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Stereolithography 3D Bioprinting
Abstract
Stereolithography (SLA) 3D bioprinting has emerged as a prominent bioprinting method addressing the requirements of complex tissue fabrication. This chapter addresses the advancement in SLA 3D bioprinting in concurrent with the development of novel photocrosslinkable biomaterials with enhanced physical and chemical properties. We discuss the cytocompatible photoinitiators operating in the wide spectrum of the ultraviolet (UV) and the visible light and high-resolution dynamic mask projection systems with a suitable illumination source. The potential of SLA 3D bioprinting has been explored in various themes, like bone and neural tissue engineering and in the development of controlled microenvironments to study cell behavior. The flexible design and versatility of SLA bioprinting makes it an attractive bioprinting process with myriad possibilities and clinical applications.
... Grigoryan et al. [23] presented the application, characterization, and development of an SLA bioprinter, which supports multi-material, minimizes bioink mixing, and yields precise regional feature alignment. Kumar and Kim [24] addressed the advancement in SLA 3D bioprinting synchronized with the fabrication of new photo-crosslinkable biomaterials with improved chemical and physical properties. SLA based on visible light is also being developed for bioprinting where normal light can be used for curing bioinks for stabilization of constructs [14]. ...
Bioprinting is fast emerging as a viable technique for organ fabrication. Though various types of bioprinting methods have been developed, the most commonly used bioprinting is extrusion-based bioprinting (EBB). Bioinks are extruded layer-by-layer forming a 3D multicellular construct and scaled up to dimensions depending upon the specific tissue to be regenerated. Among various bioinks, alginate, a natural polysaccharide, has been extensively used because of its good printability in physiologically amenable conditions. Though alginate possesses good printability properties, it promotes little cell–material interaction resulting in limited biofunctionality. Therefore, it becomes necessary to blend/modify alginate to improve the biological properties of bioink without compromising printability. This paper presents a review of the various approaches used to optimize bioprinting with alginate bioinks and their limitations.
... Stereolithography (Figure 1(g)), similar to laser-assisted method uses light irradiation to solidify bio-ink selectively via layer-by-layer process. Here, digital light projector is used to cure bio-inks and this method holds higher advantages over traditional method in printing complex pattern [25]. ...
Cardiovascular diseases (CVDs) represent a paramount global mortality concern, and their prevalence is on a relentless ascent. Despite the effectiveness of contemporary medical interventions in mitigating CVD-related fatality rates and complications, their efficacy remains curtailed by an array of limitations. These include the suboptimal efficiency of direct cell injection and an inherent disequilibrium between the demand and availability of heart transplantations. Consequently, the imperative to formulate innovative strategies for cardiac regeneration therapy becomes unmistakable. Within this context, 3D bioprinting technology emerges as a vanguard contender, occupying a pivotal niche in the realm of tissue engineering and regenerative medicine. This state-of-the-art methodology holds the potential to fabricate intricate heart tissues endowed with multifaceted structures and functionalities, thereby engendering substantial promise. By harnessing the prowess of 3D bioprinting, it becomes plausible to synthesize functional cardiac architectures seamlessly enmeshed with the host tissue, affording a viable avenue for the restitution of infarcted domains and, by extension, mitigating the onerous yoke of CVDs. In this review, we encapsulate the myriad applications of 3D bioprinting technology in the domain of heart tissue regeneration. Furthermore, we usher in the latest advancements in printing methodologies and bioinks, culminating in an exploration of the extant challenges and the vista of possibilities inherent to a diverse array of approaches.
Within the human body, the intricate network of blood vessels plays a pivotal role in transporting nutrients and oxygen and maintaining homeostasis. Bioprinting is an innovative technology with the potential to revolutionize this field by constructing complex multicellular structures. This technique offers the advantage of depositing individual cells, growth factors, and biochemical signals, thereby facilitating the growth of functional blood vessels. Despite the challenges in fabricating vascularized constructs, bioprinting has emerged as an advance in organ engineering. The continuous evolution of bioprinting technology and biomaterial knowledge provides an avenue to overcome the hurdles associated with vascularized tissue fabrication. This article provides an overview of the biofabrication process used to create vascular and vascularized constructs. It delves into the various techniques used in vascular engineering, including extrusion-, droplet-, and laser-based bioprinting methods. Integrating these techniques offers the prospect of crafting artificial blood vessels with remarkable precision and functionality. Therefore, the potential impact of bioprinting in vascular engineering is significant. With technological advances, it holds promise in revolutionizing organ transplantation, tissue engineering, and regenerative medicine. By mimicking the natural complexity of blood vessels, bioprinting brings us one step closer to engineering organs with functional vasculature, ushering in a new era of medical advancement.
