The in vitro potential of a synthetic matrix metalloproteinase (MMP)-responsive poly(ethylene glycol) (PEG)-based hydrogel as a bioactive co-encapsulation system for vascular cells and a small bioactive peptide, thymosin beta 4 (T beta 4), was examined. We show that the physical incorporation of T beta 4 in this bioactive matrix creates a three-dimensional (3D) environment conducive for human umbilical vein endothelial cell (HUVEC) adhesion, survival, migration and organization. Gels with entrapped T beta 4 increased the survival of HUVEC compared to gels without T beta 4, and significantly up-regulated the endothelial genes vascular endothelial-cadherin and angiopoietin-2, whereas von Willebrand factor was significantly down-regulated. Incorporation of T beta 4 significantly increased MMP-2 and MMP-9 secretion of encapsulated HUVEC. The gel acts as a controlled T beta 4-release system, as MMP-2 and MMP-9 enzymes trigger the release. In addition, T beta 4 facilitated HUVEC attachment and induced vascular-like network formation upon the PEG-hydrogels. These MMP-responsive PEG-hydrogels may thus serve as controlled co-encapsulation system of vascular cells and bioactive factors for in situ regeneration of ischemic tissues (c) 2009 Elsevier Ltd. All rights reserved