Microcomputed Tomography for the Microstructure Evaluation of 3D Bioprinted Scaffolds.

This study implemented the application of microcomputed tomography (micro-CT) as a characterization technique for the study and investigation of the microstructure of 3D scaffold structures produced via three-dimensional bioprinting (3DBP). The study focused on the preparation, characterization, and cytotoxicity analysis of gold nanoparticles (Au-NPs) incorporated into 3DBP hydrogels for micro-CT evaluation. The Au-NPs were characterized by using various techniques, including UV-vis spectrometry, dynamic light scattering (DLS), zeta potential measurement, and transmission electron microscopy (TEM). The characterization results confirmed the successful coating of the Au-NPs with 2 kDa methoxy-PEG and revealed their spherical shape with a mean core diameter of 66 nm. Cytotoxicity analysis using live-dead fluorescent microscopy indicated that all tested Au-NP solutions were nontoxic to AC16 cardiomyocytes in both 2D and 3D culture conditions. Scanning electron microscopy (SEM) showed distinguishable differences in image contrast and intensity between samples with and without Au-NPs, with high concentrations of Au-NPs displaying nanoparticle aggregates. Micro-CT imaging demonstrated that scaffolds containing Au-NPs depicted enhanced imaging resolution and quality, allowing for visualization of the microstructure. The 3D reconstruction of scaffold structures from micro-CT imaging using Dragonfly software further supported the improved visualization. Mechanical analysis revealed that the addition of Au-NPs enhanced the mechanical properties of acellular scaffolds, including their elastic moduli and complex viscosity, but the presence of cells led to biodegradation and reduced mechanical strength. These findings highlight the successful preparation and characterization of Au-NPs, their nontoxic nature in both 2D and 3D culture conditions, their influence on imaging quality, and the impact on the mechanical properties of 3D-printed hydrogels. These results contribute to the development of functional and biocompatible materials for tissue engineering and regenerative medicine applications.

A Semi-Three-Dimensional Bioprinted Neurocardiac System for Tissue Engineering of a Cardiac Autonomic Nervous System Model.

In this study, we designed a tissue-engineered neurocardiac model to help us examine the role of neuronal regulation and confirm the importance of neural innervation techniques for the regeneration of cardiac tissue. A three-dimensional (3D) bioprinted neurocardiac scaffold composed of a mixture of gelatin-alginate and alginate-genipin-fibrin hydrogels was developed with a 2:1 ratio of AC16 cardiomyocytes (CMs) and retinoic acid-differentiated SH-SY5Y neuronal cells (NCs) respectively. A unique semi-3D bioprinting approach was adopted, where the CMs were mixed in the cardiac bioink and printed using an anisotropic accordion design to mimic the physiological tissue architecture in vivo. The voids in this 3D structure were methodically filled in using a NC-gel mixture and crosslinked. Confocal fluorescent imaging using microtubule-associated protein 2 (MAP-2) and anticholine acetyltransferase (CHAT) antibodies for labeling the NCs and the MyoD1 antibody for the CMs revealed functional coupling between the two cell types in the final crosslinked structure. These data confirmed the development of a relevant neurocardiac model that could be used to study neurocardiac modulation under physiological and pathological conditions.

Demonstration of doxorubicin's cardiotoxicity and screening using a 3D bioprinted spheroidal droplet-based system.

