心脏移植受体的超微结构改变

目的：观察心脏移植标本的超微结构变化。方法：通过移植后心脏的心内膜活检，经固定、常规则超薄切片制备，JEM－2000EX透射电镜检查。结果：移植后的早期阶段，未见明显移植排斥反应的心脏心内膜活检显示间质水肿，偶尔可见毛细血管内皮细胞的肿胀。心肌细胞和内皮细胞内溶酶体明显增多。在早期细胞排斥反应阶段，电镜检查可发现显著的心肌细胞坏死和变性，并可确定浸润炎细胞的性质。在血管排斥反应阶段，辨认内皮细胞增生、内皮细胞损伤和基底膜的改变，在患有移植性冠状动脉病的病人，其缺血性心脏具有显著特点：肌动蛋白丝的缺失比肌球蛋白丝更明显，肌微丝相对较粗大。结论：这些改变对于进一步确诊心脏移植受体的排斥和局部缺血是有用的形态学特征。     

3D Microstructured Carbon Nanotube Electrodes for Trapping and Recording Electrogenic Cells

Electrogenic cells such as cardiomyocytes and neurons rely mainly on electrical signals for intercellular communication. Microelectrode arrays (MEAs) have been developed for long‐term recording of cell signals and stimulation of electrogenic cells under low‐cell‐stress conditions, providing new insights in the behavior of electrogenic cells and the operation of the brain. To date, MEAs are relying on flat or needle‐shaped electrode surfaces, mainly due to limitations in the lithographic processes. This paper relies on a previously reported elasto‐capillary aggregation process to create 3D carbon nanotube (CNT) MEAs. This study shows that CNTs aggregate in well‐shaped structures of similar size as cardiomyocytes are particularly interesting for MEA applications. This is because i) CNT microwells of the right diameter preferentially trap individual cardiomyocytes, which facilitates single cell recording without the need for clamping cells or signal deconvolution, and ii) once the cells are trapped inside of the CNT wells, this 3D CNT structure is used as an electrode surrounding the cell, which increases the cell–electrode contact area. As a result, this study finds that the recorded output voltages increase significantly (more than 200%). This fabrication process paves the way for future study of complex interactions between electrogenic cells and 3D recording electrodes.     

The role of intramyocardial pressure during coronary sinusinterventions: a computer model study

A model consisting of an arterial, a capillary, and a venous compartment was developed to reproduce experimentally determined hemodynamic results of the arterial and venous sides of the coronary circulation. From the model, intramyocardial flow between capillaries and veins, which is inaccessable to measurement, was then estimated. Results are given for normal perfusion and coronary sinus occlusion conditions. For both conditions, intramyocardial pressure was varied within the model in order to study its impact on intramyocardial flow. For low and moderate intramyocardial pressures under coronary sinus occlusion, retrograde flow from veins to capillaries was seen to increase with increasing intramyocardial pressure. However, for very high values of intramyocardial pressure, which are close to left ventricular pressure, retrograde flow declined.     

Surface Features of Recombinant Spider Silk Protein eADF4(κ16)‐Made Materials are Well‐Suited for Cardiac Tissue Engineering

Cardiovascular diseases causing high morbidity and mortality represent a major socioeconomic burden. The primary cause of impaired heart function is often the loss of cardiomyocytes. Thus, novel therapies aim at restoring the lost myocardial tissue. One promising approach is cardiac tissue engineering. Previously, it is shown that Antheraea mylitta silk protein fibroin is a suitable material for cardiac tissue engineering, however, its quality is difficult to control. To overcome this limitation, the interaction of primary rat heart cells with engineered Araneus diadematus fibroin 4 (κ16) (eADF4(κ16)) is investigated here, which is engineered based on the sequence of ADF4 by replacing the glutamic acid residue in the repetitive unit of its core domain with lysine. The data demonstrate that cardiomyocytes, fibroblasts, endothelial cells, and smooth muscle cells attach well to eADF4(κ16) films on glass coverslips which provide an engineered surface with a polycationic character. Moreover, eADF4(κ16) films have, in contrast to fibronectin films, no hypertrophic effect but allow the induction of cardiomyocyte hypertrophy. Finally, cardiomyocytes grown on eADF4(κ16) films respond to pro‐proliferative factors and exhibit proper cell‐to‐cell communication and electric coupling. Collectively, these data demonstrate that designed recombinant eADF4(κ16)‐based materials are promising materials for cardiac tissue engineering.     

