The implantation of any foreign material into the body automatically starts an immune reaction that serves as the first, mandatory step to regenerate tissue. The course of this initial immune reaction decides on the fate of the implant: either the biomaterial will be integrated into the host tissue to subsequently fulfill its intended function (e.g., tissue regeneration), or it will be repelled by fibrous encapsulation that determines the implant failure. Especially neutrophils and macrophages play major roles during this inflammatory response and hence mainly decide on the biomaterial's fate. For clinically relevant tissue engineering approaches, biomaterials may be designed in shape and morphology as well as in their surface functionality to improve the healing outcome, but also to trigger stem cell responses during the subsequent tissue regeneration phase. The main focus of this thesis was to unravel the influence of scaffold characteristics, including scaffold morphology and surface functionality, on primary human innate immune cells (neutrophils and macrophages) and human mesenchymal stromal cells (hMSCs) to assess their in vitro immune response and tissue regeneration capacity, respectively. The fiber-based constructs were produced either via melt electrowriting (MEW), when the precise control over scaffold morphology was required, or via solution electrospinning (ES), when the scaffold design could be neglected. All the fiber-based scaffolds used throughout this thesis were composed of the polymer poly(ε caprolactone) (PCL). A novel strategy to model and alleviate the first direct cell contact of the immune system with a peptide-bioactived fibrous material was presented in chapter 3 by treating the material with human neutrophil elastase (HNE) to imitate the neutrophil attack. The main focus of this study was put on the effect of HNE towards an RGDS-based peptide that was immobilized on the surface of a fibrous material to improve subsequent L929 cell adhesion. The elastase efficiently degraded the peptide-functionality, as evidenced by a decreased L929 cell adhesion, since the peptide integrated a specific HNE-cleavage site (AAPV-motif). A sacrificial hydrogel coating based on primary oxidized hyaluronic acid (proxHA), which dissolved within a few days after the neutrophil attack, provided an optimal protection of the peptide-bioactivated fibrous mesh, i.e, the hydrogel alleviated the neutrophil attack and largely ensured the biomaterial's integrity. Thus, according to these results, a means to protect the biomaterial is required to overcome the neutrophil attack. Chapter 4 was based on the advancement of melt electrowriting (MEW) to improve the printing resolution of MEW scaffolds in terms of minimal inter-fiber distances and a concomitant high stacking precision. Initially, to gain a better MEW understanding, the influence of several parameters, including spinneret diameter, applied pressure, and collector velocity on mechanical properties, crystallinity, fiber diameter and fiber surface morphology was analyzed. Afterward, innovative MEW designs (e.g., box-, triangle-, round , and wall-shaped scaffolds) have been established by pushing the printing parameters to their physical limits. Further, the inter-fiber distance within a standardized box-structured scaffold was successfully reduced to 40 µm, while simultaneously a high stacking precision was maintained. In collaboration with a co-worker of my department (Tina Tylek, who performed all cell-based experiments in this study), these novel MEW scaffolds have been proven to facilitate human monocyte-derived macrophage polarization towards the regenerative M2 type in an elongation-driven manner with a more pronounced effect with decreasing pore sizes. Finally, a pro-adipogenic platform for hMSCs was developed in chapter 5 using MEW scaffolds with immobilized, complex ECM proteins (e.g., human decellularized adipose tissue (DAT), laminin (LN), and fibronectin (FN)) to test for the adipogenic differentiation potential in vitro. Within this thesis, a special short-term adipogenic induction regime enabled to more thoroughly assess the intrinsic pro-adipogenic capacity of the composite biomaterials and prevented any possible masking by the commonly used long-term application of adipogenic differentiation reagents. The scaffolds with incorporated DAT consistently showed the highest adipogenic