Human induced pluripotent stem cell-derived cardiac muscle rings for biohybrid self-beating actuator.

Cardiac muscle, a subtype of striated muscle composing our heart, has garnered attention as a source of autonomously driven actuators due to its inherent capability for spontaneous contraction. However, conventional cardiac biohybrid robots have utilized planar (2D) cardiac tissue consisting of a thin monolayer of cardiac myotubes with a thickness of 3-5 µm, which can generate a limited contractile force per unit footprint. In this study, 3D cardiac muscle rings were proposed as robotic actuator units. These units not only exhibit higher contractile force per unit footprint compared to their 2D counterparts due to their increased height, but they can also be integrated into desired 3D configurations. We fabricated cardiac muscle rings from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), evaluated their driving characteristics, and verified the actuation effects by integrating them with artificial components. After the 10th day from culture, the cardiac muscle rings exhibited rhythmic spontaneous contraction and increased contractile force in response to stretching stimuli. Furthermore, after constructing a centimeter-sized biohybrid self-beating actuator with an antagonistic pair structure of cardiac muscle rings, the periodic antagonistic beating motion at its tail portion was confirmed. We believe that 3D cardiac muscle rings, possessing high contractile force and capable of being positioned within limited 3D space, can be used as potent biohybrid robotic actuators.

Pillar electrodes embedded in the skeletal muscle tissue for selective stimulation of biohybrid actuators with increased contractile distance.

Electrodes are crucial for controlling the movements of biohybrid robots, but their external placement outside muscle tissue often leads to inefficient and non-selective stimulation of nearby biohybrid actuators. To address this, we propose embedding pillar electrodes within the skeletal muscle tissue, resulting in enhanced contraction of the target muscle without affecting the neighbor tissue with a 4 mm distance. We use finite element method simulations to establish a selectivity model, correlating the VIE(volume integration of electric field intensity within muscle tissue) with actual contractile distances under different amplitudes of electrical pulses. The simulated selective index closely aligns with experimental results, showing the potential of pillar electrodes for effective and selective biohybrid actuator stimulation. In experiments, we validated that the contractile distance and selectivity achieved with these pillar electrodes exceed conventional Au rod electrodes. This innovation has promising implications for building biohybrid robots with densely arranged muscle tissue, ultimately achieving more human-like movements. Additionally, our selectivity model offers valuable predictive tools for assessing electrical stimulation effects with different electrode designs.

Millimeter-thick 3D tissues constructed by densely cellularized core-shell microfluidic bioprinting.

Recently, microfluidic bioprinting methods, which utilize microfluidic devices as printheads to deposit microfilaments, have improved printing resolution. Despite the precise placement of cells, current efforts have not succeeded in forming densely cellularized tissue within the printed constructs, which is highly desired for the biofabrication of solid-organ tissues with firm tissue consistency. In this paper, we presented a microfluidic bioprinting method to fabricate three dimension tissue constructs consisting of core-shell microfibers where extracellular matrices and cells can be encapsulated within the core of the fibers. Using the optimized printhead design and printing parameters, we demonstrated the bioprinting of core-shell microfibers into macroscale constructs and checked the viability of cells after printing. After culturing the printed tissues using the proposed dynamic culture methods, we analyzed the morphology and function of the tissues bothin vitroandin vivo. The confluent tissue morphology in the fiber cores indicates the establishment of intensive cell-cell contacts in the fiber cores, which also leads to the upregulation of the albumin-secretion function compared to the cells cultured in a 2D format. Analysis on the cell density of the confluent fiber cores indicate the formation of densely cellularized tissues with a similar level of cell density ofin-vivosolid organ tissues. In the future, better culture techniques with improved perfusion design are anticipated to enable further the fabrication of thicker tissues, which can be used as thick tissue models or implantation grafts for cell therapy.

A Cylindrical Molding Method for the Biofabrication of Plane-Shaped Skeletal Muscle Tissue.

Muscle tissues can be fabricated in vitro by culturing myoblast-populated hydrogels. To counter the shrinkage of the myoblast-populated hydrogels during culture, a pair of anchors are generally utilized to fix the two ends of the hydrogel. Here, we propose an alternative method to counter the shrinkage of the hydrogel and fabricate plane-shaped skeletal muscle tissues. The method forms myoblast-populated hydrogel in a cylindrical cavity with a central pillar, which can prevent tissue shrinkage along the circumferential direction. By eliminating the usages of the anchor pairs, our proposed method can produce plane-shaped skeletal muscle tissues with uniform width and thickness. In experiments, we demonstrate the fabrication of plane-shaped (length: ca. 10 mm, width: 5~15 mm) skeletal muscle tissue with submillimeter thickness. The tissues have uniform shapes and are populated with differentiated muscle cells stained positive for myogenic differentiation markers (i.e., myosin heavy chains). In addition, we show the assembly of subcentimeter-order tissue blocks by stacking the plane-shaped skeletal muscle tissues. The proposed method can be further optimized and scaled up to produce cultured animal products such as cultured meat.