On the Fluid Mechanics of Human Coronary Artery Stenosis

Human coronary artery stenosis can have dramatic hemodynamic effects on coronary flow rate and perfusion. To quantitatively investigate these relationships, nineteen proximal coronary arteries with varying focal stenoses (2%-98%) were dissected from fresh post mortem adult hearts and perfused with isotonic glycerol saline (n = 2.7 centipoise) at constant pressures of 30, 50, 75, 100, 150 and 200 mm Hg, while varying the distal bed resistance (rb) over the range 0.1 to 5 mm Hg/ml/min. Flow rate (Q) and arterial segment pressure drop (?P) were measured at each perfusion pressure and rb, and a permanent cast of silicone rubber was made under 100 mm Hg pressure following perfusion. Hydraulic resistance (R = ?P/Q) tended to be constant at low Q (10-30 ml/min), with resistance increasing 2 or 3 times at higher Q (30-100 + ml/min). Curves of Q vs. % stenosis showed that Q was relatively constant with stenoses less than 70-80%. With a small further increase in stenosis, however, Q decreased dramatically. Furthermore, significant reductions occurred at lower % stenoses for greater demands (lower rb), a contributing factor toward effort angina, as less severe stenoses became increasingly significant under flow demand. Elastic effects in eccentric lesions produced additional flow losses at lowered perfusion pressures. In addition, a critical relation was demonstrated between percent stenosis and the minimum coronary perfusion pressure necessary to maintain a given Q.     

Engineered Extracellular Matrices as Biomaterials of Tunable Composition and Function

Engineered and decellularized extracellular matrices (ECM) are receiving increasing interest in regenerative medicine as materials capable to induce cell growth/differentiation and tissue repair by physiological presentation of embedded cues. However, ECM production/decellularization processes and control over their composition remain primary challenges. This study reports engineering of ECM materials with customized properties, based on genetic manipulation of immortalized and death‐inducible human mesenchymal stromal cells (hMSC), cultured within 3D porous scaffolds under perfusion flow. The strategy allows for robust ECM deposition and subsequent decellularization by deliberate cell‐apoptosis induction. As compared to standard production and freeze/thaw treatment, this grants superior preservation of ECM, leading to enhanced bone formation upon implantation in calvarial defects. Tunability of ECM composition and function is exemplified by modification of the cell line to overexpress vascular endothelial growth factor alpha (VEGF), which results in selective ECM enrichment and superior vasculature recruitment in an ectopic implantation model. hMSC lines culture under perfusion‐flow is pivotal to achieve uniform scaffold decoration with ECM and to streamline the different engineering/decellularization phases in a single environmental chamber. The findings outline the paradigm of combining suitable cell lines and bioreactor systems for generating ECM‐based off‐the‐shelf materials, with custom set of signals designed to activate endogenous regenerative processes.     

脑胶质瘤的血瘤屏障超微结构观察

目的：探讨脑部胶质瘤中血瘤屏障的超微结构改变与胶质瘤细胞生物学特性之间的关系。方法：应用电镜观察13例胶质瘤患者瘤组织中血瘤屏障的超微结构改变。结果：脑胶质瘤组织中的血瘤屏障发生的一系列改造和重建：1、多数血瘤屏障中毛细血管基膜呈限局性或广泛性增厚,基板多层化,胶原纤维增生;2、多数毛细血管基膜的胶质膜侧常见大小不等,多少不一的虫蚀状空洞或血管外间隙扩大;3、毛细血管胶质膜则脚板外侧基板常不完整;4、内此细胞间紧密连接延长,偶见内皮细胞出现窗孔,内此细胞增生出芽。结论：脑胶质瘤组织中的血瘤屏障发生的一系列改造和重建的超微结构改变,可能是导致脑部胶质瘤低转移率和高侵袭性的形态学依据。     