outcome and hence provided an adipo-inductive microenvironment for hMSCs, which holds great promise for applications in soft tissue regeneration. Future studies should combine all three addressed projects in a more in vivo-related manner, comprising a co-cultivation setup of neutrophils, macrophages, and MSCs. The MEW-scaffold, particularly due to its ability to combine surface functionality and adjustable morphology, has been proven to be a successful approach for wound healing and paves the way for subsequent tissue regeneration. / Die Implantation eines Biomaterials löst stets eine Immunreaktion im Körper aus, die den ersten zwingenden Schritt zur Geweberegeneration darstellt. Der Verlauf dieser anfänglichen Immunreaktion entscheidet über das Schicksal des Implantats: Entweder wird das Biomaterial in das Wirtsgewebe integriert, um anschließend seine vorgesehene Funktion (z.B. Geweberegeneration) zu erfüllen, oder aber es findet eine Abstoßungsreaktion durch Einkapselung des Implantats statt. Insbesondere Neutrophile und Makrophagen spielen für die Immunantwort eine wichtige Rolle und entscheiden daher hauptsächlich über das Schicksal des Biomaterials. Für klinisch relevante Ansätze der Gewebezüchtung können Biomaterialien sowohl in ihrer Morphologie als auch in ihrer Oberflächenfunktionalität so gestaltet werden, dass sie zum einen die Wundheilung verbessern, zum anderen auch Stammzellreaktionen während der anschließenden Geweberegenerationsphase auslösen. Der Fokus dieser Doktorarbeit lag auf der Beurteilung des Einflusses von Morphologie und Oberflächenfunktionalität fasriger Scaffolds auf die frühe Phase der Geweberegeneration. Insbesondere wurde die in vitro-Immunantwort von primären humanen Immunzellen (Neutrophile und Makrophagen) sowie die Geweberegenerationskapazität von humanen mesenchymalen Stromazellen (hMSCs) untersucht. Die hierfür verwendeten faserbasierten Poly(ε-Caprolacton) (PCL) Scaffolds wurden entweder mittels Solution Electrospinning (ES) oder Melt Electrowriting (MEW) hergestellt. Während ES eine zufällig orientierte Faserablage zur Folge hat, erlaubt MEW eine präzise Kontrolle der Scaffold-Morphologie. Zunächst wurde eine neue Strategie zur Nachahmung und Abmilderung des ersten direkten Zellkontakts während der Immunreaktion vorgestellt. Dabei wurde die Interaktion zwischen Neutrophilen mit einem Peptid-bioaktivierten Fasermaterial untersucht (Kapitel 3), wobei der sog. Neutrophilen-Angriff mittels des Enzyms Neutrophilen Elastase (HNE) nachgeahmt wurde. Das an der Faseroberfläche immobilisierte CGGGAAPVGGRGDS-Peptid verfügte über eine spezifische HNE-Schnittstelle (AAPV-Motiv), an welcher die Elastase das Peptid effizient degradieren konnte. Das Degradationsverhalten des Enzyms wurde anschließend über L929 Zelladhärenz analysiert, welche über das RGDS-Motiv im Peptid vermittelt wurde. Im Rahmen der Arbeit konnte nachgewiesen werden, dass der Neutrophilen-Angriff und die damit einhergehende Verringerung des RGDS-Motivs zu einer reduzierten Zelladhärenz führte. Die Einbettung des Scaffolds in ein Hydrogel auf der Basis von Aldehyd-haltiger Hyaluronsäure (proxHA) bot während des Neutrophilen-Angriffs einen optimalen Schutz der Peptidfunktionalität. Um diese wiederum anschließend für Adhäsionsversuche verfügbar zu machen, konnte das Hydrogelsystem derartig eingestellt werden, dass sich dieses innerhalb weniger Tage auflöste. Auf diese Weise konnte das Hydrogel den Neutrophilen-Angriff abmildern und so die Integrität des Biomaterials weitestgehend gewährleisten. Kapitel 4 behandelt die Präzisierung der Faserablage, insbesondere die Verringerung des Faserabstands, während des MEW-Prozesses. Zunächst wurde der Einfluss verschiedener Parameter (Spinndüsendurchmesser, angelegter Luftdruck und Kollektorgeschwindigkeit) auf die mechanischen Eigenschaften, die Kristallinität, den Faserdurchmesser und die Faseroberflächenmorphologie analysiert. Durch Optimierung der Druckparameter konnten innovative MEW-Designs (u.a. mit runder Porengeometrie) gedruckt werden. Der Abstand zwischen den Fasern in einem Scaffold mit standardisierter kastenförmiger Porengeometrie wurde erfolgreich auf 40 µm reduziert, während gleichzeitig eine hohe Stapelpräzision gewährleistet wurde. In Zusammenarbeit mit einer Kollegin am Lehrstuhl (Tina Tylek, die alle zellbasierten Experimente in dieser Studie durchführte) wurde nachgewiesen, dass diese innovativen