Comprehensive Analysis of the Value of SMYD Family Members in the Prognosis and Immune Infiltration of Malignant Digestive System Tumors.

BACKGROUND: The SET and MYND domain-containing (SMYD) gene family comprises a set of genes encoding lysine methyltransferases. This study aimed to clarify the relationship between the expression levels of SMYD family members and the prognosis and immune infiltration of malignant tumors of the digestive system. METHODS: The Oncomine, Ualcan, Kaplan-Meier Plotter, cBioPortal, Metascape, and TIMER databases and tools were used to analyze the correlation of SMYD family mRNA expression, clinical stage, TP53 mutation status, prognostic value, gene mutation, and immune infiltration in patients with esophageal carcinoma (ESCA), liver hepatocellular carcinoma (LIHC), and stomach adenocarcinoma (STAD). RESULTS: In ESCA, the mRNA expression of SMYD2/3/4/5 was significantly correlated with the incidence rate, that of SMYD2/3 with the clinical stage, that of SMYD2/3/4/5 with TP53 mutation status, that of SMYD2/4/5 with overall survival (OS), and that of SMYD1/2/3/4 with relapse-free survival (RFS). In LIHC, the mRNA expression of SMYD1/2/3/4/5 was significantly correlated with the incidence rate, that of SMYD2/4/5 with the clinical stage, that of SMYD3/5 with TP53 mutation status, that of SMYD2/3/4/5 with OS, and that of SMYD3/5 with RFS. In STAD, the mRNA expression of SMYD2/3/4/5 was significantly correlated with the incidence rate, that of SMYD1/4 with the clinical stage, that of SMYD1/2/3/5 with TP53 mutation status, that of SMYD1/3/4 with OS, and that of SMYD1/3 with RFS. Furthermore, the function of SMYD family mutation-related genes in ESCA, LIHC, and STAD patients was mainly related to pathways, such as mitochondrial gene expression, mitochondrial matrix, and mitochondrial translation. The expression of SMYD family genes was significantly correlated with the infiltration of six immune cell types and eight types of immune check sites. CONCLUSION: SMYD family genes are differentially expressed and frequently mutated in malignant tumors of the digestive system (ESCA, LIHC, and gastric cancer). They are potential markers for prognostic prediction and have important significance in immunity and targeted therapy.

Manufacturing of animal products by the assembly of microfabricated tissues.

With the current rapidly growing global population, the animal product industry faces challenges which not only demand drastically increased amounts of animal products but also have to limit the emission of greenhouse gases and animal waste. These issues can be solved by the combination of microfabrication and tissue engineering techniques, which utilize the microtissue as a building component for larger tissue assembly to fabricate animal products. Various methods for the assembly of microtissue have been proposed such as spinning, cell layering, and 3D bioprinting to mimic the intricate morphology and function of the in vivo animal tissues. Some of the demonstrations on cultured meat and leather-like materials present promising outlooks on the emerging field of in vitro production of animal products.

Biofabricating murine and human myo-substitutes for rapid volumetric muscle loss restoration.

The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. However, its regenerative capability is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. Here, we propose a biofabrication approach to rapidly restore skeletal muscle mass, 3D histoarchitecture, and functionality. By recapitulating muscle anisotropic organization at the microscale level, we demonstrate to efficiently guide cell differentiation and myobundle formation both in vitro and in vivo. Of note, upon implantation, the biofabricated myo-substitutes support the formation of new blood vessels and neuromuscular junctions-pivotal aspects for cell survival and muscle contractile functionalities-together with an advanced muscle mass and force recovery. Altogether, these data represent a solid base for further testing the myo-substitutes in large animal size and a promising platform to be eventually translated into clinical scenarios.

Microfluidic Device for the Analysis of Angiogenic Sprouting under Bidirectional Biochemical Gradients.

