Guiding axonal growth in 3-D hydrogel matrices: An adhesive biochemical channel approach

Modern biomaterial design often requires incorporating spatial and temporal features into the material construct, so that cell behavior and function can be modulated for in vitro and in vivo applications. Patterning biomolecular cell responsive cues on substrates using lithographic techniques has been effective to position cell attachment and control cellular function on substrates; however, with most of the studies performed on surfaces, few have applied the strategy to three-dimensional (3-D) scaffolds for guided tissue regeneration. Given this context, the two general goals of this thesis are: (i) to develop photochemistry and photofabrication methodologies to enable the generation of isolated adhesive biochemical channels in otherwise non-adhesive 3-D hydrogel matrices and (ii) to investigate the haptotactic guidance effects of adhesion in 3-D on axonal growth.; The chemistry was devised for synthesizing an agarose polymer grafted with a caged cysteine derivative, in which the sulfhydryl was protected by a photocleavable 2-nitrobenzyl group. The resulting transparent photolabile agarose hydrogel matrices allowed the immobilization of proteins or peptides in selected volumes using light. The photofabrication of biochemical channels involves using a focused laser to release free sulfhydryl groups in 3-D hydrogel matrices in columnar shapes, and then reacting this functional template with maleimide activated oligopeptides and proteins.; Primary cell culture studies were carried out to understand the guidance effect of the adhesive biochemical channels. When chick dorsal root ganglion cells were plated on top of hydrogel matrices containing oligopeptide channels enriched with adhesive fibronectin fragment GRGDS, thick and long neurites grew into the bulk of the agarose hydrogel, accompanied by the elongated and oriented cellular assembly inside the channel. Both the neurite extension and cell migration were confined within the GRGDS geometry, exhibiting the potential for 3-D guided regeneration.; The work of this thesis offers a general way of patterning cell responsive biomolecules in transparent hydrogel matrices and confirms the adhesive biochemical channel as an appealing approach for guided nerve (and other) tissue regeneration.