MEW-Scaffolds die Polarisierung menschlicher Makrophagen in Richtung des regenerativen M2-Typs förderten. Die Makrophagen-Polarisierung ging einher mit einer Zellelongation, wobei dieser Effekt verstärkt für kleinere Porengrößen auftrat. Abschließend stand die Untersuchung der pro-adipogenen Wirkung von faserfunktionalisierten MEW-Scaffolds im Fokus (Kapitel 5), welche mit ECM-Proteinen, wie beispielsweise dezellularisiertes Fettgewebe (DAT), beschichtet wurden. Das pro-adipogene Potential dieser Materialien wurde mit Hilfe einer adipogenen Kurzzeitinduktion näher analysiert, da eine Langzeitapplikation der Differenzierungsreagenzien diesen Effekt überdeckte. Die Scaffolds mit der DAT-Beschichtung zeigten durchweg die höchste adipogene Differenzierung und boten somit für Stammzellen eine adipo-induzierende Mikroumgebung, weshalb sie für die Anwendung in der Weichgeweberegeneration sehr vielversprechend sind. An diese Arbeit anschließende Experimente sollten alle drei Projekte in einem Co-Kulturansatz von Neutrophilen, Makrophagen und MSCs kombinieren, um so einen stärkeren in vivo-Bezug herzustellen. Hierfür erweist sich das MEW-Scaffold insbesondere durch seine Kombinationsfähigkeit der Oberflächenfunktionalität und Morphologie als Ansatz für einen erfolgreichen Wundheilungsprozess und ebnet damit den Weg für eine bestmögliche Geweberegeneration.

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This thesis will explore, both generally and very specifically, the role of biomaterials in drug delivery and tissue engineering applications. A novel therapeutic conjugate to treat dry eye disease using hyaluronic acid and sulfadiazine was created and tested using a benzalkonium chloride induced dry eye model. Thermoresponsive hydrogels based around poly(n-isopropylacrylamide) were synthesized to create a potential in situ gelling cell scaffold for cell delivery to the subretinal space. Finally, a mucoadhesive micelle was developed as a platform delivery system to increase bioavailability of drug in anterior segment therapeutics. / Thesis / Doctor of Philosophy (PhD)

This thesis investigates new processing methods and bone tissue engineering applications of cross-linked cellulose nanocrystal (CNC) aerogels. Aerogels are highly porous, low-density materials that have been praised for their high surface area and interconnected pores, but criticized for their brittleness. This prompted a search for new aerogel “building blocks” to produce more flexible materials; CNCs meet this need and chemically cross-linked CNC aerogels have good compressive strength and shape recovery properties in air and liquid environments. CNCs are high aspect ratio, non-toxic and renewably-sourced nanoparticles. Literature has demonstrated CNC aerogel production using cryo-templating with controlled drying. In this work, we produce aerogels using a new scalable process called pressurized gas expansion (PGX) and compare them to conventional cryo-templated aerogels. PGX aerogels were found to have more expanded fibrillar morphology, a range of mesopore sizes and smaller macropores, in contrast to cryo-templated aerogels that had a sheet-like morphology surrounding larger macropores. Additionally, PGX aerogels had higher specific surface area and porosity, but lower compressive strength due to a lower cross-link density. While neither CNC aerogel type dispersed in water, PGX aerogels partially shrank whereas cryo-templated aerogels did not; this is attributed to their morphological differences. This work shows that new aerogel processing methods can introduce new properties and thus broaden the potential applications of CNC aerogels. One specific biomedical application was evaluated for CNC aerogels – their use as bone tissue scaffolds. Cryo-templated aerogels comprised of CNCs with different surface chemistries, either sulfate or phosphate groups, were found to have attractive chemical, physical and mechanical properties for bone tissue engineering. This work shows that both types of CNC aerogels can facilitate cell proliferation, favorable differentiation, and can nucleate uniform hydroxyapatite growth. These positive in vitro results and the bimodal pore morphology of CNC aerogels make them promising bone scaffolds for in vivo studies. / Thesis / Master of Applied Science (MASc) / Aerogels are light, porous, sponge-like materials that are essentially 99% air by volume. In this work, the aerogels are made from non-toxic plant-based nanoparticles called cellulose nanocrystals (CNCs). This thesis investigates: 1) new ways to control CNC aerogel properties and pore size through different processing methods and 2) the use of CNC aerogels to aid in the repair of damaged bones. High-resolution microscopy and nano-characterization tools show that CNC aerogels have tunable properties, which may extend their possible applications. The internal structure, sponge-like mechanical properties and biocompatibility of CNC aerogels allowed them to be successfully utilized to support bone cells and grow bone-like mineral.

