SF/PCL/KAE纳米纤维支架通过免疫微调控促进骨髓间充质干细胞成骨分化的研究

SF/PCL/KAE Nanofibrous Scaffolds Promote Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells through Immune Microregulation

目的:制备丝素蛋白/聚己内酯/山奈酚纳米纤维支架(SF/PCL/KAE),评估其对小鼠外周巨噬细胞RAW264.7极化的影响,并探究免疫微调控对骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)成骨分化的影响。方法:通过静电纺丝技术制备SF/PCL/KAE,通过扫描电子显微镜(scanning electron microscopy, SEM)、傅里叶红外光谱(fourier transform infrared spectroscopy, FTIR)和X射线衍射图谱(X-ray diffraction spectroscopy, XRD)对SF/PCL/KAE进行表征,并进行拉伸力学检测和抗菌性能评估。采用CCK-8试验和SEM观察细胞粘附状态,考察支架的细胞相容性。将脂多糖(lipopolysaccharide, LPS)处理的RAW264.7接种在支架上,检测支架的活性氧(reactive oxygen species, ROS)清除能力,并通过流式细胞术和荧光定量聚合酶链反应(real-time fluorescence quantitative PCR,RT-qPCR)检测RAW264.7的极化情况和炎症相关因子的表达。最后,将BMSCs与LPS处理的RAW264.7共培养,矿化诱导7 d采用RT-qPCR检测成骨分化相关基因的表达,并分别在第7天和第14天行碱性磷酸酶(alkaline phosphatase, ALP)染色,14 d后行茜素红染色(alizarin red staining, ARS)。结果:SEM,FTIR和XRD结果证明了纳米纤维形态和KAE的成功引入。相比SF/PCL,SF/PCL/KAE具有更好的拉伸力学性能和优异的抗金黄色葡萄球菌性能(>99%),且没有明显的细胞毒性。SF/PCL/KAE表现出良好的ROS清除能力,抑制RAW264.7向M1表型极化并促进其向M2表型极化,且下调了肿瘤坏死因子(tumor necrosis factor, TNF)-α的表达,上调了CD206、TGF-β1的表达。在用LPS处理RAW264.7模拟炎症环境中,SF/PCL/KAE可上调ALP、Ⅰ型胶原蛋白(collagen typeⅠ, COL-1)、骨形态发生蛋白-2(bone morphogenetic protein-2,BMP2)和骨桥蛋白(osteopontin, OPN)基因的表达,ALP和ARS染色也证实KAE的引入可促进BMSCs成骨分化。以上研究中,2%浓度的KAE相比于1%浓度体现出更优的机械性能、抗菌效果和骨免疫微调控性能。结论:SF/PCL/KAE具有良好的机械性能、抗菌性能和骨免疫微调控能力,可显著促进BMSCs在炎性环境中的成骨分化,具有良好的临床应用前景。

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 typeⅠ(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.