Rat forelimbs grown in the lab

The moving pictures of American soldiers who lost limbs while serving their country come across our computer screens with some regularity. However, while we celebrate the courage of these young men and women, we should also be amazed at the technological advances that provide artificial limbs for these soldiers. What if, we could grow replacement limbs in culture? Is this science fiction? Maybe not.

Biolimb from Ott lab
Biolimb from Ott lab

The photo above comes from work done in the laboratory of Harald Ott who is at the Massachusetts General Hospital in Boston has succeeded in growing rodent forelimbs in the laboratory. “We’re focusing on the forearm and hand to use it as a model system and proof of principle,” said Ott. “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.”

Modern amputees are often fitted with prosthetic limbs that have an excellent cosmetic look, but these artificial limbs don’t function as well as real limbs. Bionic replacement limbs that work well are now being made, but they look quite unnatural. Hand transplants have also been successful, but these surgeries are extremely expensive, and the recipient needs lifelong immunosuppressive drugs to prevent their body rejecting the transplanted hand.

Tissue engineered “biolimbs” would get round many of these obstacles as it only contains cells from the recipient and would, therefore, avoid the need for immunosuppression. Biolimbs would 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.

To grow rat forelimbs in the laboratory, the so-called “decel/recel” technique was used. This same technique was previously been used to build hearts, lungs and kidneys in the lab. In fact, simpler organs such as windpipes and voice box tissue have been built and transplanted into people with varying levels of success, but not without controversy.

Decel stands for decellularization is the first step. In the decel step, organs from dead donors are treated with detergents that strips the soft tissue and leaves just the “scaffold” of the organ, which consists mainly from the inert protein collagen. This retains all the intricate architecture of the original organ. In the case of the rat forearm, these collagen structures include blood vessels, tendons, muscles and bones.

The second step, the recel step, recellularizes the flesh of the organ by seeding the scaffold with the relevant cells extracted from the recipient. This scaffold is then nourished in a bioreactor, which enables the new tissue to grow and colonize the scaffold. Because none of the donor’s soft tissue remains, this bioengineered limb, or biolimb, will not be recognized as foreign and rejected by the recipient’s immune system.

As tissue engineered organs go, the forearm is much more difficult to grow that a windpipe. It has a far greater number of cell types that need to be grown. Ott began by suspending the decellularized forelimb in a bioreactor, and then plumbing the collagen artery into an artificial circulatory system to provide nutrients, oxygen and electrical stimulation to the limb. Next, Ott and his colleagues injected human endothelial cells into the collagen structures of blood vessels to recolonize the surfaces of blood vessels. This was important, because, according to Ott, this made the blood vessels more robust and prevented them from rupturing as fluids circulated through them.

Next, he injected a mixture of cells from mice that included myoblasts or muscle forming cells that would 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 then finished off the limb by coating the forelimbs with skin grafts.

But would the limb’s muscles work? In order to work, the muscles must be connected to motor nerves from the central nervous system. To try this out, Ott’s team used electrical pulses to activate the muscles and found that the rat’s paw could clench and unclench. This experiment “showed we could flex and extend the hand,” says Ott. They also attached the biolimbs to anaesthetized 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 decellularized around 100 rat forelimbs, recellularizing at least half of them, there is still a great deal of work to do, said Ott. First they need to seed the limb with bone, cartilage and other cells to see whether these structures can be grown in the biolimb. Then they must demonstrate that a nervous system will develop in these cells. Results of hand transplants have shown the re-enervation occurs by means of the recipient’s nerve tissue growing into the transplanted hand and penetrating it. These growing nerves then make connections with the appropriate muscles. Thus, Ott believes that this would enable the recipients of a transplanted biolimb to control of their new organ. However, whether this also works in regenerated limbs remains to be seen.

Ott and his colleagues have also shown that forearms from nonhuman primates can be successfully decellularized. His team has begun recolonizing the primate scaffolds with human cells that line blood vessels, which is the first step towards human-scale biolimb development. They have also started experiments using human myoblasts in rats instead of the mouse myoblasts. Considerable work is needed to perfect this technology and it will be at least a decade before the first biolimbs are ready for human testing, says Ott, which is probably an optimistic estimate.

Nonhuman primate limb
Nonhuman primate limb

“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 endeavor, 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 transplantation of biolimbs. 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.”