Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Os desenvolvimentos teóricos e experimentais na área de biomateriais têm sido aplicados diretamente a distintos campos da Medicina (odontologia, medicina regenerativa e radioterapia). Esses avanços foram concentrados tanto para diagnosticar doenças como para a quantificação de seus graus de progressão. Na perspectiva desses estudos, biomateriais estão sendo projetados e confeccionados para aplicação em diversas áreas da ciência, proporcionado avanços no radiodiagnóstico, na dosimetria para radioterapia e na calibração de equipamentos radioterápicos. Desenvolver um phantom a partir de um biomaterial se tornou um grande aliado da Medicina no tratamento de pacientes com doenças oncológicas, possibilitando melhor desempenho dos equipamentos, com a finalidade de redução dos danos causados ao tecido sadio devido ao excesso de exposição à radiação. Este trabalho utilizou polímeros: quitosana e gelatina para confecção das estruturas poliméricas e foi possível controlar as diferentes formas de produção e processamento, caracterizar e avaliar o biopolímero por técnicas físicas (ELT,MEV, e DEI) e, por consequência, analisar a aplicabilidade como phantom pulmonar de camundongo. Foi possível avaliar a morfologia dos biomateriais quantitativamente por microscopia eletrônica de varredura associada a técnica de imagem. A relevância deste trabalho se concentra em desenvolver um phantom a partir de biomateriais poliméricos que possa atuar como objeto simulador fornecendo alto contraste de imagem quando submetido a análise. Dessa forma, a escolha da técnica DEI foi satisfatória uma vez que trata-se de uma técnica de imagem de raios X de alta resolução. As imagens obtidas por DEI têm mostrado os detalhes da microestrutura interna dos biomateriais produzidos as quais possuem ≈ 10 μm de dimensão. Os phantoms confeccionados apresentaram densidade variando de 0,08 a 0,13 g/cm3. / The theoretical and experimental developments in the biomaterials area have been directly applied to different fields of Medicine (odontology, regenerative medicine and radiotherapy). These advances have focused both for diagnosing diseases such as for quantifying degrees of progression. From the perspective of these studies, biomaterials are being designed and manufactured for application in various areas of science, provided advances in diagnostic radiology, radiotherapy dosimetry and calibration of radiotherapy equipment. Develop a phantom from a biomaterial has become a great ally of medicine in the treat patients with oncological diseases, allowing better performance of the equipment in order to reduce damage to healthy tissue due to excessive exposure to radiation. This work used polymers: chitosan and gelatin, for making the polymeric structures and controlled for different types of production and processing, characterizing and evaluating the biopolymer by physical techniques (STL, SEM and DEI) and therefore analyze applicability as phantom mouse lung. It was possible to evaluate the morphology of biomaterials quantitatively by scanning electron microscopy associated with imaging technique. The relevance of this work focuses on developing a phantom from polymeric biomaterials that can act as phantom providing high image contrast when subjected to analysis. Thus, the choice of DEI technique is satisfactory since it is an imaging technique of X-ray high resolution. The images obtained by DEI have shown the details of the internal microstructure of the biomaterial produced which have ≈ 10 μm dimension. The phantoms had made density ranging from 0.08 a 0.13 g/cm3. / Tese (Doutorado)

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

Gene therapy is a potential strategy for treatment of diseases caused by a gene defect. Recent studies are involved particulary in the cure of diseases caused by single gene defect (cystic fibrosis, haemophilia, muscular dystrophy etc.). Our work is part of a project aiming at developing ex vivo non-viral gene delivery systems that could be used for the treatment of ocular and cardiovascular diseases. The gene vectors are biodegradable polymeric carriers based on poly-α-amino acids. These polyplexes should transfect target cells which are supposed to be seeded on polyimide membranes. The biodegradable polymer membrane will be implanted into the retina or used as a coating for cardiovascular prosthesis. As a cover of the implantable membranes we used polymerized methacrylamide-modified gelatin forming hydrogels and mediating a growth support for transfected cells. We focus on material bio- and immunocompatibility/immunoacceptability. The results indicated a very good bio- and immunocompatibility of the gelatin B hydrogel both in vitro and in vivo. The gelatin B hydrogel did not cause erythrocytes lysis, stimulation of proliferation (spontaneous or mitogen-induced) of mouse or human lymphoid cells, neither production of cytokines or NO in vitro. Histological examination following subcutaneous...

