Post, M. J., et. al. (2010) Effects of a combined mechanical stimulation protocol: Value for skeletal muscle tissue engineering. doi:10.1016/j.jbiomech.2010.01.039
Reviewed and edited by New Harvest Journal Club’s Tom Ben Arye and Matt Sharp, respectively.
Abstract: Skeletal muscle is an appealing topic for tissue engineering because of its variety in applications for regenerative medicine,in vitro physiological model systems, and in vitro meat production. Besides conventional biochemical cues to promote muscle tissue maturation in vitro, biophysical stimuli are necessary to reach the desired functionality and texture of the engineered tissue. Stretch, caused by active movements of the body, is an important factor present in the niche of muscle progenitor cellsin vivo. We therefore investigated the effects of uniaxial ramp stretch (2%) followed by uniaxial intermittent dynamic stretch (4%) on C2C12 and murine muscle progenitor cells in a 2D and 3D environment and found that stretch negatively influenced maturation in all cases, demonstrated by decreased expression of MRFs and sarcomere proteins at the RNA level and a delay in the formation of cross striations. We therefore conclude that the current protocol is not recommended for skeletal muscle tissue engineering purposes.
Overview: This study investigated a protocol for physically stimulating muscle cells to encourage them to mature in vitro. The protocol failed to achieve this and is not recommended for future use.
In animals, developing muscle tissues are subjected to stretching and physical stimulation. This helps them grow and strengthen into mature muscles. Tissue engineers try to mimic this process this process in-vitro so that muscle cells can be grown for regenerative medicine and for meat production. Previous studies into the effects of in vitro mechanical stimulation seem contradictory.
Experimental Setup: This study investigated the effects of mechanical stimulation/stretching on two types of undeveloped mouse muscle cell: muscle progenitor cells (MPCs) and C2C12 myoblasts. It looked at whether stretching had a different impact on these cells if they were:
(a)grown in 2-dimensional (2D) layers of one cell thick and (b)grown in 3-dimensional (3D) constructs. They were compared to cell cultures in the same conditions that weren’t stretched that acted as controls.
They also studied the effect of the substrate (substance the cells were grown on) had on cell development.
The stretching protocol involved several parts to make the conditions more natural and to let the cells adjust to the changes.
First, they seeded the cells and let them settle for 24 hours. Then they stretched the cells in ‘ramp stretch’ for 2 days. Cells were stretched constantly, with the amount of stretch increasing at constant rate up to 2%. After that, the cells were stretched intermittently for 3-hour cycles, with stretch increasing from 2% up to 6% and then back to 2% over each cycle. This cyclical stretching went on for 4 days and had breaks of 3 hours between each consecutive cycles..
In order to determine whether and how the stretching had influenced cell development, a number of factors were looked at. These were:
(a)the amount of myotube formation.
(b)the presence of cross striations.
(c)the expression of various genes/proteins associated with muscle development, such as myogenic regulatory factors (MRFs).
Results and Discussion: In the cells stretched in 2D layers, both MPCs and C2C12 cells were seen to have very similar levels of myotube formation and cross striations as the control cultures. Some MRFs were expressed in lower levels in response to stretch compared to controls, implying that the cells were actually becoming less mature. The type of coating used influenced gene expression, with different responses to collagen, ProNectin, and laminin.
In the 3D structures, the MPCs and C2C12 cells developed differently, showing different patterns of cross-striations after stretching. Intermittent stretching appeared to impede maturation of MPCs, shown by a decrease in the expression of MRFs.
Overall, the stretching protocol failed to increase maturation of the muscle cells, and is not recommended for tissue engineering.
Glossary:
Myotube: these are developing muscle fibers. Myotubes are formed by the fusion of myoblasts. Because each individual myoblast has a nucleus, a myotube has many nucleii.
Cross striations: these are ‘stripes’ in the muscle fiber. They result from the arrangement of myofibrils, which are made up of the proteins that allow the muscles to contract.
Additional information
An example video of 2D stretching.
