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International Journal of Bioprinting 3D-printed contractive pennate muscle

defect injuries increasingly challenging. Skeletal muscle in controlling the microstructure of engineered muscle
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tissue engineering (SMTE) and regenerative medicine, tissues, biofabrication of in vitro 3D skeletal muscle tissues
as significantly effective alternatives to current clinical with improved muscle fiber organization design have great
strategies, have great research value and development potential to improve muscle function.
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potential for replicating the structure and function Unlike the arrangement of muscle fibers in parallel
of natural muscle in vitro. To achieve these goals, muscles, the contraction and extension of pennate
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functionalizing the myogenic capacity of natural muscles gastrocnemius muscles in the hind limbs of frogs play
in artificial muscles is a primary research focus in this a significant role in their explosive jumping capacity
field, especially through the use of advanced materials (with a horizontal jumping distance of ~30 times their
such as nanomaterials. Moreover, tissue-engineered body length) with a high mechanical power output in
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skeletal muscle can play a role in disease modeling, drug the jumping process (Figure 1A and B). The pennate
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testing, and bio-robotics. 7,9,10 Some examples include architecture in native skeletal muscles, characterized by
in vitro models for studying myogenesis and muscle the tilted contractile force direction of the muscle fibers,
pathology, and preclinical models for drug screening is widely recognized for producing greater force despite
and toxicity testing. 11–13 Additionally, engineered having less contraction displacement. This is due to the
skeletal muscles could also serve as advanced bio- higher density of muscle fibers per unit volume compared
actuators to microrobots due to their controllability to parallel muscles (Figure 1C). In this regard, we
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and adaptability, 14–19 enabling biomimetic movements, proposed a basic process, including the design, fabrication,
such as crawling and gripping, and promoting the and evaluation of a novel in vitro skeletal muscle tissue
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development of self-powered microrobots. 9 design, that mimics the macro and microstructure of the
3D bioprinting is a powerful approach to precisely gastrocnemius muscle in frogs (Figure 2). The design and
deliver cells, biomaterials, and biological factors to modeling of muscle tissue, including its macro shape and
targeted positions. It has great potential for fabricating microstructure, were optimized through simulation based
3D-customized skeletal muscle tissues with complex on mechanical properties. Subsequently, a repeatable and
microscopic arrangements in a one-step process. For customizable technique of multi-material 3D bioprinting
example, 3D bioprinting was applied to precisely deposit technique was used to fabricate tissues with fusiform
mouse myoblasts (C2C12) in a matrix on cantilevers geometry and induced pennate myotube alignment
with 85 µm resolution, >90% cell viability, and high for optimal force generation. Next, we stimulated the
reproducibility, and the myotubes became excitable after 3D-bioprinted muscle tissues using an electrical field to
differentiation. Biomimetic human skeletal muscle induce directional differentiation of myotubes. Finally, we
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constructs were also 3D-bioprinted from mm to cm validated the controllable contractile function of the tissues
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scale, consisting of tens to hundreds of densely packed by modifying stimulation amplitudes and frequencies. This
long parallel myofiber bundles, thereby demonstrating that work would be useful for exploring design strategies and
3D-bioprinted skeletal muscle constructs can form multi- rapid fabrication techniques for the next generation of
layered bundles with aligned myofibers. 23,24 Researchers high-function in vitro skeletal muscle tissues.
also validated the feasibility of 3D-printed skeletal muscle
tissues to treat critical-sized muscle defect injuries in a 2. Materials and methods
rodent model; however, the tissues did not fully support 2.1. Design of engineered muscle tissue inspired by
the structural and functional restoration of defected the gastrocnemius muscle
muscles. Furthermore, researchers revealed that neural The design model was inspired by the gastrocnemius
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input into the bioprinted skeletal muscle construct could muscle in frogs, featuring a spindle shape that fits the
greatly improve myofiber formation, long-term survival, leg. Therefore, the engineered muscle construct was
and neuromuscular junction formation in vitro, facilitating designed with a shuttle shape in macro geometry, with
rapid innervation and maturation into organized muscle interlaced strips and microchannels (Figure 3A). In the
tissue in a rodent model. However, despite significant macrostructure, the diameter of the muscle belly was
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progress in the development of 3D-engineered skeletal defined as the major diameter (R), and the diameter of the
muscle tissues, the contractile forces generated by the muscle tendon as the minor diameter (r). Measurement of
engineered skeletal muscle tissues remain low compared the frog’s gastrocnemius muscles revealed that the ratio of
to their natural counterparts. Thus, functional skeletal the long axis to minor diameter (L/r) was 6:1, while the
muscle tissue constructs have not yet been fabricated in ratio of the major diameter to minor diameter (R/r) was 3:1.
vitro. Since the muscle fiber arrangement is highly relevant To obtain macro and microstructure designs with better
to contractile performance and 3D bioprinting is effective contractility, we adjusted the ratios and pennate angles

Volume 10 Issue 6 (2024) 246 doi: 10.36922/ijb.4371