Predicted risk of heart failure pandemic due to persistent SARS-CoV-2 infection using a three-dimensional cardiac model.

Patients with chronic cardiomyopathy may have persistent viral infections in their hearts, particularly with SARS-CoV-2, which targets the ACE2 receptor highly expressed in human hearts. This raises concerns about a potential global heart failure pandemic stemming from COVID-19, an SARS-CoV-2 pandemic in near future. Although faced with this healthcare caveat, there is limited research on persistent viral heart infections, and no models have been established. In this study, we created an SARS-CoV-2 persistent infection model using human iPS cell-derived cardiac microtissues (CMTs). Mild infections sustained viral presence without significant dysfunction for a month, indicating persistent infection. However, when exposed to hypoxic conditions mimicking ischemic heart diseases, cardiac function deteriorated alongside intracellular SARS-CoV-2 reactivation in cardiomyocytes and disrupted vascular network formation. This study demonstrates that SARS-CoV-2 persistently infects the heart opportunistically causing cardiac dysfunction triggered by detrimental stimuli such as ischemia, potentially predicting a post COVID-19 era heart failure pandemic.

Therapeutic potential of human induced pluripotent stem cell-derived cardiac tissue in an ischemic model with unloaded condition mimicking left ventricular assist device.

OBJECTIVE: This study aimed to explore the therapeutic potential of human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) in the emerging approach of bridge to recovery for severe heart failure with ventricular assist devices. We used a rat model of heterotopic heart transplantation (HTx) to mimic ventricular assist device support and heart unloading. METHODS: HiCTs were created by inserting gelatin hydrogel microspheres between cell sheets made from hiPSC-derived cardiovascular cells. Male athymic nude rats underwent myocardial infarction (MI) and were divided into the following groups: MI (loaded, untreated control), MI + HTx (unloaded, untreated control), MI + HTx + HiCT (unloaded, treated), and MI + HiCT (loaded, treated). HiCTs were placed on the epicardium of the heart in treated groups. We evaluated HiCT engraftment, fibrosis, and neovascularization using histologic analysis. RESULTS: After 4 weeks, HiCTs successfully engrafted in 5 of 6 rats in the MI + HTx + HiCT group (83.3%). The engrafted HiCT area was greater under unloaded conditions (MI + HTx + HiCT) than loaded conditions (MI + HiCT) (P < .05). MI + HTx + HiCT had a significantly smaller infarct area compared with MI and MI + HTx. The MI + HTx + MiCT group exhibited greater vascular density in the border zone than MI and MI + HTx. HiCT treatment suppressed cardiomyocyte atrophy due to left ventricular unloading (P = .001). The protein level of muscle-specific RING finger 1, an atrophy-related ubiquitin ligase, was lower in the MI + HTx + HiCT group than in MI + HTx (P = .036). CONCLUSIONS: Transplanting HiCTs into ischemic hearts under unloaded conditions promoted engraftment, neovascularization, attenuated infarct remodeling, and suppressed myocyte atrophy. These results suggest that HiCT treatment could contribute to future advancements in bridge to recovery.

Antagonist of sphingosine 1-phosphate receptor 3 reduces cold injury of rat donor hearts for transplantation.

Cold storage is widely used to preserve an organ for transplantation; however, a long duration of cold storage negatively impacts graft function. Unfortunately, the mechanisms underlying cold exposure remain unclear. Based on the sphingosine-1-phosphate (S1P) signal involved in cold tolerance in hibernating mammals, we hypothesized that S1P signal blockage reduces damage from cold storage. We used an in vitro cold storage and rewarming model to evaluate cold injury and investigated the relationship between cold injury and S1P signal. Compounds affecting S1P receptors (S1PR) were screened for their protective effect in this model and its inhibitory effect on S1PRs was measured using the NanoLuc Binary Technology (NanoBiT)-ß-arrestin recruitment assays. The effects of a potent antagonist were examined via heterotopic abdominal rat heart transplantation. The heart grafts were transplanted after 24-hour preservation and evaluated on day 7 after transplantation. Cold injury increased depending on the cold storage time and was induced by S1P. The most potent antagonist strongly suppressed cold injury consistent with the effect of S1P deprivation in vitro. In vivo, this antagonist enabled 24-hour preservation, and drastically improved the beating score, cardiac size, and serological markers. Pathological analysis revealed that it suppressed the interstitial edema, inflammatory cell infiltration, myocyte lesion, TUNEL-positive cell death, and fibrosis. In conclusion, S1PR3 antagonist reduced cold injury, extended the cold preservation time, and improved graft viability. Cold preservation strategies via S1P signaling may have clinical applications in organ preservation for transplantation and contribute to an increase in the donor pool.

