3 D Bio-Printing of Tissues and Organs.

Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education, and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues.

3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues.

Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures.

Some 3D Bioprintable Tissues

Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology.

Bioprinting

3D printing is increasingly permitting the direct digital manufacture (DDM) of a wide variety of plastic and metal items. While this in itself may trigger a manufacturing revolution, far more startling is the recent development of bioprinters. These artificially construct living tissue by outputting layer-upon-layer of living cells. Currently all bioprinters are experimental. However, in the future, bioprinters could revolutionize medical practice as yet another element of the New Industrial Convergence.

Bioprinters may be constructed in various configurations. However, all bioprinters output cells from a bioprint head that moves left and right, back and forth, and up and down, in order to place the cells exactly where required. Over a period of several hours, this permits an organic object to be built up in a great many very thin layers.

In addition to outputting cells, most bioprinters also output a dissolvable gel to support and protect cells during printing. A possible design for a future bioprinter appears below here and shown in the final stages of printing out a replacement human heart.

A future model of a 3D Bioprinter; printing a human heart.

The History of 3D Bioprinting.

• The promise of printing human organs began in 1983 when Charles Hull invented stereolithography. This special type of printing relied on a laser to solidify a polymer material extruded from a nozzle. The instructions for the design came from an engineer, who would define the 3-D shape of an object in computer-aided design (CAD) software and then send the file to the printer. Hull and his colleagues developed the file format, known as ".stl"  that carried information about the object's surface geometry, represented as a set of triangular faces.

Steriolithography.

• At first, the materials used in stereolithography weren't sturdy enough to create long-lasting objects. As a result, engineers in the early days used the process strictly as a way to model an end product -- a car part, for example -- that would eventually be manufactured using traditional techniques. An entire industry, known as rapid prototyping, grew up around the technology, and in 1986, Hull founded 3D Systems to manufacture 3-D printers and the materials to go in them.

• By the early 1990s, 3D Systems had begun to introduce the next generation of materials -- Nano  composites, blended plastics and powdered metals. These materials were more durable, which meant they could produce strong, sturdy objects that could function as finished products, not mere stepping-stones to finished products.

• It didn't take long for medical researchers to notice. What's an organ but an object possessing a width, height and depth? Couldn't such a structure be mapped in three dimensions? And couldn't a 3-D printer receive such a map and then render the organ the same way it might render a hood ornament or piece of jewelry? Such a feat could be easily accomplished if the printer cartridges sprayed out biomaterials instead of plastics.

• Scientists went on the hunt for such materials and by the late 1990s, they had devised viable techniques and processes to make organ-building a reality. In 1999, scientists at the Wake Forest Institute for Regenerative Medicine used a 3-D printer to build a synthetic scaffold of a human bladder. They then coated the scaffold with cells taken from their patients and successfully grew working organs. This set the stage for true bioprinting. 

• In 2002, scientists printed a miniature functional kidney capable of filtering blood and producing urine in an animal model.

• In 2010, Organovo -- a bioprinting company headquartered in San Diego -- printed the first blood vessel.

Today, the revolution continues. Taking center stage are the printers themselves, as well as the special blend of living inks they contain.

The Basic Concept.

The idea of 3-D printing evolved directly from a technology everyone knows: the inkjet printer. Watch the HP or Epson machine churn out a printed page, and there can be noticed that the print head, driven by a motor, moves in horizontal strips across a sheet of paper. As it moves, ink stored in a cartridge sprays through tiny nozzles and falls on the page in a series of fine drops. The drops build up to create an image, with higher-resolution settings depositing more ink than lower-resolution settings. To achieve full top-to-bottom coverage, the paper sheet, located beneath the print head, rolls up vertically.

Working mechanism of an ink-jet printer.

