Search

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.

Stereolithography (SLA) 3D printing is a rapid prototyping technique and reproducible manufacturing platform, which makes it a useful tool to develop advanced microfluidic devices for bioanalytical applications. However, limited information exists regarding the physical, chemical, and biological properties of the photocured polymers printed with SLA. This study demonstrates the characterization of a commercially available SLA 3D printed polymer to evaluate the potential presence of any time-dependent changes in material properties that may affect its ability to produce functional, capillary-action microfluidic devices. The printed polymer was analyzed with Fourier transform infrared-attenuated total reflectance, contact angle measurements, tensile test, impact test, scanning electron microscopy, and fluid flow analysis. Polymer biocompatibility was assessed with propidium iodide flow cytometry and an MTT assay for cell viability. The material characterization and biocompatibility results were then implemented to design and fabricate a self-driven capillary action microfluidic device for future use as a bioanalytical assay. This study demonstrates temporally stable mechanical properties and biocompatibility of the SLA polymer. However, surface characterization through contact angle measurements shows the polymer's wettability changes over time which indicates there is a limited postprinting period when the polymer can be used for capillary-based fluid flow. Overall, this study demonstrates the feasibility of implementing SLA as a high-throughput manufacturing method for capillary action microfluidic devices.

Parkinson's disease (PD), a progressive central nervous system disorder marked by involuntary movements, poses a significant challenge in neurodegenerative research due to the gradual degeneration of dopaminergic (DA) neurons. Early diagnosis and understanding of PD's pathogenesis could slow disease progression and improve patient management. In vitro modeling with DA neurons derived from human-induced pluripotent stem cell-derived neural progenitor cells (NPCs) offers a promising approach. These neurons can be cultured on electrospun (ES) nanofibrous polycaprolactone (PCL) scaffolds, but PCL's hydrophobic nature limits cell adhesion. We investigated the ability of ES PCL scaffolds coated with hydrophilic extracellular matrix-based biomaterials, including cell basement membrane proteins, Matrigel, and Fibrin, to enhance NPC differentiation into DA neurons. We hypothesized that fibrin-coated scaffolds would maximize differentiation based on fibrin's known benefits in neuronal tissue engineering. The scaffolds both coated and uncoated were characterized using scanning electron microscopy (SEM), transmission electron microscopy, Fourier transform infrared spectroscopy-attenuated total reflectance, and dynamic mechanical analysis to assess their properties. NPCs were seeded on the coated scaffolds, differentiated, and matured into DA neurons. Immunocytochemistry targeting tyrosine hydroxylase (TH) and SEM confirmed DA neuronal differentiation and morphological changes. Electrophysiology via microelectrode array recorded their neuronal firing. Results showed enhanced neurite extension, increased TH expression, and active electrical activity in cells on fibrin-coated scaffolds. Diluted fibrin coatings particularly promoted more pronounced neuronal differentiation and maturation. This study introduces a novel tissue-on-a-chip platform for neurodegenerative disease research using DA neurons., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

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.

Heart diseases cause over 17.9 million total deaths globally, making them the leading source of mortality. The aim of this review is to describe the characteristic mechanical, chemical and cellular properties of human cardiac tissue and how these properties can be mimicked in 3D bioprinted tissues. Furthermore, the authors review how current healthy cardiac models are being 3D bioprinted using extrusion-, laser- and inkjet-based printers. The review then discusses the pathologies of cardiac diseases and how bioprinting could be used to fabricate models to study these diseases and potentially find new drug targets for such diseases. Finally, the challenges and future directions of cardiac disease modeling using 3D bioprinting techniques are explored.

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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 → 50 mM or 25 → 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 → 50 mM in comparison with the 25 → 50 mM condition. Western Blotting showed a significant upregulation of the protein expression of the YAP1/TAZ pathway under these conditions as well (5 → 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 → 50 mM condition. The application of an ERK 1/2 inhibitor inhibited the expression of the YAP1/TAZ protein in the 5 → 50 mM condition, and this strategy may be useful in preventing and improving hyperglycemia associated cardiovascular damage and inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

