BACKGROUND: Managing large bone defects remains a significant clinical problem. We enhanced the osteogenic activity of the induced membrane (IM) by incorporating bone marrow fluid, leading to spontaneous osteogenesis (SO). We aimed to explore the application of this method in tibiofibular fusion (TFF) for reconstructing segmental tibial defects. METHODS: Forty-two rats with femoral defects were divided into seven groups (n = 6). Defects in groups A1-3 and B1-3 were filled with polymethylmethacrylate spacers while Group B4 was the control. Kirschner wires were used in Groups A1 and B1, plating was used in Groups A3 and B3-4, while the medullary canal was sealed in Groups A2 and B2. In Group A, osteogenic activity was measured using immunohistochemistry, W-B, and qRT-PCR. In Group B, the osteogenic results were measured using X-ray and gross examinations. Ten patients with 4-10 cm segmental defects of the middle and distal tibia underwent reconstruction using the IM technique, and IM and bone marrow fluid-induced SO for TFF, whose effects were assessed. RESULTS: At five weeks, Group A1 showed higher levels of BMSCs and expression of BMP-2 and TGF-β1 than Groups A2 and A3 (p <
0.05). After 12 weeks, Group B1 had more new bone at the bone end than Group B3 (p = 0.009) whereas Groups B2 and B4 did not. All tibial defects and TFF healed. The TFF site and posterior tibia healed faster than the other sides and showed quicker clinical healing (p <
0.05). All patients could fully bear weight before tibial clinical healing, with an excellent-to-good functional rate of 80% (Paley's criterion) at the 13 - to 36-month follow-up. CONCLUSION: Bone marrow fluid enhances the osteogenic activity of IMs leading to SO. TFF by SO progresses faster than tibial clinical healing, making the IM technique an effective stabilizer for faster and better functional recovery after reconstruction of segmental defects of the middle and distal tibia.