Print of the Future: Bioprinting
Over the past 20-30 years, printing industry has been experiencing real skyrocketing: the spectrum of materials became much wider (along with their possible size and thickness), printing on flat surfaces gave way to making 3D models.
And now, creating donor organs and tissues with specific printing mechanisms is no more a sci-fi plot but a tangible reality. What’s more, this phenomenon of future medicine has already become a part of Russian reality: in 2014, the first Russian 3D-bioprinter FABION was presented.
How This Technology Appeared and What It Has To Do With «Printing»
Physical and biological aspects
The idea of bioprinting is based on an assumption that self-assembling of cell structures can take place even outside of a living organism. In a living world, directed self-organization of cellular aggregates (run by surface tension forces and protein-protein interactions) is ubiquitous, including the process of cell membrane or organs’ formation.
The ability of cells to self-assembling was accidentally discovered in XIX century by a German professor Gustav Born during tadpole’s dissection. Later in 1907, it was confirmed by Peter von Wilson, who was using sea sponges’ tissues as an example.
For biological science, there’s nothing new in cellular self-assembling; same with the printing technology that is well known for European civilization since XV century, owing to German jeweler Johannes Gutenberg. However, it took until XXI century for these two ideas to merge and create something fundamentally new – due to inkjet printing and 3D printing technologies’ development.
Printing technologies: Flatness, laser and volume
Inkjet printing: In printing industry, movement towards human cells printing has been evolving more rapidly, although the conception of ink-jet printing is not new neither: perfect uniformity of drops of liquid that passes through narrow hole was discovered by Felix Savart in 1833.
A century later, in 1951, Siemens patented their first machine, created on the basis of Thompson’s discovery. This was Mingograph – a self-recorder for voltage value changes’ fixation. In order to develop a full-fledged inkjet printer that would print text and images, the issue of selective drop venting needed to be solved.
The final version of drop-on-demand technology appeared in 1979, when Canon presented an evaporative bubble system. In that model, ink drops formed on the surface of heating element near the nozzle, and ink spreading was managed with the help of paint evaporation’s condensation.
One year later, in 1980, Hewlett-Packard suggested similar printing method, and in 1984 they released the first commercially successful inkjet printer, which provided good printing quality and high resolution. After this, printers had got an ability to mix colors getting almost any hue, and then printing industry started to develop in quantum leaps.
Laser: in 1969, XEROX came up with a brilliant idea to use copy machines technology in printing. This gave rise to laser print, which mode boils down to the following: laser beam discharges part of the energy from negatively charged drum in theareas where pixels should be printed, then drum transfers the image onto paper, and with the help of a heating element the image is instantly fused onto the paper.
The prototype appeared in 1971, but the first XEROX laser printer was released to the market only in 1977.
3D printing: just in three years, from 1985 to 1988, there was a great breakthrough in print technologies, as multiple methods and techniques of creating 3D figures were suggested.
In 1985, Michael Feygin offered layer-by-layer way of formation by sintering such sheet materials as paper, polyester, film, plastic, and composites. That technology, however, had several drawbacks, as the finished product was a bit rough and easy to delaminate.
One year later, Charles W. Hull patented a machine that used stereolithography and photopolymeric plate; soon, such machines were put into serial production. Today, they still exist, but are not massively popular because of equipment and consumable products’ high prices.
In 1986, Carl Decart invented a method of selective laser sintering. The idea was similar to Feygin’s, but according to Decart, laser beam should bind layers of powders instead of sheet materials.
In 1987, Israel-based company Cubital abandoned the idea of sintering layers and decided to condense them by layer-by-layer photopolymer application and following UV-curing. The object’s empty spaces were filled with wax. However, this machine’s cost also was unreasonable; moreover, consumable polymers were poisonous.
In 1988, Scott Crump patented fused deposition modeling (FDM), where the model is produced by gradual extruding small flattened strings of molten metal or plastic to form layers. These layers were settled one by one onto the working table, forming a 3D object.