Unlike traditional stochastic scaffold fabrication techniques, additive manufacturing (AM) can be used to create tissue-specific three-dimensional scaffolds with controlled porosity and pore geometry (meso-structure). However, due to the relatively few biocompatible materials available for processing in AM machines, direct fabrication of tissue scaffolds is limited. To alleviate material limitations and improve feature resolution, a new indirect scaffold fabrication method is developed. A four step fabrication process is explored: Fused Deposition Modeling (FDM) is used to fabricate scaffold patterns of varied pore size and geometry. Next, scaffold patterns are surface treated, and then mineralized via simulated body fluid (SBF); forming a bone-like ceramic throughout the scaffold pattern. Finally, mineralized patterns are heat treated to pyrolyze the pattern and sinter the minerals. Two scaffold meso-structures are tested: "tube" and "backfill." Two pattern materials are tested [acrylonitrile butadiene styrene (ABS) and investment cast wax (ICW)] to determine which material is the most appropriate for mineralization and sintering. Mineralization is improved through plasma surface treatment and dynamic flow conditions. Appropriate burnout and sintering temperatures to remove pattern material are determined experimentally. While the "tube scaffolds" were found to fail structurally, "backfill scaffolds" were successfully created using the new fabrication process. The "backfill scaffold" meso-structure had wall thicknesses of 470 – 530 µm and internal channel diameters of 280 – 340 µm, which is in the range of appropriate pore size for bone tissue engineering. "Backfill scaffolds" alleviated material limitations, and had improved feature resolution compared to current indirect scaffold fabrication processes. / Master of Science

The anterior cruciate ligament (ACL) contributes to normal knee function, but it is commonly injured and has poor healing capabilities. Of the current treatments available for ACL reconstruction, none replicate the long-term mechanical properties of the ACL. It was hypothesized that tissue-engineered scaffolds comprised of reconstituted type I collagen fibers would have the potential to yield a more suitable treatment for ACL reconstruction. Ultra-violet (UV) radiation and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were investigated as possible crosslinking methods for the scaffolds, and EDC crosslinking was deemed more appropriate given the gains in strength and stiffness afforded to individual collagen fibers. Scaffolds were composed of 54 collagen fibers, which were made using an extrusion process, organized in accordance with a braid-twist design; the addition of a hydrogel (gelatin) to this scaffold was also investigated. The scaffolds were tested mechanically to determine ultimate tensile strength (UTS), Young's modulus, and viscoelastic properties. Scaffolds were also evaluated for the cellular activity of primary rat lateral collateral ligament (LCL) and medial collateral ligament (MCL) fibroblast cells after 7, 14, and 21 days. The crosslinked scaffolds without gelatin exhibited mechanical and viscoelastic properties that were more similar to the human ACL. Cellular activity on the crosslinked scaffolds without gelatin was observed after 7 and 21 days, but no significant increase was observed with time. Although more studies are needed, these results indicate that a braid- twist scaffold (composed of collagen fibers) has the potential to serve as a scaffold for ACL replacement. / Master of Science

Yes / Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50 Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50 Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.

