A number of treatments are being investigated for vocal fold (VF) scar, including designer implants. The aim of the present study was to validate a 3D model system for probing the effects of various bioactive moieties on VF fibroblast (VFF) behavior toward rational implant design. We selected poly(ethylene glycol) diacrylate (PEGDA) hydrogels as our base-scaffold due to their broadly tunable material properties. However) since cells encapsulated in PEGDA hydrogels are generally forced to take on rounded/stellate morphologies, validation of PEGDA gels as a 3D VFF model system required that the present work directly parallel previous studies involving more permissive scaffolds. We therefore chose to focus on hyaluronan (HA), a polysaccharide that has been a particular focus of the VF community. Toward this end, porcine VFFs were encapsulated in PEGDA hydrogels containing consistent levels of high M-w HA (HA(HMw)), intermediate M-w HA (HA(IMw)), or the control polysaccharide, alginate, and Cultured for 7 and 21 days. HAH(HMw) promoted sustained increases in active ERK1/2 relative to HA(IMw). Furthermore, VFFs in HA(IMw) gels displayed a more myofibroblast-like phenotype, higher elastin production, and greater protein kinase C (PkC) levels at day 21 than VFFs in HA(HMw) and alginate gels. The present results are in agreement with a previous 3D study of VFF responses to HA(IMw) relative to alginate in collagen-based scaffolds permissive of cell elongation, indicating that PEGDA hydrogels may serve as an effective 3D model system for probing at least certain aspects of VFF behavior. Biotechnol. Bioeng. 2009;104: 821-831. (C) 2009 Wiley Periodicals, Inc.