A Bioresponsive and Bioinstructive Protein-Engineered Hydrogel for 3D Cell Encapsulation and Neurite Growth

Neural regeneration within the central nervous system (CNS) is a critical unmet challenge as brain and CNS disorders continue to be the leading cause of disability nationwide. Common tissue engineering goals seek to customize cell-biomaterial interactions and guide cell behavior. Here we have developed a material that is both cell instructive and cell responsive, creating a dynamic interplay between cells and their engineered extracellular matrix (ECM). Using recombinant protein technology, we engineered a family of elastin-like protein hydrogels with multiple independently tunable properties. With these materials we can investigate individual and synergistic effects of elastic modulus, degradation rate, and cell adhesivity on cell behavior in a tunable microenvironment. Unlike natural matrices, the concentration of adhesive ligands such as the fibronectin-derived sequence RGD can be precisely controlled without altering the mechanical properties of the hydrogel. When dorsal root ganglion neurons were cultured in 3D in these gels, RGD ligands at 1.9 x 10⁷ ligands/μm³ promoted neuron-specific growth, and more than doubled the rate of neurite extension compared to hydrogels without the adhesive sequence. Crosslinking density was tuned to create scaffolds with elastic moduli from 0.5-2.1 kPa with constant adhesive site densities. The most compliant gels led to the greatest outgrowth from encapsulated DRGs with neurites extending over 1800 μm by day 7. In contrast, the stiffest gels permitted far fewer extensions and limited outgrowth to a maximum of 600 μm over the same time frame.

We have designed the proteins with a second set of bioactive sequences that specifically respond to changes in cell phenotype. Neural stem cells (NSCs) undergoing differentiation may change their production of the protease urokinase plasminogen activator (uPA), which has previously been found at the growth cones of extending neurites. By incorporating cell-mediated degradable subunits into the elastin-like proteins, we are able to mimic the natural remodeling of the ECM. We engineered multiple uPA target sites with different degradation kinetics into the elastin-like protein to allow neural cell-mediated control of the scaffold degradation dynamics. These crosslinked scaffolds are useful for directing the growth and differentiation of multiple cell types including clinically relevant NSCs. Adult murine NSCs were capable of proliferation and differentiation into neurons and glia when seeded on top of RGD-containing scaffolds.

This cell-regulated strategy was also used to enhance the functionality of the polymer by controlling delivery of multiple molecules with distinct release kinetics. One molecule was tethered to the matrix via a fast-degrading uPA-responsive sequence and fully released in 48 hours, while another molecule tethered by a more slowly degrading uPA-responsive sequence was continually released for more than 240 hours.

These tunable scaffolds are responsive to neural cells which may be able to specifically self-modulate the release of multiple bioactive factors while undergoing differentiation. This work demonstrates the versatility and responsiveness of our modularly-designed protein hydrogels for neural cell culture and encourages continued development as a biomaterial tissue construct for treating spinal cord injury.