Skeletal muscle differentiation of human iPSCs meets bioengineering strategies: perspectives and challenges

Although skeletal muscle repairs itself following small injuries, genetic diseases or severe damages may hamper its ability to do so. Induced pluripotent stem cells (iPSCs) can generate myogenic progenitors, but their use in combination with bioengineering strategies to modulate their phenotype has not been sufficiently investigated. This review highlights the potential of this combination aimed at pushing the boundaries of skeletal muscle tissue engineering. First, the overall organization and the key steps in the myogenic process occurring in vivo are described. Second, transgenic and non-transgenic approaches for the myogenic induction of human iPSCs are compared. Third, technologies to provide cells with biophysical stimuli, biomaterial cues, and biofabrication strategies are discussed in terms of recreating a biomimetic environment and thus helping to engineer a myogenic phenotype. The embryonic development process and the pro-myogenic role of the muscle-resident cell populations in co-cultures are also described, highlighting the possible clinical applications of iPSCs in the skeletal muscle tissue engineering field

Piezoelectric nanocomposite bioink and ultrasound stimulation modulate early skeletal myogenesis

Despite the significant progress in bioprinting for skeletal muscle tissue engineering, new stimuli-responsive bioinks to boost the myogenesis process are highly desirable. In this work, we developed a printable alginate/Pluronic-based bioink including piezoelectric barium titanate nanoparticles (nominal diameter: ∼60 nm) for the 3D bioprinting of muscle cell-laden hydrogels. The aim was to investigate the effects of the combination of piezoelectric nanoparticles with ultrasound stimulation on early myogenic differentiation of the printed structures. After the characterization of nanoparticles and bioinks, viability tests were carried out to investigate three nanoparticle concentrations (100, 250, and 500 μg mL−1) within the printed structures. An excellent cytocompatibility was confirmed for nanoparticle concentrations up to 250 μg mL−1. TEM imaging demonstrated the internalization of BTNPs in intracellular vesicles. The combination of piezoelectric nanoparticles and ultrasound stimulation upregulated the expression of MYOD1, MYOG, and MYH2 and enhanced cell aggregation, which is a crucial step for myoblast fusion, and the presence of MYOG in the nuclei. These results suggest that the direct piezoelectric effect induced by ultrasound on the internalized piezoelectric nanoparticles boosts myogenesis in its early phases

Effects of the 3D Geometry Reconstruction on the Estimation of 3D Porous Scaffold Permeability

3D scaffolds for tissue engineering typically need to adopt a dynamic culture to foster cell distribution and survival throughout the scaffold. It is, therefore, crucial to know fluids' behavior inside the scaffold architecture, especially for complex porous ones. Here we report a comparison between simulated and measured permeability of a porous 3D scaffold, focusing on different modeling parameters. The scaffold features were extracted by microcomputed tomography (μCT) and representative volume elements were used for the computational fluid-dynamic analyses. The objective was to investigate the sensitivity of the model to the degree of detail of the μCT image and the elements of the mesh. These findings highlight the pros and cons of the modeling strategy adopted and the importance of such parameters in analyzing fluid behavior in 3D scaffolds

Soft Perfusable Device to Culture Skeletal Muscle 3D Constructs in Air

Devices for in vitro culture of three-dimensional (3D) skeletal muscle tissues have multiple applications, including tissue engineering and muscle-powered biorobotics. In both cases, it is crucial to recreate a biomimetic environment by using tailored scaffolds at multiple length scales and to administer prodifferentiative biophysical stimuli (e.g., mechanical loading). On the contrary, there is an increasing need to develop flexible biohybrid robotic devices capable of maintaining their functionality beyond laboratory settings. In this study, we describe a stretchable and perfusable device to sustain cell culture and maintenance in a 3D scaffold. The device mimics the structure of a muscle connected to two tendons: Tendon−Muscle−Tendon (TMT). The TMT device is composed of a soft (E ∼ 6 kPa) porous (pore diameter: ∼650 μm) polyurethane scaffold, encased within a compliant silicone membrane to prevent medium evaporation. Two tendon-like hollow channels interface the scaffold with a fluidic circuit and a stretching device. We report an optimized protocol to sustain C2C12 adhesion by coating the scaffold with polydopamine and fibronectin. Then, we show the procedure for the soft scaffold inclusion in the TMT device, demonstrating the device’s ability to bear multiple cycles of elongations, simulating a protocol for cell mechanical stimulation. By using computational fluid dynamic simulations, we show that a flow rate of 0.62 mL/min ensures a wall shear stress value safe for cells (<2 Pa) and 50% of scaffold coverage by an optimal fluid velocity. Finally, we demonstrate the effectiveness of the TMT device to sustain cell viability under perfusion for 24 h outside of the CO2 incubator. We believe that the proposed TMT device can be considered an interesting platform to combine several biophysical stimuli, aimed at boosting skeletal muscle tissue differentiation in vitro, opening chances for the development of muscle-powered biohybrid soft robots with long-term operability in real-world environments

