The state of bioprinting

We have been covering 3D bioprinting for nearly a decade, starting back when the bioprinting industry began to expand beyond high-end systems used exclusively for academic research into a young commercial segment. Over time it became filled with new and more affordable hardware, commercial and increasingly standardized materials and the dream of the first commercial applications. Today all those aspects of the bioprinting industry are experiencing vibrant growth, pushing innovation on all three fronts and enabling a real industry to emerge.

In 2013, we had the unique opportunity to attend one of the first summer courses on bioprinting, held by Professor Jos Malda at Utrecht University. Together with other key hubs (the Wyss Institute at Harvard in Boston, POSTECH University in Pohang, Korea and more), the researchers in Utrecht have been at the forefront of bioprinting innovation and democratization. At that time, Professor Malda enabled us to gain a deeper understanding of the possibilities at hand. In February 2022, that same group presented the first-ever examples of bioprinting of functional hepatic (liver) tissue units using volumetric bioprinting technology, which seems to be one of the most viable directions for the regeneration and eventually bioengineering of complex organs and tissues. Recently, a new company, Trestle Biotherapeutics, emerged to commercialize renal (kidney) tissue grafts based on research conducted over the past decade by Professor Jennifer Lewis Lab at Harvard’s Wyss Institute. 3D Systems, one of the conventional additive manufacturing industry leaders, has also entered the market by working on bioprinting hardware (with Allevi acquisition), bioinks (collPlant collaboration) and applications (primarily lung tissue, with United Therapeutics).

Liver and kidney tissues are seen as both the most complex and most useful tissues that could be achieved with bioprinting within an acceptable time frame. Their uses are both in tissue grafting for regenerative medicine and in drug development testing (DDT). However, the road to developing these bioprinted tissues has been a difficult one, which has already had casualties. Organovo, the company that almost single-handedly created the idea of commercial bioprinting based on the bioprinting of kidney and liver tissue, was not able to bring its products to market and burned through several millions of dollars before abandoning the bioprinting segment.

Bioprinting promises to revolutionize the way we approach medicine and tissue engineering. It has the potential to address the global shortage of organ donors and transform the medical industry, offering solutions for numerous diseases and injuries. In this comprehensive article, we will explore everything you need to know about bioprinting, from the different types of bioprinters and bioinks to the various applications of this cutting-edge technology. Whether you’re a student, researcher, or just curious about this exciting field, this article will provide a comprehensive overview of bioprinting, its current state, and its future potential.

What do we mean by bioprinting?

The term 3D bioprinting (or simply bioprinting) refers to a family of additive and digital manufacturing methods that produce physical objects layer by layer, using a machine (a 3D bioprinter). Like conventional 3D printing, bioprinting creates objects based on 3D models designed in CAD software. Bioprinted objects (or constructs) are usually replicas of human or animal tissue, created through the combination of cells with other biomaterials and biocompatible materials such as polymers and ceramics.

Bioprinting was “officially” introduced in 1988 when Robert J. Klebe used an inkjet printer for printing cells. Since then, the field has continued to evolve, and new methods and techniques have been introduced–initially at an academic level and more recently at various commercial levels. Over the past two decades, researchers have concentrated on approaches to accommodate the sensitivity of live cells to stresses (friction, pressure, fluid viscosity, etc.) that manifest during the printing process for biological materials.

Bioprinting processes, like industrial AM processes, can be classified into two main categories: tool-based or indirect (scaffold-based printing) and direct (scaffold-free printing). Both of these are further divided into two categories: laser-assisted bioprinting and laser-free bioprinting, each of which includes several sub-categories.

Bioprinted constructs are intrinsically multi-material and—to some extent—continue to evolve after they have been printed, which is a noticeable contrast from 3D printing for industrial manufacture. Multiple cell types, varied scaffolds and scaffolding materials, vascularity (the necessity to provide oxygen to cells across and beneath the construct’s surface) and numerous extracellular matrix materials can all be part of functional bioprinted structures. As the cells develop and adapt, there is also a time factor to consider. This is also why some refer to advanced bioprinting as a type of 4D printing.

Applications of bioprinting

Some industrial additive manufacturing has already progressed from research and prototype to commercial end-use applications. Although 3D bioprinting is expected to follow a similar trajectory, practically all existing commercial potential for bioprinting hardware is currently focused on research applications. The significant change is that, although this research had been limited to academic institutions, some research is now starting to be conducted in the corporate sector. The largest pharmaceutical companies are still hesitant to embrace it but have started looking into it.

