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.

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.

Modular assembly of thick multifunctional cardiac patches.

In cardiac tissue engineering cells are seeded within porous biomaterial scaffolds to create functional cardiac patches. Here, we report on a bottom-up approach to assemble a modular tissue consisting of multiple layers with distinct structures and functions. Albumin electrospun fiber scaffolds were laser-patterned to create microgrooves for engineering aligned cardiac tissues exhibiting anisotropic electrical signal propagation. Microchannels were patterned within the scaffolds and seeded with endothelial cells to form closed lumens. Moreover, cage-like structures were patterned within the scaffolds and accommodated poly(lactic-co-glycolic acid) (PLGA) microparticulate systems that controlled the release of VEGF, which promotes vascularization, or dexamethasone, an anti-inflammatory agent. The structure, morphology, and function of each layer were characterized, and the tissue layers were grown separately in their optimal conditions. Before transplantation the tissue and microparticulate layers were integrated by an ECM-based biological glue to form thick 3D cardiac patches. Finally, the patches were transplanted in rats, and their vascularization was assessed. Because of the simple modularity of this approach, we believe that it could be used in the future to assemble other multicellular, thick, 3D, functional tissues.

A Stretchable and Flexible Cardiac Tissue-Electronics Hybrid Enabling Multiple Drug Release, Sensing, and Stimulation.

Replacement of the damaged scar tissue created by a myocardial infarction is the goal of cardiac tissue engineering. However, once the implanted tissue is in place, monitoring its function is difficult and involves indirect methods, while intervention necessarily requires an invasive procedure and available medical attention. To overcome this, methods of integrating electronic components into engineered tissues have been recently presented. These allow for remote monitoring of tissue function as well as intervention through stimulation and controlled drug release. Here, an improved hybrid microelectronic tissue construct capable of withstanding the dynamic environment of the beating heart without compromising electronic or mechanical functionality is reported. While the reported system is enabled to sense the function of the engineered tissue and provide stimulation for pacing, an electroactive polymer on the electronics enables it to release multiple drugs in parallel. It is envisioned that the integration of microelectronic devices into engineered tissues will provide a better way to monitor patient health from afar, as well as provide facile, more exact methods to control the healing process.

Gold Nanorod-Based Engineered Cardiac Patch for Suture-Free Engraftment by Near IR.

Although cardiac patches hold a promise for repairing the infarcted heart, their integration with the myocardium by sutures may cause further damage to the diseased organ. To address this issue, we developed facile and safe, suture-free technology for the attachment of engineered tissues to organs. Here, nanocomposite scaffolds comprised of albumin electrospun fibers and gold nanorods (AuNRs) were developed. Cardiac cells were seeded within the scaffolds and assembled into a functioning patch. The engineered tissue was then positioned on the myocardium and irradiated with a near IR laser (808 nm). The AuNRs were able to absorb the light and convert it to thermal energy, which locally changed the molecular structure of the fibrous scaffold, and strongly, but safely, attached it to the wall of the heart. Such hybrid biomaterials can be used in the future to integrate any engineered tissue with any defected organs, while minimizing the risk of additional injury for the patient, caused by the conventional stitching methods.

A microfluidic chip containing multiple 3D nanofibrous scaffolds for culturing human pluripotent stem cells.

In microfluidics-based lab-on-a-chip systems, which are used for investigating the effect of drugs and growth factors on cells, the latter are usually cultured within the device's channels in two-dimensional, and not in their optimal three-dimensional (3D) microenvironment. Herein, we address this shortfall by designing a microfluidic system, comprised of two layers. The upper layer of the system consists of multiple channels generating a gradient of soluble factors. The lower layer is comprised of multiple wells, each deposited with 3D, nanofibrous scaffold. We first used a mathematical model to characterize the fluid flow within the system. We then show that induced pluripotent stem cells can be seeded within the 3D scaffolds and be exposed to a well-mixed gradient of soluble factors. We believe that utilizing such system may enable in the future to identify new differentiation factors, investigate drug toxicity, and eventually allow to perform analyses on patient-specific tissues, in order to fit the appropriate combination and concentration of drugs.

