Bioprinted Tissues Research Articles

3D bioprinted and integrated platforms for cardiac tissue modeling and drug testing

Recent advances in biofabrication techniques, including 3D bioprinting, have allowed for the fabrication of cardiac models that are similar to the human heart in terms of their structure (e.g., volumetric scale and anatomy) and function (e.g., contractile and electrical properties). The importance of developing techniques for assessing the characteristics of 3D cardiac substitutes in real time without damaging their structures has also been emphasized. In particular, the heart has two primary mechanisms for transporting blood through the body: contractility and an electrical system based on intra and extracellular calcium ion exchange. This review introduces recent trends in 3D bioprinted cardiac tissues and the measurement of their structural, contractile, and electrical properties in real time. Cardiac models have also been regarded as alternatives to animal models as drug-testing platforms. Thus, perspectives on the convergence of 3D bioprinted cardiac tissues and their assessment for use in drug development are also presented.

Introduction to bioprinting of in vitro cancer models.

Cancer models are essential in cancer research and for new drug development pipelines. However, conventional cancer tissue models have failed to capture the human cancer physiology, thus hindering drug discovery. The major challenge is the establishment of physiologically relevant cancer models that reflect the complexity of the tumor microenvironment (TME). The TME is a highly complex milieu composed of diverse factors that are associated with cancer progression and metastasis, as well as with the development of cancer resistance to therapeutics. To emulate the TME, 3D bioprinting has emerged as a way to create engineered cancer tissue models. Bioprinted cancer tissue models have the potential to recapitulate cancer pathology and increased drug resistance in an organ-mimicking 3D environment. This review overviews the bioprinting technologies used for the engineering of cancer tissue models and provides a future perspective on bioprinting to further advance cancer research.

Recent advances in microarray 3D bioprinting for high-throughput spheroid and tissue culture and analysis.

Three-dimensional (3D) cell culture in vitro has proven to be more physiologically relevant than two-dimensional (2D) culture of cell monolayers, thus more predictive in assessing efficacy and toxicity of compounds. There have been several 3D cell culture techniques developed, which include spheroid and multicellular tissue cultures. Cell spheroids have been generated from single or multiple cell types cultured in ultralow attachment (ULA) well plates and hanging droplet plates. In general, cell spheroids are formed in a relatively short period of culture, in the absence of extracellular matrices (ECMs), via gravity-driven self-aggregation, thus having limited ability to self-organization in layered structure. On the other hand, multicellular tissue cultures including miniature tissues derived from pluripotent stem cells and adult stem cells (a.k.a. 'organoids') and 3D bioprinted tissue constructs require biomimetic hydrogels or ECMs and show highly ordered structure due to spontaneous self-organization of cells during differentiation and maturation processes. In this short review article, we summarize traditional methods of spheroid and multicellular tissue cultures as well as their technical challenges, and introduce how droplet-based, miniature 3D bioprinting ('microarray 3D bioprinting') can be used to improve assay throughput and reproducibility for high-throughput, predictive screening of compounds. Several platforms including a micropillar chip and a 384-pillar plate developed to facilitate miniature spheroid and tissue cultures via microarray 3D bioprinting are introduced. We excluded microphysiological systems (MPSs) in this article although they are important tissue models to simulate multiorgan interactions.

Application of 3D Printing Technology in Bone Tissue Engineering: A Review.

