• 3D bioprinting
• barium titanate
• skeletal muscle
• piezoelectric nanoparticles

Millions of people currently suffer from volumetric muscle loss yearly (VML). The current therapies for addressing VML reduce the symptoms deriving from the pathology, but without a significant restoration of the muscle functionality. The aim of skeletal muscle tissue engineering (SMTE) is to induce the regeneration of the skeletal muscle tissue by employing cells and biomaterials, exploiting cues that direct cell growth, differentiation, and maturation.
In the literature of SMTE researchers focus on: developing a macroscopic architecture, creating a biomimetic structure, finding the most effective protocols for external stimulation, and characterizing a promyogenic bioink to be used effectively in 3D bioprinting procedures. Efforts are missing in combining all these different aspects together.
The MIO-PRO project (“Muscoli ingegnerizzati paziente-specifici per il ripristino di canali MIOelettrici e il controllo di PROtesi”, PR19-CR-P1), funded and supported by INAIL, aims to develop 3D engineered contractile muscle tissues.
Within this framework, the objectives of this thesis are:
 To extensively characterize a promyogenic bioink doped with barium titanate piezoelectric nanoparticles, enabling future electrical stimulation mediated by ultrasound;
 To build a macroscopic and multilayered construct through 3D bioprinting;
 To provide the construct with intercalating filaments of a sacrificial bioink, for the creation of empty channels that facilitate medium diffusion.
In the thesis, a repeatable protocol for the preparation and storage of the cell-laden bioink and the sacrificial one, as well as a rheometric characterization of both materials, were defined. Moreover, the myoblast-laden bioink doped with piezoelectric nanoparticles was also characterized. Furthermore, a systematic evaluation of the effect of temperature on cell viability, and an optimization of the printing parameters for both materials were performed. Finally, a 3D macroscopic construct (20 mm x 10 mm x 2.5 mm) with intercalated filaments of both cell-laden and sacrificial bioink, alternated, was printed.
Future efforts should focus on the washing out and creation of empty channels inside the printed construct, and on the ultrasound stimulation of the samples. Another interesting aspect would be the extensive evaluation of cell alignment inside the 3D construct, both before and after the differentiation protocol, an aspect that was only preliminarily addressed in this thesis.