A novel approach for the endothelialization of xenogeneic decellularized vascular tissues by human cells utilizing surface modification and dynamic culture.

Decellularized xenogeneic vascular grafts can be used in revascularization surgeries. We have developed decellularization methods using high hydrostatic pressure (HHP), which preserves the extracellular structure. Here, we attempted ex vivo endothelialization of HHP-decellularized xenogeneic tissues using human endothelial cells (ECs) to prevent clot formation against human blood. Slices of porcine aortic endothelium were decellularized using HHP and coated with gelatin. Human umbilical vein ECs were directly seeded and cultured under dynamic flow or static conditions for 14 days. Dynamic flow cultures tend to demonstrate higher cell coverage. We then coated the tissues with the E8 fragment of human laminin-411 (hL411), which has high affinity for ECs, and found that Dynamic/hL411showed high area coverage, almost reaching 100% (Dynamic/Gelatin vs Dynamic/hL411; 58.7 ± 11.4 vs 97.5 ± 1.9%, P = 0.0017). Immunostaining revealed sufficient endothelial cell coverage as a single cell layer in Dynamic/hL411. A clot formation assay using human whole blood showed low clot formation in Dynamic/hL411, almost similar to that in the negative control, polytetrafluoroethylene. Surface modification of HHP-decellularized xenogeneic endothelial tissues combined with dynamic culture achieved sufficient ex vivo endothelialization along with prevention of clot formation, indicating their potential for clinical use as vascular grafts in the future.

Pioglitazone-incorporated microspheres targeting macrophage polarization alleviates cardiac dysfunction after myocardial infarction.

OBJECTIVES: Excessive and chronic inflammation after a myocardial infarction (MI) is associated with left ventricular remodelling and impaired cardiac function. Among inflammatory cells, macrophages play a critical role in polarizing proinflammatory M1 or the reparative M2 subtype. Pioglitazone (PGZ) is reported to regulate macrophage polarization to the M2 subtype. Our goal was to validate the therapeutic effects and the mechanisms of PGZ utilizing a drug delivery system. METHODS: Poly L-lactic-co-glycolic acid microspheres (MS) incorporating PGZ were prepared. To validate the therapeutic potential of PGZ-MS, Sprague-Dawley rats were subjected to permanent left coronary artery ligation to induce an MI. Placebo-MS (100 µg) or PGZ-MS (100 µg) was injected to the infarct region just after induction. Cardiac function and size were assessed by echocardiography. At 28 days after surgery, the rats were sacrificed, and the excised hearts were evaluated histologically. RESULTS: Sustained release of PGZ from the PGZ-MS was confirmed in vitro. PGZ-MS significantly rehabilitated cardiac dysfunction after an MI (fractional shortening: MI vs MI+placebo-MS vs MI+PGZ-MS, 24.4 ± 1.1 vs 24.3 ± 1.6 vs 32.2 ± 1.4%; P = 0.0035) with reverse remodelling. Immunohistochemical analyses revealed that PGZ-MS enhanced macrophage polarization (ratio of M2 subtype: 0.39 ± 0.03 vs 0.42 ± 0.02 vs 0.54 ± 0.02; P = 0.0004) and attenuated apoptosis of cardiomyocytes in the ischaemic border zone. CONCLUSIONS: We confirmed macrophage polarization by sustained release of PGZ, which resulted in amelioration of adverse left ventricular remodelling and cardiac dysfunction. Drug delivery system-based macrophage polarization might serve as a promising strategy in cardiac regenerative therapy for ischaemic heart disease. (241 words).

