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For years, a major obstacle has dashed the hopes of creating "replacement parts" for the human body: the lack of an internal, nourishing blood system in engineered tissues. Without it, thicker tissues can't thrive, a fact that has confined tissue engineering's practical application to thin skin, which can recruit blood vessels from underlying tissue.
Now, researchers in Professor Robert Langer's lab at MIT have used a novel cocktail of cells, including endothelial cells derived from human embryonic stem cells, to coax in vitro muscle tissue to develop its own vascular network, a process called pre-vascularization. When implanted in living mice and rats, these tissues integrated more robustly with the body's own tissues than similar implants without blood vessels.
"What's even more exciting than being able to make skeletal muscles for reconstructive surgery or to repair congenitally defective muscles, is that this is a generic approach that can be applied towards making other complex tissues. It could allow us to do really wonderful things," said collaborator Daniel Kohane, an MIT affiliate and assistant professor of pediatrics at Harvard Medical School.
The human derivation of the endothelial cells is key for clinical use in humans to avoid an immune rejection, but this was the first time that this particular type of cell had been isolated. Credit for that goes to researcher Shulamit Levenberg. For implantation in living animals, the lab used immunodeficient mice and rats that would not reject the human-derived endothelial cells.
The researchers published their work in Nature Biotechnology, available online in advance on June 19. An accompanying News and Views commentary says this "landmark paper" provides "a compelling demonstration of the benefits of pre-vascularization for engineering larger pieces of tissue."
"When I came to work with Bob Langer for my postdoc, it was my dream to vascularize a tissue," said first author Levenberg, who is now on the faculty of the biomedical engineering department at Technion in Haifa, Israel, where she completed these studies. She chose to tackle muscles, since they depend on blood vessels interspersed with muscle fibers and also serve as a model for highly vascularized organs such as the liver, heart and lung.
Levenberg theorized she needed to combine three cell types: myoblasts that form muscle fibers; endothelial cells that independently self-organize into vessel tubes; and fibroblasts that are the precursors for the smooth muscle cells that stabilize the vessel amidst the tissue's gooey extracellular matrix. "No one had tried a 3-D tri-culture scaffold before. It's hard enough to work with one cell type, let alone three," said senior author Langer, who is an MIT Institute Professor and a pioneer in tissue engineering.
In vitro experiments validated Levenberg's hypothesis. "The endothelial cells formed vessels, recruited the fibroblasts, and differentiated them into smooth muscle cells," she said. "The differentiated fibroblasts expressed the angiogenic growth factor, VEGF, which further stimulated vessel growth." The constructs measured 5mm by 5mm by 1mm.
The animal studies progressed in stages. First, the researchers implanted a muscle construct under the skin, then inserted one within a leg muscle, and finally replaced a piece of a rat's abdominal muscle with a construct, simulating a situation applicable to trauma victims, for instance. Later tissue staining showed that the implants' vessels grew into the host tissue and the host's vessels grew into the constructs.
But what good are blood vessels if they don't deliver blood? Using two non-invasive live imaging techniques (labeled lectin injected into the tail vein and a luminescent luciferase-based system), the researchers could watch the host's blood flow into the engineered vessels. About 41 percent of the constructed vessels became perfused with the hosts' blood, meaning they functioned in the living body. "That's pretty good for a first try," Levenberg asserted.
Importantly, twice as many of the cells survived in the tri-culture implants compared to implants without endothelial cells. "The myoblasts also became even longer tubes when implanted, and they began to align themselves with the host's muscle fibers," Levenberg said.
"This tri-culture system shows a whole new way of creating a vascular network in the tissue," said Langer. "We've also demonstrated another powerful use of human embryonic stem cells."
In addition to Kohane, Levenberg and Langer collaborated with Patricia D'Amore and Diane Darland at The Schepens Eye Research Institute, Evan Garfein at Brigham and Women's Hospital, Robert Marini of MIT's Division of Comparative Medicine, Richard Mulligan of Children's Hospital and Harvard Medical School, Clemens van Blitterswijk at Twente University in the Netherlands, and present and former MIT graduate students Mara Macdonald and Jeroen Rouwkema. The work was supported by the National Institutes of Health.