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Photocrosslinked hydrogels, such as methacrylate-modified gelatin (gelMA) and hyaluronic acid (HAMA), are widely utilized as tissue engineering scaffolds and/or drug delivery vehicles, but lack a suitable means for non-invasive, longitudinal monitoring of surgical placement, biodegradation, and drug release. Therefore, we developed a novel photopolymerizable X-ray contrast agent, methacrylate-modified gold nanoparticles (AuMA NPs), to enable covalent-linking to methacrylate-modified hydrogels (gelMA and HAMA) in one-step during photocrosslinking and non-invasive monitoring by X-ray micro-computed tomography (micro-CT). Hydrogels exhibited a linear increase in X-ray attenuation with increased Au NP concentration to enable quantitative imaging by contrast-enhanced micro-CT. The enzymatic and hydrolytic degradation kinetics of gelMA-Au NP hydrogels were longitudinally monitored by micro-CT for up to one month in vitro, yielding results that were consistent with concurrent measurements by optical spectroscopy and gravimetric analysis. Importantly, AuMA NPs did not disrupt the hydrogel network, rheology, mechanical properties, and hydrolytic stability compared with gelMA alone. GelMA-Au NP hydrogels were thus able to be bioprinted into well-defined three-dimensional architectures supporting endothelial cell viability and growth. Overall, AuMA NPs enabled the preparation of both conventional photopolymerized hydrogels and bioprinted scaffolds with tunable X-ray contrast for noninvasive, longitudinal monitoring of placement, degradation, and NP release by micro-CT.

Vascular restenosis is a major complication in recanalized arteries. Nanoparticles (NPs) have shown great promise as delivery systems in advancing strategies to treat such vascular anomalies. By enabling precise targeting, NPs can overcome the challenges of low drug efficacy and off-target effects. Here we present a biomimetic in vitro platform comprised of 3D bioprinting, nanomaterials, and perfusion technologies, to study the use of NP targeting to address endothelial overgrowth. We bioprinted 3D vascular channels at high fidelity, using gelatin methacrylate as bioink, with artery-like stiffness. Human endothelial cells (ECs) were used to endothelialize the printed channels. GFP-labelled superparamagnetic iron oxide NPs (SPIONs), loaded with the Rapamune anti-proliferative drug, were perfused through the bifurcated artery model at physiological rate. Computational modeling predicted greatest level of alterations in wall shear stress in the conduit’s junction with the artery, identifying this region prone to restenosis. A neodymium disc magnet was embedded in the printed tissue to attract the therapeutic SPIONs to the region of high risk. In vitro dynamic culture was conducted for 2 wks. We assessed cell viability, proliferation, and function using AlamarBlue and immunohistochemistry. Results showed significant targeted effect of NP delivery in reducing EC overgrowth. This platform enables design of precise targeting of therapeutics to treat a variety of cardiovascular diseases at a high spatial and temporal control.

Rapid advancements in 3D bioprinting have enabled the design of functionalized bioinks to create scaffolds with robust adhesive properties. These constructs are of great interest in cardiac tissue engineering, where the integration of grafted patch with the host myocardium, through sutures, staples, or adhesives, faces risks such as bleeding, cytotoxicity, and infection. We introduce the first generation of functional adhesive bioinks that can be bioprinted through various modalities to fabricate patch structures with intrinsic adhesive properties. A dopamine-modified hyaluronic acid methacrylate (HAMA-Dopa) and gelatin methacrylate (gelMA) composite bioink is used to create the patch via air and embedded bioprinting. Constructs are crosslinked onto a collagen sheet substrate simulating the host cardiac tissue ( Figure 1A-C ). We developed novel in vitro methods to assess adhesion properties of printed patch under shear, tension, or dynamic loading ( Figure 1C ). Our approach allowed for steady and precise application of stress to the adhesion interface. Embedded-printed HAMA-Dopa/gelMA scaffold showed significantly enhanced adhesion strength (1025 Pa) compared to gelMA (495 Pa) and HAMA (477 Pa) control groups under tension, and under shear (548 Pa vs. 234 and 239 Pa). The adhesion strength of air-printed HAMA-Dopa/gelMA constructs was markedly higher (10,128 Pa) than the embedded ones, possibly due to the more effective, in-situ crosslinking. We also investigated the dynamic adhesive properties in aqueous environment using an ex vivo beating heart model. Air-printed HAMA-Dopa/gelMA showed the greatest adhesion under wet conditions, tolerating 345,600 cycles. We further characterized printing fidelity, mechanical properties, swelling, and biocompatibility of HAMA-Dopa/gelMA constructs, demonstrating adequate functionality of this bioprinted adhesive scaffold for cardiac tissue engineering applications. Figure 1 . Summary of workflow to develop bioprinted adhesive scaffolds. A: Embedded (top) or air (bottom) bioprinting was used to create 3D patch geometries. B: Printing fidelity assessment and optimization. C: Different adhesion mechanisms were assessed using novel customized tools and approaches to apply tensile ( C-i ), shear ( C-ii ), and dynamic ( C-iii ) loading.

