One of the main issues in bone tissue engineering is to realize the response of the host to the engineered scaffolds. In this paper, the in-vivo healing of critical-sized bony defects by cell-free and stem cell-seeded 3D printed PLA scaffolds was studied in rat calvaria bone. First, the scaffolds were 3D printed based on a designed computer model and half of them were seeded by with bone marrow-derived mesenchymal stem cells (BMSCs). The SEM images of the surfaces of PLA and PLA+Cell scaffolds were taken for morphological analysis. All the scaffolds were implanted in the defect sites of rat calvaria bones and histological analysis was conducted after 8 and 12 weeks. The results showed that both cell-free and stem cell-seeded scaffolds exhibited superb healing compared with the empty defect controls. The histological observation revealed the formation of both new bone and connective tissues in the healing site after 8 and 12 weeks, postoperatively. The bone cells including osteoblasts and osteocytes with lacuna were also observed. The higher filled area and the higher bone formation and bone maturation were observed after 12 weeks and in particular for PLA+Cell scaffolds. Furthermore, the systemic toxicity evaluation of the scaffolds using ALT and AST tests reject any toxicity for both cell-free and stem cell-seeded scaffolds. It can be concluded that the 3D printed PLA scaffold with BMSCs seeding has well osteogenic potential to be used for bone defect healing.