Systems for the Functional Evaluation of Human Heart Tissues Derived from Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) are expected to be a promising cell source in regenerative medicine and drug discovery for the treatment of various intractable diseases. An approach for creating a 3-dimensional (3D) structure from hPSCs that mimics human cardiac tissue functions has made it theoretically possible to conduct drug discovery and cardiotoxicity tests by assessing pharmacological responses in human cardiac tissues by a screening system using a compound library. The myocardium functions as a tissue composed of organized vascular networks, supporting stromal cells and cardiac muscle cells. Considering this, the reconstruction of tissue structure by various cells of cardiovascular lineages, such as vascular cells and cardiac muscle cells, is desirable for the ideal conformation of hPSC-derived cardiac tissues. Heart-on-a-chip, an organ-on-a-chip system to evaluate the physiological pump function of 3D cardiac tissues might hold promise in medical researchs such as drug discovery and regenerative medicine. Here, we review various modalities to evaluate the function of human stem cell-derived cardiac tissues and introduce heart-on-a-chip systems that can recapitulate physiological parameters of hPSC-derived cardiac tissues.

Evaluations for surrounding tissue incorporation after implantation of synthetic vascular prostheses in animal models.

Incorporation of surrounding tissues after implantation of synthetic vascular prostheses potentially varies in accordance with implanted prostheses. To evaluate post-implant tissue incorporation, we examined surgical, histological and ultrastructural findings after implantation in animal models. Three types of commercially available prostheses were tested (Gelweave™; Group G, J Graft SHIELD NEO®; Group J and Triplex®; Group T). Prostheses were implanted into Sprague-Dawley rats subcutaneously or sutured on abdominal aorta of Japanese white rabbits. The tissues were surgically examined for adhesion and were subjected to histological evaluations for cellular and tissue infiltration and ultrastructural observations by scanning electron microscopy (SEM). Group G exhibited less tendency in adhesion formation in early phase (rat: G vs J, P < 0.0001; G vs T, P < 0.0001/rabbit: G vs J, P < 0.0001; G vs T, P = 0.059). In late phase, Group J showed highest adhesion (rat: G vs J, P = 0.0004; J vs T, P = 0.015/rabbit: G vs J, P = 0.0015; J vs T, P = 0.0044). In group G, a gap was observed between implants and surrounding tissues forming capsulation, whereas other groups exhibited tissue infiltration inside of the implants wall which were also confirmed by SEM. The tissue permeation toward the implants and adhesion was positively correlated (P < 0.0001). Surrounding tissue conformation varied in accordance with the type of prostheses. It is desirable to elucidate characteristics of each prosthesis to select suitable grafts for each patient to achieve a better surgical outcome.

Quiescence-inducing neurons-induced hypometabolism ameliorates acute kidney injury in a mouse model mimicking cardiovascular surgery requiring circulatory arrest.

