3D Printing and Biofabrication

Additive manufacturing is becoming a focus of attention owing to its unique abilities to fabricate different objects using various materials. Perhaps printing technologies are the most popular type of additive manufacturing that is gaining ground in a wide range of industrial and academic utilization. Three- and two-dimensional printing of different materials such as ceramics, plastics, and metals as well as electronic functional materials is considered as the next revolution in science and technology. Importantly, these technologies are being used extensively in medical applications. Tissue engineering, which aims to fabricate human tissues and organs, is benefiting from the reproducible, computer-controlled, and precise procedure that can be obtained by printers. Three-dimensional printings of scaffolds, cell-laden biomaterials, and cellular (scaffold-free) materials hold a great promise to advance the tissue engineering field toward the fabrication of functional tissues and organs. Here, we review the utilization of different printing technologies for various tissue engineering applications. The application of printers in tissue engineering of bones, cartilages, and tendons and ligaments is di. Moreover, an overview of the advancements in printing skeletal muscles as well as the cardiovascular system is given. Finally, future directions and challenges will be described.

At the increasing pace with which additive manufacturing technologies are advancing, it is possible nowadays to fabricate a variety of three-dimensional (3D) scaffolds with controlled structural and architectural properties. Examples span from metal cellular solids, which find application as prosthetic devices, to bioprinted constructs holding the promise to regenerate tissues and organs. These 3D porous constructs can display a variety of physicochemical and mechanical properties depending on the used material and on the design of the pore network to be created. To determine how these properties change with changing the scaffold’s design criteria, a plethora of characterization methods are applied in the biofabrication field. In this chapter, we review the most common techniques used to characterize such fabricated scaffolds by additive manufacturing technologies.

In order to scale benchtop tissue mimics into viable constructs of clinically relevant dimensions, these structures must contain internal vascular networks to support convective mass transport. Without vessels to support perfusion culture, encapsulated cells located farther than 200 μm from the outer surface of a construct will quickly die due to the diffusional limits of oxygen and small molecule nutrients. By endowing artificial tissues with hollow vessels, researchers have made exciting progress towards the longitudinal maintenance of cellular function in large, dense tissues. But the field currently lacks standardized platforms and protocols to fabricate highly vascularized constructs in a rapid and cost-effective manner, which has left the literature base to become crowded with custom apparatus and diverse technical schemes. Here we highlight some promising, contemporary strategies for the vascularization of 3D printed and engineered tissues. We discuss the advantages and limitations of various fabrication platforms in the field, making note of desirable properties such as high spatial resolution, freely tunable 3D architecture, and the presence of discrete fluidic ports. With clinical targets in mind, this overview concludes with a brief survey of progress towards fluidic integration with the circulatory system in vivo.

The design of bone tissue engineering materials and scaffold structures made thereof is a delicate task, owing to the various, sometimes contradicting requirements that must be fulfilled. The traditional approach is based on a trial-and-error strategy, which may result in a lengthy and inefficient process. Aiming at improvement of this unsatisfactory situation, computer simulations, based on sound mathematical modeling of the involved processes, have been identified as promising complement to experimental testing. After giving a brief overview of available modeling and simulation concepts, the core of this chapter is presented, namely recent examples of multiscale, continuum micromechanics-based homogenization approaches developed in relation to bone tissue engineering. First, the fundamentals of continuum micromechanics are introduced, in order to lay the groundwork for the subsequently elaborated stiffness and strength homogenization approach related to a hydroxyapatite-based granular bone tissue engineering material. For the latter, the derivation of an upscaling scheme allowing for estimating the macroscopic stiffness and the macroscopic strength is demonstrated. Finally, avenues to utilization of this method in the design process of such materials are pointed out.

