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.
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.
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.
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.
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.
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
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.
3D biopriting solutions
Allevi3D (3D Systems)
ETEC (Desktop Health)
Dr Invivo 4D
TandR Biofab bioprinter
Materials for 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.
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).
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.
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.
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
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 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.
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.
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 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.
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.
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.
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.
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.
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).
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.
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.
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.
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
From left to right, the Manufacturer, Developer and Starter series of the 3D Bioplotter
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.