Caveats: numerical requirements in graph theory based quantitation of tissue architecture.

Graph theory based methods represent one approach to an objective and reproducible structural analysis of tissue architecture. By these methods, neighborhood relations between a number of objects (e.g., cells) are explored and inherent to these methods are therefore certain requirements as to the number of objects to be included in the analysis. However, the question of how many objects are required to achieve reproducible values in repeated computations of proposed structural features, has previously not been adressed specifically. After digitising HE stained slides and storing them as grey level images, cell nuclei were segmented and their geometrical centre of gravity were computed, serving as the basis for construction of the Voronoi diagram (VD) and its subgraphs. Variations in repeated computations of structural features derived from these graphs were related to the number of cell nuclei included in the analysis. We demonstrate a large variation in the values of the structural features from one computation to another in one and the same section when only a limited number of cells (100-500) are included in the analysis. This variation decreased with increasing number of cells analyzed. The exact number of cells required to achieve reproducible values differ significantly between tissues, but not between separate cases of similar lesions. There are no significant differences between normal and malignantly changed tissues in oral mucosa with respect to how many cells must be included. For graph theory based analysis of tissue architecture, care must be taken to include an adequate number of objects; for some of the structural features we have tested, more than 3000 cells.     

The preservation of tissues for transplantation

This paper is a written version of a lecture given during the celebration of Professor Rudolf Klen’s 90th birthday. Dr. Klen played by far the major part in the introduction and the development of Tissue Banking in Europe. His concept of a tissue bank envisaged the storage of all types of cell, tissue and organ that physicians and surgeons might need for the treatment of their patients. There has been much progress towards this goal, but still the final objective remains elusive. This review of the current position starts with the recognition that some tissues are required to comprise or include cells that exhibit all the formal characteristics of life if they are to function as grafts, whereas other tissues do not. For some tissues, the preservation of mechanical properties is crucial: for others it is not. These considerations are crucial for the design of preservation methods for specific tissues: bone tendon and skin can provide useful grafts in the absence of living cells and this may even be true of cardiac valves: the crucial requirement here is that the mechanical properties remain intact. Simply freezing at around −80°C may be sufficient. In contrast, many cell systems, and all metabolizing organs do require healthy cells to function. Cryopreservation is often an effective remedy for isolated cells, for example haemopoietic stem cells, but the damaging effects of the formation of ice are sufficient to rule out this approach for whole vascularised organs and for some tissues too. The damaging mechanisms are discussed, and it is concluded that the site of ice crystallization is crucial. Cartilage has hitherto been recalcitrant, but we have recently developed a method that permits this tissue to be stored at liquid nitrogen temperatures without any ice and with the recovery of living cells and intact mechanical properties after storage. Thus, many methods are available to help develop tissue banking originally envisioned by Dr. Klen.     

FDTD analysis of a noninvasive hyperthermia system for brain tumors

ABSTRACT: BACKGROUND: Hyperthermia is considered one of the new therapeutic modalities for cancer treatment and is based on the difference in thermal sensitivity between healthy tissues and tumors. During hyperthermia treatment, the temperature of the tumor is raised to 40--4[DEGREE SIGN]C for a definite period resulting in the destruction of cancer cells. This paper investigates design, modeling and simulation of a new non-invasive hyperthermia applicator system capable of effectively heating deep seated as well as superficial brain tumors using inexpensive, simple, and easy to fabricate components without harming surrounding healthy brain tissues. METHODS: The proposed hyperthermia applicator system is composed of an air filled partial half ellipsoidal chamber, a patch antenna, and a head model with an embedded tumor at an arbitrary location. The irradiating antenna is placed at one of the foci of the hyperthermia chamber while the center of the brain tumor is placed at the other focus. The finite difference time domain (FDTD) method is used to compute both the SAR patterns and the temperature distribution in three different head models due to two different patch antennas at a frequency of 915 MHz. RESULTS: The obtained results suggest that by using the proposed noninvasive hyperthermia system it is feasible to achieve sufficient and focused energy deposition and temperature rise to therapeutic values in deep seated as well as superficial brain tumors without harming surrounding healthy tissue. CONCLUSIONS: The proposed noninvasive hyperthermia system proved suitable for raising the temperature in tumors embedded in the brain to therapeutic values by carefully selecting the systems components. The operator of the system only needs to place the center of the brain tumor at a pre-specified location and excite the antenna at a single frequency of 915 MHz. Our study may provide a basis for a clinical applicator prototype capable of heating brain tumors.     

