Dissecting the Interplay Mechanism among Process Parameters toward the Biofabrication of High-Quality Shapes in Embedded Bioprinting.

Embedded bioprinting overcomes the barriers associated with the conventional extrusion-based bioprinting process as it enables the direct deposition of bioinks in 3D inside a support bath by providing in situ self-support for deposited bioinks during bioprinting to prevent their collapse and deformation. Embedded bioprinting improves the shape quality of bioprinted constructs made up of soft materials and low-viscosity bioinks, leading to a promising strategy for better anatomical mimicry of tissues or organs. Herein, the interplay mechanism among the printing process parameters toward improved shape quality is critically reviewed. The impact of material properties of the support bath and bioink, printing conditions, cross-linking mechanisms, and post-printing treatment methods, on the printing fidelity, stability, and resolution of the structures is meticulously dissected and thoroughly discussed. Further, the potential scope and applications of this technology in the fields of bioprinting and regenerative medicine are presented. Finally, outstanding challenges and opportunities of embedded bioprinting as well as its promise for fabricating functional solid organs in the future are discussed.

Chemotherapeutics and CAR-T Cell-Based Immunotherapeutics Screening on a 3D Bioprinted Vascularized Breast Tumor Model.

Despite substantial advancements in development of cancer treatments, lack of standardized and physiologically-relevant in vitro testing platforms limit the early screening of anticancer agents. A major barrier is the complex interplay between the tumor microenvironment and immune response. To tackle this, a dynamic-flow based 3D bioprinted multi-scale vascularized breast tumor model, responding to chemo and immunotherapeutics is developed. Heterotypic tumors are precisely bioprinted at pre-defined distances from a perfused vasculature, exhibit tumor angiogenesis and cancer cell invasion into the perfused vasculature. Bioprinted tumors treated with varying dosages of doxorubicin for 72 h portray a dose-dependent drug response behavior. More importantly, a cell based immune therapy approach is explored by perfusing HER2-targeting chimeric antigen receptor (CAR) modified CD8+ T cells for 24 or 72 h. Extensive CAR-T cell recruitment to the endothelium, substantial T cell activation and infiltration to the tumor site, resulted in up to ≈70% reduction in tumor volumes. The presented platform paves the way for a robust, precisely fabricated, and physiologically-relevant tumor model for future translation of anti-cancer therapies to personalized medicine.

Intra-Operative Bioprinting of Hard, Soft, and Hard/Soft Composite Tissues for Craniomaxillofacial Reconstruction.

Reconstruction of complex craniomaxillofacial (CMF) defects is challenging due to the highly organized layering of multiple tissue types. Such compartmentalization necessitates the precise and effective use of cells and other biologics to recapitulate the native tissue anatomy. In this study, intra-operative bioprinting (IOB) of different CMF tissues, including bone, skin, and composite (hard/soft) tissues, is demonstrated directly on rats in a surgical setting. A novel extrudable osteogenic hard tissue ink is introduced, which induced substantial bone regeneration, with ≈80% bone coverage area of calvarial defects in 6 weeks. Using droplet-based bioprinting, the soft tissue ink accelerated the reconstruction of full-thickness skin defects and facilitated up to 60% wound closure in 6 days. Most importantly, the use of a hybrid IOB approach is unveiled to reconstitute hard/soft composite tissues in a stratified arrangement with controlled spatial bioink deposition conforming the shape of a new composite defect model, which resulted in ≈80% skin wound closure in 10 days and 50% bone coverage area at Week 6. The presented approach will be absolutely unique in the clinical realm of CMF defects and will have a significant impact on translating bioprinting technologies into the clinic in the future.

The Role of Concentration on Drop Formation and Breakup of Collagen, Fibrinogen, and Thrombin Solutions during Inkjet Bioprinting.

The influence of protein concentration on drop formation and breakup of aqueous solutions of fibrous proteins collagen and fibrinogen and globular protein thrombin in different concentration regimes has been investigated during drop-on-demand (DOD) inkjet printing. The capillary-driven thinning and breakup of dilute collagen, fibrinogen, and thrombin solutions, the solutions in which protein molecules are far away from each other, are predominantly resisted by inertial force. Although the capillary-driven thinning and breakup of semidilute unentangled collagen and fibrinogen solutions, the solutions in which protein molecules begin to interpenetrate each other, are predominantly resisted by inertial force on the initial onset of necking, the breakup of droplets is delayed because of the resistance of elastic force. The resistance of viscous force to the necking and breakup of both the dilute and semidilute unentangled protein solutions is negligible. Aggregates or subvisible particles (between 1 and 100 µm) constantly disrupt the formation of droplets for the semidilute unentangled protein solutions, even when their inverse Ohnesorge number (Z) is within the printability range of 4 ≤ Z ≤ 14. Although aggregates are present in the dilute protein solutions, they do not disrupt the formation of droplets.

