[EN] Abstract The main purpose of this thesis is the design and characterization of an experimental articular cartilage model. The in vitro model is composed of a macro and micro- porous Polycaprolactone scaffold with a Poly(Vinyl Alcohol) filling. The scaffold/hydrogel construct has been subjected to repeating number of freezing and thawing cycles in order to crosslink the hydrogel inside the scaffold's pores. The Poly(Vinyl Alcohol) resembles the growing cartilaginous tissue inside the scaffolds pores, as it gets denser and stiffer for each cycle of freezing and thawing. The in vitro model allows studying a variety of characteristics of the scaffold and hydrogel, revealing interesting features. The importance of water flow on the mechanical properties is studied, so as the influence of micro-porosity. It can be seen that the mechanical properties of the porous scaffolds are influenced in distinct ways by the hydrogel density and micro-porosity of the scaffold. The permeability of the scaffolds is studied and is seen independent of crosslinking density of the hydrogel inside the porous scaffolds. The experimental cartilage model has also been applied on a macro porous acrylic scaffold. The results show that the water has different effect on the mechanical properties, for macro, or macro and micro-porous scaffolds. The in vitro cartilage model has elastic modulus, aggregate modulus and permeability values in the same order as human articular cartilage. The model is useful to predict the mechanical behavior of porous scaffolds in vivo. A scaffold implant device for animal studies has been designed based on a previous patent of the research group, and implanted in two different in vivo trials in sheep. The results show that the fixation and anchoring to the subchondral bone improve the tissue repair and diminish alterations in the subchondral bone. ¿ / [ES] Resumen El objetivo principal de esta tesis doctoral es el diseño y caracterización de un modelo de cartílago articular experimental. El modelo in vitro se compone de un scaffold micro- y macroporoso de Policaprolactona con un relleno de Poli(Vinil Alcohol). El constructo scaffold/hidrogel ha sido sometido a ciclos consecutivos de congelación y descongelación con objeto de entrecruzar el hidrogel dentro de los poros del scaffold. El Poli(Vinil Alcohol) mimetiza al tejido de cartílago que se regenerará en los poros, ya que en cada ciclo de congelación y descongelación se vuelve más denso y duro. El modelo in vitro permite estudiar una gran variedad de características del scaffold e hidrogel, revelando fenómenos interesantes para la ingeniería tisular. Se ha estudiado la importancia del flujo de agua a través del scaffold en las propiedades mecánicas, así como la influencia de la microporosidad. Se ha podido constatar que la densidad del hidrogel y la microporosidad influyen de distinta forma en las propiedades mecánicas de los scaffolds porosos. Se ha estudiado la permeabilidad de los scaffolds, que ha resultado ser independiente de la densidad de entrecruzamiento del hidrogel dentro de sus poros. El modelo experimental de cartílago se ha aplicado también a un scaffold macroporoso acrílico. Los resultados muestran que el agua tiene un efecto distinto en las propiedades mecánicas de los scaffolds macroporosos y en los micro- macroporosos. El modelo de cartílago in vitro tiene valores del modulo elástico, módulo agregado y permeabilidad que son del mismo orden de magnitud que los del cartílago articular humano. El modelo permite predecir el comportamiento mecánico in vivo de scaffolds porosos. Se ha diseñado un dispositivo de implante de scaffold para experimentos en animales basado en una patente del grupo de investigación, que ha sido implantado en dos ensayos in vivo diferentes en ovejas. Los resultados muestran que la fijación y anclaje al hueso subcondral tiene un gran papel en la reparación del tejido. / [CA] Resum L'objectiu principal d'aquesta tesi doctoral és el disseny i caracterització d'un model de cartílag articular experimental. El model in vitro es compon d'un scaffold micro- i macroporós de Policaprolactona amb un farciment de Poli(Vinil Alcohol). El constructe scaffold/hidrogel ha estat sotmès a cicles consecutius de congelació i descongelació amb l'objectiu d'entrecreuar l'hidrogel dins del porus del scaffold. El Poli(Vinil Alcohol) mimetitza al teixit de cartílag que es regenerarà en el porus, ja que en