Investigating the Life Expectancy and Proteolytic Degradation of Engineered Skeletal Muscle Biological Machines

Abstract

A combination of techniques from 3D printing, tissue engineering and biomaterials has yielded a new class of engineered biological robots that could be reliably controlled via applied signals. These machines are powered by a muscle strip composed of differentiated skeletal myofibers in a matrix of natural proteins, including fibrin, that provide physical support and cues to the cells as an engineered basement membrane. However, maintaining consistent results becomes challenging when sustaining a living system in vitro. Skeletal muscle must be preserved in a differentiated state and the system is subject to degradation by proteolytic enzymes that can break down its mechanical integrity. Here we examine the life expectancy, breakdown, and device failure of engineered skeletal muscle bio-bots as a result of degradation by three classes of proteases: plasmin, cathepsin L, and matrix metalloproteinases (MMP-2 and MMP-9). We also demonstrate the use of gelatin zymography to determine the effects of differentiation and inhibitor concentration on protease expression. With this knowledge, we are poised to design the next generation of complex biological machines with controllable function, specific life expectancy and greater consistency. These results could also prove useful for the study of disease-specific models, treatments of myopathies, and other tissue engineering applications.

Simulation and Fabrication of Stronger, Larger, and Faster Walking Biohybrid Machines

“…The goal is to design these biological machines with a high degree of control, tissue‐to‐scaffold dynamics, and longevity, among other parameters. Such advances include the use of light to temporally control muscle contractions, micropatterning to achieve spatial coordination of contraction, and incorporation of biomolecules to control the degradation of the structural matrix …”

3D-printed drug testing platform based on a 3D model of aged human skeletal muscle

“…Beforehand, we performed studies of cell growth, proliferation and differentiation of human myoblasts in 2D, which showed no differences when the media were supplemented with ACA. Moreover, previous reports in the literature show beneficial aspects of using ACA to increase the lifetime of bio-actuators without loss of function 37 .…”

Bio-hybrid soft robots with self-stimulating skeletons

“…1D), leaving the whole setup first in growth medium (GM) to let the cells grow and expand, and later on in differentiation medium (DM) to allow the differentiation process [49] . Both GM and DM were supplemented with 6aminocaproic acid (ACA) to reduce the degradation of the hydrogel due to proteases [52] . The cross-linking of the hydrogel was studied over time to closely evaluate the gelation process and obtain reproducible cell-laden scaffolds for the biobots construction.…”

“…Later, the same bio-robotic device was lightcontrolled remotely by optogenetically-modified skeletal muscle cells which contract upon blue light stimulation [40] . In addition, this device demonstrated self-healing [50] , adaptability [40] , integration of motor neurons for advanced stimulation [51] , long-time preservation [52,53] , scalability [54] or their integration with micro-electrodes [55] . The integration of neuronal and skeletal muscle tissue in one single biobot has been of great interest, as it resembles the structure of native muscle to obtain improved controllability of the bio-robotic systems [24,56] .…”