Three-dimensional bioprinting in medical surgery

Introduction: 3D bioprinting technology in a few words and its evolution through the years

The need to overcome the restrictions of transplantation process such as the limited number of biocompatible donated organs, the increasing demand for organs, the transplants rejection, and/or the overall difficulties after transplantation has led the research to a new field knowing as tissue engineering and regenerative medicine. Currently, the new applications adjusted in this field include three-dimensional (3D) bioprinting technology. Bioprinted tissues and organs will be greatly expected to eliminate this increasing organ shortage crisis that predominates.

The bioprinting technology was initially introduced as a method for fabricating tissue scaffolds and it has been applied for engineering a variety of tissue constructs such as the bone, skin, and cartilage. In conventional tissue fabrication, cells are seeded onto the scaffold, which prοvides a microenvironment mimicking the properties of the real extracellular matrix (ECM), after the printing process. The emerging difficulty with this method is the inability to position different cell types in appropriate locations within the scaffold and control the cell migration. These parameters are crucial for the generation of complex tissues and/or organs in order to satisfy the transplantation requirements.

The limitations of scaffold-based tissue engineering were overcome with the evolution of 3D bioprinting, where living cells are directly disposed mixed with biomaterials in order to develop complex structures tending to mimic native tissues and/or organs. 3D bioprinting is defined as the precisely deposition of biocompatible materials and growth factors along with living cells layer-by-layer using the traditional 3D printing technology, which is mentioned as cell-laden printing. It is well established that each human tissue is consisted of multiple and different cell types organized in an accurate and complex architecture. The 3D bioprinting methods allow for the organization of multiple cell types in predesigned positions with the contribution of computer-aided design (CAD) in a totally controlled way in order to generate 3D structures that maximally imitate the native tissue/organ characteristics. ^,

The trigger which gave birth to 3D printing technology was when Charles Hull invented a 3D printing method based on stereolithography (SLA) apparatus. This major breakthrough along with the introduction of tissue engineering laid the foundations of bioprinting, setting the goal to combine cells, and biomaterials for the fabrication of tissue analogues of custom-tailored shapes and sizes. A great number of studies and observations have been conducted through the years making 3D bioprinting a very promising and challenging tool for the development of organ-on-a-chip drug screening systems and fully functional tissues and organs. ^,

3D bioprinting methodology has already been applied for scaffold fabrication meeting the needs of tissue engineering. Regarding the use of living cells in the fabrication of complex tissue constructs, by the late 1990s, scientists mentioned the development of 2D bioprinting systems combined with living cells by the use of micropositioning methods. In 2004, a scientific team with Roth at the head achieved to print cells employing an inkjet printer filled with collagen. Taking this great progress into account, Boland and his colleagues focused their research on the printability of mammalian ovary cells and as a result the first patent for inkjet bioprinting of viable cells is a reality. ^,

Mironov and his scientific team developed tube-shaped tissues (e.g., blood vessels) through printing layers of cell aggregates and gel alternatively. One year later, Mironov, Forgacs, and their colleagues brought to the forefront and applied for a bioprinting-related patent that includes self-assembling cell aggregates and modeling methods in order to produce an engineered tissue with the desired 3D structure. Inspired by this work, one of the pioneers of bioprinting, the research company Organovo, was founded in 2005, and in 2009, the first world's commercial 3D bioprinter, NovoGen MMX (Organovo, United States), was available. Several studies have been conducted resulting in the use of multicellular spheroids, the scaffold-free printing of blood vessels, and the commercialization of printed liver cells. ^, Common goal until nowadays is the evolution of 3D bioprinting process from a research tool to an efficient process for the fabrication of viable and functional tissues and organs. However, despite the significant progress of the researchers worldwide, there are few reports concerning the clinical applications of bioprinted constructs.

In general, 3D bioprinting process consists of three stages: prebioprinting, bioprinting, and postbioprinting. ^, ^, Major components that are crucial for the execution of the bioprinting process are the suitable bioinks, the appropriate bioprinting process (modality), and a functional 3D bioprinter.

In this chapter, the latest advances in the use of the 3D bioprinitng will be discussed, including the processing, the used bioprinting modalities, the criteria of the most convenient bioink's selection, and the recent clinical applications.

