Background

It can be argued that the concept of bioengineering began when Alexis Carrel and Charles Lindbergh published “The Culture of Organs” in 1938, which described the equipment and methods that made the in vitro maintenance of organs possible. The final chapter of the book mentions an ‘ultimate goal’ of increasing the speed of wound healing. From its conception in the 1980s to the present day, scientists and medical researchers alike have been investigating the exciting prospects that three-dimensional printing offers to the field of medicine. Over the course of three decades, advances in this technology have led to several famous milestones, in the process spawning the term ‘bioprinting’. In contemporary medicine, bioprinting is beginning to play a role in regenerative medicine and clinical research by providing scientists with the ability to build tissue-engineered scaffolds, prosthetic limbs, and even functioning kidneys. One of the earliest cases of bioprinting made international headlines in 1999 when the world’s first 3D printed collagen scaffold was used for bladder augmentation in dogs. Then, in 2009, researchers at Organovo Inc., a 3D bioprinting company in the United States, created the world’s first bioprinted blood vessels for hepatic tissue by printing tri-layered analogues formed of human fibroblasts (to represent the adventitia), smooth muscle cells (to represent the media), and vascular endothelial cells (to represent the intima).

Outside of regenerative medicine, bioprinting is already being used to screen new drugs and drug delivery pathways. This may speed up the rate at which new drugs are released for use by allowing pharmaceutical companies and clinical researchers alike to observe the way their drugs affect models as realistically as possible. Bioprinting is also implicated in the high attrition rate of trial drugs during pharmaceutical research, as it allows companies to quickly print and use cell lines consisting of different specialised cells. This then allows for modelling functionally complex tissues. The process is briefly described by an excerpt from a paper published in March 2016 by Atala et al. [1], highlighting the attractive opportunities provided by bioprinting groups of specialised micro-tissues rather than using traditional cell lines; bioprinted micro-tissues allow for observations of the drug’s effects on multi-cellular specialised tissues to be modelled and used during drug research. This is different to current methods which study the effects of the drugs after application, and also emphasises that the utility of bioprinting is limited to simply creating models for ease of visualisation during experiments.

Clinical need

In the United Kingdom, the clinical need for bioprinting is most evident in the field of transplantation medicine, where it is often difficult to guarantee organ availability for patients. In addition, the risk of rejecting transplanted organs requires the patient to take a life-long course of immunosuppressant drugs to prevent the body’s immune system from attacking the ‘foreign’ organ. In the 2014-2015 financial year, there were 4433 organ transplant operations registered with the NHS; these figures correlated to reports of the first drop of organ donations in the past eleven years. This is a major problem facing the NHS, with the demand for organ transplants so high and yet the supply so low. Not only is this a nationwide issue, as reported by BBC News [2], but there is a global shortage of organ donors as well, further demonstrating the need of bioprinting in transplant settings [3].

In Wales, the organ donation system is different from the rest of the UK in that a ‘deemed consent’ system is employed. This opt-out system in Wales has proven its effectiveness by increasing access to organs for those who need them. In fact, more than half of the organs transplanted earlier this year were from deemed consent [4]. Unfortunately, Wales is the first nation in the UK to adopt this system and it has yet to face the true test of time.

Developing regenerative medicine for the battlefield is another area of need, which would allow soldiers and personnel working in high-risk environments to obtain fully bioprinted organs and limbs. Further research has shown that there have been 1,645 cases of major limb amputations since the US military began recording their occurrence during the 15 years of US deployment to the Middle-East (beginning in 2001) [5]. Sufferers of these life-changing accidents may no longer find themselves with terminal conditions; instead they could be given a second chance at life with newly bioprinted organs and tissues.

The importance of 3D printing in medicine lies behind the future applications it boasts, which include significantly reducing the pressure on the organ donor registry and eliminating the risk of rejection in patients. This independent study aims to explore those potential applications for bioprinting and their relevant details in greater depth.

