*3.1. Raw Materials*

Nile tilapia skins were procured from Zhenhua Aquatic Product Company (Guangzhou, Guangdong province of China). Frozen skins were thawed with running water and removed from the residuals manually. They were chopped into small pieces and stored at −20 ◦C. Porcine collagen (PC) was obtained from Kele Biotech (Chengdu, China). All of the other reagents used were of analytical grade.

#### *3.2. Preparation of ASC*

All the procedures were carried out at 4 ◦C to minimize collagen denaturation. An acellular environment was used in the extraction process to reduce the exogenous pyrogen. The pieces of Nile tilapia skins were soaked with 0.1 M of NaOH for 24 h with continuous stirring to remove the non-collagenous proteins. Washing the fish skins repeatedly with cooled deionized water ensured that they were neutralized. Adding 0.5 M of acetic acid in a ratio of 1:50 (*w*/*v*) started extraction for two days. Then, they were centrifuged at 10,000 rpm for 30 min at 4 ◦C. NaCl was added to the collected supernatants until a concentration of 0.9 M was reached to salt out the collagen. The precipitated collagen was separated by centrifugation at 10,000 rpm for 30 min at 4 ◦C and dissolved in 0.5 M of acetic acid. Then, the solution was dialyzed for 24 h against 0.1 M of acetic acid in a dialysis membrane with a molecular weight cut-off of 50 kDa, and then for 48 h against ultra-pure water; the water was changed every eight hours. The resulting collagen was freeze-dried for three days and sealed in polythene bags until further use.