3D printing combined with design using patient image data has enabled the development of patient-specific devices. This is especially true for smaller commercial entities and academic groups due to the lower barriers for 3D printing as a manufacturing method. Such patient-specific devices can significantly advance patient care but also face significant hurdles to ensure quality since (1) the devices are built in small lots for specific niche patient markets, (2) there is inherent variability in design parameters to match specific patient anatomy and function, and (3) nontraditional groups now have the capability to readily manufacture medical devices. Following the design control paradigm with specific attention to 3D printing idiosyncrasies is one path to address quality issues in patient-specific design. We present in this chapter an example of a design control approach for quality control of 3D patient-specific devices using a recently developed airway splint as a paradigmatic example for small lot 3D printed patient-specific devices.

This chapter is focused on bioceramics for musculoskeletal regenerative medicine, with emphasis on material and manufacturing compatibility in the development of synthetic bone grafts. Bioceramics are classified into families depending on their relative bioactivity: passive, bioactive, and bioresorbable. Passive bioceramics, such as alumina and zirconia, are mainly used for load-bearing implants. Bioactive ceramics, such as bioactive glass, are useful to generate a strong bond between metallic surfaces and bone. Bioresorbable ceramics are applied to bone void filling and scaffolds for synthetic grafts. A description of bioceramics and their use in manufacturing processes is given, with major emphasis on techniques that may be useful in the fabrication of regenerative devices such as synthetic bone grafts. The manufacturing processes of interest are classified into molding, additive manufacturing, and coating techniques. The use of bioceramic-based scaffolds in bone repair animal models and clinical studies is reviewed. Finally, this chapter provides an outlook of future research directions for improved bioceramic use in synthetic bone grafts or regenerative skeletal devices.

Technological advances in medical imaging have provided healthcare professionals with powerful resources for storing, analyzing, and visualizing three-dimensional images in a variety of diagnostic tasks. Equipments for acquiring high-quality images and computer-aided tools for image interpretation play an important role in surgical planning, disease assessment, and therapy response monitoring. This chapter presents an overview of relevant aspects related to image processing and computer graphics techniques for the construction of three-dimensional models for visualization and biofabrication.

Tissue engineering represents a new field aiming at developing biological substitutes to restore, maintain, or improve tissue functions. In this approach, scaffolds provide a temporary mechanical and vascular support for tissue regeneration while tissue ingrowth is being formed. The design of optimized scaffolds for tissue engineering applications is a key topic of research, as the complex macro- and micro-architectures required for a scaffold depends on the mechanical and vascular properties and physical and molecular queues of the surrounding tissue at the defect site. One way to achieve such hierarchical designs is to create a library of unit cells, which can be assembled through a computational tool.

Besides presenting an overview scaffold designs based hyperbolic surfaces, this chapter investigates the use of two different types of triply periodic minimal surfaces, Schwarz and Schoen, in order to design better biomimetic scaffolds with high surface-to-volume ratio, high porosity, and good mechanical properties. The effect of two parametric parameters (thickness and surface radius) is also evaluated regarding its porosity and mechanical behavior.

Extrusion-based bioprinting is a powerful three-dimensional (3D) bioprinting technology that provides unique opportunities for use in organ fabrication. This technology has grown rapidly during the last decade. Extrusion-based bioprinting provides great versatility in printing various biological compounds or devices, including cells, tissues, organoids, and microfluidic devices that can be applied in basic research, pharmaceutics, drug testing, transplantation, and clinical uses. Extrusion-based bioprinting offers great flexibility in printing wide range of bioinks, including tissue spheroids, cell pellets, microcarriers, decellularized matrix components, and cell-laden hydrogels. Despite these assets, extrusion-based bioprinting has several limitations, such as inadequate control and resolution cell deposition, to create a complex tissue micro-microenvironment, shear stress-induced cell damage, and constraints associated with the current bioink materials.

