Networks of nanofibers are ubiquitous in biological systems:​ the cellular cytoskeleton is composed of actin fibers, microtubules, and intermediate filaments, while the extracellular matrix (ECM) is made up of collagen, elastin, and fibronectin fibers. The filamentous architecture in these systems impacts biological processes and imparts the ECM and cytoskeleton with unique nonlinear mechanical properties. Nanofibrillar hydrogels that recapitulate the structure and nonlinear mechanical properties of these biological nanofiber networks have tremendous potential for applications in tissue engineering and three-dimensional cell culture. My thesis is focused on using rod-shaped cellulose nanocrystal (CNCs) as building blocks for nanofibrillar hydrogels. I first present two approaches for preparing CNC-based hydrogels:​ (1) by grafting thermoresponsive gelling polymers to the CNC surface, or (2) by mixing aldehyde functionalized CNCs with a crosslinking polymer (gelatin). The second approach, CNCs crosslinked with gelatin, produced a biocompatible fibrillar hydrogel named EKGel that had a precisely tunable structure, as well as a scalable and simple synthesis. Furthermore, I demonstrated that, because of its filamentous architecture, EKGel recapitulates the unique nonlinear mechanical properties of biological nanofiber networks.

After establishing its ability to recapitulate the structure and properties of biological nanofiber networks, I explored several potential applications of EKGel. For example, through a microextrusion 3D-printing approach, EKGel can be used to prepare patterned structurally anisotropic hydrogel sheets for tissue engineering. Additionally, by modulating the composition of EKGel I was able to mimic the dynamic structural changes that occur in the ECM during tissue fibrosis, making EKGel an excellent matrix for in-vitro studies of fibrotic disease. I also describe the application of EKGel for the culture of patient-derived breast tumor organoids and demonstrate that EKGel is an improved alternative to the current gold-standard, BME. Finally, I describe the incorporation of EKGel into a microfluidic tumor spheroids-on-a-chip platform for drug screening.