Fabrication of a fibrillar β-lactoglobulin-Mumijo-nanohydroxyapatite complex for antibacterial and wound healing applications

In this study, we developed and characterized a novel multifunctional complex (fBMHA) comprising fibrillar β-lactoglobulin (BLG), Mumiju, and nanohydroxyapatite (nHAP), aimed at enhancing wound healing and tissue regeneration. Structural and physicochemical analyses using Fourier Transform Infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), zeta potential analyzer, and X-ray diffraction (XRD) confirmed a successful integration of all components into a hybrid matrix with both amorphous and -crystalline features. The MTT assay demonstrated a concentration-dependent enhancement in fibroblast viability, with maximal proliferative stimulation observed at 10 mg/mL after 48 h, and an IC₅₀ value calculated at 71 mg/mL. Flow cytometry revealed a significant shift in cell cycle dynamics: the G1 phase decreased from 64.7% to 59.4%, while the S and G2/M phases increased from 25.3% to 27.8% and 4.6% to 6.7%, respectively (<i>p</i> < 0.05), indicating enhanced proliferation. AO/EtBr staining further confirmed preserved cellular integrity with minimal nuclear fragmentation. Scratch assay results showed substantial wound closure within 48 h, supporting the complex's role in promoting cell migration and confluency. Immunofluorescence analyses revealed upregulation of E-cadherin and fibronectin, markers essential for epithelial integrity and ECM remodeling. Moreover, disk diffusion assays confirmed antibacterial activity, with inhibition zones of 22.7 ± 0.5 mm (<i>Staphylococcus aureus</i>) and 20.0 ± 0.2 mm (<i>Escherichia coli</i>). Collectively, these findings validate the fBMHA complex as a biologically safe and multifunctional therapeutic material that simultaneously promotes fibroblast proliferation, accelerates wound healing, and mitigates bacterial infection, highlighting its translational potential for advanced regenerative applications.