Cardiovascular disease is the leading global cause of death. As a possible remedy, the field of cardiac tissue engineering has emerged as a regenerative medicine approach to develop bioartificial tissue constructs that can be implanted in order to provide support to the damaged heart tissue and restore function. Currently, there is a dire clinical need for suitable biomaterials that allow for cardiac graft integration, remodeling, and endogenous tissue regeneration. A range of biomaterial strategies can be used to address this need, by incorporating natural or synthetic materials to create tissue mimics such as vascular grafts, heart valves, and cardiac patches. Cardiovascular tissue engineering mainly relies on developing biodegradable polymeric scaffolds, often supplemented with biomolecules or natural proteins, to imitate the extracellular matrix architecture and biochemical composition of native tissues, while promoting tissue integration and regeneration. Considering the highly complex architecture of targeted cardiovascular tissues, there is a need for high-precision manufacturing techniques to generate cardiac grafts in a reproducible fashion. While methods such as 3D casting, electrospinning, and self-assembly have been traditionally used in the field, several new entrants, such as additive manufacturing (3D bioprinting) techniques, have shown great promise. This review is aimed at assessing the current state of the art of biomaterials and manufacturing techniques used in cardiovascular tissue engineering.

Vascular atresia are often treated via transcatheter recanalization or surgical vascular anastomosis due to congenital malformations or coronary occlusions. The cellular response to vascular anastomosis or recanalization is, however, largely unknown and current techniques rely on restoration rather than optimization of flow into the atretic arteries. An improved understanding of cellular response post anastomosis may result in reduced restenosis. Here, an in vitro platform is used to model anastomosis in pulmonary arteries (PAs) and for procedural planning to reduce vascular restenosis. Bifurcated PAs are bioprinted within 3D hydrogel constructs to simulate a reestablished intervascular connection. The PA models are seeded with human endothelial cells and perfused at physiological flow rate to form endothelium. Particle image velocimetry and computational fluid dynamics modeling show close agreement in quantifying flow velocity and wall shear stress within the bioprinted arteries. These data are used to identify regions with greatest levels of shear stress alterations, prone to stenosis. Vascular geometry and flow hemodynamics significantly affect endothelial cell viability, proliferation, alignment, microcapillary formation, and metabolic bioprofiles. These integrated in vitro-in silico methods establish a unique platform to study complex cardiovascular diseases and can lead to direct clinical improvements in surgical planning for diseases of disturbed flow.

