Engineered Three-Dimensional Microenvironments with Starch Nanocrystals as Cell-Instructive Materials.

Naturally, cells reside in three-dimensional (3D) microenvironments composed of biopolymers that guide cellular behavior via topographical features as well as through mechanical and biochemical cues. However, most studies describing the influence of topography on cells' behavior are performed on rigid and synthetic two-dimensional substrates. To design systems that more closely resemble native microenvironments, herein we develop 3D nanocomposite hydrogels consisting of starch nanocrystals (SNCs) embedded in a gelatin matrix. The incorporation of different concentrations of SNCs (0.05, 0.2, and 0.5 wt %) results in an increase of compressive modulus when compared to hydrogels without SNCs, without affecting the swelling ratio, thus providing a tunable system. Confirming the cytocompatibility of the novel composites, the viability of encapsulated L929 fibroblasts is >90% in all hydrogels. The cellular metabolic activity and DNA content are similar for all formulations and increase over time, indicating that the fibroblasts proliferate within the hydrogels. After 4 d of culture, Live/Dead staining and F-actin/nuclei staining show that the encapsulated fibroblasts develop an elongated morphology in the hydrogels. On the other hand, encapsulated chondrogenic progenitor ATDC5 cells also maintain a viability around 90% but display a round morphology, especially in the hydrogels with SNCs, indicating a potential application of the materials for cartilage tissue engineering. We believe that topographical and mechanical cues within 3D microenvironments can be a powerful tool to instruct cells' behavior and that the developed gelatin/SNC nanocomposite warrants further study.