WP6: In vitro and in vivo tests
Task 6.1: In vitro tests (SSSA)
In vitro tests will be carried out at SSSA (The BioRobotics Institute) by using neural-like cell lines, such as the pheochromocytoma-derived cell line PC12, the human neuroblastoma cell line SH-SY5Y and a standard macrophage cell line (U937 or RAW 246.7), to assess inflammatory reaction. By means of standard tests such as WST-1, Pico-Green assay, MTT, etc., cytotoxicity and neural cell growth modulation will be assessed. In addition, gene and protein expression, cytokine production and metabolic-related indexes will be quantified by means of specific assays.
By means of these tests, important feedbacks concerning material safety and/or ability to promote or inhibit inflammatory response, neural migration, axonal sprouting, etc. will be obtained in the project course. The tests will be performed to evaluate the effects of different surface treatments, of zwitterionic and 2-oxazoline-based hydrogels, of degradable linkers and drugs / neurotrophic factors released in the cell culture environment and of piezoelectric nanoparticle-based ultrasound stimulation.
Task 6.2: In vivo tests (SSSA)
In vivo tests will be performed on Sprague-Dawley rats (200-250 g), which will be treated in accordance with the approved guidelines of SSSA (The Joint Center on Translational Neurorehabilitation).
In order to collect statistically relevant data, a total of 24 animals (12 from M12 to M17 and other 12 from M31 to M36) will be used for the evaluation of the efficacy of the different prototypes developed. In both the Task phases the animals will be divided in four groups (three rats per group). The first group will serve as control. The last group (including hydrogel, drugs and nanoparticles) will be stimulated, in the weeks following the implant, by the US set-up developed within Task 4.1. The US stimulation timeline (e.g. few minutes of stimulation, twice a day) will be properly defined within WP2.