Abstract: Objective: To prepare silk fibroin/polycaprolactone/kaempferol nanofibrous scaffolds(SF/PCL/KAE)and evaluate their effect on RAW264.7 polarization, and to investigate the effect of immune microregulation on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs). Methods: SF/PCL/KAE was prepared by electrospinning and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction spectroscopy (XRD). Tensile mechanics properties and antibacterial properties were also evaluated. CCK-8 assay and SEM observation were performed to evaluate the cytocompatibility of the scaffolds. Lipopolysaccharide (LPS)-treated RAW264.7 was seeded on scaffolds, the reactive oxygen species (ROS) scavenge ability of the scaffold was detected, and the polarization of RAW264.7 and the expression of inflammation-related factors were detected by flow cytometry and RT-qPCR. Finally, BMSCs were co-cultured with LPS-treated RAW264.7, and RT-qPCR was used to detect the expression of genes related to osteogenic differentiation after 7 days of mineralization induction, and alkaline phosphatase (ALP) staining was performed on 7days and 14 days, respectively, and alizarin red staining (ARS) was performed 14 days later. Results: SEM, FTIR, and XRD results demonstrated good morphology of the nanofibrous scaffold and the successful introduction of KAE. Compared with SF/PCL, SF/PCL/KAE has better tensile mechanical properties and excellent antimicrobial ability to S.aureus(>99%) without obvious cytotoxicity. SF/PCL/KAE showed good ROS scavenging ability, inhibited the polarization of RAW264.7 to the M1 phenotype, and promoted its polarization to the M2 phenotype, and down-regulated the expression of tumor necrosis factor-α (TNF-α) and up-regulated the expression of CD206 and TGF-β1. In the inflammatory environment simulated by RAW264.7 treated with LPS, SF/PCL/KAE up-regulated the expression of ALP, collagen type Ⅰ (COL-1), bone morphogenetic protein-2 (BMP-2), and osteopontin (OPN), and ALP and ARS staining also confirmed that the introduction of KAE could promote the osteogenic differentiation of BMSCs. In the above studies, the 2% KAE showed better mechanical properties, antibacterial effects, and bone immune micromodulation properties than 1%KAE. Conclusion: SF/PCL/KAE has good mechanical properties, antibacterial properties, and bone immune micromodulation ability, which can significantly promote the osteogenic differentiation of BMSCs in inflammatory environment.

Key words: kaempferol, antibacterial, immunomodulatory, nanofibrous