Sequential Fabrication of a Three-Layer Retina-like Structure.

Tissue engineering is considered a promising approach to treating advanced degenerative maculopathies such as nonexudative age-related macular degeneration (AMD), the leading cause of blindness worldwide. The retina consists of several hierarchical tissue layers, each of which is supported by a layer underneath. Each of these layers has a different morphology and requires distinct conditions for proper assembly. In fact, a prerequisite step for the assembly of each of these layers is the organization of the layer underneath. Advanced retinal degeneration includes degeneration of the other retina layers, including the choroid, the retinal pigmented epithelium (RPE), and the photoreceptors. Here, we report a step-by-step fabrication process of a three-layer retina-like structure. The process included the 3D printing of a choroid-like structure in an extracellular matrix (ECM) hydrogel, followed by deposition of the RPE monolayer. After the formation of the blood vessel-RPE interface, the photoreceptor cells were deposited to interact with the RPE layer. At the end of the fabrication process, each layer was characterized for its morphology and expression of specific markers, and the integration of the three-layer retina was evaluated. We envision that such a retina-like structure may be able to attenuate the deterioration of a degenerated retina and improve engraftment and regeneration. This retinal implant may potentially be suitable for a spectrum of macular degenerative diseases for which there are currently no cures and may save millions from complete blindness.

Biodegradable, Biocompatible, and Implantable Multifunctional Sensing Platform for Cardiac Monitoring.

Cardiac monitoring after heart surgeries is crucial for health maintenance and detecting postoperative complications early. However, current methods like rigid implants have limitations, as they require performing second complex surgeries for removal, increasing infection and inflammation risks, thus prompting research for improved sensing monitoring technologies. Herein, we introduce a nanosensor platform that is biodegradable, biocompatible, and integrated with multifunctions, suitable for use as implants for cardiac monitoring. The device has two electrochemical biosensors for sensing lactic acid and pH as well as a pressure sensor and a chemiresistor array for detecting volatile organic compounds. Its biocompatibility with myocytes has been tested in vitro, and its biodegradability and sensing function have been proven with ex vivo experiments using a three-dimensional (3D)-printed heart model and 3D-printed cardiac tissue patches. Moreover, an artificial intelligence-based predictive model was designed to fuse sensor data for more precise health assessment, making it a suitable candidate for clinical use. This sensing platform promises impactful applications in the realm of cardiac patient care, laying the foundation for advanced life-saving developments.

Modified ECM-Based Bioink for 3D Printing of Multi-Scale Vascular Networks.

The survival and function of tissues depend on appropriate vascularization. Blood vessels of the tissues supply oxygen, and nutrients and remove waste and byproducts. Incorporating blood vessels into engineered tissues is essential for overcoming diffusion limitations, improving tissue function, and thus facilitating the fabrication of thick tissues. Here, we present a modified ECM bioink, with enhanced mechanical properties and endothelial cell-specific adhesion motifs, to serve as a building material for 3D printing of a multiscale blood vessel network. The bioink is composed of natural ECM and alginate conjugated with a laminin adhesion molecule motif (YIGSR). The hybrid hydrogel was characterized for its mechanical properties, biochemical content, and ability to interact with endothelial cells. The pristine and modified hydrogels were mixed with induced pluripotent stem cells derived endothelial cells (iPSCs-ECs) and used to print large blood vessels with capillary beds in between.

Imageable AuNP-ECM Hydrogel Tissue Implants for Regenerative Medicine.

In myocardial infarction, a blockage in one of the coronary arteries leads to ischemic conditions in the left ventricle of the myocardium and, therefore, to significant death of contractile cardiac cells. This process leads to the formation of scar tissue, which reduces heart functionality. Cardiac tissue engineering is an interdisciplinary technology that treats the injured myocardium and improves its functionality. However, in many cases, mainly when employing injectable hydrogels, the treatment may be partial because it does not fully cover the diseased area and, therefore, may not be effective and even cause conduction disorders. Here, we report a hybrid nanocomposite material composed of gold nanoparticles and an extracellular matrix-based hydrogel. Such a hybrid hydrogel could support cardiac cell growth and promote cardiac tissue assembly. After injection of the hybrid material into the diseased area of the heart, it could be efficiently imaged by magnetic resonance imaging (MRI). Furthermore, as the scar tissue could also be detected by MRI, a distinction between the diseased area and the treatment could be made, providing information about the ability of the hydrogel to cover the scar. We envision that such a nanocomposite hydrogel may improve the accuracy of tissue engineering treatment.