The ultimate goal may be to replicate functioning organs using bioprinting technologies, but reproducing complex organs via bioprinting will require advancements far beyond the capabilities of existing technology and materials. For the foreseeable future, the relevant applications of bioprinting will be for drug development testing and—to some extent—food-related products (cellular agriculture). In terms of regenerative medicine, we may start to see some adoption of bioprinting for tissue grafts and some implants based on non-cellular biocompatible and bioresorbable polymers and ceramics.

Multi-materiality, which is still one of the major limits of all industrial 3D printing methods, is an even greater barrier in tissue and organ printing, as the body’s most complicated organs are made up of many different cell types. The need for more volumetric approaches (printing “holographically” from all sides at the same time) and speed of production are among the most obvious limitations of current technologies, though there are “simpler” biological structures that could be bioprinted, even for implantation in humans, within this decade.

Renal and hepatic tissue production is limited to research applications, where 3D printed tissues offer certain advantages over 2D printed tissues, but genuine functional organs are still a long way off. Heart regeneration (3D printed resorbable heart valves, for example) and, more recently, lung regeneration have made some headway. One of the key drivers of bioprinting for regenerative medicine applications is, of course, the enormous need for organs for transplantation.

Bioprinting studies

Nearly all cells in the body live in an extracellular matrix (ECM) and communicate with one another via biochemical and mechanical cues. Interactions between cells and between cells and the ECM form a communication network that keeps the tissue’s specificity and homeostasis. In 2D cell culture tests, the cells’ inability to acquire in vitro structural organization and connectivity might limit or impair attributes such as cellular morphology, viability, proliferation, differentiation, gene expression, stimuli response, drug metabolism and overall cell function. Preclinical cell-based drug and toxicity screening assays have weak predictive power due to these constraints. Evidence suggests that 3D cell cultures can better imitate the specificity of native tissue with physiological relevance than 2D cultures.

This is especially visible in stem cell differentiation and culture, cancer biology, medication and toxicity screening and tissue engineering. The culture of cellular aggregates in suspension without the use of matrix-based substrata has been used in some of the more basic 3D models. To accurately anticipate tissue development and morphogenesis, in vitro 3D models must imitate elements of in vitro cell behavior. Scaffolds with variable physical and biological features have been developed using a variety of materials and construction techniques to meet the needs of various cell types. For in vitro applications of 3D cell growth, naturally produced ECM-based hydrogels (collagens, elastins, fibronectins and laminins) are the most commonly employed approach.

Companies in bioprinting

Due to its potential commercial value in broad fields such as pharmaceutical discovery (drug toxicity testing), personalized medicine, tissue transplantation and other commercial segments such as cosmetics testing and alternatives to animal-derived food products, 3D bioprinting has transformed from a purely experimental and research segment to an emerging industry, accelerating significantly over the past five years (as 3D printing technologies, in general, became much more broadly available).

According to 3dpbm’s Index, there are currently at least 135 businesses working in the global bioprinting market (without including bioprinting product resellers). Among these companies, 38% are bioprinter manufacturers, 30% are biomaterials suppliers and 44% are bioprinting labs or service providers. Note that companies can belong to multiple categories and that numbers continue to increase as new companies emerge and/or are discovered by the 3dpbm Research team.

In the bioprinter hardware area, one company, BICO (previously Cellink), has been playing a major role in driving the industry’s expansion. The company began by producing and distributing bioinks and then went on to build a line of low-cost extrusion bioprinters. After introducing its first commercial products, Cellink grew at a breakneck pace, creating a community of bioprinting enthusiasts, researchers and professionals at various universities all over the world. In just a few years, the company was listed on the Swedish NASDAQ and opened a branch in the US market. Many companies have since joined the BICO family and its bio-convergence mission since 2016, with Advanced Biomatrix (biomaterials) and Nanoscribe (2PP nano 3D printing hardware) among the most notable. The company now has 14 companies that offer researchers and clinicians technologies, products and services to help them generate, understand and master biology, with a focus on 3D printing but with a wide range of applications.

Many other companies have developed significant businesses based on bioprinting hardware in the past, and many more are doing it presently, including Cellink. Some of the most well-known, long-standing system manufacturers include RegenHU, a Swiss company that was among the first to market high-end bioprinting hardware systems. Another key traditional operator in the hardware market is EnvisionTEC, a leading industrial DLP system manufacturer that was recently acquired by Desktop Metal and rebranded as ETEC/Desktop Health. ETEC’s bioplotter has been used for dozens of published studies. Other relevant names include Advanced Solutions, the company that developed a multi-axis bioprinting robot (the BioAssembly Bot) and relative software and formed a distribution partnership with GE Healthcare. Regenovo is the leading name for bioprinter manufacturing in China, with several machines on the market. Low-cost solutions-propelled companies that target hardware as their core business include Rokit and Allevi, the company that was recently acquired by 3D Systems, as well as a slew of others.

