yes / Biological hydrogels have been increasingly sought after as wound dressings or scaffolds for regenerative medicine, owing to their inherent biofunctionality in biological environments. Especially in moist wound healing, the ideal material should absorb large amounts of wound exudate while remaining mechanically competent in situ. Despite their large hydration, however, current biological hydrogels still leave much to be desired in terms of mechanical properties in physiological conditions. To address this challenge, a multi-scale approach is presented for the synthetic design of cyto-compatible collagen hydrogels with tunable mechanical properties (from the nano- up to the macro-scale), uniquely high swelling ratios and retained (more than 70%) triple helical features. Type I collagen was covalently functionalized with three different monomers, i.e. 4-vinylbenzyl chloride, glycidyl methacrylate and methacrylic anhydride, respectively. Backbone rigidity, hydrogen-bonding capability and degree of functionalization (F: 16 ± 12–91 ± 7 mol%) of introduced moieties governed the structure–property relationships in resulting collagen networks, so that the swelling ratio (SR: 707 ± 51–1996 ± 182 wt%), bulk compressive modulus (Ec: 30 ± 7–168 ± 40 kPa) and atomic force microscopy elastic modulus (EAFM: 16 ± 2–387 ± 66 kPa) were readily adjusted. Because of their remarkably high swelling and mechanical properties, these tunable collagen hydrogels may be further exploited for the design of advanced dressings for chronic wound care.

The expanding field of tissue engineering provides a new approach to regenerative medicine for common ailments such as cardiovascular disease and type-I diabetes. Biomaterials can be administered as a delivery vehicle to introduce therapeutic cells to sites of damaged or diseased tissue. A specific class of biomaterials, termed hydrogels, is suitable for this application as they can provide a biocompatible, biodegradable scaffold that mimics the physical properties of the native soft tissue. Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in the delivery of therapeutics such as cells or growth factor-releasing particles. In this study, the first aim was to determine the interactive effects between collagen-based hydrogels and additives (cells and microspheres) for cardiac regeneration. The results demonstrated that the addition of either cells or microspheres to a collagen-based hydrogel decreased its gelation time and increased its viscosity. Increased cross-linker concentrations resulted in lower cell viability. However, this cell loss could be minimized by delivering cells with the cross-linker neutralizing agent, glycine. As a potential application of these materials, the second aim of this study was to develop a hydrogel for use as an ectopic islet transplant site. Specifically, collagen-chitosan hydrogels were synthesized and characterized, with and without laminin, and tested for their ability to support angiogenic and islet cell survival and function. Matrices synthesized with lower chitosan content (20:1 collagen:chitosan) displayed greater cell compatibility for both angiogenic cells and for islets and weaker mechanical properties, while matrices with higher chitosan content (10:1 collagen:chitosan) had the opposite effect. Laminin did not affect the physical properties of the matrices, but did improve angiogenic cell and islet survival and function. Overall the proposed collagen-based hydrogels can be tailored to meet the physical property requirements for cardiac and islet tissue engineering applications and demonstrated promising cell support capabilities.

The expanding field of tissue engineering provides a new approach to regenerative medicine for common ailments such as cardiovascular disease and type-I diabetes. Biomaterials can be administered as a delivery vehicle to introduce therapeutic cells to sites of damaged or diseased tissue. A specific class of biomaterials, termed hydrogels, is suitable for this application as they can provide a biocompatible, biodegradable scaffold that mimics the physical properties of the native soft tissue. Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in the delivery of therapeutics such as cells or growth factor-releasing particles. In this study, the first aim was to determine the interactive effects between collagen-based hydrogels and additives (cells and microspheres) for cardiac regeneration. The results demonstrated that the addition of either cells or microspheres to a collagen-based hydrogel decreased its gelation time and increased its viscosity. Increased cross-linker concentrations resulted in lower cell viability. However, this cell loss could be minimized by delivering cells with the cross-linker neutralizing agent, glycine. As a potential application of these materials, the second aim of this study was to develop a hydrogel for use as an ectopic islet transplant site. Specifically, collagen-chitosan hydrogels were synthesized and characterized, with and without laminin, and tested for their ability to support angiogenic and islet cell survival and function. Matrices synthesized with lower chitosan content (20:1 collagen:chitosan) displayed greater cell compatibility for both angiogenic cells and for islets and weaker mechanical properties, while matrices with higher chitosan content (10:1 collagen:chitosan) had the opposite effect. Laminin did not affect the physical properties of the matrices, but did improve angiogenic cell and islet survival and function. Overall the proposed collagen-based hydrogels can be tailored to meet the physical property requirements for cardiac and islet tissue engineering applications and demonstrated promising cell support capabilities.