Wounds are a stern healthcare concern in the growth of chronic disease conditions as they can increase healthcare costs and complicate internal and external health. Advancements in the current and newer management systems for wound healing should be in place to counter the health burden of wounds. Researchers discovered that two-dimensional (2D) media lacks appropriate real-life detection of cellular matter as these have highly complicated and diverse structures, compositions, and interactions. Hence, innovation towards three-dimensional (3D) media is called to conquer the high-level assessment and characterization in vivo using new technologies. The application of modern wound dressings prepared from a degenerated natural tissue, biodegradable biopolymer, synthetic polymer, or a composite of these materials in wound healing is currently an area of innovation in tissue regeneration medicine. Moreover, the integration of 3D printing and nanomaterial science is a promising approach with the potential for individualized, flexible, and precise technology for wound care approaches. This review encompasses the outcomes of various investigations on recent advances in 3D-printed drug-loaded natural, synthetic, and composite nanomaterials for wound healing. The challenges associated with their fabrication, clinical application progress, and future perspectives are also addressed.
Cancerous tumors are among the most fatal diseases worldwide, claiming nearly 10 million lives in 2020. Due to their complex and dynamic nature, modeling tumors accurately is a challenging task. Current models suffer from inadequate translation between in vitro and in vivo results, primarily due to the isotropic nature of tumors and their microenvironment's relationship. To address these limitations, hydrogel-based 3D bioprinting is emerging as a promising approach to mimic cancer development and behavior. It provides precise control over individual elements' size and distribution within the cancer microenvironment and enables the use of patient-derived tumor cells, rather than commercial lines. Consequently, hydrogel bioprinting is expected to become a state-of-the-art technique for cancer research. This manuscript presents an overview of cancer statistics, current modeling methods, and their limitations. Additionally, we highlight the significance of bioprinting, its applications in cancer modeling, and the importance of hydrogel selection. We further explore the current state of creating models for the five deadliest cancers using 3D bioprinting. Finally, we discuss current trends and future perspectives on the clinical use of cancer modeling using hydrogel bioprinting.
Three-dimensional (3D) bioprinting technologies involving photopolymerizable bioinks (PBs) have attracted enormous attention in recent times owing to their ability to recreate complex structures with high resolution, mechanical stability, and favorable printing conditions that are suited for encapsulating cells. 3D bioprinted tissue constructs involving PBs can offer better insights into the tumor microenvironment and offer platforms for drug screening to advance cancer research. These bioinks enable the incorporation of physiologically relevant cell densities, tissue-mimetic stiffness, and vascularized channels and biochemical gradients in the 3D tumor models, unlike conventional two-dimensional (2D) cultures or other 3D scaffold fabrication technologies. In this perspective, we present the emerging techniques of 3D bioprinting using PBs in the context of cancer research, with a specific focus on the efforts to recapitulate the complexity of the tumor microenvironment. We describe printing approaches and various PB formulations compatible with these techniques along with recent attempts to bioprint 3D tumor models for studying migration and metastasis, cell-cell interactions, cell-extracellular matrix interactions, and drug screening relevant to cancer. We discuss the limitations and identify unexplored opportunities in this field for clinical and commercial translation of these emerging technologies.
Three-dimensional (3D) bioprinting, or additive manufacturing, is a rapid fabrication technique with the foremost objective of creating biomimetic tissue and organ replacements in hopes of restoring normal tissue function and structure. Generating the engineered organs with an infrastructure that is similar to that of the real organs can be beneficial to simulate the functional organs that work inside our bodies. Photopolymerization-based 3D bioprinting, or photocuring, has emerged as a promising method in engineering biomimetic tissues due to its simplicity, non-invasive, and spatially controllable approach. In this review, we investigated types of 3D printers, mainstream materials, photoinitiators, phototoxicity, and selected tissue engineering applications of 3D photopolymerization bioprinting.
A stereolithography‐based bioprinting platform for multimaterial fabrication of heterogeneous hydrogel constructs is presented. Dynamic patterning by a digital micromirror device, synchronized by a moving stage and a microfluidic device containing four on/off pneumatic valves, is used to create 3D constructs. The novel microfluidic device is capable of fast switching between different (cell‐loaded) hydrogel bioinks, to achieve layer‐by‐layer multimaterial bioprinting. Compared to conventional stereolithography‐based bioprinters, the system provides the unique advantage of multimaterial fabrication capability at high spatial resolution. To demonstrate the multimaterial capacity of this system, a variety of hydrogel constructs are generated, including those based on poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacryloyl (GelMA). The biocompatibility of this system is validated by introducing cell‐laden GelMA into the microfluidic device and fabricating cellularized constructs. A pattern of a PEGDA frame and three different concentrations of GelMA, loaded with vascular endothelial growth factor, are further assessed for its neovascularization potential in a rat model. The proposed system provides a robust platform for bioprinting of high‐fidelity multimaterial microstructures on demand for applications in tissue engineering, regenerative medicine, and biosensing, which are otherwise not readily achievable at high speed with conventional stereolithographic biofabrication platforms. A stereolithographic bioprinting platform for multimaterial fabrication of hydrogel constructs is presented. Dynamic patterning by a digital micromirror device synchronized with a moving stage and a microfluidic device is used to create multimaterial constructs at high fidelity.