Rheological investigation of collagen, fibrinogen, and thrombin solutions for drop-on-demand 3D bioprinting.

Collagen, fibrinogen, and thrombin proteins in aqueous buffer solutions are widely used as precursors of natural biopolymers in three-dimensional (3D) bioprinting applications. The proteins are sourced from animals and their quality may vary from batch to batch, inducing differences in the rheological properties of such solutions. In this work, we investigate the rheological response of collagen, fibrinogen, and thrombin protein solutions in bulk and at the solution/air interface. Interfacial rheological measurements show that fibrous collagen, fibrinogen and globular thrombin proteins adsorb and aggregate at the solution/air interface, forming a viscoelastic solid film at the interface. The viscoelastic film corrupts the bulk rheological measurements in rotational rheometers by contributing to an apparent yield stress, which increases the apparent bulk viscosity up to shear rates as high as 1000 s-1. The addition of a non-ionic surfactant, such as polysorbate 80 (PS80) in small amounts between 0.001 and 0.1 v/v%, prevents the formation of the interfacial layer, allowing the estimation of true bulk viscosity of the solutions. The estimation of viscosity not only helps in identifying those protein solutions that are potentially printable with drop-on-demand (DOD) inkjet printing but also detects inconsistencies in flow behavior among the batches.

Thermally-controlled extrusion-based bioprinting of collagen.

Thermally-crosslinked hydrogels in bioprinting have gained increasing attention due to their ability to undergo tunable crosslinking by modulating the temperature and time of crosslinking. In this paper, we present a new bioink composed of collagen type-I and Pluronic® F-127 hydrogels, which was bioprinted using a thermally-controlled bioprinting unit. Bioprintability and rheology of the composite bioink was studied in a thorough manner in order to determine the optimal bioprinting time and extrusion profile of the bioink for fabrication of three-dimensional (3D) constructs, respectively. It was observed that collagen fibers aligned themselves along the directions of the printed filaments after bioprinting based on the results on an anisotropy study. Furthermore, rat bone marrow-derived stem cells (rBMSCs) were bioprinted in order to determine the effect of thermally-controlled extrusion process. In vitro viability and proliferation study revealed that rBMSCs were able to maintain their viability after extrusion and attached to collagen fibers, spread and proliferated within the constructs up to seven days of culture.

Challenges in Bio-fabrication of Organoid Cultures.

Three-dimensional (3D) organoids have shown advantages in cell culture over traditional two-dimensional (2D) culture, and have great potential in various applications of tissue engineering. However, there are limitations in current organoid fabrication technologies, such as uncontrolled size, poor reproductively, and inadequate complexity of organoids. In this chapter, we present the existing techniques and discuss the major challenges for 3D organoid biofabrication. Future perspectives on organoid bioprinting are also discussed, where bioprinting technologies are expected to make a major contribution in organoid fabrication, such as realizing mass production and constructing complex heterotypic tissues, and thus further advance the translational application of organoids in tissue engineering and regenerative medicine as well drug testing and pharmaceutics.

Transplantation of Bioprinted Tissues and Organs: Technical and Clinical Challenges and Future Perspectives.

: Three-dimensional (3D) bioprinting is a revolutionary technology in building living tissues and organs with precise anatomic control and cellular composition. Despite the great progress in bioprinting research, there has yet to be any clinical translation due to current limitations in building human-scale constructs, which are vascularized and readily implantable. In this article, we review the current limitations and challenges in 3D bioprinting, including in situ techniques, which are one of several clinical translational models to facilitate the application of this technology from bench to bedside. A detailed discussion is made on the technical barriers in the fabrication of scalable constructs that are vascularized, autologous, functional, implantable, cost-effective, and ethically feasible. Clinical considerations for implantable bioprinted tissues are further expounded toward the correction of end-stage organ dysfunction and composite tissue deficits.

Bone tissue bioprinting for craniofacial reconstruction.

