Myoblast 3D bioprinting to burst in vitro skeletal muscle differentiation

Abstract

Skeletal muscle regeneration is one of the major areas of interest in sport medicine as well as trauma centers. Three-dimensional (3D) bioprinting (BioP) is nowadays widely adopted to manufacture 3D constructs for regenerative medicine but a comparison between the available biomaterial-based inks (bioinks) is missing. The present study aims to assess the impact of different hydrogels on the viability, proliferation, and differentiation of murine myoblasts (C2C12) encapsulated in 3D bioprinted constructs aided to muscle regeneration. We tested three different commercially available hydrogels bioinks based on: (1) gelatin methacrylate and alginate crosslinked by UV light; (2) gelatin methacrylate, xanthan gum, and alginate-fibrinogen; (3) nanofibrillated cellulose (NFC)/alginate-fibrinogen crosslinked with calcium chloride and thrombin. Constructs embedding the cells were manufactured by extrusion-based BioP and C2C12 viability, proliferation, and differentiation were assessed after 24 h, 7, 14, 21, and 28 days in culture. Although viability, proliferation, and differentiation were observed in all the constructs, among the investigated bioinks, the best results were obtained by using NFC/alginatefibrinogen-based hydrogel from 7 to 14 days in culture, when the embedded myoblasts started fusing, forming at day 21 and day 28 multinucleated myotubes within the 3D bioprinted structures. The results revealed an extensive myotube alignment all over the linear structure of the hydrogel, demonstrating cell maturation, and enhanced myogenesis. The bioprinting strategies that we describe here denote a strong and endorsed approach for the creation of in vitro artificial muscle to improve skeletal muscle tissue engineering for future therapeutic applications.

Alginate-Lysozyme Nanofibers Hydrogels with Improved Rheological Behavior, Printability and Biological Properties for 3D Bioprinting Applications

“…Commonly used nanomaterials include inorganic (e.g., magnetic, gold, silver [34]) nanoparticles and carbon nanotubes [35]. Nevertheless, the lack of biocompatibility of some of these materials has raised interest in using biobased nanofibrillar structures [36] particularly cellulose nanostructures, such as cellulose nanofibrils [37], cellulose nanocrystals [38] and bacterial nanocellulose [39], to reinforce alginate-based hydrogel bioinks.…”

Hydrogels: From Design to Applications in Forensic Investigations

“…Murine myoblast viability, differentiation, and proliferation were studied. The results displayed the positive fusion of myogenic cells to form a myotube within 21–28 days [100] . These types of hydrogels are not only attractive for tissue engineering or medicine but also forensic, as they can help in traumatic forensic injury cases.…”

A Guide to Polysaccharide-Based Hydrogel Bioinks for 3D Bioprinting Applications

“…The high diversity of chemical structures and properties of these plant polysaccharides opens the possibility for their application in several fields, including in the food area (e.g., as gelling agents) [ 97 ], in the biomedical field (e.g., in drug delivery systems) [ 98 ], and, more recently, in tissue engineering [ 99 ]. The interest in using some of these plant-derived polysaccharides in the formulation of bioinks for 3D bioprinting applications has also grown considerably in the later years, as summed up in Table 2 , with several works using cellulose and nanocelluloses [ 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 ] and pectin [ 119 , 120 ].…”

“…CELLINK Bioink from Cellink [ 132 ]; throughout the years, not much evolution and creativity has been observed regarding the use of NFC as a bioink component. Most recent studies still describe its combination with alginate for bioprinting of different cell lines, namely human cartilage [ 115 , 116 ], human dermis [ 117 ], bone [ 102 , 118 ], and skeletal muscle [ 103 ]. Additionally, nanocelluloses are still mainly used as reinforcing components for hydrogel bioinks due to their excellent mechanical properties.…”