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3D Bioprinting for Vascular Structures

Dr. Varsha Reddy

Intern

Mr. P. Amruth sagar

Senior Knowledge Scientist
INTRODUCTION

The WHO defines health as “A state of complete physical, mental and social wellbeing, and not merely the absence of any disease or infirmity.” The failure of physical health is, at most times, one of the major causes of death or debilitation. The idea of replaceable, artificially engineered, biocompatible body parts and organs, which might have been a pipe dream for every science fiction lover, has gradually emerged as one of the front runners in research over the last half a decade. In a world where there is a gross variation in the kind of lifestyle from person to person, there is a need for customizable parts that are both compatible and also suit the specific requirements of the individual. Three-dimensional printing provides precise control, and can be used with a large number of biological structures. Various organs can be printed and sustained in a lab grown environment, of which the tissue level in two dimensions is the easiest to achieve and can be used in drug interaction testing. However, the end game in the field is the successful bioprinting of three-dimensional organ level structures with adequate vasculature, as cells that are more than around 200 micrometers — a couple of human-hair widths — away from a source of nutrients quickly die. Therefore, creating a realistic vascular system will be necessary if researchers are to successfully build organs. Such a system can sustain the organs independent of a lab, and can be used for organ transplants; which, as futuristic as it sounds, could one day result in quasi immortality, as all failing organs could be replaced as per the requirement to further prolong one’s life as long as one wishes to.


OBJECTIVES

  • To outline the progression in the field of vascular bioprinting.
  • To identify the revolutionary researchers in the field.
  • To establish the potential uses of current trends.
  • To identify the complications with the current technologies.
  • To recognize the future needs and potential of the technology.

PROGRESSION OF THE TECHNOLOGY

Considering a timeline extending from 2012 up until the time of publishing, one of the earliest attempts at creating a branched, bio printed vascular system was made by researchers at the University of Penn in 2012, by using a rapid casting method to create 3D-printed templates of filament networks that used a combination of sucrose and glucose along with dextran for structural reinforcement and printed with RepRap, an open-source 3D printer with a custom-designed extruder and controlling software.[1]

(Source: https://news.upenn.edu/news/penn-researchers-improve-living-tissues-3d-printed-vascular-networks-made-sugar)

2015 was a watershed year in the current field- various newer technologies, methods and bio inks were created and, for the first time, a 3D printed vascular structure was successfully implanted in rat aortae, in a collaboration between Saga University and Cyfuse biomedical, Japan, using their patented 3D bioprinter known as Regenova. They used a method where Human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells (HASMC) and human normal dermal fibroblasts (HNDFB) were cultured in an appropriate medium. The resultant multicellular spheroids (MCSs) were used to assemble MCSs for constructing scaffold-free tubular tissue. Upon perfusion with a bioreactor, a tubular tissue of 1.5 mm in diameter and 7 mm in length was obtained, which was then implanted into the abdominal aorta of nude rats. The flow rate was monitored and found adequate, and furthermore, the graft compliance was noted and the rats were euthanized. [2]

Using an inkjet 3D printer with the droplet dispersion method, researchers from the Rensselaer Polytechnic Institute, New York and Harvard Medical school, Boston, cultured Human umbilical vein endothelial cells (HUVEC’s) and collagen hydrogel precursor (Rat tail, type I) was used as a scaffold material. The fabricated vasculature has a tight, confluent endothelium lining, presenting barrier function for both plasma proteins and high-molecular weight dextran molecules, but could only sustain tissue viability for a distance of up to 5mm under simulated human body conditions. [3]

EU research project ArtiVasc 3D, which comprised an interdisciplinary team of more than 20 partners and researchers led by the Fraunhofer Institute for Laser Technology, combined the freeform methods of inkjet printing and stereo lithography. With these combined processes, the researchers were able to achieve a very fine resolution for the construction of branched, porous blood vessels with layer thicknesses of about 20 microns. The researchers used mathematical simulations to develop data for the construction of branched structures, to allow for uniform blood flow. [4]

Using a process called Electrospinning, Techshot Inc., in collaboration with the Pentagon’s defense health program, have created implantable vascular grafts with stable vascular features. Stem cells collected from the patient’s own fat are printed onto a tubular scaffold. Upon complete healing, within six to nine months, the polymer tube is absorbed by the body and a natural blood vessel is left behind. This research was carried out mainly to treat war wounds of soldiers with severe injuries to their extremities. Pre-clinical trials have been completed and human clinical trials for this invention are expected to begin in the last quarter of 2017. [5]