The expanding field of tissue engineering provides a new approach to regenerative medicine for common ailments such as cardiovascular disease and type-I diabetes. Biomaterials can be administered as a delivery vehicle to introduce therapeutic cells to sites of damaged or diseased tissue. A specific class of biomaterials, termed hydrogels, is suitable for this application as they can provide a biocompatible, biodegradable scaffold that mimics the physical properties of the native soft tissue. Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in the delivery of therapeutics such as cells or growth factor-releasing particles. In this study, the first aim was to determine the interactive effects between collagen-based hydrogels and additives (cells and microspheres) for cardiac regeneration. The results demonstrated that the addition of either cells or microspheres to a collagen-based hydrogel decreased its gelation time and increased its viscosity. Increased cross-linker concentrations resulted in lower cell viability. However, this cell loss could be minimized by delivering cells with the cross-linker neutralizing agent, glycine. As a potential application of these materials, the second aim of this study was to develop a hydrogel for use as an ectopic islet transplant site. Specifically, collagen-chitosan hydrogels were synthesized and characterized, with and without laminin, and tested for their ability to support angiogenic and islet cell survival and function. Matrices synthesized with lower chitosan content (20:1 collagen:chitosan) displayed greater cell compatibility for both angiogenic cells and for islets and weaker mechanical properties, while matrices with higher chitosan content (10:1 collagen:chitosan) had the opposite effect. Laminin did not affect the physical properties of the matrices, but did improve angiogenic cell and islet survival and function. Overall the proposed collagen-based hydrogels can be tailored to meet the physical property requirements for cardiac and islet tissue engineering applications and demonstrated promising cell support capabilities.

No description available.

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.

Biological materials such as extracellular matrix scaffolds, cancer cells, and tissues are often assumed to respond elastically for simplicity; the viscoelastic response is quite commonly ignored. Extracellular matrix mechanics including the viscoelasticity has turned out to be a key feature of cellular behavior and the entire shape and function of healthy and diseased tissues, such as cancer. The interference of cells with their local microenvironment and the interaction among different cell types relies both on the mechanical phenotype of each involved element. However, there is still not yet clearly understood how viscoelasticity alters the functional phenotype of the tumor extracellular matrix environment. Especially the biophysical technologies are still under ongoing improvement and further development. In addition, the effect of matrix mechanics in the progression of cancer is the subject of discussion. Hence, the topic of this review is especially attractive to collect the existing endeavors to characterize the viscoelastic features of tumor extracellular matrices and to briefly highlight the present frontiers in cancer progression and escape of cancers from therapy. Finally, this review article illustrates the importance of the tumor extracellular matrix mechano-phenotype, including the phenomenon viscoelasticity in identifying, characterizing, and treating specific cancer types.