Post-Maturation Reinforcement of 3D-Printed Vascularized Cardiac Tissues.

Despite advances in biomaterials engineering, a large gap remains between the weak mechanical properties that can be achieved with natural materials and the strength of synthetic materials. Here, a method is presented for reinforcing an engineered cardiac tissue fabricated from differentiated induced pluripotent stem cells (iPSCs) and an extracellular matrix (ECM)-based hydrogel in a manner that is fully biocompatible. The reinforcement occurs as a post-fabrication step, which allows for the use of 3D-printing technology to generate thick, fully cellularized, and vascularized cardiac tissues. After tissue assembly and during the maturation process in a soft hydrogel, a small, tissue-penetrating reinforcer is deployed, leading to a significant increase in the tissue's mechanical properties. The tissue's robustness is demonstrated by injecting the tissue in a simulated minimally invasive procedure and showing that the tissue is functional and undamaged at the nano-, micro-, and macroscales.

A universal, multimodal cell-based biosensing platform for optimal intracellular action potential recording.

Intracellular recording of action potentials is an essential mean for studying disease mechanisms, and for electrophysiological studies, particularly in excitable cells as cardiomyocytes or neurons. Current strategies to obtain intracellular recordings include three-dimensional (3D) nanoelectrodes that can effectively penetrate the cell membrane and achieve high-quality intracellular recordings in a minimally invasive manner, or transient electroporation of the membrane that can yield temporary intracellular access. However, the former strategy requires a complicated and costly fabrication process, and the latter strategy suffers from high dependency on the method of application of electroporation, yielding inconsistent, suboptimal recordings. These factors hinder the high throughput use of these strategies in electrophysiological studies. In this work, we propose an advanced cell-based biosensing platform that relies on electroporation to produce consistent, high-quality intracellular recordings. The suggested universal system can be integrated with any electrode array, and it enables tunable electroporation with controllable pulse parameters, while the recorded potentials can be analyzed in real time to provide instantaneous feedback on the electroporation effectiveness. This integrated system enables the user to perform electroporation, record and assess the obtained signals in a facile manner, to ultimately achieve stable, reliable, intracellular recording. Moreover, the proposed platform relies on microelectrode arrays which are suited for large-scale production, and additional modules that are low-cost. Using this platform, we demonstrate the tuning of electroporation pulse width, pulse number, and amplitude, to achieve effective electroporation and high-quality intracellular recordings. This integrated platform has the potential to enable larger scale, repeatable, convenient, and low-cost electrophysiological studies.

Chondroitin sulfate-AuNRs electroactive scaffolds for on-demand release of biofactors.

Controlled release systems are often integrated into polymeric scaffolds to supply essential biofactors to trigger physiological processes in engineered tissues. Here, we report the modification of chondroitin sulfate (CS) electroactive polymer with gold nanorods (AuNRs) to create hybrid macroporous scaffolds for enhanced on-demand release of growth factors and cytokines. The mechanical properties, porosity and degradation of the hybrid scaffolds were evaluated, and the viability and functionality of seeded cardiac cells were assessed. Following, the ability to control the release of the enzyme lysozyme, and the cytokine, stromal cell-derived factor 1 (SDF-1) by applying electrical stimulation, was demonstrated. The AuNRs were able to increase the current through the scaffolds, providing an efficient on-off release profile of SDF-1, which resulted in higher migration of cells expressing CXCR4 receptor. Finally, the engineered scaffolds were transplanted in rats and SDF-1 was released daily by electrical stimulation, promoting blood vessel-forming cell infiltration and vascularization. We envision that gold nanoparticles and other conducting nanomaterials can be incorporated into different electroactive materials to improve their capabilities not only for tissue engineering applications, but for a variety of biomedical applications, where enhanced electrical stimulation is needed.

Regenerating the Injured Spinal Cord at the Chronic Phase by Engineered iPSCs-Derived 3D Neuronal Networks.