Modeling population size independent tissue epigenomes by ChIL‐seq with single thin sections

Recent advances in genome‐wide technologies have enabled analyses using small cell numbers of even single cells. However, obtaining tissue epigenomes with cell‐type resolution from large organs and tissues still remains challenging, especially when the available material is limited. Here, we present a ChIL‐based approach for analyzing the diverse cellular dynamics at the tissue level using high‐depth epigenomic data. “ChIL for tissues” allows the analysis of a single tissue section and can reproducibly generate epigenomic profiles from several tissue types, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation in cells from regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analyses performed using ChIL can elucidate in vivo cell‐type dynamics of tissues.     

A Glycosaminoglycan Based,Modular Tissue Scaffold System for Rapid Assembly of Perfusable,High Cell Density,Engineered Tissues

The limited ability to vascularize and perfuse thick, cell-laden tissue constructs has hindered efforts to engineer complex tissues and organs, including liver, heart and kidney. The emerging field of modular tissue engineering aims to address this limitation by fabricating constructs from the bottom up, with the objective of recreating native tissue architecture and promoting extensive vascularization. In this paper, we report the elements of a simple yet efficient method for fabricating vascularized tissue constructs by fusing biodegradable microcapsules with tunable interior environments. Parenchymal cells of various types, (i.e. trophoblasts, vascular smooth muscle cells, hepatocytes) were suspended in glycosaminoglycan (GAG) solutions (4%/1.5% chondroitin sulfate/carboxymethyl cellulose, or 1.5 wt% hyaluronan) and encapsulated by forming chitosan-GAG polyelectrolyte complex membranes around droplets of the cell suspension. The interior capsule environment could be further tuned by blending collagen with or suspending microcarriers in the GAG solution These capsule modules were seeded externally with vascular endothelial cells (VEC), and subsequently fused into tissue constructs possessing VEC-lined, inter-capsule channels. The microcapsules supported high density growth achieving clinically significant cell densities. Fusion of the endothelialized, capsules generated three dimensional constructs with an embedded network of interconnected channels that enabled long-term perfusion culture of the construct. A prototype, engineered liver tissue, formed by fusion of hepatocyte-containing capsules exhibited urea synthesis rates and albumin synthesis rates comparable to standard collagen sandwich hepatocyte cultures. The capsule based, modular approach described here has the potential to allow rapid assembly of tissue constructs with clinically significant cell densities, uniform cell distribution, and endothelialized, perfusable channels.     

Differential expression of keratin 19 in normal human epithelial tissues revealed by monospecific monoclonal antibodies

Summary Three monospecific monoclonal antibodies (BA16, BA17 and A53—B/A2) recognizing different epitopes of the human keratin 19 were used to determine tissue distribution of this 40 kDa keratin polypeptide. Immunohistochemical methods revealed four different staining patterns among normal human epithelial tissues: firstly, complete negativity of the epidermis, sebaceous glands, hepatocytes and other tissues; secondly, homogeneous positivity as seen for example in the gall bladder and urinary bladder epithelium, endometrium and many other epithelia; thirdly, a mosaic of positive and negative cells among mammary gland luminal cells, prostate epithelia and some other epithelia and fourthly, a more complex heterogeneous pattern found in non-keratinizing squamous epithelia and hair follicles with generally the basal layer being the most strongly or sometimes exclusively stained. The pattern seen in non-keratinizing squamous epithelia varied considerably according to the fixation method and the antibody used as well as among different donors and in different areas of the same organ. The other three staining patterns were on the other hand nearly identical with all three antibodies on both frozen sections and sections of methacarn-fixed paraffinembedded tissues. Our results provide evidence for differential expression of the human keratin 19 at the single cell level, an observation which could be exploited in the study of epithelial differentiation and pathology.     

