Ultrasound stimulation of piezoelectric ZnO films for cell growth

Tissue engineering is an approach of regenerative medicine aimed at the integration, proliferation and maturation of isolated cells in an artificial environment, often based on biocompatible materials, with the final goal of repairing or substituting a damaged organ or tissue. Among these materials, micro and nano-structured scaffolds with physical and chemical properties tunable on-demand by an external source of stimulation, also known as “smart materials”, represent promising tools for favouring a particular cellular behaviour, for instance during both proliferation and differentiation events. This Master Thesis work is focused on the fabrication and characterization of nanostructured zinc oxide (ZnO) smart piezoelectric scaffolds for bone tissue engineering that can be remotely activated by means of a mechanical stimulation provided under the form of ultrasounds (US), with the goal of generating physiologically relevant piezo-potentials favouring cell proliferation and differentiation. ZnO microparticles characterized by surface nanostructuring with flower-like morphology were synthetized and used for fabrication of films for cell culture. More in detail, ZnO particles were synthesized by means of a sol-gel hydrothermal technique and dispersed into an ethanol, water and acetic acid solution for deposition in the form of films on polyimide (PI) substrates. In order to increase uniformity and adhesion of the ZnO film to the substrate, the dispersion formulation was properly optimized and cast on PI substrates pre-treated with Argon plasma. To enhance biocompatibility of the obtained films, graphene oxide (GO) flakes, poly-2-hydroxyethil methacrylate (pHEMA) or a Gelatin/GO composite coatings were considered as physical barriers against Zn2+ release (potentially cytotoxic) and deposited atop of the ZnO scaffold surface by drop-casting method. Gelatin-based coatings were stabilized for usage at mild temperature during cell culture by crosslinking with genipin. The prepared samples were analysed in terms of chemical composition, crystal structure and morphology, by means of water contact angle and infrared spectroscopy measurements, X-Ray diffraction and field-emission scanning electron microscopy. Zn2+ release experiments were performed in vitro, to check the effectiveness of the barriers in limiting ion release. Electron paramagnetic resonance spectroscopy coupled with spin-trapping technique was used to evaluate the generation of potentially harmful Reactive Oxygen Species under US stimulation. The piezoelectric response of the prepared scaffolds under US stimulation was also evaluated. Preliminary tests of biocompatibility of the obtained substrates were conducted with SaOS-2 osteoblast-like cells, and by performing qualitative and quantitative analyses on cell viability with fluorescence microscopy and fluorimetry through Live/Dead and AlamarBlue staining, respectively. Layered smart scaffolds were successfully fabricated, characterized and optimized from the chemical and physical standpoints. ROS generation was shown to be decreased by the barrier coatings and an electrical response due to US stimulation was also demonstrated. Biological tests suggest that gelatin-GO coatings can prove useful to limit Zn2+ toxicity. Further studies on optimized samples may consider US stimulation during cell culture, to evaluate the effect of the piezoelectric activity of ZnO on cell proliferation and differentiation.