- Pre-vascularization is important for the reconstruction of dense and metabolically active myocardial tissue and its integration with the host myocardium after implantation. Herein, we demonstrate that the fabrication of micro-channels in alginate scaffold combined with the presentation of adhesion peptides and an angiogenic growth factor promote vessel-like networks in the construct, both in vitro and in vivo. Using a CO2?laser engraving system, 200??m diameter channels were formed from top to bottom of the 2?mm thick alginate scaffold, with a channel-to-channel distance of 400??m. Cells were seeded in a sequential manner onto the scaffolds: first, human umbilical vascular endothelial cells (HUVECs) were seeded and cultured for three days, then neonatal rat cardiomyocytes (CMs) and cardiofibroblasts were added at a final cell ratio of 50:35:15, respectively, and the constructs were cultivated for an additional seven days. A vessel-like network was formed within the cell constructs, wherein HUVECs were organized around the channels in a multilayer manner, while the CMs were located in-between the channels and exhibited the characteristic morphological features of a mature cardiac fiber. Acellular scaffolds with the affinity-bound basic fibroblast growth factor were implanted subcutaneously in mice. Increased cell penetration into the channeled scaffold and greater vessel density were found in comparison with the nonchanneled scaffolds. Our results thus point to the importance of micro-channels as a major structural promoter of vascularization in scaffolds, in conjunction with the sequential preculture of ECs and angiogenic factor presentation.