Classification of 3D bioprinting methods

The bioprinting could be divided into inkjet-based bioprinting, extrusion-based bioprinting, laser direct-write bioprinting, photocuring-based bioprinting, and cell ball assembling bioprinting. Among them, the inkjet-based bioprinting, the extrusion-based bioprinting, and the laser direct-writing bioprinting are the most widely used. Each method influences positively or negatively the cell viability and integrity during the process. In the sections below, the characteristics of the existing bioprinting methods are cited.

Inkjet-based bioprinting

In order to form the desired 3D structure, the inkjet-based bioprinting method utilizes bioinks, instead of ink, deposited repeatedly in the form of droplets at predesigned locations in a moving stage. ^, The desired 3D structure is printed using the conventional layer-by-layer approach as a series of consistent and fusing together droplets using the thermal heater method or the piezoelectric actuator method.

Inkjet-based bioprinting technology was introduced in 2003 and constitutes the first bioprinting attempt, which is mainly used for printing small scaffolds. Since then, Ciu, Boland, and their colleagues printed hamster ovary cells in 2005, and a few years later, in 2009, it was Boland and Cui who constructed a structure that resembles the blood vessels using human microvascular endothelial cells. The wide use of this bioprinting modality emerged from the fact that it is an inexpensive, non-time-consuming method and the printing properties (e.g., printing speed, resolution, size, position of droplets) can be easily electronically controlled.

The inkjet bioprinter can be used for printing different types of cells that allows the cell-cell interfaces printing, as well as for heterocellular tissue engineering ^, and for the creation of complex structures. ^,

On the other hand, there are some limitations that restrict its use. The inkjet bioprinters use bioinks of low viscocity due to the small squeezing force of the nozzle, resulting in a limited capability of processing high-density cells. The preference of low viscous bioinks creates structures of weak strength and limited ability for perfusion and/or implantation. In addition, denaturalization of the biomaterials, cell lysis, and damage in cell viability is possible to occur probably due to thermal or mechanical properties of the inkjet bioprinter (for example, the applied voltage in piezoelectric bioprinter is about 12–25 Hz). Finally, it is very crucial for the ink droplets to be consistent and fused immediately with each other in order to avoid deformation and mechanical instability of the constructs.

Extrusion-based bioprinting

The extrusion-based bioprinting method is considered to be the most widely used method of bioprinting. In this technique, the bioink is extruded out of a nozzle driven by pneumatic pressure ^, ^, or mechanical force via screws and pistons, in a totally controlled manner. The extrusion process produces filaments, instead of the droplets in the inkjet-based bioprinting method, resulting in the formation of a 2D model that under the appropriate processing produces the desired 3D structure. The first attempt for extrusion printing was performed in 2003 and since then plenty of reports mentioned the method in a variety of tissue applications. The bioinks used for extrusion bioprinting have high density, which permits the printing of cell-laden patterns with enhanced ability for perfusion and/or implantation. Moreover, the bioinks should have fluid-like capacities in order to be extruded through the nozzle tip. In comparison with inkjetting, the choice of biomaterials is versatile. More specifically, viscous polymers, high cell density cell-encapsulated hydrogels, and thermoplastic biomaterials can be printed. However, it is of notice that the resolution of this technique is low compared with the inkjet- and the laser-based bioprinting, the nozzle becomes often clogged and the cell viability could be damaged in case of increased extrusion pressure. It would be a very interesting challenge to find a balance between printing cells at high viscocity while protecting the cellular function and viability during extrusion. ^, It is worthy to report that it is simple and easily accessible method. An extrusion-based system can be developed in an easy way and with low cost by the suitable processing of a commercialized plotter or a desktop 3D printer.

Laser direct-write bioprinting

The laser direct-write bioprinting was first introduced in 1999, when Odde and his colleagues have used laser for cell patterning, and 1 year later, the same team reported direct writing of living cells with the use of laser as well. Since then, plenty of reports have been followed regarding the development of the laser-assisted cell printing method. This method is based on the principles of laser-induced forward transfer to transport droplets and resembles the conventional typewriters. The main components of such methods include the use of a donor slide (or ribbon), a laser pulse, and a receiver slide. The top layer of the ribbon is a transparent glass coated with a thin layer of metal so as to absorb the energy of the laser protecting the cells from direct contact. The underlayer contains the bioink. Initially, a laser pulse is applied onto the top layer resulting in the formation of microbubbles, from the glass substrate in the underlayer containing the printable biomaterial. Subsequently, the absorbed laser energy forces the mixture of cells and cell encapsulation (a kind of hydrogel) to be vaporized forming droplets of the materials containing the cells. Then, the droplets are transported to the receiver slide and the 3D object can finally be printed.