Biohybrid Microrobots

Thanks to millions of years of evolution, living beings have developed complex mechanisms for sensing, actuation, and adaptation to the surrounding environment. Biohybrid robots exploit these mechanisms by embedding living components and combining them with nonliving elements. This allows overcoming some of the issues affecting entirely artificial devices, such as difficult scalability to small scales, inability to self-heal, and possible immunogenicity. In this chapter, biohybrid microrobots based on bacteria or other single cells (e.g., sperm cells, erythrocytes, neutrophils) are described. The most relevant examples reported in the state-of-the-art are analyzed, focusing on their specific sensing and actuation mechanisms. Relying on the use of a complete and autonomous living organism, they must be considered examples of a top-down approach, which needs to find ways of adequately controlling them. Specific applications of these systems are also described, with a particular focus on clinical ones. Then, muscle-based multicellular robots are introduced. Such systems are based on a bottom-up approach, through which single contractile units (skeletal muscle cells, cardiomyocytes, or insect-derived cells) are assembled and integrated with materials supporting them, to achieve effective locomotion and other functions. The main applications and challenges related to multicellular biohybrid microrobots are described, mentioning among others, modeling issues and the need for implementing multiple degrees of freedom, yet keeping high controllability by an external user

Highly controlled and usable system for Low-Intensity Pulsed Ultrasound Stimulation of Cells

This work aims to describe the design and development of an in vitro highly controlled ultrasonic stimulation system able to guarantee, at the same time, high usability and full sterility of the tested samples. After creating the first prototype of an ultrasound-transparent three-chambers culture well, sealing tests were conducted to prove its impermeability to external contaminants and in vitro tests were carried out to verify the usability of this system for ultrasonic stimulation of cells in vitro. No statistically significant differences were found between control and tested samples during sealing tests, thus demonstrating optimal sealing ability towards external contaminants. Furthermore, the thin polystyrene membrane used to guarantee US-transparency guaranteed a good adhesion and viability of both human fibroblasts and induced pluripotent stem cells

Myoblast proliferation in a porous polyurethane matrix: first steps towards a 3D bio-hybrid actuator

Robots movement depends on artificial actuators performances which can be impaired by actuators’ efficiency limits. Muscle cells can be used for fabricating bio-hybrid actuators, overcoming these limits. We present a 3D soft porous polyurethane scaffold with an interconnected network. To fully colonize it, we functionalized it with laminin and fibronectin, assessing muscle cells proliferation. These results are preparatory for the construction of a 3D bio-hybrid system for robotics applications

Monolithic Three-Dimensional Functionally Graded Hydrogels for Bioinspired Soft Robots Fabrication

Bioinspired soft robotics aims at reproducing the complex hierarchy and architecture of biological tissues within artificial systems to achieve the typical motility and adaptability of living organisms. The development of suitable fabrication approaches to produce monolithic bodies provided with embedded variable morphological and mechanical properties, typically encountered in nature, is still a technological challenge. Here we report on a novel manufacturing approach to produce three-dimensional functionally graded hydrogels (3D-FGHs) provided with a controlled porosity gradient conferring them variable stiffness. 3D-FGHs are fabricated by means of a custom-designed liquid foam templating (LFT) technique, which relies on the inclusion of air bubbles generated by a blowing agent into the monomer-based template solution during ultraviolet-induced photopolymerization. The 3D-FGHs' apparent Young's modulus ranges from 0.37 MPa (bulky hydrogel region) to 0.09 MPa (highest porosity region). A fish-shaped soft swimmer is fabricated to demonstrate the feasibility of the LFT technique to produce bioinspired robots. Mobility tests show a significant improvement in terms of swimming speed when the robot is provided with a graded body. The proposed manufacturing approach constitutes an enabling solution for the development of macroscopic functionally graded hydrogel-based structures usable in biomimetic underwater soft robotics applications

HOTAIRM1 regulates neuronal differentiation by controlling NEUROGENIN 2 and the downstream neurogenic cascade

Neuronal differentiation is a timely and spatially regulated process, relying on precisely orchestrated gene expression control. The sequential activation/repression of genes driving cell fate specification is achieved by complex regulatory networks, where transcription factors and noncoding RNAs work in a coordinated manner. Here, we provide the molecular, functional and mechanistic characterization of the long noncoding RNA HOTAIRM1 (HOXA Transcript Antisense RNA, Myeloid-Specific 1) as a new player in neuronal differentiation. At the molecular level, we describe HOTAIRM1 neuronal isoform and identify the RNA-binding proteins HNRNPK and FUS as regulators of its biogenesis and metabolism. Functionally, we discover that HOTAIRM1 controls the expression of the proneural transcription factor NEUROGENIN 2, that is key to neuronal fate commitment and critical for brain development. We demonstrate that, during neuronal differentiation, nuclear HOTAIRM1 controls the transitory expression of NEUROGENIN 2. Mechanistically, HOTAIRM1 acts as an epigenetic regulator that, recruiting the repressive complex PRC2, contributes to limit the time-window of NEUROGENIN 2 expression. Remarkably, HOTAIRM1 also controls NEUROGENIN 2 downstream regulatory cascade contributing to the achievement of proper neuronal differentiation timing