Current strategies for regeneration of large bone fractures yield limited clinical success mainly due to poor integration and healing. Multidisciplinary approaches in design and development of functional tissue engineered scaffolds are required to overcome these translational challenges. Here, a new generation of hyperelastic bone (HB) implants, loaded with superparamagnetic iron oxide nanoparticles (SPIONs), are 3D bioprinted and their regenerative effect on large non-healing bone fractures is studied. Scaffolds are bioprinted with the geometry that closely correspond to that of the bone defect, using an osteoconductive, highly elastic, surgically friendly bioink mainly composed of hydroxyapatite. Incorporation of SPIONs into HB bioink results in enhanced bacteriostatic properties of bone grafts while exhibiting no cytotoxicity. In vitro culture of mouse embryonic cells and human osteoblast-like cells remain viable and functional up to 14 days on printed HB scaffolds. Implantation of damage-specific bioprinted constructs into a rat model of femoral bone defect demonstrates significant regenerative effect over the 2-week time course. While no infection, immune rejection, or fibrotic encapsulation is observed, HB grafts show rapid integration with host tissue, ossification, and growth of new bone. These results suggest a great translational potential for 3D bioprinted HB scaffolds, laden with functional nanoparticles, for hard tissue engineering applications.

Background: COVID-19 poses a risk to the endoscopic skull base surgeon. Significant efforts to improving safety have beenemployed, including the use of personal protective equipment (PPE), preoperative COVID-19 testing, and recently the useof a modified surgical mask barrier. The objective of our cadaveric study is to reduce the risks of pathogen transmissionwith the use of readily available and innovative equipment as a means of creating a three-dimensional (3D) printed mask inaddition to a trocar system for use during endoscopic skull base surgery. Methods: Our study presents the ventilated upper airway endoscopic procedure mask (VPM), a 3D-printed mask with ananterior aperture fitted with a surgical glove with ports designed to allow for surgical instrumentation and side ports toaccommodate suction ventilation and an endotracheal tube. As an alternative, a modified laparoscopic surgery trocar servedas a port for instruments, and, on the contralateral side, rubber tubing was used over the endoscrub sheath to create anairtight seal. On cadaveric models, various surgical approaches were performed which each device to assess for surgicalmaneuverability including septoplasty, maxillary antrostomy, total ethmoidectomy, frontal sinusotomy, and sphenoidotomy.Surgical freedom (SF) and aerosolization were tested in both modalities. Results: The VPM allowed the unimpeded performance of the above-mentioned surgical approaches, using both two- andfour-handed techniques, with excellent surgical maneuverability and access, while maintaining a continuous facial seal.Debris and smoke were ventilated with a size 8-Frazier tip suction, and ambient gas was additionally suctioned by thenegative-pressure VPM. SF with the VPM was equivalent (1.0 cm2 ) to the SF with no mask. With the presence of a posterior septectomy, the endoscopic endonasal trocar (EET) system was effective for posteriorsurgical procedures, allowing access to critical paramedian structures (clivus, sella, and tuberculum), and afforded asuperior surgical seal, but was limited in terms of visualization and maneuverability during anterior approaches. For thisreason, aerosolization studies were only performed in the posterior nasal cavity with the EET. SF was found to be reducedby 55% (SF = 0.45 cm2 ), and surgery anterior to the sphenoid rostrum was limited when using the trocar system. Aerosolization was reduced using both the VPM and EET. During anterior surgery, the VPM reduced particle spillage by 86%.During posterior surgery, the VPM reduced overall particle spillage by 71%, while the EET reduced spillage by 97%.Conclusion: The VPM mask allows for a sealed surgical barrier during endoscopic skull base surgery and may play a criticalrole in advancing skull base surgery in the COVID-19 era. The EET may be a useful alternative for binarial procedures in theposterior nasal cavity and in instances where 3D printing is not available. Additionally, considering the ongoing pandemic,PPE shortage is a serious concern. The VPM may serve as a renewable alternative with various applications. In the future,our team will be performing studies to validate these preliminary findings.

The heart is the first organ to develop in the human embryo through a series of complex chronological processes, many of which critically rely on the interplay between cells and the dynamic microenvironment. Tight spatiotemporal regulation of these interactions is key in heart development and diseases. Due to suboptimal experimental models, however, there is little knowledge on the role of microenvironmental cues in the heart development. This study investigates the use of 3D bioprinting and perfusion bioreactor technologies to create bioartificial constructs that can serve as high-fidelity models of the developing human heart. Bioprinted hydrogel-based, anatomically accurate models of the human embryonic heart tube (e-HT, day 22) and fetal left ventricle (f-LV, week 33) were perfused and analyzed both computationally and experimentally using ultrasound and magnetic resonance imaging. Results demonstrated comparable and precise flow hemodynamic patterns within the 3D space. We demonstrated human endothelial cell growth and endothelialization within the bioprinted e-HT and f-LV constructs, which varied significantly in varying cardiac geometries and under flow. This study introduces the first generation of anatomically accurate, 3D functional models of developing human heart at different stages. This platform enables precise tuning of microenvironmental factors, such as flow and geometry, thus allowing the study of normal developmental processes and underlying diseases.

