Neurite outgrowth on well-characterized surfaces: preparation and characterization of chemically and spatially controlled fibronectin and RGD substrates with good bioactivity.
Study of axonal growth and ligand-receptor interactions requires specificity and careful characterization of the biomaterial substrates to which the neurons bind. It would be impossible to predict the effects of important variables such as composition, surface density, spatial distribution, and conformation of the ligands on axonal growth of a neuron without highly specific surface characterization. Here, we compare two methods of surface modification (hereafter referred to as "Heterobifunctional Crosslinker" and "Pluronics" methods) used for immobilization of fibronectin (FN) and FN-derived, RGD-containing peptides to the substrates. We also characterized their performance in neurite outgrowth experiments. Various surface analytical techniques such as contact angle measurement, XPS, and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were used for the analysis of the substrates at each step of the two different chemistries involved. FN-patterned surfaces were created by micro-contact printing methods and confirmed by imaging TOF-SIMS, and AFM techniques. After immobilization of FN and/or FN-derived RGD-containing peptide, including the formation of micron-scale patterns of FN, the modified surfaces were plated with neurons from postnatal rat dorsal root ganglia (DRG) and incubated in serum-free medium. Both the peptide- and/or protein-modified substrates supported significantly greater neurite outgrowth than controls, and outgrowth on both substrate chemistries was inhibited by the addition of soluble RGD peptide. Patterned FN surfaces were successful in spatially controlling the neuron attachment and outgrowth.
Zhang
Zhanping
Z
Department of Chemistry & Biochemistry, 175 Brown Lab, University of Delaware, Newark, DE 19716, USA.
Yoo
Raphael
R
Wells
Matthew
M
Beebe
Thomas P
TP
Biran
Roy
R
Tresco
Patrick
P
D000327:Adsorption; D000818:Animals; D000831:Animals, Newborn; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018914:Cells, Immobilized / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003460:Crystallization / Q000379:methods*; D005353:Fibronectins / Q000737:chemistry* / Q000494:pharmacology; D008422:Materials Testing; D016501:Neurites / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure*; D009842:Oligopeptides / Q000737:chemistry* / Q000494:pharmacology; D020671:Posterior Horn Cells / Q000187:drug effects / Q000254:growth & development / Q000648:ultrastructure; D011485:Protein Binding; D051381:Rats; D023822:Tissue Engineering
D020099:Coated Materials, Biocompatible; D003432:Cross-Linking Reagents; D005353:Fibronectins; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid