Nonmulberry Silk Based Ink for Fabricating Mechanically Robust Cardiac Patches and Endothelialized Myocardium-on-a-Chip Application.

Bioprinting holds great promise towards engineering functional cardiac tissue constructs for regenerative medicine and as drug test models. However, it is highly limited by the choice of inks that require maintaining a balance between the structure and functional properties associated with the cardiac tissue. In this regard, we have developed a novel and mechanically robust biomaterial-ink based on non-mulberry silk fibroin protein. The silk-based ink demonstrated suitable mechanical properties required in terms of elasticity and stiffness (~40 kPa) for developing clinically relevant cardiac tissue constructs. The ink allowed the fabrication of stable anisotropic scaffolds using a dual crosslinking method, which were able to support formation of aligned sarcomeres, high expression of gap junction proteins as connexin-43, and maintain synchronously beating of cardiomyocytes. The printed constructs were found to be non-immunogenic in vitro and in vivo. Furthermore, delving into an innovative method for fabricating a vascularized myocardial tissue-on-a-chip, the silk-based ink was used as supporting hydrogel for encapsulating human induced pluripotent stem cell derived cardiac spheroids (hiPSC-CSs) and creating perfusable vascularized channels via an embedded bioprinting technique. We confirmed the ability of silk-based supporting hydrogel towards maturation and viability of hiPSC-CSs and endothelial cells, and for applications in evaluating drug toxicity.

3D Printed Cartilage-Like Tissue Constructs with Spatially Controlled Mechanical Properties.

Developing biomimetic cartilaginous tissues that support locomotion while maintaining chondrogenic behavior is a major challenge in the tissue engineering field. Specifically, while locomotive forces demand tissues with strong mechanical properties, chondrogenesis requires a soft microenvironment. To address this challenge, 3D cartilage-like tissue is bioprinted using two biomaterials with different mechanical properties: a hard biomaterial to reflect the macromechanical properties of native cartilage, and a soft biomaterial to create a chondrogenic microenvironment. To this end, a hard biomaterial (MPa order compressive modulus) composed of an interpenetrating polymer network (IPN) of polyethylene glycol (PEG) and alginate hydrogel is developed as an extracellular matrix (ECM) with self-healing properties, but low diffusive capacity. Within this bath supplemented with thrombin, fibrinogen containing human mesenchymal stem cell (hMSC) spheroids is bioprinted forming fibrin, as the soft biomaterial (kPa order compressive modulus) to simulate cartilage's pericellular matrix and allow a fast diffusion of nutrients. The bioprinted hMSC spheroids improve viability and chondrogenic-like behavior without adversely affecting the macromechanical properties of the tissue. Therefore, the ability to print locally soft and cell stimulating microenvironments inside of a mechanically robust hydrogel is demonstrated, thereby uncoupling the micro- and macromechanical properties of the 3D printed tissues such as cartilage.

High Concentrations of Cannabidiol Induce Neurotoxicity in Neurosphere Culture System.

Recent studies have demonstrated that cannabinoids are potentially effective in the treatment of various neurological conditions, and cannabidiol (CBD), one of the most studied compounds, has been proposed as a non-toxic option. However, the adverse effects of CBD on neurodevelopmental processes have rarely been studied in cell culture systems. To better understand CBD's influence on neurodevelopment, we exposed neural progenitor cells (NPCs) to different concentrations of CBD (1 µM, 5 µM, and 10 µM). We assessed the morphology, migration, differentiation, cell death, and gene expression in 2D and 3D bioprinted models to stimulate physiological conditions more effectively. Our results showed that CBD was more toxic at higher concentrations (5 µM and 10 µM) and affected the viability of NPCs than at lower concentrations (1 µM), in both 2D and 3D models. Moreover, our study revealed that higher concentrations of CBD drastically reduced the size of neurospheres and the number of NPCs within neurospheres, impaired the morphology and mobility of neurons and astrocytes after differentiation, and reduced neurite sprouting. Interestingly, we also found that CBD alters cellular metabolism by influencing the expression of glycolytic and ß-oxidative enzymes in the early and late stages of metabolic pathways. Therefore, our study demonstrated that higher concentrations of CBD promote important changes in cellular functions that are crucial during CNS development.

3D Bioprinted Neural-Like Tissue as a Platform to Study Neurotropism of Mouse-Adapted SARS-CoV-2.