The laser-assisted bioprinting can be regarded as a nozzle-free inkjet-based bioprinting method. As a result, the problem of nozzle clogging no longer exists and the ability to position precise patterns for a variety of cells types and biomaterials is enhanced. The laser bioprinting permits the printing of bioinks with high density and in high resolution. The printing resolution is depended on the laser's energy and pulse frequency, the speed of the laser, the thickness of the layers, the distance between the donor and the receiver slide, and the bioink's viscosity as well. ^, Moreover, it permits a single-cell manipulation and the accurate control of the cell droplets positioning.

However, there are some parameters that restrict its use including the low cell viability in the printed tissue constructs as a consequence of the laser's energy, the fact that it is an expensive method, ^, the fact that it is time consuming to spread the mixture of cells in each printing layer, and that there are no available commercialized laser-based bioprinters. It is very challenging for the applications of this method to be combined with a well-designed bioreactor or other biofabrication systems. ^,

Photocuring-based bioprinting

Photocuring-based bioprinting is a method that mimics the conventional printing of tissue engineering scaffolds where the cells are seeded onto the scaffold after the printing. In photocuring boiprinting system, the use of UV light turns light-sensitive materials to be solidified layer by layer. One layer is solidified at a time regardless of the complexity of the layer's structure. The presence of the UV light restricts the use of this technique in printing cell-laden structures due to the damage that provokes in the cells. A lot of studies have been conducted in order to achieve simultaneous printing of cells and scaffolds by using less harmful light. Examining the viability of these cells, the results were very encouraging. ^, There are two different technologies of photocuring-based bioprinting resulted from the way of scanning: the SLA technology, where the bioink is cured point by point, and the digital light projection technology, where the bioink is cured plane by plane. Many types of cells have been reported to be seeded onto the printed scaffolds and the HUVECs and HepG2 are indicative. Apart from the fact that generally this type of printing is time saving, convenient, and of high resolution, the choice of the materials and the selection of the light source should be very careful in order to avoid the cell damage.

Cell ball assembling bioprinting

A new approach has emerged in the field of tissue engineering called modular tissue engineering. Modular tissue engineering aims to recreate biomimetic macroscale structures by designing microscale structural features in order to fabricate modular tissues to use them as building blocks in order to fabricate larger tissues. ^, Between these building blocks, cell balls are used in bioprinting by a self-fusion process. ^, ^, ^, The cell ball assembling bioprinting is based on the creation of 3D structures by self-assembly and self-fusion process. More specifically, 3D constructs are manufactured by fusion of smaller discrete units. The basic blocking units involve microparticles, microfilaments, and planes. Each unit is glued together in order to fabricate a realistic 3D structure which can be achieved with different methods in inkjet-based, laser direct-writing, extrusion-based, photocuring-based bioprinting techniques. The cell ball assembling bioprinting is a scaffold-free method, which means that the fabrication of a 3D structure is based on the in vitro fusion of two adjacent cell aggregates.

Bioinks in 3D bioprinting

One of the crucial components of 3D bioprinting technology is the bioink that is used for the printing process. This bioprintable material is consisted of various biological factors such as cells, media, genes, growth factors, proteins, polymers, hydrogels, etc.

The bioinks used in 3D bioprinting for the development of cell-laden structures are divided into two categories depending on the use or not of a scaffold: the cell scaffold-based approach and the scaffold-free cell-based approach. In the cell scaffold-based approach, the bioink consists of biomaterials and living cells. The cells are encapsulated in the biomaterial scaffold which will be biodegraded forcing the cells to proliferation, growth, and conquest of the space in order to form the desired 3D structure. On the other hand, in the scaffold-free cell-based approach, the cells (cell aggregates—spheroids), in the absence of scaffold, are directly printed resembling the real embryonic development. Specific cell types are printed at a time to form the primary tissues and finally these tissues form larger and more complex tissue structures.

