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Exogenous modulation of embryonic tissue and stem cells to form nephronal structuresSebinger, David Daniel Raphael 04 July 2013 (has links) (PDF)
Renal tissue engineering and regenerative medicine represent a significant clinical objective because of the very limited prospect of cure after classical kidney treatment. Thus, approaches to isolate, manipulate and reintegrate structures or stimulating the selfregenerative potential of renal tissue are of special interest. Such new strategies go back to knowledge and further outcome of developmental biological research. An understanding of extracellular matrix (ECM) structure and composition forms thereby a particularly significant aspect in comprehending the complex dynamics of tissue regeneration. Consequently the reconstruction of these structures offers beneficial options for advanced cell and tissue culture technology and tissue engineering. In an effort to investigate the influence of natural extracellular structures and components on embryonic stem cell and renal embryonic tissue, methodologies which allow the easy application of exogenous signals on tissue in vitro on the one hand and the straight forward evaluation of decellularization methods on the other hand, were developed. Both systems can be used to investigate and modulate behaviour of biological systems and represent novel interesting tools for tissue engineering. The novel technique for culturing tissue in vitro allows the growing of embryonic renal explants in very low volumes of medium and optimized observability, which makes it predestined for testing additives. In particular, this novel culture set up provides an ideal opportunity to investigate renal development and structure formation. Further studies indicated that the set is universally applicable on all kinds of (embryonic) tissue. Following hereon, more than 20 different ECM components were tested for their impact on kidney development under 116 different culture conditions, including different concentrations and being either bound to the substrate or dissolved in the culture medium. This allowed to study the role of ECM constituents on renal structure formation. In ongoing projects, kidney rudiments are exposed to aligned matrix fibrils and hydrogels with first promising results. The insights gained thereof gave rise to a basis for the rational application of exogenous signals in regenerative kidney therapies. Additionally new strategies for decellularization of whole murine adult kidneys were explored by applying different chemical agents. The obtained whole matrices were analysed for their degree of decellularization and their residual content and composition. In a new straight forward approach, a dependency of ECM decellularization efficiency to the different agents used for decellularization could be shown. Moreover the capability of the ECM isolated from whole adult kidneys to direct stem cell differentiation towards renal cell linage phenotypes was proved. The data obtained within this thesis give an innovative impetus to the design of biomaterial scaffolds with defined and distinct properties, offering exciting options for tissue engineering and regenerative kidney therapies by exogenous cues.
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Structural Analysis of Reconstituted Collagen Type I - Heparin Cofibrils / Strukturanalyse von rekonstituierten Kollagen Typ I - Heparin KofibrillenStamov, Dimitar 25 March 2010 (has links) (PDF)
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.
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Organisationsprinzipien der extrazellulären Matrix in der Substantia nigra des Menschen und ihr Bezug zum Morbus ParkinsonKanter, Marlene 24 November 2010 (has links) (PDF)
Der Morbus Parkinson ist durch den selektiven Zelltod der dopaminergen Neurone der Substantia nigra pars compacta gekennzeichnet. Hierbei sind die verschiedenen Populationen pigmentierter Neurone innerhalb der SNc unterschiedlich stark
betroffen. Die Ursachen für diese unterschiedliche Schädigung sind noch nicht bekannt. Möglicherweise besteht aber ein Zusammenhang mit der Verteilung der extrazellulären Matrix innerhalb der Substantia nigra. Für die Untersuchung wurden immunhistochemische Methoden an Hirnschnittserien von menschlichen Kontrollgehirnen angewandt. Zur Darstellung von Komponenten der extrazellulären Matrix wurden drei verschiedene Antikörper genutzt. Dazu gehörten anti- CRTL-1, welcher das Link- Protein 1 von CSPGs detektiert, ein Aggrecan- Antikörper ( Klon HAG7D4), welcher an das Kern- Protein menschlichen Aggrecans bindet, sowie anti- Proteoglykan- Di-0S (Klon 1B5), der die Reste der Chondroitin- Sulfat- Seitenketten verschiedener Proteoglykane detektiert, die nach Verdau mit Chondroitinase ABC übrigbleiben. Zur räumlichen Orientierung und strukturellen Gliederung der Substantia nigra nach der von Damier et al. ( 1999) beschriebenen Calbindin- Methode, auf deren Grundlage die SNc in eine Calbindin-reiche Matrix und Calbindin- arme Bereiche, die sogenannten Nigrosomen, gegliedert wird, wurden benachbarte Hirnschnitte mit anti- Calbindin D₂₈K behandelt. Es zeigte sich, dass extrazelluläre Matrix in Form von perineuronalen Netzen nur an den nicht pigmentierten Neuronen der SNr und SNl vorkommt, während die pigmentierten Neurone der SNc keine perineuronalen Netze besitzen, aber von einer Vielzahl von ACs kontaktiert werden. Deren Dichte war an großen, stark Melanin- haltigen Neuronen am höchsten, sodass in der dorsalen Schicht der SNc, also in den Nigrosomen 3 und 4, besonders viel extrazelluläre Matrix detektiert werden konnte. Im ventralen Anteil der SNc war entsprechend der unterschiedlichen Zellgrößen, insbesondere in Nigrosom 1, eine heterogene Verteilung der extrazellulären Matrix
festzustellen. Zur Untersuchung über mögliche Veränderungen der extrazellulären Matrix im Verlauf des Morbus Parkinson wurden Hirnschnitte menschlicher Gehirne mit diagnostiziertem Morbus Parkinson ebenfalls mit den drei Antikörpern zur Darstellung der extrazellulären Matrix behandelt. Dabei zeigte sich, dass insgesamt
die Menge extrazellulärer Matrix verringert scheint. Eine Darstellung der
perineuronalen Netze mit anti- Proteoglykan- Di-0S (Klon 1B5) war nicht mehr möglich. Wie bereits in früheren Studien verschiedener Autoren festgestellt, waren die stärksten Auswirkungen der neurodegenerativen Prozesse im ventralen Anteil der
SNc, vor allem in Nigrosom 1, auszumachen, während die Neurone der Nigrosomen 3 und 4 im dorsalen Anteil weniger vulnerabel erscheinen. Diese Ergebnisse verstärken die Annahme, dass die extrazelluläre Matrix eine protektive Funktion für bestimmte Neuronengruppen besitzt. Bei der Parkinsonschen Erkrankung wird möglicherweise zuerst dieses Schutzsystem zerstört bevor es zum progressiven
Neuronenverlust kommt. Ungeklärt bleibt weiterhin was die Ursachen dafür sind.