In this context, the renewed interest that 3D Systems, one of the long-standing additive manufacturing market pioneers and leaders, has manifested may offer one of the clearest indications that a solid bioprinting industry and the relative market opportunity are now within reach. The company has targeted lungs as a key organ bioprinting application via a partnership with United Therapeutics and is collaborating with CollPlant on tissue scaffolds for breast reconstruction. It also acquired Allevi to build its commercial bioprinting hardware business and named former Allevi Chief Scientific Officer Taci Pereira General Manager of Bioprinting. In September 2022, 3D Systems created a new wholly-owned subsidiary, Systemic Bio, dedicated to bioprinting for drug discovery and development, and named Taci Pereira as CEO of that business.

Some companies developing bioprinting hardware technologies, such as TandR Biofab, 3Dbio, Poietis and others, are focusing on specific 3D bioprinting applications, such as implantable tissues and grafts. For example, ​​South Korean company T&R Biofab has not only pioneered its own bioprinting technology but is also developing a diverse range of applications for its platform. T&R Biofab—which stands for Tissue Engineering and Regenerative Medicine, Biofabrication—has been behind some truly groundbreaking projects.

Materials for bioprinting

Combinations of polymers, ceramics, cells, cell aggregates, growth factors, hydrogels, scaffold components, and other materials make up bioprinting inks, or bioinks. To ensure cellular viability (i.e. that cells survive the printing process), cellular bioinks must be processed with caution. Bioinks are available in a wide and quickly expanding range of different materials, with many institutions and businesses manufacturing materials in-house to meet their own unique needs. More commercial bioinks are being brought into the global market as the industry evolves and criteria become more standardized. Here you can find an overview of some key bioink companies and materials.

Biomaterials, which are designed for use in bioprinting, include a wide range of hydrogel, metallic, ceramic, polymeric, composite and cellular materials. The best printing method is determined by the physical properties of the materials. Low-viscosity materials, for example, are more appealing for bioprinting because cells thrive in a low-pressure environment. Other material parameters like pore size and interconnectivity can have an impact on the encapsulated cells.

Generally speaking, all consumables used in bioprinters for bioprinting applications are referred to as bioinks. Bioinks are sometimes used as materials that contain specific cells, distinguishing them from pure hydrogels and scaffolding materials. Bioinks are typically polymeric, although they can also be made of ceramics or metals. Bioinks are further classified as sacrificial bioinks, matrix base reagents, matrix ECM GAGs, matrix print enhancers and UV-curable bioinks.

To accommodate the encapsulated cells and, in the case of implantation, the recipient’s own tissues, the scaffold materials must be biocompatible. The implant must be cytocompatible, allowing cells to grow, adhere, proliferate and migrate while remaining safe for the host and causing no significant inflammation or immunologic rejection.

Tissue engineering scaffolds have been made from nearly all cell-free 3D printing materials, including metal, synthetic and natural polymers. To improve the mechanical strength of hard tissue repair replacements, metal and hydroxyapatite powders are typically employed as starting materials.

Hydrogels

Hydrogels are an important tool to grow and maintain cells successfully because they allow cells to grow and interact with all of their surroundings in a 3D environment. Cells grown in 3D models have proven to be more physiologically relevant, with improved cell viability, morphology, proliferation and differentiation. Because they are made up of large three-dimensional networks of polymer chains that retain a bulk of water, hydrogels are ideal materials for bioprinting. Due to their closeness to the original tissue milieu, natural polymers are commonly used in hydrogels as printable materials that enclose and print living cells. Alginate, collagen, gelatin, gelMA, fibrin and hyaluronic acid are common hydrogel materials.

Alginate is an algae-derived polysaccharide (a polymeric carbohydrate molecule). It is made up of two monosaccharides that repeat themselves. Crosslinked alginate is appealing for 3D tissue/organ printing because of its comparable structure to native ECM, great biocompatibility and ease of quick gelation. It’s also adaptable to a wide range of tissue engineering applications. Glycine, proline and hydroxyproline residues are plentiful in collagen. Collagen is the most prevalent protein in many tissues’ extracellular matrix (ECM). It creates a hydrogel under physiological circumstances by forming a triple helix. Because of the presence of cell-interactive RGD (Arginine-Glycine-Aspartic acid), which stimulates cell adhesion, collagen is also regarded as a good material for cell encapsulation.

