Biomedical Sculpture: 3D Printing Living Tissue

Sculpture has existed as a means for turning raw material into three-dimensional objects for tens of thousands of years. The oldest surviving sculpture, dated 30 000-40 0000 years old, is that of a lion-man, Löwenmensch¹. This is evidence that the inclination to create tangible, fantastical objects from one’s imagination is an intrinsic part of human society. Of course, the surrealism aspect of the sculpture is based on the lack of evidence for the existence of lion-men. Historically, sculpture involved taking a solid substance, such as rock or wood, and removing bits and pieces of the amorphous material to reveal a shape within it. As civilizations evolved, sculpture included building 3D structures from shaping malleable material, such as clay, into desired configurations. However, most sculpting requires practice, skill, access to appropriate tools, and, of course, hands-on time to actually sculpt. Wouldn’t it be wonderful if the ability to create 3D objects was more accessible? That’s where 3D printing comes in!

The concept for 3D printing objects started in the 1980s with the need to rapidly create prototypes. The initial idea involved turning liquid materials solid with light, with the very first 3D printer using UV light to cure (harden) resin layer-by-layer. The SLA-1, built by Charles Hull, was commercially available in 1988². This specific type of 3D printing is called stereolithography. It did not take very long for 3D printing technology to diversity and expand because of its incredible usefulness in a wide variety of applications. Nowadays, the most prominent type of 3D printer available does not require light to solidify individual layers, but instead melts materials that quickly cool to solidify in thin layers that can be built upon immediately. There are a few commercially available 3D printers for under $300 USD that can use plastic, and wood and metal composites as their “ink”. All the 3D printer needs to be able to “sculpt” is a computer file and the material to “print” it out of. This allows it to make replicates of the same thing, and if there is a small change that needs to be made, precise tweaking can be done using the software. As for the printing process itself, just get the printer started (after significant tweaking, testing, optimizing, and possible modifications), and then you can forget about it (almost). When you return to your printer a few hours later, voilà, the computer file has become a touchable object! Although it might be daunting for some to learn the software to be able to use 3D printers, there is a massive, supportive online community that not only helps each other learn this technology, but also works together on international projects. For an example of this, check out e-NABLE, a 3D printing community providing customizable free prosthetic hands for kids (http://enablingthefuture.org/) .

Since its invention, engineers, inventors, and scientists have been able to build a variety of unique objects and develop novel printable “inks”. For example, its customizability is being capitalized on by Adidas that is advertising its first personalized 3D printed sole for their Futurecraft shoe line. Even rocket engines have been 3D printed, including the Orbex Prime rocket which was printed in one complete piece using a lightweight carbon fibre and aluminium composite ink³. What could be cooler than printing rockets? Rocket fuel! Yes, it is possible to print rocket fuel. Surprisingly, the starting materials for rocket fuel has a consistency comparable to cookie dough, which makes it very to hard to print. It is ill-advised to use standard 3D printing methods for rocket fuel because normal printing involves heating the “ink”, which is less than ideal when printing rocket fuel. Scientists at Purdue’s School of Aeronautics and Astronauts found that vibrating the ink-dispensing nozzle allows for even flow of the rocket fuel ink while maintaining precision⁴. Being able to 3D print rocket fuel provides more control to rocket geometry and fuel combustion rates.

Biomedical technology has also adopted 3D printing as an important tool for developing novel therapies. There are few people out there who enjoy being pricked by a needle, so how awesome would it be if medicine could be administered without the need for being stabbed? An FDA-approved, renewable, biodegradable, and thermoplastic material has been used to 3D print microneedles⁵. They are so small that they cause no pain when used and the medicine is slowly released as the material biodegrades. This also eliminates the sharp biohazardous waste that is a significant negative element to consider when using traditional needles. However, the most exciting development in the biomedical community with 3D printing technology is building tissue by either printing biocompatible scaffolding for living cells to populate or directly print living cells using a “bioink”. An excellent, albeit currently unrealistic, visual example of 3D printing a biological organism in pop culture is the building of Leeloo in The Fifth Element, where her skeleton is rapidly assembled layer-by-layer. In reality, it is possible to 3D print porous customized structures to fit in places where bone needs to be replaced, and then allow the body’s natural healing processes to turn that implant into a bone-like structure to be filled by the recipient’s own cells. The building of Leeloo also implies that the tissue being deposited is alive, which is genuinely achievable with today’s 3D bioprinting technology.

A heart with all the cells washed away.

Just like the scaffolding of a building establishes its overall shape, our organs have protein scaffolding that provide them with shape. Without this protein scaffold, we would essentially be oozy blobs of cells, similar to Ditto, although without this Pokémon’s ability to morph. It is even possible to wash away all the living cells from an entire organ to leave its 3D protein structure intact in a procedure called decellularization⁶. Imaging technologies, especially magnetic resonance imaging (MRI), allows for accurate scanning of organs and structures within the body to get the dimensions required to be able to custom print a scaffold for a patient so that it fits perfectly. This is a growing branch of personalized medicine that uses biomedical engineering to treat patients. It is simpler, although not easy, to 3D print a biocompatible scaffold that will work with the patient’s cells to repair tissue. It is crucial that the printed materials won’t react negatively with or be rejected by the body. There are many biocompatible materials, called biomaterials, already being used in a variety of medical applications including implants that serve as artificial joints, thread that dissolves in the body after surgery, and the many components of pacemakers to keep the heart pumping. Some printable biomaterials include polycaprolactone (PCL), polylactic acid (PLA), olea-gum-resins with metal oxide nanoparticles, gelatin, cellulose, and silk⁷. They are used to create scaffolds that will either be put in a patient directly or seeded with living cells in a lab before being implanted. These scaffolds are nearly always porous to allow for cells to surround as much of it as possible so that the implant mimics its natural environment. Some scaffolds are even designed to be degraded by the body over time, either to release medicine slowly in a localized area or to allow the body’s natural healing processes to replace the scaffold after a little biological boost of encouragement is provided. Scientists have been able to 3D print scaffolds that are used in bone grafting and bone repair. This technique has been very significant in repairing skull or facial damage or malformation because the scaffolds can be printed with incredible precision to match a person’s facial features⁸. Similar to an artist sculpting a beautiful visage in marble, scientists are 3D printing bits of sculpture; however, their work will perfectly fit a moving, living face!

