Soft tissues such as the anterior cruciate ligament (ACL) integrate with subchondral bone through a multi-tissue interface, which functions to minimize the formation of stress concentrations and enable load transfer between two distinct types of tissue. Currently, the inability of ACL reconstruction grafts to regenerate this critical interface compromises their long term clinical outcome. To address the challenge of biological graft fixation, the objective of this thesis is to design a biomimetic scaffold that is able to promote the regeneration of the multi-cell, multi-tissue ACL-bone interface. First, scaffold design parameters were obtained from characterization of the matrix distribution and mechanical response of the native ACL-bone interface using functional imaging methods. A triphasic scaffold based on the inherent matrix organization of the interface was then fabricated, characterized and evaluated in vitro and in vivo. The scaffold was subsequently optimized based on these results. Formation of the multitissue interface was facilitated by exercising spatial control over the distribution of interface-relevant cell populations such as fibroblasts, chondrocytes and osteoblasts on the scaffold. Specifically, 3-D co-culture and tri-culture of these cell types on the stratified scaffold led to phase-specific cell distribution, which resulted in cell-specific matrix deposition on each scaffold phase. Tri-culture led to the formation of distinct yet continuous tissue regions mimicking those of the ACL-bone interface. The results of this thesis demonstrate the potential of the novel stratified scaffold for interface regeneration and biological fixation of soft tissue grafts. The biomimetic multi-phasic scaffold design and tri-culture methods developed here represent innovative departures from traditional tissue engineering approaches, which have typically focused on the formation of a single tissue type on a scaffold with uniform properties. The findings described in this thesis will lay the foundation for the development of a new generation of fixation devices, while providing fundamental insight into interface regeneration and the development of complex tissue systems with the potential to expedite the clinical translation of tissue engineered technologies.