Automated mechanical exfoliation technique: a spin pumping study in YIG/TMD heterostructures.

Spintronics devices rely on the generation and manipulation of spin currents. Two-dimensional transition-metal dichalcogenides (TMDs) are among the most promising materials for a spin current generation due to a lack of inversion symmetry at the interface with the magnetic material. Here, we report on the fabrication of Yttrium Iron Garnet(YIG)/TMD heterostructures by means of a crude and fast method. While the magnetic insulator single-crystalline YIG thin films were grown by magnetron sputtering, the TMDs, namely MoS2 and MoSe2, were directly deposited onto YIG films using an automated mechanical abrasion method. Despite the brute force aspect of the method, it produces high-quality interfaces, which are suitable for spintronic device applications. The spin current density and the effective spin mixing conductance were measured by ferromagnetic resonance, whose values found are among the highest reported in the literature. Our method can be scaled to produce ferromagnetic materials/TMD heterostructures on a large scale, further advancing their potential for practical applications.

Construction and Evaluation of a Bio-Engineered Pump to Enable Subpulmonary Support of the Fontan Circulation: A Proof-of-Concept Study.

Our objective was to create a bio-engineered pump (BEP) for subpulmonary Fontan circulation support capable of luminal endothelialization and producing a 2-6 mmHg pressure gradient across the device without flow obstruction. To accomplish this, porcine urinary bladder submucosa was decellularized to produce a urinary bladder matrix (UBM) which produced acellular sheets of UBM. The UBM was cultured with human umbilical vein endothelial cells producing a nearly confluent monolayer of cells with the maintenance of typical histologic features demonstrating UBM to be a suitable substrate for endothelial cells. A lamination process created bilayer UBM sheets which were formed into biologic reservoirs. BEPs were constructed by securing the biologic reservoir between inlet and outlet valves and compressed with a polyurethane balloon. BEP function was evaluated in a simple flow loop representative of a modified subpulmonary Fontan circulation. A BEP with a 92-mL biologic reservoir operating at 60 cycles per minute produced pulsatile downstream flows without flow obstruction and generated a favorable pressure gradient across the device, maintaining upstream pressure of 6 mm Hg and producing downstream pressure of 13 mm Hg. The BEP represents potential long-term assistance for the Fontan circulation to relieve venous hypertension, provide pulsatile pulmonary blood flow and maintain cardiac preload.

Recommendations for nomenclature and definition of cell products intended for human cardiovascular use.

Exogenous cell-based therapy has emerged as a promising new strategy to facilitate repair of hearts damaged by acute or chronic injury. However, the field of cell-based therapy is handicapped by the lack of standardized definitions and terminology, making comparisons across studies challenging. Even the term 'stem cell therapy' is misleading because only a small percentage of cells derived from adult bone marrow, peripheral blood, or adipose tissue meets the accepted haematopoietic or developmental definition of stem cells. Furthermore, cells (stem or otherwise) are dynamic biological products, meaning that their surface-marker expression, phenotypic and functional characteristics, and the products they secrete in response to their microenvironment can change. It is also important to point out that most surface markers are seldom specific for a cell type. In this article, we discuss the lack of consistency in the descriptive terminology used in cell-based therapies and offer guidelines aimed at standardizing nomenclature and definitions to improve communication among investigators and the general public.

Restoring anatomical complexity of a left ventricle wall as a step toward bioengineering a human heart with human induced pluripotent stem cell-derived cardiac cells.