Biological hydrogels such as collagen and gelatin are highly attractive materials for tissue engineering and biomedicine. Due to their excellent biocompatibility and biodegradability, they represent promising candidates in regenerative medicine, cell culture, tissue replacement and wound dressing applications. Thereby, precisely tuned material properties are indispensable for customization. High-energy electron-treatment is a highly favourable crosslinking technique to tailor the material properties. In five sub-projects, this thesis investigates the potential of high-energy electron-treatment to precisely modify collagen hydrogels, to develop thermo- as well as hydration-sensitive systems and functional surfaces from gelatin for biomedical applications. The first sub-project focusses on the modification of collagen hydrogels by electron-induced crosslinking with potential application as biomimetic extracellular matrix material. Thereby, it is shown that the material properties can be precisely tailored by adapting electron-induced crosslinking while high cytocompatibility is maintained. Within the second sub-project, an electron-crosslinking-induced shape-memory effect in gelatin is described in order to develop a thermo-responsive system. The effect is described experimentally as well as theoretically to demonstrate the fundamental physical processes. The third sub-project develops an electroncrosslinked hydration-sensitive gelatin system. The work discusses how swelling of electroncrosslinked gelatin is influenced by the pH-value and salt concentration of the swelling liquid. Thereby, response of the hydration-sensitive gelatin system can be further modified towards biological actuatoric systems. The fourth sub-project develops a two-step process to mechanically pattern gelatin surfaces. Within the first step, thin gelatin surfaces are mechanically patterned by a highly focussed electron beam. In a second step, they are stabilized by homogeneous electron-crosslinking for applications at physiological conditions. Another method to develop functional gelatin surfaces is described in the last sub-project. Here, gelatin is topographically patterned via a moulding technique. The resulting micro-structures are then stabilized via electron-crosslinking. In addition, the presented work investigates pattern transfer, long time stability at physiological conditions as well as cytocompatibility.:1 Introduction and Objective 1.1 Biomimetic ECM Models 1.2 Stimuli-Responsive Hydrogels 1.3 Functional Hydrogel Surfaces 2 General Background 2.1 Hydrogels 2.1.1 Collagen 2.1.2 Gelatin 2.2 Polymer Crosslinking 2.2.1 High-Energy Electron-Treatment of Polymers 2.2.2 Electron-Irradiation-Induced Crosslinking of Gelatin 2.3 High-Energy Electron Accelerator 3 Cumulative Part 3.1 High-Energy Electron-Induced Modification of Collagen 3.2 Thermo-Responsive Gelatin System 3.3 Hydration-Responsive Gelatin System 3.4 Mechanically Patterned Gelatin Surfaces 3.5 Topographically Patterned Gelatin Surfaces 4 Summary and Conclusion 5 Outlook Bibliography Author Contributions List of Abbreviations List of Figures Acknowledgements Scientific Curriculum Vitae Publication List Selbstständigkeitserklärung / Biologische Hydrogele wie Kollagen und Gelatine sind wichtige Materialien vor allem in biomedizinischen Anwendungsbereichen. Durch deren exzellente Biokompatibilität und biologische Abbaubarkeit werden sie vor allem bei