Three-dimensional (3D) bioprinting of hydrogel-based constructs at adequate consistency and reproducibility can be obtained through a compromise between the hydrogel's inherent instability and printing fidelity. There is an increasing demand to develop bioprinting modalities that enable high-fidelity fabrication of 3D hydrogel structures that closely correspond to the envisioned design. In this work, we performed a systematic, in-depth characterization and optimization of embedded 3D bioprinting to create 3D gelatin-methacryloyl (gelMA) structures with highly controlled fidelity using Carbopol as suspension bath. The role of various embedded printing process parameters in bioprinting fidelity was investigated using a combination of experimental and theoretical approaches. We examined the effect of rheological properties of gelMA and Carbopol at varying concentrations, as well as printing conditions on the volumetric flow rate of gelMA bioink. Printing speed was examined and optimized to successfully print gelMA into the support bath at varying Carbopol concentrations. Printing fidelity was characterized in terms of printed strand diameter, uniformity, angle, and area. The optimal Carbopol solution that retained filament shape at highest fidelity was determined. The efficacy of developed bioprinting approach was then demonstrated by fabricating 3D hydrogel constructs with varying geometries and visualized using an advanced synchrotron-based imaging technique. We also investigated the influence of the Carbopol medium on cross-linking and the resulting stiffness of gelMA constructs. Finally, in vitro cytotoxicity of the developed bioprinting approach was assessed by printing human umbilical vein endothelial cells encapsulated in the gelMA bioink. These results demonstrate the significance of the close interplay between bioink-support bath rheology and printing parameters and help to establish an optimized workflow for creating 3D hydrogel structures with high fidelity and cytocompatibility via embedded bioprinting techniques. This robust platform could further expand the application of bioprinted soft tissue constructs in a wide variety of biomedical applications.

Recent advances in 3D printing allow the generation of tissue models that provide a unique in vitro platform to recapitulate and analyze complex cardiovascular anatomies, including the developing human heart. While it is challenging to quantify the 3D blood flow dynamics in the developing fetal heart, with spatial resolution on the order of a millimeter or less, printed tissue constructs allow such 3D measurements by reconstructing multiple 2D measurements under constant flow conditions. Such functional phantoms can offer critical insights into hemodynamics in healthy and diseased states, including complicated flow conditions in congenital heart defects during the early stage of prenatal development. In this study, ultrasound-compatible tissue-mimicking phantoms were fabricated using water-soluble 3D printed fetal human heart models, i.e., an embryonic heart tube at day 22 and a fetal left ventricle at week 33 after fertilization. An ultrasound-based 3D velocimetry method was used to measure the flow velocity magnitude and direction in these anatomically-accurate phantom models of the linear heart tube and the left ventricle.

Single ventricle physiology is a complex disease state requiring multiple open-heart surgeries to achieve stable hemodynamics. For patients with abnormalities in the pulmonary arteries (PAs), these must be remedied before the patient can be a candidate for such palliations. Transcatheter techniques could rescue this subset of single ventricle patients through intervascular PA connections, allowing a high-risk population to ultimately achieve stable pulmonary blood flow. However, there is currently no in vitro platform to model transcatheter processes for anastomosis, particularly to palliate single ventricle defects. This project utilizes 3D bioprinting and perfusion bioreactor technologies to develop a functional in vitro biological device to model severely stenotic PAs of single ventricle patients. Human endothelial (ECs) & smooth muscle (SMCs) cells embedded in extracellular matrix bioink are used in a multi-material bioprinting approach to create 3D bilayer vascular structures with controlled geometry and flow. In collaboration with CHOA Cardiac Catheterization Laboratory , stent devices are deployed in the printed model to re-establish intervascular connection. Healthy, stenotic, and stented tissues are cultured via a bioreactor and analyzed for flow hemodynamics by echo PIV and 4D MR imaging. Cell viability, proliferation, and endothelialization of printed vessels, plus EC-SMC interplay were closely monitored pre- and post- anastomosis, to identify the effect of geometry and flow on cellular overgrowth. This advanced planning enables a subset of single ventricle patients, otherwise not eligible, to ultimately accept further palliative strategies.

