Extraction of Decellularized Bovine Tendon ECM

tECM was extracted as described previously [15 (link)]. Briefly, bovine Achilles tendons (2- to 3-month-old calves from Research 87, USA) were harvested, cryosectioned at 10 µm thickness (Cryotome, Thermo Fisher Scientific, USA) and decellularized using 1% Triton X-100 (Sigma-Aldrich, USA) and treatment with DNase (200 U/mL, Worthington, USA), and RNase (50 U/mL, Worthington). The decellularized tissue was extracted using 3 M urea (Sigma-Aldrich) at 4 °C for 3 days and then dialyzed (2 K molecular weight cutoff, Thermo Fisher Scientific) against phosphate-buffered saline (PBS, Santa Cruz Biotechnology, USA) at 4 °C for 2 days. The dialysate was then spin-concentrated using protein centrifugation tubes (3 K molecular weight cutoff, Thermo Fisher Scientific) and stored at − 80 °C for subsequent experimental use. Prior to supplementation in culture medium, the tECM solution was filter-sterilized using a 0.22-μm polyvinylidene fluoride (PVDF) syringe filter unit (Merck Millipore, USA).