The Impact of S‐220–Loaded Silk Hydrogels on In Vitro Neurite Outgrowth in 2 and 3 Dimensions

Silk hydrogels have attracted increasing interest as biocompatible substrates for neural cell culture and regenerative research. Recent research has shown that activation of exchange protein directly activated by cAMP 2 (Epac2) promotes neurite outgrowth in several neuronal models. Here, we investigated the effects of Bombyx mori silk hydrogel, alone or in combination with S-220, a non-hydrolysable cAMP analogue that selectively activates Epac2, on neurite outgrowth. Epac2 activation significantly and dose-dependently enhanced both the total and longest neurite length. Neurons cultured on silk hydrogel alone exhibited neurite outgrowth comparable to control neurons, indicating good biocompatibility of the hydrogel. Addition of S-220 to the culture medium with silk hydrogel modestly improved neurite morphology; however, incorporation of S-220 directly into the hydrogel did not enhance neurite outgrowth but instead reduced neurite length. A subsequently established three-dimensional cortical neuron culture model using silk hydrogel supported neurite extension and increased branching complexity beyond that observed with two-dimensional cultures, despite a shorter overall neurite length. Collectively, these findings support silk hydrogel as a permissive biomaterial substrate for in vitro cortical neuron culture. The system provides a useful platform for investigating neurite outgrowth, neuron-biomaterial interactions and the delivery of bioactive compounds in two- and three-dimensional neural cultures.