Objectives: Acute kidney injury is a serious complication after cardiovascular surgery requiring circulatory arrest. It is reported that mice can be induced into a hibernation-like hypometabolic state by stimulating a specific neuron located at the hypothalamus (quiescence-inducing neurons-induced hypometabolism [QIH]). Here, we investigated the efficacy of QIH for the amelioration of acute kidney injury in an experimental circulatory arrest using a transgenic mouse model. Methods: We genetically prepared mice in which QIH can be conditionally induced (QIH-ready mice). Mice were divided into 4 groups (n = 6 for each): QIH-ready normothermia (QN), QIH-ready hypothermia (QH), control normothermia (CN), and control hypothermia (CH). After induction of QIH, left thoracotomy and descending aorta crossclamping were conducted. After reperfusion, we collected kidneys and evaluated histologic changes and serum biochemical markers, specifically neutrophil gelatinase-associated lipocalin and cystatin C, indicating early kidney injury. Results: Normothermia showed higher tubular injury scores than those in hypothermia (QN vs QH [P = .0021] and CN vs CH [P < .001]). QN exhibited lower neutrophil gelatinase-associated lipocalin and cystatin C levels than those in CN (neutrophil gelatinase-associated lipocalin: CN vs QN: 1.51 ± 0.71 vs 0.82 ± 0.32; P = .0414 and cystatin C: 1.48 ± 0.39 vs 0.71 ± 0.26; P = .0015). There was no significant difference between QN and QH. Conclusions: QIH partly ameliorated acute kidney injury in a mouse ischemia model even in normothermia. QIH might be a promising approach to achieving sufficient kidney protection without hypothermic circulatory arrest in the future.

In vivo recellularization of xenogeneic vascular grafts decellularized with high hydrostatic pressure method in a porcine carotid arterial interpose model.

Autologous vascular grafts are widely used in revascularization surgeries for small caliber targets. However, the availability of autologous conduits might be limited due to prior surgeries or the quality of vessels. Xenogeneic decellularized vascular grafts from animals can potentially be a substitute of autologous vascular grafts. Decellularization with high hydrostatic pressure (HHP) is reported to highly preserve extracellular matrix (ECM), creating feasible conditions for recellularization and vascular remodeling after implantation. In the present study, we conducted xenogeneic implantation of HHP-decellularized bovine vascular grafts from dorsalis pedis arteries to porcine carotid arteries and posteriorly evaluated graft patency, ECM preservation and recellularization. Avoiding damage of the luminal surface of the grafts from drying significantly during the surgical procedure increased the graft patency at 4 weeks after implantation (P = 0.0079). After the technical improvement, all grafts (N = 5) were patent with mild stenosis due to intimal hyperplasia at 4 weeks after implantation. Neither aneurysmal change nor massive thrombosis was observed, even without administration of anticoagulants nor anti-platelet agents. Elastica van Gieson and Sirius-red stainings revealed fair preservation of ECM proteins including elastin and collagen after implantation. The luminal surface of the grafts were thoroughly covered with von Willebrand factor-positive endothelium. Scanning electron microscopy of the luminal surface of implanted grafts exhibited a cobblestone-like endothelial cell layer which is similar to native vascular endothelium. Recellularization of the tunica media with alpha-smooth muscle actin-positive smooth muscle cells was partly observed. Thus, we confirmed that HHP-decellularized grafts are feasible for xenogeneic implantation accompanied by recellularization by recipient cells.

Generation of Cylindrical Engineered Cardiac Tissues from Human iPS Cell-Derived Cardiovascular Cell Lineages.

The present protocol describes a method to generate cylindrical engineered cardiac tissues (ECTs) composed of cardiovascular cell lineages induced from human induced pluripotent stem cells (hiPSCs). Cardiomyocytes, endothelial cells, and vascular mural cells induced from hiPSCs are mixed with gel matrix and poured into a tissue mold with posts. By culture day 14, the mixed culture matures into a cylindrical ECT which beats spontaneously and synchronously. Cardiomyocytes align to the long axis of the ECT. The ECTs generated by the present method may be regarded as a surrogate of human myocardium and be served as researches in cardiac regenerative medicine, disease modeling, drug discovery, and cardiac toxicity tests.

Therapeutic potential of clinical-grade human induced pluripotent stem cell-derived cardiac tissues.

