Patterned glycopeptide-based supramolecular hydrogel promotes the alignment and contractility of iPSC-derived cardiomyocytes.

The functional restoration of a damaged cardiac tissue relies on a synchronized contractile capacity of exogenous and/or endogenous cardiomyocytes, which is challenging to achieve. Here, we explored the potential of the short glycopeptide diphenylalanine glucosamine-6-sulfate (FFGlcN6S) conjugated with an aromatic moiety, namely fluorenylmethoxycarbonyl (Fmoc), to enhance cardiac tissue regeneration. At physiological conditions, Fmoc-FFGlcN6S assembles into nanofibrous hydrated meshes, i.e., matrix mimicking hydrogels. These hydrogels can be further micropatterned allowing co-existence of hierarchical structures at different lenght. The patterned hydrogels support the culture of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and promote their alignment. The cultured iPSC-CMs exhibit anisotropic synchronized contractions, indicating maturation and electrical interconnectivity. Moreover, the cultures express specific cardiac markers including, connexin-43 and sarcomeric-α-actinin, confirming enhanced cell-cell crosstalk, spontaneous contractility, and efficient transmission of electrical signals. Our results showcase the potential of short amphiphilic glycopeptides to mimic physical and biochemical cues that are essential for cardiomyocytes functionality and thus, these conjugates can be used in cardiac tissue engineering and regeneration.

CMC/Gel/GO 3D-printed cardiac patches: GO and CMC improve flexibility and promote H9C2 cell proliferation, while EDC/NHS enhances stability.

In this research, carboxymethyl cellulose (CMC)/gelatin (Gel)/graphene oxide (GO)-based scaffolds were produced by using extrusion-based 3D printing for cardiac tissue regeneration. Rheological studies were conducted to evaluate the printability of CMC/Gel/GO inks, which revealed that CMC increased viscosity and enhanced printability. The 3D-printed cardiac patches were crosslinked with N-(3-dimethylaminopropyl)-n'-ethylcarbodiimide hydrochloride (EDC)/ N-hydroxysuccinimide (NHS) (100:20 mM, 50:10 mM, 25:5 mM) and then characterized by mechanical analysis, electrical conductivity testing, contact angle measurements and degradation studies. Subsequently, cell culture studies were conducted to evaluate the viability of H9C2 cardiomyoblast cells by using the Alamar Blue assay and fluorescence imaging. A high concentration of EDC/NHS (100:20 mM) led to the stability of the patches; however, it drastically reduced the flexibility of the scaffolds. Conversely, a concentration of 25:5 mM resulted in flexible but unstable scaffolds in PBS solution. The suitable EDC/NHS concentration was found to be 50:10 mM, as it produced flexible, stable, and stiff cardiac scaffolds with high ultimate tensile strength (UTS). Mechanical characterization revealed that % the strain at break of C15/G7.5/GO1 exhibited a remarkable increase of 61.03% compared to C15/G7.5 samples. The improvement of flexibility was attributed to the hydrogen bonding between CMC, Gel and GO. The electrical conductivity of 3D printed CMC/Gel/GO cardiac patches was 7.0×10-3 S/cm, demonstrating suitability for mimicking the desired electrical conductivity of human myocardium. The incorporation of 1 wt% of GO and addition of CMC concentration from 7.5 wt % to 15 wt % significantly enhanced relative % cell viability. Overall, although this research is at its infancy, CMC/Gel/GO cardiac patches have potential to improve the physiological function of cardiac tissue.

Hydrogels in cardiac tissue engineering: application and challenges.

Cardiovascular disease remains the leading cause of global mortality. Current stem cell therapy and heart transplant therapy have limited long-term stability in cardiac function. Cardiac tissue engineering may be one of the key methods for regenerating damaged myocardial tissue. As an ideal scaffold material, hydrogel has become a viable tissue engineering therapy for the heart. Hydrogel can not only provide mechanical support for infarcted myocardium but also serve as a carrier for various drugs, bioactive factors, and cells to increase myocardial contractility and improve the cell microenvironment in the infarcted area, thereby improving cardiac function. This paper reviews the applications of hydrogels and biomedical mechanisms in cardiac tissue engineering and discusses the challenge of clinical transformation of hydrogel in cardiac tissue engineering, providing new strategies for treating cardiovascular diseases.

