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Both cultured Bone Marrow Mesenchymal Stem Cells (BMSCs) and non-cultured Bone Marrow Mononuclear Cells (BM-MNCs) can improve random-pattern skin flap survival. However, whether prior culture expansion of bone marrow cells is beneficial for this therapy remains unclear. In the current study, the protective effects of BM-MNCs and BMSCs derived from identical bone marrow aspirates were compared in a random-pattern skin flap rat model. The mean skin flap survival rates were 71.6 ± 8.4% in the BM-MNC-treated group and 66.2 ± 3.1% in the BMSCtreated group, both of which were significantly higher than the control group (55.9 ± 3.4%). The protective effects were confirmed by blood perfusion analysis and vessel density assay. However, no significant difference was observed between the cell transplanted groups. These results indicate that the current method for pre-culture of BMSCs does not bring therapeutic benefits in skin flap protection. Therefore, BM-MNCs without pre-culture might be more practicable in the clinical setting.