Sequential activation of osteogenic microenvironment via composite peptide-modified microfluidic microspheres for promoting bone regeneration.

The osteogenic microenvironment (OME) significantly influences bone repair; however, reproducing its dynamic activation and repair processes remains challenging. In this study, we designed injectable porous microspheres modified with composite peptides to investigate cascade alterations in OME and their underlying mechanisms. Poly -lactic acid microfluidic microspheres underwent surface modifications through alkaline hydrolysis treatment, involving heterogeneous grafting of bovine serum albumin nanoparticles with stem cell-homing peptides (BNP@SKP) and BMP-2 mimicking peptides (P24), respectively. These modifications well-organized the actions of initial release and subsequent in situ grafting of peptides. Cellular experiments demonstrated varied degrees of chemotactic recruitment and osteogenic differentiation in mesenchymal stem cells. Further biological analysis revealed that BNP@SKP targeted the Ras/Erk axis and upregulated matrix metalloproteinase (MMP)2 and MMP9 expression, thereby enhancing initial chemotaxis and recruitment. In vivo studies validated the establishment of a dynamically regulated OME centered on the microspheres, resulting in increased stem cell recruitment, sequential activation of the differentiation microenvironment, and facilitation of in situ osteogenesis without ectopic ossification. In conclusion, this study successfully fabricated composite peptide-modified microspheres and systematically explored the mechanisms of bone formation through sequential activation of OME via heterogeneous grafting of signaling molecules. This provides theoretical evidence for biomaterials based on microenvironment regulation.