Objectives: To establish a protocol to prepare and transplant clinical-grade human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) and to evaluate the therapeutic potential in an animal myocardial infarction (MI) model. Methods: We simultaneously differentiated clinical-grade hiPSCs into cardiovascular cell lineages with or without the administration of canonical Wnt inhibitors, generated 5- layer cell sheets with insertion of gelatin hydrogel microspheres (GHMs) (HiCTs), and transplanted them onto an athymic rat MI model. Cardiac function was evaluated by echocardiography and cardiac magnetic resonance imaging and compared with that in animals with sham and transplantation of 5-layer cell sheets without GHMs. Graft survival, ventricular remodeling, and neovascularization were evaluated histopathologically. Results: The administration of Wnt inhibitors significantly promoted cardiomyocyte (CM) (P < .0001) and vascular endothelial cell (EC) (P = .006) induction, which resulted in cellular components of 52.0 ± 6.1% CMs and 9.9 ± 3.0% ECs. Functional analyses revealed the significantly lowest left ventricular end-diastolic volume and highest ejection fraction in the HiCT group. Histopathologic evaluation revealed that the HiCT group had a significantly larger median engrafted area (4 weeks, GHM(-) vs HiCT: 0.4 [range, 0.2-0.7] mm2 vs 2.2 [range, 1.8-3.1] mm2; P = .005; 12 weeks, 0 [range, 0-0.2] mm2 vs 1.9 [range, 0.1-3.2] mm2; P = .026), accompanied by the smallest scar area and highest vascular density at the MI border zone. Conclusions: Transplantation of HiCTs generated from clinical-grade hiPSCs exhibited a prominent therapeutic potential in a rat MI model and may provide a promising therapeutic strategy in cardiac regenerative medicine.

Chronic Optogenetic Pacing of Human-Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissues.

The delivery of cells into damaged myocardium induces limited cardiac regeneration due to extensive cell death. In an effort to limit cell death, our lab formulates three-dimensional matrices as a delivery system for cell therapy. Our primary work has been focused on the formation of engineered cardiac tissues (ECTs) from human-induced pluripotent stem cell-derived engineered cardiac cells. However, ECT immaturity hinders ability to fully recover damaged myocardium. Various conditioning regimens such as mechanical stretch and/or electric pacing have been used to activate maturation pathways. To improve ECT maturity, we use non-contacting chronic light stimulation using heterologously expressed light-sensitive channelrhodopsin ion channels. We transduce ECTs with an AAV packaged channelrhodopsin and chronically optically pace (C-OP) ECTs for 1 week above the intrinsic beat rate, resulting in increased ECT electrophysiological properties.

Establishment of a heart-on-a-chip microdevice based on human iPS cells for the evaluation of human heart tissue function.

Human iPS cell (iPSC)-derived cardiomyocytes (CMs) hold promise for drug discovery for heart diseases and cardiac toxicity tests. To utilize human iPSC-derived CMs, the establishment of three-dimensional (3D) heart tissues from iPSC-derived CMs and other heart cells, and a sensitive bioassay system to depict physiological heart function are anticipated. We have developed a heart-on-a-chip microdevice (HMD) as a novel system consisting of dynamic culture-based 3D cardiac microtissues derived from human iPSCs and microelectromechanical system (MEMS)-based microfluidic chips. The HMDs could visualize the kinetics of cardiac microtissue pulsations by monitoring particle displacement, which enabled us to quantify the physiological parameters, including fluidic output, pressure, and force. The HMDs demonstrated a strong correlation between particle displacement and the frequency of external electrical stimulation. The transition patterns were validated by a previously reported versatile video-based system to evaluate contractile function. The patterns are also consistent with oscillations of intracellular calcium ion concentration of CMs, which is a fundamental biological component of CM contraction. The HMDs showed a pharmacological response to isoproterenol, a ß-adrenoceptor agonist, that resulted in a strong correlation between beating rate and particle displacement. Thus, we have validated the basic performance of HMDs as a resource for human iPSC-based pharmacological investigations.

Strategies for immune regulation in iPS cell-based cardiac regenerative medicine.