The heart is a highly complex, multicellular solid organ with energy-demanding processes that require a dense vascular network, extensive cell-cell interactions, and extracellular matrix (ECM)-mediated crosstalk among heterogeneous cell populations. Here, we describe the regeneration of left ventricular (LV) wall using decellularized whole rabbit heart scaffolds recellularized exclusively with human induced pluripotent stem cell-derived endothelial cells, cardiomyocytes, and other cardiac cell types. Cells were sequentially delivered to the scaffold using an optimized endothelial cell:cardiomyocyte media. Macroscopic assessment after 60 days showed that the LV wall of recellularized hearts was anatomically restored to full thickness from base to apex and endocardium to epicardium. Histologic analysis of the recellularized LV wall revealed a heterogeneous pool of cardiac cells containing aligned cardiac troponin T-positive cells in close contact with ECM; vessels varied from large artery-like, surrounded by smooth muscle actin+ cells, to capillary-like. Vessel patency was demonstrated after perfusion of recellularized hearts transplanted into the femoral artery bed of a pig. The construct exhibited visible beating and responded to chronotropic drug administration. These results demonstrate the ability to tissue engineer a vascularized, full-thickness LV wall with an unparalleled level of microanatomical organization and multicellular composition, using decellularized ECM and human cardiomyocytes, endothelial cells, and other cardiac cell types. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrix (ECM) is a bioactive template for tissue engineering, but recellularizing acellular whole heart scaffolds is challenging. Here, we successfully revascularized and repopulated a large, full-thickness portion of a ventricle using human induced pluripotent stem cell-derived endothelial and cardiac cells. At 60 days, histologic studies showed that the microanatomical organization and cellular composition of this region was similar to that of the native heart. The recellularized heart showed visible beating and responded appropriately to heartbeat-altering drugs. Vessels surrounded by smooth muscle cells and endothelial cells supported blood flow through the vessels of a recellularized heart that was surgically connected to a pig femoral artery. These findings move this approach closer to the possibility of clinical translation.

Analysis of sex-based differences in clinical and molecular responses to ischemia reperfusion after lung transplantation.

BACKGROUND: Sex and hormones influence immune responses to ischemia reperfusion (IR) and could, therefore, cause sex-related differences in lung transplantation (LTx) outcomes. We compared men's and women's clinical and molecular responses to post-LTx IR. METHODS: In 203 LTx patients, we used the 2016 International Society for Heart and Lung Transplantation guidelines to score primary graft dysfunction (PGD). In a subgroup of 40 patients with blood samples collected before LTx (T0) and 6, 24, 48 (T48), and 72 h (T72) after lung reperfusion, molecular response to IR was examined through serial analysis of circulating cytokine expression. RESULTS: After adjustment, women had less grade 3 PGD than men at T48, but not at T72. PGD grade decreased from T0 to T72 more often in women than men. The evolution of PGD (the difference in mean PGD between T72 and T0) was greater in men. However, the evolution of IL-2, IL-7, IL-17a, and basic fibroblast growth factor levels was more often sustained throughout the 72 h in women. In the full cohort, we noted no sex differences in secondary clinical outcomes, but women had significantly lower peak lactate levels than men across the 72 h. CONCLUSIONS: Men and women differ in the evolution of PGD and cytokine secretion after LTx: Women have a more sustained proinflammatory response than men despite a greater reduction in PGD over time. This interaction between cytokine and PGD responses warrants investigation. Additionally, there may be important sex-related differences that could be used to tailor treatment during or after transplantation.

Incidence of primary graft dysfunction is higher according to the new ISHLT 2016 guidelines and correlates with clinical and molecular risk factors.

