Tesis sobre el tema "Extracellular matrix (ECM) peptides"

Diverses stratégies de fonctionnalisation de surface visent à améliorer la biocompatibilité des matériaux pour les dispositifs implantables, notamment en ingénierie tissulaire. Par exemple, le polydiméthylsiloxane (PDMS), bien qu’utilisé dans de nombreux domaines, présente des propriétés de surface défavorables à l’adhérence cellulaire. La fonctionnalisation par des protéines de la matrice extracellulaire (MEC) ou des peptides synthétiques dérivés de celles-ci permet d’améliorer l'adhérence des cellules. Bien que ces approches offrent certaines solutions, des défis tels que le coût de production et le contrôle de la présentation en 3D entravent leur manipulation. Pour répondre à ces défis, nous avons développé des particules pseudo-virales (VLPs) présentant des peptides bioactifs à leur surface. La protéine d’enveloppe CP3, dérivée du bactériophage à ARN AP205, a été modifiée génétiquement à ses extrémités N- et C-terminales pour produire des VLPs présentant des peptides d’adhésion (RGD et YIGSR) et ostéogéniques (BMP2). La bioactivité des VLPs a été testée sur du PDMS avec des cellules de myoblastes C2C12, montrant une stimulation de l'adhérence, de la migration, de la prolifération et de la différenciation cellulaires. Des VLPs hétéromériques co-exprimant les peptides RGD et YIGSR ou BMP2 ont montré une bioactivité combinée. Des comparaisons entre la fibronectine et les VLP-RGD ont révélé des similarités et des différences dans les interactions cellulaires et la formation des adhésions focales. Ces résultats démontrent que les VLPs d’AP205 peuvent servir de nano-plateformes de signalisation, avec des applications potentielles en nanomédecine et dans les biomatériaux
Scientists have explored various surface functionalization strategies to improve the biocompatibility of materials used in implantable devices, particularly in tissue engineering. For example, polydimethylsiloxane (PDMS), although used in many fields, has surface properties that are unfavorable for cell adhesion. Functionalization with extracellular matrix (ECM) proteins or synthetic peptides derived from ECM components improves cell adhesion. While these approaches offer some solutions, challenges such as production cost and control over 3D presentation limit their use. To overcome these challenges, we developed virus-like particles (VLPs) displaying bioactive peptides on their surface. The coat protein CP3, derived from the RNA bacteriophage AP205, was genetically modified at both its N- and C-termini to produce VLPs displaying adhesion peptides (RGD and YIGSR) and an osteogenic peptide (BMP2). The bioactivity of the VLPs was tested on PDMS with C2C12 myoblast cells, demonstrating enhanced cell adhesion, migration, proliferation, and differentiation. Heteromeric VLPs co-expressing RGD and YIGSR or BMP2 peptides showed combined bioactivity. By comparing focal adhesions formed by RGD VLPs and those formed by fibronectin, we elucidate both the similarities and the differences in cell interactions. These results demonstrate that AP205 VLPs can be used as nanoscale signaling platforms to stimulate multiple cell functions, with promising applications in nanomedicine and biomaterials

Glomerular filtration is a vital physiological process removing waste products from the circulation and this process occurs across the glomerular filtration barrier (GFB). The cells and extracellular matrix (ECM), which form this barrier, are exposed to forces during ultrafiltration and special adaptation is required to withstand these forces. Dysfunction in cellular adhesion machinery or ECM assembly within the GFB causes loss of selective glomerular filtration, however, the mechanisms governing these processes are poorly understood. To this end we sought to characterise the glomerular ECM and adhesion machinery using high throughput mass spectrometry (MS)-based proteomics. MS of human glomerular ECM identified a highly complex extracellular niche, revealing the potential involvement of novel ECM proteins in glomerular development and disease processes. Furthermore we identified that glomerular cells in culture had distinct ECM proteomes and interestingly, coculture experiments demonstrated that the ECM proteome was influenced by cellular crosstalk and had a closer resemblance to glomerular ECM in vivo. Protein network analyses of in vivo and in vitro ECM datasets revealed a common core of highly connected structural ECM proteins that may be important for glomerular ECM assembly. To understand how this ECM proteome altered in disease, we studied mice with mild glomerular dysfunction. Here, transcriptomic and proteomic analyses identified alterations in ECM composition and 3D electron microscopy revealed striking ultrastructural changes in glomerular ECM. MS-based proteomics was next applied to the analysis of glomerular podocyte adhesion complexes, leading to the discovery that the actin cytoskeletal regulators and trafficking machinery are recruited to adhesions sites in an ECM-ligand dependent manner. Furthermore, these differences functionally altered cell shape and adhesion strength. These same analyses were applied to podocyte cell-cell junctions, revealing an unexpected overlap of cell-ECM and cell-cell adhesion machinery. Overall, these findings demonstrate for the first time the complexity of the glomerular ECM and adhesion signalling complexes and reinforce the benefits of global, unbiased experimental approaches. In addition the results suggest that glomerular ECM composition, organisation and adhesion signalling are context dependent, and therefore, represent potential therapeutic targets.

Stem cell therapies have shown promising capabilities in regaining the functionality of scar tissue following a myocardial infarction. Biological sutures composed of fibrin have been shown to more effectively deliver human mesenchymal stem cells (hMSCs) to the heart when compared to traditional cell delivery mechanisms. While the biological sutures do show promise, improvements can be made. To enhance the fibrin sutures, we propose to incorporate native cardiac extracellular matrix (ECM) into the fibrin microthreads to produce a more in vivo-like environment. This project investigated the effects that ECM incorporation has on fibrin microthread structure, mechanics, stem cell seeding, and pro-angiogenic potential. Single microthreads composed of fibrin or fibrin and ECM were subjected to uniaxial tensile testing. It was found that the microthreads consisting of both fibrin and ECM had significantly high elastic moduli than fibrin only microthreads. Cell seeding potential was evaluated by performing a 24-hour hMSC seeding experiment using sutures of the varying microthread types. A CyQuant cell proliferation assay was used to determine the number of cells seeded onto each suture type. The results determined that there was no statistical difference between the numbers of cells seeded on the types of sutures. To examine the pro-angiogenic potential the microthreads had, a 24-hour endothelial progenitor outgrowth cell (EPOC) outgrowth assay was used. Fibrin and 15% ECM-fibrin microthreads were placed within the scratch of an EPOC culture and evaluated every 6 hours for 24 hours. We found that the 15% ECM microthreads had significantly increased the EPOC outgrowth, approximately 16% more distance travelled than fibrin microthreads and 18% more than no microthreads. Our combined results suggest that ECM does not affect hMSC attachment to biological sutures but does increase the pro-angiogenic potential of the microthreads due to their increase in guiding EPOC outgrowth.

The relations between cells and extracellular matrix seem to orchestrate tissue organization by regulating cell functions during fetal development and throughout normal adult life. Thus, focusing on the innate ability of the native ECM to better modulate cell behavior, the coating of synthetic biomaterials with cell-derived decellularized extracellular matrices is a promising approach to confer bioactivity to inert materials and direct the fate of host or transplanted cells in tissue engineering applications. This study aims to better understand ECM influence on human bone marrow stem cells and its role during the induction of an osteogenic phenotype. For this purpose decellularized matrices were prepared and examined for viability, morphology, adhesion, ALP content, mineralization and gene expression. Extracellular matrix coatings were able to delay, unlike uncoated surfaces, cell spontaneous differentiation, underlying its role in the preservation of cell stemness. Furthermore, the combination of free cell ECM with osteogenic medium resulted in a strong effect on cell differentiation. In conclusion, the ECM provides an ideal environment that promote cell adhesion and proliferation creating an ideal setting for the large-scale expansion of MSCs. In addition, it could enhance, in the presence of osteogenic factors, the cells differentiative ability providing the basis for an easier tissue-specific fate control of MSCs for therapeutic applications.

Remodeling after myocardial infarction (MI) associates with left ventricular (LV) dilation, decreased cardiac function and increased mortality. The dynamic synthesis and breakdown of extracellular matrix (ECM) proteins play a significant role in myocardial remodeling post-MI. Expression of osteopontin (OPN) increases in the heart post-MI. Evidence has been provided that lack of OPN induces LV dilation which associates with decreased collagen synthesis and deposition. Inhibition of matrix metalloproteinases, key players in ECM remodeling process post-MI, increased ECM deposition (fibrosis) and improved LV function in mice lacking OPN after MI. This review summarizes — 1) signaling pathways leading to increased expression of OPN in the heart; 2) the alterations in the structure and function of the heart post-MI in mice lacking OPN; and 3) mechanisms involved in OPN-mediated ECM remodeling post-MI.

Mechanical cues coming from the extracellular matrix (ECM) are key factors in the control of tissue homeostasis in physiology and disease. Cells can sense these physical cues and measure external resisting forces by adjusting their actomyosin cytoskeleton, which in turn regulates intracellular signalling pathways to orchestrate a proper cell response. Thus, ECM stiffness is important for many biological aspects such as proliferation, differentiation and migration. Very little is known instead, about its impact on cellular metabolism, and the molecular players involved in this process are largely unknown. Starting from an unbiased metabolomics approach, we found lipid accumulation as a general response to mechanical signals and to low tension conditions. Mechanicistically, this accumulation is associated with a decreased Lipin1 phosphatidate phosphatase localization at ER/Golgi membranes and decreased Lipin1 activity which ultimately lead to nuclear translocation and activation of SREBP1/2 transcription factors. This occurs independently of YAP/TAZ and mTOR, and in parallel to the feedback control by sterols. Led by our findings, we discovered a coherent regulation of SREBP in stiffened pathological human tissues, and identified SREBP as required for the pro-survival activity of ROCK inhibitors in embryonic stem cells. We thus propose that SREBP is a general mechanism that links the physical cell microenvironment to a key metabolic pathway
Gli stimoli meccanici provenienti dalla matrice extracellulare (ECM) sono fattori chiave nel controllo dell'omeostasi tissutale in condizioni fisiologiche e patologiche. Le cellule possono percepire questi segnali fisici e misurare le forze di resistenza esterne regolando il loro citoscheletro attraverso i filamenti di actomiosina, che a loro volta regolano le vie di segnalazione intracellulare per indurre una risposta cellulare adeguata. Pertanto, la rigidità della matrice extracellulare è importante per molti aspetti biologici come la proliferazione, la differenziazione e la migrazione. Si sa invece molto poco sull’ impatto che essa ha sul metabolismo cellulare, e i fattori molecolari coinvolti in questo processo sono in gran parte sconosciuti. Attraverso un’analisi metabolomica iniziale, abbiamo visto che le cellule tendono ad accumulare lipidi come risposta generale ai segnali meccanici e alle condizioni di bassa tensione. Abbiamo inoltre osservato che questo accumulo è associato ad un cambio di localizzazione della fosfatasi Lipin1, che diminuisce la sua affinità per le membrane del reticolo endoplasmatico e dell’apparato di Golgi e alla ridotta attività di Lipin1 che alla fine porta alla traslocazione nucleare e all'attivazione dei fattori di trascrizione SREBP1 / 2. Ciò si verifica indipendentemente dall’attività di YAP / TAZ e mTOR e in modo parallelo rispetto alla regolazione dei livelli di steroli nella cellula. Cercando una rilevanza biologica per il meccanisco da noi descritto, abbiamo inoltre scoperto che SREBP viene regolato in maniera coerente nei cheloidi, una patologia fibro-proliferativa legata a stress meccanici, e abbiamo identificato SREBP come un fattore importante e richiesto per la sopravvivenza di cellule staminali embrionali mediata dall’inibizione di ROCK. Quindi riassumendo, il nostro modello vede l’inibizione di LIPIN1 e l’attivazione di SREBP come un meccanismo generale che collega le forze fisiche derivanti dal microambiente e il metabolismo cellulare

