Background: Tissue engineering enables the generation of functional human cardiac tissue with cells derived in vitro in combination with biocompatible materials. Human-induced pluripotent stem cell-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electric pacing on the maturation of human-induced pluripotent stem cell-derived cardiac tissues. Methods: Cardiomyocytes derived from human-induced pluripotent stem cells were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subjected to different mechanical stress and electric pacing conditions. Results: The engineered human myocardium exhibits Frank-Starling–type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated increases in contractility (0.63±0.10 mN/mm ² vs 0.055±0.009 mN/mm ² for no stress), tensile stiffness, construct alignment, and cell size. Stress conditioning also increased SERCA2 (Sarco/Endoplasmic Reticulum Calcium ATPase 2) expression, which correlated with a less negative force-frequency relationship. When electric pacing was combined with static stress conditioning, the tissues showed an additional increase in force production (1.34±0.19 mN/mm ² ), with no change in construct alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 (Ryanodine Receptor 2) and SERCA2 further increased by combined static stress and electric stimulation. Conclusions: These studies demonstrate that electric pacing and mechanical stimulation promote maturation of the structural, mechanical, and force generation properties of human-induced pluripotent stem cell-derived cardiac tissues.

Recent advances in pluripotent stem cell biology and directed differentiation have identified a population of human cardiovascular progenitors that give rise to cardiomyocytes, smooth muscle, and endothelial cells. Because the heart develops from progenitors in 3D under constant mechanical load, we sought to test the effects of a 3D microenvironment and mechanical stress on differentiation and maturation of human cardiovascular progenitors into myocardial tissue. Progenitors were derived from embryonic stem cells, cast into collagen hydrogels, and left unstressed or subjected to static or cyclic mechanical stress. Compared to 2D culture, the unstressed 3D environment increased cardiomyocyte numbers and decreased smooth muscle numbers. Additionally, 3D culture suppressed smooth muscle α-actin content, suggesting diminished cell maturation. Cyclic stress-conditioning increased expression of several cardiac markers, including β-myosin heavy chain and cardiac troponin T, and the tissue showed enhanced calcium dynamics and force production. There was no effect of mechanical loading on cardiomyocyte or smooth muscle specification. Thus, 3D growth conditions favor cardiac differentiation from cardiovascular progenitors, whereas 2D conditions promote smooth muscle differentiation. Mechanical loading promotes cardiomyocyte structural and functional maturation. Culture in 3-D facilitates understanding how cues such as mechanical stress affect the differentiation and morphogenesis of distinct cardiovascular cell populations into organized, functional human cardiovascular tissue. Stem Cells 2015;33:2148–2157

The high water content of hydrogels allows these materials to closely mimic the native biological extracellular conditions, but it also makes difficult the histological preparation of hydrogel-based bioengineered tissue. Paraffin-embedding techniques require dehydration of hydrogels, resulting in substantial collapse and deformation, whereas cryosectioning is hampered by the formation of ice crystals within the hydrogel material. Here, we sought to develop a method to obtain good-quality cryosections for the microscopic evaluation of hydrogel-based tissue-engineered constructs, using polyethylene glycol (PEG) as a test hydrogel. Conventional sucrose solutions, which dehydrate cells while leaving extracellular water in place, produce a hydrogel block that is brittle and difficult to section. We therefore replaced sucrose with multiple protein-based and nonprotein-based solutions as cryoprotectants. Our analysis demonstrated that overnight incubation in bovine serum albumin (BSA), fetal bovine serum (FBS), polyvinyl alcohol (PVA), optimum cutting temperature (OCT^®) compound, and Fisher HistoPrep frozen tissue-embedding media work well to improve the cryosectioning of hydrogels. The protein-based solutions give background staining with routine hematoxylin and eosin, but the use of nonprotein-based solutions PVA and OCT reduces this background by 50%. These methods preserve the tissue architecture and cellular details with both in vitro PEG constructs and in constructs that have been implanted in vivo. This simple hydrogel cryosectioning technique improves the methodology for creation of good-quality histological sections from hydrogels in multiple applications.

This study investigates the reported ability of nanobubbles (1 micrometer in diameter). Eight hypotheses were examined. Both the theoretical and experimental results supported only one hypothesis: The apparent echogenicity of nanobubbles under both linear and nonlinear imaging is due to the presence of preexisting microbubbles, which are not reliably detected by available nanoparticle sizing methods. There was no evidence to support the other hypotheses, although the possibility of microbubble formation due to bubble aggregation/coalescence or swelling due to gas absorption in vivo could not be completely ruled out. Nanobubbles may offer advantages in terms of circulatory stability and potential for therapeutic delivery compared with microbubbles, but these advantages must be weighed against the need to use higher bubble concentrations, higher ultrasound frequencies, and/or higher intensities to achieve equivalent imaging and/or therapeutic effects.

Cavitation induced by ultrasound enhances enzymatic fibrinolysis by increasing the transport of reactants. However, the effects of cavitation need to be fully understood before sonothrombolysis can be applied clinically. In order to understand the underlying mechanisms, we examined the effects of combining ultrasound, microbubbles and thrombolytic enzymes on thrombolysis. First, we evaluated the relations between inertial cavitation and the reduction in the weight of a blood clot. Inertial cavitation was varied by changing the amplitude and duration of the transmitted acoustic wave as well as the concentration of microbubbles used to induce cavitation. Second, we studied the combined effects of streptokinase and inertial cavitation on thrombolysis. The results show that inertial cavitation increases the weight reduction of a blood clot by up to 33.9%. With linear regression fitting, the measured differential inertial cavitation dose and the weight reduction had a correlation coefficient of 0.66. Microscopically, enzymatic thrombolysis effects manifest as multiple large cavities within the clot that are uniformly distributed on the side exposed to ultrasound. This suggests that inertial cavitation plays an important role in producing cavities, while microjetting of the microbubbles induces pits on the clot surface. These observations preliminarily demonstrate the clinical potential of sonothrombolysis. The use of the differential inertial cavitation dose as an indicator of blood clot weight loss for controlled sonothrombolysis is also possible and will be further explored.

Iron is vital to living cells, playing a key role in cellular respiration, DNA synthesis, and various metabolic functions. Importantly, cancer cells have a higher dependency on iron compared to normal cells to support their rapid growth and survival. Due to this fact, tumors are more vulnerable to ferroptosis, an iron-dependent form of regulated cell death. Radiation therapy (RT), a standard treatment for many cancer patients, is known to induce ferroptosis. Ultra-high dose rate FLASH RT offers an improved therapeutic window by minimizing damage to normal tissues while preserving tumor control. However, the precise biological mechanisms behind the protective effects of FLASH RT on normal tissues remain unclear. In this study, we propose that variations in lipid peroxidation and ferroptosis, driven by intrinsic differences in iron levels between normal and cancerous tissues, contribute to this effect. Our findings show that FLASH RT increases lipid peroxidation and induces ferroptosis in tumor cells but does not significantly elevate lipid peroxidation and ferroptosis in normal tissues compared to conventional RT. To determine whether raising iron levels in normal tissues could abrogate the protective effects of FLASH, mice were fed a high-iron diet before RT. A high-iron diet before and after RT reversed the protective effect of FLASH, resulting in increased intestinal damage and lipid peroxidation. This suggests that baseline iron levels and iron-driven lipid peroxidation are critical factors in mediating the protective outcomes of FLASH RT. Overall, our study sheds light on the role of iron in modulating RT responses and provides new mechanistic insights into how FLASH RT influences normal and cancerous tissues.