3D printing under the skin

Scientists have printed an artificial ear right inside a mouse – even without surgery. A video projector and glowing nanoparticles came in handy

Alexander Ershov, Meduza

Researchers from Sichuan University conducted the world's first non-invasive 3D printing of an artificial organ – that is, recreated the three-dimensional structure of the organ right under the skin of a model animal, without the need for implantation surgery. The organ turned out to be a miniature copy of a human ear measuring only half a centimeter. Of course, there was no practical application for this particular "mini-ear". The main thing is that scientists have managed to prove that this is possible in principle. The technology required the use of the latest developments in nanochemistry and showed the current state of regenerative medicine and three-dimensional printing.

Article by Chen et al. Noninvasive in vivo 3D bioprinting is published in the journal Science Advances.

Before talking about the new technology, it should be emphasized that the problem that Chinese engineers tried to solve with its help is quite real and quite acute. Approximately one in 2-10 thousand people in the world is born with an underdeveloped auricle – this condition is called microtia.

It can be more or less pronounced, and in many cases, plastic reconstruction of the ear is required – a surgical operation is performed for this. However, any operation is associated with possible complications, so non-invasive reconstruction of the ear cartilage using new technology could become a safer option for such children. Of course, if she herself does not give complications. That remains to be seen.

"Printing through the skin" became possible thanks to infrared lasers, microscopic mirrors and special nanoparticles glowing with ultraviolet light

The main difficulty faced by scientists, and which they still managed to solve for the first time, is the opacity of the skin for ordinary visible light. Until now, it has not been encountered by specialists in regenerative medicine, but primarily by neuroscientists. The inability to see the glow of neurons through the skin, the inability to send them a light signal through the skull – all this greatly complicates most modern experiments on the brain and forces scientists to implant optical fibers in animals.

Such interfaces, of course, constrain movements, change the behavior of animals and are fraught with inflammation, infection and other problems. But since the invention of optogenetics – the excitation of neurons by exposure to light – and until now, scientists have had to deliver light to the brain only in an invasive way. And only recently have attempts begun to appear to get rid of such dependence by creating particularly photosensitive proteins, the excitation of which becomes possible directly through the skin and skull, without any "wires".

When trying to conduct non-invasive three-dimensional printing through the skin, Chinese scientists faced the same problem: although light printing is well studied and widely used in laboratories, it turns out to be very difficult to deliver this light under the skin of a model animal. Moreover, it is difficult because 3D light printing does not require any, namely short-wave, ultraviolet radiation, and it is absorbed by biological tissues especially strongly.

We managed to solve this problem as follows. Scientists penetrated through the skin with the help of infrared radiation, which passes through the tissues best, and the ultraviolet light necessary for the actual "printing" was obtained already inside, on the spot. It was extracted with the help of special nanoparticles capable of accumulating and re-emitting electromagnetic energy. This part of the experiment was made possible thanks to the recently published technology of Russian researchers from the Institute of Crystallography of the Russian Academy of Sciences, who showed exactly how the conversion of radiation on nanoparticles can be effectively applied for three-dimensional printing.

Technology of non-invasive printing of the auricle:

• individual design (A,B,C);
• ear prototype created in the laboratory (D);
• coloring of living cells inside the tissue (E);
• the ear printed under the mouse skin, immediately after the process (F)
• and a month later (G);
• histological analysis of the obtained artificial tissue with normal islands of cartilage cells – chondrocytes (H, I).

In short, the method works as follows: an animal is injected under the skin with a three-dimensional printing material consisting of modified gelatin in liquid, unpolymerized form, as well as specially grown cartilage cells and nanoparticles acting as the initiator of the reaction. Then its image is projected layer by layer on the place where you want to create a new organ. An infrared laser and a DLP projector based on microscopic mirrors were used here - exactly the same ones are used in ordinary household video projectors.

Infrared light, getting under the skin of the animal, excited nanoparticles, which then themselves became a source of radiation – only already ultraviolet. And ultraviolet light, absorbed by the modified gelatin monomers, initiated the hardening of the material exactly at the point where the light from the projector fell – there the process of assembling individual molecules into real artificial cartilage was started.

Thus, after one injection and a small laser irradiation, a three-dimensional structure of intercellular substance filled with cells characteristic of cartilage tissue – chondrocytes - was formed under the skin of animals without any incisions. Subsequent observations showed that the artificial ear under the skin of mice "felt" good and generally differed little from ordinary natural cartilage. In addition, the scientists conducted an additional experiment in which instead of cartilage cells, muscle cells derived from stem cells were used and a small fragment of muscle was restored in a similar way.

Mice with "ears on their backs" were learned to create back in the 90s, but now the same result has been obtained on a completely different level

Among all the methods of creating artificial organs (and so very young and experimental), the new technology is certainly one of the most exotic and immature. Even the creation of an artificial ear using such non–invasive printing is more of a "classic test" accepted in regenerative medicine than a uniquely suitable application for such a technology.

In general, scientists have demonstrated the creation of an auricle implant from artificial cartilage many times – without changing the object, but each time improving the technology of its manufacture. The first and most famous example is connected with the experiment of the Vacanti brothers from the Children's Hospital in Boston, during which a full-size artificial ear was implanted on the back of a mouse. The technology described in the 1997 article was significantly different from the one discussed today – then the researchers did not use three-dimensional printing, but a specially made mold in which the polymer solidified like gypsum. An important difference from the current experiment, of course, was that an artificial ear was grown first in the laboratory, and only then it was implanted in rodents. The result, however, still turned out to be very impressive for its time – to the point that photos of Vacanti mice with ear implants went viral and were distributed among opponents of GMOs as evidence of their incredible danger (although in fact gene modification was not used in this experiment).

In 2016, the same "ear test" was already passed by the technology of layer-by-layer three-dimensional printing, and not pouring into a mold. A group of researchers from the Center for Regenerative Medicine in Wake Forest, led by Anthony Atala, printed not only the ear, but also a fragment of the jaw, skull and even a small piece of muscle tissue using a printer resembling a conventional inkjet. By itself, the resulting ear did not differ much from the "Vacanti ear" – it was also based on a natural biopolymer, which included islands of cartilage cells. However, this time the printing method was much more universal with respect to different organs and individual for a particular patient. The main achievement of the scientists was the adaptation of the old technology of inkjet printing to a completely new and extremely sensitive "paint" – living cells.

Like the previous experiments, the new technology of "printing under the skin" is very similar in its end result, but it is not interesting to them, but above all its approach. Whether noninvasive printing will find its application in regenerative medicine will become clear only after more detailed and practically-oriented experiments, including on humans. However, it can already be said that the most understandable and closest application for this technology can be aesthetic surgery, where the fewer incisions, the better.

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