Academic literature on the topic 'Tissues'

Thesis (Ph.D.)--Tufts University, 2005.<br>Adviser: Gregory H. Altman. Submitted to the Dept. of Biology--Biotechnology. Includes bibliographical references (leaves 183-192). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Collagen II and the proteoglycan aggrecan are key extracellular matrix (ECM) proteins in cartilaginous tissues such as the intervertebral disc (IVD). Given the functional role that these structural and functional proteins have in the IVD, ECM in tissue engineered intervertebral disc (TE IVD) constructs needs to recapitulate native tissue. As such, there is a need to understand the structure and mechanical function of these molecules in native tissue to inform TE strategies. The aims here were to characterise aggrecan and collagen II using atomic force microscopy (AFM), size-exclusion chromatography multi angle light scattering (SEC-MALS), histology, quantitative PCR, nanomechanical and computational modelling in: (i) skeletally immature and mature bovine articular cartilage (AC) and nucleus pulposus (NP), (ii) TE IVD constructs cultured in hypoxia or treated with transforming growth factor beta [TGFÎ23] or growth differentiation factor [GDF6]), and (iii) porcine AC and NP tissue. No variation in collagen II structure was observed although the proportion of organised fibrillar collagen varied between tissues. Both intact (containing all three globular domains) and non-intact (fragmented) aggrecan monomers were isolated from both AC and IVD and TE IVD constructs. Mature intact native NP aggrecan was ~60 nm shorter (core protein length) compared to AC. In skeletally mature bovine NP and AC tissue, most aggrecan monomers were fragmented (99% and 95%, respectively) with fragments smaller and more structurally heterogeneous in NP. Similar fragmentation was observed in skeletally immature bovine AC (99.5%), indicating fragmentation occurs developmentally at an early age. Fragmentation was not a result of enhanced gelatinase activity. Aggrecan monomers isolated from notochordal cell rich porcine NP were also highly fragmented, similar to bovine NP. Application of a computational packing model suggested fragmentation may affect porosity and nutrient transfer. The reduced modulus was greater in AC than NP (497 kPa and 76.7 kPa, respectively) with the difference likely due to the organisation and abundance of ECM molecules, rather than individual structure. Growth factors (GDF6 and TGFÎ23), and not oxygen tension treated TE IVD constructs were structurally (with >95% fragmented monomers), histologically and mechanically (GDF6: 60.2 kPa; TGFÎ23; 69.9 kPa) similar to native NP tissue (76.7 kPa) and there was evidence of gelatinase activity. To conclude, these results show that the ultrastructure of intact aggrecan was tissue and cell dependent, and could be modified by manipulation of cell culture conditions, specifically GDF6 which may play a role in aggrecan glycosylation.

Continuous monitoring during tissue culture is important for the success of engineered tissue development. It is also challenging due to lack of suitable established monitoring techniques. In this study, microdialysis, a sampling technique for measuring the unbound solute concentrations in the tissues and organs of the living body, was adopted to monitor functional tissue growth in a bioreactor with explanted bovine caudal intervertebral discs (IVD) as the test tissue. Apart from cell metabolic activities, cell and tissue biological functions were investigated for the development of microdialysis for monitoring purposes. Methodologies of microdialysis with large pore size membrane probes for sampling macromolecular bio-functional markers were established. The effects of pumping methods, including 'push', 'pull' or 'push-and-pull', and the effect of the resulting transmembrane pressure on the fluid balance, and the relative recovery of small molecules and of macromolecules (proteins) were experimentally studied. The validity of the internal reference in-situ calibration was examined in detail. It was concluded that a push-and-pull system was the only effective method to eliminate fluid loss or gain. The relative recovery of small solutes was hardly affected by the applied pumping methods; however the relative recovery of macromolecules was significantly influenced by them. The in situ calibration technique using Phenol Red can provide reliable results for small molecules including glucose and lactic acid. Using lOkDa and 70kDa fluorescent dextrans as the internal standard for in situ calibration of large molecules of similar size, it was found that the pull pump system did not work well but that the push-and-pull pumping method did work well. A novel bioreactor system for in vitro IVD culture with static load and microdialysis monitoring was developed. Explanted IVDs were cultured under three different loads for up to 7 days. A single microdialysis probe with 3000 kDa membrane was inserted into each of the IVDs at a defined location. The in situ calibration technique was proved valid in the experiments and membrane fouling was not significant. The tissue metabolism and extracellular matrix turnover during 7 day culture were continuously monitored