Photo-crosslinkable hydrogels, in particular gelatin methacryloyl (GelMa), are gaining increasing importance in biofabrication and tissue engineering. While GelMa is often described as mechanically ‘tunable’, clear relationships linking the photocrosslinking conditions to reaction rates, and the resulting mechanical properties, have not been described. Meanwhile the conditions employed in the literature are disparate, and difficult to compare. In this work, in situ rheological measurements were used to quantify the relative rate of reaction of GelMa hydrogels with respect to light intensity, exposure time and photo-initiator concentration. In addition the UV degradation of the photo-initiator Irgacure 2959 was measured by UV-vis spectroscopy, and used to estimate the rate of free radical production as a function of light exposure. Using these data an expression was derived which predicts the mechanical properties of GelMa hydrogels produced across a wide range of crosslinking conditions. The model was validated through fabrication of a GelMa gradient which matched predicted properties. Human mesenchymal stem cells encapsulated in crosslinked GelMa exhibited high (>90%) viability post encapsulation, however metabolic activity over one week was influenced by the intensity of light used during crosslinking. The expressions described may be used to aid rational choices of GelMa photocrosslinking conditions, especially in cell encapsulation experiments where minimising the cytotoxic elements in the reaction is a priority.
Microfabrication methods have widely been used to control the local cellular environment on a micron scale. However, accurately mimicking the complexity of the in vivo tissue architecture while maintaining the freedom of form and design is still a challenge when co-culturing multiple types of cells on the same substrate. For the first time, we present a drop-on-demand inkjet printing method to directly pattern living cells into a cell-friendly liquid environment. High-resolution control of cell location is achieved by precisely optimizing printing parameters with high-speed imaging of cell jetting and impacting behaviors. We demonstrated the capabilities of the direct cell printing method by co-printing different cells into various designs, including complex gradient arrangements. Finally, we applied this technique to investigate the influence of the heterogeneity and geometry of the cell population on the infectivity of seasonal H1N1 influenza virus (PR8) by generating A549 and HeLa cells printed in checkboard patterns of different sizes in a medium-filled culture dish. Direct inkjet cell patterning can be a powerful and versatile tool for both fundamental biology and applied biotechnology.
We present the first cell attachable and visible light crosslinkable hydrogels based on gelatin methacryloyl (GelMA) with eosin Y (EY) photoinitiation for stereolithography 3D bioprinting. In order to develop visible a light crosslinkable hydrogel, we systematically studied five combinations of the GelMA and EY photoinitiator with various concentrations. Their mechanical properties, microstructures, and cell viability and confluency after encapsulation were investigated rigorously to elucidate the effects of the EY and GelMA macromer concentration on the characteristics of the hydrogel. Experimental results show that the compressive Young's Modulus and pore size are positively affected by the concentration of EY, while the mass swelling ratio and cell viability are negatively affected. Increasing the concentration of GelMA helps to improve the compressive Young's Modulus and cell attachment. We further employed the developed visible light-based stereolithography bioprinting system to print the patterned cell-laden hydrogels to demonstrate the bioprinting applications of the developed hydrogel. We observed good cell proliferation and the formation of a 3D cellular network inside the printed pattern at day 5, which proves the great feasibility of using EY-GelMA as the bioinks for biofabrication and tissue engineering.
Bioprinting is a method by which a cell-encapsulating bioink is patterned to create complex tissue architectures. Given the potential impact of this technology on neural research, we review the current state-of-the-art approaches for bioprinting neural tissues. While 2D neural cultures are ubiquitous for studying neural cells, 3D cultures can more accurately replicate the microenvironment of neural tissues. By bioprinting neuronal constructs, one can precisely control the microenvironment by specifically formulating the bioink for neural tissues, and by spatially patterning cell types and scaffold properties in three dimensions. We review a range of bioprinted neural tissue models and discuss how they can be used to observe how neurons behave, understand disease processes, develop new therapies and, ultimately, design replacement tissues.
