Collagen scaffolds provide a promising platform for tissue regeneration due to their ability to support cell attachment, proliferation and differentiation. In this study, we used porous freeze-dried collagen scaffolds with either uniaxially-aligned or randomly-oriented pores to investigate the formation of vascular structures in vitro. We characterised the scaffold pore structure, specific permeability, cell migration and endothelial cell self-assembly into vessel-like networks in mono- and co-culture with osteoblasts. Our work shows that improved pre-vascularisation can be achieved in uniaxially-aligned scaffolds and in a co-culture environment.