An automatic segmentation and classification framework for anti-nuclear antibody images

Autoimmune disease is a disorder of immune system due to the over-reaction of lymphocytes against one's own body tissues. Anti-Nuclear Antibody (ANA) is an autoantibody produced by the immune system directed against the self body tissues or cells, which plays an important role in the diagnosis of autoimmune diseases. Indirect ImmunoFluorescence (IIF) method with HEp-2 cells provides the major screening method to detect ANA for the diagnosis of autoimmune diseases. Fluorescence patterns at present are usually examined laboriously by experienced physicians through manually inspecting the slides with the help of a microscope, which usually suffers from inter-observer variability that limits its reproducibility. Previous researches only provided simple segmentation methods and criterions for cell segmentation and recognition, but a fully automatic framework for the segmentation and recognition of HEp-2 cells had never been reported before. This study proposes a method based on the watershed algorithm to automatically detect the HEp-2 cells with different patterns. The experimental results show that the segmentation performance of the proposed method is satisfactory when evaluated with percent volume overlap (PVO: 89%). The classification performance using a SVM classifier designed based on the features calculated from the segmented cells achieves an average accuracy of 96.90%, which outperforms other methods presented in previous studies. The proposed method can be used to develop a computer-aided system to assist the physicians in the diagnosis of auto-immune diseases.     

The impact of fabrication parameters and substrate stiffness in direct writing of living constructs

Biomolecules and living cells can be printed in high‐resolution patterns to fabricate living constructs for tissue engineering. To evaluate the impact of processing cells with rapid prototyping (RP) methods, we modeled the printing phase of two RP systems that use biomaterial inks containing living cells: a high‐resolution inkjet system (BioJet) and a lower‐resolution nozzle‐based contact printing system (PAM²). In the first fabrication method, we reasoned that cell damage occurs principally during drop collision on the printing surface, in the second we hypothesize that shear stresses act on cells during extrusion (within the printing nozzle). The two cases were modeled changing the printing conditions: biomaterial substrate stiffness and volumetric flow rate, respectively, in BioJet and PAM². Results show that during inkjet printing impact energies of about 10^?8 J are transmitted to cells, whereas extrusion energies of the order of 10^?11 J are exerted in direct printing. Viability tests of printed cells can be related to those numerical simulations, suggesting a threshold energy of 10^?9 J to avoid permanent cell damage. To obtain well‐defined living constructs, a combination of these methods is proposed for the fabrication of scaffolds with controlled 3D architecture and spatial distribution of biomolecules and cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

生物组织弹性成像方法及其新技术进展

弹性是一种描述物质物理意义的重要参数,在描述物质在热力学和动力学的变化过程中有着重要的意义。在医学上,弹性的变化往往和病变联系在一起。然而,绝大多数生物组织在他们的力学特性上所表现出的复杂性并不是弹性模量一项参数就可以完全表述的,在对于他们的粘弹性表征和流变学行为的描述中,粘滞性往往和弹性一样的重要。现在被广泛用来对生物组织机械特性表征的成像技术是弹性成像,其基本原理是给组织施加一个激励,组织会产生一个响应,而该响应的分布结合技术的处理方法,可以反映出其弹性模量等力学属性的差异。本文介绍了生物组织常见的弹性成像方法:超声弹性成像,磁共振弹性成像以及光学相干弹性成像;详细阐述了新发展起来的技术-光声弹性成像和光声粘弹成像,并讨论分析其应用前景。     

Remodelling of the angular collagen fiber distribution in cardiovascular tissues

Understanding collagen fiber remodelling is desired to optimize the mechanical conditioning protocols in tissue-engineering of load-bearing cardiovascular structures. Mathematical models offer strong possibilities to gain insight into the mechanisms and mechanical stimuli involved in these remodelling processes. In this study, a framework is proposed to investigate remodelling of angular collagen fiber distribution in cardiovascular tissues. A structurally based model for collagenous cardiovascular tissues is extended with remodelling laws for the collagen architecture, and the model is subsequently applied to the arterial wall and aortic valve. For the arterial wall, the model predicts the presence of two helically arranged families of collagen fibers. A branching, diverging hammock-type fiber architecture is predicted for the aortic valve. It is expected that the proposed model may be of great potential for the design of improved tissue engineering protocols and may give further insight into the pathophysiology of cardiovascular diseases.     

