35%) showed significant maximum increases (p < 0.0001 vs. normal) at 40 days after injection and might return to the level of the normal.