Craniofacial (CF) tissue is an architecturally complex tissue consisting of both bone and soft tissues with significant patient specific variations. Conditions of congenital abnormalities, tumor resection surgeries, and traumatic injuries of the CF skeleton can result in major deficits of bone tissue. Despite advances in surgical reconstruction techniques, management of CF osseous deficits remains a challenge. Due its inherent versatility, bioprinting offers a promising solution to address these issues. In this review, we present and analyze the current state of bioprinting of bone tissue and highlight how these techniques may be adapted to serve regenerative therapies for CF applications. Biotechnol. Bioeng. 2017;114: 2424-2431. © 2017 Wiley Periodicals, Inc.

Materials and scaffolds in medical 3D printing and bioprinting in the context of bone regeneration.

The structural and functional repair of lost bone is still one of the biggest challenges in regenerative medicine. In many cases, autologous bone is used for the reconstruction of bone tissue; however, the availability of autologous material is limited, which always means additional stress to the patient. Due to this, more and more frequently various biocompatible materials are being used instead for bone augmentation. In this context, in order to ensure the structural function of the bone, scaffolds are implanted and fixed into the bone defect, depending on the medical indication. Nevertheless, for the surgeon, every individual clinical condition in which standardized scaffolds have to be aligned is challenging, and in many cases the alignment is not possible without limitations. Therefore, in the last decades, 3D printing (3DP) or additive manufacturing (AM) of scaffolds has become one of the most innovative approaches in surgery to individualize and improve the treatment of patients. Numerous biocompatible materials are available for 3DP, and various printing techniques can be applied, depending on the process conditions of these materials. Besides these conventional printing techniques, another promising approach in the context of medical AM is 3D bioprinting, a technique which makes it possible to print human cells embedded in special carrier substances to generate functional tissues. Even the direct printing into bone defects or lesions becomes possible. 3DP is already improving the treatment of patients, and has the potential to revolutionize regenerative medicine in future.

In vitro evaluation of carbon-nanotube-reinforced bioprintable vascular conduits.

Vascularization of thick engineered tissue and organ constructs like the heart, liver, pancreas or kidney remains a major challenge in tissue engineering. Vascularization is needed to supply oxygen and nutrients and remove waste in living tissues and organs through a network that should possess high perfusion ability and significant mechanical strength and elasticity. In this paper, we introduce a fabrication process to print vascular conduits directly, where conduits were reinforced with carbon nanotubes (CNTs) to enhance their mechanical properties and bioprintability. In vitro evaluation of printed conduits encapsulated in human coronary artery smooth muscle cells was performed to characterize the effects of CNT reinforcement on the mechanical, perfusion and biological performance of the conduits. Perfusion and permeability, cell viability, extracellular matrix formation and tissue histology were assessed and discussed, and it was concluded that CNT-reinforced vascular conduits provided a foundation for mechanically appealing constructs where CNTs could be replaced with natural protein nanofibers for further integration of these conduits in large-scale tissue fabrication.

Posttranscriptional Modification to Modulate Progenitor Differentiation on Heterotypic Spheroids.

Cell aggregates are widely used to study heterotypic cellular interactions during the development of vascularization in vitro. In this study, we examined heterotypic cellular spheroids made of adipose-derived stem cells and CD34+/CD31- endothelial progenitor cells induced by the transfection of miR-148b mimic for de novo induction of osteogenic differentiation and miR-210 mimic for de novo induction of endotheliogenesis, respectively. The effect of the microRNA (miRs) mimic treatment group and induction time on codifferentiation was assessed in spheroids formed of transfected cells over the course of a 4-week culture. Based on gene and protein markers of osteogenic and endotheliogenic differentiation, as well as mineralization assays, our results showed that miRs directed cell differentiation and that progenitor maturity influenced the development of heterotypic cellular regions in aggregates. Overall, the success of coculture to create a prevascularized bone model is dependent on a number of factors, particularly the induction time of differentiation before combining the multiple cell types in aggregates. The approach that has been proposed could be valuable in creating vascularized bone tissue by employing spheroids as the building blocks of more complex issues through the use of cutting-edge methods such as 3D bioprinting.

A comprehensive review on 3D tissue models: Biofabrication technologies and preclinical applications.

