Three-dimensional (3D) printing is used to manufacture everything from guns to guitars—and even underwear. The next big thing could be 4D printing. This adds time to the mix by using smart materials that transform themselves into finished articles under different conditions.
Some of the most powerful early applications of 4D may be in biotechnology and medicine. “Stents, compression structures and drug delivery mechanisms could be printed and programmed to transform themselves directly in situ,” explains Skylar Tibbits, a researcher at Massachusetts Institute of Technology (MIT). “This would be a major advance on traditional methods of actuation, sensing and diagnostics.” In addition, he says, 4D printing “could also be used for medical equipment that responds to the patient, the doctor or environmental triggers for automatic response.”
Tibbits leads a research program at Massachusetts Institute of Technology’s (MIT) Self-Assembly Lab in collaboration with Stratasys, a prominent 3D printer manufacturer. The team is developing self-assembly technologies for large-scale structures in the physical environment.
Another area of biotechnology, the bioprinting of human tissue, is already using 3D printer technology. Gabor Forgacs of the University of Missouri-Columbia prints living cells as “ink” and using his technical breakthroughs, Organovo, a company that he helped found, has engineered several human tissue types. These include liver—the bioprinted product that is most advanced – as well as lung, vasculature, bone and skeletal muscle.
Organovo’s bioprinting process can construct tissues on the order of 1 (and up to 1.5 mm thick in some instances) that mimic key aspects of native in vivo tissues. The bioprinted tissues are created without dependence on integrated scaffolding or hydrogel components, so they have a tissue-like density and highly organized architectural features, such as intercellular tight junctions, microvascular networks, and defined compartments. The microvasculature emerges spontaneously in the tissues and prevents central necrosis as the tissues are being fed.
“The bioprinter places cells with great precision but ultimately we rely on the cells to self-assemble into tissues. So instead of trying to place cells individually, one at a time, which would be inefficient and artificial, we put them close to where they need to be in the right proportions relative to other cell types allowing them to self-assemble as they want to.”
The company’s unique bioprinters are designed and built in conjunction with various partners and contract manufacturers. The company is not selling the hardware right now but has placed bioprinters with certain partners, for example Knight Cancer Institute and Harvard Medical School, which enables Organovo to work with research partners to create new tissues at their own sites.
Eric Michael David, Chief Strategy Officer at Organovo explains that, “In terms of our liver tissue, we plan to launch an off-the-shelf product by end of 2014. In drug discovery, we are already working with pharma partners on our liver tissues, and are doing a good deal of bespoke disease modeling and toxicology work on other tissues as well.” The reason is that bioprinted liver tissue is much closer to in vivo human tissue than anything else, David says. Bioprinted liver tissue stays alive and active in vitro for 40 days. For comparison, the liver toxicology standard is to use primary liver cells (hepatocytes) plated in culture, which generally stop acting like liver cells after 48 hours.
There is also a buzz around the bioprinting of tumor microenvironments. “That’s what the pharma companies and oncology researchers want to study,” says David. “They need to know how the microenvironment surrounding a tumor affects its growth and influences metastatic disease. To enable this you need to be able to study the microenvironment in 3D.”
Where do things stand in terms of development of organ replacement therapies? “Just because you have a 3D bioprinter does not mean you can simply print out a fully functional heart right this minute,” David explains. “In the short and medium term we can print things such as a patch of cardiac muscle, a small piece of liver tissue, or organoids of the kidney or other organs. And, you can print vasculature for bypass surgical procedures, particularly small size vessels that you cannot manufacture using artificial materials.”
However, the ability to print an entire organ, such as a whole kidney, liver or heart, for transplant is “a good 10 to 15 years away at least,” according to David.
4D printing the pancreas
Ibrahim T. Ozbolat, Assistant Professor for the Mechanical and Industrial Engineering Department at the University of Iowa, is working to produce a functional, transplantable pancreas.
Ozbolat immersed himself in bioprinting during his PhD research several years ago. He began by printing scaffolds to house cells and today he works in 4D to control pancreatic cell maturation, and therefore the development of tissue, under laboratory conditions. With tens of thousands of people waiting for replacement organs and demand outweighing supply of donor organs, Ozbolat says that he hopes to make a practical contribution. “I like building things that work for humans, and we want to provide functional organs for transplantation. To do so, we go beyond 3D printing; we print tissues that fuse and mature.”
