The outermost surface properties of silk fibroin films reflect ethanol-treatment conditions used in biomaterial preparation.

Silk fibroin has attracted interest as a biomaterial, given its many excellent properties. Cell attachment to silk substrates is usually weaker than to standard culture dishes, and cells cultured on silk films or hydrogels typically form spheroids and micro-aggregates. However, too little is known about the higher order structures and behavior of fibroin under different conditions to explain the features of silk fibroin as a culture substrate. For instance, different biomaterial surfaces, with distinct effects on cell culture, can be achieved by varying the conditions of crystallization by alcohol immersion. Here, we show that treatment of fibroin film with <80% ethanol results in a jelly-like, hydrated hydrogel as the outermost surface layer; fibroblasts preferably aggregate, rather than attach individually to such a hydrogel surface, and therefore aggregate into spheroids. In contrast, a fibroin film treated with >90% ethanol has a harder surface than the <80% ethanol-treated fibroin, to which individual cells prefer to attach (and then expand on the surface), rather than to aggregate. We discuss the influence of alcohol concentration on the surface properties, based on surface analysis of the films. The surface analysis involved assessment of static and dynamic contact angles, zeta potential, changes in crystallinity and microscopic morphology of electrospun fibers, and texture changes of the outermost surface at a nanometer-scale captured by a scanning probe microscope.