Clinically, the treatment of bone defects remains a significant challenge, as it requires autogenous bone grafts or bone graft substitutes. However, the existing biomaterials often fail to meet the clinical requirements in terms of structural support, bone induction, and controllable biodegradability. In the treatment of bone defects, 3D porous scaffolds have attracted much attention in the orthopedic field. In terms of appearance and microstructure, complex bone scaffolds created by 3D printing technology are similar to human bone. On this basis, the combination of active substances, including cells and growth factors, is more conducive to bone tissue reconstruction, which is of great significance for the personalized treatment of bone defects. With the continuous development of 3D printing technology, it has been widely used in bone defect repair as well as diagnosis and rehabilitation, creating an emerging industry with excellent market potential. Meanwhile, the diverse combination of 3D printing technology with multi-disciplinary fields, such as tissue engineering, digital medicine, and materials science, has made 3D printing products with good biocompatibility, excellent osteoinductive capacity, and stable mechanical properties. In the clinical application of the repair of bone defects, various biological materials and 3D printing methods have emerged to make patient-specific bioactive scaffolds. The microstructure of 3D printed scaffolds can meet the complex needs of bone defect repair and support the personalized treatment of patients. Some of the new materials and technologies that emerged from the 3D printing industry's advent in the past decade successfully translated into clinical practice. In this article, we first introduced the development and application of different types of materials that were used in 3D bioprinting, including metal, ceramic materials, polymer materials, composite materials, and cell tissue. The combined application of 3D bioprinting and other manufacturing methods used for bone tissue engineering are also discussed in this article. Finally, we discussed the bottleneck of 3D bioprinting technique and forecasted its research orientation and prospect.

Multimaterial bioprinting approaches and their implementations for vascular and vascularized tissues

Multimaterial bioprinting is a promising technology integrating multimaterial setups into bioprinting platforms for the fabrication of multicellular, heterogeneous and functional tissue constructs. By simultaneously or sequentially dispensing different categories of materials including cell-laden hydrogels or extracellular matrix components, sacrificial materials and scaffolding polymers with hierarchical microarchitecture, multimaterial bioprinting has achieved several milestones for the native tissue biomimicry. Particularly, reconstruction of multiscale, hierarchically branched vascular networks within engineered tissues has been residing as a grand challenge for the fabrication of tissues with long term viability and functionality, which hampers the transition of engineered constructs from research to clinic. To date, various multimaterial bioprinting approaches have been proposed to address the need for fabrication of vascular or vascularized tissues. In this review, a comprehensive overview is given to demonstrate the potential of multimaterial bioprinting technology for the biofabrication of vascular and vascularized tissues. In this regard, different multimaterial bioprinting approaches developed so far are described thoroughly, following the introduction of bioprinting modalities employed in tissue engineering and organization of vascular tissues. Subsequently, multimaterial bioprinting of vascular and vascularized tissues are presented with the main emphasis on the developed fabrication strategies and applied techniques. Comparative evaluation of the multimaterial bioprinting approaches and future perspectives are also delivered to reader.

Latest impact of engineered human liver platforms on drug development.

Drug-induced liver injury (DILI) is a leading cause of drug attrition, which is partly due to differences between preclinical animals and humans in metabolic pathways. Therefore, in vitro human liver models are utilized in biopharmaceutical practice to mitigate DILI risk and assess related mechanisms of drug transport and metabolism. However, liver cells lose phenotypic functions within 1–3 days in two-dimensional monocultures on collagen-coated polystyrene/glass, which precludes their use to model the chronic effects of drugs and disease stimuli. To mitigate such a limitation, bioengineers have adapted tools from the semiconductor industry and additive manufacturing to precisely control the microenvironment of liver cells. Such tools have led to the fabrication of advanced two-dimensional and three-dimensional human liver platforms for different throughput needs and assay endpoints (e.g., micropatterned cocultures, spheroids, organoids, bioprinted tissues, and microfluidic devices); such platforms have significantly enhanced liver functions closer to physiologic levels and improved functional lifetime to >4 weeks, which has translated to higher sensitivity for predicting drug outcomes and enabling modeling of diseased phenotypes for novel drug discovery. Here, we focus on commercialized engineered liver platforms and case studies from the biopharmaceutical industry showcasing their impact on drug development. We also discuss emerging multi-organ microfluidic devices containing a liver compartment that allow modeling of inter-tissue crosstalk following drug exposure. Finally, we end with key requirements for engineered liver platforms to become routine fixtures in the biopharmaceutical industry toward reducing animal usage and providing patients with safe and efficacious drugs with unprecedented speed and reduced cost.