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Neuronal mechanisms of the adaptation of conditional visuomotor behavior / Neuronale Mechanismen für die Adaptation von konditionellem visuomotorischem VerhaltenWestendorff, Stephanie 28 October 2010 (has links)
No description available.
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The Sweet Side of the Extracellular Matrix -Rother, Sandra 01 November 2017 (has links) (PDF)
Bone fractures and pathologic conditions like chronic wounds significantly reduce the quality of life for the patients, which is especially dramatic in an elderly population with considerable multi-morbidity and lead to substantial socio-economic costs. To improve the wound healing capacity of these patients, new strategies for the design of novel multi-functional biomaterials are required: they should be able to decrease extensive pathologic tissue degradation and specifically control angiogenesis in damaged vascularized tissues like bone and skin.
Glycosaminoglycans (GAGs) like hyaluronan (HA) and chondroitin sulfate (CS) as important extracellular matrix (ECM) components are involved in several biological processes such as matrix remodeling and growth factor signaling, either by directly influencing the cellular response or by interacting with mediator proteins. This could be useful in functionalizing biomaterials, but native sulfated GAGs (sGAGs) show a high batch-to-batch variability and are limited in their availability. Chemically modified HA and CS derivatives with much more defined characteristics regarding their carbohydrate backbone, sulfate group distribution and sulfation degree are favorable to study the structure-function relationship of GAGs in their interaction with mediator proteins and/or cells and this might be used to precisely modulate activity profiles to stimulate wound healing.
By combining collagen type I as the main structural protein of the bone and skin ECM with these GAG derivatives, 2.5-dimensional (2.5D) and 3D artificial ECM (aECM) coatings and hydrogels were developed. These biomaterials as well as the respective GAG derivatives alone were compared to native GAGs and used to analyze how the sulfation degree, pattern and carbohydrate backbone of GAGs influence:
i) the activity of tissue inhibitor of metalloproteinase-3 (TIMP-3) and vascular endothelial growth factor-A (VEGF-A) as main regulators of ECM remodeling and angiogenesis,
ii) the composition and characteristics of the developed 2.5D and 3D aECMs,
iii) the enzymatic degradation of collagen-based aECMs and HA/collagen-based hydrogels,
iv) the proliferation and functional morphology of endothelial cells.
Surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA) binding studies revealed that sulfated HA (sHA) derivatives interact with TIMP-3 and VEGF-A in a sulfation-dependent manner. sHA showed an enhanced interplay with these proteins compared to native GAGs like heparin (HEP) or CS, suggesting a further impact of the carbohydrate backbone and sulfation pattern. sGAGs alone were weak modulators of the matrix metalloproteinase-1 and -2 (MMP-1 and -2) activity and did not interfere with the inhibitory potential of TIMP-3 against these proteinases during enzyme kinetic analyses. However, the formation of TIMP 3/GAG complexes reduced the binding of TIMP-3 to cluster II and IV of its endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1, mediates the up-take and degradation of TIMP-3 from the extracellular environment) in a sulfation- and GAG type-dependent manner. It is of note that the determined complex stabilities of TIMP-3 with cluster II and IV were almost identical indicating for the first time that both clusters contribute to the TIMP-3 binding. Competitive SPR experiments demonstrated that GAG polysaccharides interfere stronger with the TIMP 3/LRP-1 interplay than GAG oligosaccharides. The importance of the position of sulfation is highlighted by the finding that a sHA tetrasaccharide exclusively sulfated at the C6 position of the N-acetylglucosamine residues significantly blocked the receptor binding, while CS and HEP hexasaccharides had no detectable effects. Thus, sHA derivatives as part of biomaterials could be used to sequester and accumulate TIMP 3 in aECMs in a defined manner where sHA-bound TIMP-3 could decrease the matrix breakdown by potentially restoring the MMP/TIMP balance. GAG binding might extend the beneficial presence of TIMP-3 into wounds characterized by excessive pathologic tissue degradation (e.g. chronic wounds, osteoarthritis).
Mediator protein interaction studies with sHA coated surfaces showed the simultaneous binding of TIMP-3 and VEGF-A, even though the sHA/VEGF-A interplay was preferred. Moreover, kinetic analysis revealed almost comparable affinities of both proteins for VEGF receptor-2 (VEGFR-2), explaining their competition that mainly regulates the activation of endothelial cells. Additional SPR measurements demonstrated that the binding of sGAGs to TIMP-3 or VEGF-A decreases the binding of the respective mediator protein to VEGFR-2. Likewise, a sulfation-dependent reduction of the binding signal was observed after pre-incubation of a mixture of TIMP-3 and VEGF-A with sGAG poly- and oligosaccharides. The biological consequences of GAGs interfering with VEGF-A/VEGFR-2 and TIMP-3/VEGFR 2 were assessed in vitro using porcine aortic endothelial cells stably transfected with VEGFR 2 (PAE/KDR cells). The presence of sHA both decreased VEGF-A activity and the activity of TIMP-3 to inhibit the VEGF-A-induced VEGFR-2 phosphorylation. The same decreased activities could be observed for the migration of endothelial cells.
However, if sHA, TIMP-3 and VEGF-A were present simultaneously, sHA partially restored the TIMP-3-mediated blocking of VEGF-A activity. These findings provide novel insights into the regulatory potential of sHA during endothelial cell activation as an important aspect of angiogenesis, which could be translated into the design of biomaterials to treat abnormal angiogenesis. These sHA-containing materials might control the angiogenic response by modulating the activity of TIMP 3 and VEGF-A.
