(685f) A Cell-Biomaterial Feedback Loop for Neural Tissue Engineering

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 can be both cell instructive, as well as cell responsive, thus creating a dynamic interplay between cells and their engineered extracellular matrix. Using recombinant protein technology, we have engineered a family of elastin-like, protein-based hydrogels with multiple independently tunable properties. This allows molecular-level design of our protein polymers and the incorporation of bioactive peptide sequences directly into the polymer backbone. 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. The protein-polymers are covalently crosslinked into hydrogel scaffolds with elastic moduli from 1-100 kPa. Unlike natural matrices, the concentration of adhesive ligands such as the fibronectin-derived sequence RGD, laminin-derived YIGSR, or neural cell adhesion molecule (NCAM) domain, can be precisely controlled without altering the mechanical properties of the hydrogel. Increased RGD density was found to increase the adhesion of neuronal-like PC12 cells more than 10-fold and increase neurite outgrowth nearly 4-fold.

We have designed the proteins with a second set of bioactive sequences that specifically respond to changes in cell phenotype. By incorporating cell-mediated degradable subunits and adhesive sequences into the elastin-like proteins, we are able to mimic the natural remodeling of the extracellular matrix. We have found that neural stem cells (NSCs) undergoing differentiation increase their production of the protease urokinase plasminogen activator (uPA), which has previously been found at the growth cones of extending neurites. We engineered multiple uPA degradable bioactive sites with different degradation kinetics into the elastin protein to allow neural cell-mediated control of the scaffold degradation dynamics. This 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 greater than 240 hours.

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. 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.