Drop-on-demand inkjet printing of amphiphilic peptide I3K micropatterns on regenerated silk fibroin surfaces to promote neuronal cell attachment and alignment

Silk is a biomaterial mainly composed of two proteins: silk sericin and silk fibroin [1]. After removing the silk sericin using a process called degumming, a regenerated silk fibroin (RSF) aqueous solution can be obtained. RSF has been used previously as a scaffold in tissue engineering due to its high biocompatibility and biodegradability and by varying the production and post-treatment process, RSF can be used for various biological purposes.
However, silk biomaterials can also lack the needed biological activity to support the adhesion and proliferation of cells [2, 3]. In this study, results show that the time of the degumming process can affect the final cell attachment on RSF surfaces. This would allow us to obtain a surface that can be tuned to either promote or prevent cell attachment and thus be used for different applications, while retaining its biocompatibility. Cell attachment of neuronal PC12-Adh cells was studied using crystal violet assay on RSF surfaces obtained from two different times of degumming. Moreover, SEM images and weight-loss during the degumming process were used to check that the removal of sericin was successful.
In addition, the non-adherent RSF surface is then used as a non-fouling surface for inkjet printing. Inkjet printing is a high definition technique that allows to deposit small volumes of liquid onto a surface to form a predefined pattern. This method allows the jetting of proteins and peptides under mild conditions with no waste. A novel amphiphilic peptide Ac-IIIK-NH2 (I3K) is used as a bio-ink to create patterns that can guide cell adhesion and proliferation. I3K is dissolved in an aqueous solution and left to self-assemble to form nanotubes with the positively charged hydrophilic domain facing outwards. In this case, the positively charged I3K strongly interacts with the RSF surfaces via electrostatic interactions preventing the peptide to be redissolved into the culture media. To evaluate cell attachment, proliferation, and differentiation PC12-Adh cells were cultured on the patterns via fluorescent microscopy.