The in vitro fibrillogenesis of collagen type I in the presence of sHA derivatives led to 2.5D collagen-based aECM coatings with stable collagen contents and GAG contents that resemble the organic part of the bone ECM. A burst release of GAGs was observed during the first hour of incubation in buffer with the GAG content remaining almost constant afterwards, implying that the number of GAG-binding sites of collagen restricts the amounts of associated GAGs. Moreover, two differently sulfated HA derivatives could for the first time be incorporated into one multi-GAG aECM as verified via agarose gel electrophoresis and fluorescence measurements. This illustrates the multiple options to modify the aECM composition and thereby potentially their functionality. Atomic force microscopy showed that the presence of sHA derivatives during fibrillogenesis significantly reduced the resulting fibril diameter in a concentration- and sulfation-dependent manner, indicating an interference of the GAGs with the self-assembly of collagen monomers. In line with enzyme kinetic results, none of the GAGs as part of aECMs altered the enzymatic collagen degradation via a bacterial collagenase. Thus aECMs were proven to be biodegradable independent from their composition, which is favorable concerning a potential biomedical usage of the aECMs e.g. as implant coatings.
HA/collagen-based hydrogels containing fibrillar collagen embedded into a network of crosslinked HA and sGAGs were developed as 3D aECMs. Scanning electron microscopy demonstrated a porous structure of the gels after lyophilization, which could favor the cultivation of cells. The presence of collagen markedly enhanced the stability of the gels against the enzymatic degradation via hyaluronidase, something beneficial to clinical use as this is often limited by the generally fast breakdown of HA. Binding and release experiments with lysozyme, as positively charged model protein for e.g. pro-inflammatory cytokines, and VEGF A revealed that the sulfation of GAGs increased the protein binding capacity for pure GAG coatings and retarded the protein release from hydrogels compared to hydrogels without sGAGs. Moreover, the additional acrylation of sHA was shown to strongly reduce the interaction with both proteins when the primary hydroxyl groups were targets of acrylation. This stresses the influence of the substitution pattern on the protein binding properties of the GAG derivatives. However, hydrogel characteristics like the elastic modulus remained unaffected. The different interaction profiles of lysozyme and VEGF-A with GAGs demonstrated a protein-specific preference of different monosaccharide compositions, suggesting that the mediator protein binding could be simultaneously adjusted for several proteins by combining different GAG derivatives. This might allow the scavenging of pro-inflammatory cytokines and at the same time a binding and release of wound healing stimulating growth factors.
Since there is a growing demand for biomaterials to regenerate injured vascularized tissues like bone and skin, endothelial cells were used to examine the direct effects of solute GAGs and hydrogels containing these GAGs in vitro. In both cases, sHA strongly enhanced the proliferation of PAE/KDR cells. A VEGFR-2-mediated effect of GAGs on endothelial cells as underlying mechanism is unlikely since GAGs alone did not bind to VEGFR-2 and had no influence on VEGFR-2 phosphorylation. Other factors like GAG-induced alterations of cell-matrix interactions and cell signaling could be responsible. In accordance with SPR results, a decreased endothelial cell proliferation stimulating activity of VEGF-A was observed in the presence of solute GAGs or after binding to hydrogels compared to the respective treatment without VEGF-A. However, tube formation could be observed in the presence of solute VEGF A and GAGs and within hydrogels with sGAGs that released sufficient VEGF-A amounts over time. Overall the presence of GAGs and VEGF-A strongly promoted the endothelial cell proliferation compared to the treatment with GAGs or VEGF-A alone. Thus, HA/collagen-based hydrogels functionalized with sHA derivatives offer a promising option for the design of “intelligent” biomaterials that direct and regulate the cellular behavior instead of simply acting as inert filling material. They could be used for the controlled delivery and/or scavenging of multiple mediator proteins, thus enhancing the local availability or reducing the activity of these GAG-interacting mediator proteins, or by directly influencing the cellular response. This might be applied to a range of pathological conditions by tuning the biomaterial compositions to patient-specific needs.
However, extensive in vivo validation is required to show whether these in vitro findings could be used to control the biological activity of for instance TIMP-3 and VEGF-A, especially under the pathological conditions of extended matrix degradation and dysregulated angiogenesis.
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Analysis of the role of the atypical cadherin Fat2 during tissue elongation in the developing ovary of Drosophila melanogaster / Analyse der Rolle des atypischen Cadherins Fat2 bei der Gewebestreckung während der Ovarentwicklung von Drosophila melanogasterAurich, Franziska 29 May 2017 (has links) (PDF)
Tissue elongation is an important requirement for proper tissue morphogenesis during animal development. The Drosophila egg chamber is an excellent model to study the molecular processes underlying tissue elongation. An egg chamber is composed of germline cells that are enveloped by a somatic follicle epithelium. While the egg chamber matures, the drastic increase of the egg chamber’s volume is accompanied by a shape change from round to oval. Egg chamber elongation coincides with a circumferential alignment of F-actin filaments, microtubules, and fibrils of the extracellular matrix (ECM). Additionally, egg chambers rotate around their future long axis. It has been proposed that this rotation aligns F-actin filaments and ECM fibrils. The circumferentially aligned F-actin and ECM fibrils form a molecular corset that promotes egg chamber elongation. The atypical cadherin Fat2 is required for egg chamber rotation, the circumferential alignment of F-actin, microtubules, and ECM fibrils and for egg chamber elongation. However, the molecular mechanisms by which Fat2 influences egg chamber elongation remain unknown.
In my thesis I performed a structure-function analysis of Fat2. I generated a Fat2 version that lacks the intracellular region and a second version, which lacks both intracellular region and the transmembrane domain and tested their ability to compensate for Fat2 functions in fat2-/- mutant egg chambers. My results reveal that the intracellular region is required for the microtubule alignment, and for egg chamber rotation. In contrast, the intracellular region is not required for F-actin and ECM alignment, and for egg chamber elongation.