The limitation of inkjet printers is that they only print in two dimensions -- along the x- and y-axes. A 3-D printer overcomes this by adding a mechanism to print along an additional axis, usually labeled the z-axis in mathematical applications. This mechanism is an elevator that moves a platform up and down. With such an arrangement, the ink head can lay down material from side to side, but it can also deposit layers vertically as the elevator draws the platform down and away from the print head. Fill the cartridge with plastic, and the printer will output a three-dimensional plastic widget. Fill it with cells, and it will output a mass of cells.

Conceptually, bioprinting is really that simple. In reality, it's a bit more challenging because an organ contains more than one type of material. And because the material is living tissue, it needs to receive nutrients and oxygen. To accommodate this, bioprinting companies have modified their 3-D printers to better serve the medical community.

Bioprinting Pioneers
Several experimental bioprinters have already been built. For example, in 2002 Professor Makoto Nakamura from University of Toyama; realized that the droplets of ink in a standard inkjet printer are about the same size as human cells. He therefore decided to adapt the technology, and by 2008 had created a working bioprinter that can print out biotubing similar to a blood vessel. In time, Professor Nakamura hopes to be able to print entire replacement human organs ready for transplant.

Prof. Makoto Nakamura

Another bioprinting pioneer is Organovo. This company was set up by a research group lead by Professor Gabor Forgacs from the University of Missouri, and in March 2008 managed to bioprint functional blood vessels and cardiac tissue using cells obtained from a chicken. Their work relied on a prototype bioprinter with three print heads. The first two of these output cardiac and endothelial cells, while the third dispensed a collagen scaffold -- now termed 'bio-paper' -- to support the cells during printing.

Prof. Gabor Forgacs

Since 2008, Organovo has worked with a company called Invetech to create a commercial bioprinter called the NovoGen MMX. This is loaded with bioink spheroids that each contain an aggregate of tens of thousands of cells. To create its output, the NovoGen first lays down a single layer of a water-based bio-paper made from collagen, gelatin or other hydrogels. Bioink spheroids are then injected into this water-based material. As illustrated below, more layers are subsequently added to build up the final object. Amazingly, Nature then takes over and the bioink spheroids slowly fuse together. As this occurs, the biopaper dissolves away or is otherwise removed, thereby leaving a final bioprinted body part or tissue.

Bioprinting process in NovoGen

As Organovo have demonstrated, using their bioink printing process it is not necessary to print all of the details of an organ with a bioprinter, as once the relevant cells are placed in roughly the right place Nature completes the job. This point is powerfully illustrated by the fact that the cells contained in a bioink spheroid are capable of rearranging themselves after printing. For example, experimental blood vessels have been bioprinted using bioink spheroids comprised of an aggregate mix of endothelial, smooth muscle and fibroblast cells. Once placed in position by the bioprint head, and with no technological intervention, the endothelial cells migrate to the inside of the bioprinted blood vessel, the smooth muscle cells move to the middle, and the fibroblasts migrate to the outside.

In more complex bioprinted materials, intricate capillaries and other internal structures also naturally form after printing has taken place. The process may sound almost magical. However, as Professor Forgacs explains, it is no different to the cells in an embryo knowing how to configure into complicated organs. Nature has been evolving this amazing capability for millions of years. Once in the right places, appropriate cell types somehow just know what to do.

In December 2010, Organovo create the first blood vessels to be bioprinted using cells cultured from a single person. The company has also successfully implanted bioprinted nerve grafts into rats, and anticipates human trials of bioprinted tissues by 2015. However, it also expects that the first commercial application of its bioprinters will be to produce simple human tissue structures for toxicology tests. These will enable medical researchers to test drugs on bioprinted models of the liver and other organs, thereby reducing the need for animal tests.

In time, and once human trials are complete, Organovo hopes that its bioprinters will be used to produce blood vessel grafts for use in heart bypass surgery. The intention is then to develop a wider range of tissue-on-demand and organs-on-demand technologies. To this end, researchers are now working on tiny mechanical devices that can artificially exercise and hence strengthen bioprinted muscle tissue before it is implanted into a patient.

