We have developed a biomimetic scaffold for tissue engineering using bovine collagen with different topographic characteristics, using matrices with random or parallel-arranged collagen fibres. Scaffolds were characterized by SEM, water contact angle and mechanical test. Biocompatiblitiy studies were performed with MC3T3E1 pre-osteoblasts, and adhesion, proliferation, differentiation and mineralization were assessed. Random film showed a smooth surface, while the ordered membrane revealed a grooved structure with channels separated 265 m from each other. Water contact angle was dependent on the direction of observation and the mechanical evaluation showed a lower resistance to traction and a greater ductility for the ordered membrane. Adhesion, proliferation, alkaline phosphatase activity and mineralization were significantly improved when cells were grown on the ordered-collagen matrix. There was no significant increase in pro-inflammatory cytokine release from cells grown on both random and ordered-collagen films. This material could be useful for bone tissue regeneration.