Engineered hybrid cardiac patches with multifunctional electronics for online monitoring and regulation of tissue function.

In cardiac tissue engineering approaches to treat myocardial infarction, cardiac cells are seeded within three-dimensional porous scaffolds to create functional cardiac patches. However, current cardiac patches do not allow for online monitoring and reporting of engineered-tissue performance, and do not interfere to deliver signals for patch activation or to enable its integration with the host. Here, we report an engineered cardiac patch that integrates cardiac cells with flexible, freestanding electronics and a 3D nanocomposite scaffold. The patch exhibited robust electronic properties, enabling the recording of cellular electrical activities and the on-demand provision of electrical stimulation for synchronizing cell contraction. We also show that electroactive polymers containing biological factors can be deposited on designated electrodes to release drugs in the patch microenvironment on demand. We expect that the integration of complex electronics within cardiac patches will eventually provide therapeutic control and regulation of cardiac function.

Effect of fiber diameter on the assembly of functional 3D cardiac patches.

The cardiac ECM has a unique 3D structure responsible for tissue morphogenesis and strong contractions. It is divided into three fiber groups with specific roles and distinct dimensions; nanoscale endomysial fibers, perimysial fibers with a diameter of 1 µm, and epimysial fibers, which have a diameter of several micrometers. We report here on our work, where distinct 3D fibrous scaffolds, each of them recapitulating the dimension scales of a single fiber population in the heart matrix, were fabricated. We have assessed the mechanical properties of these scaffolds and the contribution of each fiber population to cardiomyocyte morphogenesis, tissue assembly and function. Our results show that the nanoscale fiber scaffolds were more elastic than the microscale scaffolds, however, cardiomyocytes cultured on microscale fiber scaffolds exhibited enhanced spreading and elongation, both on the single cell and on the engineered tissue levels. In addition, lower fibroblast proliferation rates were observed on these microscale topographies. Based on the collected data we have fabricated composite scaffolds containing micro and nanoscale fibers, promoting superior tissue morphogenesis without compromising tissue contraction. Cardiac tissues, engineered within these composite scaffolds exhibited superior function, including lower excitation threshold and stronger contraction forces than tissue engineered within the single-population fiber scaffolds.

Gold nanoparticle-decellularized matrix hybrids for cardiac tissue engineering.

Decellularized matrices are valuable scaffolds for engineering functional cardiac patches for treating myocardial infarction. However, the lack of quick and efficient electrical coupling between adjacent cells may jeopardize the success of the treatment. To address this issue, we have deposited gold nanoparticles on fibrous decellularized omental matrices and investigated their morphology, conductivity, and degradation. We have shown that cardiac cells engineered within the hybrid scaffolds exhibited elongated and aligned morphology, massive striation, and organized connexin 43 electrical coupling proteins. Finally, we have shown that the hybrid patches demonstrated superior function as compared to pristine patches, including a stronger contraction force, lower excitation threshold, and faster calcium transients.

Albumin fiber scaffolds for engineering functional cardiac tissues.