der Züchtung von biomimetischem Gewebe, in der Zellkultur, als Gewebeersatz in der regenerativen Medizin oder auch als Wundverband eingesetzt. In der Verwendung solcher Materialien besteht eine wesentliche Herausforderung darin, deren Eigenschaften so präzise wie möglich einzustellen, um speziell angepasste Substrate und Gewebe entwickeln zu können. Eine äußerst vorteilhafte Methode zu Adaptierung der Materialeigenschaften ist die elektronenstrahlbasierte Vernetzung, die auf die Verwendung zusätzlicher chemischer Vernetzer verzichtet. Die vorgelegte Arbeit untersucht in fünf Teilprojekten das Potential von Elektronenstrahlvernetzung zur Modifizierung von Kollagen- sowie Gelatinehydrogelen für biomedizinische Anwendungen. Das erste Teilprojekt fokussiert sich auf die Auswirkungen hochenergetischer Elektronen auf Kollagenhydrogele und deren Eigenschaften für potentielle Anwendungen als biomimetisches Modell der extrazellulären Matrix. Dabei wird gezeigt, dass sich die Materialeigenschaften in Abhängigkeit der Elektronenbestrahlung präzise einstellen lassen und dass diese Gele eine hohe Zellkompatibilität aufweisen. Das zweite Teilprojekt beschreibt den Effekt des thermischen Formgedächtnisses in Gelatine nach Elektronenstrahlvernetzung und dessen Potential für die Entwicklung biologischer Aktuatoren. Die Effizienz des Formgedächtniseffekts wird in diesem Teilprojekt ausführlich theoretisch beschrieben und mit experimentellen Untersuchungen an Gelatine verglichen. Im dritten Teilprojekt wird ein elektronenstrahlvernetztes, hydrations-responsives Gelatinesystem beschrieben. Zusätzlich wird der Einfluss von pH-Wert und Salzkonzentration der Quelllösung auf das Quellen von elektronenstrahlvernetzter Gelatine untersucht um das Reaktionsverhalten noch präziser einstellen zu können. Das vierte Teilprojekt beschreibt einen Zwei-Schritt-Prozess, bei dem dünne Gelatineschichten mittels hochenergetischer Elektronen mechanisch funktionalisiert werden können. Dabei wird in einem ersten Schritt die Oberfläche durch hoch fokussierte Elektronen mechanisch strukturiert, um im zweiten Schritt mittels homogener Elektronenstrahlvernetzung für die Anwendung unter physiologischen Bedingungen stabilisiert zu werden. Eine weitere Methode zur Funktionalisierung der Oberfläche von Gelatinehydrogelen wird im letzten Teilprojekt dieser Arbeit dokumentiert. Dabei werden topographische Mikrostrukturen auf Gelatineoberflächen aufgebracht und mittels Elektronenstrahlvernetzung stabilisiert. Dieses Teilprojekt untersucht zusätzlich den Strukturtransfer, die Langzeitstabilität unter physiologischen Bedingungen sowie die Zellkompatibilität.:1 Introduction and Objective 1.1 Biomimetic ECM Models 1.2 Stimuli-Responsive Hydrogels 1.3 Functional Hydrogel Surfaces 2 General Background 2.1 Hydrogels 2.1.1 Collagen 2.1.2 Gelatin 2.2 Polymer Crosslinking 2.2.1 High-Energy Electron-Treatment of Polymers 2.2.2 Electron-Irradiation-Induced Crosslinking of Gelatin 2.3 High-Energy Electron Accelerator 3 Cumulative Part 3.1 High-Energy Electron-Induced Modification of Collagen 3.2 Thermo-Responsive Gelatin System 3.3 Hydration-Responsive Gelatin System 3.4 Mechanically Patterned Gelatin Surfaces 3.5 Topographically Patterned Gelatin Surfaces 4 Summary and Conclusion 5 Outlook Bibliography Author Contributions List of Abbreviations List of Figures Acknowledgements Scientific Curriculum Vitae Publication List Selbstständigkeitserklärung