Procurement and Preservation of Neonatal Porcine Cardiac Tissue.

The porcine and human heart are remarkably similar in cardiac physiology and biochemistry. Translational research involving the porcine biomedical model is becoming increasingly applicable for the study of human cardiac function in health and disease. Presently, few protocols exist for collecting experimentally viable cardiac tissue from large animal models, particularly during neonatal maturation. To address this deficiency, we have developed a technique to procure and preserve ventricular tissue from neonatal piglets at day 3 (n=4) and day 30 (n=6) post-partum. Piglets were subjected to a strict sedation, anesthesia, and analgesia regimen. During surgery, cardiopulmonary indices of electrocardiogram, heart rate, systolic and diastolic blood pressure, respiration rate, peripheral O2 saturation, and end-tidal CO2 were monitored continuously to ensure normal cardiac function. Prior to cardiectomy, each heart was perfused with an intravenous administration of heparin (10 ml/kg) and ice-cold Custodiol HTK cardioplegia solution (10 ml/kg). After cardiac explantation, myocardial samples (dimensions: 1 x 1 x 1 cm) were dissected from the left and right ventricles and snap-frozen in liquid nitrogen. Analysis via SDS-PAGE and densitometry demonstrated that myofibrillar proteins are stable and undegraded. Western Blots showed full expression of protein. These results suggest that the detailed cardiac tissue procurement technique preserves the experimental viability of the cardiac tissue and prevents the degradation of myofibrillar proteins.

3D Nanofiber-Assisted Embedded Extrusion Bioprinting for Oriented Cardiac Tissue Fabrication.

Three-dimensional (3D) bioprinting technology stands out as a promising tissue manufacturing process to control the geometry precisely with cell-loaded bioinks. However, the isotropic culture environment within the bioink and the lack of topographical cues impede the formation of oriented cardiac tissue. To overcome this limitation, we present a novel method named 3D nanofiber-assisted embedded bioprinting (3D-NFEP) to fabricate cardiac tissue with an oriented morphology. Aligned 3D nanofiber scaffolds were fabricated by divergence electrospinning, which provided structural support for printing of the low-viscosity bioink and structural induction to cardiomyocytes. Cells adhered to the aligned fibers after hydrogel degradation, and a high degree of cell alignment was observed. This technology was also demonstrated as a feasible solution for multilayer cell printing. Therefore, 3D-NFEP was demonstrated as a promising method for bioprinting oriented cardiac tissue with low-viscosity bioink and is expected to be applied for structured and cardiac tissue engineering.

Multidirectional Filamented Light Biofabrication Creates Aligned and Contractile Cardiac Tissues.

Biofabricating 3D cardiac tissues that mimic the native myocardial tissue is a pivotal challenge in tissue engineering. In this study, we fabricate 3D cardiac tissues with controlled, multidirectional cellular alignment and directed or twisting contractility. We show that multidirectional filamented light can be used to biofabricate high-density (up to 60 × 106 cells mL-1) tissues, with directed uniaxial contractility (3.8x) and improved cell-to-cell connectivity (1.6x gap junction expression). Furthermore, by using multidirectional light projection, we can partially overcome cell-induced light attenuation, and fabricate larger tissues with multidirectional cellular alignment. For example, we fabricate a tri-layered myocardium-like tissue and a bi-layered tissue with torsional contractility. The approach provides a new strategy to rapidly fabricate aligned cardiac tissues relevant to regenerative medicine and biohybrid robotics.

Cellular Alignment and Matrix Stiffening Induced Changes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes.