The limitations of traditional two-dimensional (2D) cultures and animal testing, when it comes to precisely foreseeing the toxicity and clinical effectiveness of potential drug candidates, have resulted in a notable increase in the rate of failure during the process of drug discovery and development. Three-dimensional (3D) in-vitro models have arisen as substitute platforms with the capacity to accurately depict in-vivo conditions and increasing the predictivity of clinical effects and toxicity of drug candidates. It has been found that 3D models can accurately represent complex tissue structure of human body and can be used for a wide range of disease modeling purposes. Recently, substantial progress in biomedicine, materials and engineering have been made to fabricate various 3D in-vitro models, which have been exhibited better disease progression predictivity and drug effects than convention models, suggesting a promising direction in pharmaceutics. This comprehensive review highlights the recent developments in 3D in-vitro tissue models for preclinical applications including drug screening and disease modeling targeting multiple organs and tissues, like liver, bone, gastrointestinal tract, kidney, heart, brain, and cartilage. We discuss current strategies for fabricating 3D models for specific organs with their strengths and pitfalls. We expand future considerations for establishing a physiologically-relevant microenvironment for growing 3D models and also provide readers with a perspective on intellectual property, industry, and regulatory landscape.

A Versatile Photocrosslinkable Silicone Composite for 3D Printing Applications.

Embedded printing has emerged as a valuable tool for fabricating complex structures and microfluidic devices. Currently, an ample of amount of research is going on to develop new materials to advance its capabilities and increase its potential applications. Here, we demonstrate a novel, transparent, printable, photocrosslinkable, and tuneable silicone composite that can be utilized as a support bath or an extrudable ink for embedded printing. Its properties can be tuned to achieve ideal rheological properties, such as optimal self-recovery and yield stress, for use in 3D printing. When used as a support bath, it facilitated the generation microfluidic devices with circular channels of diameter up to 30 µm. To demonstrate its utility, flow focusing microfluidic devices were fabricated for generation of Janus microrods, which can be easily modified for multitude of applications. When used as an extrudable ink, 3D printing of complex-shaped constructs were achieved with integrated electronics, which greatly extends its potential applications towards soft robotics. Further, its biocompatibility was tested with multiple cell types to validate its applicability for tissue engineering. Altogether, this material offers a myriad of potential applications (i.e., soft robotics, microfluidics, bioprinting) by providing a facile approach to develop complicated 3D structures and interconnected channels.

Intraoperative bioprinting of human adipose-derived stem cells and extra-cellular matrix induces hair follicle-like downgrowths and adipose tissue formation during full-thickness craniomaxillofacial skin reconstruction.

Craniomaxillofacial (CMF) reconstruction is a challenging clinical dilemma. It often necessitates skin replacement in the form of autologous graft or flap surgery, which differ from one another based on hypodermal/dermal content. Unfortunately, both approaches are plagued by scarring, poor cosmesis, inadequate restoration of native anatomy and hair, alopecia, donor site morbidity, and potential for failure. Therefore, new reconstructive approaches are warranted, and tissue engineered skin represents an exciting alternative. In this study, we demonstrated the reconstruction of CMF full-thickness skin defects using intraoperative bioprinting (IOB), which enabled the repair of defects via direct bioprinting of multiple layers of skin on immunodeficient rats in a surgical setting. Using a newly formulated patient-sourced allogenic bioink consisting of both human adipose-derived extracellular matrix (adECM) and stem cells (ADSCs), skin loss was reconstructed by precise deposition of the hypodermal and dermal components under three different sets of animal studies. adECM, even at a very low concentration such as 2 % or less, has shown to be bioprintable via droplet-based bioprinting and exhibited de novo adipogenic capabilities both in vitro and in vivo. Our findings demonstrate that the combinatorial delivery of adECM and ADSCs facilitated the reconstruction of three full-thickness skin defects, accomplishing near-complete wound closure within two weeks. More importantly, both hypodermal adipogenesis and downgrowth of hair follicle-like structures were achieved in this two-week time frame. Our approach illustrates the translational potential of using human-derived materials and IOB technologies for full-thickness skin loss.

Evaluation of cell viability and functionality in vessel-like bioprintable cell-laden tubular channels.

Organ printing is a novel concept recently introduced in developing artificial three-dimensional organs to bridge the gap between transplantation needs and organ shortage. One of the major challenges is inclusion of blood-vessellike channels between layers to support cell viability, postprinting functionality in terms of nutrient transport, and waste removal. In this research, we developed a novel and effective method to print tubular channels encapsulating cells in alginate to mimic the natural vascular system. An experimental investigation into the influence on cartilage progenitor cell (CPCs) survival, and the function of printing parameters during and after the printing process were presented. CPC functionality was evaluated by checking tissue-specific genetic marker expression and extracellular matrix production. Our results demonstrated the capability of direct fabrication of cell-laden tubular channels by our newly designed coaxial nozzle assembly and revealed that the bioprinting process could induce quantifiable cell death due to changes in dispensing pressure, coaxial nozzle geometry, and biomaterial concentration. Cells were able to recover during incubation, as well as to undergo differentiation with high-level cartilage-associated gene expression. These findings may not only help optimize our system but also can be applied to biomanufacturing of 3D functional cellular tissue engineering constructs for various organ systems.

