Three dimensional (3D) bioprinting is the utilization of 3D printing and 3D printing–like techniques to combine cells, growth factors, and biomaterials to fabricate biomedical parts that maximally imitate natural tissue characteristics. Generally, 3D bioprinting utilizes the layer-by-layer method to deposit materials known as bioinks to create tissue-like structures that are later used in medical and tissue engineering fields. Bioprinting covers a broad range of biomaterials.

Currently, bioprinting can be used to print tissues and organs to help research drugs and pills. However, emerging innovations span from bioprinting of cells or extracellular matrix deposited into a 3D gel layer by layer to produce the desired tissue or organ. The recent explosion in popularity of 3D printing is a testament to the promise of this technology and its profound utility in research and regenerative medicine. In addition, 3D bioprinting has begun to incorporate the printing of scaffolds. These scaffolds can be used to regenerate joints and ligaments.

3D bio-printing is the process of creating cellular structures in a confined space using 3D printing technologies, where cell function and viability are retained in the printed construct. Generally, 3D bio-printing uses the layer-by-layer printing method to deposit materials sometimes referred to as bioencres to create structures similar to natural biological tissues that are then used in the fields of medical engineering and tissues 9. Bio-printing uses a wide range of materials. At present, bio-printing can be used to print tissues and organs, especially forpharmaceutical research. The first patent related to this technology was filed in the United States in 2003 and granted in 2006.

Bio-printing is at the interface of many fields: medicine, engineering, computer science, genetic engineering, etc. Biological tissues consist of hard tissues composed of organic and inorganic extracellular matrices and soft tissues formed by cells. Living cellular matter is printed from stem cells. It is deposited in droplets of biological ink which will form successive layers and which in superimposing will constitute a biological tissue in three dimensions. To produce biological ink, one can use the patient’s stem cells that one will grow (it takes millions to create a square millimeter of tissue). The stem cells are suspended in a specific medium that can be modified at room temperature. The support on which the fabric is printed is a thin layer of collagen(the most abundant protein in the human body, responsible for tissue cohesion) that could be compared to the paper of a traditional printer. In addition to cells and biomaterials, the bioprinter must also incorporate a spectrum of biochemicals (ie, chemokines, growth factors, adhesion factors, or signaling proteins) to promote an environment of survival, motility and cell differentiation.

Several stages can be distinguished when printing a fabric by 3D bio-printing. These three sequential technological steps are pretreatment, processing (printing) and post-processing:
The design is more or less identical to the original tissues and then the computer design of the model that will define how the stem cells will be printed layer by layer according to the characteristics expressed in the first step. This step is coupled with the third step which is to program the printer via specialized software that will translate the actions to be performed into the printer language. These two steps are similar to those to be done to design an object from a plastic 3D printer.
The automated printing of the fabric by the printer which differs according to the technology used.

Two key parameters in bio-printing are density and resolution. The density of the cells is that in the biological ink. If it is too low then the final phase will not be well done and the fabric will not be viable. Resolution is the precision with which cells will be placed by the printer. If the precision is not optimal then the predefined structure of the cells will not be respected and the tissue will not have the right shape, preventing at the same time the good progress of the final phase of development of the cells.

The last step is the maturation of printed fabrics. This is the phase in which the assembled cells will evolve and interact together to form a coherent and viable tissue. During the process of post-printing within a bioreactor the tissues undergo rapid maturation including the development of multi-level vascularization and innervation increasing the strength and mechanical integrity of tissues for transplantation. Placed in an incubatorthe tissues develop to form a coherent tissue. This phase begins approximately 48 hours after printing and can last several weeks depending on the size of the fabric. With the maturation phase, we can talk about 4D printing because the time dimension after printing is essential.

Bioreactors work by providing an environment conducive to tissue development by providing convective nutrients, creating a microgravity environment and promoting the circulation of the solution in the cells. There are different types of bioreactors suitable for different types of tissue, for example, compression bioreactors are ideal for cartilage tissue.

