Macrophage-Mediated Bone Formation of Scaffolds Modified with MSC-derived Extracellular Matrix Depends on Migration Inhibitory Factor Signaling Pathway

Recently the role of macrophages on osteogenesis of mesenchymal stem cells (MSCs) is brought to focus. On the other hand, the cross-talk between macrophages and MSCs confirms that the paracrine molecules derived from macrophages are also carefully regulated by MSCs. Human umbilical cord mesenchymal stem cells (hucMSCs) could reduce secretions of inflammatory factors from macrophages for improving injury healing. hucMSC-derived extracellular matrix (hucMSC-ECM) exhibit nature similar to hucMSCs. It has been demonstrated that hucMSC-ECM possess considerable effects on reducing inflammatory response of macrophages, while the role of hucMSC-ECM on the expression of macrophage-derived paracrine osteogenic molecules is unclear. Here, we presented the decalcified bone scaffolds modified by hucMSC-derived extracellular matrix (DBM-ECM) maintaining multiple soluble cytokines including macrophage migration inhibitory factor (MIF). Compared with DBM, the DBM-ECM scaffolds induced bone formation in a macrophage-depending manner by an improved heterotopic ossification in SCID mice with or without macrophage depletion. Macrophage cocultured with the DBM-ECM expressed four osteoinductive cytokines (BMP2, FGF2, TGFβ3 and OSM), which were screen out by RNA sequencing, qPCR and western blot. The conditioned medium from macrophages cocultured with the DBM-ECM improved osteogenic differentiation of hBMSCs and activated CD74/CD44 signal transduction including phosphorylation of P38 and dephosphorylation of c-jun. Furthermore, the inhibitory effects of the DBM-ECM scaffolds with knockdown of MIF on osteogenesis in vitro and in vivo revealed that macrophage-mediated osteogenesis depends on MIF/CD74/ P38 signal transduction. This study indicates the coordinated crosstalk of macrophages and MSCs play a key role on bone regeneration induced by the DBM-ECM, with the emphasis on the constructing macrophage-derived osteoimmunological microenvironment. mRNA profiles of Thp-1 derived macrophages cocultured with DBM and DBM-ECM for 3 and 7days.

近年来，巨噬细胞（macrophages）对间充质干细胞（mesenchymal stem cells, MSCs）成骨作用的调控机制成为研究焦点。另一方面，巨噬细胞与MSCs之间的细胞串扰研究表明，巨噬细胞来源的旁分泌分子同样受到MSCs的精密调控。人脐带间充质干细胞（human umbilical cord mesenchymal stem cells, hucMSCs）可通过降低巨噬细胞的炎症因子分泌水平以促进损伤修复。hucMSC衍生的细胞外基质（hucMSC-derived extracellular matrix, hucMSC-ECM）的生物学特性与hucMSCs相似。已有研究证实，hucMSC-ECM可显著抑制巨噬细胞的炎症应答，但hucMSC-ECM对巨噬细胞旁分泌成骨分子表达的调控作用仍不明确。本研究构建了经hucMSC-ECM修饰的脱钙骨支架（DBM-ECM），该支架可保留包括巨噬细胞迁移抑制因子（macrophage migration inhibitory factor, MIF）在内的多种可溶性细胞因子。相较于脱钙骨支架（DBM），DBM-ECM支架可通过巨噬细胞依赖的途径诱导骨形成：在伴或不伴巨噬细胞耗竭的重症联合免疫缺陷小鼠（severe combined immunodeficiency mice, SCID mice）模型中，其异位骨化水平均得到显著提升。通过RNA测序、实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qPCR）与蛋白质印迹（western blot）筛选发现，与DBM-ECM共培养的巨噬细胞可表达四种成骨诱导细胞因子：骨形态发生蛋白2（bone morphogenetic protein 2, BMP2）、成纤维细胞生长因子2（fibroblast growth factor 2, FGF2）、转化生长因子β3（transforming growth factor β3, TGFβ3）及制瘤素M（oncostatin M, OSM）。与DBM-ECM共培养的巨噬细胞条件培养基可促进人骨髓间充质干细胞（human bone marrow mesenchymal stem cells, hBMSCs）的成骨分化，并激活CD74/CD44信号通路，包括P38的磷酸化与c-jun的去磷酸化过程。此外，敲低MIF的DBM-ECM支架在体内外均表现出对成骨的抑制作用，这一结果证实巨噬细胞介导的成骨过程依赖于MIF/CD74/P38信号通路。本研究表明，巨噬细胞与MSCs的协同串扰在DBM-ECM诱导的骨再生过程中发挥关键作用，其核心在于构建巨噬细胞来源的骨免疫微环境（osteoimmunological microenvironment）。本数据集包含与DBM及DBM-ECM共培养3天、7天的Thp-1来源巨噬细胞的mRNA表达谱数据。