A major challenge of tissue engineering is to generate materials that combine bioactivity with stability in a form that captures the robust nature of native tissues. Here we describe a procedure to fabricate a novel hybrid extracellular matrix (ECM)-synthetic scaffold biomaterial by cell-mediated deposition of ECM within an electrospun fiber mat. Synthetic polymer fiber mats were fabricated using poly(desamino tyrosyl-tyrosine carbonate) (PDTEC) co-spun with poly(ethylene glycol) (PEG) used as a sacrificial polymer. PEG removal increased the overall mat porosity and produced a mat with a layered structure that could be peeled into separate sheets of about 50 mu m in thickness. Individual layers had pore sizes and wettability that facilitated cell infiltration over the depth of the scaffold. Confocal microscopy showed the formation of a highly interpenetrated network of cells, fibronectin fibrils, and synthetic fibersmimicking a complex ECM as observed within tissues. Decellularization did not perturb the structure of the matrix or the fiber mat. The resulting hybrid ECM-scaffold promoted cell adhesion and spreading and stimulated new ECM assembly by stem cells and tumor cells. These results identify a new technique for fabricating highly porous synthetic fibrous scaffolds and an approach to supplement them with natural biomimetic cues. (C) 2017 Wiley Periodicals, Inc.