Il est de plus en plus évident que la matrice extracellulaire (MEC), au-delà de sa fonction d’échafaudage cellulaire, génère des signaux de nature biochimique et biophysique jouant un rôle primordial au cours du processus de différenciation des cellules souches. A l’heure actuelle, plus de 15 différents facteurs extrinsèques (environnementaux), incluant l’organisation spatiale de la MEC, sa topographie, rigidité, porosité, biodégradabilité et chimie ont été identifiés comme modulateurs potentiels de la différenciation des cellules souches en lignées cellulaires spécialisées. Ainsi, il est plausible que l’intégration d’un biomatériau au sein de l’organisme dépendra largement de sa capacité à mimer les propriétés de la MEC du tissu à remplacer. Récemment, les techniques de micro-ingénierie ont émergé comme outil innovant pour découpler les différentes propriétés de la MEC et étudier l’impact individuel ou combiné de ces facteurs sur le comportement des cellules souches. De plus, ces techniques de microfabrication ont un intérêt particulier dans une perspective de reconstruction de la MEC dans tous ses aspects, in vitro. Dans ce projet de thèse, le concept de déconstruction/reconstruction de la complexité de la MEC a été appliqué pour récapituler, in vitro, plusieurs aspects inhérents à la MEC osseuse et explorer leurs effets individuels ou combinés sur la différenciation ostéoblastique des cellules souches mésenchymateuses (CSMs) humaines. Trois principales composantes ont été utilisées tout au long du projet : un matériau modèle (verre borosilicate), des séquences peptidiques mimétiques dérivées de la MEC naturelle, favorisant à la fois l’adhérence cellulaire (peptide RGD) et la différenciation ostéoblastique (peptide BMP-2) des CSMs prélevées de la moelle osseuse des patients. La première étude du projet consiste à greffer, d’une manière aléatoire, les peptides RGD et/ou BMP-2 sur la surface du matériau. Brièvement, nous avons développé trois types de matériaux bioactifs : matériaux fonctionnalisés avec le peptide RGD, matériaux fonctionnalisés avec le peptide BMP-2 et matériaux bi-fonctionnalisés avec les peptides RGD/BMP-2. La caractérisation physicochimique de ces matériaux a été réalisée en utilisant la spectrométrie photoélectrique à rayons X (XPS) pour évaluer la composition chimique de la surface, la microscopie à force atomique (AFM) pour évaluer la topographie de la surface et la microscopie à fluorescence pour confirmer la présence des peptides sur la surface et évaluer leur densité. L’objectif de cette étude est d’évaluer le potentiel individuel et synergétique de ces peptides à induire et contrôler la différentiation ostéoblastique des CSMs. Dans un premier temps, la caractérisation physicochimique nous a permis de confirmer l’immobilisation covalente des peptides sur la surface et de mesurer leur densité. En effet, la densité des peptides, mesurée sur les surfaces greffées uniquement avec le peptide RGD ou BMP-2, était de 1.8 ± 0.2 pmol/mm² et 2.2 ± 0.3 pmol/mm², respectivement. Cependant, sur les surfaces bifonctionnalisées, la densité de chaque peptide a diminué de presque la moitié, atteignant 0.7 ± 0.1 pmol/mm² pour le peptide RGD et 1 ± 0.1 pmol/mm² pour le peptide BMP-2. Ensuite, l’évaluation biologique des différents matériaux fonctionnalisés a clairement révélé que contrairement au peptide RGD, le peptide BMP-2 induit la différenciation ostéoblastique des CSMs. Cependant, le greffage simultané des peptides RGD/BMP-2 améliore significativement la différenciation des CSMs en ostéoblastes et cela malgré la diminution significative de la densité de chaque peptide sur les surfaces bi-fonctionnalisées, comparativement aux surfaces contenant qu’un seul peptide. Ces résultats montrent que les peptides RGD et BMP-2 peuvent engendrer un effet synergétique pour améliorer la différenciation ostéoblastique des CSMs. Le second chapitre de thèse vise à déterminer si la microstructuration de la surface des matériaux avec des ligands bioactifs améliore la différenciation ostéoblastique des CSMs. En effet, les peptides RGD et BMP-2 ont été greffés séparément sur la surface du matériau sous forme de micro-motifs de différentes formes mais de taille similaire. En se basant sur des précédents travaux de littérature – discutés dans le chapitre II – nous avons sélectionné trois différentes formes de motifs peptidiques (triangle, carré et rectangle) dont la surface est de 50 μm². Ces micromotifs ont été créées grâce à une technique assez répondue et facile à utiliser qui est la photolithographie. Les surfaces microstructurées ont été caractérisées avec l’interférométrie optique et la microscopie à fluorescence. Les résultats montrent que les micromotifs peptidiques ont à la fois la forme et les dimensions prédéfinies. In vitro, les résultats de différenciation cellulaire ont révélé que la distribution spatiale des ligands à l’échelle micrométrique joue un rôle très important dans l’engagement et la différenciation des CSMs en ostéoblastes. En effet, contrairement aux micromotifs peptidiques en forme de rectangles, les micromotifs triangulaires et carrés améliorent significativement l’expression des marqueurs ostéogéniques (Runx-2 et Ostéopontine) comparativement à la distribution aléatoire des peptides. Il est important de noter que ce profile d’expression des marqueurs biologiques a été observé que sur les matériaux fonctionnalisés avec le peptide BMP-2, tant dis que les matériaux fonctionnalisés avec le peptide RGD n’ont induit aucun effet spécifique sur la différenciation des CSMs et cela peu importe la forme des micromotifs peptidiques. En conclusion, cette étude a permis d’identifier un nouveau facteur extracellulaire capable de contrôler la différenciation des CSMs. De plus, nous avons démontré que la distribution spatiale des ligands à l’échelle micrométrique affecte le devenir des CSMs, dépendamment de la nature du principe actif. Finalement, la troisième étude de ce projet de thèse est une suite logique de l’étude 1 et 2, puisqu’elle consiste à greffer simultanément les peptides RGD et BMP-2 sous forme de micromotifs. En effet, ces surfaces ont été développées afin de bénéficier à la fois de l’effet synergétique des peptides RGD/BMP-2, observé dans l’étude 1 (facteur 1), et de l’effet de la distribution spatiale contrôlée des ligands, observé dans l’étude 2 (facteur 2). Les différents types de matériaux ont été caractérisés avec les mêmes techniques de caractérisation de surface mentionnées dans l’étude 2. Les résultats montrent clairement que les surfaces microstructurées sont très bien définies et correspondent à un damier de micromotifs RGD, intercalé par un damier de micromotifs BMP-2. L’évaluation de la différenciation des CSMs sur ces matériaux a révélé que la combinaison des facteurs 1 et 2 améliore significativement la différenciation des CSMs vers le lignage ostéoblastique, comparativement à l’exposition des CSMs à un seul facteur extracellulaire (1 ou 2). De plus, cette étude confirme les résultats obtenus dans l’étude 2, puisque les micromotifs triangulaires et carrés ont permis une meilleure différenciation cellulaire, comparativement aux micromotifs rectangulaires. Il est important de noter également que l’évaluation biologique des différentes surfaces biomimétiques a été réalisée dans un milieu de culture basal qui ne contient pas de facteurs ostéogéniques solubles, afin d’étudier d’une manière assez précise et fiable les interactions des CSMs avec les différents microenvironnements in vitro développés dans ce projet. En conclusion générale, les travaux effectués jusqu’à présent ont permis d’identifier deux aspects de la MEC qui influencent considérablement la différenciation ostéoblastique des CSMs. De plus, nous avons démontré que ces deux facteurs peuvent coopérer pour induire une meilleure différenciation cellulaire. Cela révèle clairement l’intérêt des techniques de micro-ingénierie pour une meilleure et plus profonde compréhension des mécanismes d’interactions des cellules souches avec leurs niches, ce qui permettra éventuellement de concevoir des produits d’ingénierie tissulaire sur-mesure. Mots clés : Microstructuration de la surface des matériaux, matrice extracellulaire biomimétique, peptides mimétiques, BMP-2, cellules souches, ostéogenèse.
It is becoming increasingly appreciated that the role of extracellular matrix (ECM) extends beyond acting as scaffolds to providing biochemical and biophysical cues, which are critically important in regulating stem cell self-renewal and differentiation. To date, more than 15 different extrinsic (environmental) factors, including the matrix spatial organization, topography, stiffness, porosity, biodegradability and chemistry have been identified as potent regulators of stem cells specification into lineage-specific progenies. Thus, it is plausible that the behavior of biomaterials inside the human body will depend to a large extent on their ability to mimic ECM properties of the tissue to be replaced. Recently, nano- and microengineering methods have emerged as an innovative tool to dissect the individual role of ECM features and understand how each element regulates stem cell fate. In addition, such tools are believed to be useful in reconstructing complex tissue-like structures resembling the native ECM to better predict and control cellular functions. In the thesis project presented here, the concept of deconstructing and reconstructing the ECM complexity was applied to reproduce several aspects inherent to the bone ECM and harness their individual or combinatorial effect on directing human mesenchymal stem cells (hMSCs) differentiation towards the osteoblastic lineage. Three main components were used throughout this project: a model material (borosilicate glass), ECM derived peptides (adhesive RGD and osteoinductive BMP-2 mimetic peptides) and bone marrow derived hMSCs. All cell differentiation experiments were performed in the absence of soluble osteogenic factors in the medium in order to precisely assess the interplay between hMSCs and the different artificial matrices developed in the current study. First, RGD and/or BMP-2 peptides were covalently immobilized and randomly distributed on glass surfaces. The objective here was to investigate the effect of each peptide as well as their combination on regulating hMSCs osteogenic differentiation. The most important funding was that RGD and BMP-2 peptides can act synergistically to enhance hMSCs osteogenesis. Then, micropatterning technique (photolithography) was introduced to control the spatial distribution of RGD and BMP-2 at the micrometer scale. The peptides were grafted individually onto glass substrates, as specific micropatterns of varied shapes (triangular, square and rectangle geometries) but constant size (50 μm² per pattern). In this second part of the project, the focus was made on investigating the role of ligands presentation in a spatially controlled manner in directing hMSCs differentiation into osteoblasts. Herein, we demonstrated that the effect of microscale geometric cues on stem cell differentiation is peptide dependent. Finally, glass surfaces modified with combined and spatially distributed peptides were used as in vitro cell culture models to evaluate the interplay between RGD/BMP-2 crosstalk and microscale geometric cues in regulating stem cell fate. In this study, we revealed that the combination of several ECM cues (ligand crosstalk and geometric cues), instead of the action of individual cues further enhances hMSCs osteogenesis. Overall, our findings provide new insights into the role of single ECM features as well their cooperation in regulating hMSCs fate. Such studies would allow the reconstruction of stem cell microenvironment in all the aspects ex vivo, which may pave the way towards the development of clinically relevant tissue-engineered constructs. Keywords: Chemical micropatterning, bioactive surfaces, mimetic peptides, BMP-2, mesenchymal stem cells, stem-cell differentiation, stem-cell niche, osteogenesis.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第21159号
農博第2285号
新制||農||1060(附属図書館)
学位論文||H30||N5133(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 和光, 教授 阪井 康能, 教授 矢﨑 一史
学位規則第4条第1項該当

Biomedical research uses hydrogels for cell culture to closely mimic cellular environments and produce more accurate results than traditional methods. Here, human placenta, a tissue rich in bio-signalling molecules, is used as a base material to create a novel type of hydrogel that can be cured by exposure to visible light (Placental-Methacryloyl; PlacMA). Effects of different preparation methods are explored while mechanical, chemical and biological properties of PlacMA are investigated. PlacMA was found to be highly tuneable and suitable for a variety of cell culture and tissue engineering applications, demonstrating its potential to advance the current techniques in biomedical research.