Biological processes are inherently dynamic, necessitating biomaterial platforms capable of spatiotemporal control over cellular organization and matrix stiffness for accurate study of tissue development, wound healing, and disease. However, most in vitro platforms remain static. In this study, a dynamic biomaterial platform comprising a stiffening hydrogel is introduced and achieved through a stepwise approach of addition followed by light-mediated crosslinking, integrated with an elastomeric substrate featuring strain-responsive lamellar surface patterns. Employing this platform, the response of human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) is investigated to dynamic stiffening from healthy to fibrotic tissue stiffness. The results demonstrate that culturing hIPSC-CMs on physiologically relevant healthy stiffness significantly enhances their function, as evidenced by increased sarcomere fraction, wider sarcomere width, significantly higher connexin-43 content, and elevated cell beating frequency compared to cells cultured on fibrotic matrix. Conversely, dynamic matrix stiffening negatively impacts hIPSC-CM function, with earlier stiffening events exerting a more pronounced hindering effect. These findings provide valuable insights into material-based approaches for addressing existing challenges in hIPSC-CM maturation and have broader implications across various tissue models, including muscle, tendon, nerve, and cornea, where both cellular alignment and matrix stiffening play pivotal roles in tissue development and regeneration.

Enhanced drug classification using machine learning with multiplexed cardiac contractility assays.

Cardiac screening of newly discovered drugs remains a longstanding challenge for the pharmaceutical industry. While therapeutic efficacy and cardiotoxicity are evaluated through preclinical biochemical and animal testing, 90 % of lead compounds fail to meet safety and efficacy benchmarks during human clinical trials. A preclinical model more representative of the human cardiac response is needed; heart tissue engineered from human pluripotent stem cell derived cardiomyocytes offers such a platform. In this study, three functionally distinct and independently validated engineered cardiac tissue assays are exposed to increasing concentrations of known compounds representing 5 classes of mechanistic action, creating a robust electrophysiology and contractility dataset. Combining results from six individual models, the resulting ensemble algorithm can classify the mechanistic action of unknown compounds with 86.2 % predictive accuracy. This outperforms single-assay models and offers a strategy to enhance future clinical trial success aligned with the recent FDA Modernization Act 2.0.

Shear-Induced Cycloreversion Leading to Shear-Thinning and Autonomous Self-Healing in an Injectable, Shape-Holding Collagen Hydrogel.

In vivo injectable extracellular matrix (ECM) derived hydrogels that are suitable for cell encapsulation have always been the holy grail in tissue engineering. Nevertheless, these hydrogels still fall short today of meeting three crucial criteria: (a) flexibility on the injectability time window, (b) autonomous self-healing of the injected hydrogel, and (c) shape-retention under aqueous conditions. Here we report the development of a collagen-based injectable hydrogel, cross-linked by cycloaddition reaction between furan and maleimide groups, that (a) is injectable up to 48 h after preparation, (b) can undergo complete autonomous self-healing after injection, (c) can retain its shape and size over several years when stored in the buffer, (d) can be degraded within hours when treated with collagenase, (e) is biocompatible as demonstrated by in vitro cell-culture, and (f) is completely resorbable in vivo when implanted subcutaneously in rats without causing any inflammation.

Electrohydrodynamic Printing of Microscale Fibrous Scaffolds with a Sinusoidal Structure for Enhancing the Contractility of Cardiomyocytes.

Mimicking the curved collagenous fibers in the cardiac extracellular matrix to fabricate elastic scaffolds in vitro is important for cardiac tissue engineering. Here, we developed sinusoidal polycaprolactone (PCL) fibrous scaffolds with commendable flexibility and elasticity to enhance the contractility of primary cardiomyocytes by employing melt-based electrohydrodynamic (EHD) printing. Microscale sinusoidal PCL fibers with an average diameter of ∼10 µm were printed to mimic the collagenous fibers in the cardiac ECM. The sinusoidal PCL fibrous scaffolds were EHD-printed in a layer-by-layer manner and exhibited outstanding flexibility and elasticity compared with the straight ones. The sinusoidal PCL scaffolds provided an elastic microenvironment for the attaching and spreading of primary cardiomyocytes, which facilitated their synchronous contractive activities. Primary cardiomyocytes also showed improved gene expression and maturation on the sinusoidal PCL scaffolds under electrical stimulation for 5 days. It is envisioned that the proposed flexible fibrous scaffold with biomimetic architecture may serve as a suitable patch for tissue regeneration and repair of damaged hearts after myocardial infarction.

