The 3D Bioprinting Revolution

by Suraj Kannan

Perhaps no technology has grown as rapidly and promised so much in the last decade as 3D printing. Although the first industrial 3D printer was built in the 1980s, improvements in design and function over the last five years have seen a dramatic rise in production and usage; indeed, forecasts predict that the sale of 3D printing products and services will reach $10.8 billion by 2021, up from $2.2 billion in 2012 (1). The customizable and fast nature of 3D printing has made it an integral tool in rapid prototyping in a variety of industrial and research settings, ranging from academic to aerospace and military. 3D printing has also increasingly seen application in producing a wide variety of objects, ranging from household tools, furniture, and utensils to cars, aircraft, and weaponry (2-4). Along the way, this new technology has prompted ethical debates in gun control and intellectual property (4, 5). With the first commercial 3D printers now appearing on the market for hobbyists, it is easy to understand the Economist’s comment that 3D printing “may have as profound an impact on the world as the coming of the factory did” (6).

A particular application of interest for 3D printing that has already shown promising leads is in the field of tissue engineering. While 3D printing has long been applied to the production of biotechnology devices, recent interest has been directed towards printing cells in customizable fashion to produce functional tissues. Taking antecedents from earlier lithographic methods as well as breakthroughs in developmental biology, bioprinting aims to develop tissues and organs that can play a role in both laboratory investigation and disease modeling as well in therapeutics. With advances coming from both large research universities such as Harvard and companies such as Organovo, bioprinting is likely to become one of the biggest areas of investment and research in this decade.

Bioprinting: A Customizable Bottom-Up Approach

The classic definition of tissue engineering, as laid out by Langer and Vacanti, is of “an interdisciplinary field that … [works] toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ” (7). Traditionally, tissue engineering has followed a top-down approach, in which a scaffold (either synthetic, natural, or from a decellularized organ) is seeded homogeneously with cells and then matured in a bioreactor (8, 9). While the strategy has yielded some of the first clinical successes of tissue engineering, it does not allow for sufficient spatial and temporal control of cells and growth factors seeded on the scaffold. Thus, the top-down approach is limited in the amount of complexity it is able to produce in synthesized tissues.

3D bioprinting instead utilizes a bottom-up approach, in which the individual components of the tissue are patterned to allow for formation of complex tissue architecture. By utilizing computer-aided design (CAD) tools, researchers can carefully control the placement of cells, materials, and morphogens to replicate the types of organization found in the human body. These strategies often draw on the self-assembly and growth factor-driven mechanisms of cells to allow for formation of functional biomimetic tissues (8).

Perhaps the most popular form of 3D bioprinting has been extrusion printing, in which filaments are forced through a nozzle to form the 3D structure (10). Thus, in this method, there is contact between the delivery mechanism and the “bio-ink.” A contact-less approach has also been developed using thermal ink-jet printing. In this method, a pulse of current is passed through the heating element of the printhead to cause formation of small ink bubbles. The resulting change in pressure causes the bubble to collapse and the ink to be ejected from the nozzle (11). Thus, the bio-ink never comes into contact with the delivery mechanism. A number of parameters must be taken into consideration with the development of 3D printers. For example, the desired resolution plays a role in determining which type of 3D bioprinter to utilize. As tissues require both macro-scale and micro-scale control, multiple techniques must be utilized to develop both gross architecture and detailed micropatterning of cells and growth factors. Similarly, selection of material, or bio-ink, is crucial. A great deal of investigation has been devoted to the discovery and development of new bio-inks, including hydrogel mixtures (used with extrusion printers) and water-based inks (for thermal ink-jet printers). Cell viability is a third factor of interest. While extensive optimization of both extrusion and thermal ink-jet printing methods has allowed for viability of up to 90% of cells following seeding, the forces and stresses that cells are placed under throughout the printing process are a topic of current research (10-13).

Early Successes and the Challenge of Vascularization

While a great deal of effort is currently dedicated towards technical manipulations of 3D printers to ensure viability, some groups have already had successes with generating functional tissues. For example, Cui et al. at the Scripps Research Institute were able to generate synthetic cartilage consisting of human chondrocytes in a polyethylene glycol (PEG) hydrogel (11). More recently, Duan et al. from Cornell University constructed aortic valve conduits composed of multiple cell types and custom cell distribution in an alginate/gelatin hydrogel (14). While these successes have proved to be exciting for the potential of 3D bioprinting, progress with 3D-printed tissue was limited by the same challenge as other tissue engineering avenues: vascularization. Without blood vessels, nutrients, oxygen, and wastes cannot diffuse throughout thick tissues, leading to cell death throughout the construct. Previously avascular tissues produced by 3D printing were thus by necessity very thin, a constraint that prevents the generation of larger-scale organs and tissues.

A recent and astonishing breakthrough in 3D-printed tissue engineering came in February 2014 from the Lewis lab at the Harvard School of Engineering and Applied Sciences (SEAS).  The team utilized a custom-build four-inkhead bioprinter as well as several novel bio-inks, including a gelatin-based ink to provide structure for the scaffold and two cell-containing inks (15). Perhaps the most novel aspect of the investigation was the use of a Pluronic-based bio-ink that undergoes a seemingly-counterintuitive solid-to-liquid phase transition when cooled below 4°C. Thus, the researchers were able to generate 3D structures with complex networks of Pluronic ink which, upon cooling, resulted in liquification of Pluronic and production of channels within the construct. These channels were subsequently endothelialized to produce vasculature. Using this technology, the Lewis group printed structures composed of patterned human umbilical vein endothelial cells and neonatal dermal fibroblasts along with custom-built vasculature. This vasculature could be in turn be perfused in a bioreactor to allow for nutrient and oxygen flow within the construct. These results speak to the possibility of using 3D bioprinting to produce tissues of complexity far greater than that produced previously by other methods of tissue engineering.

