Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers ¹. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques ². Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; ³). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.Download video file.^{(46M, mov)}     

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.     

Engineered Heart Tissue: A Novel Tool to Study the Ischemic Changes of the Heart In Vitro

Background

Understanding the basic mechanisms and prevention of any disease pattern lies mainly on development of a successful experimental model. Recently, engineered heart tissue (EHT) has been demonstrated to be a useful tool in experimental transplantation. Here, we demonstrate a novel function for the spontaneously contracting EHT as an experimental model in studying the acute ischemia-induced changes in vitro.

Methodology/Principal Findings

EHT was constructed by mixing cardiomyocytes isolated from the neonatal rats and cultured in a ring-shaped scaffold for five days. This was followed by mechanical stretching of the EHT for another one week under incubation. Fully developed EHT was subjected to hypoxia with 1% O₂ for 6 hours after treating them with cell protective agents such as cyclosporine A (CsA) and acetylcholine (ACh). During culture, EHT started to show spontaneous contractions that became more synchronous following mechanical stretching. This was confirmed by the increased expression of gap junctional protein connexin 43 and improved action potential recordings using an optical mapping system after mechanical stretching. When subjected to hypoxia, EHT demonstrated conduction defects, dephosphorylation of connexin-43, and down-regulation of cell survival proteins identical to the adult heart. These effects were inhibited by treating the EHT with cell protective agents.

Conclusions/Significance

Under hypoxic conditions, the EHT responds similarly to the adult myocardium, thus making EHT a promising material for the study of cardiac functions in vitro.     

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child''s small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols^{[2, 3]}. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel™, and NaHCO₃. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size^{[1, 4, 5]}. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures^[6]. Once the tissue has solidified at 37°C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.Open in a separate windowClick here to view.^{(86M, flv)}     

The Use of Fluorescent Probes to Assess Oxidative Processes in Isolated-Perfused Rat Heart Tissue

The formation of reactive oxygen species (ROS) in intact heart tissue has been assessed by direct ESR measurements, and indirectly by the formation of characteristic tissue products and the protective effects of various antioxidants. The development of lipid soluble esters of compounds which can be trapped intra-cellularly after hydrolysis, and which fluoresce after oxidation, has provided a new tool to investigate ROS in vitro. The utility of 2',7'-dichlorofluorescin diacetate (DCFDA) in isolated-perfused rat heart tissue was investigated in the present study. DCFDA and its deacetylated form were incubated with various levels of hydrogen peroxide or t-butylhydroperoxide (tBOOH). Conversion of the diacetate form to a fluorescent product required 4-5 h with hydrogen peroxide and up to 24 h with tBOOH. In contrast, the deacetylated form fluoresced at 80% of maximum levels 1 h after the addition of 100 mM tBOOH. DCFDA was loaded into heart tissue by infusing for lO min at a final concentration of 10,aM in Krebs-Henseleit bicarbonate buffer. After a lO min washout period, analysis of freeze-clamped heart tissue revealed that the trapped material was readily converted to a fluorescent product by tBOOH, indicating hydrolysis had occurred. Fluorescence of material trapped in heart tissue was approximately 24% of the maximum achieved after oxidation with lOOmM tBOOH. This value decreased to 18 and 13% when the loading and washout periods were from 0 to 20 or 10 to 30min of hypoxia, respectively. Similar results were obtained with the less readily oxidized dicarboxy derivative of DCFDA. Infusion of 500μM tBOOH increased the oxidation of DCFDA in heart tissue from 24 to 31%. These data demonstrate that DCFDA can be loaded into heart tissue and is capable of reflecting relative changes in the oxidative state of this organ.     

Engineered Tissue Folding by Mechanical Compaction of the Mesenchyme

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Bifurcation Analysis of Genetically Engineered Pacemaking in Mammalian Heart

Genetically engineered pacemaking in ventricular cells has been achieved by down-regulation of the time independent inward rectifying current (I _K1), or insertion of the hyperpolarisation-activated funny current (I _f). We analyse the membrane system (i.e. ionic concentrations clamped) of an epicardial Luo-Rudy dynamic cell model using continuation algorithms with the maximum conductance () of I _K1 and I _f as bifurcation parameters. Pacemaker activity can be induced either via Hopf or homoclinic bifurcations. As _K1 is decreased by ≈74%, autorhythmicity emerged via a homoclinic bifurcation, i.e., the periodicity first appear with infinitely large periods. In contrast, the insertion of _f induced periodicity via a subcritical Hopf bifurcation at _f≈ 0.25 mSμF⁻¹. Stable autorhythmic action potentials occurred at _f > 0.329 mSμF⁻¹.     