Mechanical memory based biofabrication of hierarchical elastic cardiac tissue.

Mimicking the multilayered, anisotropic, elastic structure of cardiac tissues for controlled guidiance of 3D cellular orientation is essential in designing bionic scaffolds for cardiac tissue biofabrication. Here, a hierarchically organized, anisotropic, wavy and conductive polycaprolactone/Au scaffold was created in a facile fashion based on mechanical memory during fabrication. The bionic 3D scaffold shows good biocompatibility, excellent biomimetic mechanical properties that guide myoblast alignment, support the hyperelastic behavior observed in native cardiac muscle tissue, and promote myotube maturation, which holds potential for cardiac muscle engineering and the establishment of anin vitroculture platform for drug screening.

Advancing Human iPSC-Derived Cardiomyocyte Hypoxia Resistance for Cardiac Regenerative Therapies through a Systematic Assessment of In Vitro Conditioning.

Acute myocardial infarction (MI) is a sudden, severe cardiac ischemic event that results in the death of up to one billion cardiomyocytes (CMs) and subsequent decrease in cardiac function. Engineered cardiac tissues (ECTs) are a promising approach to deliver the necessary mass of CMs to remuscularize the heart. However, the hypoxic environment of the heart post-MI presents a critical challenge for CM engraftment. Here, we present a high-throughput, systematic study targeting several physiological features of human induced pluripotent stem cell-derived CMs (hiPSC-CMs), including metabolism, Wnt signaling, substrate, heat shock, apoptosis, and mitochondrial stabilization, to assess their efficacy in promoting ischemia resistance in hiPSC-CMs. The results of 2D experiments identify hypoxia preconditioning (HPC) and metabolic conditioning as having a significant influence on hiPSC-CM function in normoxia and hypoxia. Within 3D engineered cardiac tissues (ECTs), metabolic conditioning with maturation media (MM), featuring high fatty acid and calcium concentration, results in a 1.5-fold increase in active stress generation as compared to RPMI/B27 control ECTs in normoxic conditions. Yet, this functional improvement is lost after hypoxia treatment. Interestingly, HPC can partially rescue the function of MM-treated ECTs after hypoxia. Our systematic and iterative approach provides a strong foundation for assessing and leveraging in vitro culture conditions to enhance the hypoxia resistance, and thus the successful clinical translation, of hiPSC-CMs in cardiac regenerative therapies.

Endothelial extracellular vesicles enhance vascular self-assembly in engineered human cardiac tissues.

The fabrication of complex and stable vasculature in engineered cardiac tissues represents a significant hurdle towards building physiologically relevant models of the heart. Here, we implemented a 3D model of cardiac vasculogenesis, incorporating endothelial cells (EC), stromal cells, and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) in a fibrin hydrogel. The presence of CMs disrupted vessel formation in 3D tissues, resulting in the upregulation of endothelial activation markers and altered extracellular vesicle (EV) signaling in engineered tissues as determined by the proteomic analysis of culture supernatant. miRNA sequencing of CM- and EC-secreted EVs highlighted key EV-miRNAs that were postulated to play differing roles in cardiac vasculogenesis, including the let-7 family and miR-126-3p in EC-EVs. In the absence of CMs, the supplementation of CM-EVs to EC monolayers attenuated EC migration and proliferation and resulted in shorter and more discontinuous self-assembling vessels when applied to 3D vascular tissues. In contrast, supplementation of EC-EVs to the tissue culture media of 3D vascularized cardiac tissues mitigated some of the deleterious effects of CMs on vascular self-assembly, enhancing the average length and continuity of vessel tubes that formed in the presence of CMs. Direct transfection validated the effects of the key EC-EV miRNAs let-7b-5p and miR-126-3p in improving the maintenance of continuous vascular networks. EC-EV supplementation to biofabricated cardiac tissues and microfluidic devices resulted in tissue vascularization, illustrating the use of this approach in the engineering of enhanced, perfusable, microfluidic models of the myocardium.

Exogenous ECM in an environmentally-mediated in vitro model for cardiac fibrosis.

