Extracellular matrix stiffness negatively affects axon elongation, growth cone area and F‐actin levels in a collagen type I 3D culture

神经突 生长锥 细胞外基质 轴突 细胞骨架 细胞生物学 生物物理学 化学 延伸率 体内 肌动蛋白 机械转化 神经科学 生物 体外 材料科学 生物化学 极限抗拉强度 细胞 生物技术 冶金
作者
Gaby F. Martínez,Jimena Fagetti,Gabriela Vierci,M. Mónica Brauer,Nicolás Unsain,Analía Richeri
出处
期刊:Journal of Tissue Engineering and Regenerative Medicine [Wiley]
卷期号:16 (2): 151-162 被引量:6
标识
DOI:10.1002/term.3269
摘要

Three dimensional (3D) in vitro neuronal cultures can better reproduce physiologically relevant phenotypes compared to 2D-cultures, because in vivo neurons reside in a 3D microenvironment. Interest in neuronal 3D cultures is emerging, with special attention to the mechanical forces that regulate axon elongation and sprouting in three dimensions. Type I collagen (Col-I) is a native substrate since it is present in the extracellular matrix and hence emulates an in vivo environment to study axon growth. The impact of its mechanical properties needs to be further investigated. Here, we generated Col-I 3D matrices of different mechanical stiffness and evaluated axon growth in three dimensions. Superior cervical ganglion (SCG) explants from neonatal rats were cultured in soft and stiff Col-I 3D matrices and neurite outgrowth was assessed by measuring: maximum neuritic extent; neuritic halo area and fasciculation. Axonal cytoskeletal proteins were examined. Axon elongation in stiff Col-I 3D matrices was reduced (31%) following 24 h in culture compared to soft matrices. In stiff matrices, neurites fasciculated and formed less dense halos. Consistently, almost no F-actin rich growth cones were recognized, and F-actin staining was strongly reduced in the axonal compartment. This study shows that stiffness negatively affects 3D neurite outgrowth and adds insights on the cytoskeletal responses upon mechanic interactions of axons with a 3D environment. Our data will serve to facilitate the development of model systems that are mechanically well-behaved but still mimic key physiologic properties observed in vivo.
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