Hierarchical regulation and mechanism of “open-hollow-fibrous network” structures: Osteogenic-angiogenic coupling responses of poly(γ-benzyl-L-glutamate) microspheres

PBLG HNMs feature open-hollow nanofibrous architecture and injectability. • PBLG self-assembly drives the internal ECM-like nanofibrous network. • Covalent GHK-Cu grafting affords stable osteogenic-angiogenic activity. • PBLG-GCu HNMs promote BMSC osteogenesis and HUVEC angiogenesis. Biomimetic fibrous microspheres with a high specific surface area hold substantial promise for bone tissue engineering. In this study, asymmetric open-hollow nanofibrous microspheres (HNMs) were fabricated from polypeptide poly( γ -benzyl- L -glutamate) (PBLG) via a combination of emulsion and thermally induced phase separation, yielding PBLG HNMs. To further impart biofunctionality, the copper peptide (GHK-Cu) was covalently grafted onto the microspheres to obtain osteoinductive and pro-angiogenic PBLG-GCu HNMs. The optimized microspheres exhibited an average diameter of 372 ± 102 μm, which is suitable for injectability, and an opening size of 219 ± 53 μm, enabling efficient cellular infiltration. The internal surface featured an interconnected nanofibrous network with a fiber diameter of 417 ± 78 nm, mimicking the extracellular matrix (ECM) microenvironment and providing abundant cell-interactive sites. Live/Dead staining and CCK-8 assays confirmed the cytocompatibility of the PBLG-GCu HNMs. Compared with non-functionalized PBLG HNMs, PBLG-GCu HNMs enhanced bone marrow mesenchymal stem cell (BMSC) mineralization and upregulated osteogenic gene expression, with Runx2, OPN, and OCN expression increased by 1.61-, 3.53-, and 2.29-fold, respectively. In addition, the tube formation assay verified robust angiogenic stimulation. Overall, the PBLG-GCu HNMs integrated hierarchical structural biomimicry with dual osteogenic-angiogenic bioactivity, exhibiting great potential as injectable scaffolds for repairing irregular bone defects.