Principles of application and methods of bioprinting

Cutting-edge bioprinting technology uses computer numerically controlled machines (CNC machines) which have been purpose-built to ‘print’ somatic cells into three-dimensional organoid shapes. The first iteration of such technology came in the form of a modified inkjet printer, this was only achievable after inkjet printers themselves became capable of printing to 80μm resolution. Almost all conventional commercial inkjet printers are only capable of working in two dimensions, the ‘X’ and ‘Y’ axes, the modification which makes bioprinting possible with such printers is the introduction of a ‘Z’ axis; this is achieved by adding an elevator mechanism, allowing the print head to precisely spray biomaterial in all three axes. The printer cartridges themselves were then replaced with biomaterial cartridges, which ensured the system was fed with ample supplies of living printing material.

The inkjet printing technique utilises a modified printer which is commercially available for home and office use. The aforementioned modification requires an elevating platform for the ‘Z’ axis, allowing the inkjet printer to direct a fine spray of at least 80μm resolution onto a print surface in three-dimensional space. The print head may use either piezoelectric or thermal release systems to spray the biomaterial onto the print surface. Piezoelectric based inkjet bioprinting relies on electrical conduction of the piezoelectric crystals which vibrate and cause the biomaterial at the end of the inkjet’s nozzle to break off as droplets; the size of the droplets can be controlled by the frequency of vibration. Thermal-based inkjet bioprinters require a heating element positioned in the nozzle to vaporise some of the hydrogel in the nozzle, resulting in the formation of a gas pocket, which forces the release of a droplet of biomaterial by means of pneumatic pressure. The size of the droplets can be controlled by the intensity of the heat produced by the heating element. Although one would fear that the high temperatures required in this process could significantly affect the cell viability of thermally bioprinted cells, this is not the case [9].

Laser-induced forward transfer (LIFT) is now the most commonly practised form of laser-assisted bioprinting. Laser pulses travel through a lens, focusing the pulse and directing it to a smaller localised area of an energy absorbent layer. The energy absorbent layer (often made of gold or titanium) vaporises upon contact with the focused pulse and releases a droplet of biomaterial from the cell-laden hydrogel layer beneath it. Droplet size can be changed by adjusting the proximity of the lens and thus the area which is vaporised. LIFT technology has drastically improved the survival rate of tissue constructs made by using laser-assisted bioprinting and has made it a viable, accurate and reliable method to create bioprinted structures [8].

Extrusion-based bioprinting involves the direct release of biomaterial suspended in a hydrogel through a tapered conical needle onto the print surface; this is achieved by pneumatic pressure, piston mechanisms or Archimedean screw mechanisms. Extrusion based bioprinting releases a continuous stream of biomaterial in a controlled manner, allowing the printing of large tissue structures. The thickness of the stream can be changed by alternating the needle bore, although one would expect future extrusion based bioprinters to regulate thickness by overlap printing, which could affect cell viability if the construct is large and requires a significant amount of time to finish [10].

In order to print in three-dimensional space, the bioprinter requires information regarding the shape and size of the object being printed. This is most commonly achieved by piecing digital volumetric data obtained from MRI and CT scans together; the raw data is processed by a computer which uses several mathematical formulae and algorithms to produce a three-dimensional rendering of the object in question. Once the model is complete, scientists analyse it to check for any errors with the spacing, before exporting the file as a series of codes which instruct the printer as to how it must print the object. The biomaterial is comprised of specialised cells which have been selected for the print application and is almost always kept suspended in a hydrogel to provide structural support. When working with donated organs, decellularisation is required to remove all of the donor’s cells from the organ and allow the recellularisation of the organ using the host’s own cells; this is achieved by the employment of Wnt and GSK3β inhibitors to modulate Wnt and β-catenin signals [11].

Modern Bioprinting

To this day, the concept of bioprinting has hardly changed; bioprinters have only become specialised machines rather than modified commercial printers. The most commonly found examples of bioprinters employ either Inkjet, Laser-assisted, or Extrusion methods, and their advantages and disadvantages are listed (Table 1).