The extracellular matrix (ECM) is secreted by the host tissue and is an important key for mechanisms of cell responses. The main properties of the ECM materials include biodegradability, biocompatibility, and nanostructured in a 3D fibre network. In addition, ECM is composed of important molecules like growth factors, glycosaminoglycans (GAGs), collagens, fibronectin, and lamin, while final composition depends on the native tissue. We have selected for this study ECMs from cortical bone (B-ECM) and pericardium (P-ECM) tissue. These ECMs were digested by collagenase, pepsin and trypsin. Each of these digested ECMs was used to produce PLLA-ECM based electrospun scaffolds by two different methodologies (1) non-crosslinked (NCLK) hybrid electrospun scaffolds composed of PLLA and digested ECMs and (2) PLLA-collagen electrospun scaffolds crosslinked with digested ECMs (CLK scaffolds). This research proposes the characterization of the digestion promoted by collagenase, pepsin and trypsin on the ECMs, followed by the evaluation of the potential of the digested ECMs and of the PLLA-ECM scaffolds for bone regeneration. The proteinaceous mixture, produced from the ECM digestion, had compositions, which were dependent on the type of ECM, and on the enzymatic treatment, as shown by protein quantification, GAGs quantification, TGA, SDS-page and TPEF-SHG. All the results point to an extensive digestion caused by collagenase and pepsin and a milder digestion caused by trypsin. The digested ECMs were incorporated into nanofibrous scaffolds, and the products were characterized by SEM, TGA, DSC and TPEF-SHG. The porous nanofibrous mesh from non-crosslinked scaffolds exhibited fibres without beads and a uniform diameter. However, the crosslinked scaffolds presented non-organized agglomerates around the fibres making a less porous surface. TGA and DSC suggest the incorporation of the ECMs on the scaffolds. However, the distribution of the protein on the polymer was mostly dependent on the incorporation method, as showed by TPEF-SHG. To access the biomaterial ability for bone regeneration, bone marrow mesenchymal stem cells (BMMSCs) were cultured on the scaffolds over 21 days. Osteogenic markers such as ALP activity, mineral nodule formation by ARS staining, col1a2 immunostaining, and gene expression were analysed to access how the materials could induce BMMSCs osteodifferentiation. Comparing NCLK to CLK scaffolds the key factor for osteogenesis is the release of soluble factors, showing NCLK scaffolds with a higher ability to induce mineralization than CLK scaffolds. However, when comparing the effect of the enzymatic digestion on the mineralization of the scaffolds over the days, it is possible to establish that the effect of the enzymatic treatment is also related to the type of ECM. Despite all those differences, some PLLA-ECM scaffolds exhibited potential to induce earlier mineralization, observed by the analysis of bglap, runX2, Osx, sparc and col1a2 genes as osteogenic markers.
A matriz extracelular (ECM) é secretada pela células no tecido nativo e reúne propriedades chave para respostas celulares. Entre suas principais propriedades destacam-se: biodegradabilidade, biocompatibilidade e nanoestruturada tridimensionalmente. Além disso, é rica em sinalizadores celulares tais como: fatores de crescimento, glicosaminaglicanas (GAGs), colágeno, fibronectina e laminina, no entanto sua composição depende do tecido na qual se encontra. Para este estudo, foram selecionadas ECMs provenientes de osso cortical e de pericárdio. Estas ECMs foram digeridas por colagenase, pepsina e tripsina. Cada um dos produtos de digestão foi utlizado para a produção de suportes eletrofiados de PLLA-ECM, utilizando-se dois diferentes métodos de incorporação, (1) Suportes eletrofiados híbridos de PLLA-ECM obtidos a partir da eletrofiação da co-solução em 1,1,1,3,3,3-hexafluor-2-propanol, e (2) imobilização das ECM digeridas sobre suportes eletrofiados de PLLA-colágeno. O presente trabalho propõe-se a caracterizar as ECMs digeridas e a avaliar o potencial dos suportes eletrofiados de PLLA-ECM para a regeneração óssea. A mistura proteinácea obtida a partir da digestão das ECMs, mostrou que a sua composição é dependentes do tipo de ECM e da digestão enzimática, resultado este confirmado através da quantificação de proteínas, quantificação de glicosaminoglicanas, TGA, SDS-page e TPEF-SHG. A partir destes, foi observada que a colagenase é a enzima que promove a maior degradação das ECMs, enquanto que a tripsina promove uma degradação em menor escala. As matrizes digeridas foram incorporadas no material nanoestruturado, estes foram caraterizados por SEM, TGA, DSC e TPEF-SHG. Observou-se que a malha eletrofiada a partir da co-solução de PLLA-ECM exibiu a formação de fibras de diâmetro uniforme, enquanto que os suportes imobilizados apresentaram a formação de aglomerados sólidos ao redor das fibras, originando uma malha menos porosa. As análises de TGA e DSC confirmaram a incoporação das ECMs nas malhas eletrofiadas, e através da técnica de TPEF-SHG observou-se a distribuição das proteinas no polímero. O potencial dos materiais para a regeneração óssea foi avaliado através da cultura de células tronco mesenquimais de medula óssea sobre os suportes eletrofiados durante 21 dias, e em seguida, medidas de ALP, quantificação de coloração com vermelho de alizarina, imunofluorescência com anticorpo col1a2, e expressão de gênica foram analisadas para a avaliação de como os materiais eletrofiados de PLLA-ECM induzem a osteodiferenciação. Comparando-se materiais produzidos por co-solução e os materiais imobilizados foi possível observar que a resposta osteogênica é maior nos materiais híbridos devido a liberação de fatores solúveis dos suportes eletrofiados. No entanto, comparando-se o efeito da digestão enzimática na capacidade de mineralização dos suportes , é possível observar que o efeito da digestão enzimática é dependente do tipo de ECM. Em geral, foi possível observar que os suportes eletrofiados de PLLA-ECM exibem potencial para uso em engenharia de tecidos, em específico, regeneração óssea, uma vez que apresentaram-se regulados o conjunto de genes bglap, RunX2, Osx, sparc e col1a2.

We aimed to provide optimal and controlled growth mechanisms that determine the fate of cells by designing a novel polymeric, bio-functionalised 3-dimensional artificial Nanofibrous Extracellular Matrix (NF-ECM) that recapitulates the natural microenvironment of cells. We initially reviewed current cell-expansion methods, and clinically feasible protocols for tissue engineering applications. They are often based on conventional 2-dimensional tissue culture plates that usually require xenogenic coating substrates or feeder-cells to maintain their characteristics. Propagating cells in a 3-dimensional architecture, rather than in the conventional 2-dimensional flat monolayers, can be advantageous for many regenerative applications and biological or disease modelling studies. Furthermore, such 3-dimensional culture systems might be crucial in developing a bioreactor-based design that provides finely controlled environmental conditions that would reliably propagate 3-dimensional multilayered cell organisation or spheroids on a large scale. Furthermore, the ability to expand cells in the absence of animal-derived products is a necessary condition for clinical application.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Dentistry and Oral Health
Griffith Health
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The tissue microenvironment is a complex living system containing heterogeneous mechanical and biophysical cues. Cellular components are surrounded by extracellular matrix and interstitial fluid, while transport of nutrients and biochemical factors is achieved via the vasculature. Each constituent of the tissue microenvironment can play a significant role in its ability to function normally. Many diseases including cancer have been linked with dysfunction in the tissue microenvironment; therefore an improved understanding of interaction between components of this complex system is needed. In vitro platforms mimicking the tissue microenvironment appear to provide the most promising avenue for studies of cell-cell and cell-matrix interactions as well as elucidation of the mechanisms leading to disease phenomena such as tumor metastasis. However, successful recapitulation of all three primary components of the tissue microenvironment in three dimensions has remained challenging. In particular, matching mechanical cues and biochemical transport to in vivo conditions is difficult because of lack of quantitative characterization of the physical properties and parameters of such platforms. In this work, extensive characterization of collagen I hydrogels, popular for use as extracellular matrix mimics, was performed in order to enable tuning to specific in vivo conditions. Additionally, perfusion of blood in a 3D tissue microenvironment platform fabricated using collagen hydrogels was characterized to enable future advances in in vitro modeling of the in vivo microenvironment. Finally, the tissue microenvironment platform is modified to enable biochemical gradients within the hydrogel and used to examine directed migration (chemotaxis) of human breast cancer cells in response to gradients in growth factor combined with varied stiffness and pore diameter of the extracellular matrix.
Ph. D.

The tissue microenvironment is a complex living system containing heterogeneous mechanical and biophysical cues. Cellular components are surrounded by extracellular matrix and interstitial fluid, while transport of nutrients and biochemical factors is achieved via the vasculature. Each constituent of the tissue microenvironment can play a significant role in its ability to function normally. Many diseases including cancer have been linked with dysfunction in the tissue microenvironment; therefore an improved understanding of interaction between components of this complex system is needed. In vitro platforms mimicking the tissue microenvironment appear to provide the most promising avenue for studies of cell-cell and cell-matrix interactions as well as elucidation of the mechanisms leading to disease phenomena such as tumor metastasis. However, successful recapitulation of all three primary components of the tissue microenvironment in three dimensions has remained challenging. In particular, matching mechanical cues and biochemical transport to in vivo conditions is difficult because of lack of quantitative characterization of the physical properties and parameters of such platforms. In this work, extensive characterization of collagen I hydrogels, popular for use as extracellular matrix mimics, was performed in order to enable tuning to specific in vivo conditions. Additionally, perfusion of blood in a 3D tissue microenvironment platform fabricated using collagen hydrogels was characterized to enable future advances in in vitro modeling of the in vivo microenvironment. Finally, the tissue microenvironment platform is modified to enable biochemical gradients within the hydrogel and used to examine directed migration (chemotaxis) of human breast cancer cells in response to gradients in growth factor combined with varied stiffness and pore diameter of the extracellular matrix.
Ph. D.

Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

In this study a microbial polyester, poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), and its blends were wet or electrospun into fibrous scaffolds for tissue engineering. Wet spun fiber diameters were in the low micrometer range (10-50 &
#956
m). The polymer concentration and the stirring rate affected the properties the most. The optimum concentration was determined as 15% (w/v). Electrospun fiber diameters, however, were thinner. Solution viscosity, potential, distance between the syringe tip and the collector, and polymer type affected the morphology and the thickness of beads formed on the fibers. Concentration was highly influential
as it increased from 5% to 15% (w/v) fiber diameter increased from 284 ±
133 nm to 2200 ±
716 nm. Increase in potential (from 20 to 50 kV) did not lead to the expected decrease in fiber diameter. The blends of PHBV8 with lactide-based v polymers (PLLA, P(L,DL-LA) and PLGA (50:50)) led to fibers with less beads and more uniform thickness. In vitro studies using human osteosarcoma cells (SaOs-2) revealed that wet spun fibers were unsuitable because the cells did not spread on them while all the electrospun scaffolds promoted cell growth and penetration. The surface porosities for PHBV10, PHBV15, PHBV-PLLA, PHBV-PLGA (50:50) and PHBV-P(L,DL)LA were 38.0±
3.8, 40.1±
8.5, 53.8±
4.2, 50.0±
4.2 and 30.8±
2.7%, respectively. Surface modification with oxygen plasma treatment slightly improved the cell proliferation rates. Consequently, all scaffolds prepared by electrospinning revealed a significant potential for use in bone tissue engineering applications
PHBV-PLLA blend appeared to yield the best results.

The chondroitin sulphate proteoglycan (CSPG) versican forms complexes with hyaluronan (HA), which are essential in a range of functions including cellular proliferation and migration. Four isoforms of versican result from alternative splicing. Furthermore, biological roles have been identified for the proteolytic cleavage product of versican which contains the N-terminal G1 hyaluronan binding domain. All of these versican forms have different tightly regulated tissue expression profiles. Consequently, impaired regulation is associated with a number of disease pathologies. For example the largest variants (V0/V1) have been shown to be negative indicators of disease outcome in a number of malignant cancers and are a marker of disease progression in atherosclerosis. Interestingly, the smaller versican isoform V3 which lacks CS chains has been demonstrated to have the potential to reverse disease associated phenotypes. The motivation for carrying out the work in this thesis was to try and gain a better understanding of how versican functions on a molecular scale. In this regard, the first aim was to investigate the structure of the hyaluronan binding region of versican using a construct called VG1. The structure of VG1 was analysed in the presence and absence of hyaluronan oligomers. This revealed an insight into the multi-modular structure of the versican hyaluronan binding region and demonstrated that on binding to HA, VG1 under goes a conformational change. Furthermore, the interaction between VG1 and longer lengths of hyaluronan (pHA) was investigated. This demonstrated that when VG1 binds to pHA it is does so with positive cooperativity, packing very close to neighbouring VG1 molecules along a chain of HA. One consequence of this interaction was to reorganise pHA into a helical conformation, an organisation that was confirmed by a number of solution phase techniques. The effect of this reorganisation of pHA by VG1 on HA/CD44 interactions was also assessed. Previously the interaction between CD44 (a cell surface hyaluronan receptor) and long chains of HA (>30 kDa) was shown to be irreversible; however we demonstrate that VG1 can reverse this. Furthermore, a TSG-6 enhanced CD44/interaction was also completely reversed by the addition of VG1. This provides an indication that a functional hierarchy of hyaluronan binding proteins may exist which could have important implications in understanding the function of hyaluronan complexes. Currently, we do not know whether intact versican molecules could interact with HA in the same way as VG1. However, preliminary data suggests that the CS-containing variants (i.e. V0, V1 and V2) would not, whereas V3 and versican fragments could. This work provides an exciting mechanistic insight into the function of versican variants and their breakdown products.

Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

Articular cartilage is a highly organized tissue with cellular and matrix properties that vary with depth zones. Regenerating this zonal organization has proven difficult in tissue-engineered cartilage to treat damaged cartilage. In this thesis, we evaluated the effects of culture environments that mimic aspects of the native cartilage environment on chondrocyte subpopulations. We found that decellularized cartilage matrix can improve zonal tissue-engineered cartilage. Also, chondrocytes respond to signals from bone cells and compressive stimulation in a zone-dependent manner. These results highlight the importance of a zone-specific environment to improve tissue-engineered cartilage in vitro.