BACKGROUND: Primary graft dysfunction (PGD) is the most important determinant of morbidity and mortality after lung transplantation, but its definition has evolved over the past decade. The implications of this refinement in clinical definition have not been evaluated. In this single-center study, we compared PGD incidence, risk factors, and outcomes using the 2005 and the updated-2016 International Society of Heart and Lung Transplantation guidelines for PGD grading in lung transplant patients. METHODS: In this retrospective study, we extracted data from the medical records of 127 patients who underwent lung transplantation between 1/1/2016-12/31/2018. PGD was defined as PGD3 present at 48 and/or 72 hours post-reperfusion. We used the 2005 and the updated 2016 guidelines to assess clinical risk factors, outcomes, and baseline biomarkers for PGD. RESULTS: On the basis of the 2016 and 2005 guidelines, we identified PGD in 37% and 26% of patients, respectively. PGD was significantly associated with extracorporeal life support, large body mass index, and restrictive lung disease using the 2016 but not the 2005 guidelines. Based on the 2016 guidelines, pretransplant levels of several biomarkers were associated with PGD; using the 2005 guidelines, only increased interleukin-2 levels were significantly associated with PGD. No preoperative biomarkers were associated with PGD using either guidelines after adjusting for clinical variables. Postoperative morbidity and 1-year mortality were similar regardless of guidelines used. CONCLUSIONS: Our findings suggest that refinements in the PGD scoring system have improved the detection of graft injury and associated risk factors without changing its ability to predict postoperative morbidity and mortality.

Characterization of perfusion decellularized whole animal body, isolated organs, and multi-organ systems for tissue engineering applications.

To expand the application of perfusion decellularization beyond isolated single organs, we used the native vasculature of adult and neonatal rats to systemically decellularize the organs of a whole animal in situ. Acellular scaffolds were generated from kidney, liver, lower limb, heart-lung system, and a whole animal body, demonstrating that perfusion decellularization technology is applicable to any perfusable tissue, independent of age. Biochemical and histological analyses demonstrated that organs and organ systems (heart-lung pair and lower limb) were successfully decellularized, retaining their extracellular matrix (ECM) structure and organ-specific composition, as evidenced by differences in organ-specific scaffold stiffness. Altogether, we demonstrated that organs, organ systems and whole animal bodies can be perfusion decellularized while retaining ECM components and biomechanics.

Sex-Based Differences in Autologous Cell Therapy Trials in Patients With Acute Myocardial Infarction: Subanalysis of the ACCRUE Database.

Background: Sex-based differences are under-studied in cardiovascular trials as women are commonly underrepresented in dual sex studies, even though major sex-based differences in epidemiology, pathophysiology, and outcomes of cardiovascular disease have been reported. We examined sex-based differences in patient characteristics, outcome, and BM-CD34+ frequency of the ACCRUE (Meta-Analysis of Cell-based CaRdiac studies) database involving patients with acute myocardial infarction (AMI) randomized to autologous cell-based or control treatment. Methods: We compared baseline characteristics and 1-year follow-up clinical data: composite major adverse cardiac and cerebrovascular events (primary endpoint), and changes in left ventricular ejection fraction (LVEF), end-diastolic (EDV), and end-systolic volumes (ESV) (secondary efficacy endpoint) in women and men (N = 1,252; 81.4% men). Secondary safety endpoints included freedom from hard clinical endpoints. Results: In cell-treated groups, women but not men had a lower frequency of stroke, AMI, and mortality than controls. The frequency of BM-CD34+ cells was significantly correlated with baseline EDV and ESV and negatively correlated with baseline LVEF in both sexes; a left shift in regression curve in women indicated a smaller EDV and ESV was associated with higher BM-CD34+ cells in women. Conclusions: Sex differences were found in baseline cardiovascular risk factors and cardiac function and in outcome responses to cell therapy.

Cues from human atrial extracellular matrix enrich the atrial differentiation of human induced pluripotent stem cell-derived cardiomyocytes.

New robust and reproducible differentiation approaches are needed to generate induced pluripotent stem cell (iPSC)-derived cardiomyocytes of specific subtypes in predictable quantities for tissue-specific disease modeling, tissue engineering, and eventual clinical translation. Here, we assessed whether powdered decellularized extracellular matrix (dECM) particles contained chamber-specific cues that could direct the cardiac differentiation of human iPSCs toward an atrial phenotype. Human hearts were dissected and the left ventricle (LV) and left atria (LA) were isolated, minced, and decellularized using an adapted submersion decellularization technique to generate chamber-specific powdered dECM. Comparative proteomic analyses showed chamber-specific dECM segregation, with atrial- and ventricle-specific proteins uniquely present in powdered dECM-hA and dECM-hV, respectively. Cell populations differentiated in the presence of dECM-hA showed upregulated atrial molecular markers and a two-fold increase in the number of atrial-like cells as compared with cells differentiated with dECM-hV or no dECM (control). Finally, electrophysiological data showed an increase in action potentials characteristic of atrial-like cells in the dECM-hA group. These findings support the hypothesis that dECM powder derived from human atria retained endogenous cues to drive cardiac differentiation toward an atrial fate.