to investigate the effect of different loads. Microdialysis proved to be a feasible and efficient method for multi-parameter monitoring of tissue culture. Substantial effort was directed towards the identification of functional macromolecular markers in conjunction with microdialysis sampling. Amongst several proteins sampled, chitinase-3-like protein 1 (CHI3L1), a major soluble protein secreted by cultured IVD cells in alginate beads and by cultured IVD explants was identified following its successful isolation. Then it was established as a suitable functional marker. The effect of physico-chemical and mechanical stimuli (e.g. osmolarity, pH, oxygen tension and mechanical load) on secretion of CHI3L1 by cultured IVD cells and chondrocytes in alginate beads and by cultured IVD explant were investigated. CHI3L1 release was sensitive to physico-chemical stimulation. The production of CHI3L1 was directly correlated with the cell metabolism and this could be readily monitored with microdialysis.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2013.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (p. 157-168).<br>Recent developments in computer-integrated surgery and in tissue-engineered constructs necessitate advances in experimental and analytical techniques in characterizing properties of mechanically compliant materials such as gels and soft tissues, particularly for small sample volumes. One goal of such developments is to quantitatively predict and mimic tissue deformation due to high rate impact events typical of industrial accidents and ballistic insults. This aim requires advances in mechanical characterization to establish tools and design principles for tissue simulant materials that can recapitulate the mechanical responses of hydrated soft tissues under dynamic contact-loading conditions. Given this motivation, this thesis studies the mechanical properties of compliant synthetic materials developed for tissue scaffold applications and of soft tissues, via modifying an established contact based technique for accurate, small scale characterization under fully hydrated conditions, and addresses some of the challenges in the implementation of this method. Two different engineered material systems composed of physically associating block copolymer gels, and chemically crosslinked networks including a solvent are presented as potential tissue simulants for ballistic applications, and compared directly to soft tissues from murine heart and liver. In addition to conventional quasistatic and dynamic bulk mechanical techniques that study macroscale elastic and viscoelastic properties, new methodologies are developed to study the small scale mechanical response of the aforementioned material systems to concentrated impact loading. The resistance to penetration and the energy dissipative constants are quantified in order to compare the deformation of soft tissues and mechanically optimized simulants, and to identify the underlying mechanisms by which the mechanical response of these tissue simulant candidates are modulated. Finally, given that soft tissues are biphasic in nature, atomic force microscopy enabled load relaxation experiments are utilized to develop approaches to distinguish between poroelastic and viscoelastic regimes, and to study how the anisotropy of the tissue structure affects elastic and transport properties, in order to inform the future design of tissue simulant gels that would mimic soft tissue response.<br>by Zeynep Ilke Kalcioglu.<br>Ph.D.

An essential prerequisite for the existence of multi-cellular life is the organization of cells into tissues. In this thesis, we theoretically study how large-scale tissue properties can emerge from the collective behavior of individual cells. To this end, we focus on the properties of epithelial tissue, which is one of the major tissue types in animals. We study how rheological properties of epithelia emerge from cellular processes, and we develop a physical description for the dynamics of an epithelial cell polarity. We apply our theoretical studies to observations in the developing wing of the fruit fly, Drosophila melanogaster. In order to study epithelial mechanics, we first develop a geometrical framework that rigorously describes the deformation of two-dimensional cellular networks. Our framework decomposes large-scale deformation into cellular contributions. For instance, we show how large-scale tissue shear decomposes into contributions by cell shape changes and into contributions by different kinds of topological transitions. We apply this framework in order to quantify the time-dependent deformation of the fruit fly wing, and to decompose it into cellular contributions. We also use this framework as a basis to study large-scale rheological properties of epithelia and their dependence on cellular fluctuations. To this end, we represent epithelial tissues by a vertex model, which describes cells as elastic polygons. We extend the vertex model by introducing fluctuations on the cellular scale, and we develop a method to perform perpetual simple shear simulations. Analyzing the steady state of such simple shear simulations, we find that the rheological behavior of vertex model tissue depends on the