Physical and Mechanical Characterization of Fibrin-Based Bioprinted Constructs Containing Drug-Releasing Microspheres for Neural Tissue Engineering Applications

Three-dimensional bioprinting can fabricate precisely controlled 3D tissue constructs. This process uses bioinks—specially tailored materials that support the survival of incorporated cells—to produce tissue constructs. The properties of bioinks, such as stiffness and porosity, should mimic those found in desired tissues to support specialized cell types. Previous studies by our group validated soft substrates for neuronal cultures using neural cells derived from human-induced pluripotent stem cells (hiPSCs). It is important to confirm that these bioprinted tissues possess mechanical properties similar to native neural tissues. Here, we assessed the physical and mechanical properties of bioprinted constructs generated from our novel microsphere containing bioink. We measured the elastic moduli of bioprinted constructs with and without microspheres using a modified Hertz model. The storage and loss modulus, viscosity, and shear rates were also measured. Physical properties such as microstructure, porosity, swelling, and biodegradability were also analyzed. Our results showed that the elastic modulus of constructs with microspheres was 1032 ± 59.7 Pascal (Pa), and without microspheres was 728 ± 47.6 Pa. Mechanical strength and printability were significantly enhanced with the addition of microspheres. Thus, incorporating microspheres provides mechanical reinforcement, which indicates their suitability for future applications in neural tissue engineering.

Potential of three-dimensional bioprinting in oral soft tissue engineering applications

Oral soft tissue engineering involves the reconstruction or restoration of oral and maxillofacial functions and esthetics. As an emerging technology from the early 21st century, three-dimensional (3D) bioprinting promises great application potentials in the preparation of scaffolds and engineered tissues/organs. Although oral soft tissues include dental pulp, periodontal ligament, gum, oral mucosa, and salivary glands as well as related maxillofacial skin, vascular, muscular, and neuronal tissues, the current application of 3D bioprinting in oral soft tissue-restoration is mainly limited to dental pulp-regeneration. Different bioinks are used to load dental pulp cells into the dentin matrix for restoring the dental pulp tissue; 3D bioprinting has only been reported in a few in vitro studies on periodontal ligament-reconstruction and salivary gland culture; and 3D bioprinting used towards regenerating gingival tissue/oral mucosa has not been demonstrated. The limited application of 3D bioprinting in oral soft tissue engineering is perhaps related to the complex, fine, and orderly structure of the periodontal ligament, the moist environment of the oral cavity, the small operating space, and the continuous chewing pressure. The studies on bioprinting of skin, vascular, muscular, and neuronal tissues are broad, but they are typically not oral-specific. This article introduces the current application status and prospects of 3D bioprinting in the regeneration of oral soft tissues, using cytocompatible hydrogels as bioinks.

Application of the three-dimensional bioprinting for the osteochondral tissue regeneration

The repair of osteochondral damage is clinically challenging. There are some limitations in the current technologies for osteochondral repair and regeneration, and it is still difficult for the strategies for osteochondral tissue engineering to obtain lasting and effective outcomes. The emergence and development of three-dimensional (3D) bioprinting have provided novel and powerful technical means for the reconstruction of osteochondral tissue. The controlled deposition of bioactive materials makes it possible to accurately reconstruct the heterogeneous microstructure of osteochondral tissue. This article summarizes the latest developments in the field of osteochondral bioprinting, including scaffold-based bioprinting, scaffold-free bioprinting, and in situ bioprinting. At the same time, the major considerations of osteochondral bioprinting are discussed, and current solutions and research trends are proposed, to broaden the understanding of readers about 3D bioprinting of osteochondral tissue.

Quantitative Photothermal Characterization with Bioprinted 3D Complex Tissue Constructs for Early-Stage Breast Cancer Therapy Using Gold Nanorods.

Plasmonic photothermal therapy (PPTT) using gold nanoparticles (AuNPs) has shown great potential for use in selective tumor treatment, because the AuNPs can generate destructive heat preferentially upon irradiation. However, PPTT using AuNPs has not been added to practice, owing to insufficient heating methods and tissue temperature measurement techniques, leading to unreliable and inaccurate treatments. Because the photothermal properties of AuNPs vary with laser power, particle optical density, and tissue depth, the accurate prediction of heat generation is indispensable for clinical treatment. In this report, bioprinted 3D complex tissue constructs comprising processed gel obtained from porcine skin and human decellularized adipose tissue are presented for characterization of the photothermal properties of gold nanorods (AuNRs) having an aspect ratio of 3.7 irradiated by a near-infrared laser. Moreover, an analytical function is suggested for achieving PPTT that can cause thermal damage selectively on early-stage human breast cancer by regulating the heat generation of the AuNRs in the tissue.

