3D bioprinted aged human post-infarct myocardium tissue model.

Background and Aims: Fibrotic tissue formed after myocardial infarction (MI) can be as detrimental as MI itself. However, current in vitro cardiac fibrosis models fail to recapitulate the complexities of post-MI tissue. Moreover, although MI and subsequent fibrosis is most prominent in the aged population, the field suffers from inadequate aged tissue models. Herein, an aged human post-MI tissue model, representing the native microenvironment weeks after initial infarction, is engineered using three-dimensional bioprinting via creation of individual bioinks to specifically mimic three distinct regions: remote, border, and scar. Methods: The aged post-MI tissue model is engineered through combination of gelatin methacryloyl, methacrylated hyaluronic acid, aged type I collagen, and photoinitiator at variable concentrations with different cell types, including aged human induced pluripotent stem cell-derived cardiomyocytes, endothelial cells, cardiac fibroblasts, and cardiac myofibroblasts, by introducing a methodology which utilizes three printheads of the bioprinter to model aged myocardium. Then, using cell-specific proteins, the cell types that comprised each region are confirmed using immunofluorescence. Next, the beating characteristics are analyzed. Finally, the engineered aged post-MI tissue model is used as a benchtop platform to assess the therapeutic effects of stem cell-derived extracellular vesicles on the scar region. Results: As a result, high viability (>74%) was observed in each region of the printed model. Constructs demonstrated functional behavior, exhibiting a beating velocity of 6.7 µm/s and a frequency of 0.3 Hz. Finally, the effectiveness of hiPSC-EV and MSC-EV treatment was assessed. While hiPSC-EV treatment showed no significant changes, MSC-EV treatment notably increased cardiomyocyte beating velocity, frequency, and confluency, suggesting a regenerative potential. Conclusion: In conclusion, we envision that our approach of modeling post-MI aged myocardium utilizing three printheads of the bioprinter may be utilized for various applications in aged cardiac microenvironment modeling and testing novel therapeutics.

An Experimental and Numerical Investigation of Cardiac Tissue-Patch Interrelation.

Tissue engineered cardiac patches have great potential as a regenerative therapy for myocardial infarction. Yet, the mutual interaction of cardiac patches with healthy tissue has not been completely understood. Here, we investigated the impact of acellular and cellular patches on a beating two-dimensional (2D) cardiac cell layer, and the effect of the beating of this layer on the cells encapsulated in the patch. We cultured human-induced pluripotent stem cell-derived cardiomyocytes (iCMs) on a coverslip and placed gelatin methacryloyl hydrogel alone or with encapsulated iCMs to create acellular and cellular patches, respectively. When the acellular patch was placed on the cardiac cell layer, the beating characteristics and Ca+2 handling properties reduced, whereas placing the cellular patch restored these characteristics. To better understand the effects of the cyclic contraction and relaxation induced by the beating cardiac cell layer on the patch placed on top of it, a simulation model was developed, and the calculated strain values were in agreement with the values measured experimentally. Moreover, this dynamic culture induced by the beating 2D iCM layer on the iCMs encapsulated in the cellular patch improved their beating velocity and frequency. Additionally, the encapsulated iCMs were observed to be coupled with the underlying beating 2D iCM layer. Overall, this study provides a detailed investigation on the mutual relationship of acellular/cellular patches with the beating 2D iCM layer, understanding of which would be valuable for developing more advanced cardiac patches.

Myocardial infarction from a tissue engineering and regenerative medicine point of view: A comprehensive review on models and treatments.

In the modern world, myocardial infarction is one of the most common cardiovascular diseases, which are responsible for around 18 million deaths every year or almost 32% of all deaths. Due to the detrimental effects of COVID-19 on the cardiovascular system, this rate is expected to increase in the coming years. Although there has been some progress in myocardial infarction treatment, translating pre-clinical findings to the clinic remains a major challenge. One reason for this is the lack of reliable and human representative healthy and fibrotic cardiac tissue models that can be used to understand the fundamentals of ischemic/reperfusion injury caused by myocardial infarction and to test new drugs and therapeutic strategies. In this review, we first present an overview of the anatomy of the heart and the pathophysiology of myocardial infarction, and then discuss the recent developments on pre-clinical infarct models, focusing mainly on the engineered three-dimensional cardiac ischemic/reperfusion injury and fibrosis models developed using different engineering methods such as organoids, microfluidic devices, and bioprinted constructs. We also present the benefits and limitations of emerging and promising regenerative therapy treatments for myocardial infarction such as cell therapies, extracellular vesicles, and cardiac patches. This review aims to overview recent advances in three-dimensional engineered infarct models and current regenerative therapeutic options, which can be used as a guide for developing new models and treatment strategies.

Electrically conductive 3D printed Ti3C2Tx MXene-PEG composite constructs for cardiac tissue engineering.