Hence, my findings for the first time demonstrate that egg chamber rotation is not required for F-actin and ECM fibril alignment and that egg chamber elongation can occur independently from egg chamber rotation. My work uncouples some of the parallel processes that take place during oogenesis and changes the view on the mechanisms that drive tissue elongation in this important model system. / Das Strecken von Geweben ist ein wichtiger Prozess bei der Gestaltbildung während der Entwicklung von Organismen. Die Eikammer von Drosophila ist ein hervorragendes Modellsystem, um die Gewebestreckung zu untersuchen. Eine Eikammer besteht aus Keimbahnzellen und einem einschichtigen Follikelepithel, das die Keimbahn umschließt. Während die Eikammer heranwächst durchläuft sie eine drastische Gestaltveränderung von rund nach oval. Zeitgleich zur Streckung der Eikammer weist das Follikelepithel parallel angeordnete F-actin−Filamente, Mikrotubuli und Fasern der extrazellulären Matrix (ECM) auf, welche die Eikammer ringsum umlaufen. Zudem rotieren die Eikammern um ihre zukünftige Längsachse. Bisher nahm man an, die Rotation würde für die Ausrichtung der F-actin−Filamente, Microtubuli und ECM-Fasern gebraucht werden. Die Anordnung der F-actin−Filamente und ECM-Fasern bilden dann ein molekulares Korsett, das die Gewebestreckung fördert.
Das atypische Cadherin Fat2 wird für die Rotation der Eikammern, die umlaufende Anordnung der F-actin–Filamente, Microtubuli und ECM-Fasern sowie für die Streckung der Eikammern benötigt. Die Mechanismen, mit denen Fat2 die Gewebestreckung beeinflusst, sind allerdings unbekannt. In meinem Projekt führte ich eine Struktur-Funktions-Analyse von Fat2 durch.
Ich generierte eine Version von Fat2 mit einer Deletion der kompletten intrazellulären Region und eine zweite, die weder die intrazelluläre Region noch die Transmembran-Domäne besitzt und testete, ob diese Versionen die Funktionen von Fat2 in fat2-/- mutanten Eikammern kompensieren können. Meine Ergebnisse zeigen, dass die intrazelluläre Region für die Anordnung der Mikrotubuli und für die Rotation der Eikammern gebraucht wird. Die intrazelluläre Region wird jedoch weder für die Anordnung von F-actin–Filamenten und den ECM-Fasern noch für die Streckung der Eikammer benötigt. Meine Erkenntnisse zeigen erstmalig, dass die Streckung der Eikammern ohne Rotation stattfinden kann. Meine Arbeit entkoppelt damit mehrere parallel stattfindende Prozesse während der Entwicklung der Eikammer und eröffnet einen neuen Einblick in die Mechanismen der Gewebestreckung in diesem wichtigen Modellsystem.
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Immunohistochemical Comparison of Markers for Wound Healing on Plastic-Embedded and Frozen Mucosal TissueMai, Ronald, Gedrange, Tomasz, Leonhardt, Henry, Sievers, Nicole, Lauer, Günter January 2009 (has links)
Immunohistologic investigations of wound healing in human oral mucosa require specific cell biological markers as well as consecutive small biopsies. Small specimens are ideally embedded in plastic (methylmethacrylate, MMA) resin due to their miniature size. This limits the use of antibodies for these markers. In this immunohistochemical study, the distribution of wound healing markers, e.g. cytokeratin (CK), laminin, collagen IV, vimentin, vinculin and fibronectin, were compared between semithin sections of plastic-embedded tissue and frozen sections of mucosal tissue in order to assess their use for future investigations. The antibodies against laminin, collagen IV and CK 1/2/10/11, 5/6, 13, 14, 17, 19 gave comparable staining patterns on cryostat sections of attached mucosa and on semithin sections of MMA-embedded attached mucosa. In the epithelial cell layers, the following distribution of CK immunostaining was observed: The basal cell layer was positive for CK 5/6, CK 14 and CK 19; the intermediate cell layer for CK 13, CK 17 and CK 1/2/10/11, and the superficial cell layer for CK 13 and CK 1/2/10/11. For most of these antibodies, enzyme digestion with 0.1% trypsin was adequate for demasking the antigens, except for anti-CK 14, anti-CK 17 and anti-laminin; predigestion with 0.4% pepsin in 0.01 N HCl gave similar staining results. The antibodies against vimentin, vinculin, fibronectin and CK 4 showed no affinity or a reciprocal reaction on the semithin sections. Therefore, the antibodies against CK 1/2/10/11; 5/6; 13; 14; 17, and 19, as well as the basement proteins laminin and collagen IV are deemed markers suitable on semithin sections of plastic-embedded attached oral mucosa. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Analysis of the role of the atypical cadherin Fat2 during tissue elongation in the developing ovary of Drosophila melanogasterAurich, Franziska 10 April 2017 (has links)
Tissue elongation is an important requirement for proper tissue morphogenesis during animal development. The Drosophila egg chamber is an excellent model to study the molecular processes underlying tissue elongation. An egg chamber is composed of germline cells that are enveloped by a somatic follicle epithelium. While the egg chamber matures, the drastic increase of the egg chamber’s volume is accompanied by a shape change from round to oval. Egg chamber elongation coincides with a circumferential alignment of F-actin filaments, microtubules, and fibrils of the extracellular matrix (ECM). Additionally, egg chambers rotate around their future long axis. It has been proposed that this rotation aligns F-actin filaments and ECM fibrils. The circumferentially aligned F-actin and ECM fibrils form a molecular corset that promotes egg chamber elongation. The atypical cadherin Fat2 is required for egg chamber rotation, the circumferential alignment of F-actin, microtubules, and ECM fibrils and for egg chamber elongation. However, the molecular mechanisms by which Fat2 influences egg chamber elongation remain unknown.
In my thesis I performed a structure-function analysis of Fat2. I generated a Fat2 version that lacks the intracellular region and a second version, which lacks both intracellular region and the transmembrane domain and tested their ability to compensate for Fat2 functions in fat2-/- mutant egg chambers. My results reveal that the intracellular region is required for the microtubule alignment, and for egg chamber rotation. In contrast, the intracellular region is not required for F-actin and ECM alignment, and for egg chamber elongation.
