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Building muscle from scratch is no mean feat; skeletal muscle is highly organized and requires considerable oxygenation—and thus vascularization—to survive and function. Earlier attempts to cultivate muscle tissue have focused largely on the transplantation of myoblasts, and although some of these efforts have proven partially successful, proper graft vascularization still remains an issue.

When postdoc Shulamit Levenberg first arrived in Robert Langer's lab at the Massachusetts Institute of Technology, she hoped to take advantage of his team's experience with biomaterials and cell culture to develop more effective tissue engineering approaches. “The whole idea was to find... a new way to engineer a tissue with a whole network of blood vessels inside in vitro,” says Levenberg, now at the Technion Institute in Israel. In a report in Nature Biotechnology, Levenberg, Langer and their colleagues describe an important first step—a method for the culture of vascularized, transplantable muscle tissue that could serve as a model for future tissue engineering efforts.

Their strategy relies on two important innovations. The first involves the use of a three-dimensional polymeric scaffold for mouse myoblast culture. The material is porous, providing space for cells to grow, but also biodegradable—the cells gain more space for growth as the material gradually breaks down; after transplantation, the scaffold will eventually dissolve completely. The second key feature is the coculture of two other cell types alongside the myoblasts: human endothelial cells, which form the foundation for developing vasculature, and mouse embryonic fibroblasts, which support and stimulate vascular formation through growth factor secretion.

This system, which Langer dubs 'tri-culture', proved highly effective in vitro. Within two weeks, the myoblasts had differentiated and started to assemble into the elongated, multinucleate myotubes that are the foundation of muscle tissue; after a month of tri-culture, vessel-like endothelial structures were clearly visible, surrounded by fibroblast-derived smooth muscle cells. To assess the extent to which these differentiated constructs could integrate into host tissue, Langer's group implanted their cultures into different muscular contexts in immunodeficient rodents. In all cases, the grafts continued to grow and differentiate, effectively incorporating into the host (Fig. 1). “They looked very connected with the host tissue,” says Levenberg, “and there were some areas where you could see almost no border between the implanted muscle and the host muscle. Our muscle is still not really a fully organized muscle... but we saw long fibers that were aligned in the mice.”

Figure 1: Engineered muscle grafts exhibit organization and vascularization after transplantation into abdominal muscle of nude mice.
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(a–c) These implants were immunostained for the muscle-specific proteins desmin (a) and myogenin (b), and for an endothelium-specific protein, von Willebrand factor (c).

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Notably, the grafts were well vascularized, and in many cases the newly-formed vascular tissue appeared to be functionally incorporated into the host; when recipient mice were injected with labeled lectin via the tail vein, roughly 40% of the implant-derived blood vessels in the graft were found to be perfused with the lectin. The group also noted that tri-culture–derived implants had a marked and significant increase in implant survival and functional vascularization relative to myoblast-only implants.

The authors were impressed with the level of integration demonstrated by these implants, and hope to optimize their system to improve graft organization and functionality. Most importantly, however, they express hope that this system will offer a foundation for the development of similar engineered implants for other complex, highly vascularized tissues, such as the liver and the lung.