cada cicle de congelació i descongelació es torna més dens i dur. El model in vitro permet estudiar una gran varietat de característiques del scaffold i hidrogel, posant de manifest fenòmens interessants per a l'enginyeria tissular. S'ha estudiat la importància del flux d'aigua a través del scaffold en les propietats mecàniques, així com la influència de la microporositat. S'ha pogut constatar que la densitat de l'hidrogel i la microporositat influeixen de distinta manera en les propietats mecàniques dels scaffolds porosos. S'ha estudiat la permeabilitat dels scaffolds, que ha resultat ser independent de la densitat d'entrecreuament de l'hidrogel dins dels seus porus. El model experimental de cartílag s'ha aplicat també a un scaffold macroporós acrílic. Els resultats mostren que l'aigua té un efecte distint en les propietats mecàniques dels scaffolds macroporosos i en els micro- macroporosos. El model de cartílag in vitro té valors del mòdul elàstic, mòdul agregat i permeabilitat que són del mateix ordre de magnitud que els del cartílag articular humà. El model permet predir el comportament mecànic in vivo de scaffolds porosos. S'ha dissenyat un dispositiu d'implant de scaffold per a experiments en animals basat en una patent del grup d'investigació, que ha segut implantat en dos assaigs in vivo diferents en ovelles. Els resultats mostren que la fixació i ancoratge a l'os subcondral té un gran paper en la reparació del teixit. / Vikingsson, LKA. (2015). An experimental model to mimic the mechanical behavior of a scaffold in a cartilage defect [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/53912

Science and engineering studies on biocompatible implantable materials for tissue and organ repair have recently focused on polymeric materials to serve as scaffolds for cellular integration. Cellulose in many forms has been demonstrated as potential biopolymer for tissue engineering; however, it has not been previously electrospun into a scaffold for tissue engineering applications. The overall goal of this research project was to produce electrospun cellulose acetate (CA) nanofibers with specific architectures and surface chemistries to be evaluated as scaffolds for tissue regeneration. The size and morphology of electrospun CA was impacted by polymer concentration, solvent system, and solution flow rate. The conversion of CA electrospun scaffolds into regenerated cellulose by exposure to NaOH ethanol solution was successful for scaffolds produced at polymer solution flow rate of at least 1 mL/h. The regeneration process resulted in minimal degradation of the cellulose while retaining the original fiber structure of the scaffold. In vitro cytotoxicity evaluation of the fibrous cellulose scaffolds on a culture of mouse fibroblast (L-929) cells indicated that this material posed no threat to mammalian cells. Electrospun cellulose scaffolds with different architectures and surface chemistries were designed and evaluated to enhance scaffold properties and cell adhesion. The morphology of the partially regenerated cellulose revealed only a broad diffraction peak for the scaffold material, while the fully regenerated cellulose showed a characteristic semi-crystalline cellulose II diffraction pattern. Fiber orientation and porosity of the scaffolds were controlled by electrospining CA solution onto the edge of a rotator wheel and laser microablation, respectively. Bioactivity of the scaffolds was shown to be enhanced via scaffold surface modification with either anionic or cationic functional groups. Biomimetic Ca-P crystal mineralization on electrospun cellulose fibers was produced by means of carboxymethyl cellulose (CMC) adsorption and treatments with simulated body fluid (SBF) or phosphate buffer saline (PBS) solutions. Porosity and Ca-P crystals enhanced osteoprogenitor cell adhesion on the electrospun cellulose scaffolds. Cationic modification by trimethyl ammonium betahydroxy propyl (THAMP) derivation and adsorption of extracellular matrix proteins on cellulose fibers promoted adhesion and proliferation of neural-like (PC12) and myoblast (C2C12) cells. Differentiation of myoblast cells (C2C12) towards myotubes on electrospun cellulose scaffolds was controlled by surface chemistry and mechanical properties. Together these studies showed great potential for cellulose acetate to be electrospun and converted into a viable biocompatible tissue engineering scaffolds. / Ph. D.

