The widespread clinical implementation of hamstring tendon (HT) autografts for anterior cruciate ligament (ACL) reconstruction is currently limited by the unpredictable integration of the graft with subchondral bone and a lack of devices that are capable of promoting biological fixation of HT grafts to bone. The site of HT graft fixation within the bone tunnel has been identified as the weak point in the reconstructed ACL, likely due to the failure of the graft to reestablish the physiological tendon-bone interface capable of transmitting load from the ligament to bone while minimizing stress concentration at the interface. Although a fibrovascular tissue has been shown to form at the graft-bone interface, this fibrovascular tissue is non-anatomically oriented compared to the native fibrocartilage found at direct ligament to bone insertions. Interface tissue engineering embodies a new approach for graft fixation, focusing on securing tendon grafts to bone via biological fixation wherein the complex functional interface found natively at tendon and ligament junctions with bone are regenerated at the graft insertion site into the bone tunnels. This study focuses on the in vivo evaluation of a novel biomimetic, triphasic scaffold system co-cultured with relevant cell types found at the graft-bone interface, specifically fibroblasts, chondrocytes, and osteoblasts. The scaffold is intended to promote biological fixation of HT grafts to bone by guiding the reestablishment of an anatomically-oriented and mechanically functional fibrocartilage interfacial region. It was found that the cell-seeded triphasic scaffolds supported cellular interactions as well as tissue infiltration and abundant matrix production in vivo. In addition, controlled phase-specific matrix heterogeneity was induced on the scaffold, with distinct mineral and interface-like tissue regions. The results of this study demonstrate the feasibility of multi-tissue regeneration on a single graft, as well as th- e potential of interface tissue engineering to enable the biological fixation of soft tissue grafts to bone.