Reduced Graphene-Oxide-Doped Elastic Biodegradable Polyurethane Fibers for Cardiomyocyte Maturation.

Conductive biomaterials offer promising solutions to enhance the maturity of cultured cardiomyocytes. While the conventional culture of cardiomyocytes on nonconductive materials leads to more immature characteristics, conductive microenvironments have the potential to support sarcomere development, gap junction formation, and beating of cardiomyocytes in vitro. In this study, we systematically investigated the behaviors of cardiomyocytes on aligned electrospun fibrous membranes composed of elastic and biodegradable polyurethane (PU) doped with varying concentrations of reduced graphene oxide (rGO). Compared to PU and PU-4%rGO membranes, the PU-10%rGO membrane exhibited the highest conductivity, approaching levels close to those of native heart tissue. The PU-rGO membranes retained anisotropic viscoelastic behavior similar to that of the porcine left ventricle and a superior tensile strength. Neonatal rat cardiomyocytes (NRCMs) and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on the PU-rGO membranes displayed enhanced maturation with cell alignment and enhanced sarcomere structure and gap junction formation with PU-10%rGO having the most improved sarcomere structure and CX-43 presence. hiPSC-CMs on the PU-rGO membranes exhibited a uniform and synchronous beating pattern compared with that on PU membranes. Overall, PU-10%rGO exhibited the best performance for cardiomyocyte maturation. The conductive PU-rGO membranes provide a promising matrix for in vitro cardiomyocyte culture with promoted cell maturation/functionality and the potential for cardiac disease treatment.

In Situ Synthesis and Self-Assembly of Peptide-PEG Conjugates: A Facile Method for the Construction of Fibrous Hydrogels.

Peptide-based hydrogels have gained considerable attention as a compelling platform for various biomedical applications in recent years. Their attractiveness stems from their ability to seamlessly integrate diverse properties, such as biocompatibility, biodegradability, easily adjustable hydrophilicity/hydrophobicity, and other functionalities. However, a significant drawback is that most of the functional self-assembling peptides cannot form robust hydrogels suitable for biological applications. In this study, we present the synthesis of novel peptide-PEG conjugates and explore their comprehensive hydrogel properties. The hydrogel comprises double networks, with the first network formed through the self-assembly of peptides to create a ß-sheet secondary structure. The second network is established through covalent bond formation via N-hydroxysuccinimide chemistry between peptides and a 4-arm PEG to form a covalently linked network. Importantly, our findings reveal that this hydrogel formation method can be applied to other peptides containing lysine-rich sequences. Upon encapsulation of the hydrogel with antimicrobial peptides, the hydrogel retained high bacterial killing efficiency while showing minimum cytotoxicity toward mammalian cells. We hope that this method opens new avenues for the development of a novel class of peptide-polymer hydrogel materials with enhanced performance in biomedical contexts, particularly in reducing the potential for infection in applications of tissue regeneration and drug delivery.

Self-healing of electrical damage in insulating robust epoxy containing dynamic fluorine-substituted carbamate bonds for green dielectrics.

Power systems and electrical grids are critical for the development of renewable energy. Electrical treeing is one of the major factors that lead to electrical damage in insulating dielectrics and decline in the reliability of power equipment and ultimately results in catastrophic failure. Here, we demonstrate that bulk epoxy damaged by electrical treeing is able to efficiently heal repeatedly to recover its original robust performance. The classical dilemma between the insulating properties and electrical-damage healability is overcome by dynamic fluorinated carbamate bonds. Moreover, the dynamic bond enables the epoxy to have admirable degradability, which is demonstrated to be used as an attractive green degradable insulation coating. When used as a matrix for fiber-reinforced composites, the reclaimed glass fibers after decomposing the epoxy maintained their original morphology and functionality. This design provides a novel approach for developing smart and green dielectrics to enhance the reliability, sustainability and lifespan of power equipment and electronics.

Fibrin-Enriched Cardiac Extracellular Matrix Hydrogel Promotes In Vitro Angiogenesis.

Angiogenesis is essential for cardiac repair after myocardial infarction. Promoting angiogenesis has been demonstrated as an effective approach for myocardial infarction treatment. Several different strategies for inducing myocardial angiogenesis have been explored, including exogenous delivery of angiogenic genes, proteins, microRNAs, cells, and extracellular vesicles. Various types of injectable hydrogels have been investigated for cardiac tissue repair. One of the most promising injectable hydrogels in cardiac regeneration is a cardiac extracellular matrix hydrogel that is derived from decellularized porcine myocardium. It can be delivered minimally invasively via transendocardial delivery. The safety and efficacy of cardiac extracellular matrix hydrogels have been shown in small and large animal myocardial infarction models as well as clinical trials. The main mechanisms underlying the therapeutic benefits of cardiac extracellular matrix hydrogels have been elucidated and involved in the modulation of the immune response, downregulation of pathways related to heart failure progression and fibrosis, upregulation of genes important for cardiac muscle contraction, and enhancing cardiomyocyte differentiation and maturation from stem cells. However, no potent capillary network formation induced by cardiac extracellular matrix hydrogels has been reported. In this study, we tested the feasibility of incorporating a fibrin matrix into cardiac extracellular matrix hydrogels to improve the angiogenic properties of the hydrogel. Our in vitro results demonstrate that fibrin-enriched cardiac extracellular matrix hydrogels can induce robust endothelial cell tube formation from human umbilical vein endothelial cells and promote the sprouting of human mesenchymal stem cell spheroids. The obtained information from this study is very critical toward the future in vivo evaluation of fibrin-enriched cardiac extracellular matrix hydrogels in promoting myocardial angiogenesis.

