Abstract: Engineering biomaterial surfaces with synergistic peptide combinations unlocks new possibilities for directing stem cell fate and advancing regenerative medicine. This study explores a novel multi-peptide functionalization strategy to enhance the osteoinductive potential of biomaterial surfaces. Accordingly, the existing synergy between RGD peptides and BMP-2 was leveraged by incorporating a third peptide designed to activate complementary signaling pathways, therefore enhancing the recruitment and differentiation of stem cells. This approach represents a paradigm shift in biomaterial design, taking advantage of the cooperative action of multiple ligands to optimize the cellular microenvironment. To achieve precise and reproducible surface modifications, an optimized spin-coating method was developed and employed for the first time to facilitate controlled grafting of biomolecules onto the surfaces. Unlike traditional immersion-based silanization techniques, this advanced approach enabled the achievement of a well-organized, self-assembled monolayer with superior bioactivity. Systematic optimization of the spin-coating parameters allowed for fine-tuning surface properties for subsequent peptide functionalization. Experimental results demonstrated that multi-peptide functionalized surfaces significantly outperformed single- or dual-peptide coatings in promoting osteogenic differentiation. Early upregulation of key osteogenic markers was observed through qPCR, Western blot, and immunofluorescence analyses. The introduction of additional peptides amplified stem cell signaling by targeting distinct molecular pathways, resulting in a highly osteoinductive environment. This study highlights the transformative potential of multi-peptide functionalization to orchestrate complex biological processes at the cell-material interface. This strategy paves the way for advancements in biomaterial design for regenerative medicine and tissue engineering applications by offering an efficient and adaptable approach for engineering bioactive surfaces.
