Fujihara Yuko, Hikita Atsuhiko, Takato Tsuyoshi, Hoshi Kazuto
Department of Oral-Maxillofacial Surgery and Orthodontics, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan.
Department of Cartilage and Bone Regeneration (Fujisoft), Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
J Cell Physiol. 2018 Feb;233(2):1490-1499. doi: 10.1002/jcp.26036. Epub 2017 Jul 4.
To obtain stable outcomes in regenerative medicine, understanding and controlling immunological responses in transplanted tissues are of great importance. In our previous study, auricular chondrocytes in tissue-engineered cartilage transplanted in mice were shown to express immunological factors, including macrophage migration inhibitory factor (MIF). Since MIF exerts pleiotropic functions, in this study, we examined the roles of MIF in cartilage regenerative medicine. We made tissue-engineered cartilage consisting of auricular chondrocytes of C57BL/6J mouse, atellocollagen gel and a PLLA scaffold, and transplanted the construct subcutaneously in a syngeneic manner. Localization of MIF was prominent in cartilage areas of tissue-engineered cartilage at 2 weeks after transplantation, though it became less apparent by 8 weeks. Co-culture with RAW264 significantly increased the expression of MIF in chondrocytes, suggesting that the transplanted chondrocytes in tissue-engineered cartilage could enhance the expression of MIF by stimulation of surrounding macrophages. When MIF was added in the culture of chondrocytes, the expression of type II collagen was increased, indicating that MIF could promote the maturation of chondrocytes. Meanwhile, toluidine blue staining of constructs containing wild type (Mif+/+) chondrocytes showed increased metachromasia compared to MIF-knockout (Mif-/-) constructs at 2 weeks. However, this tendency was reversed by 8 weeks, suggesting that the initial increase in cartilage maturation in Mif+/+ constructs deteriorated by 8 weeks. Since the Mif+/+ constructs included more iNOS-positive inflammatory macrophages at 2 weeks, MIF might induce an M1 macrophage-polarized environment, which may eventually worsen the maturation of tissue-engineered cartilage in the long term.
为了在再生医学中获得稳定的结果，了解和控制移植组织中的免疫反应非常重要。在我们之前的研究中，组织工程软骨中的耳廓软骨细胞移植到小鼠体内后显示出表达免疫因子，包括巨噬细胞迁移抑制因子（MIF）。由于MIF具有多效性功能，在本研究中，我们研究了MIF在软骨再生医学中的作用。我们制备了由C57BL/6J小鼠的耳廓软骨细胞、无定形胶原凝胶和聚乳酸支架组成的组织工程软骨，并将构建体以同基因方式皮下移植。移植后2周，MIF在组织工程软骨的软骨区域定位明显，但在8周时变得不那么明显。与RAW264共培养显著增加了软骨细胞中MIF的表达，表明组织工程软骨中移植的软骨细胞可通过刺激周围巨噬细胞增强MIF的表达。当在软骨细胞培养中添加MIF时，II型胶原的表达增加，表明MIF可促进软骨细胞成熟。同时，在2周时，含有野生型（Mif+/+）软骨细胞的构建体的甲苯胺蓝染色显示与MIF基因敲除（Mif-/-）构建体相比异染性增加。然而，这种趋势在8周时逆转，表明Mif+/+构建体中软骨成熟的最初增加在8周时恶化。由于Mif+/+构建体在2周时包含更多iNOS阳性炎性巨噬细胞，MIF可能诱导M1巨噬细胞极化环境，这最终可能长期恶化组织工程软骨的成熟。