3D printing for the manufacture of artificial organs has become a major subject of study in biological engineering. As 3D printing manufacturing techniques become more and more efficient, their applicability in artificial organ synthesis has become more apparent. The main advantages of 3D printingare its mass production capacity of customizable complex structures as well as the high degree of anatomical precision obtained. 3D bio-printing offers unprecedented versatility in positioning cells and biomaterials with precise control over their compositions, spatial distributions and architectural accuracy, allowing detailed or even personalized reconstruction of the final shape, structure, the microstructure and architecture of printed fabrics and organs.

Compared to non-biological 3D printing, 3D bio-printing induces additional levels of complexity, such as choice of materials, cell type, growth and differentiation factors, and technical challenges related to cell sensitivities. living and fabric construction.

Printing organs using 3D printing can be performed using a variety of techniques, each carrying specific benefits that can be tailored to specific types of production of organs.

The traditional tissue engineering approach was to seed cells on a matrix scaffold ie a solid support structure comprising an interconnected pore network. This structure must maintain the shape and mechanical properties of the synthesized tissue and assist in cell attachment by providing a substrate for cell proliferation. 3D printing technology is a recent innovation that allows simultaneous seeding of living cells and the creation of the biomaterial structure in layers.

The three most popular 3D bio-printing technologies are laser printing technology, micro extrusion technology and inkjet technology. In addition to these technologies, a team of researchers in Cambridge is developing an acoustic printer where waves vibrate the bioencre, which will cause the ejection of droplets with the precision of the size of a cell. Today, on the internet, you can find explanations on how to make your own bio-printer from an office printer type HP as presented on the site TeVido BioDevices.

Each technology has advantages and disadvantages for the printing of hard biological tissue engineering and organ. The hard tissues of the human body include bones, teeth and cartilage and consist of some types of single cells and a significant proportion of organic and inorganic extracellular matrices.

Laser printing
This latest technology required 10 years of research at INSERM in Bordeaux. This technique works on the principle of the laser. A laser is directed by means of a mirror, passes through a lens, then focuses, strikes a coverslip on which is placed a film of biological ink. During the laser / cartridge interaction drop micro drops containing cells in small numbers on the support with an accuracy of 5 microns. The printing is fast enough. Experiments have even shown that it works on mice, thanks to an in vivo impression(directly on the skin of a living being). The patterns of the cell are obtained by laser scanning at 10,000 pulses per second, each pulse generating a micro droplet. This technology is the only one with a resolution of unity (cell by cell) up to 50 cells per micro drop. This precision makes it possible to reproduce complex biological tissues in 3 dimensions, such as skin samples.

Laser printing combines resolution and density (about 108 cells / ml of biologic ink) with multiple benefits. Three of the benefits of laser bio-printing are more than 95% cell viability, reduced waste and no mechanical stress. This is due to the shortness of the pulses, a few nanoseconds, which minimizes cell warming and reduces their “stress”. However, the viability of the printed fabrics depends on the stresses exerted on the cells. It is important that the cells are the least “degraded” possible.

However, some factors remain to be improved because the machine does not yet stack many layers of cell in a well organized way, the preparation time is high and the cost of printing too.

This technology is used in particular in Tedivo Biodevices DIY printers. It is this technology that works at the University of Manchester in England. The inkjet printer works with a print head that projects micro droplets of a liquid containing cells (the bio-ink). The ejection of the droplets is caused by a thermal (heat) or piezoelectric process (electric polarization of the ink under the action of a mechanical stress). The ink is liquid at 20° but gels at a temperature of 36°. This process is the one that is most similar to that of 3D plastic printers.

This technology is the most affordable and easy to use with minimal preparation time and low cost. The printing time is low and the viability of the cells is greater than 85% but the resolution is poor, resulting in poor cell development. In addition, the density is also a difficult to manage parameter, it is often too low, or very low (about 106 cells / ml, 100 times less than for the laser printer). These disadvantages make it at the moment unsuitable for printing complex fabrics, it only serves to print patterns thanks to the cells to be printed.

The microextrusion (also called bioextrusion) is the only method that began to be industrialized by the US company Organovo with its printer Novogen MMX, developed in conjunction with the University of Missouri and development in 2005.