Doxorubicin (DOX) is a highly effective anthracycline chemotherapy agent effective in treating a broad range of life-threatening malignancies but it causes cardiotoxicity in many subjects. While the mechanism of its cardiotoxic effects remains elusive, DOX-related cardiotoxicity can lead to heart failure in patients. In this study, we investigated the effects of DOX-induced cardiotoxicity on human cardiomyocytes (CMs) using a three-dimensional (3D) bioprinted cardiac spheroidal droplet based-system in comparison with the traditional two-dimensional cell (2D) culture model. The effects of DOX were alleviated with the addition of N-acetylcysteine (NAC) and Tiron. Caspase-3 activity was quantified, and reactive oxygen species (ROS) production was measured using dihydroethidium (DHE) staining. Application of varying concentrations of DOX (0.4 µM-1 µM) to CMs revealed a dose-specific response, with 1 µM concentration imposing maximum cytotoxicity and 0.22 ± 0.11% of viable cells in 3D samples versus 1.02 ± 0.28% viable cells in 2D cultures, after 5 days of culture. Moreover, a flow cytometric analysis study was conducted to study CMs proliferation in the presence of DOX and antioxidants. Our data support the use of a 3D bioprinted cardiac spheroidal droplet as a robust and high-throughput screening model for drug toxicity. In the future, this 3D spheroidal droplet model can be adopted as a human-derived tissue-engineered equivalent to address challenges in other various aspects of biomedical pre-clinical research.

Inhibition of ERK 1/2 pathway downregulates YAP1/TAZ signaling in human cardiomyocytes exposed to hyperglycemic conditions.

Hyperglycemia-mediated cardiac dysfunction is an acute initiator in the development of vascular complications, leading to cardiac fibrosis. To investigate the effects of hyperglycemia-mediated changes in cardiomyocytes, cells were cultured in-vitro under normoglycemic (5 mM or 25 mM D-glucose) and hyperglycemic (5 â†’ 50 mM or 25 â†’ 50 mM D-glucose) conditions, respectively. After 24-h of hyperglycemic exposure, cells were collected for RNA-sequencing (RNA-seq) studies to further investigate the differentially expressed genes (DEG) related to inflammation and fibrosis in samples cultured under hyperglycemic-in comparison with normoglycemic-conditions. Western Blotting was done to evaluate the protein expression of YAP1/TAZ under hyperglycemia induced stress conditions, as it is known to be involved in fibrotic and vascular inflammatory-mediated conditions. RNA-seq revealed the DEG of multiple targets including matrix metalloproteinases and inflammatory mediators, whose expression was significantly altered in the 5 â†’ 50 mM in comparison with the 25 â†’ 50 mM condition. Western Blotting showed a significant upregulation of the protein expression of the YAP1/TAZ pathway under these conditions as well (5 â†’ 50 mM). To further probe the relationship between the inflammatory extracellular-signal-regulated kinase (ERK 1/2) and its downstream effects on YAP1/TAZ expression we studied the effect of inhibition of the ERK 1/2 signaling cascade in the 5 â†’ 50 mM condition. The application of an ERK 1/2 inhibitor inhibited the expression of the YAP1/TAZ protein in the 5 â†’ 50 mM condition, and this strategy may be useful in preventing and improving hyperglycemia associated cardiovascular damage and inflammation.

Engineering approaches for cardiac organoid formation and their characterization.

Cardiac organoids are 3-dimensional (3D) structures composed of tissue or niche-specific cells, obtained from diverse sources, encapsulated in either a naturally derived or synthetic, extracellular matrix scaffold, and include exogenous biochemical signals such as essential growth factors. The overarching goal of developing cardiac organoid models is to establish a functional integration of cardiomyocytes with physiologically relevant cells, tissues, and structures like capillary-like networks composed of endothelial cells. These organoids used to model human heart anatomy, physiology, and disease pathologies in vitro have the potential to solve many issues related to cardiovascular drug discovery and fundamental research. The advent of patient-specific human-induced pluripotent stem cell-derived cardiovascular cells provide a unique, single-source approach to study the complex process of cardiovascular disease progression through organoid formation and incorporation into relevant, controlled microenvironments such as microfluidic devices. Strategies that aim to accomplish such a feat include microfluidic technology-based approaches, microphysiological systems, microwells, microarray-based platforms, 3D bioprinted models, and electrospun fiber mat-based scaffolds. This article discusses the engineering or technology-driven practices for making cardiac organoid models in comparison with self-assembled or scaffold-free methods to generate organoids. We further discuss emerging strategies for characterization of the bio-assembled cardiac organoids including electrophysiology and machine-learning and conclude with prospective points of interest for engineering cardiac tissues in vitro.