There are many preconditions that the bioink should fulfill in order to be selected. The bioink's selection is very crucial because the success of the bioprinting process is depended on these preconditions. The properties an ideal bioink should have are determined by the characteristics of the available bioprinters as well as by their biological status. The bionk's characteristics should be also adjustable to the requirements of the desired tissue and organ and modifiable in order to achieve efficiently the regeneration of the corresponding tissues and organs. Among others, some of the most important bioinks' properties include biocompatibility, biodegradability, and printability. In the section below, we will mention the overall requirements for the selection of the appropriate bioink.

If the preferable method is the cell scaffold, there are criteria for the selection of the bioink formulation concerning the nature of the biomaterial's properties (such as printability, biodegradability, mechanical properties) and its biological status (biocompatibility, cytocompatibility) as well.

The printability of the biomaterial refers to its ability to be disposed in a totally controlled way and subsequently form a designed structure via a fusion process. It is depended on the rheological properties of the bioink such as viscosity, gelation and shear thinning properties, yield stress, and shear recovery. These properties should be optimized in order to ensure the effectiveness of printability and the mechanical strength. Before printing, the biomaterial should be in liquid state while it is placed on the basic blocking units (microparticles, microfilaments, planes). A very crucial feature of an appropriate biomaterial is its viscosity and the capacity to be tunable so as to be used by the different commercially available bioprinters and to be adjusted in the different printing conditions (e.g., temperature, shear thinning). It is considered that bioinks with higher viscosity form more stable 3D constructs when compared with lower viscous bioinks. However, the balance between the high viscosity and the printing speed requires attention so as to yield highly stable structures because the pressure applied may affect the cell viability. After printing, the printed units must glue each other through a sol-gel process, which imposes the material to be solidified/gelatinized immediately after printing in order to maintain the shape and ensure the fusion. After that, the materials are stacked together through a layer-by-layer process deposition, and it is important for them to be stacked continuously in the vertical direction to keep the predesigned architecture. Apart from the printability, there are also other properties for the selection of the most suitable bioink. Mechanical properties of the material, such as high mechanical strength, shear thinning, stiffness, and elasticity, play very important role in the integrity, the cell survival, and the maintenance of the 3D shape of the printed structure (so as the internal design of the structure does not collapse during the layer-by-layer deposition).

The mechanical strength of the printed object should mimic the properties of the native tissue in order to ensure the cell growth, proliferation, and tissue maturation. ^, Furthermore, the structure should be stabilized as soon as possible after printing within a postprinting maturation process. For example, the high viscous bioinks used in extrusion bioprinting need to have shear thinning in order to compensate the high shear stress applied during printing procedure. The major challenge for the candidate biomaterial is the ability to provide the environment which permits the cells to achieve their cellular function, retain their potency, and ensure their bioactivity after printing. The properties of this artificial environment should be indicative of cell survival just like the native cellular environment does. The candidate biomaterial should be also biodegradable, biocompatible, cytocompatible, and immunocompatible as well. The biomaterial scaffold ideally provides the environment for the cellular function. Once the cells grow and proliferate, the scaffold will be degraded, with the cells conquesting the space and producing their own ECM. It is of notice that the biomaterial and subsequently the biodegradation products should not be toxic or generally harmful for the cells disposed within the biomaterial and for the other neighboring tissues/cells. In addition, it is prohibitive to create any immunological response to the host in case of in vivo implantation. In order to be biocompatible and cytocompatible, the bioink material should also be favorable for cell adhesion and modification of the functional groups on their surface to include and deliver different biochemical signals or biomolecules. Another desirable aspect for a bioink is the capacity to create the conditions for the permeability of oxygen, nutrients, growth factors, and metabolic waste, in order to enhance cell growth and metabolic activity. ^,

The bioinks used so far and their properties

As mentioned in the section above, the suitable bioink should satisfy specific preconditions that are also depended on the requirements of the desired tissues and organs as well. The widely used bioinks include tissue spheroids, cell pellets, tissue strands, and biomaterial scaffolds with encapsulated cells. The scaffolds include hydrogels, microcarriers (MCs) and decellularized ECM.

The different human tissues have diverse properties and they are composed of different types of cells that achieve specific cellular function. In 3D bioprinting, different types of cells can be used according to the demands of the corresponding tissue. These cell's types include already differentiated adult and progenitor cells and stem cells as well. Induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), and extraembryonic cells have the ability to be bioprinted for developing a functional tissue and/or organ.

