Body Building
By Beth W. Orenstein
Radiology Today
Vol. 25 No. 2 P. 24

Researchers explore new ways to repair damaged cells with 3D printing and flexible ink.

Imagine a surgeon repairing a heart valve without having to open the patient’s chest; instead, using a 3D printer and focused ultrasound to complete the task. Or, imagine physicians 3D printing structures with cells that grow into mature tissue in a petri dish. Eventually, the tissues can be implanted to repair damaged nerves in a patient’s back. Although these scenarios sound somewhat like Star Trek episodes, both 3D printing applications are inching closer to reality.

The first is a collaborative effort by a National Institutes of Health–funded team that has designed a method of through-tissue 3D printing using ultrasoundsensitive ink. Lead authors Junjie Yao, PhD, an associate professor of biomedical engineering at Duke University, and Y. Shrike Zhang, PhD, an associate professor and associate bioengineer at Harvard Medical School and Brigham and Women’s Hospital, published their research in December 2023 in Science. Yao and Zhang met in graduate school and credit their familiarity with each other in aiding their collaboration.

Some 3D printers use light as a form of energy. Energy is needed to trigger the solidification of the printer’s “ink,” which is often a liquid, typically a resin or plastic, when solidified. However, light does not travel well through skin and organs. Rather, as light passes through the body, it scatters. Yao and Zhang recognized that to successfully “print” through biological tissues, they would need a different energy source.

They were intrigued by focused ultrasound because ultrasound waves can travel deep into tissue, sending energy far below the surface. If their idea worked, a physician could potentially build biologically relevant structures inside the human body without ever making an incision.

“The idea originated from exploring how to overcome the limitations of traditional 3D printing in medical applications, particularly in creating structures within the body,” Zhang says. “We were inspired by the precision and depth capabilities of medical ultrasound imaging and sought to apply similar principles to 3D printing.”

Their technique is based on the sonothermal effect. Temperature increases with ultrasound, as a result of the body absorbing the ultrasound waves, Yao explains. They realized that if they could precisely control the temperature increase by focusing the ultrasound waves, they could carefully guide the solidification of the injected ink, even through layers of tissue.

Inking the Deal
The researchers made an ink from a blend of materials designed to be biocompatible and responsive to ultrasound. It consists of four components: a compound that helps absorb the ultrasound waves, a microparticle that helps control viscosity, a polymer that provides structure, and a salt that absorbs heat to trigger solidification.

“It’s composed of a blend of materials that solidify upon exposure to focused ultrasound to allow specific patterns to be formed, ensuring safety and compatibility with the human body,” Zhang says.

To test their hypothesis, the researchers began trying to create 3D shapes using focused ultrasound. They filled a chamber with their novel ink and suspended a focused ultrasound transducer over it. A “matching medium” was placed between the transducer and the ink to ensure the efficient transmission of ultrasound waves. (Most ultrasound scans use a medium.) It worked. They were able to create a variety of different structures at different depths inside the ink chamber. The structures they created were of varying sizes and shapes, including a multilayered honeycomb, a branched vascular network, and models that resembled a spider.

The next step was to determine whether their technique could print through biological tissues. They used pig tissues that were up to 17 mm thick and placed them on top of the chamber that had been filled with their special ink. Sending ultrasound waves through the tissue and into the chamber below, they were able to print a variety of different structures, including a pig liver and a pig phantom. The phantom was composed of skin, fat, and muscle.

They also performed a mock surgery on an ex vivo goat heart. The researchers injected their ultrasound-sensitive ink into the goat’s left atrial appendage. Then, they used ultrasound waves to solidify the ink and effectively seal off this area of the goat’s heart. The procedure they were mimicking is known as a left atrial appendage closure. Surgeons sometimes perform this procedure with open-heart surgery on patients with atrial fibrillation who are at increased risk of stroke.

Toward Custom Interventions
Zhang says they have proven that the technique could at least augment, if not replace, surgeries that involve implantation of medical devices or tissue scaffolds, “especially in minimally invasive procedures. It has the potential to reduce the need for extensive surgical incisions.” The primary advantage of 3D printing with ultrasound-sensitive ink vs surgery, Zhang adds, “is that the minimally invasive nature reduces recovery times and complications associated with traditional surgeries. It also allows for more precise and tailored interventions, potentially improving patient outcomes.”

Zhang says more testing is needed. “Testing can be done initially in lab settings using tissue-mimicking materials,” he says. The next step will be to test animal models to evaluate biocompatibility and functionality. “Clinical trials would be the final step to ensure safety and efficacy in human applications,” Zhang says.

Focused ultrasound printing can require high levels of energy. A potential challenge is that the high energy could cause surrounding tissue to overheat. To address this issue, the researchers constructed a confocal, high-intensity ultrasound printer. Any 3D printer employing their technique would have to be specialized, Yao says. “It would have to be capable of handling biocompatible materials and equipped with advanced ultrasound technology for precise internal structuring.”