Organovo anticipates that its first artificial human organ will be a kidney. This is because, in functional terms, kidneys are one of the more straight-forward parts of the body. The first bioprinted kidney may in fact not even need to look just like its natural counterpart or duplicate all of its features. Rather, it will simply have to be capable of cleaning waste products from the blood.

Regenerative Scaffolds and Bones

A further research team with the long-term goal of producing human organs-on-demand has created the Envisiontec Bioplotter. Like Organovo's NovoGen MMX, this outputs bio-ink 'tissue spheroids' and supportive scaffold materials including fibrin and collagen hydrogels. But in addition, the Envisontech can also print a wider range of biomaterials. These include biodegradable polymers and ceramics that may be used to support and help form artificial organs, and which may even be used as bioprinting substitutes for bone.

ElvisionTec 3D - Bioplotter.

Talking of bone, a team lead by Jeremy Mao at the Tissue Engineering and Regenerative Medicine Lab at Columbia University is working on the application of bioprinting in dental and bone repairs. Already, a bioprinted, mesh-like 3D scaffold in the shape of an incisor has been implanted into the jaw bone of a rat. This featured tiny, interconnecting microchannels that contained 'stem cell-recruiting substances'. In just nine weeks after implantation, these triggered the growth of fresh periodontal ligaments and newly formed alveolar bone. In time, this research may enable people to be fitted with living, bioprinted teeth, or else scaffolds that will cause the body to grow new teeth all by itself. 

In another experient, Mao's team implanted bioprinted scaffolds in the place of the hip bones of several rabbits. Again these were infused with growth factors. As reported in The Lancet, over a four month period the rabbits all grew new and fully-functional joints around the mesh. Some even began to walk and otherwise place weight on their new joints only a few weeks after surgery. Sometime next decade, human patients may therefore be fitted with bioprinted scaffolds that will trigger the grown of replacement hip and other bones. In a similar development, a team from Washington State University have also recently reported on four years of work using 3D printers to create a bone-like material that may in the future be used to repair injuries to human bones.

In Situ Bioprinting

The aforementioned research progress will in time permit organs to be bioprinted in a lab from a culture of a patient's own cells. Such developments could therefore spark a medical revolution. Nevertheless, others are already trying to go further by developing techniques that will enable cells to be printed directly onto or into the human body in situ. Sometime next decade, doctors may therefore be able to scan wounds and spray on layers of cells to very rapidly heal them.

Healing a wound by In Situ Bioprinting: A future aspect

Already a team of bioprinting researchers lead by Anthony Alata at the Wake Forrest School of Medicine have developed a skin printer. In initial experiments they have taken 3D scans of test injuries inflicted on some mice and have used the data to control a bioprint head that has sprayed skin cells, a coagulant and collagen onto the wounds. The results are also very promising, with the wounds healing in just two or three weeks compared to about five or six weeks in a control group. Funding for the skin-printing project is coming in part from the US military who are keen to develop in situ bioprinting to help heal wounds on the battlefield. At present the work is still in a pre-clinical phase with Alata progressing his research using pigs. However, trials of with human burn victims could be a little as five years away.

The potential to use bioprinters to repair our bodies in situ is pretty mind blowing. In perhaps no more than a few decades it may be possible for robotic surgical arms tipped with bioprint heads to enter the body, repair damage at the cellular level, and then also repair their point of entry on their way out. Patients would still need to rest and recuperate for a few days as bioprinted materials fully fused into mature living tissue. However, most patients could potentially recover from very major surgery in less than a week.

Cosmetic Applications
As well as allowing keyhole bioprinters to repair organs inside a patient during an operation, in situ bioprinting could also have cosmetic applications. For example, face printers may be created. These would evaporate existing flesh and simultaneously replace it with new cells to exact patient specification. People could therefore download a face scan from the Internet and have it applied to themselves. Alternatively, some teenagers may have their own face scanned, and then reapplied every few years to achieve apparent perpetual youth.