Hence, my findings for the first time demonstrate that egg chamber rotation is not required for F-actin and ECM fibril alignment and that egg chamber elongation can occur independently from egg chamber rotation. My work uncouples some of the parallel processes that take place during oogenesis and changes the view on the mechanisms that drive tissue elongation in this important model system.:1 ABSTRACT I
2 ZUSAMMENFASSUNG II
3 TABLE OF CONTENTS III
4 LISTS 7
4.1 List of Abbreviations 7
4.2 List of figures 9
5 INTRODUCTION 11
5.1 Tissue morphogenesis during development 11
5.1.1 Tissue organization by differential cell affinity 11
5.1.2 Cell adhesion is mediated by cadherins12
5.1.3 The cytoskeleton drives cell shape changes 13
5.1.4 Planar polarity is required for tissue-level directionality 17
5.2 Models of tissue elongation 19
5.2.1 Germ-band extension in Drosophila melanogaster 19
5.2.2 Primitive streak formation in the chick embryo 21
5.2.3 Neural tube formation in Xenopus 22
5.3 Drosophila egg chamber as a model system to study tissue morphogenesis 24
5.3.1 Oogenesis in Drosophila 24
5.3.2 Egg chamber as a model for tissue elongation 27
5.3.3 Planar polarized organization of the F-actin cytoskeleton in the follicle epithelium 29
5.3.4 Egg chamber elongation requires a link between extracellular matrix and F-actin cytoskeleton 32
5.3.5 Egg chamber rotation is proposed to be a requisite for egg chamber elongation 34
5.3.6 The atypical cadherin Fat2 provides a key role during egg chamber elongation 35
6 AIMS OF THE THESIS 38
7 MATERIALS AND METHODS 39
7.1 Fly husbandry 39
7.2 Used fly stocks 39
7.3 Phenotypic markers 40
7.4 Ovary dissection for fixation 40
7.5 Antibody stainings 41
7.6 Used antibodies 42
7.7 Drug treatment 42
7.8 Microscopy of fixed samples 43
7.9 Live imaging 43
7.9.1 Imaging of the basal F-actin oscillations 43
7.9.2 Imaging of egg chamber rotation 44
7.10 Generation of the transgenic fosmid constructs 44
7.10.1 General materials required for molecular genetics in E.coli 46
7.10.2 Step 1: Amplification of the tagging cassette 47
7.10.3 Step 2: Transformation of the helper plasmid pRedFlp4 49
7.10.4 Step 3: Red-operon driven insertion of the tagging cassette 50
7.10.5 Step 4: Removal of the KanR gene 50
7.10.6 Step 5: DNA isolation and verification of the correct transgenic construct 50
7.10.7 Step 6: Integration of the transgene into the fly genome 52
7.11 Image analysis and quantifications 53
7.11.1 Statistics 53
7.11.2 Aspect ratio measurements 54
7.11.3 Quantification of GFP localization55
7.11.4 Quantification of tissue-wide Collagen IV alignment 55
7.11.5 Quantification of tissue-wide angles of F-actin and microtubules 57
7.11.6 Analysis of periodicity of F-actin oscillations 58
7.11.7 Quantification of the rotation velocity of egg chambers 60
8 RESULTS 61
8.1 Expression of full-length fat2-GFP gene fully rescues all aspects of the fat258D mutant phenotype 61
8.1.1 Expression of the fat2-GFP gene rescues the fat258D mutant egg shape and sterility 61
8.1.2 Using an ‘Alignment parameter’ SAP to quantify the directionality of cytoskeletal structures and extracellular matrix fibrils 63
8.1.3 Expression of the fat2-GFP gene rescues microtubule alignment of fat258D mutant egg chambers65
8.1.4 Expression of the fat2-GFP gene rescues F-actin and Collagen IV alignment of fat258D mutant egg chambers 67
8.2 Generation of different fat2 mutant transgenes by homologous recombineering 70
8.3 The intracellular region of Fat2 is dispensable for some specific aspects of the Fat2 functions 73
8.3.1 The egg chamber elongation is independent of the intracellular region of Fat2 73
8.3.2. Localization of Fat2 protein depends on the intracellular region of Fat2 76
8.3.3 The alignment of microtubules is dependent on the intracellular region of the protein 78
8.3.4 The intracellular region of Fat2 is required for proper early F-actin and Collagen IV fibril alignment 81
8.3.5 The intracellular region of Fat2 is required for late F-actin and Collagen IV fibril alignment 85
8.3.6 F-actin filaments and ECM fibrils co-align in fat258D mutant stage 8 egg chambers 88
8.3.7 F-actin filaments and ECM fibrils do not co-align in fat258D mutant stage 10 egg chambers 90
8.3.8 The stability of basal F-actin fibers and Collagen IV fibrils mutually depend on each other at stage 8 92
8.3.9 The contractile pulses of F-actin in stage 9 egg chambers are independent of the intracellular region of Fat293
8.3.10 The intracellular region of Fat2 is required for proper egg chamber rotation in the early developmental stages96
8.3.11 The intracellular region of Fat2 is required for proper egg chamber rotation in later developmental stages 99
9 DISCUSSION 103
9.1 Egg chamber elongation can be uncoupled from egg chamber rotation 104
9.2 Egg chamber elongation correlates with a functional molecular corset 107
9.3 Fat2 promotes egg chamber elongation by its extracellular region 109
9.4 Alternative mechanisms potentially drive egg chamber elongation 111
9.5 New model of egg chamber elongation 114
9.6 Future perspectives 116
9.7 Impact on tissue morphogenesis in general 119
10 ACKNOWLEDGEMENTS 120
11 REFERENCES 121
12 APPENDIX 134
12.1 Script for “FFTAlignment.m" 134
12.2 Script for “Test1” 143
12.3 Script for “AverageCellAlignment.m" 143 / Das Strecken von Geweben ist ein wichtiger Prozess bei der Gestaltbildung während der Entwicklung von Organismen. Die Eikammer von Drosophila ist ein hervorragendes Modellsystem, um die Gewebestreckung zu untersuchen. Eine Eikammer besteht aus Keimbahnzellen und einem einschichtigen Follikelepithel, das die Keimbahn umschließt. Während die Eikammer heranwächst durchläuft sie eine drastische Gestaltveränderung von rund nach oval. Zeitgleich zur Streckung der Eikammer weist das Follikelepithel parallel angeordnete F-actin−Filamente, Mikrotubuli und Fasern der extrazellulären Matrix (ECM) auf, welche die Eikammer ringsum umlaufen. Zudem rotieren die Eikammern um ihre zukünftige Längsachse. Bisher nahm man an, die Rotation würde für die Ausrichtung der F-actin−Filamente, Microtubuli und ECM-Fasern gebraucht werden. Die Anordnung der F-actin−Filamente und ECM-Fasern bilden dann ein molekulares Korsett, das die Gewebestreckung fördert.