Organ Printing and The Future

While 3D printing has a number of potential applications to research in basic science and cellular/tissue function, bioprinting has primarily captured the public imagination because of the role it could play in the clinical environment. Early clinical uses of 3D bioprinting have shown some success. For example, in 2012, physicians at the University of Michigan successfully utilized 3D printing to construct a synthetic trachea for three-month year old Kaiba Gionfriddo, who suffered from recurrent airway collapses (16). Other successes include printing bone to replace, as two case studies, patient jaw and skull (5). 3D bioprinting is ideal for physicians and patients alike – it allows for rapid production of tissues that can be personalized specifically for each patient (for example, by using patient MRI/CT data). While the limited clinical work thus far has involved avascular and sometimes even acellular tissues, innovations in vascularization in the lab suggest the possibility of future production of organs such as heart, lung, pancreas, and others.

Certainly, some progress in this regard has already been made. Viewers of TED will likely recall Dr. Anthony Atala’s talk, in which he printed a miniature kidney on-stage. Organovo, a San Diego company geared towards developing functional 3D-bioprinted organs, has made strides to release data on its printed liver by 2015, while others have predicted the completion of 3D-printed hearts within the decade (17). This research has also provoked a great deal of discussion over the ethics of 3D-printed tissues. These concerns range from general objections to tissue engineering and organ construction to worries about construct quality and the role of intellectual rights in the world of 3D bioprinting. In particular, the question of who can produce 3D organs must be addressed before further clinical developments can proceed.

In light of these challenges, it is perhaps too optimistic to suggest that 3D-bioprinted technology will be available for patients within the next decade, though as some isolated case studies have shown, such constructs have been successful when utilized. Technical optimizations, particularly in vascularization, cell viability, and resolution of printing, will allow for improved functionality and complexity in printed tissues. From the non-scientific perspective, leaders in ethics and policy will need to tackle some of the stickier issues regarding intellectual property and quality assurance in the generation and use of 3D-printed tissues. In spite of these obstacles, bioprinting remains perhaps the most promising avenue for pursuing the regenerative medicine of tomorrow.

Suraj Kannan ‘14 is a concentrator in Biomedical Engineering. 

Acknowledgements

Many thanks to Dr. Jennifer Lewis and David B. Kolesky, both of whom humoured my requests to hear everything about their magnificent research.

References

1. TJ McCue, “3D Printing Stock Bubble? $10.8 Billion By 2021.” Forbes. December 30, 2013. http://www.forbes.com/sites/tjmccue/2013/12/30/3d-printing-stock-bubble-10-8-billion-by-2021/
2. Alexander George, “3-D Printed Car Is as Strong as Steel, Half the Weight, and Nearing Production.” Wired. February 27 2013. http://www.wired.com/autopia/2013/02/3d-printed-car/
3. Paul Marks, “3D printing: The world’s first printed plane.” NewScientist. August 01, 2011. http://www.newscientist.com/article/dn20737-3d-printing-the-worlds-first-printed-plane.html#.Ux4SLx_LI7x
4. “Ready, Print, Fire: The regulatory and legal challenges posed by 3D printing of gun parts.” The Economist. February 16, 2013.
5. John F. Hornick, 3D Printing and the Future (or Demise) of Intellectual Property. 3D Printing 1(1), 14 – 23 (2014).
6. “Print Me a Stradivarius: How a new manufacturing technology will change the world.” The Economist. February 10, 2011.
7. Robert Langer, Joseph Vacanti, Tissue engineering. Science 260(5110), 920–926 (1993).
8. Raphaël Devillard et al., Cell Patterning by Laser-Assisted Bioprinting. Methods in Cell Biology 119, 159 – 174 (2014).
9. Bertrand Guillotin, Fabien Guillemot, Cell patterning technologies for organotypic tissue fabrication . Trends in Biotechnology 29(4), 183 – 190 (2011).
10. Cameron J. Ferris et al., Biofabrication: an overview of the approaches used for printing of living cells . Applied Microbiology and Biotechnology 97, 4243 – 4258 (2013).
11. Xiaofeng Cui et al., Thermal Inkjet Printing in Tissue Engineering and Regenerative Medicine . Recent Patents on Drug Delivery and Formulation 6(2), 149 – 155(2012).
12. Vladimir Mironov et al., Bioprinting: A Beginning. Tissue Engineering 12(4), 631 – 634 (2006).
13. Phil G. Campbell, Lee E. Weiss, Tissue engineering with the aid of inkjet printers. Expert Opinion on Biological Technology 7(8), 1123 – 1127 (2007).
14. Bin Duan et al., 3D Bioprinting of Heterogeneous Aortic Valve Conduits with Alginate/Gelatin Hydrogels . Journals of Biomedical Materials Research Part A 101(5), 1255 – 1264 (2013).
15. David B. Kolesky et al., 3D Bioprinting of Vascularized, Heterogeneous Cell-Laden Tissue Constructs . Advanced Materials (2014).
16. David A. Zopf et al., Bioresorbable Airway Splint Created with a Three-Dimensional Printer. New England Journal of Medicine 368(21), 2043 – 2045 (2013).
17. Organovo Homepage. http://www.organovo.com/
18. Liat Clark, “Bioengineer: the heart is one of the easiest organs to bioprint, we’ll do it in a decade.” Wired. November 21 2013. http://www.wired.co.uk/news/archive/2013-11/21/3d-printed-whole-heart
19. Anthony Atala, Printing a Human Kidney. TED2011. Filmed March, 2011. http://www.ted.com/talks/anthony_atala_printing_a_human_kidney

Feature image by Jonathan Juursema and is licensed under CC BY-SA 2.0