Objective: Osteochondral interface regeneration is challenging for functional and integrated cartilage repair.A variety of layered scaffolds have been used to reconstruct the complex interface.However, the permeability of the layered structure, which determines the ease of molecules penetrating through matrix, has not been widely considered in the scaffold designing.In this study, a novel bilayered scaffold with low permeability was developed and the effects of various permeability were evaluated through in vitro and in vivo assessment.Methods: Bilayered scaffolds were fabricated using articular cartilage extracellular matrix (ACECM) and hydroxyapatite (HAp), involving a porous, oriented upper layer and a dense, mineralized lower layer.Various porosity distribution and low permeability was achieved in the layers by adjustment of content of HAp and ECM.Biomechanical properties were also evaluated.The osteochondral defects were created in the trochlear groove on the unilateral femur of New Zealand white rabbits.After operation 1, 4, 12, 24 weeks, the specimens were evaluated mechanically, biochemically, and histologically.Results: Morphological observations demonstrated that a gradual interfacial region was formed with pore size varying from 128.2±20.3 to 21.2±3.1 μm.The permeability of the bilayered scaffold decreased with increasing compressive strain and HAp content.Accordingly, the optimum HAp/ACECM ratio (7w/v %) in the layer to mimic native calcified cartilage was found.This architecture have the ability to guide cellular distribution.Chondrocytes could not penetrate the interface and resided only in the upper layer, where they showed high cellularity and abundant matrix deposition.Histological results showed that hyaline-like cartilage formed and well integrated with subchondral bone.The scaffold mechanical properties were well-suited for surgical handling, fixation, and bearing osteogenic loads during bone regeneration.Conclusion: The permeability was identified as a determining factor for chondrocytes distribution in the cartilage tissue engineering.This bilayered construct with low permeability can therefore be promising candidates for osteochondral defect regeneration.