fluctuation amplitude. For small fluctuation amplitude, it behaves like a plastic material, and for high fluctuation amplitude, it behaves like a visco-elastic fluid. In addition to analyzing mechanical properties, we study the reorientation of an epithelial cell polarity. To this end, we develop a simple hydrodynamic description for polarity reorientation. In particular, we account for polarity reorientation by tissue shear, by another polarity field, and by local polarity alignment. Furthermore, we develop methods to quantify polarity patterns based on microscopical images of the fly wing. We find that our hydrodynamic description does not only account for polarity reorientation in wild type fly wings. Moreover, it is for the first time possible to also account for the observed polarity patterns in a number of genetically altered flies<br>Eine wesentliche Voraussetzung für die Existenz mehrzelligen Lebens ist, dass sich einzelne Zellen sinnvoll zu Geweben ergänzen können. In dieser Dissertation untersuchen wir, wie großskalige Eigenschaften von Geweben aus dem kollektiven Verhalten einzelner Zellen hervorgehen. Dazu konzentrieren wir uns auf Epitheliengewebe, welches eine der Grundgewebearten in Tieren darstellt. Wir stellen theoretische Untersuchungen zu rheologischen Eigenschaften und zu zellulärer Polarität von Epithelien an. Diese theoretischen Untersuchungen vergleichen wir mit experimentellen Beobachtungen am sich entwickelnden Flügel der schwarzbäuchigen Taufliege (Drosophila melanogaster). Um die Mechanik von Epithelien zu untersuchen, entwickeln wir zunächst eine geometrische Beschreibung für die Verformung von zweidimensionalen zellulären Netzwerken. Unsere Beschreibung zerlegt die mittlere Verformung des gesamten Netzwerks in zelluläre Beitrage. Zum Beispiel wird eine Scherverformung des gesamten Netzwerks auf der zellulären Ebene exakt repräsentiert: einerseits durch die Verformung einzelner Zellen und andererseits durch topologische Veränderungen des zellulären Netzwerks. Mit Hilfe dieser Beschreibung quantifizieren wir die Verformung des Fliegenflügels während des Puppenstadiums. Des Weiteren führen wir die Verformung des Flügels auf ihre zellulären Beiträge zurück. Wir nutzen diese Beschreibung auch als Ausgangspunkt, um effektive rheologische Eigenschaften von Epithelien in Abhängigkeit von zellulären Fluktuationen zu untersuchen. Dazu simulieren wir Epithelgewebe mittels eines Vertex Modells, welches einzelne Zellen als elastische Polygone abstrahiert. Wir erweitern dieses Vertex Modell um zelluläre Fluktuationen und um die Möglichkeit, Schersimulationen beliebiger Dauer durchzuführen. Die Analyse des stationären Zustands dieser Simulationen ergibt plastisches Verhalten bei kleiner Fluktuationsamplitude und visko-elastisches Verhalten bei großer Fluktuationsamplitude. Neben mechanischen Eigenschaften untersuchen wir auch die Umorientierung einer Zellpolarität in Epithelien. Dazu entwickeln wir eine einfache hydrodynamische Beschreibung für die Umorientierung eines Polaritätsfeldes. Wir berücksichtigen dabei insbesondere Effekte durch Scherung, durch ein anderes Polaritätsfeld und durch einen lokalen Gleichrichtungseffekt. Um unsere theoretische Beschreibung mit experimentellen Daten zu vergleichen, entwickeln wir Methoden um Polaritätsmuster im Fliegenflügel zu quantifizieren. Schließlich stellen wir fest, dass unsere hydrodynamische Beschreibung in der Tat beobachtete Polaritätsmuster reproduziert. Das gilt nicht nur im Wildtypen, sondern auch in genetisch veränderten Tieren

Tooth development is complex and dependent on epithelial-mesenchymal interactions involving key molecular signalling pathways. Preliminary data indicate that the pleiotropic growth factor adrenomedullin (ADM) is expressed during tooth development. Furthermore, in osteoblasts, cells which share structural and functional similarities to odontoblasts, ADM increases proliferation in vitro and can promote mineralised bone volume and strength in vivo. Immunohistochemical analysis of ADM demonstrated expression during key stages in tooth development in particular in cells responsible for signalling odontoblast differentiation and subsequently in secretory odontoblasts. Similarities with the temporo-spatial expression profile of TGF-β1 were also observed. In vitro analysis using the developmentally derived dental cell lines, MDPC-23 and OD-21, demonstrated ADM stimulated a biphasic response in dental cell numbers with peak stimulation at 10-11M and that it stimulated mineral deposition at levels comparable to that of the known mineralising agent dexamethasone. Analysis of tooth tissue volume and key mandibular measurements in Swiss mice systemically treated with ADM using techniques including micro-Computer Tomography did not identify significant differences in craniofacial mineralised tissue structures compared to sham treated controls. The data presented here along with the known pleiotropic properties of ADM indicate it may be an important regulator of tooth development particularly in the processes of cell proliferation, differentiation and mineralisation. However, in adult animals systemic ADM supplementation appears to have limited affect on mandibular bone and dentine synthesis.

