Essays on Science and SocietyREGENERATIVE MEDICINE

Modeling muscle

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Science  08 Mar 2019:
Vol. 363, Issue 6431, pp. 1051
DOI: 10.1126/science.aaw3611

Communication underlies the complexity of biological systems. Adaptive behaviors ranging from self-assembly to self-healing showcase the ability of such systems to sense and adapt to dynamic environments based on signaling between living cells. This signaling takes on many forms—biochemical, mechanical, and electrical—and uncovering it has become as much the purview of regenerative medicine as of fundamental biology. We cannot reverse-engineer native tissues if we do not understand the fundamental design rules and principles that govern their assembly from the bottom up (1).

Movement is fundamental to many living systems and driven primarily by skeletal muscle in human bodies. Disease or damage that limits the functionality of skeletal muscle severely affects human health, mobility, and quality of life. Replacing dysfunctional muscle with synthetic actuators cannot fully recover the functionality of the original tissue because the large forces that skeletal muscle produces from small volumes, combined with its adaptability to changing environmental loads, cannot currently be matched with nonbiological materials (24). There is thus a critical need to uncover the underlying structure and cell-cell communication that drives the formation, maturation, and responsive behaviors of skeletal muscle tissue.

Replacing diseased or damaged muscle in vivo with tissue engineered in vitro requires a model system that enables studying the physiology and pathology of the tissue. The scale of this model is critically important because it must enable observing cell-cell communication as well as tissue-wide coordinative functions.

For skeletal muscle, a model system no thicker than ∼500 µm permits diffusion of nutrients throughout the tissue without the need for a vascular system and enables ready visualization of tissue architecture with histological or immunofluorescent imaging. Tissue that is a few millimeters in length, moreover, generates contractile forces that can readily be observed with the naked eye and measured with a simple camera or light microscope. Because contractility is a measure of the primary functionality of skeletal muscle tissue, this serves as a mechanical marker of tissue health.

We have developed a mesoscale in vitro model of skeletal muscle that meets the requirements described above (57). The model is composed of engineered tissue coupled to a flexible three-dimensional (3D)–printed skeleton that serves as a surrogate for the skeletal system. The skeleton is printed by using a biocompatible polymer, poly(ethylene glycol) diacrylate, that can be photo-patterned into complex 3D structures with precisely tunable mechanical properties (811). This enables printing architectures with spatially varying stiffness, such as two stiff pillars (modeling tendons) connected by a flexible beam (modeling articulating joints).

Anchored around this structure is a rubber band–like ring of engineered skeletal muscle, formed by means of injection molding and polymerizing a cell-gel solution. The ring is stretched around the pillars of the printed skeleton. The biopolymer solution is composed of C2C12 myoblasts embedded in a variety of proteins that replicate the in vivo extracellular matrix. Maturation of the muscle around the skeleton drives the myoblasts to differentiate into contractile tissue, and the tensile force generated can readily be measured by the deformation of the skeleton. This straightforward measure of tissue functionality enables the biochemical and mechanical stimuli supplied to the tissue to be fine-tuned in culture, so tissue viability, maturation timeline, and force production can be readily optimized to suit the target application.

Initial studies corroborated prior observations that muscle cultured in stiffer environments and in the presence of human insulin-like growth factor (IGF-1) produced larger forces and that the presence of a protease inhibitor such as aminocaproic acid (ACA) prolonged tissue lifetime (5). More importantly, this platform generated new insights into muscle maturation and function. For example, our model offered the first proof that light-stimulation “exercise” of optogenetic skeletal muscle enhanced force production (6). Furthermore, combining optical and mechanical stimulation yielded synergistic increases in the contractile force produced by the muscle tissue, with exercised tissue producing forces 550% greater than that of a nonexercised control.

Our mesoscale model showcased the first demonstration of locomotion in an untethered system powered by engineered skeletal muscle, as electrically triggered contraction generated tissue-wide contractility and motility. The light-triggered contraction of our optogenetically modified tissue allowed for greater spatiotemporal control of the pace, degree, and location of muscle contraction, more closely replicating the in vivo environment. This enhanced spatial control enabled the first muscle-powered engineered system capable of bidirectional locomotion and rotation, previously only observed in natural biological systems.

The predominant cause of muscle loss of function in the body is mechanical damage to the tissue, and our model provided a distinctly functional system with which to measure and modulate the tissue's response to damage and subsequent healing. We showed that induced laceration irrecoverably reduces both the viability and contractility of muscle tissue but that this damage can be completely healed within 2 days if the wound site is treated with myoblasts, extracellular matrix proteins, sustained local release of IGF-1, and optically stimulated exercise (12). This information, which had not been measured or modulated in any previous model system, provides critical knowledge that will guide future integration of engineered tissue into damaged sites in vivo.

Our studies have generated substantial interest around the field of regenerative medicine while providing valuable insights into the biochemical mechanisms that govern muscle tissue function (13). They also raise questions regarding how we will train the next generation of engineers and scientists to “build with biology.” To address this need, we have developed a laboratory-based curriculum to teach biohybrid design to undergraduates and have validated that this technique teaches biofabrication and tissue engineering principles more clearly than a traditional format (14). Transferring our protocol to other scientists and laboratories has also generated new knowledge of the dynamics of neuromuscular junctions (15), muscle lifetime and proteolytic degradation (16), long-term muscle cryopreservation and revival (17), and muscle force enhancement through tissue scalability (18).

The modularity, scalability, and adaptability of skeletal muscle in vivo is unmatched by synthetic materials, generating a crucial need for replacement engineered tissue in response to disease or damage. We have developed a robust protocol for manufacturing engineered skeletal muscle that has been used to study tissue development and adaptation in vitro and that we are optimistic will eventually be used to replace and recover muscle loss of function in vivo.



Ritu Raman

Ritu Raman received her undergraduate degree from Cornell University and her Ph.D. from the University of Illinois at Urbana-Champaign. Dr. Raman is currently a postdoctoral fellow at the Massachusetts Institute of Technology, where she is developing and integrating novel responsive biohybrid materials into implantable devices.

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