In each organ the extracellular matrix has a specific architecture and composition, adapted to the functional needs of that organ. As cells are known to respond to matrix organization, biomaterials that take into account the specific architecture of the tissues to be regenerated may have an advantage in regenerative medicine. In this study we focussed on the diaphragm, an organ essential for breathing, and consisting of radial oriented skeletal muscle fibres diverging from a central tendon plate. To mimic this structure dual layered collagenous scaffolds were constructed with a radial pore orientation, prepared by inward out freezing, and reinforced by a layer of compressed collagen. Similar scaffolds with a random round pore structure were taken as controls. Scaffolds were first mildly crosslinked by formaldehyde vapour fixation for initial stabilization (13% and 17% crosslinking for the radial and control scaffolds, respectively), and further crosslinked using aqueous 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (38% and 37% crosslinking, respectively). Scaffolds were implanted into a surgically created diaphragm defect in rats and explanted after 12weeks. Macroscopically, integration of the radial scaffolds with the surrounding diaphragm was better compared with the controls. Cells had infiltrated further into the centre of the scaffolds (P=0.029) and there was a tendency of blood vessels to migrate deeper into the radial scaffolds (P=0.057, compared with controls). Elongated cells (SMA-positive) were aligned with the radial structures. In conclusion, collagenous scaffolds with a stable radial pore structure can be constructed which facilitate cellular in-growth and alignment in vivo.