Biomaterials and Regenerative Medicine Laboratory, School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, Pennsylvania.
Tissue Eng Part C Methods. 2020 Mar;26(3):156-169. doi: 10.1089/ten.TEC.2019.0309.
The primary regulators of the innate immune response to implanted biomaterials are macrophages, which change phenotype over time to regulate multiple phases of the tissue repair process. Immunomodulatory biomaterials that target macrophage phenotype are a promising approach for promoting tissue repair. Although expression of multiple markers has been widely used to characterize macrophage phenotype, the complexity of the macrophage response to biomaterials makes interpretation difficult. The aim of this study was to put forth an objective method to characterize macrophage phenotype with respect to specific biological processes or standard phenotypes of interest. We investigated the utility of gene set analyses to analyze macrophages as they respond to model biomaterials in comparison to "reference" M1 and M2a macrophage phenotypes. Primary human macrophages were seeded onto crosslinked collagen scaffolds with or without adsorption of the proinflammatory cytokine interferon-gamma (IFNg). Gene expression of a custom-curated panel of 48 genes, representing the M1 and M2a gene signatures as well as other genes important for angiogenesis and tissue repair, was quantified using NanoString on days 3, 5, and 8 of culture. A dataset of phenotype controls, consisting of M0, M1, and M2a macrophages, was used as a source of comparison and to validate the methods of characterization. Gene expression of M1 and M2a markers showed mixed upregulation and downregulation by macrophages seeded on collagen and IFNg-adsorbed collagen scaffolds, highlighting the need for more holistic analyses. Euclidean distance measurements to the reference phenotypes were unable to resolve differences between groups. In contrast, rotation gene set testing with and without gene weighting based on the genes' ability to differentiate between M1, M2a, and M0 controls, followed by gene set variation analysis, showed that collagen scaffolds inhibited the classic M1 phenotype without promoting a classic M2a phenotype, and that IFNg-adsorbed collagen scaffolds promoted the M1 phenotype and inhibited the M2a phenotype. In summary, this work demonstrates a powerful, objective methodology for characterizing the macrophage response to biomaterials in comparison to reference macrophage phenotypes. With the addition of more macrophage phenotypes with defined gene expression signatures, this method could prove beneficial for characterizing complex hybrid phenotypes.
固有免疫反应对植入生物材料的主要调节者是巨噬细胞，它随着时间的推移改变表型以调节组织修复过程的多个阶段。针对巨噬细胞表型的免疫调节生物材料是促进组织修复的一种很有前途的方法。尽管已经广泛使用多种标志物来表征巨噬细胞表型，但巨噬细胞对生物材料的反应的复杂性使得解释变得困难。本研究的目的是提出一种客观的方法来描述与特定生物过程或感兴趣的标准表型相关的巨噬细胞表型。我们研究了基因集分析在比较模型生物材料时分析巨噬细胞的效用，与“参考”M1 和 M2a 巨噬细胞表型进行比较。将原代人巨噬细胞接种到交联胶原支架上，或者在没有吸附促炎细胞因子干扰素-γ（IFNg）的情况下。使用 NanoString 在培养的第 3、5 和 8 天定量测定定制的 48 个基因的基因表达，这些基因代表了 M1 和 M2a 基因特征以及其他对血管生成和组织修复很重要的基因。使用表型对照数据集，由 M0、M1 和 M2a 巨噬细胞组成，作为比较和验证表征方法的来源。接种在胶原和 IFNg 吸附胶原支架上的巨噬细胞表现出 M1 和 M2a 标志物的混合上调和下调，这突出表明需要更全面的分析。到参考表型的欧几里得距离测量无法区分组间差异。相比之下，基于基因区分 M1、M2a 和 M0 对照的能力进行旋转基因集测试和没有基因加权，然后进行基因集变异分析，显示胶原支架抑制了经典的 M1 表型，而没有促进经典的 M2a 表型，并且 IFNg 吸附的胶原支架促进了 M1 表型并抑制了 M2a 表型。总之，这项工作证明了一种强大的、客观的方法，用于比较参考巨噬细胞表型来描述巨噬细胞对生物材料的反应。通过添加具有定义基因表达特征的更多巨噬细胞表型，这种方法可能有助于描述复杂的混合表型。