Inkjet printing is a noncontact printing technology with high resolution, high throughput, and considerable reproducibility. Instead of printing normal ink, inkjet technology is also applied in the field of biofabrication to print living cells and other biological factors. Cell viability and function were demonstrated to be sustained after printing. Besides two dimensional cell patterns, three-dimensional cell-laden hydrogel structures can also be inkjet printed through cross-linking. Special phenomena such as the temporary permeability change of cell membranes were also observed during printing procedures, thus making it possible to achieve gene transfection through inkjet printing. Inkjet-printed biomolecule patterns with gradient concentration were also used to direct cell fates. Since the diversity of bioink and the capability of fabricating complex structures, inkjet bioprinting behaves as an effective tool in the field of biofabrication. The applications of inkjet printing include but not limit to drug formulation, tissue repair, and cancer research.

The development of reproducible well-defined 3D cell models is a key challenge for the future progress in tissue engineering. The structural dimensions in natural tissue are significantly lower than 100 μm. Thus, the ability to precisely position different cells in complex 3D patterns is of essential importance, even if it is not fully determined which precision or resolution is really required.

This chapter discusses laser-based techniques for printing living cells in two- or three-dimensional patterns. One method known as laser-guided direct writing has been used to position individual cells in a cell medium bath by applying the laser optical tweezer technique.

A more common method applies the laser-induced forward transfer (LIFT) for cell printing. For this method, many different designations are used like biological laser printing (BioLP), laser-assisted bioprinting (LAB, LaBP), or matrix-assisted pulsed laser evaporation - direct write (MAPLE-DW). There are also some technical differences in the realization of cell printing with this method that are discussed in this chapter. Applications like printing of multicellular arrays, stem cell grafts, and tissue as well as in situ printing will be presented.

3D bioprinting technology is expected to revolutionize the field of medicine and health care particularly within soft tissue repair and reconstruction. Surgical needs for soft tissue repair include nose, ear, meniscus, and cartilage in joints, as well as repair of damaged nerve tissue, and repair or replacement of damaged skin. 3D bioprinting technology includes a 3D bioprinter, cells, and bioink. Novel bioinks which will be suitable for soft tissue repair need to be developed before 3D bioprinting technology can get into the clinic. Hydrogels and cell-laden hydrogels are very attractive for soft tissue application because of the similarity of mechanical properties and cell environment. The process of design and development of novel bioinks is described in detail in this chapter which includes rheology, printability, cross-linking, long-term stability in medium, cell viability, and stimulation of cells during tissue growth. The commercialization process of bioinks is also described.

Photopolymerization of hydrogels in the presence of cells is a frequently applied technique to realize tissue engineering and regeneration due to the fact that the reaction can take place under cell-friendly physiological conditions. Photopolymerization can be subdivided into three modes, including radical, cationic, and anionic photopolymerization, according to the reactive species which are formed during initiation and propagation. However, radical photoinitiators are the only species suitable for hydrogel formation since ionic photopolymerization inevitably leads to termination of the reactive species as a result of the presence of water. Hydrogels are promising materials due to their capability to absorb large amounts of water and biological fluids without dissolving, their ability to become photopolymerized in the presence of cells, and their close resemblance to the extracellular matrix of native tissue. The present chapter aims to provide an overview of commonly applied photoinitiators as well as photopolymerizable natural and synthetic polymers which are frequently used for cell encapsulation purposes.

Bioprinting has emerged over the past decade as a prominent technology in the field of tissue engineering. This enables fabrication of cell-laden hydrogels with precise control over the architecture of the scaffold and the location of cells, growth factors, and other biological cues of interest. We first discuss the challenges that exist in terms of choosing a bioprinter, ensuring mechanical support and printability of a material, and minimizing cellular stress for cell-laden prints. We then explain the different crosslinking methods commonly used in hydrogel printing and approaches to alter bioink crosslinking mechanisms. We discuss material selection for bioprinting with elaboration on common materials that have been used and a review of multi-material prints involving hydrogels. We also explore the use of a single, mixed bioink to fabricate complex but homogeneous constructs and multiple, independent bioinks to fabricate complex heterogeneous tissue constructs within the multi-material review. We conclude with a summary of the current state of the field and an outlook on future research.

