Corey Joseph M, Gertz Caitlyn C, Wang Bor-Shuen, Birrell Lisa K, Johnson Sara L, Martin David C, Feldman Eva L
Department of Neurology, The University of Michigan, Ann Arbor, MI 48109, USA.
Acta Biomater. 2008 Jul;4(4):863-75. doi: 10.1016/j.actbio.2008.02.020. Epub 2008 Mar 12.
Aligned electrospun nanofibers direct neurite growth and may prove effective for repair throughout the nervous system. Applying nanofiber scaffolds to different nervous system regions will require prior in vitro testing of scaffold designs with specific neuronal and glial cell types. This would be best accomplished using primary neurons in serum-free media; however, such growth on nanofiber substrates has not yet been achieved. Here we report the development of poly(L-lactic acid) (PLLA) nanofiber substrates that support serum-free growth of primary motor and sensory neurons at low plating densities. In our study, we first compared materials used to anchor fibers to glass to keep cells submerged and maintain fiber alignment. We found that poly(lactic-co-glycolic acid) (PLGA) anchors fibers to glass and is less toxic to primary neurons than bandage and glue used in other studies. We then designed a substrate produced by electrospinning PLLA nanofibers directly on cover slips pre-coated with PLGA. This substrate retains fiber alignment even when the fiber bundle detaches from the cover slip and keeps cells in the same focal plane. To see if increasing wettability improves motor neuron survival, some fibers were plasma etched before cell plating. Survival on etched fibers was reduced at the lower plating density. Finally, the alignment of neurons grown on this substrate was equal to nanofiber alignment and surpassed the alignment of neurites from explants tested in a previous study. This substrate should facilitate investigating the behavior of many neuronal types on electrospun fibers in serum-free conditions.
排列整齐的电纺纳米纤维可引导神经突生长，并可能被证明对整个神经系统的修复有效。将纳米纤维支架应用于不同的神经系统区域需要事先在体外使用特定的神经元和神经胶质细胞类型对支架设计进行测试。这最好在无血清培养基中使用原代神经元来完成；然而，尚未实现原代神经元在纳米纤维基质上的这种生长。在此，我们报告了聚（L-乳酸）（PLLA）纳米纤维基质的开发，该基质能在低接种密度下支持原代运动神经元和感觉神经元的无血清生长。在我们的研究中，我们首先比较了用于将纤维固定到玻璃上以保持细胞浸没并维持纤维排列的材料。我们发现聚（乳酸-共-乙醇酸）（PLGA）能将纤维固定到玻璃上，并且对原代神经元的毒性比其他研究中使用的绷带和胶水小。然后，我们设计了一种通过将PLLA纳米纤维直接静电纺丝在预涂有PLGA的盖玻片上制成的基质。即使纤维束从盖玻片上分离，该基质仍能保持纤维排列，并使细胞保持在同一焦平面。为了观察增加润湿性是否能提高运动神经元的存活率，一些纤维在细胞接种前进行了等离子体蚀刻。在较低接种密度下，蚀刻纤维上的存活率降低。最后，在该基质上生长的神经元的排列与纳米纤维排列相当，并且超过了先前研究中测试的外植体神经突的排列。这种基质应有助于在无血清条件下研究许多神经元类型在电纺纤维上的行为。
