The effect of mechanical stress on enthesis homeostasis in a rat Achilles enthesis organ culture model.

Tendons and ligaments are jointed to bones via an enthesis that is essential to the proper function of the muscular and skeletal structures. The aim of the study is to investigate the effect of mechanical stress on the enthesis. We used ex vivo models in organ cultures of rat Achilles tendons with calcaneus including the enthesis. The organ was attached to a mechanical stretching apparatus that can conduct cyclic tensile strain. We made the models of 1-mm elongation (0.5 Hz, 3% elongation), 2-mm elongation (0.5 Hz, 5% elongation), and no stress. Histological evaluation by Safranin O staining and Toluidin Blue and Picro Sirius red staining was conducted. Expression of sex-determining region Y-box 9 (Sox9), scleraxis (Scx), Runt-related transcription factor 2 (Runx2), and matrix metalloproteinase 13 (Mmp13) were examined by real-time polymerase chain reaction and immunocytochemistry. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling and live/dead staining and was conducted for evaluation of the apoptosis and cell viability. The structure of the enthesis was most maintained in the model of 1-mm elongation. The electronic microscope showed that the enthesis of the no stress model had ill-defined borders between fibrocartilage and mineralized fibrocartilage, and that calcification of mineralized fibrocartilage occurred in the model of 2-mm elongation. Sox9 and Scx was upregulated by 1-mm elongation, whereas Runx2 and Mmp13 were upregulated by 2-mm elongation. Apoptosis was inhibited by low stress. The results of this study suggested that 1-mm elongation can maintain the structure of the enthesis, while 2-mm elongation promotes degenerative changes.