To model or to function? Answering the million-dollar question of 3D bioprinting and regenerative medicine with leading experts in bioengineering and biomaterials.
According to the Organ Procurement and Transplantation Network, 17 people die each day while waiting for an organ transplant, and in 2020, only 39,035 of the 108,494 people who needed organ transplants received them. These figures alone show how short the supply of organs available for transplantations is, which is not surprising considering that donors are the only viable source of organs, and organs aren’t exactly reproducible. Or are they?
Though once a far-fetched dream, creating artificial organs has now become a tangible reality with the help of three-dimensional (3D) bioprinting, an emerging field that is soon to lead the future of regenerative medicine.
In “Bioprinting: An Organic Solution,” an online conference hosted by SGInnovate and CATALYST on the 13th of July 2021, Dr. Riccardo Levato from the University Medical Center Utrecht and Dr. Javier Fernandez from the Singapore University of Technology and Design shed light on the potential for bioprinting to become the latest ground-breaking solution in bioengineering and usher in the era of personalised medicine.
What is Bioprinting?
Bioprinting, as its name suggests, is a form of additive manufacturing that turns the blueprints of a digital file into 3D biomedical parts. The process follows standard 3D printing methods, but instead of using plastic or synthetic materials, bioprinting creates structures of tissues in a layer-by-layer manner using a printable ink known as “bio-ink”.
Bio-ink is a biomaterial made from a mixture of cell types collected from patients amid viscous materials like alginate or gelatine. When layered, this ink can adhere together to create structures that closely resemble the composition of natural tissues at both cellular and geometrical levels.
In principle, with the right tools and the correct approach, bioprinting can mould living cells into virtually any shape conceivable. This versatility is what gives 3D bioprinting its widespread applicability – from creating 3D models for surgeons to train with before surgeries, to replacing humans and animals in drug trials.
However, of its various potential uses, 3D bioprinting is most commonly associated with the production of artificial organs, and for good reason. 3D bioprinting allows for the creation of artificial organs that imitate their natural counterparts from cells of patients. Theoretically, this would allow clinicians to transplant printed organs without having to worry about implant rejections and so reduce the dependence on organ donations.
But, putting theory into practice cannot be more challenging. Unlike conventional 3D printing, the “finished” bioprinted structure cannot be readily used as an organ replacement.
“The object that comes out of the printer is not ready to be used yet, it is not an actual organ. It may look like such but it does not behave as that,” said Dr. Levato.
Where the Problem Lies: An Organ or a Painting of the Organ?
There exists a fine line between models and organs. But even with the advanced tools available to create precise and accurate compositions of tissues, that line has yet to be crossed. To date, scientists still struggle to capture the shape-to-function relationship of organ and tissue models, or in other words, bring these accurate models to become functional organs.
In most cases, the resulting models assembled from bioprinting still require additional guided maturation in order to function properly and stably as an organ should. For example, researchers still need to closely monitor multi-tissue and cell interactions, nutrient supply, mechanical performance, and the physiological scale of printed models of muscle tissues before they can be used.
During Dr. Fernandez’s early works with micro-masonry, otherwise known as Bio-Legos, his team encountered the same problem. Micro-masonry involves reforming groups of cells into block shapes and assembling them using capillary forces, which worked as “glue”. Like Lego blocks, they can be assembled to create structures by following certain geometries.
In their mission to mission to reproduce organs, they were successful in positioning, aggregating, and maintaining the health of the Lego cells. However, their final model could not be considered functioning tissues or organs as they lacked the complex cellular interconnections observed in real organs.
“Putting the cells in the right place is only half of the story. That’s not an organ,” said Dr. Fernandez. “If we place cells in the right place, without those interconnections, without the interactions between the cells, we are not really building an organ, we are more like representing an organ. It’s kind of like a painting of an organ rather than a functional one.”
Knowing the Difference, Now Piecing the Puzzle
Establishing this key difference between model and organ is the first step in guiding research to produce functional organoids. The next challenge was to determine whether it is possible to reproduce complex cellular interactions like vascular systems using a 3D printer at all.
After a series of experiments to create de novo cardiomyocytes from embryonic stem cells, Dr. Fernandez and his team were able to gain some valuable insights into what makes an environment optimal to trigger the development of the embryonic system and vascular networks. With more studies done to uncover these networks, it would be possible to reproduce them in the future with more precise tools.
However, as of now, the fact of the matter is that human organs are incredibly complex, and current bioprinting technologies have yet to reproduce that complexity. But even if current technologies are still limited to creating organoids and models, that does not make bioprinted organs any less useful.
Seeing the Bigger Picture: What Biomimetic Environments Can Offer
When studying the underlying mechanisms of drugs and diseases, scientists need to isolate single or several pathways from the various processes occurring in an organ. However, isolating and replicating these pathways can be extremely difficult when using cultured cells as there are many other processes simultaneously occurring in the cells.
Fortunately, as rightfully pointed out by Dr. Fernandez, “Bioprinting is really good in doing that. Bioprinting allows us to make a replica of an environment in the body.”
