Muscles in the frame

The porous polymer frame allowed the mice to grow muscle cells more efficiently

Vera Sysoeva, N+1

Biologists and materials scientists have proposed using a porous biocompatible material as a framework in tissue engineering. The precursors of muscle cells, which were attached to a framework of porous polycaprolactone fibers, developed well and formed muscle tissue in mice. Muscle "patches" built using this technology helped the animals overcome trauma and loss of a large amount of muscle. The work was published in Advanced Materials (Jin et al., Functional Skeletal Muscle Regeneration with Thermally Drawn Porous Fibers and Reprogrammed Muscle Progenitors for Volumetric Muscle Injury).

As a result of injuries, infectious diseases or surgical removal of tumors, patients can lose quite voluminous areas of muscle tissue. The treatment involves surgical intervention: muscle flaps of the same person are transplanted to the damaged place. Such a procedure, accordingly, requires an additional surgical site on the human body, which is quite painful. In addition, this approach does not always lead to effective restoration of muscle functions and innervation of the transplanted tissue.

Transplanting myogenic progenitor cells seems to be a more promising strategy. Such cells can be obtained in the laboratory from fibroblasts – connective tissue cells. To do this, it is enough to stimulate them with signaling molecules (transcription factors) characteristic of muscle tissue cells.

In order for the transplanted cells to form real muscles, it is necessary to precisely control their location and direction of growth. To do this, cells can be inserted into a synthetic framework that reproduces the properties of the natural "skeleton" of the tissue – the extracellular matrix – and forms 3D structures similar to natural muscle tissue. However, unlike the natural extracellular matrix, synthetic scaffolds often lack specific recognition sites for cells, and as a result, cells do not bind well to it.

As a result of a joint study, scientists from Yonsei University and the Massachusetts Institute of Technology, led by Seung-Woo Cho and Polina Anikeeva, proposed using a hybrid structure formed by an extracellular matrix, a synthetic framework made of polycaprolactone material and muscle progenitor cells.

The bioengineered design proposed by the authors: muscle progenitor cells and extracellular matrix on a frame of polycaprolactone fibers as an implant.

First, the researchers prepared polycaprolactone fibers in two versions: with and without pores. Composite sheets of polycaprolactone with sodium chloride crystals were rolled into tubes 50 micrometers thick and dried. Since salt crystals with a diameter of 25-30 micrometers could not completely fit into a sheet of polycaprolactone, they formed pores when dried. During the drying process, the thickness of polycaprolactone became even thinner (about 45 micrometers).

Sheets (left) and fibers (right) made of polycaprolactone. For comparison, images of a smooth material and a porous one, which was used in the study, are given.

Then the scientists obtained an extracellular matrix released from the cells from pig muscles: cells were split in the muscle tissue using the surfactant sodium lauryl sulfate, as a result of which 97.4 percent of the cells were removed. Components such as collagen, proteoglycans and glycoproteins remained in the matrix.

After soaking synthetic fibers and sheets in a solution of such an extracellular matrix (five milligrams per milliliter), a mesh structure was found on the surface of porous polycaprolactone using microscopy. The extracellular matrix has successfully attached itself to the scaffold.

Extracellular matrix fixed on a sheet (top) and fibrous (bottom) frame made of polycaprolactone.

To test biocompatibility, the researchers implanted a polycaprolactone scaffold with an extracellular matrix on it in mice. After three days and two weeks, the authors conducted a histological analysis, as a result of which they did not find necrosis or an inflammatory response of the body in the area of the implant. Then the researchers went directly to the creation of muscle implants. Muscle progenitor cells were planted on synthetic fibers and sheets of polycaprolactone. On a porous frame in the form of fibers, cells began to grow in an orderly manner, and this is very important for the regeneration of muscle tissue.

The authors also tested the effectiveness of such artificial muscles on mice, in which 75 percent of the quadriceps femoral muscle was previously removed. It is impossible to recover from such an injury without surgery. Then the scientists implanted structures made of fibrous or leafy frame and muscle progenitor cells at the site of damage. As a control group, mice were used, which received a skeleton without cells on it as an implant. There was also a group of animals in which myogenic cells were planted not on a polycaprolactone frame, but in a gel. Four weeks after implantation, the researchers observed the first signs of tissue regeneration in all groups except the control group and in mice with an implant on a gel frame. Myotubes and muscle fibers grew on fibrous polycaprolactone, and non-muscle cells mainly appeared on the sheets. A good result was also the innervation of tissues and the appearance of blood vessels in them. After ten weeks, mature muscle fibers formed on the fibrous frame. A mixture of mature and growing fibers appeared on the leaf base, and only fat deposits appeared in the control group.

At the end of the work, the scientists tested how bioengineered muscles helped mice restore the motor functions of the limbs. The researchers measured the stride length of the animals, the grip strength of the paws and the maximum speed of the rotating rod at which it can stay on it (by analogy with rotating drums on playgrounds, only mice run on it with all four paws). Three weeks after implantation, these indicators were still reduced compared to those received before the injury, and by the fifth week the step length had already begun to increase. The implants on the muscle frame helped the mice to stay on the simulator, which rotated at a speed of 10 revolutions per minute, 1.31 ± 0.27 times longer than before the injury. Mice that had no injuries showed a similar result – 1.33±0.32 times longer. Thus, the hybrid design proposed by the authors helped the mice recover after a serious injury.

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