The ability to design and develop tissue and organ-specific bioinks with tailored biophysical and chemical properties has the potential to advance 3D bioprinting applications from the basic science realm to a truly clinical suite of bioengineering tools. In the field of cardiovascular tissue engineering, such bioinks could be used to improve cardiac disease treatment outcomes, serve as functional implantable tissues to restore lost function in the heart and/or the vasculature, as well as the next-generation drug screening platforms. Specifically, design and fabrication of nanomaterial-enhanced cardiac bioinks can facilitate the design and fabrication of novel biocompatible materials which are tailored to specific organ and pathologies. Using various nano-biomaterials, specific chemical and mechanical properties of natural and synthetic bioinks can be tailored to generate complex structures which resemble the structural hierarchy and chemical composition of the native heart tissues. Versatile and tunable bioinks that can support advanced construct design through functionalization with nanomaterials and other small molecules will have important implications for next-generation bioprinting and biofabrication of cardiovascular tissues. Here, we cover some of the more promising aspects of nanomaterial-functionalized bioinks, targeted specifically toward cardiovascular applications.

Background Tetralogy of Fallot with major aortopulmonary collateral arteries is a heterogeneous form of pulmonary artery (PA) stenosis that requires multiple forms of intervention. We present a patient‐specific in vitro platform capable of sustained flow that can be used to train proceduralists and surgical teams in current interventions, as well as in developing novel therapeutic approaches to treat various vascular anomalies. Our objective is to develop an in vitro model of PA stenosis based on patient data that can be used as an in vitro phantom to model cardiovascular disease and explore potential interventions. Methods and Results From patient‐specific scans obtained via computer tomography or 3‐dimensional (3D) rotational angiography, we generated digital 3D models of the arteries. Subsequently, in vitro models of tetralogy of Fallot with major aortopulmonary collateral arteries were first 3D printed using biocompatible resins and next bioprinted using gelatin methacrylate hydrogel to simulate neonatal vasculature or second‐order branches of an older patient with tetralogy of Fallot with major aortopulmonary collateral arteries. Printed models were used to study creation of extraluminal connection between an atretic PA and a major aortopulmonary collateral artery using a catheter‐based interventional method. Following the recanalization, engineered PA constructs were perfused and flow was visualized using contrast agents and x‐ray angiography. Further, computational fluid dynamics modeling was used to analyze flow in the recanalized model. Conclusions New 3D‐printed and computational fluid dynamics models for vascular atresia were successfully created. We demonstrated the unique capability of a printed model to develop a novel technique for establishing blood flow in atretic vessels using clinical imaging, together with 3D bioprinting–based tissue engineering techniques. Additive biomanufacturing technologies can enable fabrication of functional vascular phantoms to model PA stenosis conditions that can help develop novel clinical applications.

Purpose of Review Tissue engineering has expanded into a highly versatile manufacturing landscape that holds great promise for advancing cardiovascular regenerative medicine. In this review, we provide a summary of the current state-of-the-art bioengineering technologies used to create functional cardiac tissues for a variety of applications in vitro and in vivo. Recent Findings Studies over the past few years have made a strong case that tissue engineering is one of the major driving forces behind the accelerating fields of patient-specific regenerative medicine, precision medicine, compound screening, and disease modeling. To date, a variety of approaches have been used to bioengineer functional cardiac constructs, including biomaterial-based, cell-based, and hybrid (using cells and biomaterials) approaches. While some major progress has been made using cellular approaches, with multiple ongoing clinical trials, cell-free cardiac tissue engineering approaches have also accomplished multiple breakthroughs, although drawbacks remain. Summary This review summarizes the most promising methods that have been employed to generate cardiovascular tissue constructs for basic science or clinical applications. Further, we outline the strengths and challenges that are inherent to this field as a whole and for each highlighted technology.