Few clinical solutions exist for cardiac fibrosis, creating the need for a tunable in vitro model to better understand fibrotic disease mechanisms and screen potential therapeutic compounds. Here, we combined cardiomyocytes, cardiac fibroblasts, and exogenous extracellular matrix (ECM) proteins to create an environmentally-mediated in vitro cardiac fibrosis model. Cells and ECM were combined into 2 types of cardiac tissues- aggregates and tissue rings. The addition of collagen I had a drastic negative impact on aggregate formation, but ring formation was not as drastically affected. In both tissue types, collagen and other ECM did not severely affect contractile function. Histological analysis showed direct incorporation of collagen into tissues, indicating that we can directly modulate the cells' ECM environment. This modulation affects tissue formation and distribution of cells, indicating that this model provides a useful platform for understanding how cells respond to changes in their extracellular environment and for potential therapeutic screening.

Electroactive Nanomaterials for the Prevention and Treatment of Heart Failure: From Materials and Mechanisms to Applications.

Heart failure (HF) represents a cardiovascular disease that significantly threatens global well-being and quality of life. Electroactive nanomaterials, characterized by their distinctive physical and chemical properties, emerge as promising candidates for HF prevention and management. This review comprehensively examines electroactive nanomaterials and their applications in HF intervention. It presents the definition, classification, and intrinsic characteristics of conductive, piezoelectric, and triboelectric nanomaterials, emphasizing their mechanical robustness, electrical conductivity, and piezoelectric coefficients. The review elucidates their applications and mechanisms: 1) early detection and diagnosis, employing nanomaterial-based sensors for real-time cardiac health monitoring; 2) cardiac tissue repair and regeneration, providing mechanical, chemical, and electrical stimuli for tissue restoration; 3) localized administration of bioactive biomolecules, genes, or pharmacotherapeutic agents, using nanomaterials as advanced drug delivery systems; and 4) electrical stimulation therapies, leveraging their properties for innovative pacemaker and neurostimulation technologies. Challenges in clinical translation, such as biocompatibility, stability, and scalability, are discussed, along with future prospects and potential innovations, including multifunctional and stimuli-responsive nanomaterials for precise HF therapies. This review encapsulates current research and future directions concerning the use of electroactive nanomaterials in HF prevention and management, highlighting their potential to innovating in cardiovascular medicine.

Graphene-enhanced PCL electrospun nanofiber scaffolds for cardiac tissue engineering.

Cardiovascular diseases, particularly myocardial infarction, have significant healthcare challenges due to the limited regenerative capacity of injured heart tissue. Cardiac tissue engineering (CTE) offers a promising approach to repairing myocardial damage using biomaterials that mimic the heart's extracellular matrix. This study investigates the potential of graphene nanopowder (Gnp)-enhanced polycaprolactone (PCL) scaffolds fabricated via electrospinning to improve the properties necessary for effective cardiac repair. This work aimed to analyze scaffolds with varying graphene concentrations (0.5%, 1%, 1.5%, and 2% by weight) to determine their morphological, chemical, mechanical, and biocompatibility characteristics. The results presented that incorporating graphene improves PCL scaffolds' mechanical properties and cellular interactions. The optimal concentration of 1% graphene significantly enhanced mechanical properties and biocompatibility, promoting cell adhesion and proliferation. These findings suggest that Gnp-enhanced PCL scaffolds at this concentration can serve as a potent substrate for CTE providing insights into designing more effective biomaterials for myocardial restoration.

A comprehensive experimental analysis of the local passive response across the healthy porcine left ventricle.

