Tracking cellular uptake, intracellular trafficking and fate of nanoclay particles in human bone marrow stromal cells
Tracking cellular uptake, intracellular trafficking and fate of nanoclay particles in human bone marrow stromal cells
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追踪人骨髓基质细胞中纳米粘土颗粒的细胞摄取、细胞内运输和命运
作者：
Mousa M
粘土纳米颗粒，特别是合成蒙脱石，由于其在生物材料设计中作为聚合物的交联剂和作为生长因子递送的蛋白质释放改性剂的效用，在组织工程和再生医学领域中引起了人们的兴趣。此外，最近的研究表明，对反应性干细胞和祖细胞群的成骨分化的直接影响。然而，对纳米粘土生物活性的机制，特别是纳米粘土-干细胞相互作用中涉及的细胞过程知之甚少。在这项研究中，我们采用共聚焦显微镜，电感耦合等离子体质谱和透射电子显微镜跟踪粘土纳米粒子和人骨髓基质细胞（hBMSCs）之间的相互作用。特别是，我们研究了纳米颗粒的细胞摄取机制和摄取动力学，细胞内运输途径和命运的内吞纳米粘土。我们发现纳米粘土颗粒以μ m大小的聚集体存在于细胞表面，通过网格蛋白介导的内吞作用进入hBMSCs，并且它们的摄取动力学在纳米粘土添加的第一周期间随时间线性增加。内吞的颗粒内体/溶酶体隔室内观察到，我们发现的证据，细胞内降解的纳米粘土和胞吐作用，以及自噬体活性的增加。抑制剂研究表明，内吞作用所需的纳米粘土上调碱性磷酸酶活性，但没有观察到类似的依赖性自噬。这项研究的性质纳米粘土干细胞的相互作用，特别是细胞内处理的nanosilicate，可以提供深入了解纳米粘土生物活性的机制，并通知成功利用粘土纳米粒子在生物材料设计。
Clay nanoparticles, in particular synthetic smectites, have generated interest in the field of tissue engineering and regenerative medicine due to their utility as cross-linkers for polymers in biomaterial design and as protein release modifiers for growth factor delivery. In addition, recent studies have suggested a direct influence on the osteogenic differentiation of responsive stem and progenitor cell populations. Relatively little is known however about the mechanisms underlying nanoclay bioactivity and in particular the cellular processes involved in nanoclay-stem cell interactions. In this study we employed confocal microscopy, inductively coupled plasma mass spectrometry and transmission electron microscopy to track the interactions between clay nanoparticles and human bone marrow stromal cells (hBMSCs). In particular we studied nanoparticle cellular uptake mechanisms and uptake kinetics, intracellular trafficking pathways and the fate of endocytosed nanoclay. We found that nanoclay particles present on the cell surface as μm-sized aggregates, enter hBMSCs through clathrin-mediated endocytosis, and their uptake kinetics follow a linear increase with time during the first week of nanoclay addition. The endocytosed particles were observed within the endosomal/lysosomal compartments and we found evidence for both intracellular degradation of nanoclay and exocytosis as well as an increase in autophagosomal activity. Inhibitor studies indicated that endocytosis was required for nanoclay upregulation of alkaline phosphatase activity but a similar dependency was not observed for autophagy. This study into the nature of nanoclay-stem cell interactions, in particular the intracellular processing of nanosilicate, may provide insights into the mechanisms underlying nanoclay bioactivity and inform the successful utilisation of clay nanoparticles in biomaterial design.