The printer the researchers designed uses two ultrasound transducers that are aligned in a crossbeam pattern. This allows two ultrasound wavefronts to overlap, which reduces the energy needed from each transducer. It also improves the resolution and speed of the 3D printer.

The researchers still face several obstacles in bringing their idea to a clinical setting. “Key obstacles include ensuring the technology’s precision and safety in a clinical setting, obtaining regulatory approvals, and integrating it into existing medical procedures and equipment,” Zhang says.

Yao says that if their technology ultimately proves feasible, “it could take from several years to a decade to undergo thorough preclinical testing, clinical trials, and regulatory processes before it becomes widely available in medical practice.”

In addition to replacing some open surgeries, the researchers see it being used in tissue engineering, targeted drug delivery, and, potentially, in creating diagnostic tools or devices within the body. Building entire organs is a more distant goal, Zhang says. “This technology could contribute to creating organlike structures or scaffolds that promote tissue regeneration and repair,” he says.

New Possibilities
Reaction to their work has been overwhelmingly positive. “There is tremendous excitement about the potential applications in medicine and interest from various medical and technological sectors,” Zhang says.

Xuanhe Zhao, PhD, a professor of mechanical, civil, and environmental engineering at the Massachusetts Institute of Technology, says he has been impressed by the idea and the results so far. “While the biocompatibility and printing resolution of the technology need further improvement and validation, there are definitely possibilities of real applications and impact in the future,” Zhao says.

The next steps are to refine the technology and conduct extensive testing and trials. While the technology is still a prototype that requires further optimization, “We are collaborating with medical professionals and regulatory bodies to bring this innovation eventually to clinical use,” Zhang says.

In a press release issued by the National Institutes of Health’s National Institute of Biomedical Imaging and Bioengineering, Randy King, PhD, a program director in the division of applied science and technology, says, “This potential new application, built on years of technology advancements, could set the stage for something previously thought impossible: through-tissue 3D ultrasound printing.”

— Beth W. Orenstein of Northampton, Pennsylvania, is a freelance medical write and regular contributor to Radiology Today.

Advancing Regenerative Medicine
Just as ultrasound-sensitive inks for 3D printing may someday help replace surgery, researchers in the lab of Jeffrey Hartgerink, PhD, at Rice University in Houston are 3D-printing peptide inks to advance regenerative medicine. Adam Farsheed, a Rice University bioengineering graduate student, is the lead author of a study that appeared in January 2023 in Advanced Materials. The researchers were able to 3D-print well-defined structures using a self-assembling peptide ink.

Amino acids make up proteins in the human body. Twenty amino acids occur naturally. Farsheed explains that amino acids can be linked together into larger chains, much like Legos. Once the chains contain more than 50 amino acids, they are called proteins. When they are shorter than 50, they are called peptides. “In our work,” Farsheed says, “we used peptides as our base material in our 3D printing inks.”

The multidomain peptides the researchers designed were hydrophobic (repelling water) on one side and hydrophilic (dissolving with water) on the other. When placed in water, one of the molecules would flip itself on top of another. This created a hydrophobic “sandwich.” The sandwiches stacked onto one another and formed long fibers. The fibers formed a hydrogel, a water-based material with a gelatinous texture. The potential applications for this material include tissue engineering, drug delivery, and cancer treatments, Farsheed says.

Previous work in the Hartgerink lab has shown that multidomain peptides can safely be implanted in the body. Farsheed thought of going in a different direction and seeing whether multidomain peptides could be an ideal 3D printer ink. The fact that they self-assemble was what gave him the idea, he says.

Turning the (Pep)tide
Multidomain peptides are soft and similar to Jell-O. So how could they be shaped with a 3D printer? Farsheed thought that instead of chemically modifying the material to make it more amenable to 3D printing, he would simply add more material. He increased the concentration about fourfold, and it worked extremely well, he says.

The structures were printed with either positively or negatively charged multidomain peptides. When immature muscle cells were placed on the structures, they behaved differently, depending on the charge. Those with a negative charge remained balled up on the substrate, while the cells on the positively charged material spread out and began to mature. “It shows that we can control cell behavior using both structural and chemical complexity,” Farsheed said.

Farsheed, who began his research three years ago, says the next step is to create models of different sorts of injuries and see whether using peptides as 3D printing ink allows researchers to grow mini-nerves outside of the body. “Then you could use our material for nerve regeneration,” Farsheed says. “You would implant it in the body, which is similar to procedures that are done today for some sorts of nerve injuries. This would be a replacement nerve.”

The process could have other applications, as well, Farsheed says. “Instead of trying to build nerve tissue, you could just build muscle and use that as a kind of replacement following an injury.” Farsheed also sees a role for this technology in cancer treatment, possibly testing or delivering treatments and repairing damaged organs. The hope is that the 3D printed peptides can be used to create personalized structures that are tailored towards the injuries they are trying to repair, he says.

Feedback about the work has been extremely positive, as well, Farsheed says. The concept has gotten a great deal of attention in the media, and he continues to present the work in both academic and commercial settings. Farsheed says, “We are hoping to publish the 2.0 and 3.0 versions of this work” in the first half of this year.