Synthesis of Enzyme-Degradable, Peptide-Cross-Linked Dextran Hydrogels Ste ´phane G. Le ´vesque ²,§ and Molly S. Shoichet ²,‡,§, * Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Department of Chemistry, University of Toronto, 80 St. George St., Toronto, Ontario M5S 3H6, and Institute of Biomaterials and Biomedical Engineering, 164 College Street, Toronto, Ontario M5S 3G9, University of Toronto . Received July 12, 2006; Revised Manuscript Received December 20, 2006 Hydrogels derived from synthetic polymers have been previously engineered to degrade under the activity of matrix metalloproteinases (MMPs). It is believed that these systems can act as extracellular-matrix (ECM) equivalents mimicking the degradation and remodeling of the ECM through the activity of cell-secreted enzymes. In this study, MMP-sensitive hydrogels derived from dextran were developed. In order to avoid the incorporation of hydrolyzable esters often introduced in dextran modification strategies, the polysaccharide was modified with p-maleimidophenyl isocyanate (PMPI) thereby introducing maleimide functionalities in the backbone and resulting in dextran derivatized with p-maleimidophenyl isocyanate (Dex-PMPI). This strategy was favored to separate out the effects of random hydrolysis and enzymatic digestion in the degradation of the dextran hydrogels. A peptide cross-linker, derived from collagen and susceptible to gelatinase A (MMP-2) digestion, was synthesized with bifunctional cysteine termini and used to cross-link the Dex-PMPI. These hydrogels were found to be hydrolytically stable for more than 200 days yet degraded either within 30 h when exposed to bacterial collagenase or within 16 days when exposed to human MMP-2, demonstrating enzymatic-mediated digestion of the peptide cross- links. Further modification of the cross-linked hydrogels with laminin-derived peptides enhanced cell adhesion and survival, demonstrating the potential of these materials for use in tissue engineering applications. INTRODUCTION One aspect of tissue engineering is the development of multicomponent scaffolds that can elicit specific biological functions. Ideally, these scaffolds would act as extracellular- matrix (ECM) equivalents, mimicking the cellular environment as closely as possible. The natural ECM is not only a physical support for the cells but also plays a key role in signal transduction by presenting adhesion molecules and serving as a reservoir for other molecules, such as cytokines, that influence growth and cell function. The dynamic interactions between cell and ECM have been mimicked in scaffold design where both physical and chemical stimuli have been incorporated to guide tissue regeneration both spatially and temporally (1-4); how- ever, these scaffolds have not been optimized for nerve repair strategies. Most biodegradable synthetic polymers studied for tissue- engineering applications, such as polycaprolactone, polylactide, polyglycolide, and poly(lactide-co-glycolide), rely on random ester hydrolysis of the backbone chain instead of tailoring the degradation to specific cellular activity. Over the years, advances in molecular biology have provided a better understanding of the cellular environment which has been used to design scaffolds that mimic the degradation and the remodeling of the ECM. In this environment, growth, repair, and development are controlled by cell-secreted and cell-activated enzymes, such as matrix metalloproteinases (MMPs) and plasmin. These processes are regulated through enzymatic degradation and de noVo synthesis of ECM components, making the degradation of this natural support dynamic. Due to their involvement in tissue remodeling, the activity of these enzymes is highly localized in the cellular periphery and is tightly regulated. MMPs, which are calcium-requiring and zinc-dependent endopeptidases, constitute one of the major families of protein- ases playing key roles in the responses of cells to their environment (5). They have the ability to hydrolyze one or several components of the ECM, as well as nonmatrix proteins thereby influencing cell migration, proliferation, differentiation, and death by both modifying the cellular microenvironment and regulating the activity of biological molecules. MMPs are secreted as inactive zymogens, and their activity is highly regulated at the transcriptional and posttranscriptional level. Expressed during development (6, 7), most MMPs have been found to be produced at very low or undetectable levels in the adult central nervous system (CNS); however, they are up- regulated following spinal cord injury (8, 9) and may play a beneficial role in CNS repair strategies (5, 9). MMPs are expressed in the growth cones of numerous vertebrate neurons (10-12) and may regulate axonal guidance (13-15). Muir and co-workers previously reported that MMP-2 activity facilitated neurite extension of dorsal root ganglia (DRG) neurons within a reconstituted ECM (11) and promoted axonal growth by degrading inhibitory chondroitin sulfate proteoglycans (15). MMP-2 is therefore highly relevant to the enzymatic degradation of ECM analogues designed for neuronal applications. Hubbell and West introduced the concept of mimicking the dynamic remodeling of the ECM through the development of telechelic peptide-poly(ethylene glycol) (PEG)-peptide block copolymers which were degraded either by plasmin or bacterial collagenase (16). Different hydrogels sensitive to proteases such as MMPs (3, 4, 17) and plasmins (17, 18) have been developed for various tissue engineering applications, mimicking some fundamental aspects of cell-ECM interactions by taking advantage of the time- and location-dependent ECM degradation activity exhibited during cellular outgrowth. * To whom correspondence should be addressed: Molly Shoichet, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College St., Rm. 514, Toronto, Ontario, M5S 3E1. Phone: 416-978-1460; fax: 416-9784317; e-mail: molly@ecf.utoronto.ca. ² Department of Chemical Engineering and Applied Chemistry. ‡ Department of Chemistry. § Institute of Biomaterials and Biomedical Engineering. 874 Bioconjugate Chem. 2007, 18, 874-885 10.1021/bc0602127 CCC: $37.00 © 2007 American Chemical Society Published on Web 04/03/2007