Pancreatic cancer continues to have one of the poorest prognoses amongst all cancers, with a 95% mortality rate. Standard of care chemotherapy has failed to provide significant clinical benefits, which has led to the development of targeted agents against validated signalling pathways. However, to date the approach of targeted agents alone, or in combination with traditional chemotherapeutics, has failed to significantly improve the prognosis for pancreatic cancer patients. The current standard of care chemotherapy for advanced pancreatic cancer provides only a modest increase in survival of several months. Models that improve the predictive potential of drug discovery programs and gain greater insights into the complexity of tumour biology are therefore urgently required. To better understand the mechanisms influencing the anti-cancer activities of current and novel therapies, we have developed a 3D in vitro micro-tumour cell culture model. Current in vitro models utilising cell monolayer cultures are unable to recapitulate the biological and physiological complexities of the in vivo pancreatic tumour microenvironment and may be poor predictors of drug efficacy. Pancreatic adenocarcinomas are characterised as having a highly dense and poorly vascularised stroma that is made up of extracellular matrix (ECM) components and host cells. This complex tumour microenvironment has been implicated in the chemoresistance profiles observed in pancreatic tumours.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Eskitis Institute for Cell and Molecular Therapies
Science, Environment, Engineering and Technology
Full Text

Orientador: Willian Fernando Zambuzzi
Resumo: Modelos in vitro têm facilitado a análise da fisiologia celular sob condições experimentais diversas; trata-se de modelo alternativo ao uso de animais de experimentação, que vem sendo difundido e aceito amplamente em pesquisa científica. Além disso, estes modelos têm levado à avanços significativos na compreensão das interações mútuas e adaptativas entre células e substratos. Neste trabalho, nosso objetivo foi analisar eventos moleculares responsáveis pela adaptação de preosteoblastos em substrato orgânico composto por componentes da Matriz Extracelular (MEC), sob condição de diferenciação celular. Metodologicamente, pré-osteoblastos (MC3T3-E1 50x103 células/ml) foram semeados sobre uma fina camada de Matrigel® gelificada e mantidos por 10 dias (37oC, 5% de CO2 em ambiente úmido) sob condição de diferenciação (meio de cultivo contendo 50 µg de ácido ascórbico e 10 mM de ß- glicerofosfato), com renovação do meio de cultivo a cada 3 dias. Alterações morfológicas foram monitoradas em microscópio invertido e mecanismos moleculares acompanhados pela análise global da atividade de quinases, através de arranjo de peptídeos (Pepchip). Curiosamente, nossos resultados mostraram mudanças morfológicas significantes durante adaptação celular as quais foram acompanhadas pela atuação de vias de sinalização celular distintas, responsáveis pela sobrevivência (Eixo PI3K-Akt) e proliferação (Eixo Retinoblastoma-ciclinas) celulares, além de proteínas envolvidas com metabolismo energético e comun... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In vitro models have been proposed to analyze cellular physiology under various experimental conditions. It is an alternative model instead using experimental animals that have been widespread and widely accepted in scientific research. Moreover, it has led to significant advances in the understanding of mutual and adaptive interactions between cells and substrates. In this work, our aim was to analyze adaptive events of osteoblasts cultured on an organic substrate composed by components of the extracellular matrix (ECM) in the first 10 days of cultivation on differentiation condition. Methodologically, pre-osteoblasts (MC3T3-E1 pre-osteoblasts, 50x103 cells / ml) were seeded on a thin gelified Matrigel® layer and maintained for 10 days under standard cell culture conditions (37 ° C, 5% CO2 in a humid environment) under differentiation conditions (culture media containing 50 ug of ascorbic acid and 10 mM beta-glycerophosphate). The culture medium was changed every 3 days. Morphological changes were monitored using an inverted microscope and molecular mechanisms followed by comprehensive analysis of kinase activity by peptides arrangement (Pepchip). Interestingly, our results showed significant morphological changes during cell adaptation which were accompanied by distinct signaling pathways involving proteins responsible for survival (PI3K-Akt axis) and cell proliferation (Retinoblastoma-cyclins axis), in addition of proteins involved in energy metabolism and cellular communi... (Complete abstract click electronic access below)
Mestre

En aquesta Tesi, un nou biomaterial de disseny composat per seqüències peptídiques repetitives i amfifíliques, que per autoensamblatge forma xarxes de nanofibres (i hidrogels), AcN-RADARADARADARADA-CONH2, s´ha utilitzat com a anàleg de la matriu extracel·lular per al manteniment, proliferació i diferenciació cel·lular. Aquest pèptid s'ha funcionalititzat amb motius biològicament actius procedents de proteïnes de la matriu extracel·lular incloent laminina-1 i colàgen IV. El scaffold peptídic autoensamblant RAD16-I i els seus derivats biològicament actius s´han caracteritzat i provat utilitzant diferents sistemes cel·lulars com pot ser les cèl·lules d'aorta humanes (HAEC), hepatocits madurs i la línea progenitora de fetge (Lig-8). La proteòlisi d'aquest pèptid s'ha avaluat utilitzant tripsina com a enzim proteolític, i els fragments resultants s'han analitzat per MALDI-TOF i AFM. Així mateix, la segona generació de biomaterials basats en el RAD16-I s'ha provat tant amb HAEC com amb hepatocits madurs. Amb aquests sistemes hem demostrat que el desenvolupament d'una matriu biomiètica reforça, a la vegada que manté, les funcions específiques de cada teixit. En particular, els resultats obtinguts en diferenciació, proliferació i manteniment de la funció cel·lular utilitzant pèptids sintètics autoensamblants són comparables amb els resultats que s'obtenen utilitzant matrius biològiques (Colàgen I i Matrigel). Això indica que els nostres anàlegs de la matriu extracel·lular poden substituir als materials naturals, i suggereix l'ús d'aquests materials intel·ligents amb capacitat instructiva en aplicacions terapèutiques. Així mateix s'ha provat que l'ús d'aquests pèptids auto-ensamblants és eficient en la construcció d'un nínxol de cèl·lules mare. Hem sigut capaços de controlar la cinètica cel·lular (de simètrica a assimètrica) induint diferenciació funcional, a la vegada que es mantenia una petita proporció de cèl·lules no diferenciades. Aquests resultats indiquen clarament que hem sigue capaços d'obtenir un nínxol on cèl·lules primitives (Lig-8) es diferencien adquirint funcions d'hepatocits madurs. Hem desenvolupat una plataforma de biomaterials que es podrien utilitzar per la funcionalització amb innumerables biomolècules amb capacitat d'induir processos biològics com la diferenciació, proliferació i funció metabòlica. Aquests biomaterials, preveiem que tindran un gran impacte a l'àrea terapèutica i biología regenerativa.
En esta Tesis, un nuevo biomaterial de diseño compuesto por secuencias peptídicas repetitivas y amfifílicas que por autoensamblaje forma redes de nanofibras (e hidrogeles), AcN-RADARADARADARADA-CONH2 (RAD16-I), se ha utilizado como análogo de la matriz extracelular para el mantenimiento, proliferación y diferenciación celular. Este péptido se ha funcionalizado con motivos biológicamente activos procedentes de proteínas de la matriz extracelular incluyendo laminina-1 y colágeno IV. El scaffold peptídico autoensamblante RAD16-I y sus derivados biológicamente activos se han caracterizado y probado utilizando diferentes sistemas celulares como puede ser células endoteliales de aorta humanas (HAEC), hepatocitos maduros y la línea progenitora de hígado Lig-8. La proteólisis de este péptido se ha evaluado utilizando tripsina como enzima proteolítico, y los fragmentos resultantes se han analizado por MALDI-TOF y AFM. Asimismo, la segunda generación de biomateriales basados en el RAD16-I se ha probado tanto con HAEC como hepatocitos maduros. Con estos sistemas hemos demostrado que el desarrollo de una matriz biomimética refuerza a la vez que mantiene las funciones específicas de cada tejido. En particular, los resultados obtenidos en diferenciación, proliferación y mantenimiento de la función celular utilizando los péptidos sintéticos auto-ensamblantes son comparables con los resultados que se obtienen usando matrices biológicas (Colágeno I y Matrigel). Esto indica que nuestros análogos de la matriz extracelular pueden reemplazar a los materiales naturales, y sugiere el uso de estos materiales inteligentes con capacidad instructiva en aplicaciones terapéuticas. Asimismo, se ha probado que el uso de estos péptidos auto-ensamblantes es eficiente en la construcción de un nicho de células madre. Hemos sido capaces de controlar la cinética celular (de simétrica a asimétrica) induciendo diferenciación funcional, a la vez que se mantenía una pequeña proporción de células no diferenciadas. Estos resultados indican claramente que hemos sido capaces de obtener un nicho donde células primitivas (Lig-8) se diferencian adquiriendo funciones de hepatocitos maduros. Hemos desarrollado una plataforma de biomateriales que se podrían utilizar para la funcionalización con innumerables biomoléculas con capacidad de inducir procesos biológicos como la diferenciación, proliferación y función metabólica. Estos biomateriales preveemos que tendrán un gran impacto en el área terapéutica y biología regenerativa.
In this Thesis, a new designed biomaterial made out of short repetitive amphiphilic peptide sequence AcN-RADARADARADARADA-CONH2 (RAD16-I) that self-assembles forming nanofiber networks (hydrogel scaffold) has been used as synthetic extracellular matrix analog for cell maintenance, proliferation and differentiation. This peptide has been functionalized with biological active motifs from extracellular matrix proteins including laminin-1 and collagen IV. The prototypic self-assembling peptide scaffold RAD16-I and its biologically active derivatives have been characterized and tested using several cellular systems such as human aortic endothelial cells (HAEC), mature hepatocytes and a putative liver progenitor cell line, Lig-8. The proteolysis of the peptide RAD16-I has been evaluated using trypsin as a proteolytic enzyme and the resulting fragments have been analyzed by MALDI-TOF and AFM. Moreover the second generation of RAD16-I-based biomaterials have been tested using HAEC and mature hepatocytes. With these systems we have shown that the development of a biomimetic matrix enhances as well as maintain tissue-specific functions. In particular, the results obtained in cell differentiation, proliferation and maintenance of cell function using the synthetic self-assembling peptide matrices, are comparable with the results obtained using natural biological matrices counterparts (Collagen-I and Matrigel). This indicates that our extracellular matrix analogs can replace the use of naturally-derived materials and suggests the use of these smart biomaterials with instructive capacity for cells in therapeutics. Moreover, the use of the self-assembling peptide RAD16-I in the recreation of a stem-cell niche proved to be highly efficient. We were able to control stem-cell kinetics (from symmetric to assymetric) inducing functional differentiation while maintaining a small proportion of undifferentiated cells. This striking results clearly indicate that we were able to obtain a stem-cell niche where primitive cells (Lig-8) undergo differentiation acquiring mature hepatic functions. We have developed a biomaterial platform that can be used for functionalization with innumerable biomolecules, with capacity to induce biological processes like differentiation, control of proliferation, metabolic function, etc. These biomaterials will have a strong impact in therapeutics and regenerative biology.