Researchers at the Lawrence Livermore National Laboratory used a combination of 3D printing and biologically compatible material, and the environment is engineered to enable human capillaries to develop on their own. This process takes a while, so initially, tubes are printed out of cells and other biomaterials to deliver essential nutrients to the surrounding printed environment. Eventually, the self-assembled capillaries are able to connect with the bio-printed tubes and deliver nutrients to the cells on their own, enabling these structures to function like they do in the body, resembling a “spaghetti bowl.” This research leverages the body's ability for self-directed growth, and the result is something rather akin to human physiology. [6]

A team at Harvard University in Cambridge, Massachusetts, printed thick tissue with a rudimentary vascular system and have managed to keep it alive for weeks. The team used three different inks: silicone to give a basic shape; a bio ink infused with pluripotent stem cells that would turn into the tissue; and Pluronic, which is a gel at room temperature, but a liquid when cooled. They successfully printed tissue which was 1 centimeter thick and kept it alive for a period of over 6 weeks. While the vasculature for this was rudimentary, the same principle can be applied to create more complex vascular networks. [7]

In 2016, a newer 3D bioprinting method by Cyfuse Medical called the Kenzan Method, which is a unique form of bioprinting, which uses tiny vertical spikes on which cell clusters (spheroids) are skewered and kept in position, rather than use a bio ink. A research team led by Saga University’s Professor Shigeki Morita has used the Kenzan Method to create tubular 3D printed blood vessel structures, 2 cm by 5 mm. When these tubular structures have formed on the skewers, a special broth is added which enables the cells to develop and the blood vessels to form. The Saga University scientists believe that their 3D bio printing method could help to treat patients recovering from myocardial infarctions. [8]

(Source: Cyfusebio.com)

For the first time, at the end of 2016, cardiac and vascular structures were 3D-printed in a zero gravity environment, using adult human stem cells, by a team of scientists working for the NASA, and as a collaboration between SpaceX contractor Techshot, bio printing experts nScrypt and bio-ink innovators Bioficial Organs. The project saw researchers take to the skies in a Zero Gravity Corporation parabolic flight, to test out the printer prototype. The combined expertise resulted in a bioprinter capable of printing extremely fine lines in space. The major advantages to this method are: firstly, the ability to use finer print tips and lower viscosity bio-inks (which enable faster printing) that contain only the biological materials needed to create a healthy organ; and secondly, the absence of gravity enables easier printing of hollow structures, which do not collapse upon themselves. 3D printing in space has the potential to be the biggest game changer in this field, as it could be the answer to both the organ shortage and creating vasculature for printed organs. The technology can also be used to enable people to stay longer in space. [9][10]

Researchers at the University of California, San Diego in the Nano biomaterials, Bioprinting, and Tissue Engineering Laboratory directed by Prof Shaochen Chen, have successfully manufactured branched vasculature, and the technology used has resulted in a network featuring branching vessels that become successively smaller, similar to those found in living tissue. The method involves printing tiny (4 millimeters × 5 millimeters, 600 micrometers thick), pre-designed vascular networks that turn into vessel-like structures made of the same cells as natural blood vessels. The new method, called microscale continuous optical bioprinting (μCOB), involves embedding a mix of different kinds of live cells into a hydrogel and then using ultraviolet light and mirrors to heat up and solidify 3D patterns within the hydrogel in vivo. While they are able to transmit blood, they are currently still unable to transport waste and other nutrients. [11][12]

Researchers in China have successfully transplanted blood vessels into Rhesus monkeys after bioprinting them. A special kind of bio-ink, being marketed as “Biosynsphere,” which is derived from stem cells acquired from fat tissue of the monkeys was used; this is safer and has low risk of rejection. A team led by Dr. Kang Yujian, replaced a 2 centimeter part of the abdominal artery in 30 Rhesus monkeys. The stem cells were able to grow all the required cells for a blood vessel. Post-surgical observation after a few months showed complete integration with pre-existing vasculature. The ability of Biosynsphere to develop collagen is a major breakthrough in the field of printing vascular structures. Sichuan Revotek manufactured the ink to be compatible with their blood vessel-printing 3D bioprinter that uses cloud computing; and together, they have the ability to completely overhaul the vascular 3D printing industry. [13][14]

(Source: https://wowscience.io/tag/sichuan-revotek/ and http://edition.cnn.com/2017/01/10/health/china-3d-printed-blood-vessels/)

COMPANIES AND THEIR FUNDING

Unsurprisingly, China and Japan lead the charts in terms of raising funding, increased research as well as resultant patent filings. One of the major players is Cyfuse, erstwhile known as CyfuseBio, which has collaborated with Saga University, based out of Japan, in developing a 3D printer known as Regenova, which uses the Kenzan method. The Kenzan method was invented by prof. Koich Nakayama (Saga University) and is a globally patented intellectual property. The projects are funded by a mixture of private and public funding; up to 2 billion Yen was raised by 12 investors, including venture capital funds and corporate investors. [15] An undisclosed sum is also sponsored by the Japan Agency for Medical Research and Development (AMED).

