To serve as artificial matrices for therapeutic cell transplantation synthetic hydrogels must incorporate mechanisms enabling localized cell-mediated degradation that allows cell spreading and migration. responsible for this activity and the ability of optimized gel formulations to support long-term cell survival and migration. Fibroblast spreading exhibited a biphasic response to HA concentration required a minimum HA molecular weight decreased with increasing PEGdA concentration and was independent of hydrolytic degradation at early time points. Increased gel turbidity was observed in semi-IPNs but not in copolymerized hydrogels containing methacrylated HA that did not support cell spreading; suggesting an underlying mechanism of polymerization-induced phase separation resulting in HA-enriched defects within the network structure. PEGdA/HA semi-IPNs were also able to support cell spreading at relatively high levels of mechanical properties (~10 kPa elastic modulus) compared to alternative hybrid hydrogels. In order to support long-term cellular remodeling the degradation rate of the PEGdA component was optimized by preparing blends of three different PEGdA macromers with varying susceptibility to hydrolytic degradation. Optimized semi-IPN formulations supported long-term survival of encapsulated fibroblasts and sustained migration in a gel-within-gel encapsulation model. These results demonstrate that PEGdA/HA semi-IPNs provide dynamic microenvironments that can support Pseudoginsenoside-F11 3D cell survival spreading and migration for a Pseudoginsenoside-F11 variety of cell therapy applications. [9 11 Despite their success there are several limitations to peptide-based hybrid hydrogels. First oligopeptides are difficult to synthesize in large quantities and expensive while most tissue defects requiring cell-based therapy are relatively large . In addition most oligopeptides are linear sequences of amino acids only possessing primary structure resulting in reduced degradation kinetics relative to the native macromolecules from which they are derived . Consequently gel formulations that TSPAN13 support cellular activity are frequently prepared at low polymer concentrations and crosslinking densities severely limiting their mechanical properties [9 18 This has led several groups to explore screening alternative peptide sequences and strategies for increasing the number of degradable sites [10 21 Alternatively the use of intact or modified naturally-derived macromolecules to form hybrid hydrogels offers several benefits including substantially lower cost and preservation of native structure potentially supporting higher rates of enzymatic degradation and greater diversity of bioactivity . For example PEGylated fibrinogen derivatives have been used to prepare hybrid hydrogels with improved control over mechanical properties and degradation rate compared to native fibrin that have been used for orthopaedic neural and cardiovascular applications [25-29]. Hybrid hydrogels based on chemically-modified HA crosslinked with reactive PEG derivatives have been investigated as degradable adhesion barriers and vocal fold augmentation materials [30-32]. While the above studies have used co-polymer networks our group has recently investigated the possibilities of semi-interpenetrating polymer networks (semi-IPNs) composed of hydrolytically degradable PEG-diacrylates (PEGdA) and native HA [33-35]. We Pseudoginsenoside-F11 have previously shown that these hydrogels support increased cell spreading and proliferation relative to fully synthetic networks that is dependent on Pseudoginsenoside-F11 cellular hyaluronidase activity. The objective of the present study was to systematically examine the effects of PEGdA/HA semi-IPN network composition on cell spreading. 3D spreading of encapsulated fibroblasts exhibited a biphasic response Pseudoginsenoside-F11 to HA concentration required a minimum HA molecular weight decreased with increasing PEGdA concentration and was independent of hydrolytic degradation at early time points. Incorporation of native HA increased gel turbidity suggesting a potential mechanism of microphase separation resulting in HA-enriched defects in the network structure. Finally semi-IPNs with optimized PEGdA degradation rate and HA formulation supported sustained 3D cell migration in a gel-within-gel encapsulation model. 2 Materials and Methods 2.1 Synthesis of PEGdA macromers with ester linkages containing variable alkyl spacers Three different types of PEGdA macromers with varying susceptibility to hydrolytic.