In 2016-2018, chitosan (Chi) based bendable bioplatform was fabricated for cell adhesion, spreading, proliferations and electrical stimulation. In particular, during the first year of the fellowship, a bioresorbable, biodegradable, biocompatible and readily conformable Chi substrate was achieved. Major activities in first year of the fellowship were focused on WP-1 and WP-2 for achieving Deliverables D1 to Deliverables 6.
Chi powder was solubilized in acidic aqueous solution (1% acetic acid) and casted on petri dishes and allowed for air drying overnight to yield thin, clear, conformable substrate with smooth surface topography. Aqueous solubility was overcome with crosslinking process while stability in acetone utilized in dissolving sacrificial layer during metallization process. Compatibility of the substrate was assessed with HDF cell adhesion and proliferation. The ultra-thin Chi substrate was fabricated on silicon wafer was used for metallization to realize Au micro-gap electrodes on it.
In vitro biocompatibility assay showed poor adhesion, spreading and proliferation of cell on glutaraldehyde cross-linked scaffold. Whereas, farm adhesion, well spreading and enhance proliferation was noticed when glutaraldehyde cross-linked substrate were blocked in 1M Gly. Thus, the interdigitated platform was functionalized with adhesion molecule (RGD peptide) which results farm cell adhesion and enhanced spreading and proliferation.
In the second year of this fellowship, we realized electrophysiological state of living cell and thereby explored the possibilities of regulating neuronal differentiation mechanism using electrical stimulation from external sources. For this, conductivity of the membrane was achieved with establishing a nanoscale conductive Au layer (15nm) on it and thus the Chi substrate was prepared for electrical stimulation study.
Major activities in second year of the fellowship were focused on WP-2 partly, WP-3 and WP-4 for achieving Deliverables D7 to Deliverables 10:
The cell immobilized Chi substrate was subjected to electrochemical investigation using CV. The CV obtained from cell cultured Chi-GO substrate showed a quasi-reversible redox peak with cathodic peak (Ipc) at +300mV and anodic peak (Ipa) at -300mV. The absence of such peak from a device without cell confirms that redox is originated from the immobilized HDF. The redox peak showed stability to scan rates and scan cycle. This indicates that the electrical signal is stable and repeatable. Considering stability and repeatability redox peak, together with mechanical properties discussed earlier, the device is suitable for in vivo application.
This device is able to monitor cyto-physiologic state by analyzing and quantifying the redox signal. Besides, the microscopic imaging technique was employed for the morphologic investigation of neurite differentiation. Images obtained with phase contrast microscopy showed neurites structure of a differentiated live neuron whereas the scanning microscopic images from fixed cell showed the more details neurite differentiation. The molecular characterization of nerite specific markers has not done yet because of time limitation of the project. I am still continuing the work in my home institute for the molecular confirmations of the electrically stimulated neurite differentiation.
Apart from these research activities, I was involved with the routine activities of the BEST group by attending scheduled group discussions, data meeting and seminar and symposium. I have disseminated part of my research output through attending IEEE sensors conference 2017 and 2018 and European researcher’s nights 2016 and 2017. A couple of my research articles are on the publication process.