In this paper, we developed a spheroid culture device that can trap a spheroid in the trapping site sandwiched by two extracellular matrix gels located at the upper and lower side of the spheroid. This device can form different biochemical gradients by applying target biochemicals separately in upper and lower channels, allowing us to study the angiogenic sprouting under various biochemical gradients in different directions. In the experiments, we confirmed the trapping of the spheroids and demonstrate the investigation on the direction and extent of angiogenic sprouts under unidirectional or bidirectional biochemical gradients. We believe our device can contribute to understanding the pathophysiological phenomena driven by chemical gradients, such as tissue development and tumor angiogenesis.

Luer-lock valve: A pre-fabricated pneumatic valve for 3D printed microfluidic automation.

3D printed microfluidic devices are made of stiff and easy-to-fatigue materials and hence are difficult to have robust pneumatic valves. In this work, we describe a type of prefabricated polydimethylsiloxane (PDMS) valves, named the "Luer-lock" valve, which can be incorporated in 3D printed microfluidic devices utilizing the Luer-lock mechanism. Luer-lock design has been adopted for fluidic connections worldwide; it is facile, reliable, and inexpensive. To take advantage of the Luer-lock design, we added "valve ports" to our 3D printed microfluidic devices; prefabricated PDMS valve modules could be embedded into these valve ports, in a leak-free manner, by screwing tight the Luer-locks. In the experiment, we succeeded in fabricating pneumatic valves with a footprint diameter of 0.8 mm and verified the functionality of these valves with a shut-off pressure of 140 mbar and a maximal switching frequency of ∼1 Hz. As a demonstration, we show the serial encoding of core-shell hydrogel microfibers using the Luer-lock valves. Since the Luer-lock valves can be mass-produced and the CAD model of Luer-locks can be easily distributed, we believe that our approach has the potential to be easily adopted by researchers around the globe.

3D culture of functional human iPSC-derived hepatocytes using a core-shell microfiber.

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB, HNF4α, ASGPR1, CYP2C19, and CYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either as in vitro models for drug screening or as implantation grafts to treat liver failure.

Cell-laden microfibers fabricated using µl cell-suspension.

Current microfluidic methods for cell-laden microfiber fabrication generally require larger than 100 µl of cell-suspensions. Since some 'rare' cells can be only acquired in small amounts, the preparation of >100 µl cell-suspensions with high-cell density can be both expensive and time consuming. Here, we present a facile method capable of fabricating cell-laden microfibers using small-volume cell-suspensions. The method utilizes a 3D-printed coaxial microfluidic device featured with a 'luer-lock inlet' to effectively load cell-suspensions in a deterministic volume (down to 5 µl) with a low sample-loss. In experiments, we demonstrate the formation of fibrous tissues consisting of various kinds of cells. Investigations on the morphology and function of the encapsulated cells show the viability of the cells is not significantly affected by the fabrication process, and also indicate the potential of using our method to perform quantitative assays on fiber-shaped tissues, while reducing the overall material and time consumption.

The bioprinting roadmap.

This bioprinting roadmap features salient advances in selected applications of the technique and highlights the status of current developments and challenges, as well as envisioned advances in science and technology, to address the challenges to the young and evolving technique. The topics covered in this roadmap encompass the broad spectrum of bioprinting; from cell expansion and novel bioink development to cell/stem cell printing, from organoid-based tissue organization to bioprinting of human-scale tissue structures, and from building cell/tissue/organ-on-a-chip to biomanufacturing of multicellular engineered living systems. The emerging application of printing-in-space and an overview of bioprinting technologies are also included in this roadmap. Due to the rapid pace of methodological advancements in bioprinting techniques and wide-ranging applications, the direction in which the field should advance is not immediately clear. This bioprinting roadmap addresses this unmet need by providing a comprehensive summary and recommendations useful to experienced researchers and newcomers to the field.

3D Biofabrication Using Living Cells for Applications in Biohybrid Sensors and Actuators.

In this paper, we highlight the concept of biohybrid sensors and actuators built by incorporating living cells into artificial systems. Instead of using the materials extracted from cells, these approaches utilize cells to dynamically generate functional materials and to provide the native intracellular environment for the proper functioning of the materials. By incorporating the functional cells into artificial devices/chips, the cell-based biohybrid approaches can be applied to create portable odorant sensors with high sensitivity and to create biohybrid muscle actuators for applications in both drug screening and soft robotics.

Bottom-up biofabrication using microfluidic techniques.