Ultrastructural basis for the transition of cell death mode from apoptosis to necrosis in menadione-treated osteosarcoma 143B cells

Time-dependent ultrastructural changes of menadione-treated human osteosarcoma 143B cells were correlated with those in their stainability to Annexin V and propidium iodide (PI). Populations of both apoptotic (Annexin V(+)/PI(-)) and necrotic (Annexin V(+)/PI(+)) cells, judged by flow cytometry, began to increase at 2 h after menadione treatment. The former reached a maximum at 6 h followed by abrupt decreases thereafter, while the latter continued to increase. Electron microscopically, cells obtained at 6 h after the menadione treatment consisted of mixed populations of cells with typical apoptotic features and those with a mixture of apoptotic and necrotic features, while cells obtained at 8-24 h consisted exclusively of cells with a mixture of apoptotic and necrotic features. Thus, necrotic cells, as judged by flow cytometry, were in a transitional state of cell death mode from apoptosis to necrosis and are thus designated as 'intermediate cells'. Lack of apoptotic bodies, judged by flow cytometric analysis on sub-G1 nuclei and by electron microscopy in menadione-treated cells, suggested that the transition of cell death mode from apoptosis to necrosis occurred before the apoptotic processes were completed. Effects of N-acetylcysteine and Z-VAD-fmk on menadione-induced ultrastructural changes were also studied.     

Rapid Magnetically Directed Assembly of Pre-Patterned Capillary-Scale Microvessels

Capillary scale vascularization is critical to the survival of engineered 3D tissues and remains an outstanding challenge for the field of tissue engineering. Current methods to generate micro-scale vasculatures such as 3D printing, two photon hydrogel ablation, angiogenesis, and vasculogenic assembly face challenges in rapidly creating organized, highly vascularized tissues at capillary length-scales. Within metabolically demanding tissues, native capillary beds are highly organized and densely packed to achieve adequate delivery of nutrients and oxygen and efficient waste removal. Here, two existing techniques are adopted to fabricate lattices composed of sacrificial microfibers that can be efficiently and uniformly seeded with endothelial cells (ECs) by magnetizing both lattices and ECs. Ferromagnetic microparticles are incorporated into microfibers produced by solution electrowriting and fiber electropulling. By loading ECs with superparamagnetic iron oxide nanoparticles, the cells could be seeded onto magnetized microfiber lattices. Following encapsulation in a hydrogel, the capillary templating lattice is selectively degraded by a bacterial lipase that does not impact mammalian cell viability or function. This study introduces a novel approach to rapidly producing organized capillary networks within metabolically demanding engineered tissue constructs which should have broad utility in the fields of tissue engineering and regenerative medicine.     

Measurement of Ventricular Volume by Intracardiac Impedance: Theoretical and Empinrcal Approaches

The most widely used equation, V = pL2/R, is developed for the computation of ventricular volume from catheter based impedance measurements. The assumptions implicit in this derivation are examined and found to be generally invalid. An empirical discrete resistor model is described which includes the impedance of the myocardial tissue and the adjoining ventricular blood volume. Once the parameters of this model are determined for individual canine hearts, the model predicts stroke volume from measured impedances. Due to the difficulty involved in determining the parameters of the empirical model, a numerical model is developed which solves the equation V ?a V U + F = 0 in a three-dimensional volume. This model is then used to determine the effect of parallel tissue resistance, catheter position, and contraction of the other ventricle on volumes computed by intracardiac impedance. Parallel tissue resistance is found to have the greatest impact on absolute volume measurements. However, stroke volume computations are relatively unaffected by any of the three factors.     