According to the WHO, foodborne derived enteric infections are a global disease burden and often manifest in diseases that can potentially reach life threatening levels, especially in developing countries. These diseases are caused by a variety of enteric pathogens and affect the gastrointestinal tract, from the gastric to the intestinal to the rectal tissue. Although the complex mucosal structure of these organs is usually well prepared to defend the body against harmful agents, specialised pathogens such as Salmonella enterica can overcome the intestinal defence mechanism. After ingestion, Salmonella are capable of colonising the gut and establishing their proliferative niche, thereby leading to inflammatory processes and tissue damage of the host epithelium. In order to understand these processes, the scientific community in the last decades mostly used cell line based in vitro approaches or in vivo animal studies. Although these approaches provide fundamental insights into the interactions between bacteria and host cells, they have limited applicability to human pathology. Therefore, tissue engineered primary based approaches are important for modern infection research. They exhibit the human complexity better than traditional cell lines and can mimic human-obligate processes in contrast to animal studies. Therefore, in this study a tissue engineered human primary model of the small intestinal epithelium was established for the application of enteric infection research with the exemplary pathogen Salmonella Typhimurium. To this purpose, adult stem cell derived intestinal organoids were used as a primary human cell source to generate monolayers on biological or synthetic scaffolds in a Transwell®-like setting. These tissue models of the intestinal epithelium were examined for their comparability to the native tissue in terms of morphology, morphometry and barrier function. Further, the gene expression profiles of organotypical mucins, tight junction-associated proteins and claudins were investigated. Overall, the biological scaffold-based tissue models showed higher similarity to the native tissue - among others in morphometry and polarisation. Therefore, these models were further characterised on cellular and structural level. Ultrastructural analysis demonstrated the establishment of characteristic microvilli and tight-junction connections between individual epithelial cells. Furthermore, the expression pattern of typical intestinal epithelial protein was addressed and showed in vivo-like localisation. Interested in the cell type composition, single cell transcriptomic profiling revealed distinct cell types including proliferative cells and stem cells, progenitors, cellular entities of the absorptive lineage, Enterocytes and Microfold-like cells. Cells of the secretory lineage were also annotated, but without distinct canonical gene expression patterns. With the organotypical polarisation, protein expression, structural features and the heterogeneous cell composition including the rare Microfold-like cells, the biological scaffold-based tissue model of the intestinal epithelium demonstrates key requisites needed for infection studies with Salmonella. In a second part of this study, a suitable infection protocol of the epithelial tissue model with Salmonella Typhimurium was established, followed by the examination of key features of the infection process. Salmonella adhered to the epithelial microvilli and induced typical membrane ruffling during invasion; interestingly the individual steps of invasion could be observed. After invasion, time course analysis showed that Salmonella resided and proliferated intracellularly, while simultaneously migrating from the apical to the basolateral side of the infected cell. Furthermore, the bacterial morphology changed to a filamentous phenotype; especially when the models have been analysed at late time points after infection. The epithelial cells on the other side released the cytokines Interleukin 8 and Tumour Necrosis Factor α upon bacterial infection in a time-dependent manner. Taken together, Salmonella infection of the intestinal epithelial tissue model recapitulates important steps of the infection process as described in the literature, and hence demonstrates a valid in vitro platform for the investigation of the Salmonella infection process in the human context. During the infection process, intracellular Salmonella populations varied in their bacterial number, which could be attributed to increased intracellular proliferation and demonstrated thereby a heterogeneous behaviour of Salmonella in individual cells. Furthermore, by the application of single cell transcriptomic profiling, the upregulation of Olfactomedin-4 (OLFM4) gene expression was detected; OLFM4 is a protein involved in various functions including cell immunity as well as proliferating signalling pathways and is often used as intestinal stem cell marker. This OLFM4 upregulation was time-dependent, restricted to Salmonella infected cells and seemed to increase with bacterial mass. Investigating the OLFM4 regulatory mechanism, nuclear factor κB induced upregulation could be excluded, whereas inhibition of the Notch signalling led to a decrease of OLFM4 gene and protein expression. Furthermore, Notch