Carboxymethylation of Silk Fibroin and Injectable Microgel Assembly Prepared from Thereof

Silk fibroin (SF) from silkworm cocoons is a fibrous protein and found its application in various fields such as tissue engineering, drug delivery, soft electronics, etc., owing to its biocompatibility. SF can be fabricated into various different forms, such as films, macro-to-nano fibers, scaffolds, and hydrogels. Among these forms, SF hydrogel can be made by physical and chemical crosslinking. Physical crosslinking of SF is based on the formation of β-sheet structure, which results in a brittle mechanical behavior of the hydrogel. On the other hand, chemical crosslinking of SF through enzymatic crosslink or photo-crosslink results in elastic SF hydrogel. In 3D printing, various kinds of natural polymers are used as ink, and, in many cases, 3D constructs are formed upon the gelation of the polymer right after its extrusion. SF was also considered as a candidate natural polymer in 3D printing, but the inherent low viscosity of SF aqueous solution and its slow gelation kinetic were the technical hurdles. Therefore, blending with other 3D printable polymers or introducing photo-crosslinkable functional groups were suggested as a solution for SF. Meanwhile, microgel assembly has been applied in the 3D printing field. Microgels can be assembled into a macro-sized hydrogel through various interactions between the microgel particles. If the microgel assembly was formed by a reversible dynamic bonding, such microgel assembly can flow upon shear stress and recover back into a viscoelastic solid state when the stress is removed.<br/>The aim of this study is to prepare a suitable SF microgel assembly for 3D printing. Many previous studies have been reported on preparing SF microgels, but they had to dissolve the SF fiber. However, large amounts of chemicals are required during this dissolution, and time-consuming dialysis should be followed. We have skipped this step by conducting direct modification of SF in its fiber state. Carboxymethylation was performed directly on the SF fiber, which resulted in a rod-like microgel. The carboxymethylation has been done primarily on the tyrosine of SF. The negative charges of carboxymethyl groups allowed high water uptake and swelling by the repulsive forces, but the dissolution was prevented by the intact β-sheet structure working as a physical crosslinker. By controlling the concentration and the degree of carboxymethylation of SF microgel, optimum conditions for microgel assembly formation were established. Rheological studies confirmed the injectability of SF microgel assembly, in which gel-like behavior under a low shear rate but flowability under a high shear rate. Finally, we have successfully built a standalone 3D construct of SF microgel assembly by extrusion 3D printer. The prepared SF microgel would have high potential in tissue engineering, and enhancing the structural stability of the 3D construct of SF microgel is currently underway.