Photoacoustic Imaging of Embryonic Stem Cell‐Derived Cardiomyocytes in Living Hearts with Ultrasensitive Semiconducting Polymer Nanoparticles

Human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) have become promising tools to repair injured hearts. To achieve optimal outcomes, advanced molecular imaging methods are essential to accurately track these transplanted cells in the heart. In this study, it is demonstrated for the first time that a class of photoacoustic nanoparticles (PANPs) incorporating semiconducting polymers (SPs) as contrast agents can be used in the photoacoustic imaging (PAI) of transplanted hESC‐CMs in living mouse hearts. This is achieved by virtue of two benefits of PANPs. First, strong photoacoustic (PA) signals and specific spectral features of SPs allow PAI to sensitively detect and distinguish a small number of PANP‐labeled cells (2000) from background tissues. Second, the PANPs show a high efficiency for hESC‐CM labeling without adverse effects on cell structure, function, and gene expression. Assisted by ultrasound imaging, the delivery and engraftment of hESC‐CMs in living mouse hearts can be assessed by PANP‐based PAI with high spatial resolution (≈100 µm). In summary, this study explores and validates a novel application of SPs as a PA contrast agent to track labeled cells with high sensitivity and accuracy in vivo, highlighting the advantages of integrating PAI and PANPs to advance cardiac regenerative therapies.     

A novel approach to dual excitation ratiometric optical mapping of cardiac action potentials with di-4-ANEPPS using pulsed LED excitation

We developed a new method for ratiometric optical mapping of transmembrane potential (V(m)) in cardiac preparations stained with di-4-ANEPPS. V(m)-dependent shifts of excitation and emission spectra establish two excitation bands (<481 and >481 nm) that produce fluorescence changes of opposite polarity within a single emission band (575-620 nm). The ratio of these positive and negative fluorescence signals (excitation ratiometry) increases V(m) sensitivity and removes artifacts common to both signals. We pulsed blue (450 ± 10 nm) and cyan (505 ± 15 nm) light emitting diodes (LEDs) at 375 Hz in alternating phase synchronized to a camera (750 frames-per-second). Fluorescence was bandpass filtered (585 ± 20 nm). This produced signals with upright (blue) and inverted (cyan) action potentials (APs) interleaved in sequential frames. In four whole swine hearts with motion chemically arrested, fractional fluorescence for blue, cyan, and ratio signals was 1.2 ± 0.3%, 1.2 ± 0.3%, and 2.4 ± 0.6%, respectively. Signal-to-noise ratios were 4.3 ± 1.4, 4.0 ± 1.2, and 5.8 ± 1.9, respectively. After washing out the electromechanical uncoupling agent, we characterized motion artifact by cross-correlating blue, cyan, and ratio signals with a signal with normal AP morphology. Ratiometry improved cross-correlation coefficients from 0.50 ± 0.48 to 0.81 ± 0.25, but did not cancel all motion artifacts. These findings demonstrate the feasibility of pulsed LED excitation ratiometry in myocardium.     

Thoracic electrical impedance tomographic measurements during volume controlled ventilation-effects of tidal volume and positive end-expiratory pressure

The aim of the study was to analyze thoracic electrical impedance tomographic (EIT) measurements accomplished under conditions comparable with clinical situations during artificial ventilation. Multiple EIT measurements were performed in pigs in three transverse thoracic planes during the volume controlled mode of mechanical ventilation at various tidal volumes (V(T)) and positive end-expiratory pressures (PEEP). The protocol comprised following ventilatory patterns: 1) V(T)(400, 500, 600, 700 ml) was varied in a random order at various constant PEEP levels and 2) PEEP (2, 5, 8, 11, 14 cm H2O) was randomly modified during ventilation with a constant V(T). The EIT technique was used to generate cross-sectional images of 1) regional lung ventilation and 2) regional shifts in lung volume with PEEP. The quantitative analysis was performed in terms of the tidal amplitude of the impedance change, reflecting the volume of delivered gas at various preset V(T) and the end-expiratory impedance change, revealing the variation of the lung volume at various PEEP levels. The results showed: 1) an increase in the tidal amplitude of the impedance change, proportional to the delivered V(T) at all constant PEEP levels, 2) a rising end-expiratory impedance change, with PEEP reflecting an increase in gas volume, and 3) a PEEP-dependent redistribution of the ventilated gas between the planes. The generated images and the quantitative results indicate the ability of EIT to identify regional changes in V(T) and lung volume during mechanical ventilation.     