Gelatin Promotes Cell Retention Within Decellularized Heart Extracellular Matrix Vasculature and Parenchyma.

INTRODUCTION: Recellularization of organ decellularized extracellular matrix (dECM) offers a potential solution for organ shortage in allograft transplantation. Cell retention rates have ranged from 10 to 54% in varying approaches for reseeding cells in whole organ dECM scaffolds. We aimed to improve recellularization by using soluble gelatin as a cell carrier to deliver endothelial cells to the coronary vasculature and cardiomyocytes to the parenchyma in a whole decellularized rat heart. METHODS: Rat aortic endothelial cells (RAECs) were perfused over decellularized porcine aorta in low (1%) and high (5%) concentrations of gelatin to assess attachment to a vascular dECM model. After establishing cell viability and proliferation in 1% gelatin, we used 1% gelatin as a carrier to deliver RAECs and neonatal rat cardiomyocytes (NRCMs) to decellularized adult rat hearts. Immediate cell retention in the matrix was quantified, and recellularized hearts were evaluated for visible contractions up to 35 days after recellularization. RESULTS: We demonstrated that gelatin increased RAEC attachment to decellularized porcine aorta; blocking integrin receptors reversed this effect. In the whole rat heart gelatin (1%) increased retention of both RAECs and NRCMs respectively, compared with the control group (no gelatin). Gelatin was associated with visible contractions of NRCMs within hearts (87% with gelatin vs. 13% control). CONCLUSIONS: Gelatin was an effective cell carrier for increasing cell retention and contraction in dECM. The gelatin-cell-ECM interactions likely mediated by integrin.

In vivo Hemodynamic Evaluation of an Implantable Left Ventricular Assist Device in a Long-term Anti-coagulation Regimen.

Left ventricular assist devices (LVADs) are used to treat patients with severe (New York Heart Association class IV) heart failure. Thrombosis and bleeding are severe LVAD-related complications; thus, an effective anticoagulation regimen is crucial for successful postoperative management. The CH-VAD™ (CH Biomedical, Inc.) is a small, implantable, full-support (>5 L/min) LVAD with a centrifugal flow pump that has a fully magnetically levitated rotor, which confers superior hemocompatibility. In this study, the CH-VAD™ was implanted in two calves to evaluate its hemocompatibility and to establish an anticoagulation regimen for future GLP (good laboratory practice) studies. Heparin infusion was used during the surgery, and during postoperative management, the proper dosage of warfarin was given orally to maintain an international normalized ratio (INR) between 2.0 and 3.0. Pump performance, animal condition, and hematology results were recorded throughout the study (approximately 60 days). The results show that under the established anticoagulation regimen, the CH-VAD™ was well tolerated in the bovine model, with no significant thrombus or thromboembolic lesion formation in distal end organs. Low plasma free hemoglobin levels suggest that the device did not cause hemolysis. These results and the experience gained pave the way for future GLP studies.

Tissue-engineered human embryonic stem cell-containing cardiac patches: evaluating recellularization of decellularized matrix.