In recent years attempts to engineer contracting cardiac patches were focused on recapitulation of the myocardium extracellular microenvironment. We report here on our work, where for the first time, a three-dimensional cardiac patch was fabricated from albumin fibers. We hypothesized that since albumin fibers' mechanical properties resemble those of cardiac tissue extracellular matrix (ECM) and their biochemical character enables their use as protein carriers, they can support the assembly of cardiac tissues capable of generating strong contraction forces. Here, we have fabricated aligned and randomly oriented electrospun albumin fibers and investigated their structure, mechanical properties, and chemical nature. Our measurements showed that the scaffolds have improved elasticity as compared to synthetic electrospun PCL fibers, and that they are capable of adsorbing serum proteins, such as laminin leading to strong cell-matrix interactions. Moreover, due to the functional groups on their backbone, the fibers can be chemically modified with essential biomolecules. When seeded with rat neonatal cardiac cells the engineered scaffolds induced the assembly of aligned cardiac tissues with high aspect ratio cardiomyocytes and massive actinin striation. Compared to synthetic fibrous scaffolds, cardiac cells cultured within aligned or randomly oriented scaffolds formed functional tissues, exhibiting significantly improved function already on Day 3, including higher beating rate (P = 0.0002 and P < 0.0001, respectively), and higher contraction amplitude (P = 0.009 and P = 0.003, respectively). Collectively, our results suggest that albumin electrospun scaffolds can play a key role in contributing to the ex vivo formation of a contracting cardiac muscle tissue.

Customizable Hydrogel Coating of ECM-Based Microtissues for Improved Cell Retention and Tissue Integrity.

Overcoming the oxygen diffusion limit of approximately 200 µm remains one of the most significant and intractable challenges to be overcome in tissue engineering. The fabrication of hydrogel microtissues and their assembly into larger structures may provide a solution, though these constructs are not without their own drawbacks; namely, these hydrogels are rapidly degraded in vivo, and cells delivered via microtissues are quickly expelled from the area of action. Here, we report the development of an easily customized protocol for creating a protective, biocompatible hydrogel barrier around microtissues. We show that calcium carbonate nanoparticles embedded within an ECM-based microtissue diffuse outwards and, when then exposed to a solution of alginate, can be used to generate a coated layer around the tissue. We further show that this technique can be fine-tuned by adjusting numerous parameters, granting us full control over the thickness of the hydrogel coating layer. The microtissues' protective hydrogel functioned as hypothesized in both in vitro and in vivo testing by preventing the cells inside the tissue from escaping and protecting the microdroplets against external degradation. This technology may provide microtissues with customized properties for use as sources of regenerative therapies.

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.

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.

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.

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.

An immunoconjugate of anti-CD24 and Pseudomonas exotoxin selectively kills human colorectal tumors in mice.

BACKGROUND & AIMS: Effective and selective treatment options are needed for patients with colorectal cancer (CRC). The CD24 mucin-like glycoprotein is overexpressed in CRCs; monoclonal antibodies (mAbs) against CD24 inhibit tumor cell growth in vitro and in vivo. Based on the tumor-specific expression of CD24, we investigated the potential of anti-CD24 SWA11 mAb, to deliver a cytotoxic agent into CRC cells. METHODS: We conjugated SWA11 to a Pseudomonas exotoxin derivative (PE38) via an Fc-binding ZZ domain from Staphylococcal protein A (which binds the Fc domain of mouse IgG2a immunoglobulins) to generate the immunotoxin SWA11-ZZ-PE38; IgG-ZZ-PE38 was used as control. Human HT-29 and COLO320 (CD24-positive) and HCT116 (CD24-negative) CRC cell lines were assayed for immunotoxin binding, cytotoxicity, viability, and apoptosis. Toxicity and antitumor efficacy were tested in mice. RESULTS: The immunotoxin preserved the affinity and specificity of SWA11, bound and selectively killed CD24-expressing CRC cells via apoptosis. IC(50) values ranged from 20 to 50 ng/mL-several orders of magnitude lower than that of the mAb alone. The immunotoxins were not toxic to mice at the maximum dose of 0.75 mg/kg. Growth of HT-29 xenograft tumors was significantly reduced in mice given SWA11-ZZ-PE38 (by 78%) compared to untreated mice. CONCLUSIONS: Anti-CD24 SWA11 mAb can deliver a PE exotoxin derivative to CRC cells and cause them to undergo apoptosis, without toxicity to normal tissues. This immunotoxin might be developed as a therapeutic treatment for patients with CRC.

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.

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.

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.