Vascularization of tissue engineered cartilage - Sequential in vivo MRI display functional blood circulation

Establishing functional circulation in bioengineered tissue after implantation is vital for the delivery of oxygen and nutrients to the cells. Native cartilage is avascular and thrives on diffusion, which in turn depends on proximity to circulation. Here, we investigate whether a gridded three-dimensional (3D) bioprinted construct would allow ingrowth of blood vessels and thus prove a functional concept for vascularization of bioengineered tissue. Twenty 10 × 10 × 3-mm 3Dbioprinted nanocellulose constructs containing human nasal chondrocytes or cell-free controls were subcutaneously implanted in 20 nude mice. Over the next 3 months, the mice were sequentially imaged with a 7 T small-animal MRI system, and the diffusion and perfusion parameters were analyzed. The chondrocytes survived and proliferated, and the shape of the constructs was well preserved. The diffusion coefficient was high and well preserved over time. The perfusion and diffusion patterns shown by MRI suggested that blood vessels develop over time in the 3D bioprinted constructs; the vessels were confirmed by histology and immunohistochemistry. We conclude that 3D bioprinted tissue with a gridded structure allows ingrowth of blood vessels and has the potential to be vascularized from the host. This is an essential step to take bioengineered tissue from the bench to clinical practice.

3D Printing: Advancement in Biogenerative Engineering to Combat Shortage of Organs and Bioapplicable Materials.

Organ or cell transplantation is medically evaluated for end-stage failure saving or extending the lives of thousands of patients who are suffering from organ failure disorders. The unavailability of adequate organs for transplantation to meet the existing demand is a major challenge in the medical field. This led to day-day-increase in the number of patients on transplant waiting lists as well as in the number of patients dying while on the queue. Recently, technological advancements in the field of biogenerative engineering have the potential to regenerate tissues and, in some cases, create new tissues and organs. In this context, major advances and innovations are being made in the fields of tissue engineering and regenerative medicine which have a huge impact on the scientific community is three-dimensional bioprinting (3D bioprinting) of tissues and organs. Besides this, the decellularization of organs and using this as a scaffold for generating new organs through the recellularization process shows promising results. This review discussed about current approaches for tissue and organ engineering including methods of scaffold designing, recent advances in 3D bioprinting, organs regenerated successfully using 3D printing, and extended application of 3D bioprinting technique in the field of medicine. Besides this, information about commercially available 3D printers has also been included in this article.Lay SummaryToday’s need for organs for the transplantation process in order to save a patient’s life or to enhance the survival rate of diseased one is the prime concern among the scientific community. Recent, advances in the field of biogenerative engineering have the potential to regenerate tissues and create organs compatible with the patient’s body. In this context, major advances and innovations are being made in the fields of tissue engineering and regenerative medicine which have a huge impact on the scientific community is three-dimensional bioprinting (3D bioprinting) of tissues and organs. Besides this, the decellularization of organs and using this as a scaffold for generating new organs through the recellularization process shows promising results. This review dealt with the current approaches for tissue and organ engineering including methods of scaffold designing, recent advances in 3D bioprinting, organs regenerated successfully using 3D printing, and extended application of 3D bioprinting technique in the field of medicine. Furthermore, information about commercially available 3D printers has also been included in this article.

Short review on formulation of the bioinks for tendon and ligament 3D bioprinting

This review focuses on advances in the field of tendon bioprinting and ligament tissue engineering, as well as a discussion of the various mechanisms underlying the tendon and ligament tissue. All methods (cellular, non-cellular and both) aim to find complex living tissues capable of growing and maturing, using the same basic technology. To date, co-printing of cellular and non-cellular material has been identified as the most promising method for muscle applications because materials with good performance and good mechanical strength can be integrated within the same structure. Bioprinting publications for tendon and ligament applications are a developing field, and a detailed discussion of current challenges and future ideas is also presented in this review.

Tunable Human Myocardium Derived Decellularized Extracellular Matrix for 3D Bioprinting and Cardiac Tissue Engineering.