Three-dimensional ultrastructural distribution of cytoplasmic interdigitation between endothelium and pericyte of capillary in human granulation tissue by serial section reconstruction method

Two- and three-dimensional electron microscopic observations in the capillaries with angiogenetic sprout of human granulation tissue revealed the cytoplasmic interdigitations (CID) between the endothelium and the pericyte, which were composed of unit of cytoplasmic projection and an indentation. Two types of interdigitation were observed: the interdigitation composed of cytoplasmic projection from the pericyte plugging into the endothelial indentation, CID(P-E); the interdigitation which had a reverse mode of composition of the cytoplasmic projection and indentation to the former one, CID(E-P). We investigated three-dimensional distribution of the two kinds of interdigitation in two cases of the capillaries with angiogenetic sprout. The two kinds of interdigitation exhibited a remarkable difference in the three-dimensional distribution. The CID(P-E) was rather uniformly distributed over the parent capillary endothelial area (6.4 per endothelium in average) and was not observed at all in the angiogenetic area; the other type of cytoplasmic interdigitation, CID(E-P), was located in only the angiogenetic sprouting endothelial area (3.5 per endothelium in average). The cytoplasmic interdigitation between the endothelium and the pericyte in vivo was the only site for cell contact and seemed to be involved in a conducting function which is biochemically proved to exist in vitro between the two cells.     

Electric-field-induced volume and membrane ionic permeabilitychanges of red blood cells

When an external electric field (EF) is applied to red blood cells (RBCs), the RBCs are observed to undergo a swelling action. The swelling may or may not lead to hemolysis, depending on the EF strength. An objective verification of this swelling is by measuring the RBC mean corpuscular volume (MCV). In this study, the RBC's were exposed to the appropriate EF strength to induce swelling, but caused minimal hemolysis. The MCV was measured. The change in the erythrocyte membrane ionic permeability as a result of the EF exposure was also determined, as an objective verification of presumed membrane conductance change concomitant with the swelling. The fluxes of cations K⁺, Na ⁺, and Ca⁺⁺ and anion Cl^- were measured. The results showed that red cell MCV was indeed increased after EF application. The EF also altered the membrane ionic conductance to allow ions to flow down their respective concentration gradient across the membrane. Without a counterbalancing ionic pressure gradient, hemoglobin colloidal pressure inevitably drew H₂O in, thus producing the observed swelling     

Optimization of Coronary Blood Flow During Cardiopulmonary Resuscitation (CPR)

In order to improve coronary blood flow during cardiopulmonary resuscitation (CPR), it is necessary to increase the aortic-right atrial pressure gradient. In the closed cardiovascular system, this gradient should be primarily determined by the level of cardiac output. Alternating simultaneous compression and decompression of the thorax and abdomen should result in improved cardiac output and coronary blood flow over present methods. Based on computer simulation results, this previously untried method was found to improve coronary flow three times the level predicted for standard CPR. Other recently proposed CPR strategies were also evaluated but resulted in significantly lower coronary perfusion levels. In all cases, coronary blood flow varied directly with cardiac output. The use of simultaneous compression and decompression appears to be a promising CPR procedure which warrants further consideration.     

Discontinuous capillary segments in the extensor digitorum longus muscle of aged BUF/Mna rats

We examined the structural changes of capillaries in the extensor digitorum longus muscle of aged (25 months) male BUF/Mna rats, which caused severe muscle weakness of hind legs during aging. The aged muscle mostly consisted of bundles of muscle fibres 15-35 microm in diameter (type 1). In some small areas, however, muscle bundles contained small muscle fibres mainly 15-25 microm in diameter (type 2), possibly indicating that these small fibres are in the course of regeneration after necrosis. Examination of serial ultrathin sections revealed some remarkable changes of capillaries in the type 2 muscle bundle. In some capillaries, the vascular lumen became discontinuous by several close contacts of opposed endothelial cells in their course, forming plural closed vascular segments. Moreover, a solitary endothelial cell was often observed within a scaffold of basal lamina, which remained after destruction of endothelial cells. The segmentation of capillaries and the occurrence of the scaffolds of basal laminae are considered to indicate the degenerative process of capillaries. In some instances, on the other hand, endothelial cells closely apposed each other with no vascular lumen for distances of up to 100 microm, and some capillaries had a narrow vascular lumen (1-3 microm diameter) for a long distance (approximately 300 microm), probably indicating that these structures are in the course of regenerating capillaries to remodel the capillary networks around the muscle fibres. Pericytic processes circularly arranged outside the endothelium at the slit-like and narrow vascular lumen, like hoops, possibly preventing the rupture of the newly-formed vascular lumen from the increased blood flow and/or blood pressure. In addition, the occasional occurrence of capillaries with fenestrations may participate to increase the supply of nutrients and oxygen to the regenerating muscle fibres. The present findings suggest that the capillary networks in the extensor digitorum longus muscle of aged BUF/Mna rats are remodelled following the regeneration of muscle fibres after necrosis, and that on occasion, neighbouring endothelial cells may closely contact with each other both in the degenerative and regenerative processes of capillaries.     