This work provides a comprehensive characterization of porcine myocardial tissue, combining true biaxial (TBx), simple triaxial shear (STS) and confined compression (CC) tests to analyze its elastic behavior under cyclic loads. We expanded this study to different zones of the ventricular free wall, providing insights into the local behavior along the longitudinal and radial coordinates. The aging impact was also assessed by comparing two age groups (4 and 8 months). Resulting data showed that the myocardium exhibits a highly nonlinear hyperelastic and incompressible behavior. We observed an anisotropy ratio of 2-2.4 between averaged peak stresses in TBx tests and 1-0.59-0.40 orthotropy ratios for normalised fiber-sheet-normal peak stresses in STS tests. We obtained a highly incompressible response, reaching volumetric pressures of 2-7 MPa for perfused tissue in CC tests, with notable differences when fluid drainage was allowed, suggesting a high permeability. Regional analysis showed reduced stiffness and anisotropy (20-25%) at the apical region compared to the medial, which we attributed to differences in the fiber field dispersion. Compressibility also increased towards the epicardium and apical regions. Regarding age-related variations, 8-month animals showed stiffer response (at least 25% increase), particularly in directions where the mechanical stress is absorbed by collagenous fibers (more than 90%), as supported by a histological analysis. Although compressibility of perfused tissue remained unchanged, permeability significantly reduced in 8-month-old animals. Our findings offer new insights into myocardial properties, emphasizing on local variations, which can help to get a more realistic understanding of cardiac mechanics in this common animal model. STATEMENT OF SIGNIFICANCE: In this work, we conducted a comprehensive analysis of the passive mechanical behavior of porcine myocardial tissue through biaxial, triaxial shear, and confined compression tests. Unlike previous research, we investigated the variation in mechanical response across the left ventricular free wall, conventionally assumed homogeneous, revealing differences in terms of stiffness and compressibility. Additionally, we evaluated age-related effects on mechanical properties by comparing two age groups, observing significant variations in stiffness and permeability. To date, there has been no such in-depth exploration of myocardial elastic response and compressibility considering regional variations along the wall and may contribute to a better understanding of the cardiac tissue's passive mechanical response.

Latest progress of self-healing hydrogels in cardiac tissue engineering.

Cardiovascular diseases represent a significant public health challenge and are responsible for more than 4 million deaths annually in Europe alone (45% of all deaths). Among these, coronary-related heart diseases are a leading cause of mortality, accounting for 20% of all deaths. Cardiac tissue engineering has emerged as a promising strategy to address the limitations encountered after myocardial infarction. This approach aims to improve regulation of the inflammatory and cell proliferation phases, thereby reducing scar tissue formation and restoring cardiac function. In cardiac tissue engineering, biomaterials serve as hosts for cells and therapeutics, supporting cardiac restoration by mimicking the native cardiac environment. Various bioengineered systems, such as 3D scaffolds, injectable hydrogels, and patches play crucial roles in cardiac tissue repair. In this context, self-healing hydrogels are particularly suitable substitutes, as they can restore structural integrity when damaged. This structural healing represents a paradigm shift in therapeutic interventions, offering a more native-like environment compared to static, non-healable hydrogels. Herein, we sharply review the most recent advances in self-healing hydrogels in cardiac tissue engineering and their potential to transform cardiovascular healthcare.

Insights into the prospects of nanobiomaterials in the treatment of cardiac arrhythmia.

Cardiac arrhythmia, a disorder of abnormal electrical activity of the heart that disturbs the rhythm of the heart, thereby affecting its normal function, is one of the leading causes of death from heart disease worldwide and causes millions of deaths each year. Currently, treatments for arrhythmia include drug therapy, radiofrequency ablation, cardiovascular implantable electronic devices (CIEDs), including pacemakers, defibrillators, and cardiac resynchronization therapy (CRT). However, these traditional treatments have several limitations, such as the side effects of medication, the risks of device implantation, and the complications of invasive surgery. Nanotechnology and nanomaterials provide safer, effective and crucial treatments to improve the quality of life of patients with cardiac arrhythmia. The large specific surface area, controlled physical and chemical properties, and good biocompatibility of nanobiomaterials make them promising for a wide range of applications, such as cardiovascular drug delivery, tissue engineering, and the diagnosis and therapeutic treatment of diseases. However, issues related to the genotoxicity, cytotoxicity and immunogenicity of nanomaterials remain and require careful consideration. In this review, we first provide a brief overview of cardiac electrophysiology, arrhythmia and current treatments for arrhythmia and discuss the potential applications of nanobiomaterials before focusing on the promising applications of nanobiomaterials in drug delivery and cardiac tissue repair. An in-depth study of the application of nanobiomaterials is expected to provide safer and more effective therapeutic options for patients with cardiac arrhythmia, thereby improving their quality of life.