Sponge-like matrices with a specific 3D structural design resembling the actual extracellular matrix (ECM) of a particular tissue show significant potential for the regeneration and repair of a broad range of damaged anisotropic tissues. The manipulation of the structure of collagen scaffolds using a freeze-drying technique was explored in this work as an intrinsically biocompatible way of tailoring the inner architecture of the scaffold. The research was focused on the influence of temperature gradients, imposed during the phase of crystallisation of collagen suspensions, upon the degree of anisotropy in the microstructures of the scaffolds produced. Moulding technology was employed to achieve differences in heat transfer rates during the freezing processes. For this purpose various moulds with different configurations were developed with a view to producing uni-axial and multidirectional temperature gradients across the sample during this process. SEM analysis of different cross-sections (longitudinal and horizontal) of scaffolds revealed that highly aligned matrices with axially directed pore architecture were obtained where single unidirectional temperature gradient was induced. Alteration of freezing conditions by the introduction of multi-temperature gradients allowed collagen scaffolds to be produced with complex pore orientations, and anisotropy in pore size and alignment.