Made available in DSpace on 2016-08-17T18:39:42Z (GMT). No. of bitstreams: 1 4169.pdf: 1866763 bytes, checksum: 38d468ca7232537d6952ee24e68d0593 (MD5) Previous issue date: 2011-08-29 / Universidade Federal de Minas Gerais / Due to limited availability of autogenous bone and of the risks associated with the use of bone allografts, new synthetic materials have been developed in order to replace the bone tissue lost due to trauma or pathological process. The bioactive materials in the form of scaffolds are synthetic materials promising for bone grafting. Several studies suggest that these biomaterials are able to stimulate the proliferation of osteoblasts and osteogenesis at the site of fracture. However, the feasibility of these biomaterials to a clinical application requires the investigation of their biocompatibility. In this context, this study aimed to evaluate the biocompatibility of a scaffold synthesized from a fully crystallized glass-ceramic bioactive quaternary system P2O5-Na2O-CaO-SiO2 (Biosilicate®), through histopathological analysis of the biomaterial implanted in the subcutaneous tissue of rats and the cytotoxicity and genotoxicity analysis of the biomaterial in cell cultures (OSTEO-1 and L929 cells). Histopathologic analysis of the biomaterial was performed using 65 Wistar rats male (210- 260 g), randomly divided into two groups, Control group (n = 3 animals per period) and Biosilicate group (n = 10 animals per period), evaluated at 7, 15, 30, 45 and 60 days after surgery. The animals of Biosilicate group underwent surgery and received a subcutaneous implant of Biosilicate® scaffolds. The animals of Control group underwent surgery but did not receive any biomaterial implant. The cytotoxicity analysis was performed to assess the effect of products leaching from Biosilicate® scaffolds (extracts) on cellular proliferation (MTT). The extracts were evaluated in various concentrations (100, 50, 25 and 12.5%) in experimental periods of 24, 72 and 120 hours in two cell lines (OSTEO-1 and L929). The genotoxicity analysis (comet assay) was performed to assess DNA damage in cells OSTEO-1 and L929 grown in contact with the Biosilicate® scaffolds in different periods of 24, 72 and 96 horas. The statistical analysis of parametrics data was performed by analysis of variance (ANOVA) followed by Tukey post-hoc and the analysis of nonparametrics data was performed by Mann-Whitney test. Both statistical tests were performed with a significance level of 5%. The results of histopathological analysis showed that the animals of the Control group did not present inflammation process, necrotic tissue and fibrous tissue. The animals of Biosilicato group showed a granulation tissue after 7 days of implantation. In the other periods (15, 30, 45 and 60 days) a chronic inflammation process of foreign body, marked by the presence of fibrous tissue and giant cells was observed. No infection or necrotic tissue was observed in any animal. In the analysis of cytotoxicity, it was observed that extracts of Biosilicato® scaffolds did not have any significant effect in reducing cell proliferation OSTEO-1 and L929, and that lower concentrations of the extracts (12.5 and 25%) stimulated the proliferation of both cells in periods of 72 and 120 hours. The analysis of genotoxicity showed that the Biosilicate® scaffolds did not induce DNA damage in the cell lines tested in all experimental periods. The results of this study showed that the Biosilicate® scaffolds presented biocompatibility in vivo and in vitro. / Devido a limitada disponibilidade de osso autógeno