Cell therapy using induced pluripotent stem cell-derived neurons is considered a promising approach to regenerate the injured spinal cord (SC). However, the scar formed at the chronic phase is not a permissive microenvironment for cell or biomaterial engraftment or for tissue assembly. Engineering of a functional human neuronal network is now reported by mimicking the embryonic development of the SC in a 3D dynamic biomaterial-based microenvironment. Throughout the in vitro cultivation stage, the system's components have a synergistic effect, providing appropriate cues for SC neurogenesis. While the initial biomaterial supported efficient cell differentiation in 3D, the cells remodeled it to provide an inductive microenvironment for the assembly of functional SC implants. The engineered tissues are characterized for morphology and function, and their therapeutic potential is investigated, revealing improved structural and functional outcomes after acute and chronic SC injuries. Such technology is envisioned to be translated to the clinic to rewire human injured SC.

Bioengineering approaches to treat the failing heart: from cell biology to 3D printing.

Successfully engineering a functional, human, myocardial pump would represent a therapeutic alternative for the millions of patients with end-stage heart disease and provide an alternative to animal-based preclinical models. Although the field of cardiac tissue engineering has made tremendous advances, major challenges remain, which, if properly resolved, might allow the clinical implementation of engineered, functional, complex 3D structures in the future. In this Review, we provide an overview of state-of-the-art studies, challenges that have not yet been overcome and perspectives on cardiac tissue engineering. We begin with the most clinically relevant cell sources used in this field and discuss the use of topological, biophysical and metabolic stimuli to obtain mature phenotypes of cardiomyocytes, particularly in relation to organized cytoskeletal and contractile intracellular structures. We then move from the cellular level to engineering planar cardiac patches and discuss the need for proper vascularization and the main strategies for obtaining it. Finally, we provide an overview of several different approaches for the engineering of volumetric organs and organ parts - from whole-heart decellularization and recellularization to advanced 3D printing technologies.

Injectable Nanocomposite Implants Reduce ROS Accumulation and Improve Heart Function after Infarction.

In a myocardial infarction, blood supply to the left ventricle is abrogated due to blockage of one of the coronary arteries, leading to ischemia, which further triggers the generation of reactive oxygen species (ROS). These sequential processes eventually lead to the death of contractile cells and affect the integrity of blood vessels, resulting in the formation of scar tissue. A new heart therapy comprised of cardiac implants encapsulated within an injectable extracellular matrix-gold nanoparticle composite hydrogel is reported. The particles on the collagenous fibers within the hydrogel promote fast transfer of electrical signal between cardiac cells, leading to the functional assembly of the cardiac implants. The composite hydrogel is shown to absorb reactive oxygen species in vitro and in vivo in mice ischemia reperfusion model. The reduction in ROS levels preserve cardiac tissue morphology and blood vessel integrity, reduce the scar size and the inflammatory response, and significantly prevent the deterioration of heart function.

Nanoengineered Peptide-Based Antimicrobial Conductive Supramolecular Biomaterial for Cardiac Tissue Engineering.

Owing to their dynamic nature and ordered architecture, supramolecular materials strikingly resemble organic components of living systems. Although short-peptide self-assembled nanostructured hydrogels are regarded as intriguing supramolecular materials for biotechnology, their application is often limited due to their low stability and considerable challenge of combining other desirable properties. Herein, a di-Fmoc-based hydrogelator containing the cell-adhesive Arg-Gly-Asp (RGD) fragment that forms a mechanically stable, self-healing hydrogel is designed. Molecular dynamics simulation reveals the presence of RGD segments on the surface of the hydrogel fibers, highlighting their cell adherence capacity. Aiming to impart conductivity, the 3D network of the hydrogel is further nanoengineered by incorporating polyaniline (PAni). The composite hydrogels demonstrate semiconductivity, excellent antibacterial activity, and DNA binding capacity. Cardiac cells grown on the surface of the composite hydrogels form functional synchronized monolayers. Taken together, the combination of these attributes in a single hydrogel suggests it as an exceptional candidate for functional supramolecular biomaterial designed for electrogenic tissue engineering.

3D Tissue and Organ Printing-Hope and Reality.