Face Printers: A cosmetic application of Bioprinters

The idea of having the cells of your face slowly burnt away by a laser and reprinted to order may sound like a nightmare that nobody would ever choose to endure. However, as we all know, many people today go under the knife to achieve far less cosmetically. When the technology is available to create them, face printers -- let alone printers capable of printing new muscles without the hassle of exercise -- therefore very likely to find a market.

Bioprinting Implications

As bioprinters enter medical application, so replacement organs will be output to individual patient specification. As every item printed will be created from a culture of a patient's own cells, the risk of transplant organ rejection should be very low indeed.

Together with developments in nanotechnology and genetic engineering, bioprinting may also prove a powerful tool for those in pursuit of life extension. Mainstream bioprinting will also inevitably drive further the New Industrial Convergence, with doctors, engineers and computer scientists all increasingly learning to manipulate living tissue at its most basic cellular level.

Bioprinter Components
The basic parts of a Bioprinter:

A Basic Bioprinter

1. Print head mount:- On a bioprinter, the print heads are attached to a metal plate running along a horizontal track. The x-axis motor propels the metal plate (and the print heads) from side to side, allowing material to be deposited in either horizontal direction.
2. Elevator:- A metal track running vertically at the back of the machine, the elevator, driven by the z-axis motor, moves the print heads up and down. This makes it possible to stack successive layers of material, one on top of the next.
3. Platform:-A shelf at the bottom of the machine provides a platform for the organ to rest on during the production process. The platform may support a scaffold, a petri dish or a well plate, which could contain up to 24 small depressions to hold organ tissue samples for pharmaceutical testing. A third motor moves the platform front to back along the y-axis.
4. Reservoirs:- The reservoirs attach to the print heads and hold the biomaterial to be deposited during the printing process. These are equivalent to the cartridges in your inkjet printer.
5. Print heads/syringes:- A pump forces material from the reservoirs down through a small nozzle or syringe, which is positioned just above the platform. As the material is extruded, it forms a layer on the platform.
6. Triangulation sensor:-A small sensor tracks the tip of each print head as it moves along the x-, y- and z-axes. Software communicates with the machine so the precise location of the print heads is known throughout the process.
7. Microgel:- Unlike the normal printer ink at home, bioink is alive, so it needs food, water and oxygen to survive. This nurturing environment is provided by a microgel -- think gelatin enriched with vitamins, proteins and other life-sustaining compounds. Researchers either mix cells with the gel before printing or extrude the cells from one print head, microgel from the other. Either way, the gel helps the cells stay suspended and prevents them from settling and clumping.
8. Bioink:- Organs are made of tissues, and tissues are made of cells. To print an organ, a scientist must be able to deposit cells specific to the organ she hopes to build. For example, to create a liver, she would start with hepatocytes -- the essential cells of a liver -- as well as other supporting cells. These cells form a special material known as bioink, which is placed in the reservoir of the printer and then extruded through the print head. As the cells accumulate on the platform and become embedded in the microgel, they assume a three-dimensional shape that resembles a human organ.

The Solved Problem
When researchers built 3-D printers capable of depositing bioink and forming living masses of cells, they celebrated a major achievement. Then they immediately began to tackle the next big problem: How can bioprinting produce an organ for a specific person? 

To accomplish this, a medical team needs to collect data about the organ in question -- its size, shape and placement in the patient's body. Then team members need to concoct a bioink using cells taken from the patient. This ensures that the printed organ will be compatible genetically and won't be rejected once it's transplanted in the patient's body.

For simple organs, such as bladders, researchers don't print the living tissue directly. Instead, they print a 3-D scaffold made of biodegradable polymers or collagen. To determine the exact shape of the scaffold, they first build a 3-D model using computer-aided design (CAD) software. They usually define the exact x-, y- and z-coordinates of the model by taking scans of the patient using computerized tomography (CT) or magnetic resonance imaging (MRI) technology.

Next, researchers get the cells they need by taking a biopsy of the patient's bladder. They then place the cell samples in a culture, where they multiply into a population sufficiently large enough to cover the scaffold, which provides a temporary substrate for the cells to cling to as they organize and strengthen. Seeding the scaffold requires time-consuming and painstaking handwork with a pipette. It generally takes about eight weeks before such artificial bladders are ready for implantation. When doctors finally place the organ in the patient, the scaffold has either disappeared or disappears soon after the surgery.