The generation of 3D tissue constructs with multiple cell types and matching mechanical properties remains a challenge in cardiac tissue engineering. Recently, 3D bioprinting has become a powerful tool to achieve these goals. Decellularized extracellular matrix (dECM) is a common scaffold material due to providing a native biochemical environment. Unfortunately, dECM’s low mechanical stability prevents usage for bioprinting applications alone. In this study, we developed bioinks composed of decellularized human heart ECM (dhECM) with either gelatin methacryloyl (GelMA) or GelMA-methacrylated hyaluronic acid (MeHA) hydrogels dual crosslinked with UV light and microbial transglutaminase (mTGase). We characterized the bioinks’ mechanical, rheological, swelling, printability, and biocompatibility properties. Composite GelMA–MeHA–dhECM (GME) hydrogels demonstrated improved mechanical properties by an order of magnitude compared to the GelMA–dhECM (GE) hydrogels. All hydrogels were extrudable and compatible with human induced pluripotent stem cell derived cardiomyocytes (iCMs) and human cardiac fibroblasts (hCFs). Tissue-like beating of the printed constructs with striated sarcomeric alpha-actinin and connexin 43 expression was observed. The order of magnitude difference between the elastic modulus of these hydrogel composites offers applications in in vitro modeling of the myocardial infarct boundary. Here, as a proof of concept, we created an infarct boundary region with control over the mechanical properties along with the cellular and macromolecular content through printing iCMs with GE bioink and hCFs with GME bioink.

Microfluidic Printing of Tunable Hollow Microfibers for Vascular Tissue Engineering.

Bioprinting of vascular tissues holds great potential in tissue engineering and regenerative medicine. However, challenges remain in fabricating biocompatible and versatile scaffolds for the rapid engineering of vascular tissues and vascularized organs. Here, we report novel bioink-enabled microfluidic printing of tunable hollow microfibers for the rapid formation of blood vessels. By compositing biomaterials including sodium alginate, gelatin methacrylate (GelMA), and glycidyl-methacrylate silk fibroin (SilkMA), we prepared a novel composite bioink with excellent printability and biocompatibility. This composite bioink can be printed into hollow microfibers with tunable dimensions using a microfluidic co-axial printing. After seeding human umbilical vein endothelial cells (HUVEC) into the hollow chambers via a microfluidic prefusion device, these cells can adhere to, grow, proliferate, and then cover the internal surface of the printed hollow scaffolds to form vessel-like tissue structures within three days. By combining the unique composite bioink, microfluidic printing of vascular scaffolds, and microfluidic cell seeding and culturing, our strategy can fabricate vascular-like tissue structures with high viability and tunable dimension within three days. The presented method may engineer in vitro vasculatures for the broad applications in basic research and translational medicine including in vitro disease models, tissue microcirculation, and tissue transplantation.

Blood Flow Within Bioengineered 3D Printed Vascular Constructs Using the Porcine Model.

Recently developed biofabrication technologies are enabling the production of three-dimensional engineered tissues containing vascular networks which can deliver oxygen and nutrients across large tissue volumes. Tissues at this scale show promise for eventual regenerative medicine applications; however, the implantation and integration of these constructs in vivo remains poorly studied. Here, we introduce a surgical model for implantation and direct in-line vascular connection of 3D printed hydrogels in a porcine arteriovenous shunt configuration. Utilizing perfusable poly(ethylene glycol) diacrylate (PEGDA) hydrogels fabricated through projection stereolithography, we first optimized the implantation procedure in deceased piglets. Subsequently, we utilized the arteriovenous shunt model to evaluate blood flow through implanted PEGDA hydrogels in non-survivable studies. Connections between the host femoral artery and vein were robust and the patterned vascular channels withstood arterial pressure, permitting blood flow for 6 h. Our study demonstrates rapid prototyping of a biocompatible and perfusable hydrogel that can be implanted in vivo as a porcine arteriovenous shunt, suggesting a viable surgical approach for in-line implantation of bioprinted tissues, along with design considerations for future in vivo studies. We further envision that this surgical model may be broadly applicable for assessing whether biomaterials optimized for 3D printing and cell function can also withstand vascular cannulation and arterial blood pressure. This provides a crucial step toward generated transplantable engineered organs, demonstrating successful implantation of engineered tissues within host vasculature.

