Anatomical implant region – a critical determinant for the osteogenic potency of small extracellular vesicles and rhBMP-2

Purpose: Mechanical stabilization is crucial for bone healing, yet complex fractures, particularly osteoporotic or comminuted, remain challenging due to impaired implant osseointegration, resulting in implant loosening or non-unions. This study investigated whether co-application of small extracellular vesicles (sEVs) derived from human umbilical cord mesenchymal stromal cells (hUC-MSC-sEVs) with a low dose of recombinant human bone morphogenetic protein 2 (rhBMP-2) could enhance screw implant osseointegration. Methods: A novel small animal model was established to evaluate the effect of anatomical femur regions on screw integration. Six weeks postoperatively, bone formation and bone–implant contact were assessed by micro-computed tomography and descriptive histology. Biomechanical stability was determined using pull-out tests. Results: Outcomes differed significantly between the proximal and distal implant locations, with no improvements in osseointegration observed in the distal region. In the proximal region, application of hUC-MSC-sEVs alone did not significantly improve osseointegration, whereas local application of 1.5 µg rhBMP-2 resulted in measurable biomechanical improvements. No additive or synergistic effects were observed when sEVs were co-administered with rhBMP-2. Descriptive histology supported these findings, demonstrating the most pronounced bone formation at the proximal site following rhBMP-2 treatment. Conclusion: hUC-MSC-sEVs did not enhance screw implant osseointegration and slightly reduced new bone formation. In contrast, a low dose of rhBMP-2 (1.5 µg) promoted implant integration, with no additive effect when combined with sEVs. Notably, the osteogenic effect of rhBMP-2 was observed only at the proximal femoral site, indicating that anatomical location critically influences implant osseointegration. These findings highlight the importance of considering anatomical region when evaluating osteoinductive treatments and implant materials in small animal models.