Denatured collagen is also used to make gelatin. This substance is widely utilized in the food, pharmaceutical and cosmetic industries as a gelling agent. Fibronectin, vimentin, vitronectin and RGD peptides are all common proteins in gelatin that induce cell attachment via integrin receptors.

Due to their acceptable biological features and customizable physical characteristics, gelatin methacryloyl (GelMA), hydrogels have been widely exploited for numerous biomedical purposes. Due to the inclusion of cell-attaching and matrix metalloproteinase-sensitive peptide motifs, GelMA hydrogels closely mirror several important features of the original ECM, allowing cells to proliferate and spread in GelMA-based scaffolds. GelMA is also adaptable in terms of processing. GelMA-based hydrogels are effective in a variety of tissue engineering applications, including bone, cartilage, and cardiac and vascular tissue engineering, to name a few. Aside from tissue engineering, GelMA hydrogels are used in fundamental cell research, cell signaling, medication and gene delivery and bio-sensing.

Hyaluronic acid (HA) is a biological material that is made up of a linear polysaccharide component of the ECM. For 3D tissue/organ printing applications, this material exhibits high biocompatibility, viscoelasticity, hydrophilicity and biodegradability. Chemically conjugating methacrylate groups to generate a gel via free radical polymerization under UV exposure is a frequent modification of HA.

Cells, organoids and spheroids

The printing of tissue and organs relies heavily on cell printing. The strict printing conditions, however, limit the bioink materials available. Furthermore, biomaterial stiffness, functional groups and surface shape have an impact on cellular behavior. For bioprinted ink, cells are frequently enclosed in biodegradable hydrogels that imitate a tissue-like environment. The properties of hydrogels can protect inner cells from the shear stress created during the printing process, allowing them to maintain bio-functions such as stem cell self-renewal and multi-lineage differentiation potency.

Bioprinting processes can use a variety of cell types. The size and morphology of the cell or cell aggregate, as well as its ability to be transmitted through the printing process in a healthy form, are the most common limitations. Temperature, shear stresses, acceleration and deceleration should all be considered from the standpoint of the cell and other fragile components of the bioink.

Cellular spheroids are basic 3D models that may be made from a variety of cell types and form spheroids due to adhering cells’ inherent desire to aggregate. Embryoid bodies, mammospheres, tumor spheroids, hepatospheres and neurospheres are all examples of spheroids.

In vitro, adult tissue stem cells can generate self-organizing 3D organoids. Organoids are self-organizing 3D structures that grow in vitro, embedded in an extracellular matrix (ECM), and resemble their organ of origin, similar to evolved spheroids. They can be made from a range of tissues and cell sources, including primary tissue explants, cell lines, multipotent adult stem cells, pluripotent embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

Bioresorbable polymers

In 3D bioprinting, there are a few different synthetic polymers that are widely employed. Polycaprolactone (PCL), poly(Lactic-co-Glycolide) (PLGA), polyethylene glycol (PEG) and Poloxamer 407 (Pluronic F127) are only a few of them. In an attempt to increase cellular responses, blends of synthetic and natural polymers can be combined

Because of its low melting temperature (59–64°C) and ease of printing processing, PCL is the most extensively utilized in microextrusion procedures. PCL is also non-toxic, biocompatible and has a hydrolysis-induced bulk erosion/biodegradation profile, allowing the structure’s shape to be preserved before disintegration. PCL is a tissue engineering scaffolding material that may be used to 3D print scaffolds for 3D tissue and organs as a supporting framework to ensure shape fidelity in printed cell–laden constructions.

By altering the polymerization ratio between PLA and PGA, PLGA is a biocompatible thermoplastic that allows for controlled degradation. The most common application of PLGA in 3D bioprinting is as a biopaper substrate on which cells can be stacked to build high-resolution 3D tissue structures utilizing laser bioprinting techniques. Stand-alone PLA is one of the most often used materials in low-cost 3D printing, but it also has use in bioprinting as a biodegradable and biocompatible thermopolymer.

PEG (Polyethylene glycol) is a hydrophilic, biocompatible, FDA-approved polymer with a wide range of uses in biomedicine. Because it is water-soluble, PEG plays a role as a representative sacrificial material for manufacturing complicated 3D constructions. Before creating physical or chemical networks with PEG as a bioink, the polymer should be chemically changed. The chemically modified PEG is commonly crosslinked via photoinitiator (PI)-induced polymerization under UV exposure, which is a critical component for gel formation.

Poloxamer 407 is a water-insoluble surfactant that belongs to the poloxamer family of copolymers. Poloxamer 407 is a triblock copolymer made up of a hydrophobic polypropylene glycol block in the middle and two hydrophilic polyethylene glycol blocks on either side. The two PEG blocks are around 101 repeat units long, while the propylene gycol block is approximately 56 repeat units long. Pluronic F127 is the marketing name for the chemical developed by BASF.