3D bioprinting and microscale organization of vascularized tissue constructs using collagen-based bioink.

Bioprinting three-dimensional (3D) tissue equivalents have progressed tremendously over the last decade. 3D bioprinting is currently being employed to develop larger and more physiologic tissues, and it is of particular interest to generate vasculature in biofabricated tissues to aid better perfusion and transport of nutrition. Having an advantage over manual culture systems by bringing together biological scaffold materials and cells in precise 3D spatial orientation, bioprinting could assist in placing endothelial cells in specific spatial locations within a 3D matrix to promote vessel formation at these predefined areas. Hence, in the present study, we investigated the use of bioprinting to generate tissue-level capillary-like networks in biofabricated tissue constructs. First, we developed a bioink using collagen type-1 supplemented with xanthan gum (XG) as a thickening agent. Using a commercial extrusion-based multi-head bioprinter and collagen-XG bioink, the component cells were spatially assembled, wherein the endothelial cells were bioprinted in a lattice pattern and sandwiched between bioprinted fibroblasts layers. 3D bioprinted constructs thus generated were stable, and maintained structural shape and form. Post-print culture of the bioprinted tissues resulted in endothelial sprouting and formation of interconnected capillary-like networks within the lattice pattern and between the fibroblast layers. Bioprinter-assisted spatial placement of endothelial cells resulted in fabrication of patterned prevascularized constructs that enable potential regenerative applications in the future.

3D Bioprinting-Based Vascularized Tissue Models Mimicking Tissue-Specific Architecture and Pathophysiology for in vitro Studies.

A wide variety of experimental models including 2D cell cultures, model organisms, and 3D in vitro models have been developed to understand pathophysiological phenomena and assess the safety and efficacy of potential therapeutics. In this sense, 3D in vitro models are an intermediate between 2D cell cultures and animal models, as they adequately reproduce 3D microenvironments and human physiology while also being controllable and reproducible. Particularly, recent advances in 3D in vitro biomimicry models, which can produce complex cell structures, shapes, and arrangements, can more similarly reflect in vivo conditions than 2D cell culture. Based on this, 3D bioprinting technology, which enables to place the desired materials in the desired locations, has been introduced to fabricate tissue models with high structural similarity to the native tissues. Therefore, this review discusses the recent developments in this field and the key features of various types of 3D-bioprinted tissues, particularly those associated with blood vessels or highly vascularized organs, such as the heart, liver, and kidney. Moreover, this review also summarizes the current state of the three categories: (1) chemical substance treatment, (2) 3D bioprinting of lesions, and (3) recapitulation of tumor microenvironments (TME) of 3D bioprinting-based disease models according to their disease modeling approach. Finally, we propose the future directions of 3D bioprinting approaches for the creation of more advanced in vitro biomimetic 3D tissues, as well as the translation of 3D bioprinted tissue models to clinical applications.

Publisher's Note: “Biomechanical factors in three-dimensional tissue bioprinting” [Appl. Phys Rev. 7, 041319 (2020)

Publisher's Note: “Biomechanical factors in three-dimensional tissue bioprinting” [Appl. Phys Rev. <b>7</b>, 041319 (2020)

Tissue Engineering Through 3D Bioprinting to Recreate and Study Bone Disease.

The applications of 3D bioprinting are becoming more commonplace. Since the advent of tissue engineering, bone has received much attention for the ability to engineer normal bone for tissue engraftment or replacement. While there are still debates on what materials comprise the most durable and natural replacement of normal tissue, little attention is given to recreating diseased states within the bone. With a better understanding of the cellular pathophysiology associated with the more common bone diseases, these diseases can be scaled down to a more throughput way to test therapies that can reverse the cellular pathophysiology. In this review, we will discuss the potential of 3D bioprinting of bone tissue in the following disease states: osteoporosis, Paget’s disease, heterotopic ossification, osteosarcoma, osteogenesis imperfecta, and rickets disease. The development of these 3D bioprinted models will allow for the advancement of novel therapy testing resulting in possible relief to these chronic diseases.