Mohammadnejad Leila, Conrady Hannah, Mangold Madeline, Kimmerle-Mueller Evi, Hechler Annika, von Ohle Christiane, Rupp Frank, Krajewski Stefanie
Department of Medical Materials Science & Technology, Institute for Biomedical Engineering, University Hospital Tübingen.
Department of Conservative Dentistry and Periodontology, University Hospital Tübingen.
J Vis Exp. 2024 Dec 6(214). doi: 10.3791/67180.
Foreign body reaction (FBR), an immune-mediated complex healing process, plays a crucial role in integrating implants into the body. Macrophages, as the first line of immune system interaction with implant surfaces, play a bidirectional role in modulating the inflammation-regeneration balance. For a deep understanding and the evaluation of the reactions between implant materials and immune responses, reliable in vitro methods and protocols are pivotal. Among different in vitro models, primary monocyte-derived macrophages (MDMs) present an excellent model for investigating macrophage-implant interactions. We have implemented an experimental protocol to evaluate the polarization of MDMs into M1 (classically activated) and M2 (alternatively activated) macrophages on implant surfaces. We isolated blood monocytes from healthy donors and differentiated them into macrophages using macrophage colony-stimulating factor (M-CSF). Differentiated macrophages were cultured on implant surfaces and polarized into M1 and M2 subtypes. M1 polarization was achieved in the presence of interferon (IFN)-γ and lipopolysaccharide (LPS), while M2 polarization was performed in a medium containing interleukin (IL)-4 and IL-13. We evaluated macrophage phenotypes by Enzyme-linked Immunosorbent Assay (ELISA), confocal laser scanning microscopy (CLSM), and quantitative real-time PCR (qRT-PCR) based on panels of secreted cytokines, cell surface markers, and expressed genes. The extracted RNA was transformed into complementary DNA (cDNA), and qRT-PCR was used to quantify mRNA related to M1 and M2 macrophages. Accordingly, M1 macrophages have been characterized by higher expression of proinflammatory Tumor necrosis factor (TNF-α) cytokine and CCR7 surface marker compared to M2 macrophages, which exhibited higher levels of CD209 and CCL13. Consequently, CCR7 and CD209 were identified as specific and reliable markers of M1 and M2 macrophage subtypes by immunostaining and visualizing by CLSM. Further confirmation was achieved by ELISA detecting elevated TNF-ɑ level in M1 and increased CCL13 in M2 cells. The proposed markers and experimental setup can be used effectively to assess the immunomodulatory potential of implants.
异物反应（FBR）是一种免疫介导的复杂愈合过程，在将植入物整合到体内方面起着关键作用。巨噬细胞作为免疫系统与植入物表面相互作用的第一道防线，在调节炎症 - 再生平衡中发挥着双向作用。为了深入理解和评估植入材料与免疫反应之间的相互作用，可靠的体外方法和方案至关重要。在不同的体外模型中，原代单核细胞衍生的巨噬细胞（MDM）是研究巨噬细胞 - 植入物相互作用的优秀模型。我们实施了一个实验方案，以评估MDM在植入物表面极化为M1（经典活化）和M2（替代活化）巨噬细胞的情况。我们从健康供体中分离出血液单核细胞，并使用巨噬细胞集落刺激因子（M - CSF）将其分化为巨噬细胞。将分化后的巨噬细胞培养在植入物表面，并极化为M1和M2亚型。在干扰素（IFN）-γ和脂多糖（LPS）存在的情况下实现M1极化，而在含有白细胞介素（IL）-4和IL - 13的培养基中进行M2极化。我们通过酶联免疫吸附测定（ELISA）、共聚焦激光扫描显微镜（CLSM）和基于分泌细胞因子、细胞表面标志物和表达基因的定量实时PCR（qRT - PCR）来评估巨噬细胞表型。提取的RNA转化为互补DNA（cDNA），并使用qRT - PCR定量与M1和M2巨噬细胞相关的mRNA。因此，与M2巨噬细胞相比，M1巨噬细胞的特征在于促炎肿瘤坏死因子（TNF - α）细胞因子和CCR7表面标志物的表达更高，而M2巨噬细胞表现出更高水平的CD209和CCL13。因此，通过免疫染色和CLSM可视化，CCR7和CD209被鉴定为M1和M2巨噬细胞亚型的特异性和可靠标志物。通过ELISA检测M1中升高的TNF - α水平和M2细胞中升高 的CCL13水平进一步证实了这一点。所提出的标志物和实验设置可有效地用于评估植入物的免疫调节潜力。
