Preparation of NAg-CCS

Bovine type-Ι collagen and chitosan at a mass ratio of 9:1 were dissolved in 0.5 M of acetic acid solution to form a 0.5% (w/v) solution, and was kept at 4 °C until use. The nanosilver solution was added to the collagen/chitosan hybrid scaffold (CCS) hydrogel to achieve different concentrations (0, 2, 5, 10 and 20 ppm) of the NAg-CCS solution. In total, 3.2 ml of NAg-CCS was carefully poured on a flat plate (homemade model, 4.0 × 4.0 × 0.2 cm). The composite was incubated at 4 °C for 24 hours, frozen at −25 °C for three hours, and lyophilized for 24 hours to produce a hybrid scaffold. The porous CCS without the nanosilver was prepared and treated as a control, as previously described. All samples of CCS and NAg-CCS were cross-linked with EDAC/NHS, as previously described⁷. Before implantation in the animal experiment, the scaffolds were irradiated with a γ-ray (25 KGy) for 24 hours.