2.2. Collagen-chitosan scaffold preparation

Chitosan (Sigma) was dissolved at 5 mg/mL in 2% acetic acid with mechanical stirring and then blended with 5 mg/mL rat tail collagen type I solution (BD Biosciences, Bedford, MA) at a 9:1 collagen:chitosan ratio [20, 29]. A 0.1% final concentration of glutaraldehyde, which served as a homobifunctional crosslinker, was added, along with a 10 mg/mL concentration of PLGA/PEG microspheres. The aqueous scaffold solution was covered and stirred for 3 days at 4°C. Scaffold solution aliquots were pipetted into polystyrene plates, frozen at -80°C overnight, and then subjected to lyophilization for 3 days. Scaffolds used for in vitro tests were prepared directly on 96-well cell culture plates with 100 μL of solution; in vivo scaffolds were prepared using 6-well cell culture plates with 5 mL of solution. All scaffolds were sterilized by immersion in 70% ethanol for a minimum of 12 hours and then rinsed with sterile phosphate-buffered saline (PBS) multiple times under a laminar flow hood to remove excess ethanol and neutralize pH. Scaffolds were immediately seeded following sterilization for all experiments.