Collagen / chitosan hybrid sponge as a scaffold for cell culture

Abstract

The aim of this study is to obtain a three-dimensional collagen type I/chitosan scaffold for seeding the cells cultured in vitro, and promotion of cell adhesion and proliferation. Materials and methods used to obtain a collagen I/chitosan hybrid scaffold were bovine tendons that after mincing have been processed with 0,05M Na2 HPO4 solution for 4 days, followed by enzymatic digestion with pepsin 100 mg per 1gr. of tendon, EDTA and acetic acid for 24 hours at 4°C. Then collagen was purified by precipitation with 1.8 M NaCl, followed by acetic acid dissolution and dialysis in bags with 12000 Da pore size by a large volume of 0.02 M Na2HPO4 solution, until pH of collagen solution become neutral or weak base, then it was frozen at -60 °C and allowed to thaw at room temperature. Collagen is separated from the remaining liquid by centrifugation at 1000 g for 10 min. The obtained collagen is dissolved with acetic acid to a concentration of 1%, then freeze-dried (EVD-12; Unicryo MCL-60). Obtained sponge was treated with 0.25% chitosan solution for 24 hours, then washed with distilled water on a vibrator, frequently changing the water. After that the collagen/chitosan sponge is freeze-dried and cross-linked at room temperature in a vapor chamber with 12.5% glutaraldehyde (SERVA) for 24 hours. Results: Pore size in native collagen sponge vary between 50 and 200μ, but in the case of hybrid collagen/chitosan sponge, pore size vary between 30 and 100μ. Conclusion. The obtaining method of a hybrid collagen/chitosan scaffold for cell seeding is effective. The sponge size and microscopic structure allow its utilisation in filling tissue defects and tissue engineering.