Inledning: Sjuka barn behöver anpassad vård och säkra, effektiva och väldokumenterade läkemedel. Förskrivning och uttag av preparat för pediatriska populationen ökar, men en tydlig uppskattning på problematik finns inte. Problem kan uppstå, när en lämplig beredning saknas, när redan registrerade läkemedel saknar avdelade doser för barn eller är tillgängliga enbart som en tablett med vuxen dos. Varje barn sägs vara en individ med unika läkemedelsomsättning, metabolism och biverkningspanorama, vilket komplicerar behandling. Lösningen på detta är i många fall ett extemporeläkemedel eller ett licenspreparat, men långa ledtider och dålig tillgänglighet kan medföra svårigheter att kunna ge rätt terapi. Syftet med denna studie är att i) kartlägga behov och befintliga lösningar, ii) testa handhållna pulverdispenser (HPD) Quantos, som en lämplig metod för fasta beredningar för att tillhandahålla individuella läkemedelsdoser till barn i de fall godkända läkemedel inte räcker.  Metod: Databassökning, intervjuer av hälso-sjukvårdspersonal, samt laborativt arbete för att omformulera registrerade läkemedel i tablettformer till individanpassade doser i hårdgelatin-kapslar med hjälp av Mettler-Toledos handhållna pulverdoseringsinstrument HPD Quantos. Resultat: Litteraturstudien och intervjuer överensstämmer med varandra: behov av barnanpassade läkemedel finns. HPD Quantos kan vara en alternativ metod för fasta beredningar för att tillhandahålla mängderför uppdosering med en femte- och/ eller en sjättedel av en tablett. Slutsats: För att ombesörja behoven för barnanpassade doser på ett sjukhus, måste HPD Quantos automatiseras till en inbyggd doseringsstation. Detta kommer att säkerställa dosering, dölja obehaglig smak, samt minska arbetsmiljörisken vid exponering av toxiska läkemedel.

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Toward the next generation of nerve guidance conduits (NGCs), novel biomaterials and functionalization concepts are required to address clinical demands in peripheral nerve regeneration (PNR). As a biological polymer with bioactive motifs, gelatinous peptides are promising building blocks. In combination with an anhydride-containing oligomer, a dual-component hydrogel system (cGEL) was established. First, hollow cGEL tubes were fabricated by a continuous dosing and templating process. Conduits were characterized concerning their mechanical strength, in vitro and in vivo degradation and biocompatibility. Second, cGEL was reformulated as injectable shear thinning filler for established NGCs, here tyrosine-derived polycarbonate-based braided conduits. Thereby, the formulation contained the small molecule LM11A-31. The biofunctionalized cGEL filler was assessed regarding building block integration, mechanical properties, in vitro cytotoxicity, and growth permissive effects on human adipose tissue-derived stem cells. A positive in vitro evaluation motivated further application of the filler material in a sciatic nerve defect. Compared to the empty conduit and pristine cGEL, the functionalization performed superior, though the autologous nerve graft remains the gold standard. In conclusion, LM11A-31 functionalized cGEL filler with extracellular matrix (ECM)-like characteristics and specific biochemical cues holds great potential to support PNR.

