杏吧原创

IT MIGHT look like an amputated rat forelimb, but the photo above 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.

鈥淲e鈥檙e focusing on the forearm and hand to use it as a model system and proof of principle,鈥� says of Massachusetts General Hospital in Boston, who grew the limb. 鈥淏ut the techniques would apply equally to legs, arms and other extremities.鈥�

You鈥檝e got to hand it to them (Image: B J Jank, Ott Laboratory)

鈥淭his is science fiction coming to life,鈥� says at the University of Vermont College of Medicine in Burlington, who works on lung regeneration. 鈥淚t鈥檚 a very exciting development, but the challenge will be to create a functioning limb.鈥�

聯This is science fiction coming to life. It鈥檚 a very exciting development, but challenges remain聰

Many amputees receive artificial replacements that look fine cosmetically, but don鈥檛 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.

鈥淭his is the first attempt to make a biolimb, and I鈥檓 not aware of any other technology able to generate a composite tissue of this complexity,鈥� says Ott.

(Image: B J Jank, Ott Laboratory)

The technique behind the rat forelimb 鈥� dubbed 鈥渄ecel/recel鈥� 鈥� has previously been used to build hearts, lungs and kidneys 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 鈥�Rocky road to replacement organs鈥�).

In the first, decel step 鈥� short for decellularisation 鈥� organs from dead donors are treated with detergents that strip off the soft tissue, leaving just the 鈥渟caffold鈥� 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鈥檚 soft tissue remains, the new organ won鈥檛 be recognised as foreign and rejected by the recipient鈥檚 immune system.

聯As none of the donor鈥檚 soft tissue remains, the new limb won鈥檛 be rejected by the recipient聰

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.

(Image: B J Jank, Ott Laboratory)

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 (Biomaterials, ).

But would the limb鈥檚 muscles work? To find out, the team used electrical pulses to activate the muscles and found that the rat鈥檚 paw could clench and unclench. 鈥淚t showed we could flex and extend the hand,鈥� 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鈥檛 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鈥檚 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.

Primate arms could be next (Image: B J Jank, Ott Laboratory)

鈥淚t鈥檚 a notable step forward, and based on sound science, but there are some technical challenges that Harald鈥檚 group has to tackle,鈥� 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. 鈥淥f 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鈥檛 collapse and cause clots,鈥� he says. 鈥淏ut this is really an engineering approach, taking known fundamental principles of biology and applying them as an engineer would.鈥�

Others are more critical. 鈥淔or a complex organ like the hand, there are so many tissues and compartments that this definitely will not be a feasible protocol,鈥� says Oskar Aszmann of the Medical University of Vienna in Austria, inventor of a bionic hand that people can control through their own thoughts. 鈥淎lso, 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.鈥�

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. 鈥淚f you took about 5 grams, the size of a finger, you could grow it into human skeletal myoblasts,鈥� he says.

With 1.5 million amputees in the US alone, this regeneration work is important, says Ott. 鈥淎t present, if you lose an arm, a leg or soft tissue as part of cancer treatment or burns, you have very limited options.鈥�

This article appeared in print under the headline 鈥淕rowth of the world鈥檚 first biolimb鈥