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.

3D Bioprinting in Skeletal Muscle Tissue Engineering

“…They observed high cell compartmentalization in each half of the extruded fiber. After 5 d of culture, good spatial organization with myotube formation in the C2C12 printed half and fibroblast compartmentalization in the opposite side was observed . To avoid cell deposition in the ink and to allow longer bioprinting, materials that act as surfactants have also been added into the inks.…”

“…Janus‐like compartmentalization of the two cell types remained after 5 d of culture. In vitro analysis at day 21 of culture showed aligned, multinucleated, fully striated myotubes with abundant MHC . Moreover, after 7 d in vitro culture the constructs were implanted subcutaneously in mice and were retrieved after 28 d. The analysis showed complete maturation of tightly packed fully striated myotubes.…”

“…d) Microfluidic printing head used to precisely deposit heterogeneous janus‐like hydrogel fibers containing myoblasts and fibroblasts. Adapted with permission . Copyright 2017, Elsevier.…”

Microfluidic Platforms toward Rational Material Fabrication for Biomedical Applications

“…Fourth, the bioprinting of low‐viscosity cell‐laden bioinks via a microfluidic nozzle holds promise in forming thick 3D multicellular constructs for tissue engineering . Through microfluidic‐assisted bioprinting, multicellular constructs with well‐defined geometries and appropriately arranged cell organization resembling both the macroscopic shapes and complicated anatomy of human tissues/organs have been established . Equipped with microfluidic nozzles with multichannel or coaxial multichannel designs, bioinks with tunable compositions and multicellular incorporation could be printed in extrusion‐based methods (Figure e), which offer robust strategies for the rapid and continuous construction of thick tissue‐like structures with hierarchical and heterogeneous architectures .…”

Functional characterization of 3D contractile smooth muscle tissues generated using a unique microfluidic 3D bioprinting technology

“…Previous research into bioprinting of muscle tissues has demonstrated some advantages of 3D bioprinted tissues when compared to cast hydrogels, 34 namely induction of differentiation markers such as myosin heavy chain and sarcomere formation for skeletal muscle cells. Printed dECM-containing skeletal muscle constructs develop larger myotubes with a greater number of acetylcholine receptors compared to collagen printed muscle constructs.…”