IT MIGHT look like an amputated rat forelimb, but the photo is of something much more exciting: the limb has been grown in the lab from living cells. It may go down in history as the first step to creating real, biologically functional limbs for amputees.
(Image: B J Jank, Ott Laboratory)
"We're focusing on the forearm and hand to use it as a model system and proof of principle," says Harald Ott of Massachusetts General Hospital in Boston, who grew the limb. "But the techniques would apply equally to legs, arms and other extremities."
"This is science fiction coming to life," says Daniel Weiss at the University of Vermont College of Medicine in Burlington, who works on lung regeneration. "It's a very exciting development, but the challenge will be to create a functioning limb."
Many amputees receive artificial replacements that look fine cosmetically, but don't function as well as real limbs. And while bionic replacement limbs that work well are now being made, they look unnatural. Hand transplants have also been successful, but the recipient needs lifelong immunosuppressive drugs to prevent their body rejecting the hand.
A biolimb would get round many of these obstacles as it only contains cells from the recipient so would avoid the need for immunosuppression. It should also look and behave naturally.
"This is the first attempt to make a biolimb, and I'm not aware of any other technology able to generate a composite tissue of this complexity," says Ott.
The technique behind the rat forelimb - dubbed "decel/recel" - has previously been used to build hearts in the lab. Simpler organs such as windpipes and voicebox tissue have been built and transplanted into people with varying levels of success, but not without controversy (see "Rocky road to replacement organs").
In the first, decel step - short for decellularisation - organs from dead donors are treated with detergents that strip off the soft tissue, leaving just the "scaffold" of the organ, built mainly from the inert protein collagen. This retains all the intricate architecture of the original organ. In the case of the rat forearm, this included the collagen structures that make up blood vessels, tendons, muscles and bones.
In the second recel step the flesh of the organ is recellularised by seeding the scaffold with the relevant cells from the recipient. The scaffold is then nourished in a bioreactor, enabling new tissue to grow and colonise the scaffold.
Because none of the donor's soft tissue remains, the new organ won't be recognised as foreign and rejected by the recipient's immune system.
A forearm is much more difficult to create in this way than a windpipe, say, as a number of different cell types need to be grown. Ott began by suspending the decellularised forelimb in a bioreactor, plumbing the collagen artery into an artificial circulatory system to provide nutrients, oxygen and electrical stimulation to the limb. He then injected human endothelial cells into the collagen structures of blood vessels to recolonise the surfaces of blood vessels. This was important, he says, because it made the vessels more robust and prevented them from rupturing as fluids circulated.
Next, he injected a mixture of cells from mice that included myoblasts, the cells that grow into muscle, in the cavities of the scaffold normally occupied by muscle. In two to three weeks, the blood vessels and muscles had been rebuilt. Ott finished off the limb by coating the forelimbs with skin grafts.
But would the limb's muscles work? To find out, the team used electrical pulses to activate the muscles and found that the rat's paw could clench and unclench. "It showed we could flex and extend the hand," says Ott. They also attached the biolimbs to anaesthetised healthy rats and saw that blood from the rat circulated in the new limb. However, they didn't test for muscle movement or rejection.
While they have decellularised around 100 rat forelimbs, recellularising at least half of them, there is still much work to do, says Ott. First they need to seed the limb with bone, cartilage and other cells to see whether these can be regenerated. Then they must demonstrate that a nervous system will develop. Results of hand transplants show that this happens through the recipient's nerve tissue penetrating into the hand, he says, enabling them to build up control of the new organ. Whether this also works in regenerated limbs remains to be seen.
Ott and his colleagues have also shown that a primate forearm can be successfully decellularised (see photo, below). His team have begun recolonising the primate scaffolds with human cells that line blood vessels, the first step towards human-scale biolimb development, and have started experiments using human myoblasts in rats instead of the mice ones. But considerable extra work is needed and it will be at least a decade before the first biolimbs are ready for human testing, says Ott.
"It's a notable step forward, and based on sound science, but there are some technical challenges that Harald's group has to tackle," says Steve Badylak of the University of Pittsburgh in Pennsylvania, who has used grafts built on scaffolds made from pig muscle to rebuild damaged leg muscles in 13 people. "Of these, the circulation is probably the biggest challenge, and making sure even the tiniest capillaries are successfully lined with endothelial cells so that they don't collapse and cause clots," he says. "But this is really an engineering approach, taking known fundamental principles of biology and applying them as an engineer would."
Others are more critical. "For a complex organ like the hand, there are so many tissues and compartments that this definitely will not be a feasible protocol," says Oskar Aszmann of the Medical University of Vienna in Austria, inventor of a bionic hand that people can control through their own thoughts. "Also, the hand must be innervated by thousands of nerves to have meaningful function, and that is at this point an insurmountable problem. So although this is a worthy endeavour, it must at this stage remain in the academic arena, not as a clinical scenario."
In humans, Ott envisages organ donation schemes being extended to include limbs. Cells for regenerating blood vessels could come from minor vessels supplied by the recipient, while muscle cells could come from biopsies from large muscles, such as in the thigh. "If you took about 5 grams, the size of a finger, you could grow it into human skeletal myoblasts," he says.
With 1.5 million amputees in the US alone, this regeneration work is important, says Ott. "At present, if you lose an arm, a leg or soft tissue as part of cancer treatment or burns, you have very limited options."
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