e dos riscos associados ao uso de osso alógeno, novos materiais sintéticos vêm sendo desenvolvidos com o objetivo de substituir o tecido ósseo perdido em decorrência de traumatismos ou processos patológicos. Os materiais bioativos na forma de scaffolds são materiais sintéticos promissores para enxertia óssea. Vários estudos sugerem que estes biomateriais são capazes de estimular a proliferação de osteoblastos e a osteogênese no local da fratura. No entanto, a viabilização destes biomateriais a uma aplicação clínica requer o emprego de testes que avaliem a sua biocompatibilidade. Dentro deste contexto, o presente estudo teve como objetivo avaliar a biocompatibilidade do scaffold sintetizado a partir de uma vitrocerâmica bioativa totalmente cristalizada do sistema quaternário P2O5-Na2O-CaO-SiO2 (Biosilicato®), por meio da análise histopatológica do biomaterial implantado no tecido subcutâneo de ratos, e pelas análises de citotoxicidade e genotoxicidade do biomaterial em cultura de células da linhagem OSTEO-1 e L929. A análise histopatológica do biomaterial foi realizada utilizando 65 ratos machos da linhagem Wistar (210-260 g), distribuídos aleatoriamente em dois grupos, Controle (n = 3 animais por período) e Biosilicato (n = 10 animais por período), avaliados em períodos distintos de 7, 15, 30, 45 e 60 dias. Os animais do grupo Biosilicato foram submetidos a uma cirurgia no tecido subcutâneo e receberam um implante de scaffold de Biosilicato®. Os animais do grupo Controle foram submetidos à mesma cirurgia, mas não receberam o implante do biomaterial. A análise de citotoxicidade foi realizada para avaliar os efeitos dos produtos da lixiviação dos scaffolds de Biosilicato® (extratos) na proliferação celular pelo ensaio MTT. Os extratos foram avaliados em várias concentrações (100, 50, 25 e 12,5%) em períodos experimentais de 24, 72 e 120 horas, utilizando duas linhagens celulares (OSTEO-1 e L929). A análise de genotoxicidade (ensaio cometa) foi realizada para avaliar os danos no DNA de células OSTEO-1 e L929 cultivadas em contato com scaffolds de Biosilicato® em períodos distintos de 24, 72 e 96 horas. A análise estatística dos dados paramétricos foi realizada pelo teste de variância (ANOVA), seguido do post-hoc de Tukey, e a análise dos dados não paramétricos foi realizada pelo teste de Mann-Whitney. Ambos os testes estatísticos foram realizados com nível de significância de 5%. Os resultados da análise histopatológica demonstraram que os animais do grupo Controle não apresentaram processo inflamatório, tecido necrótico ou tecido fibroso. Já os animais do grupo Biosilicato apresentaram um tecido de granulação após 7 dias de implantação e nos demais períodos (15, 30, 45 e 60 dias) apresentaram um processo inflamatório crônico de corpo estranho, marcado pela presença de tecido fibroso e células gigantes multinucleadas. Em todos os animais avaliados não foi evidenciado foco de infecção ou tecido necrótico. Na análise de citotoxicidade foi observado que os extratos dos scaffolds de Biosilicato® não possuem efeito significativo na redução da proliferação de células OSTEO-1 e L929, e que as menores concentrações dos extratos (12,5 e 25%) estimularam a proliferação de ambas às células nos períodos de 72 e 120 horas. Na análise de genotoxicidade foi evidenciado que os scaffolds de Biosilicato® não induzem danos do DNA de células de ambas às linhagens testadas em todos os períodos experimentais. Os resultados obtidos neste estudo demonstraram que os scaffolds de Biosilicato® apresentaram biocompatibilidade em experimentos in vivo e in vitro.