title: Microfluidic-enhanced 3D bioprinting of aligned myoblast-laden hydrogels leads to functionally organized myofibers in vitro and in vivo.
abstract: We present a new strategy for the fabrication of artificial skeletal muscle tissue with functional morphologies based on an innovative 3D bioprinting approach. The methodology is based on a microfluidic printing head coupled to a co-axial needle extruder for high-resolution 3D bioprinting of hydrogel fibers laden with muscle precursor cells (C2C12). To promote myogenic differentiation, we formulated a tailored bioink with a photocurable semi-synthetic biopolymer (PEG-Fibrinogen) encapsulating cells into 3D constructs composed of aligned hydrogel fibers. After 3-5 days of culture, the encapsulated myoblasts started migrating and fusing, forming multinucleated myotubes within the 3D bioprinted fibers. The obtained myotubes showed high degree of alignment along the direction of hydrogel fiber deposition, further revealing maturation, sarcomerogenesis, and functionality. Following subcutaneous implantation in the back of immunocompromised mice, bioprinted constructs generated organized artificial muscle tissue in vivo. Finally, we demonstrate that our microfluidic printing head allows to design three dimensional multi-cellular assemblies with an exquisite compartmentalization of the encapsulated cells. Our results demonstrate an enhanced myogenic differentiation with the formation of parallel aligned long-range myotubes. The approach that we report here represents a robust and valid candidate for the fabrication of macroscopic artificial muscle to scale up skeletal muscle tissue engineering for human clinical application.
authors.affiliation: Tissue Engineering Lab, Università Campus Bio-Medico di Roma, Rome, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Costantini
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Testa
authors.affiliation: Tissue Engineering Lab, Università Campus Bio-Medico di Roma, Rome, Italy.
authors.forename: Pamela
authors.initials: P
authors.lastname: Mozetic
authors.affiliation: Department of Chemistry, Sapienza University of Rome, Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Barbetta
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Fuoco
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Ersilia
authors.initials: E
authors.lastname: Fornetti
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Tamiro
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Bernardini
authors.affiliation: Warsaw University of Technology, Faculty of Materials Science and Engineering, Warsaw, Poland.
authors.forename: Jakub
authors.initials: J
authors.lastname: Jaroszewicz
authors.affiliation: Warsaw University of Technology, Faculty of Materials Science and Engineering, Warsaw, Poland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Święszkowski
authors.affiliation: Tissue Engineering Lab, Università Campus Bio-Medico di Roma, Rome, Italy.
authors.forename: Marcella
authors.initials: M
authors.lastname: Trombetta
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Castagnoli
authors.affiliation: Department of Biomedical Engineering, Techion Institute, Haifa, Israel.
authors.forename: Dror
authors.initials: D
authors.lastname: Seliktar
authors.affiliation: Institute of Physical Chemistry, Polish Academy of Sciences, 01224 Warsaw, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Garstecki
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Gianni
authors.initials: G
authors.lastname: Cesareni
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Cannata
authors.affiliation: Tissue Engineering Lab, Università Campus Bio-Medico di Roma, Rome, Italy. Electronic address: a.rainer@unicampus.it.
authors.forename: Alberto
authors.initials: A
authors.lastname: Rainer
authors.affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy. Electronic address: cesare.gargioli@uniroma2.it.
authors.forename: Cesare
authors.initials: C
authors.lastname: Gargioli
mesh_terms: D000818:Animals; D062028:Bioprinting / Q000295:instrumentation* / Q000379:methods; D002460:Cell Line; D004867:Equipment Design; D020100:Hydrogels / Q000737:chemistry*; D056656:Lab-On-A-Chip Devices; D008297:Male; D016513:Mice, SCID; D024510:Muscle Development; D032446:Myoblasts / Q000166:cytology* / Q000637:transplantation; D066330:Printing, Three-Dimensional / Q000295:instrumentation; D023822:Tissue Engineering / Q000295:instrumentation* / Q000379:methods; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D020100:Hydrogels
keywords: Artificial muscle; Microfluidic enhanced 3D bioprinting; Myogenic precursor cells; Myotubes; PEG-Fibrinogen hydrogel