Nature builds living organisms in a bottom-up fashion, starting from the expression of genetic information on a cellular level, to the proliferation, differentiation, and self-assembly of cells into tissues/organs during embryo development and wound-healing processes. To mimic this bottom-up approach, it is essential to handle and manipulate small-scale biomaterials using specific technologies, such as microfluidic techniques. Microfluidics provides the tool-sets that deal with the behavior, precise control and manipulation of small amounts of fluids. Since the handling performed in aqueous environment guarantees the well-preserved bioactivities of biomaterials, microfluidic techniques show unique and critical advantages for biofabrication purposes. In this review, we report the recent progress on the bottom-up biofabrication using microfluidic techniques, such as the fabrication of microtissues, bioprinting of cell-laden tissue scaffolds, and organ-on-a-chip. As future perspectives, the deeper convergence of bottom-up approaches with top-down approaches as well as microfluidic-enabled vascularization of thick tissues is anticipated to have enormous impacts on the field of biofabrication.

Protective effects of fucoidan against hyperoxic lung injury via the ERK signaling pathway.

High oxygen mechanical ventilation is widely used to treat various lung diseases; however, it may result in hyperoxia, which induces inflammation and lung injury. Fucoidan is an extract of the seaweed Fucus vesiculosus, which has previously been reported to exert effects against diabetic nephropathy. The present study is the first, to the best of our knowledge, to investigate the protective effects of fucoidan against hyperoxic lung injury. Balb/c mice were ventilated with 100% oxygen, with or without the atomization inhalation of fucoidan, for 36 h. Hyperoxia reduced the body weight and increased the relative lung weight of the mice. In addition, cell quantity and differentiation were determined using a hemocytometer, hyperoxia increased the total number of cells, and the number of macrophages, neutrophils and lymphocytes in the bronchoalveolar lavage fluid. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) demonstrated that hyperoxia also increased the mRNA expression levels of cluster of differentiation (CD)68, F4/80, CD64 and CD19 in lung tissue, and induced lung morphological alterations. Furthermore, western blotting assay demonstrated that hyperoxia increased the expression levels of interleukin (IL)­1, IL­6 and tumor necrosis factor (TNF)­α, and the phosphorylation of extracellular signal­regulated kinase (ERK)1/2. Conversely, hyperoxia­induced inflammation and morphological alterations were significantly attenuated in the mice treated with fucoidan. Atomization inhalation of fucoidan also reduced the hyperoxia­induced expression of IL­1, IL­6 and TNF­α, and the phosphorylation of ERK1/2. These findings suggested that fucoidan may attenuate hyperoxic lung injury via the ERK1/2 signaling pathway.

Telmisartan suppresses cardiac hypertrophy by inhibiting cardiomyocyte apoptosis via the NFAT/ANP/BNP signaling pathway.

Telmisartan, a type of angiotensin II (Ang II) receptor inhibitor, is a common agent used to treat hypertension in the clinic. Hypertension increases cardiac afterload and promotes cardiac hypertrophy. However, the ventricular Ang II receptor may be activated in the absence of hypertension. Therefore, telmisartan may reduce cardiac hypertrophy by indirectly ameliorating hypertensive symptoms and directly inhibiting the cardiac Ang II receptor. Nuclear factor of activated T­cells (NFAT) contributes to cardiac hypertrophy via nuclear translocation, which induces a cascade of atrial natriuretic peptide (ANP) and brain/B­type natriuretic peptide (BNP) expression and cardiomyocyte apoptosis. However, NFAT-mediated inhibition of cardiac hypertrophy by telmisartan remains poorly understood. The present study demonstrated that telmisartan suppressed cardiomyocyte hypertrophy in a mouse model of cardiac afterload and in cultured cardiomyocytes by inhibiting NFAT nuclear translocation, as well as by inhibiting ANP and BNP expression and cardiomyocyte apoptosis, in a dose­dependent manner. The present study provides a novel insight into the potential underlying mechanisms of telmisartan-induced inhibition of cardiomyocyte hypertrophy, which involves inhibition of NFAT activation, nuclear translocation and the ANP/BNP cascade.

Fucoidan exerts protective effects against diabetic nephropathy related to spontaneous diabetes through the NF-κB signaling pathway in vivo and in vitro.

