Design a hydrogel scaffold to guide axonal growth in 3-D

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The spatial arrangement of biomolecules plays a critical role in manipulating cell motility and function. In designing a synthetic analog for nerve tissue regeneration, we believe that the analog's 3-dimensional character must be considered. Therefore, we first attempt to create a 3-D patterned matrix with alternating cell permissive/non-permissive regions to achieve axonal guidance in vitro. In order to achieve this, chitosan based hydrogel will be initially modified by cell non-permissive polyethylene glycol (PEG) via photo-labile nitrobenzyl groups, to render the hydrogel matrix cell non-permissive. The cell non-permissive PEG will then be selectively removed in selected regions using photochemistry and replaced by cell permissive peptides. Focused two-photon laser will be used to activate the photoreaction. Due to the quantum characteristics of photons, the wavelength of the two-photon laser is double that of the conventional single photon laser to activate the same photo-reaction. This red-shift of wavelength makes the process more adaptable for peptide immobilization. The study will be further advanced to create a 3-D patterned matrix with alternating cell permissive/non-permissive regions in combination with a concentration gradient of biomolecules to provide better nerve guidance in 3-D. It has long been speculated that a bio-mimetic device incorporating both haptotactic cues (alternating cell permissive and non-permissive regions) and chemotactic cues (concentration gradient of biomolecules) will promote and guide axonal growth after spinal cord injuries; however the means to present both cues in 3-D has been elusive. In this study, we aim to better delineate the interaction of cells and materials in vitro in 3-D, in an attempt to lay the foundation for the design of a device for implantation. The proposed project is novel in that this is the first study to incorporate both haptotactic cues and chemotactic cues to guide axonal growth within a 3-D hydrogel construct. Ultimately, we aim to overcome spinal cord injuries and anticipate that the fundamental knowledge created herein will lead to strategies to overcome other central nervous system disorders, such as brain injuries, where regeneration is critical.