Decellularized cardiac extracellular matrix scaffolds with preserved composition and architecture can be used in tissue engineering to reproduce the complex cardiac extracellular matrix. However, evaluating the extent of cardiomyocyte repopulation of decellularized cardiac extracellular matrix scaffolds after recellularization attempts is challenging. Here, we describe a unique combination of biochemical, biomechanical, histological, and physiological parameters for quantifying recellularization efficiency of tissue-engineered cardiac patches compared with native cardiac tissue. Human embryonic stem cell-derived cardiomyocytes were seeded into rat heart atrial and ventricular decellularized cardiac extracellular matrix patches. Confocal and atomic force microscopy showed cell integration within the extracellular matrix basement membrane that was accompanied by restoration of native cardiac tissue passive mechanical properties. Multi-electrode array and immunostaining (connexin 43) were used to determine synchronous field potentials with electrical coupling. Myoglobin content (~60%) and sarcomere length measurement (>45% vs 2D culture) were used to evaluate cardiomyocyte maturation of integrated cells. The combination of these techniques allowed us to demonstrate that as cellularization efficiency improves, cardiomyocytes mature and synchronize electrical activity, and tissue mechanical/biochemical properties improve toward those of native tissue.

Ticagrelor Improves Remodeling, Reduces Apoptosis, Inflammation and Fibrosis and Increases the Number of Progenitor Stem Cells After Myocardial Infarction in a Rat Model of Ischemia Reperfusion.

BACKGROUND/AIMS: We assessed the effects of ticagrelor, aspirin and prasugrel, started 7days after myocardial ischemia-reperfusion injury on remodeling, inflammation and fibrosis in the rat. We examined whether ticagrelor can affect the number of progenitor cells in the border zone. Ticagrelor, started 24h after myocardial ischemia-reperfusion injury, attenuates the decrease in heart function and adverse remodeling, an effect which is blocked by aspirin. METHODS: Rats underwent 40min ischemia followed by reperfusion. Oral dosing with vehicle, ticagrelor (300mg/kg/d), aspirin (20mg/kg/d), their combination or prasugrel (15mg/kg/d) started 7days after infarction. Echocardiography was used to assess systolic function. Heart tissue were analyzed by rt-PCR, immunoblotting, ELISA and immunohistochemistry 2weeks after infarction. RESULTS: Both ticagrelor and aspirin attenuated the decrease in systolic function and remodeling, an effect that was blocked by their combination. Ticagrelor and aspirin attenuated the increase in ANP, BNP, collagen-I and collagen-III. Again, the effect was blocked by their combination. Ticagrelor increased c-Kit, Sca-1, Ki-67, CD34, attenuated the decrease in CD105 mRNA levels, and attenuated the increase in CD31, whereas aspirin increased Ki-67, suppressed the increase in CD31 and attenuated the decrease in CD105 mRNA levels. Prasugrel did not display any effects. CONCLUSION: Ticagrelor attenuated adverse remodeling and deterioration of left ventricular systolic function despite starting treatment after the myocardial ischemia-reperfusion injury is completed. Aspirin had similar effects; however, when combined with ticagrelor, the protective effects were significantly attenuated. Ticagrelor increased the levels of several markers of stem cells and regeneration, suggesting cardiac healing by recruiting regenerative cells into the infarct.

In Vivo Restoration of Myocardial Conduction With Carbon Nanotube Fibers.

BACKGROUND: Impaired myocardial conduction is the underlying mechanism for re-entrant arrhythmias. Carbon nanotube fibers (CNTfs) combine the mechanical properties of suture materials with the conductive properties of metals and may form a restorative solution to impaired myocardial conduction. METHODS: Acute open chest electrophysiology studies were performed in sheep (n=3). Radiofrequency ablation was used to create epicardial conduction delay after which CNTf and then silk suture controls were applied. CNTfs were surgically sewn across the right atrioventricular junction in rodents, and acute (n=3) and chronic (4-week, n=6) electrophysiology studies were performed. Rodent toxicity studies (n=10) were performed. Electrical analysis of the CNTf-myocardial interface was performed. RESULTS: In all cases, the large animal studies demonstrated improvement in conduction velocity using CNTf. The acute rodent model demonstrated ventricular preexcitation during sinus rhythm. All chronic cases demonstrated resumption of atrioventricular conduction, but these required atrial pacing. There was no gross or histopathologic evidence of toxicity. Ex vivo studies demonstrated contact impedance significantly lower than platinum iridium. CONCLUSIONS: Here, we show that in sheep, CNTfs sewn across epicardial scar acutely improve conduction. In addition, CNTf maintain conduction for 1 month after atrioventricular nodal ablation in the absence of inflammatory or toxic responses in rats but only in the paced condition. The CNTf/myocardial interface has such low impedance that CNTf can facilitate local, downstream myocardial activation. CNTf are conductive, biocompatible materials that restore electrical conduction in diseased myocardium, offering potential long-term restorative solutions in pathologies interrupting efficient electrical transduction in electrically excitable tissues.

