Researchers at Duke University recently engineered a muscle bundle that mirrors naturally-occurring muscle — they proved it by inserting it into a mouse and watching the muscle grow through a literal window. The engineered tissue may help those with muscle injuries, or even serve as a biological model to replace animal studies.

Mark Juhas, Duke University graduate student and lead author of the study, said that the muscle fibers’ close biological function to mammalian muscle fibers holds promise for novel studies in muscle function, disease and repair.

“We will be able to study human diseases by creating these muscles in vitro,” Juhas said.

The study, titled “Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo,” was overseen by Juhas’ principle investigator Nenad Bursac and published in PNAS last month.

To engineer the skeletal muscle Juhas harvested rats’ satellite cells, which are a subset of stem cells that are predestined to become muscle cells and are responsible for muscle development and regeneration. The cells were later placed in a medium that promoted the growth of muscle cells so that the cells developed in a test tube — that is, in vitro.

“We place the cells into a mold that has a gel mixture that is conducive for the satellite cells to fuse and we have it anchored on both ends to create tension to align the cells,” Juhas said.

A muscle cell, also known as a myocyte, has a long, tubular shape and myofibrils within it that generate a contracting force. Juhas said that once a muscle bundle is developed, some satellite cells remain in the muscle for future growth and repairs. In his studies, the satellite cells from the rats successfully developed into myofibrils in a process known as myogenesis.

Electric pulses applied to the artificial muscle fibers revealed that the fibers were 10 times stronger than those of any previously engineered muscles. The strength and similarity of the engineering muscles to naturally-occurring muscles holds promise for their use in drug studies.

According to Juhas, the researchers also tested the muscle bundles’ ability to regenerate themselves by exposing the bundles to a cardiotoxin found in snake venom in order to induce damage.

“You do not want to totally destroy all of the muscle with the cardiotoxin, or else you would not be able to regrow it,” Juhas said. “You want to rupture enough myofibrils so it will be able to regrow itself.”

After allowing the muscle to recover for 10 days, tests found that the muscle was able to restore itself to its pre-injury conditions. Even more incredible, the post-injury muscle was able to exert a force similar to the pre-injury muscle.

These promising results moved the bioengineered muscle forward to another test: transplantation. The researchers inserted the lab-grown muscle fibers into the backs of live mice and placed a glass chamber over the area of insertion. Through the glass window, the researchers could observe the muscles growing by a fluorescent label that flashed brighter as the muscles grew stronger.

Juhas said that after two weeks of in vivo development in the mice, new blood vessels formed in the engineered muscles and that the engineered tissue was then able to generate an output 3.2 times the force of the muscle bundle when it was in vitro.

“The muscle remodels and regenerates on the host as it did in vitro. The addition of all of the vasculature and nutrients create better striations, stronger cells and a threefold force change,” Juhas said.

Juhas said that his team’s research could help deepen our understanding of the intricacies of muscles, but that more steps need to be taken to validate the model, which may not hold up as well in human studies.

“The first step is being able make human muscle. Afterwards one needs to be able to show that it behaves in such a way based on what is already known about muscle regeneration. These are the next steps to validate this model. It is tricky,” Juhas said. “Even when you have people doing all of these studies on rats we wonder how applicable it is for humans.”

Despite the uncertain future of these muscle fibers in treating human disease, their creation poses a major breakthrough in the stem cell community and the future of biomedical engineering. Juhas and his team are now working on improving the functional and structural properties of bioengineered skeletal muscle.