	Inkjet	Laser-assisted	Extrusion
Pros	Capable of continuous printing	Quality of final product is almost always near perfect	Individual cell printing is appropriate for creating cell lines or small structures
	Capable of drop-on-demand	Does not jam because it is used to cut things	Individual cell printing is often fast and products are made efficiently
	Continuous printing is good for large-scale industry operations	Never touches printing material, minimising chance of contact error	Cell-laden hydrogels are appropriate for printing large, complicated organoids
	Drop-on-demand printing allows precision and high-quality products		Using hydrogels allows cells to be provided with the necessary proteins, vitamins and essential biochemical compounds needed to sustain life
	Drop-on-demand printing operates with less material wastage		Hydrogels are appropriate for printing time-consuming operations, as they will need to keep cells alive
Cons	Continuous printing is not precise, resulting in reduced quality	Expensive to procure laser technology	Takes the longest to print objects
	Continuous printing wastes more material	Power costs are high due to laser usage	Requires more preparation of material in advance of operation
	Drop-on-demand printing consumes more time and thus takes longer to print

Table 1
Advantages and disadvantages of each approach for 3D bioprinting

Discussion

Progress

The most recent advances in bioprinting have come from the Wake Forest Institute of Regenerative Medicine, under the leadership of Professor Anthony Atala, who is often regarded as the pioneer of bioprinting. In February of 2016, scientists working with Dr. Atala demonstrated the possibility of printing replacement tissue for patients by developing a next-generation bioprinter capable of fabricating structurally stable, human-sized tissue-based objects of any desired shape. Originally, bioprinted tissue structures were limited to a maximum size of 200 microns (0.2 millimetres) because of the inversely proportional relationship between diffusion distance and the rate of diffusion. As a result, Dr. Atala’s development occurred as his team of researchers were trying to find a solution to the “problem of diffusion.”

The researchers wanted to print the structures in such a way that would encourage them to develop vascularity, thus allowing blood vessels and oxygen delivery systems to form. To test this concept, they printed bone, ear and muscular tissue suspended in bio-degradable microgels, with each printed organ containing a series of lattices formed of microchannels designed to allow in oxygen and nutrients from an external source (the body). This was done with the aim to keep the structures alive long enough for them to develop their own blood vessels. Amazingly, the prints developed functionality and formed a system of blood vessels; the results obtained by the scientists provide significant evidence for the feasibility of printing biological constructs and surgically implanting them into patients.

The more famous cases of bioprinting which made international headlines includes the story of Luke Massella, who in 2001 received one of the world’s first human transplants of a bioprinted bladder. Luke had been diagnosed with renal failure at the age of 10; he became one of the first few people in the world to receive the treatment, becoming famous overnight. The process in which the bladder was made started with a collagen scaffold of a bladder, which was then immersed in a growth medium seeded with healthy urothelial cells of the patient. The newly coated organ was then kept in an “oven-like device” (a bioreactor) at a constant temperature of 37°C and an oxygen concentration of 95%, allowing the cells to multiply and develop the structure into a fully functioning organ.

Further developments have been made since 2001. In a TED talk hosted in 2011, Dr. Atala talked about stem cells and their future uses in regenerative medicine. His team at the Wake Forest Institute had managed to extract pluripotent stem cells from amniotic and placental fluid and had eventually differentiated them into cardiac cells [3]. Replicating this on a larger scale may allow people to print their own specialised organs using bio-inks seeded with these stem cells; possibly negating the use of organ donation and the risk of organ rejection from transplants altogether. Combining 3D printing with stem cell transdifferentiation may provide an improved solution as compared to traditional treatment methods.
Challenges