This printer works with two printheads. One deposits the gel and the other the cells. The cells are pushed into a micro syringe and deposited using a needle. The layers are alternately deposited, a hydrogel layer (water mixture) followed by a layer of cells. The hydrogel is used to structure the assembly of cell layers, similar to scaffolding. The hydrogelis then dissolved during the ripening phase, allowing the cells to fuse together. Bioextrusion makes it possible to obtain a high density but with an average resolution (ranging from 5 micrometers to a few millimeters wide). The preparation time is average compared to other techniques but with a higher printing time (very slow). The cost of this type of printer is medium and the viability (ability to “survive” after printing and during the maturation phase) of the cells is between 40 and 80%, this rate is low compared to other technologies and this aspect remains to be improved.

Hybrid techniques
These technologies today have limited possibilities but some researchers are looking at “hybrid printers”. This technique remains at the testing stage, but in the United States researchers have succeeded in coupling cell printing and deposition of biodegradable polymer (substance composed of molecules characterized by the repetition of one or more atoms or groups of atoms, which may be natural, synthetic or artificial) forming cartilage.

There are different bioprinters on the market. Prices range from $ 10,000 for BioBot 1 to $ 200,000 for EnvisionTec’s 3D-Bioplotter. The Aether 1 bio-printer is expected to be marketed from 2017 for the price of 9,000 USD. In practice, researchers often develop their own experimental bio-printers.

3D bioprinting generally follows three steps, pre-bioprinting, bioprinting, and post-bioprinting.

Pre-bioprinting is the process of creating a model that the printer will later create and choosing the materials that will be used. One of the first steps is to obtain a biopsy of the organ. Common technologies used for bioprinting are computed tomography (CT) and magnetic resonance imaging (MRI). To print with a layer-by-layer approach, tomographic reconstruction is done on the images. The now-2D images are then sent to the printer to be made. Once the image is created, certain cells are isolated and multiplied. These cells are then mixed with a special liquefied material that provides oxygen and other nutrients to keep them alive. In some processes, the cells are encapsulated in cellular spheroids 500μm in diameter. This aggregation of cells does not require a scaffold, and are required for placing in the tubular-like tissue fusion for processes such as extrusion.

In the second step, the liquid mixture of cells, matrix, and nutrients known as bioinks are placed in a printer cartridge and deposited using the patients’ medical scans. When a bioprinted pre-tissue is transferred to an incubator, this cell-based pre-tissue matures into a tissue.

3D bioprinting for fabricating biological constructs typically involves dispensing cells onto a biocompatible scaffold using a successive layer-by-layer approach to generate tissue-like three-dimensional structures. Artificial organs such as livers and kidneys made by 3D bioprinting have been shown to lack crucial elements that affect the body such as working blood vessels, tubules for collecting urine, and the growth of billions of cells required for these organs. Without these components the body has no way to get the essential nutrients and oxygen deep within their interiors. Given that every tissue in the body is naturally composed of different cell types, many technologies for printing these cells vary in their ability to ensure stability and viability of the cells during the manufacturing process. Some of the methods that are used for 3D bioprinting of cells are photolithography, magnetic bioprinting, stereolithography, and direct cell extrusion.

The post-bioprinting process is necessary to create a stable structure from the biological material. If this process is not well-maintained, the mechanical integrity and function of the 3D printed object is at risk. To maintain the object, both mechanical and chemical stimulations are needed. These stimulations send signals to the cells to control the remodeling and growth of tissues. In addition, in recent development, bioreactor technologies have allowed the rapid maturation of tissues, vascularization of tissues and the ability to survive transplants.

Bioreactors work in either providing convective nutrient transport, creating microgravity environments, changing the pressure causing solution to flow through the cells, or add compression for dynamic or static loading. Each type of bioreactor is ideal for different types of tissue, for example compression bioreactors are ideal for cartilage tissue.

Bioprinting approach
Researchers in the field have developed approaches to produce living organs that are constructed with the appropriate biological and mechanical properties. 3D bioprinting is based on three main approaches: Biomimicry, autonomous self-assembly and mini-tissue building blocks.

