Zuidema Jonathan M, Hyzinski-García María C, Van Vlasselaer Kristien, Zaccor Nicholas W, Plopper George E, Mongin Alexander A, Gilbert Ryan J
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY 12208, USA.
Biomaterials. 2014 Feb;35(5):1439-49. doi: 10.1016/j.biomaterials.2013.10.079. Epub 2013 Nov 15.
Bioengineered fiber substrates are increasingly studied as a means to promote regeneration and remodeling in the injured central nervous system (CNS). Previous reports largely focused on the ability of oriented scaffolds to bridge injured regions and direct outgrowth of axonal projections. In the present work, we explored the effects of electrospun microfibers on the migration and physiological properties of brain astroglial cells. Primary rat astrocytes were cultured on either fibronectin-coated poly-L-lactic acid (PLLA) films, fibronectin-coated randomly oriented PLLA electrospun fibers, or fibronectin-coated aligned PLLA electrospun fibers. Aligned PLLA fibers strongly altered astrocytic morphology, orienting cell processes, actin microfilaments, and microtubules along the length of the fibers. On aligned fibers, astrocytes also significantly increased their migration rates in the direction of fiber orientation. We further investigated if fiber topography modifies astrocytic neuroprotective properties, namely glutamate and glutamine transport and metabolism. This was done by quantifying changes in mRNA expression (qRT-PCR) and protein levels (Western blotting) for a battery of relevant biomolecules. Interestingly, we found that cells grown on random and/or aligned fibers increased the expression levels of two glutamate transporters, GLAST and GLT-1, and an important metabolic enzyme, glutamine synthetase, as compared to the fibronectin-coated films. Functional assays revealed increases in glutamate transport rates due to GLT-1 mediated uptake, which was largely determined by the dihydrokainate-sensitive GLT-1. Overall, this study suggests that aligned PLLA fibers can promote directed astrocytic migration, and, of most importance, our in vitro results indicate for the first time that electrospun PLLA fibers can positively modify neuroprotective properties of glial cells by increasing rates of glutamate uptake.
生物工程纤维基质作为促进受损中枢神经系统（CNS）再生和重塑的一种手段，正受到越来越多的研究。先前的报道主要集中在定向支架桥接受损区域和引导轴突投射生长的能力上。在本研究中，我们探讨了电纺微纤维对脑星形胶质细胞迁移和生理特性的影响。将原代大鼠星形胶质细胞培养在纤连蛋白包被的聚-L-乳酸（PLLA）薄膜、纤连蛋白包被的随机取向PLLA电纺纤维或纤连蛋白包被的取向PLLA电纺纤维上。取向PLLA纤维强烈改变星形胶质细胞的形态，使细胞突起、肌动蛋白微丝和微管沿纤维长度方向排列。在取向纤维上，星形胶质细胞在纤维取向方向上的迁移速率也显著增加。我们进一步研究了纤维拓扑结构是否会改变星形胶质细胞的神经保护特性，即谷氨酸和谷氨酰胺的转运及代谢。这是通过定量一系列相关生物分子的mRNA表达变化（qRT-PCR）和蛋白质水平（蛋白质印迹法）来实现的。有趣的是，我们发现与纤连蛋白包被的薄膜相比，在随机和/或取向纤维上生长的细胞增加了两种谷氨酸转运体GLAST和GLT-1以及一种重要代谢酶谷氨酰胺合成酶的表达水平。功能测定显示，由于GLT-1介导的摄取，谷氨酸转运速率增加，这在很大程度上由对二氢 kainate敏感的GLT-1决定。总体而言，本研究表明取向PLLA纤维可促进星形胶质细胞的定向迁移，最重要的是，我们的体外研究结果首次表明，电纺PLLA纤维可通过提高谷氨酸摄取速率来正向改变胶质细胞的神经保护特性。