3D bioprinting techniques have shown great promise in various fields of tissue engineering and regenerative medicine. Yet, creating a tissue construct that faithfully represents the tightly regulated composition, microenvironment, and function of native tissues is still challenging. Among various factors, biomechanics of bioprinting processes play fundamental roles in determining the ultimate outcome of manufactured constructs. This review provides a comprehensive and detailed overview on various biomechanical factors involved in tissue bioprinting, including those involved in pre, during, and post printing procedures. In preprinting processes, factors including viscosity, osmotic pressure, and injectability are reviewed and their influence on cell behavior during the bioink preparation is discussed, providing a basic guidance for the selection and optimization of bioinks. In during bioprinting processes, we review the key characteristics that determine the success of tissue manufacturing, including the rheological properties and surface tension of the bioink, printing flow rate control, process-induced mechanical forces, and the in situ cross-linking mechanisms. Advanced bioprinting techniques, including embedded and multi-material printing, are explored. For post printing steps, general techniques and equipment that are used for characterizing the biomechanical properties of printed tissue constructs are reviewed. Furthermore, the biomechanical interactions between printed constructs and various tissue/cell types are elaborated for both in vitro and in vivo applications. The review is concluded with an outlook regarding the significance of biomechanical processes in tissue bioprinting, presenting future directions to address some of the key challenges faced by the bioprinting community.

3D bioprinting is a form of additive manufacturing tailored toward creating biological constructs with precise spatial control. As an extension of conventional 3D printing with a variety of materials such as polymers, ceramics, and metals, 3D bioprinting focuses on building viable, biomimetic products that can be used to replicate, improve, or substitute functional tissues. Driven by the field of tissue engineering, advancements in 3D bioprinting have enabled greater print resolutions, more customizable bioinks, and faster biomanufacturing speeds, which are critical when handling delicate biological substances. To date, researchers and engineers have creatively employed 3D bioprinting to combat cardiovascular disease, the most prevalent cause of death in the Western world. In the realm of cardiovascular medicine, 3D bioprinting has seen manifold applications including surgical models, cardiac patches, computational and theoretical models, heart valves, and stents. These technologies vary in terms of their extent of development, ranging from in vitro modeling to clinical therapies. While surgical models are most widely used in a clinical setting, other bioprinted models are rapidly developing with promising results. Overall, this chapter focuses on the clinical applications of 3D bioprinting aimed toward understanding, augmenting, or replacing cardiovascular tissues and organs.

Three-dimensional (3D) cardiac tissue bioprinting sits at the crossroads of cardiovascular biology, additive manufacturing, and materials science and engineering. As such this area of research requires know-how from each of the above fields and has to address some significant challenges when it comes to translational success and novel developments. Of specific interest are advances in bioink formulation, where biochemical and mechanical properties of hydrogels must be precisely tailored to produce cardiac-specific bioinks that could support functional heart tissue for diverse in vitro and in vivo applications. Hydrogel-based bioinks must fulfill several key biophysical and biochemical requirements, before, during, and post-printing processes. This chapter explores in depth the parameters that are necessary for a successful bioink solution that can support cardiac tissue structure and function.

In pluripotent stem cell differentiation, embryoid bodies (EBs) provide a three-dimensional [3D] multicellular precursor in lineage specification. The internal structure of EBs is not well characterized yet is predicted to be an important parameter to differentiation. Here, we use custom SU-8 molds to generate transparent lithography-templated arrays of polydimethylsiloxane (LTA-PDMS) for high throughput analysis of human embryonic stem cell (hESC) EB formation and internal architecture. EBs formed in 200 and 500 μm diameter microarray wells by use of single cells, 2D clusters, or 3D early aggregates were compared. We observe that 200 μm EBs are monocystic versus 500 μm multicystic EBs that contain macro, meso and microsized cysts. In adherent differentiation of 500 μm EBs, the multicystic character impairs the 3D to 2D transition creating non-uniform monolayers. Our findings reveal that EB core structure has a size-dependent character that influences its architecture and cell population uniformity during early differentiation.