Ceramics

Because of their outstanding mechanical properties, osteoconductivity and compatibility with bones, ceramics such as tricalcium phosphate (TCP), HA/hydroxyapatite), ZrO2 (zirconia) and SiO2 (silicate) are commonly utilized in bone tissue engineering. The most often utilized ceramic for bone tissue engineering is hydroxyapatite (HA, not to be confused with hyaluronic acid, which is also HA). HA can be employed in a variety of forms in 3D bioprinting technologies, including powder, slurry and granule. The fluidity required for 3D printing techniques can be achieved by granulating HA or mixing it with other polymer solutions. For the coalescence of powdered HA particles and even the inclusion of cells, a polymer solution is frequently utilized as a liquid binder. Because HA is abundantly present in human teeth and bones, it makes the material and related ceramics appealing materials for building scaffolds with strong mechanical qualities similar to actual bone.

Bioprinting technologies

Material jetting was the basis for the earliest bioprinting technology. This method is related to 3D printing material jetting methods like Stratasys’ PolyJet or 3D Systems’ MultiJet Printing, which are akin to traditional 2D inkjet printing. Early bioprinters were modified 2D inkjet printers that sprayed biological ingredients onto a moving Z-axis platform to create stacked 3D layers. Today most bioprinters on the market are based on extrusion technologies, however the most high-end systems integrate multiple (laser-assisted and laser-free) processes.

Scaffolded and non-scaffolded

Scaffolds provide the mechanical support and physical framework that allow cells to adhere, grow and sustain their physiological capabilities. For cells to adhere, proliferate, differentiate and secrete extracellular matrix, a scaffold must provide good biocompatibility or cytocompatibility. Bioactive compounds abound in the ECM. Traditional fabrication methods for three-dimensional scaffolds, on the other hand, lack fine control over internal structural characteristics and topology. On the other hand, several polymer additive manufacturing technologies, ranging from material extrusion to stereolithography and even selective laser sintering, can be used to create sophisticated interfacial tissue engineering scaffolds.

Scaffolds, however, remain instruments for bioprinting constructions in an indirect manner. As a result, they have geometrical limitations that scaffold-free bioprinting techniques can overcome. If living cells are directly printed onto a substrate, the supporting structure can be generated automatically thanks to natural cell activities. In such cases, scaffold-free bioprinting may be a viable option for generating complicated vascular systems within bioprinted constructs.

Laser-assisted bioprinting

Bioprinting processes, like industrial 3D printing, can be further classified into those that use a laser to initiate a polymerization (hardening) reaction and those that do not (and generally use heat or pressure). These are also known as LAB (laser-aided bioprinting) and LFB (laser-free bioprinting).

Direct laser-assisted bioprinting employs a laser source similar to that used in laser stereolithography (SLA) to direct living cells in droplet form on a substrate to digitally predetermined places. After transferring cells from the ribbon, the receiving substrate contains a biopolymer or cell culture media to maintain cellular adherence and proliferation. Long and direct laser light contact with cells, on the other hand, results in limited cell survival. LIFT (Laser Induced Forward Transfer) and LGDW (Laser Guided Direct Writing) are two LAB approaches, however, stereolithography is the most significant LAB technology for commercial development today. This is the same approach that Charles Hull, the pioneer of 3D Systems, invented in 1986 and commercialized shortly thereafter. SL can be used to 3D print light-sensitive scaffolding materials as well as directly photopolymerize polymers including cellular material in some circumstances.

In bioprinting, stereolithography is divided into various subcategories. Microstereolithography (MSTL) is a technique for fabricating 3D freeform objects at micrometer scales, which uses optical components to shrink the diameter of the laser beam. Another method is projection-based micro stereolithography (pMSTL), which uses DLP 3D printing technology to fabricate microstructures. Polymers, responsive hydrogels, shape memory polymers and biomaterials are examples of materials used in this process. Two-Photon Polymerization (2PP) is a laser-based 3D printing process that employs two-photon absorption (2PA) and a laser to start a chemical reaction that induces the polymerization of a photosensitive material, similar to stereolithography but much more detailed (to nanometric scale). It is also utilized for bioprinting applications. Of all the 3D printing techniques, 2PP has the highest resolution. Researchers have been able to build 3D habitats for cell adhesion and proliferation using it.

Volumetric bioprinting

The scalability of traditional bioprinting and additive manufacturing technologies is limited by their printing velocity, as lengthy biofabrication processes impair cell functionality. Volumetric bioprinting overcomes such limitations by bioprinting clinically relevant sized, anatomically shaped constructs, in a time frame ranging from seconds to tens of seconds.