Das atypische Cadherin Fat2 wird für die Rotation der Eikammern, die umlaufende Anordnung der F-actin–Filamente, Microtubuli und ECM-Fasern sowie für die Streckung der Eikammern benötigt. Die Mechanismen, mit denen Fat2 die Gewebestreckung beeinflusst, sind allerdings unbekannt. In meinem Projekt führte ich eine Struktur-Funktions-Analyse von Fat2 durch.
Ich generierte eine Version von Fat2 mit einer Deletion der kompletten intrazellulären Region und eine zweite, die weder die intrazelluläre Region noch die Transmembran-Domäne besitzt und testete, ob diese Versionen die Funktionen von Fat2 in fat2-/- mutanten Eikammern kompensieren können. Meine Ergebnisse zeigen, dass die intrazelluläre Region für die Anordnung der Mikrotubuli und für die Rotation der Eikammern gebraucht wird. Die intrazelluläre Region wird jedoch weder für die Anordnung von F-actin–Filamenten und den ECM-Fasern noch für die Streckung der Eikammer benötigt. Meine Erkenntnisse zeigen erstmalig, dass die Streckung der Eikammern ohne Rotation stattfinden kann. Meine Arbeit entkoppelt damit mehrere parallel stattfindende Prozesse während der Entwicklung der Eikammer und eröffnet einen neuen Einblick in die Mechanismen der Gewebestreckung in diesem wichtigen Modellsystem.:1 ABSTRACT I
2 ZUSAMMENFASSUNG II
3 TABLE OF CONTENTS III
4 LISTS 7
4.1 List of Abbreviations 7
4.2 List of figures 9
5 INTRODUCTION 11
5.1 Tissue morphogenesis during development 11
5.1.1 Tissue organization by differential cell affinity 11
5.1.2 Cell adhesion is mediated by cadherins12
5.1.3 The cytoskeleton drives cell shape changes 13
5.1.4 Planar polarity is required for tissue-level directionality 17
5.2 Models of tissue elongation 19
5.2.1 Germ-band extension in Drosophila melanogaster 19
5.2.2 Primitive streak formation in the chick embryo 21
5.2.3 Neural tube formation in Xenopus 22
5.3 Drosophila egg chamber as a model system to study tissue morphogenesis 24
5.3.1 Oogenesis in Drosophila 24
5.3.2 Egg chamber as a model for tissue elongation 27
5.3.3 Planar polarized organization of the F-actin cytoskeleton in the follicle epithelium 29
5.3.4 Egg chamber elongation requires a link between extracellular matrix and F-actin cytoskeleton 32
5.3.5 Egg chamber rotation is proposed to be a requisite for egg chamber elongation 34
5.3.6 The atypical cadherin Fat2 provides a key role during egg chamber elongation 35
6 AIMS OF THE THESIS 38
7 MATERIALS AND METHODS 39
7.1 Fly husbandry 39
7.2 Used fly stocks 39
7.3 Phenotypic markers 40
7.4 Ovary dissection for fixation 40
7.5 Antibody stainings 41
7.6 Used antibodies 42
7.7 Drug treatment 42
7.8 Microscopy of fixed samples 43
7.9 Live imaging 43
7.9.1 Imaging of the basal F-actin oscillations 43
7.9.2 Imaging of egg chamber rotation 44
7.10 Generation of the transgenic fosmid constructs 44
7.10.1 General materials required for molecular genetics in E.coli 46
7.10.2 Step 1: Amplification of the tagging cassette 47
7.10.3 Step 2: Transformation of the helper plasmid pRedFlp4 49
7.10.4 Step 3: Red-operon driven insertion of the tagging cassette 50
7.10.5 Step 4: Removal of the KanR gene 50
7.10.6 Step 5: DNA isolation and verification of the correct transgenic construct 50
7.10.7 Step 6: Integration of the transgene into the fly genome 52
7.11 Image analysis and quantifications 53
7.11.1 Statistics 53
7.11.2 Aspect ratio measurements 54
7.11.3 Quantification of GFP localization55
7.11.4 Quantification of tissue-wide Collagen IV alignment 55
7.11.5 Quantification of tissue-wide angles of F-actin and microtubules 57
7.11.6 Analysis of periodicity of F-actin oscillations 58
7.11.7 Quantification of the rotation velocity of egg chambers 60
8 RESULTS 61
8.1 Expression of full-length fat2-GFP gene fully rescues all aspects of the fat258D mutant phenotype 61
8.1.1 Expression of the fat2-GFP gene rescues the fat258D mutant egg shape and sterility 61
8.1.2 Using an ‘Alignment parameter’ SAP to quantify the directionality of cytoskeletal structures and extracellular matrix fibrils 63
8.1.3 Expression of the fat2-GFP gene rescues microtubule alignment of fat258D mutant egg chambers65
8.1.4 Expression of the fat2-GFP gene rescues F-actin and Collagen IV alignment of fat258D mutant egg chambers 67
8.2 Generation of different fat2 mutant transgenes by homologous recombineering 70
8.3 The intracellular region of Fat2 is dispensable for some specific aspects of the Fat2 functions 73
8.3.1 The egg chamber elongation is independent of the intracellular region of Fat2 73
8.3.2. Localization of Fat2 protein depends on the intracellular region of Fat2 76
8.3.3 The alignment of microtubules is dependent on the intracellular region of the protein 78
8.3.4 The intracellular region of Fat2 is required for proper early F-actin and Collagen IV fibril alignment 81
8.3.5 The intracellular region of Fat2 is required for late F-actin and Collagen IV fibril alignment 85
8.3.6 F-actin filaments and ECM fibrils co-align in fat258D mutant stage 8 egg chambers 88
8.3.7 F-actin filaments and ECM fibrils do not co-align in fat258D mutant stage 10 egg chambers 90
8.3.8 The stability of basal F-actin fibers and Collagen IV fibrils mutually depend on each other at stage 8 92
8.3.9 The contractile pulses of F-actin in stage 9 egg chambers are independent of the intracellular region of Fat293
8.3.10 The intracellular region of Fat2 is required for proper egg chamber rotation in the early developmental stages96
8.3.11 The intracellular region of Fat2 is required for proper egg chamber rotation in later developmental stages 99
9 DISCUSSION 103
9.1 Egg chamber elongation can be uncoupled from egg chamber rotation 104
9.2 Egg chamber elongation correlates with a functional molecular corset 107
9.3 Fat2 promotes egg chamber elongation by its extracellular region 109
9.4 Alternative mechanisms potentially drive egg chamber elongation 111
9.5 New model of egg chamber elongation 114
9.6 Future perspectives 116
9.7 Impact on tissue morphogenesis in general 119
10 ACKNOWLEDGEMENTS 120
11 REFERENCES 121
12 APPENDIX 134
12.1 Script for “FFTAlignment.m" 134
12.2 Script for “Test1” 143
12.3 Script for “AverageCellAlignment.m" 143
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The Sweet Side of the Extracellular Matrix -: Glycosaminoglycans in Matrix Remodeling, Endothelial Cell Activation and Functional BiomaterialsRother, Sandra 19 October 2017 (has links)
Bone fractures and pathologic conditions like chronic wounds significantly reduce the quality of life for the patients, which is especially dramatic in an elderly population with considerable multi-morbidity and lead to substantial socio-economic costs. To improve the wound healing capacity of these patients, new strategies for the design of novel multi-functional biomaterials are required: they should be able to decrease extensive pathologic tissue degradation and specifically control angiogenesis in damaged vascularized tissues like bone and skin.