Fabrication of Thermoresponsive Hydrogel Scaffolds with Engineered Microscale Vasculatures

Precise fabrication of microscale vasculatures (MSVs) has long been an unresolved challenge in tissue engineering. Currently, light-assisted printing is the most common approach. However, this approach is often associated with an intricate fabrication process, high cost, and a requirement for specific photoresponsive materials. Here, thermoresponsive hydrogels are employed to induce volume shrinkage at 37 °C, which allows for MSV engineering without complex protocols. The thermoresponsive hydrogel consists of thermosensitive poly(N-isopropylacrylamide) and biocompatible gelatin methacrylate (GelMA). In cell culture, the thermoresponsive hydrogel exhibits an apparent volume shrinkage and effectively triggers the creation of MSVs with smaller size. The results show that a higher concentration of GelMA blocks the shrinkage, and the thermoresponsive hydrogel demonstrates different behaviors in water and air at 37 °C. The MSVs can be effectively fabricated using the sacrificial alginate fibers, and the minimum MSV diameter achieved is 50 µm. Human umbilical vein endothelial cells form endothelial monolayers in the MSVs. Osteosarcoma cells maintain high viability in the thermoresponsive hydrogel, and the in vivo experiment shows that the MSVs provide a site for the perfusion of host vessels. This technique may help in the development of a facile method for fabricating MSVs and demonstrates strong potential for clinical application in tissue regeneration.     

Changes in the K+ current-density of MCF-7 cells during progression through the cell cycle: possible involvement of a h-ether.a-gogo K+ channel.

MCF-7 cells express voltage-activated K+ channels. In the present study, we used the patch-clamp and RT-PCR techniques to investigate the involvement of these channels during the cell cycle progression. The outward rectifier current (IK) recorded during depolarization was almost completely suppressed by the classical K+ channel blocker tetraethylammonium (TEA) in MCF-7 cells. TEA also inhibited cell proliferation, as measured with 3H-thymidine incorporation. Moreover, profound changes were observed in both the resting membrane potential (RMP) and IK during the release from the G0/G1 phase of the cell cycle. MCF-7 cells arrested in G0/G1 were depolarized (-26.3 +/- 10 mV, n = 30) and IK-density was small (9.4 +/- 5.6 pA/pF, n = 60) compared to cells progressing in the G1 phase (RMP = -60 +/- 7.9 mV; n = 35 and IK-density = 30.2 +/- 8.5 pA/pF; n = 76). IK was highly sensitive to Mg2+, astemizole and TEA (10 mM). Extracellular perfusion of 5 mM Mg2+ dramatically slowed the activation and perfusion of 2 microM astemizole inhibited both IK (20 +/- 3%) and cell proliferation (23%). Moreover, the h-EAG mRNA expression was modulated during the cell cycle. Thus, these data suggested that h-EAG K+ channels play a role in controlling the proliferation and/or cell cycle.     

低强度氦氖激光调整红细胞变形性的机理研究

目的红细胞变形能力对于微循环血流的维持起着至关重要的作用.细胞和临床研究表明,低强度He-Ne激光可以调整红细胞的变形性.本文根据刘承宜等人提出的低强度激光的生物信息假说来研究低强度激光对红细胞变形性的调整机制.