(Credit: Cyfusebio.com)

Sichuan Revotek, a Chinese company, is the only company to have developed a 3D printer made especially customizable to the printing of blood vessels, using a specifically made bio-ink. Based out of Chengdu in China, the health care startup has raised 33 million dollars in funding from a private real estate firm that wished to branch out. [14]

In general, Chinese provinces have created unique zones where new business ventures, especially technology startups and medical research firms, can seek funding opportunities due to a country-wide campaign to encourage entrepreneurship. Extensive funding opportunities and an abundance of highly qualified manpower explain their dominance in the field.

Academically, the University of California, San Diego campus has been a spearhead in the field. They were granted a 1.5 million dollar grant in 2015 from the NIH, specifically for the purpose of recreating vascularized tissue. [16] This grant, along with other private investments, has helped UC along in the goal.

As recently as April 2017, Dr. Yi Hong, engineer and assistant professor in the University Of Texas at Arlington, from the Department of Bioengineering, has been awarded a grant. Dr. Hong received an R21 grant from the National Institute of Health (NIH), for $211,000, to develop materials to make 3D printed blood vessels for children suffering from vascular defects. Dr. Hong, who has been on research grants totaling over $850,000 in his career, will develop materials that are elastic and can be formed into patient-specific, feasible blood vessels that can also be used in a 3D printer. Dr. Guohao Dai, who is his partner at Northeastern University, will 3D-print the blood vessels using Dr. Hong’s materials. [17]

Bio 3D-printing of vasculature in space is a collaboration between NASA, SpaceX contractor Techshot (which provides the vehicle or craft necessary for the parabolic flight), bioprinting experts nScrypt (who manufactured the 3D printer to be specifically compatible for space) and bio-ink innovators Bioficial Organs. The details of the funds involved are a closely guarded secret, due to the project’s association with the government.

(Source: https://3dprintingindustry.com/news/3d-bioprinting-zero-gravity-82894/)

Other companies and universities working in this area are:

  • ArtiVasc 3D, by the Fraunhofer Institute for Laser Technology ILT, sponsored by over 20 companies and subsidized under adequate government grants.
  • Cellink, a Swedish company that manufactures bio printers and bioinks. Completely customizable printers can be assembled by them. The company is both privately owned and funded, with the major share being shared between Erik Gatenholm (40.4%) and Hector Daniel Avila Martinez (26.8%).
  • Rokit, a South Korean company, was recently given a $3 million government grant. The company collaborating with the Korean Institute of Science and Technology (KIST), Seoul National University, Bundang Hospital, Hanyang University, and Korea Institute of Machinery and Materials (KIMM).
  • BioBots, a startup, which recently raised over $300,000 on equity crowdfunding platform FundersClub, is concentrating mainly on manufacturing affordable desktop 3D bioprinters. However, the company is well renowned for its bioinks, priced at a $1000, which can be used with other printers as well.