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The first approach of bioprinting is called biomimicry. The main goal of this approach is to create fabricated structures that are identical to the natural structure that are found in the tissues and organs in the human body. Biomimicry requires duplication of the shape, framework, and the microenvironment of the organs and tissues. The application of biomimicry in bioprinting involves creating both identical cellular and extracellular parts of organs. For this approach to be successful, the tissues must be replicated on a micro scale. Therefore, it is necessary to understand the microenvironment, the nature of the biological forces in this microenvironment, the precise organization of functional and supporting cell types, solubility factors, and the composition of extracellular matrix.

Autonomous self-assembly
The second approach of bioprinting is autonomous self-assembly. This approach relies on the physical process of embryonic organ development as a model to replicate the tissues of interest. When cells are in their early development, they create their own extracellular matrix building block, the proper cell signaling, and independent arrangement and patterning to provide the required biological functions and micro-architecture. Autonomous self-assembly demands specific information about the developmental techniques of the tissues and organs of the embryo. There is a “scaffold-free” model that uses self-assembling spheroids that subjects to fusion and cell arrangement to resemble evolving tissues. Autonomous self-assembly depends on the cell as the fundamental driver of histogenesis, guiding the building blocks, structural and functional properties of these tissues. It demands a deeper understanding of how embryonic tissues mechanisms develop as well as the microenvironment surrounded to create the bioprinted tissues.

The third approach of bioprinting is a combination of both the biomimicry and self-assembly approaches, which is called mini tissues. Organs and tissues are built from very small functional components. Mini-tissue approach takes these small pieces and manufacture and arrange them into larger framework.

Akin to ordinary ink printers, bioprinters have three major components to them. These are the hardware used, the type of bio-ink, and the material it is printed on (biomaterials). “Bio-ink is a material made from living cells that behaves much like a liquid, allowing people to “print” it in order to create a desired shape. To make bio-ink, scientists create a slurry of cells that can be loaded into a cartridge and inserted into a specially designed printer, along with another cartridge containing a gel known as bio-paper.”

In bioprinting, there are three major types of printers that have been used. These are inkjet, laser-assisted, and extrusion printers. Inkjet printers are mainly used in bioprinting for fast and large-scale products. One type of inkjet printer, called drop-on-demand inkjet printer, prints materials in exact amounts, minimizing cost and waste. Printers that utilize lasers provide high-resolution printing; however, these printers are often expensive. Extrusion printers print cells layer-by-layer, just like 3D printing to create 3D constructs. In addition to just cells, extrusion printers may also use hydrogels infused with cells.

The field of regenerative medicine has made considerable progress in recent decades in its ability to produce functional substitutes for biological tissues. Although for more than a decade, living cells and biomaterials (usually hydrogels) have been printed through bio-printing, 29 conventional approaches based on extracellular matrices and microengineering remain limited in their ability to produce tissues with precise biomimetic properties.

In 2013, Organovo produced a human liver through bio-printing techniques. The body, however, was not suitable for transplantation and was primarily used as a means of screening for drugs 30.

Use of bio-printing in 2017
Bioprinting already makes it possible to create living structures. Cellular living matter is printed in many laboratories around the world, cell tissues are viable and bio-printing does not affect cell differentiation. Some of the technologies have been applied in medical treatments with some success. 3D bio-printing has already been used for the production and transplantation of several tissues, including multilayer skin, bone, vascular grafts, tracheal prostheses, cardiac tissues and cartilage structures.

The printing of complex organs is the subject of intense research around the world. For example for the heart, pancreas, liver or kidneys. Beginning 2017, this research had not yet led to transplantation.

In May 2017, researchers used bioimpression to produce mouse ovaries. Sterile mice implanted with the artificial ovary were able to ovulate, deliver, and feed normally healthy baby mice. The study is the first to achieve such a result with the help of 3-D printing.

Current advances for the skin.
The researchers managed to print different structures and cell types: multilayers of keratinocytes (cells of the superficial layer of the skin and superficial body growths: nails, hair, hair) and collagen.

