title: Boron nitride nanotube-functionalised myoblast/microfibre constructs: a nanotech-assisted tissue-engineered platform for muscle stimulation.
abstract: In this communication, we introduce boron nitride nanotube (BNNT)-functionalised muscle cell/microfibre mesh constructs, obtained via tissue engineering, as a three-dimensional (3D) platform to study a wireless stimulation system for electrically responsive cells and tissues. Our stimulation strategy exploits the piezoelectric behaviour of some classes of ceramic nanoparticles, such as BNNTs, able to polarize under mechanical stress, e.g. using low-frequency ultrasound (US). In the microfibre scaffolds, C2C12 myoblasts were able to differentiate into viable myotubes and to internalize BNNTs, also upon US irradiation, so as to obtain a nanotech-assisted 3D in vitro model. We then tested our stimulatory system on 2D and 3D cellular models by investigating the expression of connexin 43 (Cx43), as a molecule involved in cell crosstalk and mechanotransduction, and myosin, as a myogenic differentiation marker. Cx43 gene expression revealed a marked model dependency. In control samples (without US and/or BNNTs), Cx43 was upregulated under 2D culture conditions (10.78 ± 1.05-fold difference). Interactions with BNNTs increased Cx43 expression in 3D samples. Cx43 mRNA dropped in 2D under the 'BNNTs + US' regimen, while it was best enhanced in 3D samples (3.58 ± 1.05 vs 13.74 ± 1.42-fold difference, p = 0.0001). At the protein level, the maximal expressions of Cx43 and myosin were detected in the 3D model. In contrast with the 3D model, in 2D cultures, BNNTs and US exerted a synergistic depletive effect upon myosin synthesis. These findings indicate that model dimensionality and stimulatory regimens can strongly affect the responses of signalling and differentiation molecules, proving the importance of developing proper in vitro platforms for biological modelling.
authors.affiliation: Department of Surgical, Medical and Molecular Pathology and Emergency Medicine, University of Pisa, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Danti
authors.affiliation: Istituto Italiano di Tecnologia (IIT), Centre for Micro-BioRobotics@SSSA, Pontedera, PI, Italy.
authors.forename: Gianni
authors.identifier: http://orcid.org/0000-0003-1192-3647
authors.initials: G
authors.lastname: Ciofani
authors.affiliation: Department of Innovative Technologies, University of Applied Sciences and Arts of Southern Switzerland (SUPSI), Manno, Switzerland.
authors.forename: Gianni
authors.initials: G
authors.lastname: Pertici
authors.affiliation: Department of Clinical and Experimental Medicine, University of Pisa, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Moscato
authors.affiliation: Department of Surgical, Medical and Molecular Pathology and Emergency Medicine, University of Pisa, Italy.
authors.forename: Delfo
authors.initials: D
authors.lastname: D'Alessandro
authors.affiliation: Department of Clinical and Experimental Medicine, University of Pisa, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Ciabatti
authors.affiliation: Department of Chemistry and Industrial Chemistry, University of Pisa, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Chiellini
authors.affiliation: Institute of Matter Structure, Tor Vergata Research Area, CNR, Rome, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: D'Acunto
authors.affiliation: Istituto Italiano di Tecnologia (IIT), Centre for Micro-BioRobotics@SSSA, Pontedera, PI, Italy.
authors.forename: Virgilio
authors.initials: V
authors.lastname: Mattoli
authors.affiliation: Department of Surgical, Medical and Molecular Pathology and Emergency Medicine, University of Pisa, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Berrettini
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis; D001896:Boron Compounds / Q000737:chemistry*; D002460:Cell Line; D018031:Connexin 43 / Q000096:biosynthesis; D005786:Gene Expression Regulation; D040542:Mechanotransduction, Cellular; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000378:metabolism*; D009218:Myosins / Q000096:biosynthesis; D043942:Nanotubes; D023822:Tissue Engineering; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D000943:Antigens, Differentiation; D001896:Boron Compounds; D018031:Connexin 43; C017282:boron nitride; D009218:Myosins
keywords: C2C12 cells; boron nitride nanotubes (BNNTs); connexin 43 (Cx43); muscle stimulation; myotubes; piezoelectricity; poly-l-lactic acid (PLLA) scaffold; three-dimensional (3D) model