Cardiac regenerative therapy is expected to be a promising therapeutic option for the treatment of severe cardiovascular diseases. Artificial tissues or organoids made from cardiovascular cell lineages differentiated from human induced pluripotent stem cells (iPSCs) are expected to regenerate the damaged heart. Even though immune rejection rarely occurs when iPSC-derived graft and the recipient have the same HLA type, in some cases, such as tissue transplantation onto hearts, the HLA matching would not be sufficient to fully control immune rejection. The present review introduces recent immunomodulatory strategies in iPSC-based transplantation therapies other than MHC matching including the induction of immune tolerance through iPSC-derived antigen-presenting cells, simultaneous transplantation of syngeneic mesenchymal stem cells, and using the universal donor cells such as gene editing-based HLA modulation in iPSCs to regulate T cell compatibility. In addition, we present future perspectives for proper adjustment of immunosuppression therapy after iPSC-derived tissue/organoid-based cardiac regenerative therapies by identifying biomarkers monitoring immune rejection.

Accuracy of spiked cell counting methods for designing a pre-clinical tumorigenicity study model.

BACKGROUND: Evaluations for the tumorigenicity of transplantation of stem cell products is mandatory for clinical application. It is of importance to establish a system to accurately quantify contaminated tumorigenic cells regardless of the format of stem cell product. In the present report, we aimed to examine the accuracy of the quantification of tumorigenic cell numbers with commonly used 2 methods, quantitative polymerase chain reaction (qPCR) and flow cytometry (FCM) using experimental models of stem cell products spiked with tumorigenic cells. METHODS: Human mesenchymal stem cells (hMSCs) and melanoma Mewo-Luc cells constitutively expressing luciferase were used. We stained Mewo-Luc cells with a cell linker then spiked onto hMSC suspensions and hMSC sheets. We validated the accuracy of 10-fold serial dilution technique for Mewo-Luc cell suspension using a Coulter counter. The samples spiked with Mewo-Luc cells were subjected to qPCR and FCM analyses, respectively for the quantification of Mewo-Luc cells. RESULTS: Ten-fold serial dilutions of Mewo-Luc cells were performed accurately with small deviation. In samples spiked with or less than 100 cells in hMSC suspensions, and samples spiked with or less than 1,000 cells in hMSC sheets showed significantly higher cell numbers in calculations by FCM, respectively (suspensions; qPCR vs FCM: 100 cells: 59 ± 25 vs 232 ± 35 cells, p = 0.022/10 cells: 21 ± 7 vs 114 ± 27 cells, p = 0.030, sheets; qPCR vs FCM: 1,000 cells: 1723 ± 258 vs 5810 ± 878 cells, p = 0.012/100 cells: 110 ± 18 vs 973 ± 232 cells, p = 0.012/10 cells: 20 ± 6 vs 141 ± 36 cells, p = 0.030). CONCLUSION: Differences in accuracy between quantification methods should be considered in designing a tumorigenicity study model.

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair.

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure.

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

Management of retrosternal adhesion after median sternotomy by controlling degradation speed of a dextran and ε-poly (L-lysine)-based biocompatible glue.

OBJECTIVE: Retrosternal adhesion after median sternotomy possibly raises the risk of cardiac injury at resternotomy. A biodegradable glue "Lydex" is composed of food additives, dextran and ε-poly (L-lysine), and the degradation speed can be controlled by the composition. In the present study, we evaluated the preventative effect of Lydex on retrosternal adhesion and the relationship between degradation speed and the progression of retrosternal fibrosis. METHODS: Japanese white rabbits are subjected to median sternotomy. Lydex 1, 2 and 3 were loaded at the retrosternal space of rabbits in allocated groups before sternal closure, respectively (n = 11 for each group). Retrosternal adhesion was macroscopically evaluated after surgery. Retainment of Lydex, retrosternal fibrosis and the infiltration of macrophages are histologically evaluated, respectively. RESULTS: All Lydex groups exhibited less retrosternal adhesion at 4 weeks after loading compared to unloaded control. The degradation speed of Lydex varied according to the compositions. Lydex with faster degradation (Lydex 2 or Lydex 3) showed lower progression of retrosternal fibrosis compared to that with slower degradation (Lydex 1) [fibrosis ratio: control vs Lydex 1 vs Lydex 2 vs Lydex 3: 0.60 ± 0.15 vs 0.18 ± 0.17 vs 0.00 ± 0.00 vs 0.00 ± 0.00, P = 0.0005 (Lydex 1 vs Lydex 2), P = 0.0005 (Lydex 1 vs Lydex 3)]. Retrosternal infiltrations of macrophages in Lydex 1 and Lydex 3 groups are not higher compared to that in unloaded control. CONCLUSIONS: The degradation speed of Lydex could be controlled according to the compositions. The degradation speed affected the progression of retrosternal fibrosis.