Mother mouse with her green glowing baby

One of the most commonly used materials as a base for printing biological objects, called bioprinting, are hydrogels. As the name suggests, one of their main ingredients is water, which is very biocompatible, but water itself is quite tricky to print. The other main ingredient, is another biological molecule, gelatin. Essentially, scientists are printing with flavourless, colourless jelly. The wonderful thing about water is that it is possible to dissolve other compounds in it that can help change the properties of the scaffold being printed or provide nutrients to cells. One of the most recent phenomenal applications of this technology is the 3D printing of functional ovaries⁹. The ovaries themselves were not printed, however the porous, hydrogel scaffold was. The geometry of the scaffold has to be perfect for the cells seeded within it to recognize it as a safe place to grow. If cells are not comfortable where they are, they will either die or malfunction. Mouse follicular cells (the cells that become eggs/ova ready for fertilization) were injected into 3D printed hydrogel scaffolds and then implanted in female mice, replacing their ovaries. Natural mating occurred and female mice with the 3D printed “ovaries” were able to successfully give birth to baby mice! It should be pointed out that the follicular cells used were from mice that glowed green using the green fluorescence protein (GFP) (Click here to see my article on GFP), causing the baby mice to glow green also. This is a convenient visual cue to determine that the baby mice were indeed from the 3D printed ovary and not from a residual ovary hanging out in their non-glowing mother’s uterus. Although this technique is still years away from being used in clinical trials, it provides hope to women with ovarian diseases, including cancer, who would want to have children. Being able to 3D a custom ovary, seeded with cells from the mother, would completely remove the need for an ovarian organ transplant and all the complications that come with the procedure.

What about printing with ink that is alive? It is possible! In fact, scientists at MIT have created a 3D printed living “tattoo”¹⁰. The idea behind this tattoo is to develop a living sensor that can detect environmental chemicals, including pollutants, as well as changes in temperature. It is made by using hydrogel to suspend genetically programmed bacteria that will light up to indicate a change they have detected in their surroundings. Bacterial cells are much hardier than human cells, so they are able to better survive the 3D printing process; however, scientists are working towards printing with living animal cells, including human cells. One of the biggest issues in working with human cells is that they require nutrients, warmth, and oxygen to stay alive and this can be tricky to maintain when 3D printing objects takes between tens of minutes to hours. The next step in 3D printing is called one-step volumetric additive manufacturing, or, more commonly called, holographic 3D printing. This technique is a modern take on stereolithography, by using materials that harden when exposed to specific light frequencies. The big change is that it uses holographic patterns of laser fields to cut out the desired pattern on a cube of material¹¹. Instead of an artist cutting away excess material to create a sculpture, a computer is using lasers! With this exciting technique, it only takes seconds to “print” an object. The start-up company Prellis Biologics is using this technique to make capillaries, which are one-cell-thick blood vessels vital for transporting nutrients and removing waste in organs. Without these pathways to transport blood, 3D printing larger organs will not be possible as they require constant nutrition and oxygen in order to stay alive.

Holographic printing - in nearly real time

Ingenious scientists and engineers from many different fields are working together to improve 3D printing for tissue engineering. The next frontier for 3D printing in the tissue engineering field would be to print using living cells to custom-make functional organs. Science isn’t there yet, so if you haven’t already, please consider being a organ donor in your area, because it is the only option available now for those in need. Hopefully one day, it won’t be.

References/More Information

1. Der Löwenmensch: Geschichte-Magie-Mythos. Museum Ulm. Lion-man .
2. Leo Gregurić. Making History: History of 3D printing – when was 3D printing invented? All3DP Dec 2018. .
3. Orbex unveils prime rocket at new facility in Scotland. Orbex News Release .
4. McClain MS, et al. Additive manufacturing of ammonium perchlorate composite propellant with high solids loadings. Proceedings of the Combustion Institute 2019 37: 3135-3142.
5. Luzuriaga MA, et al. Biodegradable 3D printed polymer microneedles for transdermal drug delivery. Lab Chip 2018 18: 1223-1230.
6. Ott HC, et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med 2008 14: 213-221.
7. Tappa K and Jammalamadaka U. Novel biomaterials used in medical 3D printing . Journal of Functional Biomaterials 2018.
8. Maroulakos M, et al. Applications of 3D printing on craniofacial bone repair: a systematic review. J Dent 2019 80: 1-14.
9. Laronda M, et al. A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice. Nature Communications 2017 8: 15261.
10. Jennifer Chu. Engineers 3-D print a “living tattoo”: New technique 3D prints programmed cells into living devices for first time . MIT News On Campus and Around the World.
11. Shusteff M, et al. One-step volumetric additive manufacturing of complex polymer structures. Science Advances 2017 3(12): eaao5496.