Tissue engineering and regenerative medicine have met great scientific, medical, and technological advances in the past decade. Most methods combine scaffolds, such as polymers, and living cells to make implantable structures that will integrate and heal the host’s tissues. More recently, alternative scaffold-free approaches have started to emerge. This chapter provides an overview of the current scaffold-free systems, advantages, challenges, methods, and applications. Scaffold-free tissue artificially produced in the lab using patients’ own cells has already been successfully used in heart and blood vessel regeneration at a small scale. New techniques and approaches are being developed, not only in terms of assembling cells and structures but also in terms of new equipment, namely for 3D bioprinting. Both primary and stem or iPSC-derived cells are used to assemble artificial tissues that are currently being tested in vivo and in vitro. These engineered constructs have numerous applications, such as regenerative medicine, disease models, and drug testing.

Three-dimensional (3D) bioprinting is an emerging field that holds promise for creating functional living tissues and organs. Bioprinting enables to fabricate structurally complex 3D tissue constructs by precise positioning and spatially separated patterns of multiple types of cells, biomaterials, and bioactive molecules within a single construct. With recent advances in bioprinting strategies, 3D bioprinting has been applied in various research areas, including tissue engineering and regenerative medicine, biology, physiology, drug discovery, and cancer/stem cell research. In tissue engineering and regenerative medicine, many types of 3D tissue constructs have been bioprinted to generate functional tissues for implantation, with the ultimate goal of clinical use. In addition, 3D bioprinting has been used as a tool to create in vitro tissue/organ models for drug discovery and cancer research, enabling deeper understanding of physiological phenomena of specific tissues/organs and more accurate prediction of drug or toxicity responses. In this chapter, we discuss recent applications of 3D bioprinting; first to create tissues and organs for the purposes of tissue engineering and regenerative medicine and then as platforms for in vitro tissue/organ models in drug discovery/toxicity testing and cancer research. We also discuss current challenges and future perspectives for practical applications of 3D bioprinting.

The patenting of bioprinting techniques to make tissues and organs has quietly been going on for more than a decade. Anyone seeking to develop a product or service based upon bioprinting must be concerned about the patent landscape. This chapter will first discuss the various options available to protect the different aspects of bioprinting. Then, it will provide a summary of the landscape of utility patents that protect innovation in each of the three stages of bioprinting: (i) bioimaging + CAD + blueprint, (ii) bi-oink + bio-paper + bioprinter, and (iii) maturogens + biomonitoring + bioreactor. Lastly, the chapter will discuss certain exceptions to patent infringement for the development of bioprinted tissues and organs.

After breaking out from the confines of purely academic research, 3D bioprinting technology is quickly developing as a commercial industry and exhibiting the qualities of a mature market with immense potential. We are currently witnessing not only growth in the number of companies and their geographical reach, but also the market’s segmentation. The main models of 3D bioprinting technology commercialization seem to be selling bioprinters and bioinks, services of bioprinting 3D functional tissue constructs – including for drug discovery and disease modeling – selling software, and technological consulting. As the industry advances, so does the legal regulation of the relevant issues. A number of companies are already successfully monetizing the technology and are able to raise financing through various paths. In the near future, we should expect the start of industry consolidation. At this stage of the technology development, rivalries within the industry do not represent a significant threat. The industry is currently characterized by stakeholders joining efforts in order to expedite its advancement and reach the commercial application stage. To accomplish this, the industry must overcome a number of significant hurdles, including achieving the standardization of bioprinting methods, software, and materials.

The bioprinters are robotic devices, which enable 3D bioprinting. In this chapter, we provide classification of already existing commercially available 3D bioprinters and outline basic principles of their construction and functionalities. The emerging trends in the design and development of 3D bioprinters, perspectives of creation of new types of commercial 3D bioprinters based on new physical principles, including in situ bioprinters, as well as completely integrated organ biofabrication lines or “human organ factories” will be also discussed.