The N-terminal lectin-like domain of thrombomodulin reduces acute lung injury without anticoagulant effects in a rat cardiopulmonary bypass model.

OBJECTIVES: Systemic inflammation evoked by cardiopulmonary bypass (CPB) leads to acute lung injury (ALI) and respiratory failure. Although recombinant human soluble thrombomodulin (rTM) consists of three domains (D1-3), is reported to attenuate systemic inflammation through the N-terminal lectin-like domain (D1), anticoagulant domain (D2) may exacerbate coagulopathy after CPB. We investigated the potential of selective D1 against CPB-mediated ALI free from anticoagulant effects using a rat CPB model. METHODS: Rats were divided into three groups: control (CPB alone, n = 5), D1 (CPB + D1, n = 4), and D123 (CPB + D123, n = 6). D1 or D123 was administrated to the rats of each group before CPB establishment. Blood samples are collected before, during and after CPB. Blood coagulability was assessed by a coagulation analyzer. Lung samples are collected at 1 h after the termination of CPB for histological analyses. RESULTS: D123 group exhibited significantly prolonged glass beads-activated clotting time with heparinase after CPB compared to that in control group, whereas no significant prolongation in control and D1 group (control vs. D1 vs. D123: 65.4 ± 9.2 vs. 65.3 ± 10.9 vs. 83.5 ± 4.6 s, p = 0.036 [control vs. D123], 0.99 [control vs. D1]) indicating the absence of anticoagulant activities of D1. Histological studies revealed less congestion, edema, inflammation, and hemorrhage in both D1 and D123 groups compared to those in control group indicating protective effects of both D1 and D123 against ALI mediated by CPB. CONCLUSIONS: N-terminal lectin-like domain of rTM may reduce the risk of ALI without anticoagulant effects.

Novel device prototyping for endoscopic cell sheet transplantation using a three-dimensional printed simulator.

INTRODUCTION: Considering higher risks of candidates for cardiac regenerative therapy with compromised cardiac function, it is anticipated to develop less invasive surgical procedures. In the present study, we aimed to develop a prototype of totally endoscopic cell sheet delivery device and evaluate the surgical technique for epicardial cell sheet placement using three-dimensional (3D) printed simulators based on human computed tomography data. METHODS: We designed an endoscopic cell sheet delivery device with outer and inner frame with self-expandable applicator which can be opened in thoracic cavity. We launched spout line to provide liquids on the applicator surface and tension line to gently bend the applicator dorsally. We prepared human mesenchymal stem cell (MSC) sheets and compared wet/dry conditions of 3D printed heart/porcine heart and applicator to identify suitable conditions for cell sheet transplantation. Finally we validated the feasibility of endoscopic transplantation to anterior and lateral wall of left ventricle using 3D printed simulators. RESULTS: Moist condition of both 3D printed heart/porcine heart surface and applicator at transplantation yielded highest successful rate (100%, p = 0.0197). For both endoscopic transplantation sites, MSC sheets were successfully deployed. The procedure duration was 157 ± 23 s for anterior wall and 123 ± 13 s for the lateral wall in average, respectively. CONCLUSIONS: We developed a novel prototype of endoscopic cell sheet delivery device for minimally-invasive cardiac regenerative therapy utilizing a 3D printed simulator. The commercialization of the prototype may provide a safe minimally-invasive method to deliver potential cardiac regenerative therapy in the future.