Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada.
Neuroscience and Mental Health Faculty, Lee Kong China School of Medicine, Nanyang Technological University, Singapore 308232, Singapore.
Cells. 2023 Mar 18;12(6):934. doi: 10.3390/cells12060934.
Biophysical parameters such as substrate topography and stiffness have been shown independently to elicit profound effects on neuronal differentiation and maturation from neural progenitor cells (NPCs) yet have not been investigated in combination. Here, the effects of various micrograting and stiffness combinations on neuronal differentiation and maturation were investigated using a polyacrylamide and N-acryloyl-6-aminocaproic acid copolymer (PAA-ACA) hydrogel with tunable stiffness. Whole laminin was conjugated onto the PAA-ACA surface indirectly or directly to facilitate long-term mouse and human NPC-derived neuron attachment. Three micrograting dimensions (2-10 µm) were patterned onto gels with varying stiffness (6.1-110.5 kPa) to evaluate the effects of topography, stiffness, and their interaction. The results demonstrate that the extracellular matrix (ECM)-modified PAA-ACA gels support mouse and human neuronal cell attachment throughout the differentiation and maturation stages (14 and 28 days, respectively). The interaction between topography and stiffness is shown to significantly increase the proportion of β-tubulin III (TUJ1) positive neurons and microtubule associated protein-2 (MAP2) positive neurite branching and length. Thus, the effects of topography and stiffness cannot be imparted. These results provide a novel platform for neural mechanobiology studies and emphasize the utility of optimizing numerous biophysical cues for improved neuronal yield in vitro.
生物物理参数，如基质的形貌和硬度，已经被证明可以独立地对神经祖细胞（NPCs）的神经元分化和成熟产生深远的影响，但尚未结合起来进行研究。在这里，使用具有可调硬度的聚丙烯酰胺和 N-丙烯酰基-6-氨基己酸共聚物（PAA-ACA）水凝胶研究了各种微图形和硬度组合对神经元分化和成熟的影响。全层粘连蛋白被间接或直接共轭到 PAA-ACA 表面上，以促进长期的小鼠和人 NPC 衍生神经元的附着。三种微图形尺寸（2-10 µm）被图案化到具有不同硬度（6.1-110.5 kPa）的凝胶上，以评估形貌、硬度及其相互作用的影响。结果表明，细胞外基质（ECM）修饰的 PAA-ACA 凝胶在分化和成熟阶段（分别为 14 天和 28 天）均支持小鼠和人神经元细胞的附着。形貌和硬度之间的相互作用显著增加了β-微管蛋白 III（TUJ1）阳性神经元和微管相关蛋白-2（MAP2）阳性神经突分支和长度的比例。因此，形貌和硬度的影响不能赋予。这些结果为神经机械生物学研究提供了一个新的平台，并强调了优化许多生物物理线索以提高体外神经元产量的实用性。