Association between BRAF (V600E) mutation and clinicopathological features of papillary thyroid carcinoma: a Brazilian single-centre case series

ABSTRACT Objectives: We aimed to investigate the prevalence of the BRAF (V600E) mutation in consecutive cases of papillary thyroid carcinoma (PTC) in patients diagnosed and treated at the Hospital Sao Rafael (Salvador, BA, Brazil) and evaluate its association with clinical and pathological characteristics of PTC. Subjects and methods: We retrospectively enrolled in the study a total of 43 consecutive PTC patients who underwent total thyroidectomy. We performed DNA extraction from formalin-fixed paraffin-embedded (FFPE) tumour tissue samples. Polymerase chain reaction (PCR) and direct sequencing were used to determine BRAF (V600E) mutation status. Univariate and multivariate logistic regression analyses were employed to identify independent associations. Results: The prevalence of BRAF (V600E) mutation was 65.1% (28/43). A high frequency of older patients (p value: 0.004) was observed among the BRAF-mutated PTC group and, in contrast, a low frequency of concurrent Hashimoto's thyroiditis (HT) (p value: 0.011) was noted. Multivariate analysis confirmed that older age (OR: 1.15; 95% CI: 1.00 - 1.33; p value: 0.047) and HT (OR: 0.05; 95% CI: 0.006-0.40; p value: 0.005) were independent factors associated with BRAF (V600E) mutation. Conclusion: We found a high prevalence of BRAF (V600E) mutation in PTC cases. Older age and no concurrent HT were independently associated with BRAF (V600E) mutation.

Association between BRAF (V600E) mutation and clinicopathological features of papillary thyroid carcinoma: a Brazilian single-centre case series.

OBJECTIVES: We aimed to investigate the prevalence of the BRAF (V600E) mutation in consecutive cases of papillary thyroid carcinoma (PTC) in patients diagnosed and treated at the Hospital Sao Rafael (Salvador, BA, Brazil) and evaluate its association with clinical and pathological characteristics of PTC. SUBJECTS AND METHODS: We retrospectively enrolled in the study a total of 43 consecutive PTC patients who underwent total thyroidectomy. We performed DNA extraction from formalin-fixed paraffin-embedded (FFPE) tumour tissue samples. Polymerase chain reaction (PCR) and direct sequencing were used to determine BRAF (V600E) mutation status. Univariate and multivariate logistic regression analyses were employed to identify independent associations. RESULTS: The prevalence of BRAF (V600E) mutation was 65.1% (28/43). A high frequency of older patients (p value: 0.004) was observed among the BRAF-mutated PTC group and, in contrast, a low frequency of concurrent Hashimoto's thyroiditis (HT) (p value: 0.011) was noted. Multivariate analysis confirmed that older age (OR: 1.15; 95% CI: 1.00 - 1.33; p value: 0.047) and HT (OR: 0.05; 95% CI: 0.006-0.40; p value: 0.005) were independent factors associated with BRAF (V600E) mutation. CONCLUSION: We found a high prevalence of BRAF (V600E) mutation in PTC cases. Older age and no concurrent HT were independently associated with BRAF (V600E) mutation.