OBSTACLES
1. At a vascular level:-
  • The major obstacle to bioprinting vasculature is recreating the micro vessels, especially at the capillary level, including the extensive branching that is naturally seen.
  • To exactly replicate the strength, ability to maintain lumen patency, viability and elasticity of natural vessels, even when empty.
  • To recreate vasculature that can be repaired if required, and their ease of attachment into the existing system in order to maintain continuity.
2. At the organ level:-
  • There are multiple contrasting theories for printing vasculature. While some debate that it is easier to print a larger structure to serve as an organ leaving hollows for the vessels, the converse has also been argued. Both methods have attained a modicum of success so far, but there remains to be seen the development of a method that can manage to print organs with vessels, successfully and viably.
  • The life of 3D-printed vessels has so far only been managed up to 6 weeks. However, for the purpose to be fruitful, the vessels should have the ability to survive and regenerate as the surrounding vessels do. This is especially essential if the technology has to be used to vascularize entire organ systems.
  • Tissue death occurs at a distance of 200 micrometers from the nearest source of blood supply. The lack of technology in creating that level of microcirculation is the biggest hindrance to the progression of de novo organ printing. An organ cannot survive without adequate vascularization, hence there is a great need for technology that enables the printing of patent, viable capillaries vasculature, including capillaries.
  • To attain organ level vascularization, the circulation needs to be at a three-dimensional level. So far, the technology has only been able to achieve vascularization at a two-dimensional level.
THE FUTURE SCENARIO
  • 3D-printing of the entire vasculature of a patient pre-operatively, so as to be able to pre-emptively prevent any surgical complications is a current application. Using CT and MRI-imaging scans to recreate three dimensional images and printing them out to accurately recreate the vascular structures, thus enabling surgeons to precisely treat aneurysms and the like.
  • Bioprinted vessels can be made, in a way as to avoid blockage completely, with the addition of anti-thrombolytic structures or materials. Another method being tested is the usage of acellular materials and natural polymer composition, which through multiple modes can be used to form modified blood vessels. Currently, the adaptability of materials like collagen, silk fibroin or polylactic acid is being tested. [18] Such vessels may very well be lifesaving in the case of patients with hypertension and those suffering from cardiac conditions.
  • Organ system integration needs to be achieved. It does not suffice to merely have a vascular system for an organ; the real test lies in the ability of bio printed vessels to integrate and bond with the preexisting vasculature.
  • Fully functional, viable vasculature, as close to nature as possible, which are also affordable as a technology; and those that do not cause any adverse reactions, fatal or otherwise, is something that needs to be worked on with urgency.
  • Customizable vascular structures, which can be made according to patients’ requirements, printed using cells from the patient, are operable, can be replaced or repaired with ease in the future, and do not require an extremely long time to be created, or have long waiting lists for the procedure, will essentially bring about a lot of changes in the health care industry; especially in battle scenarios and road traumas; where time is of the essence.
  • The most anticipated development from said technology is integrating a fully vascularized, viable, printed organ system into an animate body and having it carry on functioning as any other biological tissue. This development will eliminate the need for transplant lists, waiting for organ donors, deaths caused due to lack of organs donated; and may also completely wipe out the thriving illegal organ-trading industry.
CONCLUSION

All things considered, this field of study is only growing exponentially. Newer advancements in 3D printers, bio-inks, adjuvant materials, etc. are coming in hard and fast. While alternative methods; such as the recent research by the Worcester Polytechnic Institute (WPI), which involved decellularising a spinach leaf and perfusing it with blood using the plant’s own vascular system, resulting, over a course of 21 days, in functional contractile heart tissue;[19] have their merits, the customization that can be achieved with 3D-printing is unparalleled.
A future in which everyone can live a life of longevity, renewability and sustained good health, is not far-off. While everlasting life may not be everyone’s cup of tea, the ability to live a healthy life, for as long as one is alive, is definitely something to ponder. This is not to say that athanasia can be achieved, or that it is even a morally acceptable scenario; however, a world of mostly able-bodied individuals, as opposed to people being indisposed, is not something to scoff at. A world may soon be changed, as science and technology grows in leaps and bounds; all that now remains to be seen is whether the monkeys and the rats win the race.

References:
  1. https://news.upenn.edu/news/penn-researchers-improve-living-tissues-3d-printed-vascular-networks-made-sugar
  2. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136681
  3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112057/
  4. http://www.artivasc.eu/en/publication-and-press/press-release-2015-08-28.html
  5. http://www.techshot.com/news.php
  6. https://www.llnl.gov/news/researchers-3d-print-living-blood-vessels
  7. https://3dprint.com/123181/3d-printed-vascular-tissues/
  8. http://www.cyfusebio.com/en/
  9. http://www.engineering.com/3DPrinting/3DPrintingArticles/ArticleID/12481/First-Vascular-Structures-3D-Bioprinted-in-Zero-Gravity.aspx
  10. http://www.techshot.com/news.php
  11. http://www.medgadget.com/2017/03/scientists-3d-print-microscopic-vascular-networks-building-living-tissues.html
  12. https://www.theengineer.co.uk/light-activated-3d-printing-produces-branching-blood-vessels/
  13. http://edition.cnn.com/2017/01/10/health/china-3d-printed-blood-vessels/
  14. https://3dprint.com/158499/revotek-3d-printed-blood-vessels/
  15. https://www.cyfusebio.com/cyfuse-closes-1-4-billion-yen-series-b-funding-round-to-launch-overseas-sales-of-bio-3d-printer-and-accelerate-development-of-3d-tissue-engineering-technology.html
  16. http://ucsdnews.ucsd.edu/feature/how_3d_printing_could_one_day_save_lives
  17. https://3dprint.com/170564/uta-3d-printed-blood-vessels/
  18. https://www.google.com/patents/CN104771783A
  19. http://splice-bio.com/transforming-spinach-leaves-into-beating-human-hearts/