Spatial distribution and network morphology of epicardial, endocardial, interstitial, and Purkinje cell-associated elastin fibers in porcine left ventricle.

Cardiac extracellular matrices (ECM) play crucial functional roles in cardiac biomechanics. Previous studies have mainly focused on collagen, the major structural ECM in heart wall. The role of elastin in cardiac mechanics, however, is poorly understood. In this study, we investigated the spatial distribution and microstructural morphologies of cardiac elastin in porcine left ventricles. We demonstrated that the epicardial elastin network had location- and depth-dependency, and the overall epicardial elastin fiber mapping showed certain correlation with the helical heart muscle fiber architecture. When compared to the epicardial layer, the endocardial layer was thicker and has a higher elastin-collagen ratio and a denser elastin fiber network; moreover, the endocardial elastin fibers were finer and more wavy than the epicardial elastin fibers, all suggesting various interface mechanics. The myocardial interstitial elastin fibers co-exist with the perimysial collagen to bind the cardiomyocyte bundles; some of the interstitial elastin fibers showed a locally aligned, hinge-like structure to connect the adjacent cardiomyocyte bundles. This collagen-elastin combination reflects an optimal design in which the collagen provides mechanical strength and elastin fibers facilitate recoiling during systole. Moreover, cardiac elastin fibers, along with collagen network, closely associated with the Purkinje cells, indicating that this ECM association could be essential in organizing cardiac Purkinje cells into "fibrous" and "branching" morphologies and serving as a protective feature when Purkinje fibers experience large deformations in vivo. In short, our observations provide a structural basis for future in-depth biomechanical investigations and biomimicking of this long-overlooked cardiac ECM component.

Enhancing CRISPR/Cas gene editing through modulating cellular mechanical properties for cancer therapy.

Genome editing holds great potential for cancer treatment due to the ability to precisely inactivate or repair cancer-related genes. However, delivery of CRISPR/Cas to solid tumours for efficient cancer therapy remains challenging. Here we targeted tumour tissue mechanics via a multiplexed dendrimer lipid nanoparticle (LNP) approach involving co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA and sgRNA (siFAK + CRISPR-LNPs) to enable tumour delivery and enhance gene-editing efficacy. We show that gene editing was enhanced >10-fold in tumour spheroids due to increased cellular uptake and tumour penetration of nanoparticles mediated by FAK-knockdown. siFAK + CRISPR-PD-L1-LNPs reduced extracellular matrix stiffness and efficiently disrupted PD-L1 expression by CRISPR/Cas gene editing, which significantly inhibited tumour growth and metastasis in four mouse models of cancer. Overall, we provide evidence that modulating the stiffness of tumour tissue can enhance gene editing in tumours, which offers a new strategy for synergistic LNPs and other nanoparticle systems to treat cancer using gene editing.

Regional-specific meniscal extracellular matrix hydrogels and their effects on cell-matrix interactions of fibrochondrocytes.

Decellularized meniscal extracellular matrix (ECM) material holds great potential for meniscus repair and regeneration. Particularly, injectable ECM hydrogel is highly desirable for the minimally invasive treatment of irregularly shaped defects. Although regional-specific variations of the meniscus are well documented, no ECM hydrogel has been reported to simulate zonally specific microenvironments of the native meniscus. To fill the gap, different (outer, middle, and inner) zones of porcine menisci were separately decellularized. Then the regionally decellularized meniscal ECMs were solubilized by pepsin digestion, neutralized, and then form injectable hydrogels. The hydrogels were characterized in gelation behaviors and mechanical properties and seeded with bovine fibrochondrocytes to evaluate the regionally biochemical effects on the cell-matrix interactions. Our results showed that the decellularized inner meniscal ECM (IM) contained the greatest glycosaminoglycan (GAG) content and the least collagen content compared with the decellularized outer meniscal ECM (OM) and middle meniscal ECM (MM). The IM hydrogel showed lower compressive strength than the OM hydrogel. When encapsulated with fibrochondrocytes, the IM hydrogel accumulated more GAG, contracted to a greater extent and reached higher compressive strength than that of the OM hydrogel at 28 days. Our findings demonstrate that the regionally specific meniscal ECMs present biochemical variation and show various effects on the cell behaviors, thus providing information on how meniscal ECM hydrogels may be utilized to reconstruct the microenvironments of the native meniscus.

In vitro comparison of harvesting site effects on cardiac extracellular matrix hydrogels.