Les maladies cardiovasculaires sont une des causes de mortalités les plus élevées mondialement. Parmi celles-ci, on retrouve l’infarctus du myocarde, qui n’a pour traitement que la transplantation cardiaque. Or, dû à la faible quantité de donneur, une solution alternative est recherchée. De ce fait, l’ingénierie tissulaire permet le développement de tissus et d’implants thérapeutiques tels les patchs cardiaques, qui peuvent être bioimprimés. Or, une des limitations actuelles de l’utilisation d’une telle stratégie est la vascularisation de tissu bioimprimés. Dans cette étude, la bioimpression 3D a été utilisée afin de bioimprimer des patchs perfusables de gélatine méthacrylate (GelMA) à utiliser potentiellement pour le tissu cardiaque. Il a été possible de développer une bioencre pouvant être utilisée pour une application dans le tissu cardiaque, d’évaluer l’imprimabilité de l’encre et de bioimprimer de patchs standards et perfusables. Pour ce faire, GelMA a été synthétisé et les propriétés mécaniques ont été évaluées pour finalement sélectionner une encre de 10 % GelMA, ayant un module de Young approprié pour le tissu cardiaque, de 23,7±5,1 kPa. Par la suite, les processus d’impression, standard et coaxial, de patchs standards et perfusables ont pu être optimisés. Finalement, des patchs perfusables de GelMA 10% et gélatine 2% ont pu être imprimés avec une viabilité cellulaire élevée, jusqu’à 79,7±8,7 % et 83,5±5,7 % obtenue aux jours 1 et 7 de culture respectivement, avec des fibroblastes 3T3. La présence de canaux vides et la perfusabilité des patchs démontrent le potentiel de cette méthode pour éventuellement bioimprimer des patchs cardiaques vascularisés épais. / Cardiovascular diseases are a leading cause of death worldwide. Myocardial infarction captures a significant segment of this population, and the end-stage myocardial infarction can only be treated by heart transplantation. However, due to the scarcity donors, tissue engineering has been considered as an alternative solution. Tissue engineering allows the development of tissues and therapeutic implants such as cardiac patches. However, one of the main hurdles in the use of such a strategy is the vascularization of bioprinted tissue. In this study, 3D bioprinting was used to bioprint perfusable gelatin methacrylate (GelMA) patches for a potential use in cardiac tissue. This work consists in the development of a bioink that can be used for the cardiac tissue, the evaluation of the printability of the ink, and the final bioprinting of standard and perfusable patches. For this purpose, GelMA was synthesized and a final concentration of 10 % was selected as it showed an appropriate Young's modulus for cardiac tissue, of 23.7±5.1 kPa, while maintaining high biocompatibility. Subsequently, the printing process of standard and perfusable patches could be optimized with the use of GelMA and gelatin inks. Finally, 10% GelMA and 2% gelatin vascularized patches could be printed with high cell viability, of up to 79,7±8,7 % and 83,5±5,7 % on days 1 and 7 of culture respectively for 3T3 fibroblasts. Additionally, the presence of hollow channels of the perfusable patches demonstrates the potential of this method to be eventually applied to the bioprinting of thick vascularized cardiac patches.

Three-dimensional printing technologies exhibit tremendous potential in the advancing fields of tissue engineering and regenerative medicine due to the precise spatial control over depositing the biomaterial. Despite their widespread utilization and numerous advantages, the development of suitable novel biomaterials for extrusion-based 3D printing of scaffolds that support cell attachment, proliferation, and vascularization remains a challenge. Multi-material composite hydrogels present incredible potential in this field. Thus, in this work, a multi-material composite hydrogel with a promising formulation of chitosan/gelatin functionalized with egg white was developed, which provides good printability and shape fidelity. In addition, a series of comparative analyses of different crosslinking agents and processes based on tripolyphosphate (TPP), genipin (GP), and glutaraldehyde (GTA) were investigated and compared to select the ideal crosslinking strategy to enhance the physicochemical and biological properties of the fabricated scaffolds. All of the results indicate that the composite hydrogel and the resulting scaffolds utilizing TPP crosslinking have great potential in tissue engineering, especially for supporting neo-vessel growth into the scaffold and promoting angiogenesis within engineered tissues.

Yes / Infection is the major reason for guided tissue regeneration/guided bone regeneration (GTR/GBR) membrane failure in clinical application. In this work, we developed GTR/GBR membranes with localized drug delivery function to prevent infection by electrospinning of poly(ε-caprolactone) (PCL) and gelatin blended with metronidazole (MNA). Acetic acid (HAc) was introduced to improve the miscibility of PCL and gelatin to fabricate homogeneous hybrid nanofiber membranes. The effects of the addition of HAc and the MNA content (0, 1, 5, 10, 20, 30, and 40 wt.% of polymer) on the properties of the membranes were investigated. The membranes showed good mechanical properties, appropriate biodegradation rate and barrier function. The controlled and sustained release of MNA from the membranes significantly prevented the colonization of anaerobic bacteria. Cells could adhere to and proliferate on the membranes without cytotoxicity until the MNA content reached 30%. Subcutaneous implantation in rabbits for 8 months demonstrated that MNA-loaded membranes evoked a less severe inflammatory response depending on the dose of MNA than bare membranes. The biodegradation time of the membranes was appropriate for tissue regeneration. These results indicated the potential for using MNA-loaded PCL/gelatin electrospun membranes as anti-infective GTR/GBR membranes to optimize clinical application of GTR/GBR strategies.