Durch ihren Einfluß auf die Genexpression reguliert die zirkadiane Uhr physiologische Funktionen vieler Organe. Obwohl der zugrundeliegende allgemeine Uhrmechanismus gut untersucht ist, bestehen noch viele Unklarheiten über die gewebespezifische Regulation zirkadianer Gene. Neben ihrer gemeinsamen 24-h-Periode im Expressionsmuster unterscheiden diese sich darin, zu welcher Tageszeit sie am höchsten exprimiert sind und in welchem Gewebe sie oszillieren. Mittels Überrepräsentationsanalyse lassen sich Bindungsstellen von Transkriptionsfaktoren identifizieren, die an der Regulation ähnlich exprimierter Gene beteiligt sind. Um diese Methode auf zirkadiane Gene anzuwenden, ist es nötig, Untergruppen ähnlich exprimierter Gene genau zu definieren und Vergleichsgene passend auszuwählen. Eine hierarchische Methode zur Kontrolle der FDR hilft, aus der daraus entstehenden Menge vieler Untergruppenvergleiche signifikante Ergebnisse zu filtern. Basierend auf mit Microarrays gemessenen Zeitreihen wurde durch Promotoranalyse die gewebespezifische Regulation von zirkadianen Genen zweier Zelltypen in Mäusen untersucht. Bindungsstellen der Transkriptionsfaktoren CLOCK:BMAL1, NF-Y und CREB fanden sich in beiden überrepräsentiert. Diesen verwandte Transkriptionsfaktoren mit spezifischen Komplexierungsdomänen binden mit unterschiedlicher Stärke an Motivvarianten und arrangieren dabei Interaktionen mit gewebespezifischeren Regulatoren (z.B. HOX, GATA, FORKHEAD, REL, IRF, ETS Regulatoren und nukleare Rezeptoren). Vermutlich beeinflußt dies den Zeitablauf der Komplexbildung am Promotor zum Transkriptionsstart und daher auch gewebespezifische Transkriptionsmuster. In dieser Hinsicht sind der Gehalt an Guanin (G) und Cytosin (C) sowie deren CpG-Dinukleotiden wichtige Promotoreigenschaften, welche die Interaktionswahrscheinlichkeit von Transkriptionsfaktoren steuern. Grund ist, daß die Affinitäten, mit denen Regulatoren zu Promotoren hingezogen werden, von diesen Sequenzeigenschaften abhängen.<br>A circadian clock in peripheral tissues regulates physiological functions through gene expression timing. However, despite the common and well studied core clock mechanism, understanding of tissue-specific regulation of circadian genes is marginal. Overrepresentation analysis is a tool to detect transcription factor binding sites that might play a role in the regulation of co-expressed genes. To apply it to circadian genes that do share a period of about 24 hours, but differ otherwise in peak phase timing and tissue-specificity of their oscillation, clear definition of co-expressed gene subgroups as well as the appropriate choice of background genes are important prerequisites. In this setting of multiple subgroup comparisons, a hierarchical method for false discovery control reveals significant findings. Based on two microarray time series in mouse macrophages and liver cells, tissue-specific regulation of circadian genes in these cell types is investigated by promoter analysis. Binding sites for CLOCK:BMAL1, NF-Y and CREB transcription factors are among the common top candidates of overrepresented motifs. Related transcription factors of BHLH and BZIP families with specific complexation domains bind to motif variants with differing strengths, thereby arranging interactions with more tissue-specific regulators (e.g. HOX, GATA, FORKHEAD, REL, IRF, ETS regulators and nuclear receptors). Presumably, this influences the timing of pre-initiation complexes and hence tissue-specific transcription patterns. In this respect, the content of guanine (G) and cytosine (C) bases as well as CpG dinucleotides are important promoter properties directing the interaction probability of regulators, because affinities with which transcription factors are attracted to promoters depend on these sequence characteristics.