Ethical challenges with 3D bioprinted tissues and organs.

3D Bioprinting is fast advancing to offer capabilities to process living cells into geometrically and functionally complex tissue and organ substitutes. As bioprinted constructs are making their way into clinic, the bioprinting community needs to consider the responsible innovation and translation of the bioprinted tissues and organs.

Advances in Gelatin Bioinks to Optimize Bioprinted Cell Functions.

Gelatin is a widely utilized bioprinting biomaterial due to its cell-adhesive and enzymatically cleavable properties, which improve cell adhesion and growth. Gelatin is often covalently cross-linked to stabilize bioprinted structures, yet the covalently cross-linked matrix is unable to recapitulate the dynamic microenvironment of the natural extracellular matrix (ECM), thereby limiting the functions of bioprinted cells. To some extent, a double network bioink can provide a more ECM-mimetic, bioprinted niche for cell growth. More recently, gelatin matrices are being designed using reversible cross-linking methods that can emulate the dynamic mechanical properties of the ECM. This review analyzes the progress in developing gelatin bioink formulations for 3D cell culture, and critically analyzes the bioprinting and cross-linking techniques, with a focus on strategies to optimize the functions of bioprinted cells. This review discusses new cross-linking chemistries that recapitulate the viscoelastic, stress-relaxing microenvironment of the ECM, and enable advanced cell functions, yet are less explored in engineering the gelatin bioink. Finally, this work presents the perspective on the areas of future research and argues that the next generation of gelatin bioinks should be designed by considering cell-matrix interactions, and bioprinted constructs should be validated against currently established 3D cell culture standards to achieve improved therapeutic outcomes.

Bioengineering and Clinical Translation of Human Lung and its Components.

Clinical lung transplantation has rapidly established itself as the gold standard of treatment for end-stage lung diseases in a restricted group of patients since the first successful lung transplant occurred. Although significant progress has been made in lung transplantation, there are still numerous obstacles on the path to clinical success. The development of bioartificial lung grafts using patient-derived cells may serve as an alternative treatment modality; however, challenges include developing appropriate scaffold materials, advanced culture strategies for lung-specific multiple cell populations, and fully matured constructs to ensure increased transplant lifetime following implantation. This review highlights the development of tissue-engineered tracheal and lung equivalents over the past two decades, key problems in lung transplantation in a clinical environment, the advancements made in scaffolds, bioprinting technologies, bioreactors, organoids, and organ-on-a-chip technologies. The review aims to fill the lacuna in existing literature toward a holistic bioartificial lung tissue, including trachea, capillaries, airways, bifurcating bronchioles, lung disease models, and their clinical translation. Herein, the efforts are on bridging the application of lung tissue engineering methods in a clinical environment as it is thought that tissue engineering holds enormous promise for overcoming the challenges associated with the clinical translation of bioengineered human lung and its components.

Synergistic coupling between 3D bioprinting and vascularization strategies.

Three-dimensional (3D) bioprinting offers promising solutions to the complex challenge of vascularization in biofabrication, thereby enhancing the prospects for clinical translation of engineered tissues and organs. While existing reviews have touched upon 3D bioprinting in vascularized tissue contexts, the current review offers a more holistic perspective, encompassing recent technical advancements and spanning the entire multistage bioprinting process, with a particular emphasis on vascularization. The synergy between 3D bioprinting and vascularization strategies is crucial, as 3D bioprinting can enable the creation of personalized, tissue-specific vascular network while the vascularization enhances tissue viability and function. The review starts by providing a comprehensive overview of the entire bioprinting process, spanning from pre-bioprinting stages to post-printing processing, including perfusion and maturation. Next, recent advancements in vascularization strategies that can be seamlessly integrated with bioprinting are discussed. Further, tissue-specific examples illustrating how these vascularization approaches are customized for diverse anatomical tissues towards enhancing clinical relevance are discussed. Finally, the underexplored intraoperative bioprinting (IOB) was highlighted, which enables the direct reconstruction of tissues within defect sites, stressing on the possible synergy shaped by combining IOB with vascularization strategies for improved regeneration.