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TISSUE ENGINEERING

Made-to-measure organs

Stem cells aid in producing trachea and blood vessels with less risk of transplant rejection

054-056_Celulas-troncoNOVO_232Imagine a home renovation so drastic it includes removing paint and plaster from the walls, laying bare the bricks that make up its structure. This metaphor is useful for understanding the ongoing projects being carried out at the Cell Engineering Laboratory (LEC) coordinated by Elenice Deffune, a hematologist and hemotherapist at São Paulo State University (Unesp) in Botucatu. Instead of paint and cement, the researchers’ work involves removing the cells that line hollow structures in the body such as trachea and blood vessels. This procedure, known as decellularization, is the first step of a more extensive transformation: the production of replacement organs and tissues made ​​up of cells that have the genetic characteristics of the recipient.

Using this strategy, Dr. Matheus Bertanha, a vascular surgeon at LEC, is developing a potential therapeutic alternative for circulatory problems caused by atherosclerosis. In atherosclerosis, fat and calcium plaques accumulate within arterial walls and obstruct, even if only partially, the passage of blood. When this blockage is severe enough to cause symptoms, its treatment involves surgical procedures to restore circulation. In more extreme cases, an artery or vein segment removed from another part of a person’s own body is implanted, which creates a bypass—or bridge—which restores normal blood flow. Cardiac surgeons generally implant a segment of the saphenous vein, taken from the leg, into the heart of an individual who has obstructed coronary arteries. A similar procedure is done by vascular surgeons to treat blockages in arteries of the leg.

But it is not always possible to perform this procedure, however. According to the medical literature, 30% of patients requiring a graft to create the coronary bridges do not have blood vessels with the right characteristics for this procedure. It is also estimated, says Dr. Bertanha, that one in ten people for whom vascular grafts in the legs are indicated face the same problem. “Some have veins measuring less than 2.5 mm in diameter, which prevents their use,” he explains. “Other people are already on the second bridge and have no more vessels available,” he says. In such cases, one solution is to use an artificial bridge, made ​​of synthetic material. But artificial ones may have a short useful life because they become obstructed more easily. Another possibility is to obtain vessels from living donors, which is not always feasible due to immunological incompatibility, which may lead to rejection of the implant.

Dr. Bertanha is working on an alternative, which is still experimental, to try to overcome the lack of someone’s own blood vessels and the risk of obstruction of the synthetic materials. In tests on rabbits, he first extracts natural vessels—specifically veins—from a donor animal. Then the segment to be transplanted into a different animal undergoes a chemical bath with detergents that eliminate cells from the vessel walls. The aim of this decellularization process is to prevent the recipient’s body from unleashing an attack against the implanted organ. What remains from this process is a tubular structure—a scaffold—composed of collagen fibers, the protein making up the body’s supporting tissues.

Then, Dr. Bertanha seeds inside the vessel a special type of cell taken from the body of the recipient: mesenchymal stem cells. Extracted from the adipose tissue of the animal transplant recipient, these cells are capable of converting themselves into cells that have the characteristics typical of blood vessels. They are cultured in the laboratory until reaching the desired quantity—about 100,000 cells for experiments on small animals—and then glued onto the inside of the collagen tube with the aid of a gel. “The presence of the recipient’s own cells in the segment to be implanted minimizes the need to use immunosuppressive drugs to prevent rejection,” explains Elenice Deffune, who served as Dr. Bertanha’s advisor for his master’s work.

In a recently completed experiment Dr. Bertanha compared the performance of four types of implant. The animals in the first group received a vena cava segment taken directly from another animal, without having gone through the decellularization process, while the second group received only decellularized veins. The third group received vein segments that had gone through the decellularization process followed by repopulation with stem cells from another animal. And finally, the fourth group received decellularized vein segments that contained mesenchymal stem cells taken from the recipients themselves.

Rejection and regeneration
As anticipated, there was an intense inflammatory response and a strong rejection of the transplanted vessels in the first group, while the second group experienced only a mild inflammatory response. Use of a collagen tube repopulated with stem cells from another animal did not spark an immediate rejection. The cells differentiated to form the endothelium, the layer lining the inside of blood vessels, and covered a large part of the tube. One month later, however, there was significant inflammation.

Only the animals in the fourth group showed neither rejection nor significant inflammation, even one month after surgery and without the use of immunosuppressive drugs. What most surprised Bertanha was the behavior of the implanted stem cells. “In addition to having covered more than 50% of the vessel, they attracted other stem cells present in the recipient’s body,” he says. The unanticipated result was the formation of new blood vessels (angiogenesis). “In principle, this surprise is good because angiogenesis can help the new vessel integrate into the surrounding tissue,” he says. “But we will have to investigate whether or not this process is pathogenic.” Bertanha plans to carry out further tests on animals while also starting work with human stem cells; he is already thinking of future experiments.

