A porous microstructure of multi-layered BCP-ZrO2 bone substitutes was fabricated using the sponge replica method in which the highly interconnected structure was immobilized with collagen via ethyl(dimethylaminopropypcarbodiimide/N-hydroxysuccinimide crosslinking. Their struts are combined with a three-layered BCP/BCP-ZrO2/ZrO2 microstructure. Collagen fibers were firmly attached to the strut surface of the BCP-ZrO2 scaffolds. With control of the three-layered microstructure and collagen immobilization, the compressive strength of the scaffolds increased significantly to 6.8 MPa compared to that of the monolithic BCP scaffolds (1.3 MPa). An in vitro study using MU, confocal observation, and real-time polymer chain reaction analysis demonstrated that the proliferation and differentiation of the pre-osteoblast-like MC3T3-E1 cells was improved due to the collagen incorporation. Remarkable enhancement of bone regeneration was observed without any immunological reaction in the femurs of rabbits during 1 and 5 months of implantation. Furthermore, the interfaces between new bone and the scaffold struts bonded directly without any gaps. (C) 2015 Published by Elsevier B.V.