Breast cancer is a leading contributor to the burden of disease in Australia. Fortunately, the recent introduction of diverse therapeutic strategies have improved the survival outcome for many women. Despite this, the clinical management of breast cancer remains problematic as not all approaches are sufficiently sophisticated to take into account the heterogeneity of this disease and are unable to predict disease progression, in particular, metastasis. As such, women with good prognostic outcomes are exposed to the side effects of therapies without added benefit. Furthermore, women with aggressive disease for whom these advanced treatments would deliver benefit cannot be distinguished and opportunities for more intensive or novel treatment are lost. This study is designed to identify novel factors associated with disease progression, and the potential to inform disease prognosis. Frequently overlooked, yet common mediators of disease are the interactions that take place between the insulin-like growth factor (IGF) system and the extracellular matrix (ECM). Our laboratory has previously demonstrated that multiprotein insulin-like growth factor-I (IGF-I): insulin-like growth factor binding protein (IGFBP): vitronectin (VN) complexes stimulate migration of breast cancer cells in vitro, via the cooperative involvement of the insulin-like growth factor type I receptor (IGF-IR) and VN-binding integrins. However, the effects of IGF and ECM protein interactions on the dissemination and progression of breast cancer in vivo are unknown. It was hypothesised that interactions between proteins required for IGF induced signalling events and those within the ECM contribute to breast cancer metastasis and are prognostic and predictive indicators of patient outcome. To address this hypothesis, semiquantitative immunohistochemistry (IHC) analyses were performed to compare the extracellular and subcellular distribution of IGF and ECM induced signalling proteins between matched normal, primary cancer, and metastatic cancer among archival formalin-fixed paraffin-embedded (FFPE) breast tissue samples collected from women attending the Princess Alexandra Hospital, Brisbane. Multivariate Cox proportional hazards (PH) regression survival models in conjunction with a modified „purposeful selection of covariates. method were applied to determine the prognostic potential of these proteins. This study provides the first in-depth, compartmentalised analysis of the distribution of IGF and ECM induced signalling proteins. As protein function and protein localisation are closely correlated, these findings provide novel insights into IGF signalling and ECM protein function during breast cancer development and progression. Distinct IGF signalling and ECM protein immunoreactivity was observed in the stroma and/or in subcellular locations in normal breast, primary cancer and metastatic cancer tissues. Analysis of the presence and location of stratifin (SFN) suggested a causal relationship in ECM remodelling events during breast cancer development and progression. The results of this study have also suggested that fibronectin (FN) and ¥â1 integrin are important for the formation of invadopodia and epithelial-to-mesenchymal transition (EMT) events. Our data also highlighted the importance of the temporal and spatial distribution of IGF induced signalling proteins in breast cancer metastasis; in particular, SFN, enhancer-of-split and hairy-related protein 2 (SHARP-2), total-akt/protein kinase B 1 (Total-AKT1), phosphorylated-akt/protein kinase B (P-AKT), extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (ERK1/2) and phosphorylated-extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (P-ERK1/2). Multivariate survival models were created from the immunohistochemical data. These models were found to fit well with these data with very high statistical confidence. Numerous prognostic confounding effects and effect modifications were identified among elements of the ECM and IGF signalling cascade and corroborate the survival models. This finding provides further evidence for the prognostic potential of IGF and ECM induced signalling proteins. In addition, the adjusted measures of associations obtained in this study have strengthened the validity and utility of the resulting models. The findings from this study provide insights into the biological interactions that occur during the development of breast tissue and contribute to disease progression. Importantly, these multivariate survival models could provide important prognostic and predictive indicators that assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy. The outcomes of this study further inform the development of new therapeutics to aid patient recovery. The findings from this study have widespread clinical application in the diagnosis of disease and prognosis of disease progression, and inform the most appropriate clinical management of individuals with breast cancer.

The growing incidences of coronary artery bypass graft surgeries have triggered a need to engineer a viable small diameter blood vessel substitute. An ideal tissue engineered vascular graft should mimic the microenvironment of a native blood vessel, while providing the adequate compliance post-implantation. Current vascular graft technologies lack the ability to promote vascular ECM deposition, leading to a compliance mismatch and ultimately, graft failure. Hence, in order to engineer suitable vascular grafts, this thesis describes the synthesis and characterization of novel elastin mimetic peptides, EM-19 and EM-23, capable of promoting vascular ECM deposition within a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel. By combining the material properties of a synthetic and bio-inspired polymer, a suitable microenvironment for cell growth and ECM deposition can be engineered, leading to improved compliance. As such, characterization of EM-19 and EM-23 was conducted in human vascular smooth muscle cell (SMC) cultures, and the peptides self-assembled with a growing elastic matrix. After grafting the peptides onto the surface of PEG-DA hydrogels, EM-23 increased SMC adhesion by 6000% over PEG-RGDS hydrogels, which have been the gold standard of cell adhesive PEG scaffolds. Moreover, EM-23 grafted surfaces were able to promote elastin deposition that was comparable to tissue cultured polystyrene (TCPS) surface even though TCPS had roughly 4.5 times more SMCs adhered. Once translated to a 3D model, EM-23 also stimulated increased elastin deposition and improved the mechanical strength of the scaffold over time. Moreover, degradation studies suggested that EM-23 may serve as a template that not only promotes ECM deposition, but also allows ECM remodeling over time. The characterization studies in this thesis suggest that this peptide is an extremely promising candidate for improving vascular ECM deposition within a synthetic substrate, and that it may be beneficial to incorporate EM-23 within polymeric scaffolds to engineer compliant vascular grafts.

Glycosaminoglycans (GAGs) are the most prevalent hetero-polysaccharides in the body. They are usually attached to a protein core, forming proteoglycans (PGs). PGs play crucial roles in a wide range of biological processes including cell growth, migration and differentiation. Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and the extracellular matrix (ECM), which surrounds cells and is formed by a network of fibrous proteins, glycoproteins and proteoglycans. In mammalian cells heparan sulfate (HS) is cleaved by a single β-glucuronidase: heparanase. Cleavage of HS-chains changes the integrity of the ECM and releases a variety of bioactive molecules such as growth factors, cytokines and enzymes. Heparanase is implicated in cancer and inflammation and inhibition of heparanase has been shown to reduce tumour metastasis and angiogenesis. Heparanase is therefore regarded as a promising target for anti-cancer and anti-inflammatory drug design. As the three dimensional structure of human heparanase is still not available, the progress in specific inhibitor development has been impeded.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
Full Text

Synthetic biomaterials are constantly being developed and play central roles in contemporary strategies in regenerative medicine and tissue engineering as artificial extracellular microenvironments. Such scaffolds provide 2D- and 3D-support for interaction with cells and thus convey spatial and temporal control over their function and multicellular processes, such as differentiation and morphogenesis. A model fibrillar system with tunable viscoelastic properties, comprised of 2 native ECM components like collagen type I and the GAG heparin, is presented here. Although the individual components comply with the adhesive, mechanical and bioinductive requirements for artificial reconstituted ECMs, their interaction and structural characterization remains an intriguing conundrum. The aim of the work was to analyze and structurally characterize a xenogeneic in vitro cell culture scaffold reconstituted from two native ECM components, collagen type I and the highly negatively charged glycosaminoglycan heparin. Utilizing a broad spectrum of structural analysis it could be shown that pepsin-solubilized collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape, with a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of GAG concentrations. The experiments imply a pronounced impact of the nucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with atelocollagen. Heparin is assumed to stabilize the collagen-GAG complexes and to enhance their parallel accretion during cofibrillogenesis, furthermore corroborated by the heparin quantitation data showing the GAG to be intercalated as a linker molecule with a specific binding site inside the cofibrils. In addition, the exerted morphogenic effect of the GAG, appears to be influenced by factors as degree of sulfation, charge, and concentration. Further detailed structural analysis of the PSC-heparin gels using TEM and SFM showed a hierarchy involving 3 different structural levels and banding patterns in the system: asymmetric segment longspacing (SLS) fibrils and symmetric segments with an average periodicity (AP) of 250 - 260 nm, symmetric fibrous longspacing (FLS IV) nanofibrils with AP of 165 nm, and cofibrils exhibiting an asymmetric D-periodicity of 67 nm with a striking resemblance to the native collagen type I banding pattern. The intercalation of the high negatively charged heparin in the cofibrils was suggested as the main trigger for the hierarchical formation of the polymorphic structures. We also proposed a model explaining the unexpected presence of a symmetric and asymmetric form in the system and the principles governing the symmetric or asymmetric fate of the molecules. The last section of the experiments showed that the presence of telopeptides and heparin both had significant effects on the structural and mechanical characteristics of in vitro reconstituted fibrillar collagen type I. The implemented structural analysis showed that the presence of telopeptides in acid soluble collagen (ASC) impeded the reconstitution of D-periodic collagen fibrils in the presence of heparin, leaving behind only a symmetric polymorphic form with a repeating unit of 165 nm (FLS IV). Further x-ray diffraction analysis of both telopeptide-free and telopeptide-intact collagen fibrils showed that the absence of the flanking non-helical termini in pepsin-solubilized collagen (PSC) resulted in a less compact packing of triple helices of atelocollagen with an increase of interhelical distance from 1.0 to 1.2 nm in dried samples. The looser packing of the triple helices was accompanied by a decrease in bending stiffness of the collagen fibrils, which demonstrated that the intercalated heparin cannot compensate for the depletion of telopeptides. Based on morphological, structural and mechanical differences between ASC and PSC-heparin fibrils reported here, we endorsed the idea that heparin acts as an intrafibrillar cross-linker which competed for binding sites at places along the atelocollagen helix that are occupied in vivo by telopeptides in the fibrillar collagen type I. The performed studies are of particular interest for understanding and gaining control over a rather versatile and already exploited xenogeneic cell culture system. The reconstituted cofibrils with their unusual morphology and GAG intercalation – a phenomenon not reported in vivo – are expected to exhibit interesting biochemical behavior as a biomaterial for ECM scaffolds. Varying the experimental conditions, extent of telopeptide removal, and heparin concentration provides powerful means to control the kinetics, structure, dimensions, as well as mechanical properties of the system which is particularly important for predicting a certain cell behavior towards the newly developed matrix. The GAG intercalation could be interesting for studies with required long-term 'release upon demand' of the GAG, as well as native binding and stabilization of growth factors, cytokines, chemokines, thus providing a secondary tool to control cell signaling and fate, and later on tissue morphogenesis
Synthetische Biomaterialien werden stetig weiterentwickelt und spielen als künstliche Mikroumgebungen eine zentrale Rolle in den modernen Strategien der regenerativen Medizin und des Tissue Engineerings. Solche sogenannten Scaffolds liefern eine 2D- und 3D-Struktur zur Interaktion mit Zellen und üben somit eine räumliche und zeitliche Kontrolle auf ihre Funktion und multizelluläre Prozesse aus, wie die Differenzierung und Morphogenese. Obwohl häufig die adhäsiven, mechanischen und bioinduzierenden Eigenschaften von Einzelkomponenten aus natürlichen Bestandteilen der extrazellulären Matrix (ECM) rekonstituierten Trägerstrukturen bekannt sind, bleiben die funktionalen und strukturellen Auswirkungen in Mehrkomponentensystemen eine faszinierende Fragestellung. Das Ziel der Arbeit war die Analyse und die strukturelle Charakterisierung einer xenogenen in vitro Zellkultur-Trägerstruktur, die aus den zwei nativen ECM Komponenten Kollagen Typ I und das stark negativ geladene Glykosaminoglykan (GAG) Heparin rekonstituiert wurde. Unter Nutzung eines breiten Spektrums von Methoden zur strukturellen Analyse konnte gezeigt werden, dass im Beisein von Heparin rekonstituierte Pepsin-gelöste Kollagen Typ I Fibrillen eine ungewöhnlich dicke und gerade Form, mit nichtlinearen Abhängigkeiten der Größenverteilung, des Breite-zu-Länge Verhältnises und der Morphologie für eine Reihe von GAG Konzentrationen, aufweisen. Die Experimente deuten auf eine besondere Wirkung der Nukleierungsphase auf die Kofibrillmorphologie hin, als Folge der starken elektrostatischen Inteaktionen Heparins mit Atelokollagen. Es wird angenommen, dass Heparin die Komplexe aus Kollagen-GAG stabilisiert, die parallele Anlagerung während der Kofibrillogenese verbessert und dass überdies, belegt durch Heparin Quantitätsdaten, als Verbindungsmolekül mit einer spezifischen Anbindungsstelle innerhalb der Kofibrillen eingelagert wird. Darüber hinaus scheint der ausgeübte morphogene Effekt des GAGs Heparins von Faktoren wie Grad der Sulfatierung, Ladung und Konzentration abzuhängen. Weitere detailierte Strukturanalysen der PSC - Heparin Gele mit TEM und SFM zeigten eine Hierarchie mit drei unterschiedlichen strukturellen Ebenen und Bandmustern im System: asymmetrisch segmentierte, weitabständige Fibrillen (SLS) und symmetrische Segmente mit einem AP von 250-260 nm, symmetrische fibrose weitabständige (FLS IV) Nanofibrillen mit einem AP von von 165 nm und Kofibrillen asymmetrischer D-Periodizität von 67 nm, die eine erstaunliche Ähnlichkeit zum natürlichen Kollagen Typ I Bandmuster haben. Die Einlagerung des sehr negativ geladenen Heparins in die Kofibrillen wurde als Hauptauslöser der hierarchischen Formation der polymorphen Strukturen betrachtet. Wir schlugen ebenso ein Model vor, welches sowohl das unerwartete Vorhandensein symmetrischer und asymmetrischer Formen im System als auch die Regeln erklärt, die das symmetrische oder asymmetrische Schicksal der Moleküle steuern. Der letzte Abschnitt der Experimente zeigte, dass die Anwesenheit der Telopeptide und Heparins eine signifikante Wirkung auf die strukturellen und mechanischen Charakteristika der in vitro rekonstituierten Kollagen Typ I Fibrillen hatte. Die durchgeführten Strukturanalysen zeigten außerdem, dass die Anwesenheit der Telopeptide in säurelöslichem Kollagen (ASC) die Rekonstitution D-periodischer Kollagenfibrillen mit Heparin verhinderte, sodass nur symmetrisch polymorphe Formen mit einer Wiederholeinheit von 165 nm möglich waren (FLS IV). Weitere Messungen der Telopeptid-freien und Telopeptid-intakten Kollagenfibrillen mit Röntgendiffraktometrie ergaben, dass die Abwesenheit der nicht-helix-strukturierten Enden in Pepsin-gelöstem Kollagen (PSC) zu einer weniger kompakten Anordnung der Tripelhelices von Atelokollagen führte. Der interhelix Abstand erhöhte sich von 1,0 zu 1,2 nm für getrocknete Proben. Das zeigt, dass die losere Anordnung der Tripelhelices einhergeht mit der Verringerung der Biege-Elastizitäts-module der Kollagenfibrillen,. Basierend auf den hier vorgestellten morphologischen, strukturellen und mechanischen Unterschieden zwischen ASC und PSC-Heparin Fibrillen wird die Idee unterstützt, dass Heparin als intrafibrillärer Vernetzer fungiert und an Bindungsstellen der Helix bindet, welche in vivo bei Kollagen Typ I Fibrillen durch Telopeptide besetzt sind. Die durchgeführten Studien sind von besonderem Interesse für das Verständnis und die Steuerung eines sehr vielseitigen und bereits verwendeten xenogenes Zellkultursystem für das Tissue Engineering. Von den rekonstituierten Kofibrillen mit ihrer ungewöhnlichen Morphologie und GAG Einlagerung - ein in vivo nicht bekanntes Phänomen - erwartet man, dass sie ein intressantes biochemisches Verhalten als Biomaterial für ECM Scaffolds zeigen. Variationen der experimentellen Bedingungen, des Ausmaßes der Telopeptidentfernung und der Heparinkonzentration liefern vielfältige Möglichkeiten um die Kinetik, Struktur, Dimension sowie die mechanischen Eigenschaften des Systems zu kontrollieren. Damit sollte es möglich sein, ein bestimmtes Zellverhalten gegenüber der neu entwickelten Matrix vorherzusagen. Die GAG-Einlagerung bietet interessante Optionen für eine langfristige Freisetzung des GAGs 'on demand', sowie die native Bindung und Stabilisierung von Wachstumsfaktoren, Cytokinen, Chemokinen, womit zusätzlich Zellsignalisierung und -schicksal und später Gewebemorphogenese kontrolliert werden kann