Tissue engineered cardiac patches have great potential as a therapeutic treatment for myocardial infarction (MI). However, for successful integration with the native tissue and proper function of the cells comprising the patch, it is crucial for these patches to mimic the ordered structure of the native extracellular matrix and the electroconductivity of the human heart. In this study, a new composite construct that can provide both conductive and topographical cues for human induced pluripotent stem cell derived cardiomyocytes (iCMs) is developed for cardiac tissue engineering applications. The constructs are fabricated by 3D printing conductive titanium carbide (Ti3C2Tx) MXene in pre-designed patterns on polyethylene glycol (PEG) hydrogels, using aerosol jet printing, at a cell-level resolution and then seeded with iCMs and cultured for one week with no signs of cytotoxicity. The results presented in this work illustrate the vital role of 3D-printed Ti3C2Tx MXene on aligning iCMs with a significant increase in MYH7, SERCA2, and TNNT2 expressions, and with an improved synchronous beating as well as conduction velocity. This study demonstrates that 3D printed Ti3C2Tx MXene can potentially be used to create physiologically relevant cardiac patches for the treatment of MI. STATEMENT OF SIGNIFICANCE: As cardiovascular diseases and specifically myocardial infarction (MI) continue to be the leading cause of death worldwide, it is critical that new clinical interventions be developed. Tissue engineered cardiac patches have shown significant potential as clinical therapeutics to promote recovery following MI. Unfortunately, current constructs lack the ordered structure and electroconductivity of native human heart. In this study, we engineered a composite construct that can provide both conductive and topographical cues for human induced pluripotent stem cell derived cardiomyocytes. By 3D printing conductive Ti3C2Tx MXene in pre-designed patterns on polyethylene glycol hydrogels, using aerosol jet printing, at a cell-level resolution, we developed tissue engineered patches that have the potential for providing a new clinical therapeutic to combat cardiovascular disease.

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.

Breast cancer models: Engineering the tumor microenvironment.

The mechanisms behind cancer initiation and progression are not clear. Therefore, development of clinically relevant models to study cancer biology and drug response in tumors is essential. In vivo models are very valuable tools for studying cancer biology and for testing drugs; however, they often suffer from not accurately representing the clinical scenario because they lack either human cells or a functional immune system. On the other hand, two-dimensional (2D) in vitro models lack the three-dimensional (3D) network of cells and extracellular matrix (ECM) and thus do not represent the tumor microenvironment (TME). As an alternative approach, 3D models have started to gain more attention, as such models offer a platform with the ability to study cell-cell and cell-material interactions parametrically, and possibly include all the components present in the TME. Here, we first give an overview of the breast cancer TME, and then discuss the current state of the pre-clinical breast cancer models, with a focus on the engineered 3D tissue models. We also highlight two engineering approaches that we think are promising in constructing models representative of human tumors: 3D printing and microfluidics. In addition to giving basic information about the TME in the breast tissue, this review article presents the state-of-the-art tissue engineered breast cancer models. STATEMENT OF SIGNIFICANCE: Involvement of biomaterials and tissue engineering fields in cancer research enables realistic mimicry of the cell-cell and cell-extracellular matrix (ECM) interactions in the tumor microenvironment (TME), and thus creation of better models that reflect the tumor response against drugs. Engineering the 3D in vitro models also requires a good understanding of the TME. Here, an overview of the breast cancer TME is given, and the current state of the pre-clinical breast cancer models, with a focus on the engineered 3D tissue models is discussed. This review article is useful not only for biomaterials scientists aiming to engineer 3D in vitro TME models, but also for cancer researchers willing to use these models for studying cancer biology and drug testing.

Dual Crosslinked Gelatin Methacryloyl Hydrogels for Photolithography and 3D Printing.

Gelatin methacryloyl (GelMA) hydrogels have been used in tissue engineering and regenerative medicine because of their biocompatibility, photopatternability, printability, and tunable mechanical and rheological properties. However, low mechanical strength limits their applications in controlled drug release, non-viral gene therapy, and tissue and disease modeling. In this work, a dual crosslinking method for GelMA is introduced. First, photolithography was used to pattern the gels through the crosslinking of methacrylate incorporated amine groups of GelMA. Second, a microbial transglutaminase (mTGase) solution was introduced in order to enzymatically crosslink the photopatterned gels by initiating a chemical reaction between the glutamine and lysine groups of the GelMA hydrogel. The results showed that dual crosslinking improved the stiffness and rheological properties of the hydrogels without affecting cell viability, when compared to single crosslinking with either ultraviolet (UV) exposure or mTGase treatment. Our results also demonstrate that when treated with mTGase, hydrogels show decreased swelling properties and better preservation of photolithographically patterned shapes. Similar effects were observed when three dimensional (3D) printed and photocrosslinked substrates were treated with mTGase. Such dual crosslinking methods can be used to improve the mechanical properties and pattern fidelity of GelMA gels, as well as dynamic control of the stiffness of tissue engineered constructs.