Tissue development in vitro is a dynamic process that continues until the tissue dies or is sacrificed. “We print tissues directly and integrate blood vessels. It takes 10 to 15 days for maturation,” Ozbolat explains. He is clear about the limitations and unique challenges of his approach but they don’t faze him. “There are many challenges in today’s bioprinting technology. You have to work in a sterile environment, obviously, and you need to be in control of every step of the process.”
One rather fraught area of focus is the media for transferring cells from the bioprinter onto a surface or petri dish. “Hydrogels are popular for this,” he explains. “They provide a friendly environment, preserving the cells in their original phenotype. The limitation is the number of cells you can encapsulate in the gel and the fact that the cells are entrapped and unable to proliferate or interact with other cells easily.” That’s a problem because cells need to interact to produce a matrix. If they can’t collaborate sufficiently and efficiently, matrix formation is limited, which affects tissue generation. “You can’t create functional tissues just by printing living cells in hydrogels,” Ozbolat explains. “But, if you then transplant that printed tissue construct into a human body it is possible to get some tissue generation. The body provides the ultimate bioreactor conditions for generating tissues.”
Hydrogels do not degrade instantaneously; some are capable of “decrosslinking” but if they are left to degrade naturally they will take a long time to do so. They also may produce toxins that damage the cells.
From cells to organs
Printing organs provides a clear aim for those working on cell and tissue production. The big challenge with organs, however, is bioprinting blood vessels to create the necessary vascular networks.
“Without the ability to print blood vessels you can produce very thin structures—less than 1 mm thick. But, when you can introduce a vascular network you can go beyond.” says Ozbolat. “Yes, printing blood vessels is a challenge, but we are able to do it. We make quite simple networks at the moment, with the hope that we can extend these into multi-scale blood vessel networks sometime soon; these are early days, but we are moving forward.”
Let’s not beat around the bush: bioprinting is expensive. The hardware, raw material, growth factors and experiment conditons all come with high costs. It is also labor-intensive. “We need about 300 million cells to print small tissues,” explains Ozbolat, “and it takes a long time to expand that many cells, maybe a couple of weeks. Some cell types won’t proliferate and grow quickly and require growth factors and protein to encourage growth.”
Then you have to deal with cell sensitivity: some are very resilient and some are very fragile. “We’ll use resilient cells when we try out the system, for example, fibroblasts, cartilage and bone cells before moving to fragile cells,” he says. “You can experiment with cancer cells because they are resilient enough, but liver and pancreas cells are really sensitive.” During the bioprinting process there is always a risk of cell damage. Creating mechanically stronger, coherent structures is a key goal. Transferring a structure from printer to incubator is a challenge. Then, you need an efficient perfusion system in an incubator or a bioreactor to feed the tissue. “Several manufacturers have stepped forward to produce custom bioreactors for this purpose,” says Ozbolat. One thing that he avoids is using support material for creating blood vessels because you cannot remove such structures and hook up the vessels to the connector of the bioreactor. Using support material requires that the tissue constructs are kept without media until they reach sufficient tissue maturation.
Ozbolat thinks laser printers won’t be ideal for organ printing; their limit is printing cells, he says. These bioprinters are not without technical issues, either. “The nozzles can clog. You may not be able to clear it, meaning you have to change it completely, which may be result in stopping the processand restarting from scratch.”
Added to the hardware issues is the inability to automate the process completely.“We print soft material, which can collapse unexpectedly. Or the structure loses its original shape during the process. You can’t automate the process like you can when printing plastic fibres, for example,” says Ozbolat.
Despite the immensity of the challenge, progress is being made. “We’ve made small prototype tissues with vascular networks, which we’ll expand into larger, scaled-up versions.” This is proving successful for pancreas cells, but the challenge is integrating stem cells to drive the production of insulin-producing beta cells. “We are making progress and I think we’ll be able to produce a small transplantable pancreas with blood vessels 12 months from now. This will be in the sub-centimetre scale rather than a small organoid structure,” says Ozbolat.
The future of 4D printing currently rests in the hands of a few individuals. None of them want to overplay the potential – there is a need to be transparent about what can be achieved with the technology in the short, medium and long term, to ensure credibility with funding agencies, investors, partners and clients. But the potential is there for 3D and 4D printing to make major contributions to drug development, medical devices and tissue and organ replacement therapy over the coming decade.