After a few decades of technologies’ growing and improving, it no longer seemed crazy to ask this fair question:
«If we're able to print furniture, music instruments, shoes, or weapon, why not try to print a human, or at list their “spare parts”?»
Apparently, this expression is a bit exaggerated. However, a problem of donor organs shortage is increasingly urgent, and with constant improving of modern transpantology methods, no wonder that scientists are actively looking for alternative source of transplants. Thereby, the idea to “print” them is most opportune.
Bioengineer Thomas Boland in 2000 translated these words into action as he upgraded Lexmark and HP printers and for the first time tested live cell printing.
Thomas Boland and the first bioprinter
He used the printers with sufficiently big nozzle diameter in order to preserve cells while printing. The software should have ensured necessary control over temperature and viscosity.
In 2003, this method was patented, although two-dimensional monolayer printing was just an initial step on a way to printing organs, and there were still more questions than answers. First, human organ is not a plane figure but a 3D object, and second, it is not homogeneous as it contents different types of cells, vessels, nerves, forming an integrated and coherent whole.
Despite the fact that many of these questions are not solved yet, field-test of live cell printing opened a new chapter in the history of printing: we live in an era in which “bioprinting” turned from an abstract theoretical idea into a practical concept.
Bioprinting: new look at printing process
No matter how distant bioprinting and polygraphy may seem, live cell printing remain committed to the roots. It consists of same elements, only using digital organ model instead of text, cells instead of ink, hydrogels and polymer substrates instead of paper… And, finally, printing press is replaced with bioprinter, which in its latest version is more a robotized mechanism rather than a “press”. The technology itself is also being enhanced, basing on the level of progress that was achieved by polygraphy engineers; every technique they invented is being tested on a live material and properly modernized.
Bioprinting techniques: inkjet, extrusion, laser
Inkjet printing became the most demanded in bioprinting as the most common printing method. Technologically, it does not differ much from its classic version, although the ink is replaced with biological material, and the “paper” here is electronically managed pallet with hydrogel substrate (natural or synthetic), which is used to fixate the “ink”.
Depending on the liquid's features, there are two ways of drop flow: ultrasonic and thermal. Each one has its restrictions related to the material’s viscosity and possibility of damaging living cells. Another moment is that inkjet technology’s spray material is a liquid, while bioprinting should produce a stable three-dimensional structure with sufficient cells’ density. Therefore, it becomes necessary to introduce an extra step of structure seaming, which takes a long time and slows printing down.
At that, inkjet method has several obvious advantages inherited from its polygraphy ancestor: printer’s simple construction, useful software, low cost, high resolution and printing speed, gradient option (available by changing droplets’ size and density). Last but not least, this technique is compatible with various biological materials, which allows preserving high percentage of the cells' viability.
Inkjet products took their place in bioprinting since they are the ideal choice for skin covering and cartilage reconstruction, and due to high speed, cells can been put directly to an affected area.
Faster cells seaming can be achieved by UV-beams expositioning or adding chemical substances. So, it is highly possible that in near future, traumatologists, orthopedic surgeons, and combustiologists (medical professionals working in burn centers) will have compact bioprinters in their toolkit to “fix” skin, cartilages, tendons and bones.
Microextrusion printing is based on an extrusion technology borrowed from 3D-printing: it uses continuous beads of material spreading them onto x-, y-, and z-axis. Such printers are suitable for various bioprinting materials including hydrogel, biocompatible polymers, and live cellular spheroids. With applying 3D-printing technique in bioprinting, it is possible to increase cells' physical density and create a full-fledged three-dimensional object.
It may seem that microextrusion printing has all the benefits for recreating organic 3D objects, but there’s a reverse of the coin as well: with both mechanical or pneumatic material supply, cells’ survival rate is lower than in inkjet printing. Supply system depressurizing or nozzle diameter increasing can be used in order to reduce devastating effect, but all of this significantly lowers resolution and printing speed.