Fucoidan, an extract of the seaweed, Fucus vesiculosus, has been widely investigated for its antioxidant effects. However, to date and to the best of our knowledge, pathological studies on the effects of fucoidan against diabetic nephropathy (DN) related to spontaneous diabetes have not been carried out. DN is one of the most serious microvascular complications of diabetes. Therefore, in the present study, the effects of fucoidan against DN related to spontaneous diabetes were investigated in vitro and in vivo. Goto-Kakizaki (GK) rats were allowed free access to standard rat food with or without fucoidan for 13 weeks, and Wistar rats were used as controls. Fucoidan did not show any cytotoxicity on glomerular mesangial cells (GMCs) which were separated from rat kidneys. Fasting blood glucose levels were measured using a blood glucose meter, blood urea nitrogen (BUN) and serum creatinine (Cr) levels were measured using an automatic biochemistry analyzer and urine protein levels were measured using an ELISA kit. Collagen Ⅳ levels in the renal cortex were measured using an ELISA kit, and the expression levels of transforming growth factor-ß1 (TGF-ß1) and fibronectin (FN) in the renal cortex and GMCs, and nuclear factor-κB (NF-κB) in GMCs were determined by western blot analysis. Fasting blood glucose, BUN, serum Cr, urine protein and collagen Ⅳ levels, and the expression of TGF-ß1 and FN, as well as NF-κB p65 nuclear translocation all significantly increased in the GK rats compared with the control Wistar rats. The increase in the fasting blood glucose, BUN, serum Cr, urine protein and collagen Ⅳ levels in the renal cortex was reversed in the GK rats which were orally administered fucoidan. The oral administration of fucoidan also decreased the expression of TGF-ß1 and FN in the renal cortex and GMCs, as well as the nuclear translocation of NF-κB p65 in the GMCs. Taken together, the data from our in vitro and in vivo experiments indicate that fucoidan attenuates hyperglycemia and prevents or impedes the development of DN related to spontaneous diabetes by attenuating the activation of the NF-κB signaling pathway.

Antioxidant capacity responsible for a hypocholesterolemia is independent of dietary cholesterol in adult rats fed rice protein.

Dietary cholesterol and aging are major risk factors to accelerate oxidation process for developing hypercholesterolemia. The major aim of this study is to elucidate the effects of rice protein on cholesterol level and oxidative stress in adult rats fed with and without cholesterol. After 2 weeks of feeding, hepatic and plasma contents of cholesterol, reduced glutathione (GSH), oxidized glutathione (GSSG), malondialdehyde (MDA) and protein carbonyl (PCO) were measured. In liver, total antioxidative capacity (T-AOC), activities of antioxidant enzymes (total superoxide dismutase, T-SOD; catalase, CAT), glutathione metabolizing enzyme activities and gene expression levels (γ-glutamylcysteine synthetase, γ-GCS; glutathione reductase, GR; glutathione peroxidase, GPx) were determined. Under cholesterol-free/enriched dietary condition, T-AOC, activities of T-SOD and CAT, glutathione metabolism related enzymes' activities and mRNA levels (γ-GCS, GR and GPx) were effectively stimulated by rice proteins as compared to caseins. Compared with caseins, rice proteins significantly increased hepatic and plasma GSH contents, whereas hepatic and plasma accumulations of MDA, PCO and GSSG were significantly reduced by rice protein-feedings. As a result, the marked reductions of cholesterol in the plasma and in the liver were observed in adult rats fed rice proteins with and without cholesterol. The present study demonstrates that the hypocholesterolemic effect of rice protein is attributable to inducing antioxidative response and depressing oxidative damage in adult rats fed cholesterol-free/enriched diets. Results suggest that the antioxidant capability involved in the hypocholesterolemic action exerted by rice protein is independent of dietary cholesterol during adult period.

Hypocholesterolemic effect of rice protein is due to regulating hepatic cholesterol metabolism in adult rats.

Aging is one of major risk factors for developing hypercholesterolemia. To elucidate the cholesterol-lowering mechanism exerted by rice protein (RP), the effects on hepatic cholesterol outputs and cholesterol metabolism related enzymes were investigated in adult rats, which were fed by casein (CAS) and RP without cholesterol in diets. After 2 weeks of feeding, the significant cholesterol-lowering effect was observed in adult rats fed by RP compared to CAS. The hepatic total- and VLDL-cholesterol secretions into circulation were significantly depressed in RP group, whereas biliary outputs of bile acids and cholesterol were effectively stimulated by RP-feeding, causing an increase in fecal sterol excretion compared to CAS. As a result, the apparent cholesterol absorption was significantly inhibited by RP. RP-feeding significantly increased the activity and gene expression of cholesterol 7α-hydroxylase, whereas acyl-CoA:cholesterol acyltransferase-2 activity and gene expression were significantly decreased by RP as compared with CAS. Neither activity nor gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase of RP did differ from CAS in the liver. The present study demonstrates that rice protein can prevent hypercholesterolemia through modifying hepatic cholesterol metabolism under cholesterol-free dietary condition. The findings suggest that hypocholesterolemic action induced by rice protein is attributed in part to the inhibition of cholesterol absorption during the adult period.