No description available.

Le rachitisme vitamino-résistant hypophosphatémique (RVRH) est une maladie génétique rare causée par des mutations du gène PHEX. La perte de fonction de la protéine PHEX conduit à l’augmentation du FGF23, une hormone circulante qui agit sur le rein et entraîne une perte systémique de phosphate. Le squelette rachitique des patients atteints de RVRH présente des déformations osseuses et une ostéomalacie. La dentine hypominéralisée des patients est à l’origine d’abcès dentaires fréquents, mais le statut parodontal des patients RVRH est mal connu, de même que leur risque de développer une parodontite pouvant aboutir à la perte des dents. La fonction et le substrat de la protéine PHEX ne sont pas identifiés avec exactitude. Il a été montré in vitro que PHEX avait la capacité d’interagir et de dégrader des protéines membres de la famille des SIBLINGs comme MEPE ou OPN, toutes les deux impliquées dans la régulation de la minéralisation des tissus osseux et dentinaires, mais on ne sait pas si in vivo les défauts de minéralisation observés résultent principalement de l’hypophosphatémie systémique ou bien également des effets directs de l’absence de PHEX sur les protéines régulatrices de la minéralisation. L’objectif de cette thèse a consisté à s’intéresser à la physiopathologie du parodonte dans le RVRH ainsi qu’à déterminer quel était l’impact de la mutation de PHEX dans un modèle de biominéralisation humaine où les conditions de concentration en phosphate pouvaient être ajustées et normalisées. Nous avons d’abord analysé le statut parodontal de 34 patients RVRH dans une étude clinique cas-témoins et ainsi montré que les malades dont la supplémentation en phosphate et vitamine D était tardive ou incomplète présentaient une fréquence et une sévérité accrues de maladie parodontale. Le phénotype parodontal du RVRH a alors été étudié sur des échantillons humains et sur le modèle murin du RVRH, la souris HYP. Nous avons réalisé un modèle d’égression dentaire de façon à permettre une apposition du cément cellulaire, ainsi qu’un modèle de résorption et de réparation osseuses parodontales afin de caractériser l’impact du RVRH sur la physiopathologie parodontale. Nos résultats ont montré que le phénotype parodontal et sa physiopathologie étaient très perturbés dans le rachitisme vitamino-résistant hypophosphatémique et chez la souris HYP, nous avons aussi pu mettre en évidence que le rôle pathologique majeur joué par l’ostéopontine dans le tissu osseux au cours du RVRH ne pouvait pas être généralisé aux autres tissus minéralisés du parodonte. De façon à identifier le rôle de PHEX dans la minéralisation matricielle locale indépendamment de la phosphatémie systémique, nous avons ensemencé des matrices de collagène dense avec des cellules primaires humaines issues de patients RVRH comparés à des contrôles que nous avons cultivés pendant 24 jours en conditions ostéogéniques avec des concentrations en phosphate identiques. Nos résultats ont montré que malgré une concentration normale en phosphate, la perte de fonction de la protéine PHEX entraînait une diminution de la quantité et de la qualité de la phase minérale et une accumulation et une dégradation pathologiques de la protéine OPN. Les contributions originales de ce travail de thèse doctorale ont consisté à démontrer sur le plan clinique et biologique la susceptibilité accrue du rachitisme hypophosphatémique lié à l’X quant au risque de développer une maladie parodontale, ainsi qu’à apporter la preuve d’un rôle pathologique de l’absence de PHEX indépendant de la phosphatémie sur des cultures primaires humaines. / X-linked hypophosphatemia (XLH) is a rare X-linked dominant disorder caused by inactivating mutations in the PHEX gene. The impairment of PHEX protein leads to an increase in FGF23, a circulating factor that causes systemic loss of phosphate. The rachitic skeleton of patients with XLH displays short stature and osteomalacia. Dental defects include poorly mineralized dentin and spontaneous dental abscesses. Little is known about the periodontal condition of XLH and if patients are more prone to develop periodontitis, eventually leading to tooth loss. Although the exact function and substrate of PHEX are not known, it has been shown in vitro that PHEX could interact with SIBLING proteins such as MEPE or OPN, both involved in the regulation of bone and dentin mineralization, but it is not yet clear if the defects in the calcified extracellular matrices of XLH are caused by systemic hypophosphatemia only, or also by local consequences of the absence of PHEX. The aim of this doctoral dissertation was to explore the pathobiology of the XLH periodontium and to determine the impact of PHEX deficiency at the local level in a model of human biomineralization where phosphate supply could be adjusted and normalized. We first examined 34 adults with XLH in a case-control study and observed that periodontitis frequency and severity were increased in individuals with late or incomplete supplementation in phosphate and vitamin D analogs. The periodontium was then analyzed in XLH dental roots and further characterized in the Hyp mouse, the murine model of XLH. We performed a model of tooth movement adaptation leading to the formation of cellular cementum and a model of periodontal breakdown and repair to investigate the impact of XLH on the pathobiology of periodontal tissues. Our results showed strongly affected XLH/Hyp periodontal phenotype and impaired pathobiology and suggested that the key role played by OPN in bone could not be generalized to other periodontal mineralized tissues. In order to determine the role of PHEX in local human mineralization, dense collagen gels were seeded with primary human dental pulp cells harvested from XLH patients displaying PHEX mutations and age-matched healthy individuals. Cell-seeded gels were cultured up to 24 days under osteogenic conditions and controlled phosphate medium concentrations. Our results showed that despite normal phosphate concentrations, PHEX deficiency led to decreased quantity and quality of the mineral phase and a pathologic accumulation and processing of OPN. Overall the original contributions of this doctoral dissertation consist in the demonstration of a higher susceptibility of XLH patients to periodontitis and in the evidence of a local effect of PHEX deficiency in the pathologic intrinsic mineralization from XLH osteogenic cells.