The realization of personalized medicine through human induced pluripotent stem cell (iPSC) technology can be advanced by transcriptomics, epigenomics, and bioinformatics that inform on genetic pathways directing tissue development and function. When possible, population diversity should be included in new studies as resources become available. Previously we derived replicate iPSC lines of African American, Hispanic-Latino and Asian self-designated ethnically diverse (ED) origins with normal karyotype, verified teratoma formation, pluripotency biomarkers, and tri-lineage in vitro commitment. Here we perform bioinformatics of RNA-Seq and ChIP-seq pluripotency data sets for two replicate Asian and Hispanic-Latino ED-iPSC lines that reveal differences in generation of contractile cardiomyocytes but similar and robust differentiation to multiple neural, pancreatic, and smooth muscle cell types. We identify shared and distinct genes and contributing pathways in the replicate ED-iPSC lines to enhance our ability to understand how reprogramming to iPSC impacts genes and pathways contributing to cardiomyocyte contractility potential.

Three-dimensional (3D) cardiac tissue bioprinting occupies a critical crossroads position between the fields of materials engineering, cardiovascular biology, 3D printing, and rational organ replacement design. This complex area of research therefore requires expertise from all those disciplines and it poses some unique considerations that must be accounted for. One of the chief hurdles is that there is a relatively limited systematic organization of the physical and chemical characteristics of bioinks that would make them applicable to cardiac bioprinting. This is of great significance, as heart tissue is functionally complex and the in vivo extracellular niche is under stringent controls with little room for variability before a cardiomyopathy manifests. This review explores the critical parameters that are necessary for biologically relevant bioinks to successfully be leveraged for functional cardiac tissue engineering, which can have applications in in vitro heart tissue models, cardiotoxicity studies, and implantable constructs that can be used to treat a range of cardiomyopathies, or in regenerative medicine.

The human genome with all its ethnic variations contributes to differences in human development, aging, disease, repair and response to medical treatments and is an exciting area of research and clinical study. The availability of well-characterized ethnically diverse stem cell lines is limited and has not kept pace with other advances in stem cell research. Here we derived xenofree ethnically diverse-human induced pluripotent stem cell (ED-iPSC) lines from fibroblasts obtained from individuals of African American, Hispanic-Latino, Asian and Caucasian ethnic origin and have characterized the lines under a uniform platform for comparative analysis. Derived ED-iPSC lines are low passage number and evaluated in vivo by teratoma formation and in vitro by high throughput microarray analysis of EB formation and early differentiation for tri-lineage commitment to endoderm, ectoderm and mesoderm. These new xenofree ED-iPSC lines represent a well-characterized valuable resource with potential for use in future research in drug discovery or clinical investigations.

In pluripotent stem cell differentiation, embryoid bodies (EBs) provide a three-dimensional [3D] multicellular precursor in lineage specification. The internal structure of EBs is not well characterized yet is predicted to be an important parameter to differentiation. Here, we use custom SU-8 molds to generate transparent lithography-templated arrays of polydimethylsiloxane (LTA-PDMS) for high throughput analysis of human embryonic stem cell (hESC) EB formation and internal architecture. EBs formed in 200 and 500 μm diameter microarray wells by use of single cells, 2D clusters, or 3D early aggregates were compared. We observe that 200 μm EBs are monocystic versus 500 μm multicystic EBs that contain macro, meso and microsized cysts. In adherent differentiation of 500 μm EBs, the multicystic character impairs the 3D to 2D transition creating non-uniform monolayers. Our findings reveal that EB core structure has a size-dependent character that influences its architecture and cell population uniformity during early differentiation.