3D bioprinting offers a simplified solution for the engineering of complex tissue parts for in-vitro drug discovery or, in-vivo implantation. However, significant amount of challenges exist in 3D bioprinting of neural tissues, as these are sensitive cell types to handle via extrusion bioprinting techniques. We assessed the feasibility of bioprinting human neural progenitor cells (NPCs) in 3D hydrogel lattices using a fibrinogen-alginate-chitosan bioink, previously optimized for neural-cell growth, and subsequently modified for structural support during extrusion printing, in this study. The original bioink used in this study was made by adding optimized amounts of high- and medium-viscosity alginate to the fibrinogen-chitosan-based bioink and making it extrudable under shear pressure. The mechanically robust 3D constructs promoted NPC cluster formation and maintained their morphology and viability during the entire culture period. This strategy may be useful for co-culturing of NPCs along with other cell types such as cardiac, vascular, and other cells during 3D bioprinting., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In this study, we developed three-dimensional (3D) printed annular ring-like scaffolds of hydrogel (gelatin-alginate) constructs encapsulated with a mixture of human cardiac AC16 cardiomyocytes (CMs), fibroblasts (CFs), and microvascular endothelial cells (ECs) as cardiac organoid models in preparation for investigating the role of microgravity in cardiovascular disease initiation and development. We studied the mechanical properties of the acellular scaffolds and confirmed their cell compatibility as well as heterocellular coupling for cardiac tissue engineering. Rheological analysis performed on the acellular scaffolds showed the scaffolds to be elastogenic with elastic modulus within the range of a native in vivo heart tissue. The microstructural and physicochemical properties of the scaffolds analyzed through scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy-attenuated total reflectance (ATR-FTIR) confirmed the mechanical and functional stability of the scaffolds for long-term use in in vitro cell culture studies. HL-1 cardiomyocytes bioprinted in these hydrogel scaffolds exhibited contractile functions over a sustained period of culture. Cell mixtures containing CMs, CFs, and ECs encapsulated within the 3D printed hydrogel scaffolds exhibited a significant increase in viability and proliferation over 21 days, as shown by flow cytometry analysis. Moreover, via the expression of specific cardiac biomarkers, cardiac-specific cell functionality was confirmed. Our study depicted the heterocellular cardiac cell interactions, which is extremely important for the maintenance of normal physiology of the cardiac wall in vivo and significantly increased over a period of 21 days in in vitro . This 3D bioprinted "cardiac organoid" model can be adopted to simulate cardiac environments in which cellular crosstalk in diseased pathologies like cardiac atrophy can be studied in vitro and can further be used for drug cytotoxicity screening or underlying disease mechanisms.

In this study, three types of electrospun scaffolds, including furfuryl-gelatin (f-gelatin) alone, f-gelatin with polycaprolactone (PCL) in a 1:1 ratio, and coaxial scaffolds with PCL (core) and f-gelatin (sheath), were developed for tissue engineering applications. Scaffolds were developed through single nozzle electrospinning and coaxial electrospinning, respectively, to serve as scaffolds for cardiac tissue engineering. Uniform fibrous structures were revealed in the scaffolds with significantly varying average fiber diameters of 760 ± 80 nm (f-gelatin), 420 ± 110 nm [f-gelatin and PCL (1:1)], and 810 ± 60 nm (coaxial f-gelatin > PCL) via scanning electron microscopy. The distinction between the core and the sheath of the fibers of the coaxial f-gelatin > PCL electrospun fibrous scaffolds was revealed by transmission electron microscopy. Thermal analysis and Fourier transformed infrared (FTIR) spectroscopy revealed no interactions between the polymers in the blended electrospun scaffolds. The varied blending methods led to significant differences in the elastic moduli of the electrospun scaffolds with the coaxial f-gelatin > PCL revealing the highest elastic modulus of all scaffolds (164 ± 3.85 kPa). All scaffolds exhibited excellent biocompatibility by supporting the adhesion and proliferation of human AC16 cardiomyocytes cells. The biocompatibility of the coaxial f-gelatin > PCL scaffolds with superior elastic modulus was assessed further through adhesion and functionality of human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, thereby demonstrating the potential of the coaxially spun scaffolds as an ideal platform for developing cardiac tissue-on-a-chip models. Our results demonstrate a facile approach to produce visible light cross-linkable, hybrid, biodegradable nanofibrous scaffold biomaterials, which can serve as platforms for cardiac tissue engineered models., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)