How to 3-D print tissue-repairing implants inside live mice

Researchers have found a way to print complex 3D structures inside live mice (Nat. Biomed. Eng. 2020, DOI: 10.1038/s41551-020-0568-z). By injecting a light-sensitive bioink and then shooting infrared pulses at it from outside the body, they made scaffolds in the brain, skin, and muscle that could be loaded with stem cells to aid tissue regeneration. This could lead to a noninvasive technique to repair injured or diseased tissues and organs inside the body without requiring surgery.

In recent years, scientists have used 3-D bioprinting to make implantable skin, blood vessels, and heart and liver tissues. They can also print porous scaffolds that are loaded with stem cells and implanted in the body, where they form new tissue. These printed tissues could help regenerate damaged skin and cartilage or patch diseased heart and lung tissue, but they must be surgically implanted.

“There’s no way to escape from surgery so far,” says Nicola Elvassore, a chemical engineer at the University of Padova. Opening up the body for surgery entails risks, such as bacterial infections. Elvassore and his team wanted to figure out how to print inside organs such as the eye without the need for surgery.

Elvassore and his colleagues turned to intravital microscopy, which is used to image cells a few millimeters deep within live animals. Researchers first inject the animal with dyes that fluoresce when illuminated with two photons of near-infrared (NIR) light. Elvassore says his team realized they could use that light for other purposes besides imaging. They wondered, “why not use that energy to crosslink polymers?”

So the team searched for polymers that were biocompatible and that would link together to form hydrogels in response to NIR wavelengths. First they chose a coumarin compound called 7-hydroxycoumarin-3-carboxylic acid that links together via click chemistry when excited by two NIR photons. Then they bound the coumarin to biocompatible polyethylene glycol or gelatin polymers to make a bioink solution.

To print tissue scaffolds inside the body, they injected the bioink into live mice, then shone NIR light in the desired printing pattern using a commercially available two-photon microscope. The polymers in the bioink crosslink to form a hydrogel. By scanning the focused NIR light layer by layer, the researchers created 3D hydrogel objects inside the body. They could make tiny structures under the skin, in muscle, and in the brain—all without surgery.

They also showed that these printed hydrogel stuctures could help form new tissue. To demonstrate this, the team added stem cells to their bioinks, and printed them in parallel lines that mimic the aligned strands of fibers in muscle tissue. Inside the body, these grew into muscle fibers aligned in the same direction as the natural muscle.

Maling Gou of Sichuan University and his colleagues recently reported a similar in vivo 3D bioprinting technology, but they use a different light source and bioink. Gou says their technique, which uses a micromirror device to vary the NIR lightwaves, is faster (Sci. Adv. 2020, DOI: 10.1126/sciadv.aba7406). The new two-photon method, meanwhile, allows higher resolution. These in vivo printing techniques are good non-invasive alternatives to conventional bioprinting for organ repair or reconstruction that requires surgery, he says.

Elvassore says the team did not observe any side effects in mice in these experiments. The biggest limitation is that body tissue absorbs NIR light, so the technique only works up to depths of 2–3 millimeters. New intravital microscopy technology currently in development could help overcome that. “But for now this method could be perfect for repairs to the cornea, for example,” he says.

It could also be used to regenerate skin and cartilage such as in the ears or nose, says Samad Ahadian, a materials scientist at the Terasaki Institute for Biomedical Innovation. Adapting the technology to reconstruct more complicated tissues like heart or liver will be a challenge.