Three-dimensional (3D) bioprinting is an emerging, groundbreaking strategy in tissue engineering, allowing the fabrication of living constructs with an unprecedented degree of complexity and accuracy. While this technique greatly facilitates the structuring of native tissue-like architectures, many challenges still remain to be faced. In this review, the fruits of recent research that demonstrate how advanced bioprinting technologies, together with inspiring creativity, can be used to address these challenges are presented and discussed. Next, the future of the field is discussed, in terms of expected developments, as well as possible directions toward the realization of the vision of fully functional, engineered tissues, and organs. Last, a few hypothetical scenarios for the role 3D bioprinting may play in future tissue engineering are depicted, with an emphasis on its impact on tomorrow's regenerative medicine.

One-Step 3D Printing of Heart Patches with Built-In Electronics for Performance Regulation.

Three dimensional (3D) printing of heart patches usually provides the ability to precisely control cell location in 3D space. Here, one-step 3D printing of cardiac patches with built-in soft and stretchable electronics is reported. The tissue is simultaneously printed using three distinct bioinks for the cells, for the conducting parts of the electronics and for the dielectric components. It is shown that the hybrid system can withstand continuous physical deformations as those taking place in the contracting myocardium. The electronic patch is flexible, stretchable, and soft, and the electrodes within the printed patch are able to monitor the function of the engineered tissue by providing extracellular potentials. Furthermore, the system allowed controlling tissue function by providing electrical stimulation for pacing. It is envisioned that such transplantable patches may regain heart contractility and allow the physician to monitor the implant function as well as to efficiently intervene from afar when needed.

Transparent support media for high resolution 3D printing of volumetric cell-containing ECM structures.

3D bioprinting may revolutionize the field of tissue engineering by allowing fabrication of bio-structures with a high degree of complexity, fine architecture and heterogeneous composition. The printing substances in these processes are mostly based on biomaterials and living cells. As such, they generally possess weak mechanical properties and thus must be supported during fabrication in order to prevent the collapse of large, volumetric multi-layered printouts. In this work, we characterize a uniquely formulated media used to support printing of extracellular matrix-based biomaterials. We show that a hybrid material, comprised of calcium-alginate nanoparticles and xanthan gum, presents superb qualities that enable printing at high resolution of down to 10 microns, allowing fabrication of complex constructs and cellular structures. This hybrid also presents an exclusive combination of desirable properties such as biocompatibility, high transparency, stability at a wide range of temperatures and amenability to delicate extraction procedures. Moreover, as fabrication of large, volumetric biological structures may require hours and even days to accomplish, we have demonstrated that the hybrid medium can support prolonged, precise printing for at least 18 h. All these qualities make it a promising support medium for 3D printing of tissues and organs.

Injectable Cardiac Cell Microdroplets for Tissue Regeneration.

One of the strategies for heart regeneration includes cell delivery to the defected heart. However, most of the injected cells do not form quick cell-cell or cell-matrix interactions, therefore, their ability to engraft at the desired site and improve heart function is poor. Here, the use of a microfluidic system is reported for generating personalized hydrogel-based cellular microdroplets for cardiac cell delivery. To evaluate the system's limitations, a mathematical model of oxygen diffusion and consumption within the droplet is developed. Following, the microfluidic system's parameters are optimized and cardiac cells from neonatal rats or induced pluripotent stem cells are encapsulated. The morphology and cardiac specific markers are assessed and cell function within the droplets is analyzed. Finally, the cellular droplets are injected to mouse gastrocnemius muscle to validate cell retention, survival, and maturation within the host tissue. These results demonstrate the potential of this approach to generate personalized cellular microtissues, which can be injected to distinct regions in the body for treating damaged tissues.

Unusual Two-Step Assembly of a Minimalistic Dipeptide-Based Functional Hypergelator.

Self-assembled peptide hydrogels represent the realization of peptide nanotechnology into biomedical products. There is a continuous quest to identify the simplest building blocks and optimize their critical gelation concentration (CGC). Herein, a minimalistic, de novo dipeptide, Fmoc-Lys(Fmoc)-Asp, as an hydrogelator with the lowest CGC ever reported, almost fourfold lower as compared to that of a large hexadecapeptide previously described, is reported. The dipeptide self-assembles through an unusual and unprecedented two-step process as elucidated by solid-state NMR and molecular dynamics simulation. The hydrogel is cytocompatible and supports 2D/3D cell growth. Conductive composite gels composed of Fmoc-Lys(Fmoc)-Asp and a conductive polymer exhibit excellent DNA binding. Fmoc-Lys(Fmoc)-Asp exhibits the lowest CGC and highest mechanical properties when compared to a library of dipeptide analogues, thus validating the uniqueness of the molecular design which confers useful properties for various potential applications.