The procedure above works because bladder tissue only contains two types of cells. Organs like kidneys and livers have a far more complex structure with a greater diversity of cell types. While it would be easy enough to print a scaffold, it would be almost impossible to recreate the three-dimensional structure of the tissue manually. A bioprinter, however, is ideally suited to complete such a time-consuming, detail-oriented task.

Bioprinting Process for Printing Organs

• Organovo’s bioprinting process centers on the identification of key architectural and compositional elements of a target tissue, and the creation of a design that can be utilized by a bioprinter to generate that tissue in the laboratory environment. 

• Once a tissue design is established, the first step is to develop the bioprocess protocols required to generate the multi-cellular building blocks—also called bio-ink—from the cells that will be used to build the target tissue.

• The bio-ink building blocks are then dispensed from a bioprinter, using a layer-by-layer approach that is scaled for the target output. Bio-inert hydrogel components may be utilized as supports, as tissues are built up vertically to achieve three-dimensionality, or as fillers to create channels or void spaces within tissues to mimic features of native tissue.

• The bioprinting process can be tailored to produce tissues in a variety of formats, from micro-scale tissues contained in standard multi-well tissue culture plates, to larger structures suitable for placement onto bioreactors for biomechanical conditioning prior to use.

Bioprinter Printing a Human Heart.

Bioprinting Process for Printing 3D Models

• To make a 3D model using this technique, a manufacturer loads a substance, usually plastic, into a mini-fridge-sized machine. He also loads a 3-D design of the model he wants to make. 

• When he tells the machine to print, it heats up and, using the design as a set of instructions, extrudes a layer of melted plastic through a nozzle onto a platform.

• As the plastic cools, it begins to solidify, although by itself, it's nothing more than a single slice of the desired object. 

• The platform then moves downward so a second layer can be deposited on the first. The printer repeats this process until it forms a solid object in the shape of a certain 3D model.

3D Model of A Nose.

In industrial circles, this is known as additive manufacturing because the finished product is made by adding material to build up a three-dimensional shape. It differs from traditional manufacturing, which often involves subtracting a material, by way of machining, to achieve a certain shape. Additive manufacturers aren't limited to using plastic as their starting material. Some use powders, which are held together by glue or heated to fuse the powder together. 

Basic steps to print a complex organ:

1. First, doctors make CT or MRI scans of the desired organ.
2. Next, they load the images into a computer and build a corresponding 3-D blueprint of the structure using CAD software.
3. Combining this 3-D data with histological information collected from years of microscopic analysis of tissues, scientists build a slice-by-slice model of the patient's organ. Each slice accurately reflects how the unique cells and the surrounding cellular matrix fit together in three-dimensional space.
4. After that, it's a matter of hitting File > Print, which sends the modeling data to the bioprinter.
5. The printer outputs the organ one layer at a time, using bioink and gel to create the complex multicellular tissue and hold it in place.
6. Finally, scientists remove the organ from the printer and place it in an incubator, where the cells in the bioink enjoy some warm, quiet downtime to start living and working together. For example, liver cells need to form what biologists call "tight junctions," which describes how the cell membrane of one cell fuses to the cell membrane of the adjacent cell. The time in the incubator really pays off -- a few hours in the warmth turns the bioink into living tissue capable of carrying out liver functions and surviving in a lab for up to 40 days.
7. The final step of this process -- making printed organ cells behave like native cells -- has been challenging. 

• Some scientists recommend that bioprinting be done with a patient's stem cells. After being deposited in their required three-dimensional space, they would then differentiate into mature cells, with all of the instructions about how to "behave." 
• Then, of course, there's the issue of getting blood to all of the cells in a printed organ. Currently, bioprinting doesn't offer sufficient resolutions to create tiny, single-cell-thick capillaries. But scientists have printed larger blood vessels, and as the technology improves, the next step will be fully functional replacement organs, complete with the vascularization necessary to remain alive and healthy.