应用冷Schiff组织化学法检测人体组织脂质过氧化反应的研究

自由基引发的生物膜不饱和脂肪酸脂质过氧化反应涉及多种疾病过程,多年来检测脂质过氧化反应一直沿用生物化学（如硫代巴比妥酸法测定丙二醛）或生物物理技术（如分光光度法测定共轭双烯）。自从冷Schiff组织化学染色技术用于自由基研究以来,使形态学方法研究脂质过氧化反应成为可能,当前,应用冷Schiff组织化学法进行组织细胞的脂质过氧化反应检测大多限于动物实验研究,本研究对多种人体离体新鲜组织的冰冻切片应用冷Schiff组织化学法进行检测,未发现被组织存在组织化学水平上的脂质过氧化反应;胆对被测组织人为施加氧化攻击（用Fe-NADPH促氧化剂孵育）后,肝、肾及胃的泌酸细胞与其它组织相比呈现较明显的脂质过氧化反应;皮肤、脂肪组织几科不出现脂质过氧化反应;甲状腺C细胞、肌肉、骨骼等与钙代谢、贮存及利用相关的组织也出现较明显的脂质过氧化反应。结论：冷Schiff组织化学方法检测人体组织脂持过氧化反应具有简便易行、同时可以形态学定位的优点,在医学生物学、肿瘤学及老年医学研究中具有应用前景。     

High Resolution Helium Ion Scanning Microscopy of the Rat Kidney

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization.     

Determination of metallothionein in tissues by radioimmunoassay and by cadmium saturation method

The diversity of reported basal metallothionein (MT) values in animal tissues has made it necessary that the presently available methods be further developed and compared for their accuracy, reproducibility, sensitivity, and specificity. A radioimmunoassay (RIA) to quantitate MT in animal tissues was developed and its performance compared to that of a cadmium saturation method. The procedure is more accurate and reliable but no more time-consuming than other techniques in current use. It offers the advantages of greater specificity and sensitivity thus enabling the determination of basal levels of MT in tissues and the analysis of small samples, for example, biopsies, cultured cells, in vitro protein synthesis, etc. The use of a polyclonal antiserum is advantageous in that total MT can be determined in any tissue from a variety of animal species. Both nonspecific and specific interference in the assay can be eliminated by heat treatment of the sample followed by a short preincubation with cadmium. The sensitivity of the RIA is 10 ng MT/g tissue. The cadmium saturation assay is unsuitable for the measurement of low levels of MT due to its nonspecific nature and its detection limit (10 micrograms MT/g tissue) but it is useful where large amounts of the protein are present in a tissue. Difficulties arising in the analysis of MT by both methods are discussed and solutions are offered. The basal levels of MT in the brain, kidney, liver, lung, muscle, pancreas, small intestine, and spleen of rats are determined by RIA and are shown to be generally lower than the currently accepted values measured by other techniques.     

Mesenchymal stem cells: biological characteristics and potential clinical applications

Mesenchymal stem cells (MSC) are clonogenic, non-hematpoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages, for example, osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages, for example, neuronal-like cells. Several methods are currently available for isolation of the MSC based on their physical and physico-chemical characteristics, for example, adherence to plastics or other extracellular matrix components. Because of the ease of their isolation and their extensive differentiation potential, MSC are among the first stem cell types to be introduced in the clinic. Several studies have demonstrated the possible use of MSC in systemic transplantation for systemic diseases, local implantation for local tissue defects, as a vehicle for genes in gene therapy protocols or to generate transplantable tissues and organs in tissue engineering protocols. Before their widespread use in therapy, methods allowing the generation of large number of cells without affecting their differentiation potential as well as technologies that overcome immunological rejection (in case allogenic transplantation) must be developed.     

In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)

Endopolyploidy, i.e. amplification of the genome in the absence of mitosis, occurs in many plant species and happens along with organ and cell differentiation. Deciphering the functional roles of endopolyploidy is hampered by the fact that polyploid tissues generally comprise cells with various ploidy levels. In some fleshy fruits (amongst them tomato fruit) the ploidy levels present at the end of development range from 2C to 256C in the same tissue. To investigate the temporal and spatial distribution of endopolyploidy it is necessary to address the DNA content of individual nuclei in situ. Conventional methods such as fluorometry or densitometry can be used for some tissues displaying favorable characteristics, e.g. small cells, small nuclei, organization in a monolayer, but high levels of varying polyploidy are usually associated with large sizes of nuclei and cells, in a complex three dimensional (3-D) organization of the tissues. The conventional methods are inadequate for such tissue, becoming semi-quantitative and imprecise. We report here the development of a new method based on fluorescent in situ bacterial artificial chromosome hybridizations that allows the in situ determination of the DNA ploidy level of individual nuclei. This method relies on the counting of hybridization signals and not on intensity measurements and is expected to provide an alternative method for mapping endopolyploidy patterns in mature, 3-D organized plant tissues as illustrated by the analysis of ploidy level and cell size in pericarp from mature green tomato fruit.     

