Cross-Linking Electrospun Type II Collagen Tissue Engineering Scaffolds with Carbodiimide in Ethanol

Abstract

In trying to assess the structural integrity of electrospun type II collagen scaffolds, a modified but new technique for cross-linking collagen has been developed. Carbodiimides have been previously used to cross-link collagen in gels and in lyophilized native tissue specimens but had not been used for electrospun mats until recently. This cross-linking agent, and in particular 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), is of extreme interest, especially for tissue-engineered scaffolds composed specifically of native polymers (e.g., collagen), because it is a zero-length cross-linking agent that has not been shown to cause any cytotoxic reactions. The unique aspect of the cross-linking protocol in this study involves the use of ethanol as the solvent for the cross-linking agent, because the pure collagen electrospun mats immediately disintegrate when placed in an aqueous solution. This study examines 2 concentrations of EDC with and without the addition of N-hydroxysuccinimide to the reaction (which has been shown to result in higher cross-linking yields in aqueous solutions) to test the hypothesis that the use of EDC in a nonaqueous solution will cross-link electrospun type II collagen fibrous matrices in a comparable manner to typical glutaraldehyde fixation protocols. The use of EDC is compared with the cross-linking effects of glutaraldehyde via mechanical testing (uniaxial tensile testing) and biochemical testing (analysis of the percentage of free amino groups). The stress-strain curves of the cross-linked samples demonstrated uniaxial tensile behavior more characteristic of native tissue than do the dry, untreated samples. The heated, 50% glutaraldehyde cross-linking protocol resulted in a mean peak stress of 0.76 MPa, a mean strain at break of 127.30%, and a mean tangential modulus of 0.89 MPa; mean values for the samples treated with the EDC protocols ranged from 0.35 to 0.60 MPa for peak stress, from 111.83 to 159.23% for strain at break, and from 0.57 to 0.92 MPa for tangential modulus. Low and high concentrations (20 mM and 200 mM, respectively) of EDC alone were comparable in extent of cross-linking (29% and 29%, respectively) to the heated 50% glutaraldehyde cross-linking protocol (30% cross-linked).