Fibronectin is an extracellular multidomain glycoprotein that directs and regulates a variety of cell processes such as proliferation, development, haemostasis, embryogenesis, and wound healing. As a major component of blood, fibronectin exists as a soluble disulphide linked dimer, but it can also be incorporated into an insoluble cross-linked fibrillar network to form a major component of the extracellular matrix. Fibronectin is composed of an extended chain of module repeats termed Fn1, Fn2, and Fn3 that bind to a wide range of transmembrane receptors and extracellular matrix components, including collagen. The gelatin binding domain of fibronectin was first isolated as a 45kDa proteolytic fragment and has since been found to be composed of six modules: 6Fn1-1Fn2-2Fn2-7Fn1-8Fn1-9Fn1 (in this notation nFX represents the nth type X module in the native protein). This domain has been reported to bind to both collagen and denatured collagen (gelatin), but with 10-100 times higher affinity to the latter; it can be purified to homogeneity on a gelatin affinity column. In the work presented here, fragments of the gelatin binding domain are expressed in P. pastoris, purified to homogeneity, and investigated at the molecular level. Through a dissection approach, surface plasmon resonance (SPR) is used to characterise the recombinantly produced protein, to accumulate more information about the function of the full domain. NMR is used to assess the folding of the protein fragments at atomic resolution. In particular, the secondary structure of 8Fn1-9Fn1 is mapped using inter-strand NOEs, which suggests that the construct takes the fold of a pair of typical Fn1 modules. Gelatin affinity chromatography is used to confirm that both Fn1 and Fn2 modules contribute to gelatin binding, possibly in two clusters (1Fn2-2Fn2 and 8Fn1-9Fn1). The 7Fn1 module may perform a structural role in linking together these two interaction sites, in the same way as suggested for 6Fn1, which is thought to act in a structural manner to enhance the binding of 1Fn2-2Fn2 to gelatin. Three carbohydrate moieties are found on this domain, one on 2Fn2 and two on 8Fn1. Here, by means of expressing different protein length fragments, and by site directed mutagenesis, the role of each sugar chain is investigated independently. The sugar chain on 2Fn2 does not appear to promote binding to collagen, nor does the first sugar chain on 8Fn1 (N-linked to N497), implying another role for these sugars such as protection from proteolysis. However, the presence of at least a single GlcNAc sugar residue on the second sugar chain site on 8Fn1 (N- linked to N511) is essential for full affinity binding to collagen. Direct binding of the 8Fn1-9Fn1 module pair to collagen is assessed with a short collagen peptide and the binding is monitored by NMR. The peptide appears to bind, predominantly to the final strand of 8Fn1, the first β- strand of 9Fn1, and the linker between the two modules, with μM affinity. A model for bound peptide is proposed. The highly conserved amino acid motif Ile-Gly-Asp (IGD) is found on four of the nine N-terminal Fn1 modules of fibronectin. Tetrapeptides containing the IGD were demonstrated to promote the migration of fibroblast cells into a native collagen matrix. Two of these “bioactive” IGD motifs are found within the gelatin binding domain, one on 7Fn1 and one on 9Fn1. In this study, the motif in the 8Fn1-9Fn1 module pair is shown to be located in a tightly constrained loop within 9Fn1. By site directed mutagenesis, the IGD motifs of 7Fn1 and 9Fn1 are subjected to single amino acid substitutions, and their ability to stimulate cell migration assessed in our assay. By NMR, the fold of the IGD mutant proteins is found to be unaffected by the mutation with respect to the wild type, with the exception of small perturbations around the substitution site. While the wild type module is able to stimulate fibroblast migration, the mutant proteins show reduced or negligible bioactivity. The larger fragments show far more potency in stimulating fibroblast migration, with 8Fn1-9Fn1 (one IGD motif) 104 times more potent than the IGD peptide, and the full gelatin binding domain (two IGD motifs) 106 times more potent than the 8Fn1-9Fn1. Potential mechanisms for this enormous enhancement of the IGD potency in different contexts are discussed.