Glycosaminoglycans (GAGs) like hyaluronan (HA) and chondroitin sulfate (CS) as important extracellular matrix (ECM) components are involved in several biological processes such as matrix remodeling and growth factor signaling, either by directly influencing the cellular response or by interacting with mediator proteins. This could be useful in functionalizing biomaterials, but native sulfated GAGs (sGAGs) show a high batch-to-batch variability and are limited in their availability. Chemically modified HA and CS derivatives with much more defined characteristics regarding their carbohydrate backbone, sulfate group distribution and sulfation degree are favorable to study the structure-function relationship of GAGs in their interaction with mediator proteins and/or cells and this might be used to precisely modulate activity profiles to stimulate wound healing.
By combining collagen type I as the main structural protein of the bone and skin ECM with these GAG derivatives, 2.5-dimensional (2.5D) and 3D artificial ECM (aECM) coatings and hydrogels were developed. These biomaterials as well as the respective GAG derivatives alone were compared to native GAGs and used to analyze how the sulfation degree, pattern and carbohydrate backbone of GAGs influence:
i) the activity of tissue inhibitor of metalloproteinase-3 (TIMP-3) and vascular endothelial growth factor-A (VEGF-A) as main regulators of ECM remodeling and angiogenesis,
ii) the composition and characteristics of the developed 2.5D and 3D aECMs,
iii) the enzymatic degradation of collagen-based aECMs and HA/collagen-based hydrogels,
iv) the proliferation and functional morphology of endothelial cells.
Surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA) binding studies revealed that sulfated HA (sHA) derivatives interact with TIMP-3 and VEGF-A in a sulfation-dependent manner. sHA showed an enhanced interplay with these proteins compared to native GAGs like heparin (HEP) or CS, suggesting a further impact of the carbohydrate backbone and sulfation pattern. sGAGs alone were weak modulators of the matrix metalloproteinase-1 and -2 (MMP-1 and -2) activity and did not interfere with the inhibitory potential of TIMP-3 against these proteinases during enzyme kinetic analyses. However, the formation of TIMP 3/GAG complexes reduced the binding of TIMP-3 to cluster II and IV of its endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1, mediates the up-take and degradation of TIMP-3 from the extracellular environment) in a sulfation- and GAG type-dependent manner. It is of note that the determined complex stabilities of TIMP-3 with cluster II and IV were almost identical indicating for the first time that both clusters contribute to the TIMP-3 binding. Competitive SPR experiments demonstrated that GAG polysaccharides interfere stronger with the TIMP 3/LRP-1 interplay than GAG oligosaccharides. The importance of the position of sulfation is highlighted by the finding that a sHA tetrasaccharide exclusively sulfated at the C6 position of the N-acetylglucosamine residues significantly blocked the receptor binding, while CS and HEP hexasaccharides had no detectable effects. Thus, sHA derivatives as part of biomaterials could be used to sequester and accumulate TIMP 3 in aECMs in a defined manner where sHA-bound TIMP-3 could decrease the matrix breakdown by potentially restoring the MMP/TIMP balance. GAG binding might extend the beneficial presence of TIMP-3 into wounds characterized by excessive pathologic tissue degradation (e.g. chronic wounds, osteoarthritis).
Mediator protein interaction studies with sHA coated surfaces showed the simultaneous binding of TIMP-3 and VEGF-A, even though the sHA/VEGF-A interplay was preferred. Moreover, kinetic analysis revealed almost comparable affinities of both proteins for VEGF receptor-2 (VEGFR-2), explaining their competition that mainly regulates the activation of endothelial cells. Additional SPR measurements demonstrated that the binding of sGAGs to TIMP-3 or VEGF-A decreases the binding of the respective mediator protein to VEGFR-2. Likewise, a sulfation-dependent reduction of the binding signal was observed after pre-incubation of a mixture of TIMP-3 and VEGF-A with sGAG poly- and oligosaccharides. The biological consequences of GAGs interfering with VEGF-A/VEGFR-2 and TIMP-3/VEGFR 2 were assessed in vitro using porcine aortic endothelial cells stably transfected with VEGFR 2 (PAE/KDR cells). The presence of sHA both decreased VEGF-A activity and the activity of TIMP-3 to inhibit the VEGF-A-induced VEGFR-2 phosphorylation. The same decreased activities could be observed for the migration of endothelial cells.
However, if sHA, TIMP-3 and VEGF-A were present simultaneously, sHA partially restored the TIMP-3-mediated blocking of VEGF-A activity. These findings provide novel insights into the regulatory potential of sHA during endothelial cell activation as an important aspect of angiogenesis, which could be translated into the design of biomaterials to treat abnormal angiogenesis. These sHA-containing materials might control the angiogenic response by modulating the activity of TIMP 3 and VEGF-A.