组织工程皮肤生物反应器的研究与设计

目的设计一套生物反应器,能针对不同支架材料———细胞复合物进行构建组织工程皮肤。方法根据皮肤的自身生长特点和不同支架材料-细胞复合物的特性,模拟皮肤的生长环境和力学环境,通过生物反应器解决组织工程皮肤构建中支架的装夹和气液界面问题。结果生物反应器由控制系统和生物反应器主体两部分构成,能提供对多种皮肤细胞复合物的动态培养。结论皮肤生物反应器能够满足不同组织工程皮肤产品的需要。能够形成气液界面和模拟生物力学的刺激。     

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.     

Development of Small Diameter Nanofiber Tissue Engineered Arterial Grafts

The surgical repair of heart and vascular disease often requires implanting synthetic grafts. While synthetic grafts have been successfully used for medium-to-large sized arteries, applications for small diameter arteries (<6 mm) is limited due to high rates of occlusion by thrombosis. Our objective was to develop a tissue engineered vascular graft (TEVG) for small diameter arteries. TEVGs composed of polylactic acid nanofibers with inner luminal diameter between 0.5 and 0.6 mm were surgically implanted as infra-renal aortic interposition conduits in 25 female C17SCID/bg mice. Twelve mice were given sham operations. Survival of mice with TEVG grafts was 91.6% at 12 months post-implantation (sham group: 83.3%). No instances of graft stenosis or aneurysmal dilatation were observed over 12 months post-implantation, assessed by Doppler ultrasound and microCT. Histologic analysis of explanted TEVG grafts showed presence of CD31-positive endothelial monolayer and F4/80-positive macrophages after 4, 8, and 12 months in vivo. Cells positive for α-smooth muscle actin were observed within TEVG, demonstrating presence of smooth muscle cells (SMCs). Neo-extracellular matrix consisting mostly of collagen types I and III were observed at 12 months post-implantation. PCR analysis supports histological observations. TEVG group showed significant increases in expressions of SMC marker, collagen-I and III, matrix metalloproteinases-2 and 9, and itgam (a macrophage marker), when compared to sham group. Overall, patency rates were excellent at 12 months after implantation, as structural integrity of these TEVG. Tissue analysis also demonstrated vessel remodeling by autologous cell.     

Tunable Collagen I Hydrogels for Engineered Physiological Tissue Micro-Environments

Collagen I hydrogels are commonly used to mimic the extracellular matrix (ECM) for tissue engineering applications. However, the ability to design collagen I hydrogels similar to the properties of physiological tissues has been elusive. This is primarily due to the lack of quantitative correlations between multiple fabrication parameters and resulting material properties. This study aims to enable informed design and fabrication of collagen hydrogels in order to reliably and reproducibly mimic a variety of soft tissues. We developed empirical predictive models relating fabrication parameters with material and transport properties. These models were obtained through extensive experimental characterization of these properties, which include compression modulus, pore and fiber diameter, and diffusivity. Fabrication parameters were varied within biologically relevant ranges and included collagen concentration, polymerization pH, and polymerization temperature. The data obtained from this study elucidates previously unknown fabrication-property relationships, while the resulting equations facilitate informed a priori design of collagen hydrogels with prescribed properties. By enabling hydrogel fabrication by design, this study has the potential to greatly enhance the utility and relevance of collagen hydrogels in order to develop physiological tissue microenvironments for a wide range of tissue engineering applications.     

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade¹. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development². The method proposed in the following protocol uses microscopic magnetic resonance elastography (μMRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues³. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner⁴. Recently, μMRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques⁵. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map⁶. The resulting measurements at each voxel have been shown to strongly correlate (R²>0.9914) with data collected using dynamic mechanical analysis⁷. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.     

组织工程心脏瓣膜的研究进展

组织工程心脏瓣膜(tissue engineering heart valve,TEHV)理论上能克服机械瓣及生物瓣的不足,具有广阔的发展前景。目前组织工程心脏瓣膜的研究主要集中在瓣膜支架材料的选取及制备、种子细胞的选择和种子细胞的种植及培养等三方面。本文将分别就这三方面研究进展进行介绍,分析目前存在的问题,并对其应用进行展望。     