In 2010, the Bordeaux laboratory managed to print bone cells (to renew and consolidate bone tissue) directly on the skull of a living mouse with a small hole. In the case of printing directly on the patient we speak of printing in vivo. The researchers used the same principle to print a bone part and a part of skin by removing mesenchymal cells printed later. Mesenchymal cells can produce several types of cells belonging to skeletal tissues, such as cartilage, bone and fat. They are found in the mesenchyme of the embryo and in very small quantities in the adult. The doctorFabien Guillemot commented on the first tests on mice: “The results obtained are very conclusive. The printed cells retained all their functions and multiplied until two months after printing. The first subjects showed signs of healing. Same result for the Hannover Laser Center in Germany: the fabric repairs the wound of the animal without any rejection.

The American company Organovo markets printed skin samples for medical research. These functional organic tissues are used by pharmaceutical companies to test the effects of treatments and their impact on diseases. The company is also printing models of diseased tissue to better understand diseases and their evolution. The goal is also to test the effectiveness of drug molecules and reduce the cost of clinical trials. Large cosmetic groups also use samples to assess the toxicity of care before marketing and to find an alternative to animal testing that has been banned in Europe since 2013.

Current advances for vital organs
New techniques have been developed to overcome the problem of the vascularization of printed tissues. One technique actually prints eg soft tissues containing collagen and other biological fibers in a hydrogel holder. The printed fabric is then recovered by melting the support without damaging the cells and the structure. Following this principle, models of femur, coronary arteries, blood vessels and an embryo heart have already been successfully printed. These cell tissues are necessary to oxygenate the organs but have not yet tested on humans and do not allow complete vascularization of organs such as the liver,lungs or heart.

Thanks to advances in the field of vasculature, it is now possible to create miniature organs. Organovo, for example, has experimented with printing various types of complex tissue such as lung and heart muscle pieces. She managed to make a piece of kidney (1 mm thick by 4 mm wide) that survived 5 days out of the laboratory. They also created a reconstituted human liver that remained functional for 40 days. This liver sample (3mm 2 by 0.5 mm thick) was able to produce enzymes, proteins and cholesterolThis multiplies by the life of the organ through the exchanges that may have occurred. Similarly, Chinese researchers develop kidneys whose lifespan is currently limited to 4 months.

“We need to continue research and gather more information, but the fact that the tissue behaves like a liver suggests that it will continue to behave as such when it starts to be tested with drugs. Says Keith Murphy, CEO of Organovo. The company Organovo recently commercializes liver tissue that remains functional for at least 42 days. These organ samples are intended for medical research. But to date, none of these parts has yet been integrated with living organisms.

In October 2016, Harvard researchers bio-printed the world’s first on-chip heart with integrated sensors. The device, which is a micro-physiological system, mimics the behavior of human tissue. This completion is the most sophisticated organ-on-chip, including in comparison with the lungs, languages and on-chip intestines also produced by this team. The development of this bio-printed organ-on-chip application could reduce the dependence of medical research on animal testing.

Other bodies
Researchers at the University of Cambridge, England, announced their ability to recreate nerve cells in a rat ‘s retina through a bio – printer. The printer is capable of associating ganglion cell cartridges and glial cell cartridges from rat stem cells. This transplant has allowed an animal to recover much of its visual acuity while eliminating the risk of rejection. And in April 2013, scientists at Princeton University made the impression of a bionic ear: it combines organic cells and nanoparticlesto an antenna molded in cartilage. The ear thus produced can hear radio frequencies inaudible with a natural human ear.

Scientists at Columbia University are working on the creation of bio-printed teeth and joints. This team, for example, implanted an incisor created from a 3D structure printed in the jaw of a rat. In two months, the implant has allowed the growth of ligaments that support newly formed teeth and bones. The research team also implanted bio-printed hip bones on rabbits, which started walking with their new joints after a few weeks.