Although there have been many recent scientific breakthroughs in the field of regenerative medicine, there are still challenges to be addressed prior to widespread clinical implementation of this technology. One such challenge is time, since the new bioprinter showcased by Dr. Atala is capable of printing a functioning kidney in 7 hours [3]. Bioprinters of the future will need to be able to print functioning organs in a shorter amount of time, as Dr. Atala and his team envision a generation of machines capable of printing directly into the patient, reducing the time taken to keep an operating theatre active and also granting more people access to treatment at a faster rate than before. It is also important to keep the bioprinted tissue alive while the rest of the tissue is being printed. Because it takes time to print a complete organ, the tissue requires a continuous blood and nutrient supply to remain alive while the entirety of the organ prints. Current stem cell research is also contributing to advancements in this field, and thus an increased biological understanding of pluripotency and totipotency is necessary in order to make bioprinted organs capable of recapitulating their natural counterparts as closely as possible.

Additionally, current technological limitations are inhibiting the refinement of this new field. As the technology improves, the ambitions which guide it also grow, requiring solutions to address the challenges of printing organs that are both structurally and functionally complex. An example of one such challenge was documented by the US National Institute of Health [6], a project which involved the 3D printing of a functional ear. One of the challenges encountered by the researchers was overcoming the mechanical and thermal issues which arise from integrating biological and electrical materials. The researchers found that they could print the specially designed ear with silver nanoparticles to form the antenna and calf cells which would later develop into cartilage. The antenna increases auditory detection of radio frequencies, allowing the patient to possess an enhanced audible frequency range. In future, this research can lead to bioprinted eyes, providing patients with the ability to regain their sight.

CONCLUSION

The field of bioprinting and regenerative medicine shows a lot of promise for the future. Advanced prostheses, creating models for teaching anatomy and physiology, and printing fully functional human-sized organs. It has the potential to improve the situation regarding the availability of organs and organ transplants, ultimately benefitting the patient. However, it is also clear that more research is needed in this exciting field and that the possibility of seeing bioprinting in hospitals around the globe may very well become an eventuality.

References

[1] http://www.bbc.com/news/uk-wales-politics-36520627. BBC News. [Online].; 2016

[2] IRODaT. International Registry in Organ Donation and Transplantation Newsletter. [Online].: IRODaT; 2016

[3] Atala, Anthony. Printing a human kidney Juan Enriquez. TED, 3 March 2011. Filmed interview. 1 March 2016.

[4] Atala A, Shafiee A. Printing Technologies for Medical Applications. Trends in Molecular Medicine. 2016 March; 22(3): p. 254-265.

[5] Fischer H. A Guide to U.S. Military Casualty Statistics: Operation Freedom’s Sentinel, Operation Inherent Resolve, Operation New Dawn, Operation Iraqi Freedom, and Operation Enduring Freedom. Congressional Research Service Report. Washington D.C.:; 2015.

[6] Gallagher J. http://www.bbc.com/news/health-33560433. [Online].; 2015

[7] Manoor MS, Jiang Z, James T, Kong YL, Malatesta KA, Soboyejo WO, et al. 3D Printed Bionic Ears. Nano Letters. 2013 May 1; 13(6): p. 2634-2639.

[8] Bakhshinejad A, D’Souza RM. A Brief Comparison Between Available. In Great Lakes Biomedical Conference; 2015; Milwaukee. p. 2-3.

[9] Lee VK, Dias A, Ozturk MS, Chen K, Tricomi B, Corr DT, et al. 3D Bioprinting and 3D Imaging for Stem Cell Engineering. In Turksen K, editor. Stem Cell Biology and Regenerative Medicine: Bioprinting in Regenerative Medicine.: Springer International Publishing; 2015. p. 35-36.

[10] Pati F, Jang J, Lee JW, Cho DW. Extrusion Bioprinting. In Atala A, Yoo JJ, editors. Essentials of 3D Biofabrication and Translation.: Elsevier Science & Technology Books; 2015. p. 123-152.

[11] Jung JP, Bhuiyan DB, Ogle BM. Biomed Central: Biomaterials Research. [Online].; 2016 [cited 2017 January 12. Available from: https://biomaterialsres.biomedcentral.com/articles/10.1186/s40824-016-00….

Article photo credit: Andreas Kofner