Fundamental physical cellular constraints drive self‐organization of tissues

Morphogenesis is driven by small cell shape changes that modulate tissue organization. Apical surfaces of proliferating epithelial sheets have been particularly well studied. Currently, it is accepted that a stereotyped distribution of cellular polygons is conserved in proliferating tissues among metazoans. In this work, we challenge these previous findings showing that diverse natural packed tissues have very different polygon distributions. We use Voronoi tessellations as a mathematical framework that predicts this diversity. We demonstrate that Voronoi tessellations and the very different tissues analysed share an overriding restriction: the frequency of polygon types correlates with the distribution of cell areas. By altering the balance of tensions and pressures within the packed tissues using disease, genetic or computer model perturbations, we show that as long as packed cells present a balance of forces within tissue, they will be under a physical constraint that limits its organization. Our discoveries establish a new framework to understand tissue architecture in development and disease.     

Support vector machine classification and validation of cancer tissue samples using microarray expression data

MOTIVATION: DNA microarray experiments generating thousands of gene expression measurements, are being used to gather information from tissue and cell samples regarding gene expression differences that will be useful in diagnosing disease. We have developed a new method to analyse this kind of data using support vector machines (SVMs). This analysis consists of both classification of the tissue samples, and an exploration of the data for mis-labeled or questionable tissue results. RESULTS: We demonstrate the method in detail on samples consisting of ovarian cancer tissues, normal ovarian tissues, and other normal tissues. The dataset consists of expression experiment results for 97,802 cDNAs for each tissue. As a result of computational analysis, a tissue sample is discovered and confirmed to be wrongly labeled. Upon correction of this mistake and the removal of an outlier, perfect classification of tissues is achieved, but not with high confidence. We identify and analyse a subset of genes from the ovarian dataset whose expression is highly differentiated between the types of tissues. To show robustness of the SVM method, two previously published datasets from other types of tissues or cells are analysed. The results are comparable to those previously obtained. We show that other machine learning methods also perform comparably to the SVM on many of those datasets. AVAILABILITY: The SVM software is available at http://www.cs. columbia.edu/ approximately bgrundy/svm.     

Fiber alignment directs cell motility over chemotactic gradients

The ability of tissue engineered scaffolds to direct cell behavior is paramount for scaffold design. Cell migration can be directed by various methods including chemical, adhesive, mechanical, and topographical cues. Electrospinning has emerged as a popular method to control topography and create fibrous scaffolds similar to that found in extracellular matrix. One major hurdle is limited cell infiltration and several studies have explored methods to alter electrospun materials to increase scaffold porosity; however, uniform cell distributions within scaffolds is still limited. Towards this, we investigated the motility of HUVECs on a model system of electrospun hyaluronic acid fibers under a gradient of VEGF and found that topographical cues dominate cell motility direction. Using time‐lapse microscopy, cell aspect ratio, and migration angle were measured; cells were directed in a chemical gradient and/or on aligned electrospun fibers. Measurements of the persistence time demonstrated an additive effect of the chemical gradient and fiber alignment. However, when fibers were aligned perpendicular to a chemical gradient, cells were directed by fiber alignment and there was no effect of the chemical gradient. These results suggest that topographical cues may be more influential than chemical cues in directing cell motility and should be considered in material design. Biotechnol. Bioeng. 2013; 110: 1249–1254. © 2012 Wiley Periodicals, Inc.     

Large-scale profiling of Rab GTPase trafficking networks: the membrome

Rab GTPases and SNARE fusion proteins direct cargo trafficking through the exocytic and endocytic pathways of eukaryotic cells. We have used steady state mRNA expression profiling and computational hierarchical clustering methods to generate a global overview of the distribution of Rabs, SNAREs, and coat machinery components, as well as their respective adaptors, effectors, and regulators in 79 human and 61 mouse nonredundant tissues. We now show that this systems biology approach can be used to define building blocks for membrane trafficking based on Rab-centric protein activity hubs. These Rab-regulated hubs provide a framework for an integrated coding system, the membrome network, which regulates the dynamics of the specialized membrane architecture of differentiated cells. The distribution of Rab-regulated hubs illustrates a number of facets that guides the overall organization of subcellular compartments of cells and tissues through the activity of dynamic protein interaction networks. An interactive website for exploring datasets comprising components of the Rab-regulated hubs that define the membrome of different cell and organ systems in both human and mouse is available at http://www.membrome.org/.