Supplementary Material for: Macrophages and Fibroblasts Differentially Contribute to Tattoo Stability

Background: Little information is available about the complexity and function of skin cells contributing to the high stability of tattoos. It has been shown that dermal macrophages play an important role in the storage and maintenance of pigment particles. By contrast, the impact of dermal fibroblasts, forming the connective tissue of the skin, on the stability of the tattoo is not known. Method: In this study, we compared the cell number and the particle load in dermal macrophages versus dermal fibroblasts, isolated from tail skin of tattooed mice. Results: Microscopic analysis revealed that both cell populations contained the tattoo particles, although in largely different amounts. A small number of macrophages with high side scatter intensity contained a large quantity of pigment particles, whereas a high number of dermal fibroblasts harbored only a few pigment particles. Using the CD64dtr mouse model that allows for selective, diphtheria toxin-mediated depletion of macrophages, we have previously shown that macrophages hold the tattoo in place by capture-release and recapture cycles. In the tattooed skin of macrophage-depleted mice, the content of pigment particles in fibroblasts did not change; however, the total number of fibroblasts carrying particles increased. Conclusion: The present study demonstrates that dermal macrophages and fibroblasts contribute in different ways to the tattoo stability and further improves our knowledge on tattoo persistence.背景：目前关于参与维持纹身高稳定性的皮肤细胞的复杂性与功能的相关研究信息较为匮乏。已有研究证实，真皮巨噬细胞（dermal macrophages）在色素颗粒的储存与维持过程中发挥着重要作用。与之相对，构成皮肤结缔组织的真皮成纤维细胞（dermal fibroblasts）对纹身稳定性的影响尚不明晰。方法：本研究针对从接受纹身的小鼠尾部皮肤中分离得到的真皮巨噬细胞与真皮成纤维细胞，对二者的细胞数量及颗粒负载量进行了对比分析。结果：显微镜分析显示，两类细胞群均含有纹身色素颗粒，但二者的颗粒承载量差异显著。少量具有高侧向散射强度（side scatter intensity）的巨噬细胞含有大量色素颗粒，而数量众多的真皮成纤维细胞仅携带少量色素颗粒。此前，我们借助可通过白喉毒素选择性清除巨噬细胞的CD64dtr小鼠模型（CD64dtr mouse model），证实巨噬细胞可通过捕获-释放-再捕获循环将纹身色素固定于皮肤原位。在巨噬细胞被清除的纹身小鼠皮肤中，成纤维细胞内的色素颗粒含量未发生改变，但携带色素颗粒的成纤维细胞总数有所增加。结论：本研究证实，真皮巨噬细胞与成纤维细胞通过不同途径参与维持纹身稳定性，进一步加深了我们对纹身持久性的认知。