The effects of neuroinvasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) become clinically relevant due to the numerous neurological symptoms observed in Corona Virus Disease 2019 (COVID-19) patients during infection and post-COVID syndrome or long COVID. This study reports the biofabrication of a 3D bioprinted neural-like tissue as a proof-of-concept platform for a more representative study of SARS-CoV-2 brain infection. Bioink is optimized regarding its biophysical properties and is mixed with murine neural cells to construct a 3D model of COVID-19 infection. Aiming to increase the specificity to murine cells, SARS-CoV-2 is mouse-adapted (MA-SARS-CoV-2) in vitro, in a protocol first reported here. MA-SARS-CoV-2 reveals mutations located at the Orf1a and Orf3a domains and is evolutionarily closer to the original Wuhan SARS-CoV-2 strain than SARS-CoV-2 used for adaptation. Remarkably, MA-SARS-CoV-2 shows high specificity to murine cells, which present distinct responses when cultured in 2D and 3D systems, regarding cell morphology, neuroinflammation, and virus titration. MA-SARS-CoV-2 represents a valuable tool in studies using animal models, and the 3D neural-like tissue serves as a powerful in vitro platform for modeling brain infection, contributing to the development of antivirals and new treatments for COVID-19.

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes' functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups' brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% ± 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% ± 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells' native microenvironment, useful to researchers that aim to understand astrocytes' functionality and their relation to the mechanisms involved in neurological diseases.

3D culture models to study SARS-CoV-2 infectivity and antiviral candidates: From spheroids to bioprinting.

The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is receiving worldwide attention, due to the severity of the disease (COVID-19) that resulted in more than a million global deaths so far. The urgent need for vaccines and antiviral drugs is mobilizing the scientific community to develop strategies for studying the mechanisms of SARS-CoV-2 infection, replication kinetics, pathogenesis, host-virus interaction, and infection inhibition. In this work, we review the strategies of tissue engineering in the fabrication of three-dimensional (3D) models used in virology studies, which presented many advantages over conventional cell cultures, such as complex cytoarchitecture and a more physiological microenvironment. Scaffold-free (spheroids and organoids) and scaffold-based (3D scaffolding and 3D bioprinting) approach allow the biofabrication of more realistic models relevant to the pandemic, to be used as in vitro platforms for the development of new vaccines and therapies against COVID-19.

Strategies to use fibrinogen as bioink for 3D bioprinting fibrin-based soft and hard tissues.

Fibrin gel has been widely used for engineering various types of tissues due to its biocompatible nature, biodegradability, and tunable mechanical and nanofibrous structural properties. Despite their promising regenerative capacity and extensive biocompatibility with various tissue types, fibrin-based biomaterials are often notoriously known as burdensome candidates for 3D biofabrication and bioprinting. The high viscosity of fibrin (crosslinked form) hinders proper ink extrusion, and its pre-polymer form, fibrinogen, is not capable of maintaining shape fidelity. To overcome these limitations and empower fibrinogen-based bioinks for fibrin biomimetics and regenerative applications, different strategies can be practiced. The aim of this review is to report the strategies that bring fabrication compatibility to these bioinks through mixing fibrinogen with printable biomaterials, using supporting bath supplemented with crosslinking agents, and crosslinking fibrin in situ. Moreover, the review discusses some of the recent advances in 3D bioprinting of biomimetic soft and hard tissues using fibrinogen-based bioinks, and highlights the impacts of these strategies on fibrin properties, its bioactivity, and the functionality of the consequent biomimetic tissue. Statement of Significance Due to its biocompatible nature, biodegradability, and tunable mechanical and nanofibrous structural properties, fibrin gel has been widely employed in tissue engineering and more recently, used as in 3D bioprinting. The fibrinogen's poor printable properties make it difficult to maintain the 3D shape of bioprinted constructs. Our work describes the strategies employed in tissue engineering to allow the 3D bioprinting of fibrinogen-based bioinks, such as the combination of fibrinogen with printable biomaterials, the in situ fibrin crosslinking, and the use of supporting bath supplemented with crosslinking agents. Further, this review discuss the application of 3D bioprinting technology to biofabricate fibrin-based soft and hard tissues for biomedical applications, and discuss current limitations and future of such in vitro models.

Tacrolimus- and Nerve Growth Factor-Treated Allografts for Neural Tissue Regeneration.

Treatment of injured peripheral nerves, especially long-distance nerve defects, remains a significant challenge in regenerative medicine due to complex biological conditions and a lack of biomaterials for effective nerve reconstruction. Without proper treatment, nerve injury leads to motor and sensory dysfunction. Here, we have developed an efficacious nerve allograft treated with a dual drug containing acrolimus and nerve growth factor to bridge the nerve gap and achieve rapid neural tissue recovery without immunological rejection. The recovery of the structure, activity, and function of rats treated with the dual drug-treated allograft was investigated by walking track analysis and electrophysiological measurement. The sciatic functional index was measured to be -3.0 after a 12-week treatment. The nerve conduction velocity, peak latency, and peak amplitude of the nerve action potentials demonstrate the functional recovery of the nerve. To study the synergistic effect of the dual drug on the growth of neurites, a neural cell hypoxia model was created. The dual drug exhibited a high efficiency in promoting the growth of nerve cells under the nerve injury-induced hypoxic condition. The dual drug could protect the cells against antioxidative damage from hypoxia by the expression of heat shock protein, hypoxia-inducible factor, ß-tubulin, and vimentin.