In parallel to Bertanha’s work, Thaiane Cristine Evaristo, a biomedical doctoral student of the surgeon Dr. Daniele Cataneo, is using decellularization and recellularization procedures at LEC to produce trachea for transplantation. She is developing a decellularization protocol different from those adopted by teams working elsewhere in the world—one that is potentially cheaper.

In other countries, researchers often use enzymes obtained from animals or through genetic engineering to eliminate donor cells from the trachea. Although effective, this strategy is expensive. It can cost up to €80,000 to decellularize a single trachea. This cost, not to mention the surgery and the hospital stay, makes trachea transplants in humans prohibitively expensive.

Seeking an alternative, Evaristo and Deffune decided to subject trachea removed from donors to a sequence of chemical and physical treatments that produce a result similar to that obtained with enzymes. First, they surgically removed the trachea and bathed it in a powerful detergent, which helps to break down the cell membranes. Then they used a press to gently compress it before putting it through a few cycles of freezing and thawing and immersion in a liquid agitated by ultrasonic vibrations. Finally, the trachea spent a period exposed to light emitting diodes (LED).

Reconditioned parts
The cell-free tracheas obtained through this technique were tested in rabbits, and the results were promising. There was no transplant rejection and the rabbits survived for a period whose human equivalent is 10 years. Based on these results, Deffune suggested to Vanderlei Bagnato, a physicist at the University of São Paulo (USP) in São Carlos, that his group develop equipment to incorporate all of these technical stages. They have recently applied for a patent for the equipment, the prototype of which is under development.

While work on the equipment continues, the Botucatu group is preparing the next phase of testing, which will use pigs, a necessary step before the start of human trials. In addition to analyzing the effectiveness of trachea decellularization and recellularization techniques, the group plans, in the coming years, to test artificial trachea made ​​from a new technology, to be developed in partnership with the Institute for Technological Research (IPT) and the University of São Paulo (USP) Heart Institute (InCor), both based in the city of São Paulo, and the Renato Archer Center for Information Technology, in the city of Campinas, São Paulo State.

The collaboration means that LEC will provide IPT with human proteins to be used in the production of nanostructured tissue. At the Renato Archer Center, plates of this nanotissue will feed a 3D printer, which will carve new trachea. Once ready, they will be sent to LEC for the recellularization step. “We want to see whether or not this option proves to be as good as using natural trachea,” says Deffune. “Maybe the future of transplants lies in these new materials.

There is a worldwide demand for trachea transplants. Tracheas are needed to replace those of children born with a narrowing of this tube that carries air from the nose to the lungs—a disease known as congenital atresia of the trachea, which affects three children in every 100,000 live births. In addition, patients who spend long periods of time in the hospital using breathing apparatus need tracheas. “Hospital das Clínicas in São Paulo has a waiting list of about 300 people in need of a tracheal transplant,” says Deffune. “In many cases these are young adults who were injured in traffic accidents.”

A review of international medical literature shows that approximately 30 people have received  tracheal implants obtained through cell engineering on an experimental basis. But the results are unknown as they are still being analyzed. “Cell engineering may provide real hope for patients with chronic injuries to organs for which a therapeutic approach is currently difficult,” says Deffune. In her view, it is important to invest in making artificial trachea and blood vessels, since it is difficult to get these structures in their natural form, which is dependent on donor organs. “Sometimes, I compare our method to reconditioning parts, which recovers used ones and gets them ready for transplant,” says Deffune. “Making artificial tracheas would open the door to working with brand-new parts for the recellularization process.”

Regarding cell engineering in Brazil, Nance Nardi, a biologist at  Lutheran University of Brazil, in the southern state of Rio Grande do Sul, says that research in this area began with blood vessels and trachea because of the relative simplicity of these structures. “There are already studies with more complex organs, such as the liver, but most are in preliminary stages,” he says. Nardi sees the growing mastery of the decellularization process as one of the keys to progress made ​​by LEC. “Removing the cells from a scaffold without compromising their integrity is still something very difficult,” she says. “Their work has achieved a good response, but it may take some time before these procedures become common in operating rooms.”

Project
Ex-vivo structuring of blood vessels from the differentiation of rabbit stem cells (No. 2010/52549-8); Grant mechanism: Regular Research Grant; Principal investigator: Elenice Deffune (Unesp); Investment: R$61,883.41 (FAPESP).

Scientific articles
BERTANHA, M. et al. Tissue-engineered blood vessel substitute by reconstruction of endothelium using mesenchymal stem cells induced by platelet growth factorsJournal of Vascular Surgery. V. 59, No. 6, p. 1677-85. 2014.
BERTANHA, M. et al. Morphofunctional characterization of decellularized vena cava as tissue engineering scaffolds. Experimental Cell Research. V. 326 No. 1, p. 103-11. 2014.

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