Fabrication of Engineered Tendon Extracellular Matrix

A typical process for the fabrication of the tECM-DTSs is presented in Fig. 1. First, the DTSs substrate was fabricated using our previously published protocol [29 (link)]. In short, the Achilles tendons of adult beagle dogs were decellularized through the following procedures: repetitive freeze/thaw treatment, frozen section with a thickness of 300 μm and nuclease treatment (including DNase 150 IU/ml and RNase 100 μg/ml) for 12 h at 37°C. Following washing in 50 ml of 0.1 M PBS (3 × 30 min), the DTSs were lyophilized and sterilized with ethylene oxide (EO). Then, TDSCs were seeded on the top surface of DTSs substrate at 1 × 10⁵ cells per cm² and cultured in complete medium supplemented with 20% fetal bovine serum (FBS). After reaching 90% confluence, 50 μM of L-ascorbic acid phosphate (Sigma) was added for additional culture period of 8 days. At the end of 15-day culture period, the composites of TDSCs-DTSs were re-decellularized as described previously with minor alteration [15 (link)], using 0.5% Triton X-100 supplemented with 20 mM ammonium hydroxide (NH₄OH) at 37°C for 15 min, followed by 100 U/ml DNase I at 37°C for 2 h. Finally, the tECM modified DTSs (hereafter referred to as tECM-DTSs) were washed in 50 ml of 0.1 M PBS (6 × 30 min), frozen at –80°C or lyophilized and sterilized by EO for subsequent use.