There is a wide variety of biomaterials that can be used. There are two categories of biomaterials used in 3D bioprinting. The first category involves the natural and the second one the synthetic polymers. Natural biomaterials are subdivided according to their origin in these coming from proteins (silk, collagen, fibrin, gelatin) and those based on polysaccharides (hyaluronan, alginate, agarose, chitosan). Among them, the hydrogels are the most prominent mainly due to their unique properties, which satisfy the bioink's requirements (high water content, biodegradable, ideal for cell survival, adjustable mechanical and modifiable chemical properties, able to yield high resolution during printing). In addition, the fact that they mimic the properties of living tissues make them suitable in the field of tissue engineering and regenerative medicine.

Even though an ideal bioink should not need any physical, chemical, or photocross-linking modifications to become cell-friendly, natural and synthetic hydrophilic materials can be physically or chemically crosslinked to form hydrogels. Even though, natural biomaterials are more preferable due to their cytocompatibility, their weak structure and the fact that they are degraded in a fast pace make the cross-linking necessary to enhance their mechanical strength. Physical, chemical, or covalent and enzymatic cross-linking are the main mechanisms of hydrogel gelation. Physical cross-linking (e.g., ionic) is performed during bioprinting and results in ignorable viscosity fluctuations but contributes in the fabrication of relatively weaker structures and cross-linking treatment can be reversible. Chemical cross-linking needs considerable time of gelation that restricts the formation of multilayered structures. Furthermore, it creates toxic crosslinkers that must be removed completely before the implantation. ^, ^, Alternatively, photocross-linking (e.g., under light irradiation) is a way for instantaneous cross-linking, although the effect of the light and the time of exposure on cells need further consideration. Even though enzymatic cross-linking is more preferable biologically, it is expensive and has a negative effect on the practicality of the process. However, the cross-linking mechanism is possible to affect negatively the cellular compatibility and the homogeneity of the biomaterial. ^,

The bioinks also should be affordable and abundant. Unfortunately, cell-laden bioinks as well as some natural hydrogels such as collagen and hyaluronan acid are relatively expensive. In general, printable biomaterials are limited with the synthetic polymers to be more available in comparison with natural polymers.

Synthetic polymers

The high mechanical strength, the ability of the mechanical properties to be totally controlled, the tunable chemical properties (response to PH and temperature, adjust their molecular weight and functional groups) and their printability are indicative advantages that support their use over the natural materials. ^, However, the disadvantage that limits their use is the inability to ensure the cellular proliferation/differentiation and promote the interactions between cells. Among synthetic polymers, Pluronic and poly(ethylene glycol) (PEG) are the most preferable.

Pluronics, or poloxamers, are a class of synthetic block copolymers that consist of hydrophilic poly(ethylene oxide) and hydrophobic poly(propylene oxide), arranged in an A-B-A triblock structure. The amphiphilic property enables this group to interact with hydrophobic surfaces and biological membranes. The characteristic that allows its use in 3D bioprinting is the capacity to form self-assembling gels at room temperature and flow at 10°C. There are two reports that mention the use of Pluronic for the fabrication of 3D structures. Wu and his colleagues printed microchannel with Pluronic in photopolymerizable hydrogel matrix in order to develop biomimetic microvascular structures. Müller and his team, trying to develop more stable constructs, used acrylated Pluronic for the development of UV crosslinked constructions.

PEG, which is a water-soluble polymer, constitutes one of the most widely used synthetic materials in 3D bioprinting technology. It is suitable for cell encapsulation and it can become more compatible by been functionalized with cell adhesion motifs. ^, It can be modified with acrylate groups in order to achieve satisfying printability. ^, For example, in extrusion-based bioprinting, the PEG-diacrylate and PEG-methacrylate polymers are usually used. ^, ^,

PEG can be combined with a variety of other materials to achieve increased mechanical properties in 3D printing constructs. The application of PEG is limited by not being biodegradable. However, there are synthetic biomaterials suitable for bioprinting applications such as poly(lactic acid), poly(lactic-co-glycolic acid), and poly(ε-caprolactone) (PCL). Among them, PCL is more applicable due to its low melting temperature (60°C), whereas the use of polylactone-based polymers is restricted because of the release of acidic products during degradation, which are inflammatory for the tissues. Recently, it was developed a photopolymerizable, thermosensitive, biodegradable biomaterial consisted of poly(N-(2-hydroxy-propyl)methacrylamide lactate) and PEG.

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here