Abstract
Guiding neuronal cell growth is desirable for neural tissue engineering but is very challenging. In this work, a self-assembling ultra-short surfactant-like peptide I3K which possesses positively charged lysine head groups, and hydrophobic isoleucine tails, was chosen to investigate its potential for guiding neuronal cell growth. The peptides were able to self-assemble into nanofibrous structures and interact strongly with silk fibroin (SF) scaffolds, providing a niche for neural cell attachment and proliferation. SF is an excellent biomaterial for tissue engineering. However neuronal cells, such as rat PC12 cells, showed poor attachment on pure regenerated SF (RSF) scaffold surfaces. Patterning of I3K peptide nanofibers on RSF surfaces significantly improved cellular attachment, cellular density, as well as morphology of PC12 cells. The live / dead assay confirmed that RSF and I3K have negligible cytotoxicity against PC12 cells. Atomic force microscopy (AFM) was used to image the topography and neurite formation of PC12 cells, where results revealed that self-assembled I3K nanofibers can support the formation of PC12 cell neurites. Immunolabelling also demonstrated that coating of I3K nanofibers onto the RSF surfaces not only increased the percentage of cells bearing neurites but also increased the average maximum neurite length. Therefore, the peptide I3K could be used as an alternative to poly-l-lysine for cell culture and tissue engineering applications. As micro-patterning of neural cells to guide neurite growth is important for developing nerve tissue engineering scaffolds, inkjet printing was used to pattern self-assembled I3K peptide nanofibers on RSF surfaces for directional control of PC12 cell growth. The results demonstrated that inkjet-printed peptide micro-patterns can effectively guide the cell alignment and organization on RSF scaffold surfaces, providing great potential for nerve regeneration applications.
Highlights
The human nervous system is composed of the central nervous system (CNS) and the peripheral nervous system (PNS), which can be impaired by injuries, such as trauma and car accidents, as well as diseases, including Alzheimer’s disease, Parkinson’s disease, strokes and brain tumours.[1,2,3]
As inkjet printing is a non-contact technique, crosscontamination of the final product is significantly reduced. It was used as a micro-patterning technique to pattern selfassembled peptide nanofibers on regenerated silk fibroin (RSF) surface to guide the growth of neuronal PC12 cells in this study
RSF scaffolds have been extensively used in tissue engineering applications.[23]
Summary
The human nervous system is composed of the central nervous system (CNS) and the peripheral nervous system (PNS), which can be impaired by injuries, such as trauma and car accidents, as well as diseases, including Alzheimer’s disease, Parkinson’s disease, strokes and brain tumours.[1,2,3] Regeneration of both damaged CNS and PNS is challenging in tissue engineering.[3,4,5] It is vitally important to develop well-defined functional scaffolds for nerve tissue regeneration to help guide neural cell attachment, alignment, spreading and proliferation.[6,7,8]. Micro-patterning technology, which has already attracted significant attention, can enable the geometric control of neuronal cell alignment.[9,10,11,12] Lithography, including ultraviolet lithography (UVL), soft lithography (SL) and electron-beam lithography (EBL), is a traditional technology for micro-patterning proteins onto substrates.[13,14] Compared to UVL and EBL, SL is a convenient technique,[15] which has been widely used to micro-pattern neuronal cells.[9,10,12] For example, micro-patterned polydimethylsiloxane (PDMS) has been shown to enhance the attachment, alignment, spreading, proliferation, neurite formation and elongation of neuronal cells.[12] SL needs to be operated in a highstandard clean room, and samples can be contaminated during fabrication.[16,17] Inkjet printing, on the other hand, is a costeffective and flexible micro-patterning technique which is capable of patterning complex geometries at high precision.[13,14] as inkjet printing is a non-contact technique, crosscontamination of the final product is significantly reduced It was used as a micro-patterning technique to pattern selfassembled peptide nanofibers on regenerated silk fibroin (RSF) surface to guide the growth of neuronal PC12 cells in this study. Cells attached along the inkjet-printed peptide nanofiber patterns, demonstrating that inkjet printing is a promising technique to pattern scaffolds for geometrical guidance of neuronal cell growth as well as investigation of neurite development and formation in vitro.[42]
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