Cardiac extracellular matrix (cECM) derived hydrogel has been investigated to treat myocardial infarction through animal studies and clinical trials. The tissue harvesting site commonly selects porcine left ventricle (LV) because heart attack majorly takes place in LV. However, little is known about whether the region of cardiac tissue harvesting is critical for downstream applications. In this work, in vitro studies to compare cECM hydrogels derived from adult porcine whole heart (WH), LV, and right ventricle (RV) were performed. The cECM from WH has similar chemical composition compared with cECM from LV and RV. All three types of cECM hydrogels share many similarities in terms of their microstructure, gelation time, and mechanical properties. WH-derived cECM hydrogels have larger variations in storage modulus (G') and complex modulus (G*) compared with the other two types of cECM hydrogels. Both human cardiomyocytes and mesenchymal stem cells could maintain high cell viability on all hydrogels without significant difference. In terms of above results, the cECM hydrogels from WH, LV and RV exhibited similarity in material properties and cell response in vitro. Thus, future fabrication of cECM hydrogels from WH would increase the yield, which would decrease processing time and production cost.

In situ ornamenting poly(ε-caprolactone) electrospun fibers with different fiber diameters using chondrocyte-derived extracellular matrix for chondrogenesis of mesenchymal stem cells.

Biomimetic instructive tissue engineering scaffolds are critical for achieving successful tissue regeneration. In the present study, we developed a novel scaffold via ornamenting poly(ε-caprolactone) (PCL) electrospun fibers with a chondrocyte-derived extracellular matrix (ECM)-coating, which was applied for chondrogenesis of mesenchymal stem cells (MSCs). PCL fibrous films with different fiber diameters (1282±121 nm, 549±61 nm and 285±38 nm) were first prepared via electrospinning. Rabbit articular chondrocytes (rACs) were cultured on PCL fibrous scaffolds, followed by a decellularization treatment to generate decellularized ECM (dECM)-coated PCL scaffolds (dECM/PCL). Rabbit bone marrow-derived MSCs (rMSCs) were then seeded onto these scaffolds and adhesion, proliferation and chondrogenic differentiation were evaluated. dECM/PCL scaffolds displayed distinct surface microstructural features with varying fiber diameters and fibrous mesh-like ECM with more developed collagen fibers was observed on nanofibers. On dECM/PCL scaffolds, rMSCs tended to spread more at 24 h post-seeding and proliferated better within 7 d compared to those on uncoated PCL scaffolds. Based on analysis of gene expression, rMSCs underwent the best chondrogenic differentiation on dECM/PCL scaffolds of 549-nm fibers. Collectively, such dECM/PCL composite scaffolds are very promising for cartilage tissue regeneration.

Layer-by-layer assembled polyelectrolytes on honeycomb-like porous poly(ε-caprolactone) films modulate the spatial distribution of mesenchymal stem cells.

Cellular behaviors can be affected by both surface chemistry and topography of biomaterials substrates. The object of the present study was to investigate how pore structure and bioactive molecules regulate the adhesion and proliferation of rabbit bone marrow-derived mesenchymal stem cells (rMSCs) in synergy. Poly(ε-caprolactone) (PCL) films with a honeycomb-like porous structure were fabricated via a breath-figure method, and then further coated with bioactive molecules including four combinations of polyelectrolytes (GEL/CS, GEL/HA, CHI/CS and CHI/HA) via a layer-by-layer self-assembly (LBL) process. rMSCs were seeded on these films to evaluate both adhesion and proliferation. It was shown that a uniform honeycomb-like porous structure with a pore size of 18.87±3.25µm was obtained in the films. Bioactive molecules were proven to be successfully deposited on the films, the amount of which increased with the number of assembled layer. Both surface pore structure and assembled molecules play significant roles on the adhesion and proliferation of rMSCs. Notably, the spatial distribution of cells (either entrapped in pores or spreading over pores) on porous films was also dictated by the identity of the assembled molecules. These findings might be fundamental to design bioactive scaffolds for tissue engineering.

Fabrication of PLGA/MWNTs composite electrospun fibrous scaffolds for improved myogenic differentiation of C2C12 cells.

Electrically conducting scaffolds have attracted tremendous attention in skeletal muscle tissue engineering. In this paper, poly(lactic-co-glycolic acid) (PLGA)/multi-wall carbon nanotubes (MWNTs) composite fibrous scaffolds were fabricated using the electrospinning technique. The physical properties of the composite fibers were characterized and proliferation and differentiation of C2C12 cells on these scaffolds were examined. It was found that the addition of MWNTs modulated the physical properties of PLGA fibers including morphology, fiber diameter, degradation, tensile strength and electrical conductivity, depending on the amount of MWNTs. These fibrous scaffolds were cytocompatible and supported the proliferation of C2C12 cells. Importantly, C2C12 cells showed more mature myotube formation on PLGA/MWNTs composite fibrous scaffolds compared to PLGA scaffolds. These results indicate that PLGA/MWNTs composite electrospun fibers have great potential in skeletal muscle tissue engineering.