Imagine a future without eye banks, where patients needing new corneas can have them 3-D printed using their own cells, and the corneas are tailor-made according to their exact geometric specifications so patients can achieve perfect vision without the need for long-term reliance on immunosuppressants. Researchers in Belgium are working to make this a reality. A group at the University of Antwerp in collaboration with the Catholic University of Leuven has been working on creating 3-D printed corneas since 2014. “What we are trying to do is create a cornea that would mimic what we see in vivo,” said Nadia Zakaria, MD, PhD, University of Antwerp. She explained that the cornea is ideal for mimicking ex vivo because “the spatial orientation of its layers and the way the collagen fibers are parallel to each other allow the cornea to be biochemically robust and quite tough.”

 

The fact that it’s without blood vessels and simpler than a lot of the other organ tissues being tissue engineered, like the kidney, helps, she said. “To create something so complex and large and for it to be functioning is more difficult,” Dr. Zakaria said. “But if we look at the cornea, it’s a much simpler tissue, and it is at the surface so it would be easier to follow up on whether it functions correctly. It seems to be the ideal tissue to start tissue engineering with.” Still, the process of creating a 3-D print of a cornea is complex, requiring a multidisciplinary team with different expertise.

 

Two full-time PhD students, Steffi Matthyssen, University of Antwerp, and Rory Gibney, University of Leuven, are working on this project. The right tools The first consideration for the team was acquiring the appropriate material for the research. “One of the early problems was sourcing recombinant human collagen,” Dr. Zakaria said. Each of the recombinant human collagen has pros and cons, she said. “So we’ve been looking at different types of collagen and how they orient as well.” Separate from the project, her team is working to develop collagen mimicking peptides with May Griffith, PhD, Department of Ophthalmology, University of Montreal, and her team. Just as important in the research is the technical aspect of 3-D printing. Dr. Zakaria explained that there are extrusion-based 3-D printers, where material “squeezes” through a nozzle and layers are built upon each other. There are also aerosol 3-D jet printers, which are used for printing electronics because of their ability to print with very high resolution. The latter is what the research team is currently using.

 

“We want to go with something that has a very high resolution rather than extrusion-based printers because the resolution is just not there, so it’s bulky and the tissue would not mimic anything close to what we want for good vision,” she said. When they first started printing with the collagen “bio-ink,” their collaborators, Eleonora Ferraris, PhD, Department of Mechanical Engineering, and her team at the Catholic University of Leuven, had to “modify the 3-D printer to be able to print effectively and reproducibly with the collagen,” Dr. Zakaria said. It was the first time this had been done. The aerosol 3-D jet printer was designed to print electronics, not biological samples. “The cornea in itself is only about half a millimeter thick and 9 mm in diameter, but printing with high resolution means that in order to obtain a sample of about 100 microns, approximately 300 layers of bio-ink needs to be deposited, which can take up to 2 hours of machine time,” Dr. Zakaria said. “Being able to print is one thing, but the collagen also needs to be crosslinked so that it has good mechanical properties and can withstand the intraocular pressure without being too brittle or losing its biocompatibility.”

 

The team had to work in close collaboration with biomaterial specialist Jennifer Patterson, PhD, and her team at the Department of Materials Engineering, Catholic University of Leuven, in order to develop such protocols. “We have been able to print with recombinant human collagen type III, and we have been able to show that we can print up to about 100 μm so these are still very thin, but they are up to 280 layers of collagen,” she said. When they print, they are able to control the orientation of the collagen fibers, she added. Once the team has proof of principle, they will build on this further. The goal for now is to move to full thickness and develop corneas that are up to 400–450 μm thick for transplantation in animals. “Eventually we’ll get to humans, but there’s still quite a long way to go,” Dr. Zakaria said.

 

Next steps Animal testing is expected to take place early next year, and they will look to determine whether the printed collagen cells that they’ll be implanting will maintain transparency, whether they will degrade and if they do, how long it takes to degrade, and whether they induce inflammation, she said. While some research groups also working on tissue engineering the cornea use a molding method, Dr. Zakaria said that the advantage of using a 3-D printer is it’s more cost efficient. The collagen material used is expensive.