Ethanol dehydration is a common step in both scaffold manufacturing and tissue processing, yet the influence of ethanol on collagen is not well understood. This study examined the effects of dehydration, via ethanol treatment and air drying, on collagen structure, behavior, mechanics, and rehydration capacity. Multiple material characterization methods were used including Fourier Transform infrared spectroscopy (FTIR), Raman spectroscopy, scanning electron microscopy, thermogravimetric analysis, small/medium angle x-ray scattering, volumetric swelling analysis, and tensile testing. Ethanol dehydration removed bulk water from scaffolds, making them stronger and stiffer, but also showed loss of molecular water. This molecular water appears to act as a collagen stabilizer, resulting in less thermally stable scaffolds. The loss of molecular water is also evident in the molecular d-spacing. Secondary structure of scaffolds was also altered by ethanol, resulting in significantly enhanced rehydration capacity. Bulk water, both before and after rehydration, largely determined mechanical properties, which did not correlate with other structural measures such as FTIR. While rehydration largely returned collagen spacing to pre-ethanol treated state, structural alterations seen in FTIR cannot be recovered. These results have implications for not only collagen scaffolds, but in many tissue engineering and processing applications.
Arnold et al. (Sun,) studied this question.