In 2019, a team of researchers from the Utrecht Medical Center and the École Polytechnique Fédéral Lausanne (EPFL) demonstrated the bioprinting of large living tissue constructs by processing cell-friendly hydrogel-based bioresins with a volumetric, visible light laser-based printer. The scientists developed a custom bioprinter specifically for this project with the goal of establishing a spinoff company exclusively dedicated to commercializing these applications. The authors of the study included bioprinting pioneers such as Professors Jos Malda and Riccardo Levato from the Utrecht Medical Center.

Whereas all current bioprinting relies on the layer-by-layer deposition and assembly of repetitive building blocks, (typically cell-laden hydrogel fibers or voxels, single cells, or cellular aggregates), the volumetric method described leverages an optical-tomography-inspired printing approach, based on visible light projection, to generate cell-laden tissue constructs with high viability from gelatin-based photoresponsive hydrogels. This method enables the creation of free-form architectures, difficult to reproduce with conventional printing. These include anatomically correct trabecular bone models with embedded angiogenic sprouts and meniscal grafts. In 2022, the same group was able to achieve ultra-fast (<20 seconds) volumetric bioprinting of large scale (>1 cm3) engineered liver units, that are functional and able to perform key toxin elimination processes that natural livers perform in our body. The ability to bioprint such large functional units of the liver will open new opportunities for regenerative medicine and drug development testing (DDT).

Laser-free bioprinting

Material jetting (inkjet or MJ) and material extrusion (MEX) 3D printing are two families of technologies in Laser Free Bioprinting (LFB) that are traceable to industrial AM processes. The fundamental difference between these two techniques is that in material jetting, the print head contains several microscopic nozzles, whereas in extrusion 3D printing, each material is extruded and deposited by only one nozzle (or at most two or three). Material jetting bioprinters, like material jetting 3D printers for industrial manufacturing, are based on inkjet desktop printers. Micrometer-sized orifices and a print head that can be operated by thermal, piezoelectric or solenoid valves are used in 3D inkjet printers. The bioink is forced through the opening that leads to the printer head by a pressure pulse generated in the tank.

Inkjet, acoustic-droplet-ejection and micro-valve bioprinting are the three different technologies used in droplet-based bioprinting. Sonic bioprinting creates droplets using acoustic waves. A solenoid pump is used to eject droplets in micro-valve bioprinting. Continuous InkJet (CIJ), Drop-On-Demand (DoD) and ElectroHydroDynamic (EHD) jetting are the three sub-families of inkjet bioprinting. Drop-on-demand bioprinting uses heat or piezoelectric actuators (or electrostatic forces) to generate the droplets, whereas drop-on-demand bioprinting does not. Electrohydrodynamic jet (EHD) bioprinting, on the other hand, uses high-voltage electricity.

Material extrusion, also known as fused deposition modeling (FDM) or fused filament fabrication (FFF), is a method of laying down stacked layers of material by forcing a viscous liquid or molten material through a nozzle. A thermoplastic polymer filament, such as polylactic acid (PLA) or thermosets, cell suspensions and UV curing photopolymers can be extruded.

Instead of droplets, as with DoD methods, the printer creates a continuous stream that is placed on the substrate. The pressure extrusion of liquids, pastes or dispersions is used in pressure-assisted bioprinting. Extrusion bioprinters can create parts utilizing materials with a wide range of viscosities employing piston, pneumatic or screw-based methods. Extrusion methods are slower in general, but they can provide high cell survival rates, making them excellent for hard tissue engineering.

Extrusion bioprinting systems can contain multiple printheads to extrude different materials, such as scaffolding and cellular materials (for example, the MHDS or Multi Head Deposition System developed by Postech University researchers). Dr. Atala’s team at the Wake Forest Institute for Regenerative Medicine (WFIRM) presented an integrated multi-head tissue-organ printer (ITOP) in 2016, which was designed to produce stable, human-scale tissue constructs of any shape.

Other methods of bioprinting

Electrospinning is a versatile 3D printing technology that involves ejecting an electrically charged viscoelastic polymer solution onto a collector in order to create fibers. A strong electric field generated by a high voltage between a polymer solution output and the collector guides the charged polymer solution’s travel path. This method can create ultrafine fibers with dimensions ranging from a few micrometers to a few nanometers.

