Influence of Cross-Linking Method and Disinfection/Sterilization Treatment on the Structural, Biophysical, Biochemical, and Biological Properties of Collagen-Based Devices.

Disinfection/sterilization is an essential step during the manufacturing process of any implantable medical device. Cross-linking is also required for biopolymers to control resistance to degradation and enhance mechanical integrity. To date, there is still no single disinfection/sterilization treatment and cross-linking method that can be used universally for collagen-based devices. Herein, we assessed the influence of ethylene oxide, ethanol, gamma irradiation, and gas plasma disinfection/sterilization on the structural, biophysical, biochemical, and biological properties of self-assembled collagen films cross-linked with 4-arms polyethylene glycol succinimidyl glutarate and genipin. Microscopy analysis revealed that gas plasma treatment induced the most profound differences in the non-cross-linked and 4-arms polyethylene glycol succinimidyl glutarate cross-linked collagen films. Gas plasma also significantly increased the swelling ratio of the non-cross-linked and the 4-arms polyethylene glycol succinimidyl glutarate cross-linked films. Non-cross-linked and gas plasma treated 4-arms polyethylene glycol succinimidyl glutarate collagen films exhibited the lowest resistance to collagenase degradation and denaturation temperature. Between the non-cross-linked groups, the gas plasma treatment resulted in the collagen films with the lowest stress at break, strain at break, force at break, and Young's modulus values. Within the 4-arms polyethylene glycol succinimidyl glutarate groups, the ethylene oxide treatment resulted in collagen films with the lowest stress at break, strain at break, force at break, and Young's modulus values. Within the genipin groups, the gas plasma treatment resulted in collagen films with the lowest stress at break, strain at break, force at break, and Young's modulus values. Proliferation, metabolic activity, and viability of human skin fibroblasts were not affected as a function of cross-linking method and disinfection/sterilization treatment. However, proliferation, metabolic activity, and viability of THP1 cells were significantly reduced as a function of the cross-linking method, but they were not affected as a function of the disinfection/sterilization treatment. Overall, our data illustrate that the cross-linking method and the disinfection/sterilization treatment differentially affect the structural, biophysical, biochemical, and biological properties of collagen-based devices, and thus, they should be optimized according to the clinical indication.