Anaerobic Co-Culture of Mesenchymal Stem Cells and Anaerobic Pathogens - A New In Vitro Model System

BackgroundHuman mesenchymal stem cells (hMSCs) are multipotent by nature and are originally isolated from bone marrow. In light of a future application of hMSCs in the oral cavity, a body compartment with varying oxygen partial pressures and an omnipresence of different bacterial species i.e. periodontitis pathogens, we performed this study to gain information about the behavior of hMSC in an anaerobic system and the response in interaction with oral bacterial pathogens.Methodology/Principal FindingsWe established a model system with oral pathogenic bacterial species and eukaryotic cells cultured in anaerobic conditions. The facultative anaerobe bacteria Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were studied. Their effects on hMSCs and primary as well as permanent gingival epithelial cells (Ca9-22, HGPEC) were comparatively analyzed. We show that hMSCs cope with anoxic conditions, since 40% vital cells remain after 72 h of anaerobic culture. The Ca9-22 and HGPEC cells are significantly more sensitive to lack of oxygen. All bacterial species reveal a comparatively low adherence to and internalization into hMSCs (0.2% and 0.01% of the initial inoculum, respectively). In comparison, the Ca9-22 and HGPEC cells present better targets for bacterial adherence and internalization. The production of the pro-inflammatory chemokine IL-8 is higher in both gingival epithelial cell lines compared to hMSCs and Fusobacterium nucleatum induce a time-dependent cytokine secretion in both cell lines. Porphyromonas gingivalis is less effective in stimulating secretion of IL-8 in the co-cultivation experiments.Conclusions/significanceHMSCs are suitable for use in anoxic regions of the oral cavity. The interaction with local pathogenic bacteria does not result in massive pro-inflammatory cytokine responses. The test system established in this study allowed further investigation of parameters prior to set up of oral hMSC in vivo studies.研究背景 人间充质干细胞（human mesenchymal stem cells，hMSCs）天然具备多向分化潜能，最初从骨髓中分离获得。鉴于人间充质干细胞未来有望应用于口腔这一氧分压波动且遍布多种细菌（即牙周病致病菌）的体腔环境，本研究旨在探究人间充质干细胞在厌氧体系中的行为模式，以及其与口腔致病菌相互作用时的应答反应。 材料与方法/主要结果 本研究构建了厌氧培养条件下共培养口腔致病菌与真核细胞的模型体系。选取兼性厌氧菌具核梭杆菌（Fusobacterium nucleatum）、牙龈卟啉单胞菌（Porphyromonas gingivalis）以及伴放线聚集杆菌（Aggregatibacter actinomycetemcomitans）作为研究对象，比较分析它们对人间充质干细胞、原代及永生化牙龈上皮细胞（Ca9-22、HGPEC）的作用效果。结果显示，人间充质干细胞可耐受缺氧环境：经72小时厌氧培养后仍有40%的活细胞留存；而Ca9-22与HGPEC细胞对缺氧环境的敏感性显著更高。所有受试致病菌对人间充质干细胞的黏附与内化率均相对较低（分别为初始接种量的0.2%与0.01%）；相较而言，Ca9-22与HGPEC细胞更易成为致病菌黏附与内化的靶标。两种牙龈上皮细胞系的促炎性趋化因子白细胞介素8（IL-8）分泌水平均高于人间充质干细胞；具核梭杆菌可在两种细胞系中诱导产生时间依赖性的细胞因子分泌，而牙龈卟啉单胞菌在共培养实验中刺激IL-8分泌的效果相对较弱。 结论与意义 本研究结果表明，人间充质干细胞可适用于口腔内的缺氧区域；其与局部致病菌的相互作用并不会引发剧烈的促炎性细胞因子应答；本研究构建的实验体系可为后续开展口腔间充质干细胞体内研究的参数优化提供参考依据。