Osteogenesis enhancement by immobilized DOPA-BMP-2 in combination with ultrasonic stimulation

Bone morphogenetic protein-2 (BMP-2) plays a crucial role in regulating osteogenic differentiation and is widely used in tissue engineering. However, its clinical application is hindered by the high-dose administration of soluble BMP-2, thereby causing adverse effects and rapid degradation. Immobilizing BMP-2 on biomaterials offers an effective approach for achieving spatial and temporal control of growth factors while sustaining their bioactivity. In addition to growth factor signaling, mechanical forces regulate cellular behavior and interact with BMP-2 signaling pathways, potentially influencing cellular functions. In this study, we aim to investigate how the spatial presentation of BMP-2 affects osteogenic responses and its interaction with ultrasound stimulation. We observe that immobilized recombinant adhesive BMP-2 (DOPA-BMP-2) exhibits greater osteoinductive potential than its soluble counterpart, as indicated by the increased ALP activity and accelerated mineralization (93.9% <i>vs.</i> 77.5% on Day 14). Low-intensity pulsed ultrasound (LIPUS)-a mechanical stimulus-enhances osteogenic differentiation only when combined with immobilized DOPA-BMP-2, as evidenced by the upregulated ALP activity (71.7 <i>vs.</i> 58.1 mU μg_protein ^-1) and larger mineralized area (90.4% <i>vs.</i> 72.6% on Day 7), whereas no significant effect is observed with soluble DOPA-BMP-2. Furthermore, cytoskeleton remodeling and focal adhesion formation are significantly enhanced exclusively under the combined treatment of immobilized DOPA-BMP-2 and LIPUS. These findings suggest that the enhanced osteogenic differentiation induced by immobilized BMP-2 and ultrasound may involve mechanotransduction pathways mediated by focal adhesion and cytoskeleton remodeling. This study supports the further development of BMP-2-functionalized biomaterials and biophysical therapy as a combined treatment for tissue engineering applications.