Magnetic 3D bioprinting is a technique for assembling cells into 3D structures or cultures using biocompatible magnetic nanoparticles. Nano3D (n3D) Biosciences (now owned by Greiner-One Bio) developed a nanoshuttle, which is a magnetic nanoparticle assembly made up of gold, magnetic iron oxide and poly-L-lysine that aids in adherence to the cell membrane through electrostatic interactions. External magnetic forces can be used to arrange the cells tagged with the magnetic nanoshuttle into particular 3D patterns that imitate tissue structure and function. When compared to extrusion techniques, the magnetic approach is substantially faster.

Prof. Koich Nakayama of Saga University devised the Kenzan technique, and Japan-based Biomedical K.K. was granted exclusive rights to utilize it on its Regenova bioprinter. In this approach, spheroids, or cellular aggregates, with several tens of thousands of cells per spheroid, are cultivated. Then, without any additional support material, spheroids are inserted directly in thin needle arrays and allowed to combine with adjacent spheroids to form a linked structure. The cellular spheroids can be positioned in any desired three-dimensional configuration with proper alignment. Finally, growing linked cellular spheroids in a bioreactor encourages cell self-organization and produces a 3D tissue with the appropriate function and quality.

Applications of bioprinting

Organ and tissue loss or failure is a tough and costly problem in healthcare. This also means that bioprinting’s potential to generate functional organs for implantation is the single most important opportunity for additive manufacturing’s long-term future. In fact, analysts rarely consider bioprinting technology when projecting future revenues from AM, despite the fact that it has the potential to cover as much as 2% of the whole manufacturing business within the next two decades. This is also because commercial organ production applications are far beyond the realm of any practical analysis. Nonetheless, bioprinting applications in tissue regeneration that do not revolve around fully functional complex organs have a sizable market.

The scarcity of organs around the world, on the other hand, is a significant motivator for tissue engineering research, particularly the design of a cell-scaffold-microenvironment to stimulate the regeneration of many types of tissue, including skin, cartilage, bone, tendon and cardiac tissue.

Skin & bones

In tissue engineering bioprinting allows for the creation of constructs with greater resolution and complexity than is possible with traditional lab approaches.  Bioprinting has become a common method for fabricating cartilage tissue engineering scaffolds from a wide range of materials, including ceramics and nanomaterials.

Cartilage

Cartilage is a flexible connective tissue that is critical for elasticity and smooth motion in everyday human activities. It is composed of cells called chondrocytes, surrounded by a gel-like matrix made up of proteins and carbohydrates. Unlike bone, cartilage does not have a blood supply and therefore does not have the ability to repair itself quickly. This characteristic made it an ideal candidate for early bioprinting experimental applications since it would not complex require capillarization.

In the process to induce chondrogenesis (how cartilage forms), cartilage regeneration in tissue/organ printing procedures include appropriate cell sources (mesenchymal stem cells or MSCs, adipose-derived stromal stem cells or ASCs and chondrocytes), hydrogels (collagen type I and II, gelatin, hyaluronic acid, alginate) and growth factors (GFs).

The rebuilding or regeneration of neocartilage tissue using 3D bioprinting techniques has received a lot of attention but has so far not produced viable commercial applications implantable in humans. Bioprinting of cartilage in intervertebral discs, menisci and knees remains largely confined to academic research with no significant commercial applications currently in line to be approved for use in humans. Some success has been obtained in terms of implementing scaffolds to support tracheal cartilage regrowth using polymer (PCL) 3D printed structures.

Skin

Skin, the body’s largest organ, exhibits a complex structure consisting of three predominant layers (epidermis, dermis and hypodermis). Engineering multi-layer skin architecture that conforms to the native skin structure is a difficult, if not unattainable, goal to achieve with present tissue engineering methods, as is restoring all of the native skin’s functions.

In recent years, there have been significant advancements in skin bioprinting field, leading to the development of more complex and sophisticated skin tissue models. Researchers have successfully bioprinted multi-layer skin constructs that mimic the structure and composition of native skin, complete with epidermis, dermis, and subcutaneous layers. This has opened up new avenues for studying skin diseases and testing new treatments. Furthermore, skin bioprinting has also been used in the development of skin substitutes for burn victims and other patients with skin injuries. These bioprinted skin constructs have shown promising results in early clinical trials, offering a potential solution for the shortage of donor skin and the associated ethical concerns. Overall, skin bioprinting is a rapidly evolving field that holds great promise for advancing our understanding of skin biology and improving patient care.

Although a number of skin replacements exist, there have been no solutions that recapitulate the chemical, mechanical and biological roles that exist within native skin. Just recently, a team of researchers from the University of Birmingham used a method called suspended layer additive manufacturing (SLAM) to produce a continuous tri-layered implant, which closely resembles human skin. Through careful control of the bioink composition, gradients (chemical and cellular) were formed throughout the printed construct. Culture of the model demonstrated that over 21 days, the cellular components played a key role in remodeling the supporting matrix into architectures comparable with those of healthy skin. The researchers believe that these implants can facilitate healing, commencing from the fascia, up toward the skin surface—a mechanism recently shown to be key within deep wounds.