Laser-assisted printing was originally developed to transfer metals, but now it’s being used successfully with biomaterials in tissues- and organ-engineering applications. LAB machines operate in the following way: with the help of focused laser beams, cell material is transfered from laser-absorptive plate (made with gold or titanium) to receiver plate driven by high-pressure bubble. Such mechanism does not content nozzles, which helps to avoid blinding and use the material of any viscosity.
Bioprinting methods. Source: Murphy SV, Atala A. 3D
LAB machine can print with live cells with a minor effect on their vital capacity. But to get a sufficient form consistency, fast kinetics of gel formation is required. Another challenge lies in cells' exact positioning. Furthermore, there are trace amounts of metals left in end product because of laser-absorptive plate evaporation while printing.
In Russian bioprinting practice, LAB has proven to be the most productive method. In 2014, 3D Bioprinting Solutions Laboratory presented the first Russian 3D-bioprinter FABION, which uses two-photon polarization technology to build scaffolds – three-dimensional matrices, providing a mechanical carcass for live cells. Due to this technical and engineering trick, FABION can print functional three-dimensional tissues and organs, and nowadays, it surpasses other commercial bioprinters in many respects.
How this method works:
Hydrogel with live cells is placed on a top plate that contains no metal (in order to avoid metal presence in an end product). Laser pulse creates a shockwave that transfers cells to the bottom layer, where the printing object is applied.
In organ engineering, laser technology’s advantages are obvious, since they allow working with biomaterial of any viscosity or density and achieve cells’ physiological solidity, – one of bioprinting’s key objectives.
Achievements, prospects, objectives:
Despite all existing restrictions of each technique listed above, 3D-printers have already become an important bioengineering tool, actively used in various ways. Today, it seems to be dozens of medicine directions where bioprinters can make substantial changes for the better. Almost every month, artificial tracheas are printed with live cells, allowing many patients to breathe. In 2006, by applying cell material to a model of an organ, seven bladders were printed and then transplanted to the patients. However, the technology is still far from creating full-fledged organ replacement due to existing problems with vascularization and innervation: it can recreate only primitively organized tissues, not able to assume complex functions as an integral system. Those tissues are mostly used to test medications for toxic property and to simulate diseases.
In 2013, an American bioprint company Organovo managed to produce a liver tissue. Although it remained functional only within five days, this experience marked the beginning of this organ's bioprinting research. The most recent development in this field is a liver tissue made by Chinese specialists, which is able to perform its functions for 4 months. Printed cartilages were successfully tested in Sweden, and a thyroid produced with Russian bioprinter was transplanted to a mouse.
Bioprint technology's further development depends on whether it will become possible to form a vascular network inside printed organs. There are several achievements made in this direction: Organovo Laboratory’s specialists got three-dimensional liver fragments made of three cell types, containing vascular network. By 2030, they are planning to produce the first full-fledged functional printed kidney.
Among other things, it is worth mentioning that bioprint not just helps to fulfill the dream of “eternal health” when “worn out” organ is easily replaced with a fresh one. Beyond that, it is an essential tool for toxicology and pharmacology, since it allows studying the effects of medicals directly on a required organ instead of clinical trials groups or animals.
Bioprint technology, being totally safe and eco-friendly, is also a potential hot commodity in the meat, leather and fur production. Cosmetic companies have already seen this opportunity in vivo: Organovo prints leather for cosmetics testing, protecting customers from possible side effects and also protecting animals from maltreatment and killing.
Besides, improving printing mechanisms and producing perfect substrates with live cells, there is also a number of steps that should be done in engineering field, such as developing the software for modeling and print managing, increasing the resolution and printing speed, or creating aseptic conditions for a printing process.
Apparently, it is time for professional polygraphy to accommodate biologists and physiologists, because no one understands the laws of drops’ behavior better than those who deal with it everyday. Realizing the great prospects of the industry’s development, IQDEMY company this year opened their own research laboratory, which, inter alia, is working with bioprint technology. In this field, laboratory’s specialists study the technology of printing with live cells, improve bioprinters’ engineering solutions, and also develop the software for printing objects modeling and precise three-dimensional live cells positioning.