Enzymatically triggered in situ gelation of a multifunctional peptide for localized controlled release and conformal fracture repair.

Abstract

Irregular fracture edges and gaps present significant challenges to bone healing. As versatile scaffolds for bone regeneration, hydrogels can effectively guide and facilitate tissue repair. Compared to preformed hydrogels, in situ gelation via a responsive sol-gel transition at the defect site offers superior flexibility, adaptability, and conformity, minimizing void cavities and establishing a more favorable environment for healing complex fractures. Peptides, as biogenic building blocks, can self-assemble into hierarchically structured hydrogels under specific stimuli. Given that alkaline phosphatase (ALP) is a key enzyme in bone mineralization and is highly expressed at the fracture site, we developed a phosphorylated peptide (NfFpYO) that undergoes ALP-triggered dephosphorylation, enabling direct in situ gelation on irregular fracture surfaces. Our results show that the resulting nanofibrous three-dimensional scaffold, functionalized with Gly-Pro-Hyp (GPO) motifs, promotes the migration, proliferation, and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. Moreover, the resulting nanofibrous hydrogel, which presents peptide-bound naproxen and fluorinated motifs, was associated with a less inflammatory and more repair-supportive macrophage-associated marker profile, enhanced osteogenic marker expression, and improved radiographic and histological fracture repair in a murine model. This study adapts a promising ALP-instructed peptide self-assembly strategy to the fracture-repair microenvironment through a multifunctional precursor design, providing a local shape-conformal and bioactive hydrogel platform with potential for complex fracture repair.