Introduction: To induce sufficient new bone formation, high doses of bone morphogenetic protein-2 (BMP-2) are applied in regenerative medicine that often induce serious side effects. Therefore, improved treatment strategies are required. Here, we investigate whether the delivery of BMP-2 lyophilized in the presence of trehalose reduced the dose of BMP-2 required for bone regeneration. 
Materials and methods: A new growth factor delivery system was fabricated using BMP-2-loaded TiO₂ nanotubes by lyophilization with trehalose (TiO₂-Lyo-Tre-BMP-2). We measured BMP-2 release characteristics, bioactivity, and stability, and determined the effects on the osteogenic differentiation of bone marrow stromal cells in vitro. Additionally, we evaluated the ability of this formulation to regenerate new bone around implants in rat femur defects by micro-computed tomography (micro-CT), sequential fluorescent labelling, and histological analysis. 
Results: Compared with absorbed BMP-2-loaded TiO₂ nanotubes (TiO₂-BMP-2), TiO₂-Lyo-Tre-BMP-2 exhibited sustained release, consistent bioactivity, and higher stability of BMP-2, and resulted in greater osteogenic differentiation of BMSCs. Eight weeks post-operation, TiO₂-Lyo-Tre-BMP-2 nanotubes, with various dosages of BMP-2, regenerated larger amounts of new bone than TiO₂-BMP-2 nanotubes. 
Conclusion: Our findings indicate that delivery of BMP-2 lyophilized with trehalose may be a promising method to reduce the dose of BMP-2 and avoid the associated side effects.
Keywords: bone morphogenetic protein-2, dose reduction, delivery system, trehalose, lyophilization, TiO₂ nanotubes, BMP-2, regenerative medicine, surface modification