The in vitro fibrillogenesis of collagen type I in the presence of sHA derivatives led to 2.5D collagen-based aECM coatings with stable collagen contents and GAG contents that resemble the organic part of the bone ECM. A burst release of GAGs was observed during the first hour of incubation in buffer with the GAG content remaining almost constant afterwards, implying that the number of GAG-binding sites of collagen restricts the amounts of associated GAGs. Moreover, two differently sulfated HA derivatives could for the first time be incorporated into one multi-GAG aECM as verified via agarose gel electrophoresis and fluorescence measurements. This illustrates the multiple options to modify the aECM composition and thereby potentially their functionality. Atomic force microscopy showed that the presence of sHA derivatives during fibrillogenesis significantly reduced the resulting fibril diameter in a concentration- and sulfation-dependent manner, indicating an interference of the GAGs with the self-assembly of collagen monomers. In line with enzyme kinetic results, none of the GAGs as part of aECMs altered the enzymatic collagen degradation via a bacterial collagenase. Thus aECMs were proven to be biodegradable independent from their composition, which is favorable concerning a potential biomedical usage of the aECMs e.g. as implant coatings.
HA/collagen-based hydrogels containing fibrillar collagen embedded into a network of crosslinked HA and sGAGs were developed as 3D aECMs. Scanning electron microscopy demonstrated a porous structure of the gels after lyophilization, which could favor the cultivation of cells. The presence of collagen markedly enhanced the stability of the gels against the enzymatic degradation via hyaluronidase, something beneficial to clinical use as this is often limited by the generally fast breakdown of HA. Binding and release experiments with lysozyme, as positively charged model protein for e.g. pro-inflammatory cytokines, and VEGF A revealed that the sulfation of GAGs increased the protein binding capacity for pure GAG coatings and retarded the protein release from hydrogels compared to hydrogels without sGAGs. Moreover, the additional acrylation of sHA was shown to strongly reduce the interaction with both proteins when the primary hydroxyl groups were targets of acrylation. This stresses the influence of the substitution pattern on the protein binding properties of the GAG derivatives. However, hydrogel characteristics like the elastic modulus remained unaffected. The different interaction profiles of lysozyme and VEGF-A with GAGs demonstrated a protein-specific preference of different monosaccharide compositions, suggesting that the mediator protein binding could be simultaneously adjusted for several proteins by combining different GAG derivatives. This might allow the scavenging of pro-inflammatory cytokines and at the same time a binding and release of wound healing stimulating growth factors.
Since there is a growing demand for biomaterials to regenerate injured vascularized tissues like bone and skin, endothelial cells were used to examine the direct effects of solute GAGs and hydrogels containing these GAGs in vitro. In both cases, sHA strongly enhanced the proliferation of PAE/KDR cells. A VEGFR-2-mediated effect of GAGs on endothelial cells as underlying mechanism is unlikely since GAGs alone did not bind to VEGFR-2 and had no influence on VEGFR-2 phosphorylation. Other factors like GAG-induced alterations of cell-matrix interactions and cell signaling could be responsible. In accordance with SPR results, a decreased endothelial cell proliferation stimulating activity of VEGF-A was observed in the presence of solute GAGs or after binding to hydrogels compared to the respective treatment without VEGF-A. However, tube formation could be observed in the presence of solute VEGF A and GAGs and within hydrogels with sGAGs that released sufficient VEGF-A amounts over time. Overall the presence of GAGs and VEGF-A strongly promoted the endothelial cell proliferation compared to the treatment with GAGs or VEGF-A alone. Thus, HA/collagen-based hydrogels functionalized with sHA derivatives offer a promising option for the design of “intelligent” biomaterials that direct and regulate the cellular behavior instead of simply acting as inert filling material. They could be used for the controlled delivery and/or scavenging of multiple mediator proteins, thus enhancing the local availability or reducing the activity of these GAG-interacting mediator proteins, or by directly influencing the cellular response. This might be applied to a range of pathological conditions by tuning the biomaterial compositions to patient-specific needs.
However, extensive in vivo validation is required to show whether these in vitro findings could be used to control the biological activity of for instance TIMP-3 and VEGF-A, especially under the pathological conditions of extended matrix degradation and dysregulated angiogenesis.
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Screening a chemically defined extracellular matrix mimetic substrate library to identify substrates that enhance substratemediated transfectionHamann, Andrew, Thomas, Alvin K., Kozisek, Tyler, Farris, Eric, Lück, Steffen, Zhang, Yixin, Pannier, Angela K. 19 May 2022 (has links)
Nonviral gene delivery, though limited by inefficiency, has extensive utility in cell therapy, tissue engineering, and diagnostics. Substrate-mediated gene delivery (SMD) increases efficiency and allows transfection at a cell-biomaterial interface, by immobilizing and concentrating nucleic acid complexes on a surface. Efficient SMD generally requires substrates to be coated with serum or other protein coatings to mediate nucleic acid complex immobilization, as well as cell adhesion and growth; however, this strategy limits reproducibility and may be difficult to translate for clinical applications. As an alternative, we screened a chemically defined combinatorial library of 20 different extracellular matrix mimetic substrates containing combinations of (1) different sulfated polysaccharides that are essential extracellular matrix glycosaminoglycans (GAGs), with (2) mimetic peptides derived from adhesion proteins, growth factors, and cell-penetrating domains, for use as SMD coatings. We identified optimal substrates for DNA lipoplex and polyplex SMD transfection of fibroblasts and human mesenchymal stem cells. Optimal extracellular matrix mimetic substrates varied between cell type, donor source, and transfection reagent, but typically contained Heparin GAG and an adhesion peptide. Multiple substrates significantly increased transgene expression (i.e. 2- to 20-fold) over standard protein coatings. Considering previous research of similar ligands, we hypothesize extracellular matrix mimetic substrates modulate cell adhesion, proliferation, and survival, as well as plasmid internalization and trafficking. Our results demonstrate the utility of screening combinatorial extracellular matrix mimetic substrates for optimal SMD transfection towards application- and patient-specific technologies.
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