3D printed oxidized alginate-gelatin bioink provides guidance for C2C12 muscle precursor cell orientation and differentiation via shear stress during bioprinting

Abstract

Biofabrication can be a tool to three-dimensionally (3D) print muscle cells embedded inside hydrogel biomaterials, ultimately aiming to mimic the complexity of the native muscle tissue and to create in-vitro muscle analogues for advanced repair therapies and drug testing. However, to 3D print muscle analogues of high cell alignment and synchronous contraction, the effect of biofabrication process parameters on myoblast growth has to be understood. A suitable biomaterial matrix is required to provide 3D printability as well as matrix degradation to create space for cell proliferation, matrix remodelling capacity, and cell differentiation. We demonstrate that by the proper selection of nozzle size and extrusion pressure, the shear stress during extrusion-bioprinting of mouse myoblast cells (C2C12) can achieve cell orientation when using oxidized alginate-gelatin (ADA-GEL) hydrogel bionk. The cells grow in the direction of printing, migrate to the hydrogel surface over time, and differentiate into ordered myotube segments in areas of high cell density. Together, our results show that ADA-GEL hydrogel can be a simple and cost-efficient biodegradable bioink that allows the successful 3D bioprinting and cultivation of C2C12 cells in-vitro to study muscle engineering.

Mechanical properties of cell- and microgel bead-laden oxidized alginate-gelatin hydrogels

“…This multi-modal mechanical assessment of oxidized alginate-gelatin hydrogels incorporating cells and PAAm microgel bead fillers has important applications for various tissue engineering applications. 10,75,84,85,104…”

“…28 The applicability of oxidized alginate-gelatin (ADA-GEL) hydrogels has been demonstrated in several TE applications, 75 including bioprinting approaches embedding cells. 60,[82][83][84][85][86][87] However, the influence of cells embedded in ADA-GEL hydrogels on the detailed resulting complex mechanical characteristics still remains unknown.…”

“…ADA-GEL hydrogels consist of ionically crosslinked oxidized alginate polysaccharide combined with reversibly, covalently bound gelatin, which contrasts with fibrous, network-building collagen or fibrin-based hydrogels, on which the impact of cell-fillers has been investigated before. 50 We have shown in previous studies by second harmonic generation (SHG) imaging that ADA-GEL does not possess highly ordered fibrillar structures, 84,88 hence can be assumed as a non-fibrillar hydrogel. Its non-fibrous crosslinking structure renders ADA-GEL as an excellent candidate to study the effect of cellular fillers on the mechanical properties of non-fibrous hydrogel matrices, especially as this hydrogel proved its potential for TE 75,76 and biofabrication 82,84,85,89,90 applications.…”

Comparison of Hydrogels for the Development of Well-Defined 3D Cancer Models of Breast Cancer and Melanoma

“…Although alginate itself has poor printing properties due to low shape fidelity, a pre-crosslinking step with CaCO 3 and D-Glucono-δ-lactone can significantly improve the printing results without increasing the polymer density [64]. Due to the gelation properties of gelatin, ADA-GEL has also been shown to be printable [65]. Bioprinted HA-SH gels have also been produced by using, for example, the photo initiator Irgacure 2959 [66].…”

Bioengineeredin vitroskeletal muscles as new tools for muscular dystrophies preclinical studies

“…In order to mimic the extracellular environment and the native cellular morphology, the main bioengineering strategy is focused on the 3D encapsulation of muscular cell precursors in biocompatible materials. In the last years, 3D bioprinting, [43][44][45][46][47][48][49][50][51][52][53][54] hydrogel molding, [55][56][57][58][59] and microporous scaffolds [60][61][62] have been implemented to fabricate skeletal muscle tissues.…”