Tissue Carboxylesterases and Chlorpyrifos Toxicity in the Developing Rat

Young animals are more sensitive than adults to the neurotoxic effects of some organophosphorus insecticides. Many investigators attribute this difference in sensitivity to the immaturity of the detoxification capacity of preweanling rats. Chlorpyrifos [O,O-diethylO-(3,5,6-trichloro-2-pyridyl)phosphorothionate] is an organophosphorus insecticide that demonstrates considerable age-related sensitivity. The carboxylesterases are a group of related enzymes that detoxify organophosphorus insecticides by stoichiometrically binding these molecules before they can inhibit acetylcholinesterase. This study presents in vitro and in vivo evidence demonstrating that the carboxylesterases are critical for explaining the age-related sensitivity of chlorpyrifos. The data show that the fetal rat and the postnatal day 17 (PND17) rat pup have fewer molecules of carboxylesterase (less activity), less sensitive molecules of carboxylesterase, and a larger proportion of chlorpyrifos-insensitive molecules of carboxylesterase. An in vitro mixing experiment, using adult striatum as a source of acetylcholinesterase and liver homogenates as a source of carboxylesterase, demonstrates that the adult liver carboxylesterases are superior to the PND17 liver carboxylesterases for detoxifying chlorpyrifos. In the in vivo experiments the time course profiles of carboxylesterase and cholinesterase activity following a maximum tolerated dose of chlorpyrifos also suggest that the carboxylesterases of the PND17 rat were less capable of detoxifying chlorpyrifos. Carboxylesterase activity in the preweanling rat was not as severely inhibited as in the adult, but decrements in cholinesterase activity as a result of chlorpyrifos treatment were comparable. These in vitro and in vivo findings support the previously proffered postulate that the carboxylesterases are critical for determining the age-related sensitivity of chlorpyrifos. In addition, these detailed experiments allow us to propose that the detoxification potential of these enzymes is multifaceted, and depends on the (1) amount of activity (i.e., number of molecules), (2) affinity for the insecticide or metabolite, and (3) amount of carboxylesterase activity that is refractory to inhibition by the insecticide or metabolite.     

Functional Analysis of the Engineered Cardiac Tissue Grown on Recombinant Spidroin Fiber Meshes

In the present study, we examined the ability of the recombinant spidroin to serve as a substrate for the cardiac tissue engineering. For this purpose, isolated neonatal rat cardiomyocytes were seeded on the electrospun spidroin fiber matrices and cultured to form the confluent cardiac monolayers. Besides the adhesion assay and immunostaining analysis, we tested the ability of the cultured cardiomyocytes to form a functional cardiac syncytium by studying excitation propagation in the cultured tissue with the aid of optical mapping. It was demonstrated that recombinant spidroin fiber meshes are directly suitable for the adherence and growth of the cardiomyocytes without additional coating with the attachment factors, such as fibronectin.     

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro¹ and in vivo^2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits^4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula^6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.⁸ for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage⁹ using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice¹⁰ with minimal tissue damage.     

盆腔结缔组织炎大鼠模型的层黏连蛋白表达

目的建立混合菌液致SD大鼠盆腔结缔组织炎模型,研究大鼠盆腔黏连组织的病理学改变与层黏连蛋白表达。方法用注射器将细菌混悬液0．5mL,注人大鼠盆腔,并结合子宫穿孔手术,建立SD大鼠盆腔结缔组织炎模型。采用Philips分级评分法对大鼠盆腔黏连程度分级评分,切取黏连组织,用多聚甲醛固定,进行病理检测,并采用免疫组化法测定层黏连蛋白表达情况。结果模型组大鼠盆腔黏连Philips分级评分以Ⅲ级为主,与正常对照组比较差异极显著。HE染色镜检模型组可见大量炎细胞浸润,脓肿形成,伴有多少不等的纤维组织增生。免疫组化染色镜检模型组可见层黏连蛋白高表达,纤维结缔组织增生程度越高,层黏连蛋白表达越多。结论盆腔注射混合菌液并结合子宫穿孔手术,可作为建立大鼠盆腔结缔组织炎模型的方法。层黏连蛋白在盆腔结缔组织炎发病中起重要作用,参与整个炎症过程并维持盆腔黏连组织纤维化,可作为判断盆腔结缔组织炎的炎症和纤维化程度的参考指标。     

Changes in the Peritubular Tissue of Rat Testis after Cadmium Treatment

The present study demonstrates histological and immunohistochemical changes in the peritubular testicular tissue of rat testis after application of cadmium chloride. After 5-day cadmium exposure, advanced deterioration of the boundary testicular tissue, mainly oedema, disarrangement of collagen fibres and peritubular cells, dilatation and thrombosis of blood vessels were observed. Changes in the boundary tissue were accompanied with desquamation of the germinal epithelium. Immunohistochemically, positive reaction for α-smooth muscle actin and desmin in tunica media of large testicular blood vessels basically was not affected. No reaction for vimentin was seen in endothelial cells of blood capillaries, whereas positive reaction presented only these cells in large blood vessels. The myofibroblasts positively reacting for desmin and α-smooth muscle actin form a single incomplete layer in the lamina propria of seminiferous tubules. Vimentin reactivity in the myofibroblasts and in the supporting Sertoli cells as well as Leydig cells in damaged testicular tissue was not observed. An increase in fibroblasts and free inflammatory cells positive for vimentin in the peritubular space on the peripheric area of the testis was observed.