A helping hand

3D bioprinting holds huge untapped potential within medicine. While 3D printing is already widely used – with applications ranging from rapid medical device prototyping to custom-designed prosthetics – it is through bioprinting that the industry is likely to see the biggest advances.

In simple terms, 3D bioprinting refers to 3D printing with living cells. Rather than using metal or plastic, the bioprinter deposits these cells in layers to develop an artificial tissue or organoid. This tissue can be used for medical research, as it models real tissue in the body. It might also be used for skin grafts for burns victims or as an organ replica allowing surgeons to practice ahead of time.

Most speculatively, but most excitingly, it could one day be used to 3D print entire organs, bringing new hope to transplant patients who’d otherwise spend years on a waiting list. That day appears to be coming closer. In 2019, researchers at Tel Aviv University engineered the world’s first bioprinted heart – complete with blood vessels, ventricles and chambers – from a patient’s own biological materials.

“Maybe, in ten years, there will be organ printers in the finest hospitals around the world and these procedures will be conducted routinely,” said lead researcher Professor Tal Dvir, who added that he planned to transplant the heart into animal models.

In another breakthrough, scientists in Chicago recently mapped the location of structural proteins in a pig ovary. They were able to engineer an artificial ovary, which, when implanted into a sterile mouse, enabled that mouse to become pregnant.

One day, artificial ovaries of this kind could benefit girls who undergo fertility-damaging childhood cancer treatments.

A versatile tool

As Joshua Hunsberger, chief technology officer at the RegenMed Development Organization (ReMDO), explains, 3D bioprinting has scope to become a tremendously versatile tool. “Its applications could range from bioprinting organoids for drug screening and personalised medicine to bioprinting scaffolds for tissue engineering, to bioprinting bioinks for wound healing,” he says. “3D bioprinting could also assist regenerative medicine manufacturing by being able to prototype customised containment vessels to aid in cell expansion or tissue maturation processes that incorporate natural scaffold biomaterials and cells.”

By way of analogy, Hunsberger suggests thinking of Lego; give someone 1,000 Lego building blocks and he or she could probably build a million different objects.

“With 3D bioprinting, you are enabling an industry to bioprint cells and biomaterials together to build new tissues and organs that could have many, many different applications, some of which we may not even have imagined,” he says. “For instance, imagine being able to bioprint inside the body to repair or replace a damaged tissue or organ? I think this is one potential area that will really accelerate the integration of regenerative medicine into standards of care.”

If medical technologists succeed in doing this, 3D bioprinting will surely be worthy of the hype. The difficulty for the time being is that suitable bioinks – the materials used in the printing process – are hard to come by.

“Bioinks need to be highly printable while providing a robust and cell-friendly microenvironment,” says Dr Akhilesh K Gaharwar, associate professor in the Department of Biomedical Engineering at Texas A&M University. “However, current bioinks lack sufficient biocompatibility, printability, structural stability and tissue-specific functions to translate this technology to preclinical and clinical trials.”

Watch the thixotropy

While a multitude of 3D bioprinting techniques have been developed – including laser-assisted printing, inkjet printing and extrusion-based printing – the process is often fraught with technical difficulties.

The inks used need to have the right consistency, as well as being compatible with the printer itself. For the most part, researchers opt for extrusion-based methods, in which a computer-guided nozzle extrudes the bioink and deposits it in certain shapes. This relies on substances called hydrogels, which carry the cells, provide structural support and are able to control various cellular functions. Hydrogels are biocompatible and biodegradable, and a number of different varieties are available.

Unfortunately, there is often a trade-off between the viability of the cells and the printability of the hydrogel. Designers may be forced to compromise on the latter. But there may be a third option: “Advanced bioinks employ numerous strategies to elevate printability and cellular compatibility simultaneously,” says Gaharwar. “They also protect the encapsulated cells without compromising the printability or print fidelity.”

Gaharwar’s research group is leading the charge to develop inks of this kind. Their inks, known as nanoengineered ionic-covalent entanglement (NICE) bioinks, combine two different reinforcement techniques to produce stronger structures. One of these techniques is nanoreinforcement, in which trillions of tiny particles called nanoclays work like magnets to hold the ink together.

