We developed a new method for the preparation of mechanically strong collagen gels by combining successively basic gel formation, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking and lyophilization. Gels cross-linked three times with this method showed stronger mechanical properties (G': 3730±2060 Pa, G″: 288±35 Pa) than a conventional gel that was sequentially cross-linked with EDC once (G': 226±70 Pa, G″: 21±4.4 Pa), but not as strong as the same gel with heating for 30 min at 80°C (G': 7010±830 Pa, G″: 288±35 Pa) reported in our previous paper. The conventional collagen gel was cross-linked with EDC once, heated once, and then subjected twice to a lyophilization-gel formation-cross-linking cycle to give three-cycled gel 2. This gel had the strongest mechanical properties (G': 40,200±18,000 Pa, G″: 3090±1400 Pa, Young's modulus: 0.197±0.069 MPa) of the gels tested. These promising results suggest possible applications of the gels as scaffolds in tissue engineering research.
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