O Lopap é um ativador de protrombina da lagarta L. obliqua, pertence à família das lipocalinas e apresenta atividade antiapoptótica. O Lopap foi obtido na forma recombinante (rLopap), na levedura P. pastoris, por metodologia escalonável, e sua atividade foi avaliada in vitro e in vivo. O tratamento com rLopap reduziu o tempo de sangramento em animais anticoagulados com enoxaparina. Por outro lado, um peptídeo derivado do Lopap, designado antiapoptotic peptide (AP), foi capaz de induzir a síntese de colágeno em cultura de fibroblastos e na derme de animais. A região correspondente a AP apresentou propriedades físicas e estruturais semelhantes a seqüências relacionadas em outras lipocalinas com atividade antiapoptótica. Estes resultados abrem perspectivas para aplicações do Lopap, como uma molécula procoagulante, e de AP, através de sua ação na modulação celular, como um componente cosmético, no reparo e remodelamento tecidual e em disfunções que envolvem morte celular e perda de colágeno.
Lopap is a prothrombin activator from the L. obliqua caterpillar, belongs to the lipocalin family, and displays antiapoptotic activity. Lopap was obtained in the recombinant form (rLopap) in the P. pastoris yeast, by a scaled up methodology, and its activity was evaluated in vitro and in vivo. Treatment with rLopap reduced the bleeding time in animals anticoagulated with enoxaparin. On the other hand, a Lopap-derived peptide, designated antiapoptotic peptide (AP), was able to induce collagen synthesis in fibroblast culture and in the animal dermis. The region corresponding to AP had similar physical and structural properties when compared with other antiapoptotic lipocalins. These results open perspectives for the use of Lopap, as a procoagulante molecule, and the use of AP, based on its cell modulation effects, as a cosmetic component, aiding tissue repair and in dysfunctions involving cell death and loss of collagen.

Synthetic biomaterials are constantly being developed and play central roles in contemporary strategies in regenerative medicine and tissue engineering as artificial extracellular microenvironments. Such scaffolds provide 2D- and 3D-support for interaction with cells and thus convey spatial and temporal control over their function and multicellular processes, such as differentiation and morphogenesis. A model fibrillar system with tunable viscoelastic properties, comprised of 2 native ECM components like collagen type I and the GAG heparin, is presented here. Although the individual components comply with the adhesive, mechanical and bioinductive requirements for artificial reconstituted ECMs, their interaction and structural characterization remains an intriguing conundrum. The aim of the work was to analyze and structurally characterize a xenogeneic in vitro cell culture scaffold reconstituted from two native ECM components, collagen type I and the highly negatively charged glycosaminoglycan heparin. Utilizing a broad spectrum of structural analysis it could be shown that pepsin-solubilized collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape, with a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of GAG concentrations. The experiments imply a pronounced impact of the nucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with atelocollagen. Heparin is assumed to stabilize the collagen-GAG complexes and to enhance their parallel accretion during cofibrillogenesis, furthermore corroborated by the heparin quantitation data showing the GAG to be intercalated as a linker molecule with a specific binding site inside the cofibrils. In addition, the exerted morphogenic effect of the GAG, appears to be influenced by factors as degree of sulfation, charge, and concentration. Further detailed structural analysis of the PSC-heparin gels using TEM and SFM showed a hierarchy involving 3 different structural levels and banding patterns in the system: asymmetric segment longspacing (SLS) fibrils and symmetric segments with an average periodicity (AP) of 250 - 260 nm, symmetric fibrous longspacing (FLS IV) nanofibrils with AP of 165 nm, and cofibrils exhibiting an asymmetric D-periodicity of 67 nm with a striking resemblance to the native collagen type I banding pattern. The intercalation of the high negatively charged heparin in the cofibrils was suggested as the main trigger for the hierarchical formation of the polymorphic structures. We also proposed a model explaining the unexpected presence of a symmetric and asymmetric form in the system and the principles governing the symmetric or asymmetric fate of the molecules. The last section of the experiments showed that the presence of telopeptides and heparin both had significant effects on the structural and mechanical characteristics of in vitro reconstituted fibrillar collagen type I. The implemented structural analysis showed that the presence of telopeptides in acid soluble collagen (ASC) impeded the reconstitution of D-periodic collagen fibrils in the presence of heparin, leaving behind only a symmetric polymorphic form with a repeating unit of 165 nm (FLS IV). Further x-ray diffraction analysis of both telopeptide-free and telopeptide-intact collagen fibrils showed that the absence of the flanking non-helical termini in pepsin-solubilized collagen (PSC) resulted in a less compact packing of triple helices of atelocollagen with an increase of interhelical distance from 1.0 to 1.2 nm in dried samples. The looser packing of the triple helices was accompanied by a decrease in bending stiffness of the collagen fibrils, which demonstrated that the intercalated heparin cannot compensate for the depletion of telopeptides. Based on morphological, structural and mechanical differences between ASC and PSC-heparin fibrils reported here, we endorsed the idea that heparin acts as an intrafibrillar cross-linker which competed for binding sites at places along the atelocollagen helix that are occupied in vivo by telopeptides in the fibrillar collagen type I. The performed studies are of particular interest for understanding and gaining control over a rather versatile and already exploited xenogeneic cell culture system. The reconstituted cofibrils with their unusual morphology and GAG intercalation – a phenomenon not reported in vivo – are expected to exhibit interesting biochemical behavior as a biomaterial for ECM scaffolds. Varying the experimental conditions, extent of telopeptide removal, and heparin concentration provides powerful means to control the kinetics, structure, dimensions, as well as mechanical properties of the system which is particularly important for predicting a certain cell behavior towards the newly developed matrix. The GAG intercalation could be interesting for studies with required long-term 'release upon demand' of the GAG, as well as native binding and stabilization of growth factors, cytokines, chemokines, thus providing a secondary tool to control cell signaling and fate, and later on tissue morphogenesis.
Synthetische Biomaterialien werden stetig weiterentwickelt und spielen als künstliche Mikroumgebungen eine zentrale Rolle in den modernen Strategien der regenerativen Medizin und des Tissue Engineerings. Solche sogenannten Scaffolds liefern eine 2D- und 3D-Struktur zur Interaktion mit Zellen und üben somit eine räumliche und zeitliche Kontrolle auf ihre Funktion und multizelluläre Prozesse aus, wie die Differenzierung und Morphogenese. Obwohl häufig die adhäsiven, mechanischen und bioinduzierenden Eigenschaften von Einzelkomponenten aus natürlichen Bestandteilen der extrazellulären Matrix (ECM) rekonstituierten Trägerstrukturen bekannt sind, bleiben die funktionalen und strukturellen Auswirkungen in Mehrkomponentensystemen eine faszinierende Fragestellung. Das Ziel der Arbeit war die Analyse und die strukturelle Charakterisierung einer xenogenen in vitro Zellkultur-Trägerstruktur, die aus den zwei nativen ECM Komponenten Kollagen Typ I und das stark negativ geladene Glykosaminoglykan (GAG) Heparin rekonstituiert wurde. Unter Nutzung eines breiten Spektrums von Methoden zur strukturellen Analyse konnte gezeigt werden, dass im Beisein von Heparin rekonstituierte Pepsin-gelöste Kollagen Typ I Fibrillen eine ungewöhnlich dicke und gerade Form, mit nichtlinearen Abhängigkeiten der Größenverteilung, des Breite-zu-Länge Verhältnises und der Morphologie für eine Reihe von GAG Konzentrationen, aufweisen. Die Experimente deuten auf eine besondere Wirkung der Nukleierungsphase auf die Kofibrillmorphologie hin, als Folge der starken elektrostatischen Inteaktionen Heparins mit Atelokollagen. Es wird angenommen, dass Heparin die Komplexe aus Kollagen-GAG stabilisiert, die parallele Anlagerung während der Kofibrillogenese verbessert und dass überdies, belegt durch Heparin Quantitätsdaten, als Verbindungsmolekül mit einer spezifischen Anbindungsstelle innerhalb der Kofibrillen eingelagert wird. Darüber hinaus scheint der ausgeübte morphogene Effekt des GAGs Heparins von Faktoren wie Grad der Sulfatierung, Ladung und Konzentration abzuhängen. Weitere detailierte Strukturanalysen der PSC - Heparin Gele mit TEM und SFM zeigten eine Hierarchie mit drei unterschiedlichen strukturellen Ebenen und Bandmustern im System: asymmetrisch segmentierte, weitabständige Fibrillen (SLS) und symmetrische Segmente mit einem AP von 250-260 nm, symmetrische fibrose weitabständige (FLS IV) Nanofibrillen mit einem AP von von 165 nm und Kofibrillen asymmetrischer D-Periodizität von 67 nm, die eine erstaunliche Ähnlichkeit zum natürlichen Kollagen Typ I Bandmuster haben. Die Einlagerung des sehr negativ geladenen Heparins in die Kofibrillen wurde als Hauptauslöser der hierarchischen Formation der polymorphen Strukturen betrachtet. Wir schlugen ebenso ein Model vor, welches sowohl das unerwartete Vorhandensein symmetrischer und asymmetrischer Formen im System als auch die Regeln erklärt, die das symmetrische oder asymmetrische Schicksal der Moleküle steuern. Der letzte Abschnitt der Experimente zeigte, dass die Anwesenheit der Telopeptide und Heparins eine signifikante Wirkung auf die strukturellen und mechanischen Charakteristika der in vitro rekonstituierten Kollagen Typ I Fibrillen hatte. Die durchgeführten Strukturanalysen zeigten außerdem, dass die Anwesenheit der Telopeptide in säurelöslichem Kollagen (ASC) die Rekonstitution D-periodischer Kollagenfibrillen mit Heparin verhinderte, sodass nur symmetrisch polymorphe Formen mit einer Wiederholeinheit von 165 nm möglich waren (FLS IV). Weitere Messungen der Telopeptid-freien und Telopeptid-intakten Kollagenfibrillen mit Röntgendiffraktometrie ergaben, dass die Abwesenheit der nicht-helix-strukturierten Enden in Pepsin-gelöstem Kollagen (PSC) zu einer weniger kompakten Anordnung der Tripelhelices von Atelokollagen führte. Der interhelix Abstand erhöhte sich von 1,0 zu 1,2 nm für getrocknete Proben. Das zeigt, dass die losere Anordnung der Tripelhelices einhergeht mit der Verringerung der Biege-Elastizitäts-module der Kollagenfibrillen,. Basierend auf den hier vorgestellten morphologischen, strukturellen und mechanischen Unterschieden zwischen ASC und PSC-Heparin Fibrillen wird die Idee unterstützt, dass Heparin als intrafibrillärer Vernetzer fungiert und an Bindungsstellen der Helix bindet, welche in vivo bei Kollagen Typ I Fibrillen durch Telopeptide besetzt sind. Die durchgeführten Studien sind von besonderem Interesse für das Verständnis und die Steuerung eines sehr vielseitigen und bereits verwendeten xenogenes Zellkultursystem für das Tissue Engineering. Von den rekonstituierten Kofibrillen mit ihrer ungewöhnlichen Morphologie und GAG Einlagerung - ein in vivo nicht bekanntes Phänomen - erwartet man, dass sie ein intressantes biochemisches Verhalten als Biomaterial für ECM Scaffolds zeigen. Variationen der experimentellen Bedingungen, des Ausmaßes der Telopeptidentfernung und der Heparinkonzentration liefern vielfältige Möglichkeiten um die Kinetik, Struktur, Dimension sowie die mechanischen Eigenschaften des Systems zu kontrollieren. Damit sollte es möglich sein, ein bestimmtes Zellverhalten gegenüber der neu entwickelten Matrix vorherzusagen. Die GAG-Einlagerung bietet interessante Optionen für eine langfristige Freisetzung des GAGs 'on demand', sowie die native Bindung und Stabilisierung von Wachstumsfaktoren, Cytokinen, Chemokinen, womit zusätzlich Zellsignalisierung und -schicksal und später Gewebemorphogenese kontrolliert werden kann.