Creating a biomimetic environment allows scientists to study specific events occurring inside a cell. To demonstrate, Dr. Fernandez and his team reproduced the environment of breast cancer and compared the growth and movement of invasive and non-invasive cancers. By combining their model with machine learning, they successfully discovered critical differences between metastatic and non-metastatic cancers.
Additionally, “bioprinting has the chance [to be] not only useful for putting things inside the patient, but is also extremely useful, at this point, to move medicine out of the patient.” Rather than prescribing medications and therapeutics to patients without knowledge of possible side effects, clinicians can test these drugs before administration on biomimetic models grown from cells of patients.
Where We’re At: Organoids and Tissue Engineering
Today, scientists have begun to merge 3D bioprinting with other bioengineering techniques to reconstruct damaged tissues and organs. In one of his major projects published in 2017, Dr. Levato demonstrated the process of bone, cartilage, and joint restoration. He began by first “printing the organic scaffolds where cells are seeded afterwards” – a standard example of how bioprinting is merged with tissue engineering.
Using an example of a warmblood horse with a tuber coxae defect, he showed how a bioprinted ceramic material, which can mimic the composition of bones, can act as a scaffold for cells to be cultured. The cultured cells can then differentiate into bone cells and be implanted on the affected bone. In time, new tissues can be regenerated and the scaffold can be digested by multi-nucleated cells. Because the material is biodegradable, it leaves no trace of itself as the area where the scaffold was first implanted is invaded by new bone cells.
Besides organ and tissue reconstruction, “we can also think of technologies that actually include cells in the printing material. In this example, we worked with gelatine-based hydrogels in which stemmed liver organoids.” Organoids are micro-scaled aggregates of cells that can self-assemble and create multiple cell types that mimic the function of a certain organ.
Although these organoids have yet to demonstrate the capacity for transplantation, they can express a variety of important mature hepatic markers, like cytochromes, that are involved in the metabolism of drugs, making them useful indicators of drug metabolism.
Similar projects in wound repair, bone reconstruction, and heart tissue models are currently in the pipeline. To cater to the growing interest and resources being poured into this enterprise, researchers have been seeking ways to improve bioprinters to deliver faster, more precise, and more accurate performance.
Where We’re Going: Speeding up and Smoothing Out 3D Bioprinters
One of the major challenges in 3D bioprinting is speed. Currently, most printers still work in a layer-by-layer fashion, which makes the process slow and prolongs the time that cells are grown in sub-optimal culture environments. When cultured cells grow outside of their optimal culture environment, they tend to lose some of their metabolic activity and may even die.
To accelerate the process of bioprinting, Dr. Levato and colleagues developed a new, rapid hologram-based technology that eliminates the need for layering.
“We project layered light through a digital micromirror device onto a rotating vial containing our gel with cells. The different projections that we get from topographic patterns make a sort of a hologram,” Dr. Levato stated. Where the hologram appears, the combinations of projections support the crosslinking of the gel. The rest of the material can then be washed out, leaving the final biomodel.
This dynamic process allows the structures up to four cubic centimetre to be printed in less than 25 seconds, as compared to conventional exclusion methods and digital light processing, which can take any time between five minutes and several hours. Additionally, their printer can produce very smooth features without the pixelated edges typically seen in conventional printing methods. The team has reached a resolution in the range of 100 microns or less, which is comparable to exclusion-based printing.
In their recent proof-of-concept work, they demonstrated the technology’s ability to recreate a complex trabecular bone structure using a Micro-CT scan. After filling the structure with gel, they were able to add muscular cells in the porous networks as well. These cells eventually formed the capillaries that penetrated the gel and attached to the bone cells, thereby integrating into the tissues.
The same technology is currently being explored in other consortiums, with much interest in its applications for printing soft tissues like pancreatic organoids and creating an in vitro model to test the new drugs.
The ongoing ENLIGHT project, for one, seeks to develop volumetric printing to create a 3D-printed pancreas to test new diabetes medications. Volumetric printing is a stark upgrade of conventional bioprinters. By leveraging tomographic printing, it can print centimetre-scaled structures, which usually takes hours, in mere minutes.
Bioprinting in the Next 10–20 Years to Come
According to Dr. Fernandez, the most feasible application of bioprinting in the near future would be bone reconstruction. He foresees that society is more likely to adjust to and accept the concept of reconstructed bones much more quickly as compared to artificial organs, especially as many experiments in prostheses are already well underway. Moreover, the materials needed to build bones are also more flexible for fine-tuning and the mechanics to adhere and embed cells are adjustable, thus making it a promising field to “put his money”.
Dr. Ricardo agreed, and in addition, highlighted the bright prospects of bioprinted cartilage and skin. They are relatively simpler to construct as compared to complex organs like livers and hearts, which would require further improvements in not only printing technologies but also biomaterials.
- Levato, R. (2021, July 13). Keynote Presentation [Webinar]. Bioprinting: An Organic Solution.
- Fernandez, J. (2021, July 13). Keynote Presentation [Webinar]. Bioprinting: An Organic Solution.