Two-photon polymerization (2PP) leverages the two-photon absorption (TPA) of near-infrared (NIR) radiation for additive manufacturing with sub-diffraction limit resolution within the bulk of a photosensitive material. This technology draws heavily on photosensitive polymers from the microelectronics industry, which were not optimized for TPA or for biocompatibility. 2PP with sub 100. nm resolution has been repeatedly demonstrated; however, this level of fabrication resolution comes at the expense of long fabrication times. Manufacturing of medical devices beyond surface texturing would be prohibitively slow using the current state of the art 2PP technology. Current research into TPA-sensitive photopolymers with good biocompatibility and holographic projections using spatial light modulators address current technological limitations by providing materials specifically formulated for biological applications and by making better use of available laser power for applications in which nanoscale resolution is not required.
Bioprinting as an enabling technology for tissue engineering possesses the promises to fabricate highly mimicked tissue or organs with digital control. As one of the biofabrication approaches, bioprinting has the advantages of high throughput and precise control of both scaffold and cells. Therefore, this technology is not only ideal for translational medicine but also for basic research applications. Bioprinting has already been widely applied to construct functional tissues such as vasculature, muscle, cartilage, and bone. In this review, the authors introduce the most popular techniques currently applied in bioprinting, as well as the various bioprinting processes. In addition, the composition of bioink including scaffolds and cells are described. Furthermore, the most current applications in organ and tissue bioprinting are introduced. The authors also discuss the challenges we are currently facing and the great potential of bioprinting. This technology has the capacity not only in complex tissue structure fabrication based on the converted medical images, but also as an efficient tool for drug discovery and preclinical testing. One of the most promising future advances of bioprinting is to develop a standard medical device with the capacity of treating patients directly on the repairing site, which requires the development of automation and robotic technology, as well as our further understanding of biomaterials and stem cell biology to integrate various printing mechanisms for multi-phasic tissue engineering.
Photocrosslinkable materials have been frequently used for constructing soft and biomimetic hydrogels for tissue engineering. Although, ultraviolet (UV) light is commonly used for photocrosslinking such materials, its use has been associated with several biosafety concerns such as DNA damage, accelerated aging of tissues, and cancer. Here we report an injectable visible light crosslinked gelatin-based hydrogel for myocardium regeneration. Mechanical characterization revealed that the compressive moduli of the engineered hydrogels could be tuned in the range of 5-56 kPa, by changing the concentrations of the co-initiator and co-monomer in the precursor formulation. In addition, the average pore sizes (26-103 μm) and swelling ratio (7-13%) were also shown to be tunable by varying the hydrogel formulation. In vitro studies showed that visible light crosslinked GelMA hydrogels supported the growth and function of primary cardiomyocytes (CMs). In addition, the engineered materials were shown to be biocompatible in vivo, and could be successfully delivered to the heart after myocardial infarction in an animal model to promote tissue healing. The developed visible light crosslinked hydrogel could be used for the repair of various soft tissues such as the myocardium, and for the treatment of cardiovascular diseases with enhanced therapeutic functionality.
In this paper, we present a multicomponent microenvironment consisting of proteinaceous networks with submicron-sized features optionally embedded into a photoresist microscaffold. By two-photon direct laser writing, free-standing 3D proteinaceous microstructures were fabricated for cell culture application, demonstrated with NIH/3T3 fibroblast cells. A Young's modulus of megapascal-order contributes to the challenge of structural sustainability of the proteinaceous microstructures for experiments as well as sequential fabrication steps. We propose to embed proteinaceous networks into a mechanically robust photoresist microscaffold. We investigate the limits of this 3D microfabrication of embedded proteinaceous networks and demonstrate the embedment of two different proteinaceous networks within one microscaffold. Performing cell culture of PC12 cells, we observe cell adhesion and cell motility on embedded proteinaceous networks of collagen type-IV mixed with bovine serum albumin into a photoresist microscaffold. The ability to structure proteinaceous elements for 3D spatial control of microenvironment might be a key feature in cell culture to decouple environmental cues to control cellular behavior.
Living tissues rely heavily on vascular networks to transport nutrients, oxygen and metabolic waste. However, there still remains a need for a simple and efficient approach to engineer vascularized tissues. Here, we created prevascularized tissues with complex three-dimensional (3D) microarchitectures using a rapid bioprinting method - microscale continuous optical bioprinting (μCOB). Multiple cell types mimicking the native vascular cell composition were encapsulated directly into hydrogels with precisely controlled distribution without the need of sacrificial materials or perfusion. With regionally controlled biomaterial properties the endothelial cells formed lumen-like structures spontaneously in vitro. In vivo implantation demonstrated the survival and progressive formation of the endothelial network in the prevascularized tissue. Anastomosis between the bioprinted endothelial network and host circulation was observed with functional blood vessels featuring red blood cells. With the superior bioprinting speed, flexibility and scalability, this new prevascularization approach can be broadly applicable to the engineering and translation of various functional tissues.