Engineering Smart Hybrid Tissues with Built-In Electronics.

One of the major hurdles faced in tissue engineering is the inability to monitor and control the function of an engineered tissue following transplantation. Recent years have seen major developments in the field by integrating electronics within engineered tissues. Previously, the most common types of devices integrated into the body used to be pacemakers and deep brain stimulation electrodes that are stiff and non-compliant; the advent of ultra-thin and flexible electronics has brought forth a significant expansion of the field. Recent developments have enabled interfacing electronics onto, into, and within all tissues and organs with minimal adverse reactions. These have introduced the ability to engineer tissues with built-in electronics that allow for remote monitoring and regulation of tissue function. In this review, we discuss the development of technologies that allowed for the formation of tissue-electronics hybrids and give an overview of the existing examples of these hybrid "cyborg" tissues.

Collagenase Nanoparticles Enhance the Penetration of Drugs into Pancreatic Tumors.

Overexpressed extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDAC) limits drug penetration into the tumor and is associated with poor prognosis. Here, we demonstrate that a pretreatment based on a proteolytic-enzyme nanoparticle system disassembles the dense PDAC collagen stroma and increases drug penetration into the pancreatic tumor. More specifically, the collagozome, a 100 nm liposome encapsulating collagenase, was rationally designed to protect the collagenase from premature deactivation and prolonged its release rate at the target site. Collagen is the main component of the PDAC stroma, reaching 12.8 ± 2.3% vol in diseased mice pancreases, compared to 1.4 ± 0.4% in healthy mice. Upon intravenous injection of the collagozome, ∼1% of the injected dose reached the pancreas over 8 h, reducing the level of fibrotic tissue to 5.6 ± 0.8%. The collagozome pretreatment allowed increased drug penetration into the pancreas and improved PDAC treatment. PDAC tumors, pretreated with the collagozome followed by paclitaxel micelles, were 87% smaller than tumors pretreated with empty liposomes followed by paclitaxel micelles. Interestingly, degrading the ECM did not increase the number of circulating tumor cells or metastasis. This strategy holds promise for degrading the extracellular stroma in other diseases as well, such as liver fibrosis, enhancing tissue permeability before drug administration.

Composite of Peptide-Supramolecular Polymer and Covalent Polymer Comprises a New Multifunctional, Bio-Inspired Soft Material.

Peptide-based supramolecular hydrogels are utilized as functional materials in tissue engineering, axonal regeneration, and controlled drug delivery. The Arg-Gly-Asp (RGD) ligand based supramolecular gels have immense potential in this respect, as this tripeptide is known to promote cell adhesion. Although several RGD-based supramolecular hydrogels have been reported, most of them are devoid of adequate resilience and long-range stability for in vitro cell culture. In a quest to improve the mechanical properties of these tripeptide-based gels and their durability in cell culture media, the Fmoc-RGD hydrogelator is non-covalently functionalized with a biocompatible and biodegradable polymer, chitosan, resulting in a composite hydrogel with enhanced gelation rate, mechanical properties and cell media durability. Interestingly, both Fmoc-RGD and Fmoc-RGD/chitosan composite hydrogels exhibit thixotropic properties. The utilization of the Fmoc-RGD/chitosan composite hydrogel as a scaffold for 2D and 3D cell cultures is demonstrated. The composite hydrogel is found to have notable antibacterial activity, which stems from the inherent antibacterial properties of chitosan. Furthermore, the composite hydrogels are able to produce ultra-small, mono-dispersed, silver nanoparticles (AgNPs) arranged on the fiber axis. Therefore, the authors' approach harnesses the attributes of both the supramolecular-polymer (Fmoc-RGD) and the covalent-polymer (chitosan) component, resulting in a composite hydrogel with excellent potential.