A laminina contém peptídeos que podem ser liberados por proteólise. Nosso laboratório estuda os efeitos de peptídeos da laminina em biologia tumoral. Neste trabalho, verificamos se C16 (cadeia g1) estimularia invasão e atividade de invadopódios em células de carcinoma epidermóide (CAL27) e fibrossarcoma (HT1080). C16 promoveu aumento na taxa de invasão e atividade de invadopódios em ambas às linhagens celulares, comparado ao peptídeo controle C16SX. Microscopia em time-lapse demonstrou que C16 induz aumento na atividade de invadopódios em função do tempo. C16 estimula fosforilação de Src e ERK 1/2, e inibição da via ERK reduz invasão e atividade de invadopódios relacionados ao peptídeo. C16 conjugado à rodamina foi encontrando decorando a membrana de células CAL27, sugerindo possível interação com receptores. Diminuição dos níveis de integrina b1 reduzem atividade de invadopódios em amostras tratadas com C16. Nossos dados sugerem que C16 regula invasão e atividade de invadopódios em células CAL27 e HT1080, provavelmente por meio de Src, ERK e integrina b1.
Laminin harbors bioactive peptides released upon tumor-induced proteolysis. Our Laboratory has been studying laminin peptides effects in tumor biology. Here we addressed whether C16 (g1 chain) would regulate invasion and invadopodia activity in cell lines from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). C16 increased invasion rate and invadopodia activity compared to control peptide (C16SX). Through time-lapse microscopy, we observed that C16 stimulated invadopodia activity overtime. We searched for signaling pathways related to peptide effects. C16 stimulated Src and ERK 1/2 phosphorylation, and ERK signaling cascade inhibition decreased C16-induced invasion and invadopodia. Next, we addressed how C16 would interact with tumor cells. Rhodamine-conjugated C16 was found decorating CAL27 cell membrane, suggesting an interaction with receptors. Knockdown of b1 integrin reduced invadopodia activity of C16-treated cells. We propose that C16 regulates invasion and invadopodia activity of CAL27 and HT1080 cells through Src, ERK and b1 integrin.

Bacillus subtilis est une bactérie à Gram positif ubiquitaire vivant dans différents environnements terrestres et aquatiques. Différents polymères extracellulaires entrant dans la composition de la matrice des biofilms à B. subtilis ont été décrits. Des polysaccharides sont à la base de ces propriétés mécaniques, la viscoélasticité étant modulée par la teneur du biofilm en différents polymères extracellulaires comme les protéines amyloïdes et l’ADN extracellulaire.L’objectif de ce travail était d’étudier le rôle des exopolymères dans les biofilms à B. subtilis en utilisant la souche type de l’espèce CIP52.65T et différentes autres souches sauvages, cliniques et mutantes. La composition de la matrice varie en fonction de la présence de saccharose dans le milieu de culture, ainsi les effets d’une supplémentation du milieu Trypticase Soja (TS) en saccharose (20% p/v) ont été étudiés sur la croissance planctonique, la production de polymères de matrice et la formation de biofilm pour toutes ces souches de B. subtilis. Enfin, parmi les protéines de la matrice, B. subtilis produit une protéine formant des fibres amyloïdes appelée TasA. Son rôle exact dans le biofilm reste encore mal connu. Le but de cette seconde étude était de mieux comprendre le mécanisme d'auto-assemblage de TasA et de comprendre son rôle dans la matrice. En regroupant toutes les caractérisations effectuées sur les biofilms et sur les peptides amyloïdes, la conception de matrice biomimétique a permis d’effectuer de première approche sur les propriétés mécaniques de celle-ci, en reproduisant des matrices artificielles à base d’exopolysaccharides (lévane), de peptides amyloïdes et d’ADN
Bacillus subtilis is a ubiquitous gram-positive bacterium that lives in different terrestrial and aquatic environments. Various extracellular polymers involved in the composition of the B. subtilis biofilm matrix have been described. Polysaccharides are the basis of these mechanical properties, the viscoelasticity being modulated by the content of the biofilm in different extracellular polymers such as amyloid proteins and extracellular DNA.The aim of this work was to study the role of exopolymers in B. subtilis biofilms using the type CIP52.65T strain and various other wild, clinical and mutant strains. The composition of the matrix varies according to the presence of sucrose in the culture medium, so the effects of supplementation of the medium Trypticase Soy (TS) sucrose (20% w/v) were studied on the planktonic growth, matrix polymer production and biofilm formation for all these B. subtilis strains. Finally, among the proteins in the matrix, B. subtilis produces an amyloid-forming protein called TasA. Its exact role in the biofilm remains poorly understood. The purpose of this second study was to better understand the self-assembly mechanism of TasA and to understand its role in the matrix. By grouping all the characterizations carried out on the biofilms and the amyloid peptides, the biomimetic matrix design made it possible to carry out a first approach on the mechanical properties of this one, by reproducing artificial matrices based on exopolysaccharides (levan), amyloid peptides and DNA

Crucial to a cell’s ability to migrate is the organization of its plasma membrane and associated proteins in a polarized manner to interact with and respond to its surrounding environment. Cells interact with the extracellular matrix (ECM) through specialized contact sites, including podosomes and invadopodia. Tumor cells use F-actin-rich invadopodia to degrade ECM and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction and degradation. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II,reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV increases the number of F-actin punctae, which are highly dynamic and co-localize with markers of podosomes and invadopodia. Endogenous SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases the average amount of matrix degradation; RNAi-mediated downregulation of SV decreases degradation. Cortactin, an essential component of both podosomes and invadopodia, binds SV sequences in vitro and contributes to the formation of EGFP-SV induced punctae. Additionally, SV affects cortactin localization,which could provide a mechanism for SV action at invadopodia. The formation of cholesterol-rich membrane rafts is one method of plasma membrane organization. A property of membrane rafts is resistance to extraction with cold Triton X-100 and subsequent flotation to low buoyant densities. The actin cytoskeleton has been implicated in many signaling events localized to membrane rafts, but interactions between actin and raft components are not well characterized. Our laboratory isolated a heavy detergent resistant membrane fraction from neutrophils, called DRM-H, that contains at least 23 plasma membrane proteins. DRM-H is rich in cytoskeletal proteins, including fodrin, actin, myosin II, as well as supervillin. DRM-H also contains proteins implicated in both raft organization and membrane-mediated signaling. DRM-H complexes exhibit a higher buoyant density than do most DRMs (referred to as DRM-L), which are deficient in cytoskeletal proteins. By using similar purification methods, I find that COS-7 cells also contain cytoskeleton-associated DRMs. In addition, when transfected into COS-7 cells, estrogen receptor (ER)α associates with DRM-H, while ERβ is seen in both DRM-L and DRM-H populations, suggesting a role for DRM-H in nongenomic estrogen signaling. Thus, the cytoskeleton-associated DRM-H not limited to hematopoietic cells and could constitute a scaffold for membrane raftcytoskeleton signaling events in many cells. Taken together, our results show that SV is a component of cytoskeleton-associated membrane rafts as well as podosomes and invadopodia, and that SV plays a role in invadopodial function. SV, with its connections to both membrane rafts and the cytoskeleton, is well situated to mediate cortactin localization, activation state, and/or dynamics of matrix metalloproteases at the ventral cell surface for proper matrix degradation through invadopodia. The molecular dissection of invadopodia formation and function may contribute to a greater understanding of in vivo invasion, and thus, tumor cell metastasis.

Este trabalho investigou, pela primeira vez, a influência do CH na modulação do metabolismo e proliferação de fibroblastos dérmicos humanos (FDHs) derivados de áreas fotoprotegida e fotoexposta, cultivados em modelo de monocamada. Além disto, foram investigados os efeitos da suplementação com CH na secreção de colágeno tipo I, em modelo de cultura 3D de equivalente dérmico, derivado de matriz produzida exclusivamente por FDHs. O tratamento com CH não influenciou a proliferação celular dos fibroblastos derivados de ambas as áreas, porém modulou expressivamente o metabolismo dos FDHs cultivados em monocamada, elevando o conteúdo de pró-colágeno I e colágeno I e diminuindo a atividade de metaloproteinases de matriz (MMP) 1 e 2. Concentrações menores de CH foram suficientes para estimular as células de área fotoexposta, sugerindo efeitos mais pronunciados do CH nestas células. Este estudo é uma contribuição importante para compreensão dos efeitos biológicos do CH nas células da pele e viabilidade do seu uso como ingrediente funcional de suplementos alimentares.
This study investigated, for the first time, the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from sun-protected and sun-exposed body sites, cultured in monolayer in vitro model. Moreover, CH effects on the secretion of type I collagen were investigated in dermal equivalent 3D model derived from dermal matrix produced exclusively by HDFs. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of procollagen I and collagen I and decreasing metalloproteinase activity (MMP) 1 and 2. These effects were confirmed in the human dermal equivalent model. Lower concentrations of CH were enough to stimulate sun-exposed-derived HDFs, suggesting more pronounced effect in these cells. This study presents an important contribution to understanding the biological effects of CH in skin cells and viability of its use as a functional ingredient in food supplements.

BACKGROUND AND AIMS: Congenital Hepatic Fibrosis (CHF) is caused by mutations in PKHD1, a gene encoding for fibrocystin, a protein of unknown function, expressed in cholangiocyte cilia and centromers. In CHF, biliary dysgenesis is accompanied by severe progressive portal fibrosis and portal hypertension. The mechanisms responsible for portal fibrosis in CHF are unclear. The αvβ6 integrin mediates local activation of TGFβ1 and is expressed by reactive cholangiocytes during cholestasis. To understand the mechanisms of fibrosis in CHF we studied the expression of αvβ6 integrin and its regulation in Pkhd1del4/del4 mice. METHODS: In Pkhd1del4/del4 mice we studied, at different ages (1-12 months): a) portal fibrosis (Sirius Red) and portal hypertension (spleen weight/body weight); b) αvβ6 mRNA and protein expression (RT-PCR, IHC); c) α-SMA and TGFβ1 mRNA expression (RT-PCR); d) portal inflammatory infiltrate (IHC for CD45 and FACS analysis of whole liver infiltrate); f) cytokines secretion from cultured monolayers of primary cholangiocytes (Luminex assay); g) cytokine effects on monocyte/macrophage proliferation (MTS assay) and migration (Boyden chamber); h) TGFβ1 and TNFα effects on β6 integrin mRNA expression by cultured cholangiocytes before and after inhibition of the TGFβ receptor type II (TGFβRII); i) TGFβ1 effects on collagen type I (COLL1) mRNA expression by cultured cholangiocytes. RESULTS: Pkhd1del4/del4 mice showed a progressive increase in αvβ6 integrin expression on biliary cyst epithelia. Expression of αvβ6 correlated with portal fibrosis (r=0.94, p<0.02) and with enrichment of a CD45+ve cell infiltrate in the portal space (r=0.97, p<0.01). Gene expression of TGFβ1 showed a similar age-dependent increase. FACS analysis showed that 50-75% of the CD45+ve cells were macrophages (CD45/CD11b/F4/80+ve). Cultured polarized Pkhd1del4/del4 cholangiocytes secreted from the basolateral side significantly increased amounts of CXCL1 and CXCL10 (p<0.05). Both cytokines were able to stimulate macrophage migration (p<0.05). Basal expression of β6 mRNA by cultured Pkhd1del4/del4 cholangiocytes (0.015±0.002 2^-dCt) was potently stimulated by the macrophage-derived cytokines TGFβ1 (0.017±0.002 2^-dCt, p<0.05) and TNFα (0.018±0.003 2^-dCt, p<0.05). Inhibition of TGFβRII completely blunted TGFβ1 (0.014±0.003 2^-dCt, p<0.05) but not TNFα effects (0.017±0.001 2^-dCt, p=ns) on β6 mRNA. COLL1 mRNA expression by cultured Pkhd1del4/del4 cholangiocytes (0.0009±0.0003 2^-dCt) was further and significantly increased after TGFβ1 stimulation (0.002±0.0005 2^-dCt, p<0.05). CONCLUSIONS: Pkhd1del4/del4 cholangiocytes possess increased basolateral secretory functions of chemokines (CXCL1, CXCL10) able to orchestrate macrophage homing to the peribiliary microenvironment. In turn, by releasing TGFβ1 and TNFα, macrophages up-regulate αvβ6 integrin in Pkhd1del4/del4 cholangiocytes. αvβ6 integrin activates latent TGFβ1, further increasing the fibrogenic properties of cholangiocytes.

