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Objectives: The goal of this study was to prepare lyophilized platelet-rich fibrin (L-PRF) and analyze the combined use of L-PRF and osteogenic bone marrow mesenchymal stem cell (BMSC) sheet fragments for bone tissue engineering via in vivo injection. Methods: First, fresh PRF (F-PRF) was lyophilized to prepare L-PRF, the characteristics of which were examined through gross morphological, and histological and microstructural observations. In addition, the kinetics of growth factor release from L-PRF and F-PRF were also determined by enzyme-linked immunosorbent assay (ELISA). Subsequently, after assessing the proliferation and osteogenic differentiation of BMSCs exposed to L-PRF or F-PRF in vitro, we subcutaneously injected BMSC sheet fragments with L-PRF or F-PRF into nude mice and assessed bone formation through microcomputed tomography and histological analyses. Results: We observed that L-PRF released growth factors that favored BMSC proliferation and osteogenic differentiation in vitro. The combined use of L-PRF and osteogenic BMSC sheet fragments enabled bone tissue regeneration in vivo, and no significant difference between the F-PRF and L-PRF groups was observed (P = 0.24). Conclusions: The results of this study demonstrate that the combined use of L-PRF and osteogenic BMSC sheets may have potential in the fabrication of engineered bone.