Uses for 3-D Organs

• Replacing parts of the skeleton is one area being revolutionized by 3-D printing. Printing entire bones for placement in the body. Doctors in the Netherlands have already created a lower mandible on a 3-D printer and implanted the jaw -- made from bioceramic-coated titanium -- in a patient suffering from a chronic bone infection.

• Some dentists now take an intra-oral scan of a patient's teeth and send the scan to a lab that fashions a porcelain bridge using a 3-D printer. 

• Prosthetic manufacturers also have changed their approach to designing artificial limbs. Now, many are able to print fairings -- prosthetic limb covers -- that mold perfectly to a person's anatomy, giving the wearer a more comfortable fit. 

• Scientists have also successfully printed cartilaginous structures, such as ears and tracheas. To make the former, bioengineers take a 3-D scan of a patient's ear, design a mold using CAD software and then print it out. Then they inject the mold with cartilage cells and collagen. After spending some time in an incubator, the ear comes out, ready for attachment to the patient. 

• A trachea can be made in a similar fashion. In 2012, doctors at the University of Michigan printed a sleeve, made from a 3-D model generated from a CT scan, to wrap and support a baby's trachea, which had been rendered weak and floppy by a rare defect.

• Skin -- the body's largest organ -- may be the first item on the list. Researchers at the Wake Forest Institute for Regenerative Medicine already have developed a complete system to print skin grafts. The system includes a scanner to map a patient's wound and a purpose-built inkjet printer that lays down the cells, proteins and enzymes necessary to form human skin. The goal is to build portable printers for use in field hospitals, where doctors can output skin directly onto patients.

• 3-D organs will play an important role in education and drug development. 

• They might even factor into the development of food and clothing products (lab-grown meat and leather).

• Some medical schools have invested in 3-D printing technology to create surgical models of organs from CT or MRI images. This allows students to practice on hearts, livers and other structures that look and feel just like the real thing. 

• Having access to such lifelike tissues also benefits pharmaceutical companies, which can test candidate drugs to see their effects. 

• Organovo houses several printers capable of printing out three-dimensional models of liver, kidney and cancer tissues. These aren't full organs meant to live indefinitely. Instead, they're "organs on a chip" -- small, biologically active tissue samples designed to respond as native tissues would.

👉 Please Watch Our 3D Bio-Printing Videos (Part 1 & Part 2) from Our YouTube Channel Below:-

1. Part 1 Video:-

2. Part 2 Video:-

Article Prepared By:-

👉 Gaston Ravin Dias 

References:

• 3D Printing. 2017. Bio printing - Bioprinting - Human tissue - Biotech. [ONLINE] Available at: https://3dprinting.com/bio-printing/. [Accessed 07 November 2017].
• ExplainingTheFuture.com : Bioprinting. 2017. ExplainingTheFuture.com : Bioprinting. [ONLINE] Available at: http://explainingthefuture.com/bioprinting.html. [Accessed 07 November 2017].
• Organovo. 2017. Bioprinting Process - Organovo. [ONLINE] Available at: http://organovo.com/science-technology/bioprinting-process/. [Accessed 07 November 2017].
• Research & Development. 2017. A New Era for 3D Bioprinting Breakthroughs. [ONLINE] Available at: https://www.rdmag.com/article/2017/06/new-era-3d-bioprinting-breakthroughs. [Accessed 07 November 2017].
• The Guardian. 2017. Could 3D printing solve the organ transplant shortage? | Technology | The Guardian. [ONLINE] Available at: https://www.theguardian.com/technology/2017/jul/30/will-3d-printing-solve-the-organ-transplant-shortage. [Accessed 07 November 2017].
• Wyss Institute. 2017. 3D Bioprinting of Living Tissues. [ONLINE] Available at: https://wyss.harvard.edu/technology/3d-bioprinting/. [Accessed 07 November 2017].