Sprouting endothelial cells (ECs) use soluble and insoluble cues to guide migration and expand the existing vascular network to meet changing trophic needs of the tissue during angiogenesis. A noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique was used to optically image endothelial sprouting morphogenesis in three dimensional (3D) collagen matrices with simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Sprout advancement and lumen expansion companying with ECM alteration were the synergistic results of membrane-associated matrix metalloproteinase and cell traction evidenced by proteinase inhibition and Rho-associated kinase (p160ROCK) inhibition experiments. These physical EC-ECM interactions suggest that ECM mechanical properties may influence angiogenic responses. In a 3D angiogenesis model, we measure angiogenic responses as a function of collagen matrix stiffness by inducing collagen cross-linking with microbial transglutaminase (mTG). Collagen matrices stiffen with both mTG treatment and incubation time as evidenced with biaxial mechanical test results and collagen TPF intensity increases with mTG treatment and that the ratio of TPF/SHG correlates with biaxial tested mechanical stiffness. SHG and optical coherence microscopy (OCM) are further used to show that other physical properties of the matrix do not change with mTG treatment, thus providing the same density but different stiffness with which to measure angiogenic responses. Stiffer matrices promote angiogenesis with more invading sprouts that invade deeper. No differences in lumen size were observed between control and mTG stiffened 3D cultures, but there was evidence of greater matrix remodeling in stiffer gels using NLOM. Results of this study show angiogenic responses are enhanced with increasing stiffness and suggest that these properties may be used in tissue engineering and regenerative medicine applications to engineer angiogenesis.

The extracellular matrix (ECM) provides mechanical and biochemical support to tissues and cells. It is crucial for cell attachment, differentiation, and migration, as well as for ailment-associated processes such as angiogenesis, metastases and cancer development. An approach to study these phenomena is through emulation of the ECM by synthetic gels constructed of natural polymers, such as collagen and fibronectin, or simple but tunable materials such as poly(ethylene glycol) (PEG) crosslinked with short peptide sequences susceptible to digestion by metalloproteases and cell-binding domains. Our lab uses PEG gels to study cell behavior in three dimensions (3D). Although this system fosters cell attachment and crosslinking peptides mentioned, the regenerative process of the ECM has not been mimicked yet in 3D synthetic gels. In an attempt to build in this functionality to PEG-based gels, I performed phage display to identify short oligopeptides that bind either collagen or fibronectin to assess them as potential nucleation points for crosslinking elements in order to emulate the in vivo reconstitution process. A phage display is a library of random oligopeptides expressed on a M13 bacteriophage that allows identification of a phenotype and a genotype with a single screening step. This inexpensive strategy could yield a short oligopeptide with high specificity. I identified the conditions under which phage display is compatible with our targets, and I isolated and identified five peptide candidates for fibronectin binding and two for collagen. Future work includes assessing whether these candidates could facilitate the formation of cell-created crosslinking in 3D synthetic hydrogels.

Indiana University-Purdue University Indianapolis (IUPUI)
The proliferation and differentiation of osteoblasts are influenced by mechanical and geometrical growth environments. A specific aim of my thesis was the elucidation of signaling pathways involved in mechanical stimulation and geometric alterations of the extracellular matrix (ECM). A pair of questions addressed herein was (a) Does mechanical stimulation modulate translational regulation through the phosphorylation of eukaryotic initiation factor 2 (eIF2)? (b) Do geometric alterations affect the phosphorylation patterns of mitogen-activated protein kinase (MAPK) signaling? My hypothesis was mechanical stress enhances the proliferation and survival of osteoblasts through the reduction in phosphorylation of eIF2, while 3-dimensional (3D) ECM stimulates differentiation of osteoblasts through the elevation of phosphorylation of p38 MAPK. First, mechanical stimulation reduced the phosphorylation of eIF2. Furthermore, flow pre-treatment reduced thapsigargin-induced cell mortality through suppression of phosphorylation of protein kinase RNA-like ER kinase (Perk). However, H2O2-driven cell mortality, which is not mediated by Perk, was not suppressed by mechanical stimulation. Second, in the ECM geometry study, the expression of the active (phosphorylated) form of p130Cas, focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) was reduced in cells grown in the 3D matrix. Conversely, phosphorylation of p38 MAPK was elevated in the 3D matrix and its up-regulation was linked to an increase in mRNA levels of dentin matrix protein 1 and bone sialoprotein. In summary, our observations suggest the pro-survival role of mechanical stimulation and the modulation of osteoblastic fates by ECM geometry.

Výborný, Karel. Vývoj materiálů na bázi extracelulární matrix pro léčbu centrálního nervového systému. [Development of extracellular-matrix scaffolds for CNS repair]. Praha, 2020. 105 stran, 3 přílohy. Dizertační práce (Ph.D.). Univerzita Karlova, 2. lékařská fakulta, Ústav experimentální medicíny, AV ČR v.v.i. Abstract Brain and spinal cord injury are a serious traumata and despite intensive research, there is still no effective treatment for patients. One of the studied approaches is to use various biomaterials to repair the damaged neural tissue. The aim of this thesis is to study the regenerative and neurotrophic effects of injectable extracellular matrix (ECM) hydrogels prepared by decellularization from porcine tissue (brain, spinal cord, bladder) and human umbilical cord (UC) in reconstruction of damaged neural tissue of the brain and spinal cord in rats. We characterized ECM hydrogels in terms of mechanical and rheological properties, structure and composition, cell adhesion, migration and proliferation. In a model of spinal cord hemisection, we compared the regenerative effect of ECM hydrogels derived from porcine spinal cord and urinary bladder. We found no benefits of tissue specific ECM prepared from the tissue of neural origin in terms of its neurotrophic properties in vitro or in vivo....

It would be a great advantage in reconstructive surgery to have an off-the-shelf biomaterial to promote regeneration and volume augmentation following soft tissue damage. With this long-term objective, human adipose tissue (fat) is an abundant and accessible source of extracellular matrix (ECM) for bioscaffold fabrication. The main goal of the current research project was to optimize the established 5-day detergent-free decellularization protocol developed by the Flynn group, by shortening it to a maximum of 3 days, while achieving comparable results in terms of cell and lipid extraction with preservation of the ECM. The effectiveness of the optimized protocol was assessed by examination of the decellularized adipose tissue (DAT) and its characteristic biological properties, including in vitro bioactivity assays with human adipose-derived stem cells (ASCs) to measure adipogenic potential, as well as in vivo testing of scaffold biocompatibility. In the optimized approach, the addition of mechanical processing steps including repeated pressing and centrifugation were shown to enhance cell extraction. Fibrous ultrastructure was observed under scanning electron microscopy (SEM) for the original and optimized protocols. The preservation of collagen fibres was assessed with picro-sirius red staining and confirmed by high hydroxyproline content. Enhanced preservation of glycosaminoglycans (GAGs) was determined for the optimized protocol. Residual DNA content was higher in the DAT scaffolds processed with the optimized protocol, including larger DNA fragments that were not typically observed in the samples treated with the original protocol, which incorporated additional enzymatic treatment stages with DNase, RNase and lipase. However, no residual nuclei were visualized through DAPI staining for both protocols. Enhanced removal of DNA was achieved with electron beam (e-beam) sterilization. E-beam sterilization caused some changes in the fine fibrous structure of the ECM, but did not negatively affect the adipo-conductive potential in vitro. In comparison to the original protocol, DAT produced via the optimized protocol exhibited similar adipo-conductive properties in vitro. The in vivo biocompatibility study over a 16 week period using an immunocompetent Wistar rat model showed promising results. DAT implants produced with the original and optimized protocols promoted adipogenesis and angiogenesis, gradually being remodelled to resemble mature adipose tissue.
Thesis (Master, Chemical Engineering) -- Queen's University, 2014-01-30 12:25:22.044

碩士
國立成功大學
醫學檢驗生物技術學系
105
Folate is an essential nutrient for mammals during pregnancy. There are many disease, including anemia, neural tube defects and cancer correlating to the folate deficiency. Previously, we observed in the folate-deficient zebrafish larvae that increased activity of cathepsin L, a protease crucial for maintaining ECM integrity, has contributed the defective formation of swim bladder. In the current study, we attempt to confirm the role of inflammatory cells in the FD-induced loss of swim bladder and search from an herbal extracts library for the remedy with potential to intervene the ECM protease activity. Angiogenesis is an important process involving in physiological tissues development and pathological neoplasia. The activity and balance between protease and antiprotease are crucial for angiogenesis since they mediate the degradation of blood vessel basement and extracellular matrix (ECM), an essential step during angiogenesis. We used the transgenic fish with fluorescent vessels, Tg(fli1: EGFP), and the folate deficient larvae with defective swim bladder, Tg(zhsp70:EGFP-γGH), as our in vivo screening platforms. Our results showed that repressing macrophage development and inhibiting TNF-α activity aggravated the swim bladder malformation caused by folate deficiency. We found that fourteen out of the sixty-six extracts screened, including Spilanthes acmella (Linn) Murr, Achillea millefolium, Platycodon grandifloras, Mesona chinensis and Toona sinensis inhibited larval vessels development. The anti-angiogenesis activity of the selected extracts was further evaluated with the zebrafish tumor transplant model injected with non-small cell lung cancer H1299 cells. We found Spilanthes acmella (Linn) Murr and Toona sinensis could decreased angiogenesis on 1 dpi (day post injection) in xenograft model. In conclusion, our result suggested that phagocytes might involve in the development of swim bladder, and the Spilanthes acmella (Linn) Murr have the potential to regular the homeostasis of protease and modulate the ECM integrity.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily of proteins, circulates in blood in a complex with vitronectin (VN). These two proteins are also found localized together in the extracellular matrix in many different pathophysiological conditions. Both of these proteins are involved with a number of physiologically important processes. Though PAI-1 is a well-known inhibitor of serine proteases, more emphasis is now geared towards its protease independent functions. VN, on the other hand, is a binding protein that exists in the circulation in a preferred monomeric conformation. However, in the extracellular matrix, VN exists as multimer with altered conformation. Though the exact reason for such conformational alterations and compartmentalization is unknown, there are a number of biomolecules, including PAI-1 that are proposed to cause such alterations. In last few years, sufficient experimental evidence has been gathered to confirm this protease- independent effect of PAI-1 by which it induces multimerization of VN in a concentration-dependent fashion. It has been observed also that PAI-1 remains associated with this multimeric complex for several hours. A major focus of this dissertation work was to extend our understanding of the mechanism of the interaction between these proteins and to explore the physiological relevance of the multimeric complexes formed by their interaction on cellular adhesion and migration. In our study, emphasis has been given to the presence of an appropriate microenvironment so that the role of the multimeric complexes could be investigated in a relevant biological setting. Our findings indicate the importance of the surrounding microenvironment in establishing the specific role of the VN/PAI-1 complex in cell-matrix interactions. In a previous study from our lab, it was found that vitronectin knock-out mice were more resistant to Candida infection compared to wild type C57Bl/6 mice. One of the goals of this dissertation work was to provide a mechanistic explanation for their increased survival of the vitronectin knock-out mice upon Candida infection. Another important aspect of this work was to establish biophysical methods for understanding the structural changes that happen in PAI-1 naturally or due to ligand binding.