Bones

Because the nature of hard tissues is simple and primarily formed of inorganic materials, bone regeneration, along with cartilage regeneration, is the most established field utilizing printing technology. Many manufacturing processes have been used to manufacture a range of biomaterials for the construction of bone scaffolds; however, 3D bioprinting allows for more precise control of the structural and mechanical features of artificial scaffolds than other technologies. In the clinic, innovative, stable and resorbable hard tissue and organ repair materials generated with 3D bioprinting technology are needed.

Ceramics such as tricalcium phosphate (TCP) and hydroxyapatite (HA) are commonly utilized in bone tissue engineering. They are biocompatible materials similar in composition to natural bone. In 3D printing, hydroxyapatite is usually combined with a monomeric binding agent and then cured layer by layer to build up the final shape of the implant. This process allows for the creation of implants that are tailored to the specific needs of each patient, providing a better fit and increased comfort compared to traditional implant manufacturing methods.

It’s worth noting, however, that 3D printing of hydroxyapatite for bone implants is still a relatively new technology, and its use in actual cases is far from standardized. Nevertheless, the potential benefits of 3D printing in this area are significant, and the technology is expected to play an increasingly important role in the development of customized bone implants in the future. Possible applications include tibial osteotomy wedges, intervertebral cages, cranial implants, general bone substitute, general spinal implants and general orthopedic implants.

Cosmetic testing

The testing of pharmaceuticals and cosmetics is another area where modified skins are desperately needed, especially since animal testing is no longer permitted or about to be outlawed in many countries. Given this increased need, 3D bioprinting is a potential method for producing biomimetic cellular skin substitutes quickly and reliably, meeting both clinical and industrial needs.

Cosmetics firms are extremely interested in today’s advanced 3D printing and bioprinting applications, such as 3D printed tissue and even hair follicles, especially in Europe, where animal testing for cosmetics was outlawed in 2013.

Cosmetics giant L’Oréal and Poietis, a French biotech startup, signed an exclusive research partnership to bioprint follicles capable of sprouting hair. Not only may this lead to more effective hair product testing, but it could also increase our understanding of how hair works, paving the way for potential biologic remedies for adult hair loss.

Last year the EpiDerm Phototoxicity Test was accepted as part of Test Guideline No. 498 In vitro Phototoxicity: Reconstructed Human Epidermis Phototoxicity Test Method by the Organization of Economic Cooperation and Development (OECD). EpiDerm is produced by MatTek Life Sciences, a subsidiary of CELLINK. This effort is in line with the Group’s goals to reduce and eventually replace animal testing by developing technologies and methods that are more human-relevant and provide a more accurate prediction of human clinical responses. This was the fourth OECD test guideline validation for MatTek’s in vitro tissue models following validations for Skin Irritation (OECD TG 439), Skin Corrosion (OECD TG 431) and Eye Irritation (OECD TG 492).

Agriculture on a cellular level

Cellular agriculture, an interdisciplinary area of study at the junction of health and farming, could gain considerably from 3D printing’s ability to add complex shapes to lab-grown meats and dairy products, despite its current nexus with 3D bioprinting technology. Cellular agriculture companies intend to advances in tissue engineering, material sciences, bioengineering and synthetic biology to create new ways to make existing agricultural goods such as milk, meat, and perfumes (and even rhino horn) from cells and microbes.

Professor Mark Post’s cultured burger from 2013, which established a proof of concept for cultured meat, is the first example of a cellular agriculture product. The cost of generating an edible lab-grown burger-size product has been steadily falling, from several hundred thousand dollars to a few hundred and even less, however productivity remains low and far from being able to address mass market demand.

Dozens of companies around the world are working to introduce cellular agriculture meat-substitute (or alt-meat) products at some level. Not all of them are producing these foods using 3D printing technology and not all of them are using actual cells (in some cases the proteins are obtained from vegetable sources). The ones that do use 3D printing are mostly based in Israel: Aleph Farms, Savor Eat, Meat-Tech and Redfine Meat are all implementing bioprinting-related extrusion processes and workflows to reduce cost and accelerate production of their alt-meat products.

*This article was originally published in 3dpbm’s Medical/Bioprinting AM Focus 2022. It has been updated to reflect the latest developments.

**Some of the article’s generic content has been optimized using the ChatGPT AI

***The author of this article owns stocks in some of the companies mentioned