Bone tissue engineering is a promising solution for advanced bone defects reconstruction after severe trauma. In bone tissue engineering, scaffold in three-dimensional (3D) structure is a crucial component for cells growth, migration and infiltration. 3D printing technique is well suited to manufacture scaffold, since the technique can fabricate scaffolds in highly complex design under well internal structural control. In current study, 3D printing technique was utilized to produce polylactic acid (PLA) scaffolds. BMSCs were seeded onto selected scaffold with either hydrogel mixed or not and cultivated in vitro to investigate the osteogenic potential in each group. After osteogenic incubation in vitro, BMSC-seeded scaffolds were implanted onto rat cranium defects and observed bone regeneration after 12 weeks of implantation. Our results demonstrated that BMSCs were able to seed onto 3D printed PLA scaffolds under high resolution observation. Re-al-time PCR analysis showed their osteogenic ability, which could be further improved after BMSCs mixing with hydrogel. In vivo study, it showed significantly increase bone regeneration when rats’ cranium defects implanted with hydrogel mixed BMSC-seeded scaffold than other control and those without cell or hydrogel groups. This study showed that 3D printed PLA scaffold is a feasible option for BMSCs cultivation and osteogenic differentiation. After mixing with hydrogel, BMSC-seeded 3D printed scaffold can facilitate bone regeneration.