Immobilized laminin concentration gradients on electrospun fiber scaffolds for controlled neurite outgrowth

Abstract

Neuronal process growth is guided by extrinsic environmental cues such as extracellular matrix (ECM) proteins. Recent reports have described that the growth cone extension is superior across gradients of the ECM protein laminin compared to growth across uniformly distributed laminin. In this work, the authors have prepared gradients of laminin on aligned electrospun nanofibers for use as substrates for neuronal growth. The substrates therefore presented both topographical and chemical guidance cues. Step gradients were prepared by the controlled robotic immersion of plasma-treated polycaprolactone fibers reacted with N-hydroxysuccinimide into the protein solution. The gradients were analyzed using x-ray photoelectron spectroscopy and confocal laser scanning microscopy. Gradients with a dynamic range of protein concentrations were successfully generated and neurite outgrowth was evaluated using neuronlike pheochromocytoma cell line 12 (PC12) cells. After 10 days of culture, PC12 neurite lengths varied from 32.7 ± 14.2 μm to 76.3 ± 9.1 μm across the protein concentration gradient. Neurite lengths at the highest concentration end of the gradient were significantly longer than neurite lengths observed for cells cultured on samples with uniform protein coverage. Gradients were prepared both in the fiber direction and transverse to the fiber direction. Neurites preferentially aligned with the fiber direction in both cases indicating that fiber alignment has a more dominant role in controlling neurite orientation, compared to the chemical gradient.

Microtechnologies for studying the role of mechanics in axon growth and guidance

“…As mentioned earlier, self-assembling peptide nanofiber gels now provide independent control on the matrix stiffness (Sur et al, 2013 ). Effects of topography (maybe also matrix stiffness) and of haptotactic cues can also be compared in 3D by culturing cells on an electrospun nanofiber gel with a gradient of surface-bound laminin (Zander and Beebe, 2014 ). When the gradient was parallel to the nanofibers, PC12 neurites grew preferentially up the laminin gradient; however, when the gradient was imposed in the perpendicular direction, they were unaffected.…”

Fabrication and characterization of electrospun laminin‐functionalized silk fibroin/poly(ethylene oxide) nanofibrous scaffolds for peripheral nerve regeneration

“…Therefore, the plasma treatment has the main impact on decrease in hydrophobicity of SF/PEO scaffolds compared to LN functionalization. It was suggested that the plasma treatment of SF/PEO nanofibers cause partial decomposition of the scaffolds surfaces and induce O‐containing functional groups such as hydroxyl and carboxylic acid groups . These induced carboxylic acids alongside the aspartic (Asp) and glutamic (Glu) acids of SF could bind to the amine groups of LN and form amide bonds.…”

“…LN, one of the major components of ECM basement membrane, is a family of heterotrimeric glycoproteins (500–1000 kDa) which includes multi‐domain “cross‐like” structure of three polypeptide chains (i.e., a, b, and c) which characterize the functions and properties of the LN . LN contains the well‐characterized peptide sequences of RGD (Arg‐Gly‐Asp) and YIGSR (Tyr‐Ile‐Gly‐Ser‐Arg) presented in a‐chain, which has the function of promoting cell adhesion and migration, as well as the sequence of IKVAV (Ile‐Lys‐Val‐Ala‐Val) which is presented near the C‐terminal end of a1‐chain and promote neurite outgrowth . In addition to the capability of LN to modulate cell adhesion and stimulate neurite growth through its specific peptide sequences, it can essentially contribute in integrin signaling .…”

The Effect of Laminin Surface Modification of Electrospun Silica Nanofiber Substrate on Neuronal Tissue Engineering

“…Therefore, novel nontoxic and biocompatible inorganic-based materials have also been examined and explored as a desirable alternative [ 20 ]. Among several methods for the preparation of ECM-mimicking scaffold materials [ 12 , 21 ], electrospinning is a convenient technique typically used in the preparation of nanofibers for in vitro experiments [ 20 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ]. However, if materials need to be mass produced for clinical use, other possible methods such as pressure-coupled infusion gyration [ 31 ] can be considered.…”

“…For example, the proliferation of Schwann cells on the laminin-modified PCL-chitosan nanofiber substrate was tracked for 4 days [ 25 ], and on a laminin-modified PLCL core-shell nanofiber substrate for 7 days [ 26 ]. The neurite extensions of neuron-like PC12 cells on laminin-modified PLLA nanofiber substrates were observed for 5 days [ 27 ], and on a covalently-bonded laminin-PCL nanofiber for 10 days [ 28 ]. The neurite extension of DRG on a laminin-PCL blend nanofiber was traced for 4 days [ 29 ] and on a laminin-modified PLLA nanofiber for 6 days [ 30 ].…”