Although breakthroughs have been made in the production of printable organs, clinical implementation, particularly with regard to complex organs, requires further research and development. The cellular proliferation required for biological printing is conducted in an artificial and controlled environment that lacks markers and natural biological processes. The absence of these properties often inhibits the development of appropriate morphology and cell differentiation. When present, these conditions would allow the printed organ to mimic the conditions in vivo more precisely.and to adopt a structure and an adequate functioning contrary to a biological growth conceived as a simple scaffold formed of cells 34. Some of the technical challenges to be solved include:

The vascularization: While it is possible to create basic cellular tissues like skin 35, for example, it is impossible to create complex organs. Indeed, scientists can not recreate blood vessels like capillaries because they are long, thin and tubular and the accuracy of the printers is too low. The impression of any organ is therefore impossible because the cells would not be fed with oxygen and glucose and would die very quickly. In addition, the cell skin tissue printed until now are not vascularized and therefore not suitable for grafting. Cell tissues need to be vascularized as soon as their thickness exceeds 400 microns.
The nervous system: The nervous system presents a great complexity. Without the nerves, the muscles created can not be operated and can not be grafted.
The pluripotent cells: bio-printing requires a large amount of pluripotent cells.
The survival time of printed cells: For now, printed fabrics do not live very long because they are not in their natural environment. For example, the company Organovo managed to print a miniature kidney of 4 mm by 1 mm but it remained alive only 5 days.
The price: The cost of high-end functional biological printers remains very expensive, so they can hardly be acquired by small research laboratories or hospitals. Indeed, a biological printer costs several hundreds of thousands of euros.
The complex organization of organs: For example, a kidney is made up of one million nephrons that provide blood filtration and urine production. Each nephron consists of multiple subunits such as glomeruli themselves made up of four types of cells… This organization is very complex to print layer by layer.
The severity: Even with the highest known biological technique for printing, scientists are forced to print tissues layer by layer because of gravity, which greatly complicates the formation of large bodies would collapse under their own weight and deform molecular structures.
Scientific knowledge: This is probably the biggest obstacle to the development and printing of complex organs. The lack of global knowledge on the human body is felt in several areas such as the nervous system or the morphogenesis of the body.

Recent developments

In April 2017, a research team from the University of California succeeded in producing vascularized tissue with complex three-dimensional microarchitectures using the so-called “microscopic continuous optical bio-impression” (μCOB) bio-printing method. In vivo implantation of the printed tissues demonstrated the survival and progressive formation of the endothelial network in the prevascularized tissue.

Scientists are forced to print cell organs and tissues in successive layers of cells because of gravity. According to them, if one prints the organs in pseudo state of weightlessness for example by means of a magnetic field, the cells could be placed correctly and without deformation.

Professor Vladimir Mironov and his team of researchers have reached agreements for tests to be carried out aboard the International Space Station.

To counter this phenomenon of gravity, the team of Professor Adam Feinbergon had the idea to deposit the cells in a cube of Hydrogel (water-based gelatinous cube). The cells thus deposited remain in suspension in the hydrogel which gives them time to create sufficient cellular connections so that the organ created does not deform. The gel melts in water at body temperature (37 ° C). Once connections are established, it is sufficient to plunge the hydrogel cube in water at 37 ° C to recover the intact formed organ.

Complex Organization
Led by Fabien Guillemot, the team of researchers at INSERM Bordeaux aims to recreate a functional kidney. For that, they decided not to print it layer by layer, but piece by piece [precision needed]. Indeed, the complex organization of the kidney making it impossible to print layer by layer, the team of INSERM first wants to create glomeruli which could then be assembled to make nephrons, themselves assembled to make a functional kidney.

Pluripotent cells
In 2012, Japanese researcher Shinya Yamanaka successfully created functional pluripotent stem cells from differentiated cells such as skin cells. Indeed, after 7 years of research and testing on mice, the Japanese researcher discovered that by taking the genes that encode the non-differentiation of pluripotent stem cells and placing them in the genetic makeup of the differentiated cell, this last becomes pluripotent. This discovery earned him the Nobel Prize in Medicine. As a result, it is possible to create a pluripotent stem cell culture specific to an individual without even a bone marrow sample.

These differentiated cells reprogrammed into stem cells are designated as iPS cells of the English induced pluripotent stem cells or pluripotent stem cells induced in French.

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