Three-dimensional (3D) highly porous poly (DL-lactic-co-glycolic acid)/tricalcium phosphate (PLGA/TCP) scaffolds were fabricated using a rapid prototyping technique (RP). The biopolymer carriers (4mm×4mm×4mm) subsequently were coated with collagen type I (Col) to produce PLGA/TCP/Col composites and utilized as an extracellular matrix for a cell-based strategy of bone tissue engineering. Autologous bone marrow stromal cells (BMSCs) harvested from New Zealand white rabbits were cultured under an osteogenic condition (BMSCs-OB) followed by seeding into the structural highly porous PLGA/TCP/Col composites (i.e. PLGA/TCP/Col/BMSCs-OB). Scanning electron microscopy observation found that the RP-based scaffolds had appropriate microstructure, controlled interconnectivity and high porosity. Modification of the scaffolds with collagen type I (PLGA/TCP/Col) essentially increased the affinity of the carriers to seeding cells, and PLGA/TCP/Col composites were well biocompatible with BMSCs-OB. The PLGA/TCP/Col/BMSCs-OB constructs were then subcutaneously implanted in the back of rabbits compared to controls with autologous BMSCs suspension and carriers alone. As a result, histological new bone formation was observed only in the experimental group with PLGA/TCP/Col/BMSCs-OB constructs 8 weeks after implantation. In the control group with scaffold alone only biodegradation of the carriers was found. Therefore, these results validate our bio-manufacturing methods for a new bone graft substitute.