“These linkages are disrupted when the bioink flows out of the printer, but reform in seconds after the bioink stops moving, effectively turning the ink back into a solid,” says Gaharwar.

This solves a common problem with bioink development – namely getting the viscosity right. The bioink will need to have low viscosity during the extrusion process, but it will also need to solidify rapidly once it’s printed to maintain the desired shape. Hunsberger states this challenge in simpler terms: “When you squeeze a tube of toothpaste, the paste is extruded out from the pressure and is then able to maintain its shape on your toothbrush. If a bioink is too much like a liquid it will simply form a puddle after being bioprinted. If the bioink is too firm it will clog the printing nozzle. This is called thixotropy – change of physical properties in response to stress.”

Potential custom options

In the lab, Gaharwar’s inks have been used to print ears, blood vessels, cartilage and even bone. One day, they might be used for bone regeneration, bringing new treatment options to patients with arthritis, bone fractures, dental infections and craniofacial defects.

“The promise of using a 3D bioprinter to print custom bone tissues is gaining interest from researchers and clinicians, since managing bone defects and injuries through traditional treatments tends to be slow and expensive,” he says. “Our group is also focused on engineering 3D bioprinted tissue models to study various diseases and predict the efficacy of novel therapeutic interventions, potentially reducing or eliminating animal subjects.”

Hunsberger’s organisation, ReMDO, has an even more ambitious goal. A non-profit working with industry and academia, it is leading a project to develop a universal bioink. “This will be an out-ofthe- box bioink that could be used across different bioprinting platforms (extrusion, ink jet) and compatible with supporting different cell types (endothelial cells or cardiac cells),” he says. “By the end of our programme, we will have a prototype product that could be commercialised by any one of our industry collaborators.”

As well as getting the thixotropy right, the ink will also need to be tunable, both from a biomechanical and biochemical perspective. “The biomechanical tuning allows us to bioprint structures at different resolutions, and mimic stiffness ranging from bone and cartilage to soft tissues like liver, brain, and heart,” says Hunsberger. “Biochemical tuning ensures that the correct environmental needs are contained within the bioink to support the cells and tissues being bioprinted.”

One should not understate the difficulties associated with designing a universal bioink. As Gaharwar points out, the human body is highly heterogeneous, so it’s tricky to design a substance that will work across all tissues and organs.

“Each bioink needs to have a custom formulation for specific cell or tissue types,” he says. “Specifically, cells in different tissues have a certain niche, which needs to be recapitulated to facilitate tissue and organ growth. This is the current challenge in the field of bioprinting and thus it is not possible to have a common bioink formulation.”

A universal bioink

Nonetheless, the ReMDO group, along with its consortium members, is taking the challenge in its stride. A major advantage of the ReMDO Universal Bioink is that it will be tested and validated by many different users. This means a widespread set of needs, and an array of commercial and therapeutic applications, will be built into the programme early on.

“This bioink will, therefore, be able to be tuned to meet numerous applications ranging from bioprinting organoids for drug screening, disease modelling and personalised medicine to bioprinting scaffolds, to bioprinting hydrogels for applications with wound healing,” says Hunsberger. “We envision this ReMDO Universal Bioink being a foundational building block that [the] industry will use to fund future programmes to progress beyond the capabilities of this Universal Bioink Program Version 1.0.”

Beyond the Universal Bioink project, ReMDO has an entire advanced biomanufacturing initiative under way, which may be of interest to a number of prospective industry collaborators. Hunsberger also serves as executive director of the Regenerative Medicine Manufacturing Society, which seeks to ensure a smooth transition of regenerative medicine therapies to market. Among other goals, it is aiming to develop a set of standards for 3D bioprinting – evaluating the current landscape in this area and working with others in the field to arrive at solutions. While it’s early days for 3D bioprinting, the technology is emerging quickly and generating a significant buzz throughout the industry. Time will tell whether attempts to develop a ‘universal’ bioink will reap the rewards.

“I think 3D bioprinting is the future of regenerative medicine and having a universal bioink will only enable this technology to be even more prevalent,” says Hunsberger.