inhibition resulted in decreased filamentous Salmonella formation. Taken together, by the use of the introduced primary epithelial tissue model, a heterogeneous intracellular bacterial behaviour was observed and a so far overlooked host cell response – the expression of OLFM4 by individual infected cells – could be identified; although Salmonella Typhimurium is one of the best-studied enteric pathogenic bacteria. This proves the applicability of the introduced tissue model in enteric infection research as well as the importance of new approaches in order to decipher host-pathogen interactions with higher relevance to the host. / Nach Angaben der WHO stellen lebensmittelbedingte Darminfektionen eine globale Krankheitslast dar und äußern sich häufig in Krankheiten, die potenziell lebensbedrohliche Ausmaße annehmen können, insbesondere in Entwicklungsländern. Diese Krankheiten werden durch eine Vielzahl von enterischen Erregern verursacht und betreffen den Magen-Darm-Trakt, vom Magen über den Darm bis zum Enddarm. Obwohl die komplexe Schleimhautstruktur dieser Organe in der Regel gut darauf vorbereitet ist, den Körper vor schädlichen Reagenzien zu schützen, können spezialisierte Erreger wie Salmonella enterica den Abwehrmechanismus des Darms überwinden. Nach der Nahrungsaufnahme sind Salmonellen in der Lage, den Darm zu kolonisieren und ihre proliferative Nische zu etablieren, was letztlich zu entzündlichen Prozessen und Gewebeschäden des Wirtsepithels führt. Um diese Prozesse zu verstehen, hat die Wissenschaft in den letzten Jahrzehnten hauptsächlich auf Krebslinien basierende in vitro-Ansätze oder in vivo-Tierstudien verwendet. Obwohl diese Ansätze grundlegende Erkenntnisse über die Wechselwirkungen zwischen Bakterien und Wirtszellen lieferten, sind sie nur begrenzt auf die Pathologie des Menschen übertragbar. Daher sind Tissue engineering und primärzellbasierte Ansätze für die moderne Infektionsforschung wichtig. Sie spiegeln die menschliche Komplexität besser wider als Ansätze mit Krebszellen und können im Gegensatz zu Tierversuchen human-obligate Prozesse nachbilden. Daher wurde in dieser Studie ein tissue engineered humanes Primärmodell des Dünndarmepithels für die Anwendung in der enterischen Infektionsforschung am Beispiel des Erregers Salmonella Typhimurium etabliert. Zu diesem Zweck wurden aus adulten Stammzellen gewonnene Darmorganoide als primäre humane Zellquelle verwendet, um 2D-Monolayer auf biologischen oder synthetischen Trägestrukturen in einer Transwell®-ähnlichen Umgebung zu erzeugen. Die so erzeugten Gewebemodelle des Darmepithels wurden auf ihre Vergleichbarkeit mit dem nativen Gewebe in Bezug auf Morphologie, Morphometrie und Barrierefunktion untersucht. Weiterhin wurde die Genexpression von organtypischen Muzinen, Tight Junction-assoziierten Proteinen und Claudinen sowie das Expressionsmuster der Tight Junction-Proteine untersucht. Insgesamt wiesen die auf biologischen Matrizes basierenden Gewebemodelle eine größere Ähnlichkeit mit dem nativen Gewebe auf - unter anderem in Bezug auf Morphometrie und Polarisation -, weshalb diese Modelle auf zellulärer und struktureller Ebene tiefgehender charakterisiert wurden. Die ultrastrukturelle Analyse zeigte die Ausbildung charakteristischer Mikrovilli und Tight-Junction-Verbindungen zwischen einzelnen Epithelzellen. Darüber hinaus wurden die Expressionsmuster typischer Darmepithelproteine untersucht, die eine in vivo ähnliche Lokalisation aufwiesen. Im Hinblick auf die Zelltypenzusammensetzung ergab die Analyse des Transkriptoms auf Einzel-Zell-Ebene definierte Zelltypen. Dies waren Zellen mit proliferativem Profil, Stammzellen und Vorläuferzellen, und Zellen der absorptiven Linie, Enterozyten und Microfold-Zellen. Zellen der sekretorischen Linie wurden ebenfalls annotiert, jedoch ohne eindeutige kanonische Genexpression. Mit der organotypischen Polarisierung, der Proteinexpression, den strukturellen Merkmalen und der heterogenen Zellzusammensetzung, einschließlich der seltenen Microfold-Zellen, weist das auf einer biologischen Matrix basierende Gewebemodell des Darmepithels die wichtigsten Voraussetzungen für Infektionsstudien mit Salmonellen auf. Im zweiten Teil dieser Studie wurde ein geeignetes Infektionsprotokoll für das Epithelgewebemodell mit Salmonella Typhimurium erstellt, gefolgt von der Untersuchung der wichtigsten Merkmale des Infektionsprozesses. Salmonella hafteten an den epithelialen Mikrovilli und verursachten während der Invasion das typische Membran-Kräuseln; interessanterweise konnten die Schritte der Invasion einzeln beobachtet werden. Nach der Invasion zeigte die Zeitverlaufsanalyse der Infektion, dass die Salmonellen intrazellulär lokalisierten und replizierten, während sie gleichzeitig von der apikalen zur basolateralen Seite der infizierten Zelle migrierten. Darüber hinaus veränderte sich die Morphologie der Bakterien in der Spätphase der Infektion zu einem filamentösen Phänotyp. Die Epithelzellen auf der anderen Seite setzten nach der bakteriellen Infektion zeitabhängig die Zytokine Interleukin 8 und Tumor-Nekrose-Faktor-α frei. Insgesamt rekapituliert die Salmonelleninfektion des intestinalen Epithelgewebemodells wichtige Schritte des Infektionsprozesses, wie sie in der Literatur beschrieben sind und stellt somit eine valide in vitro Plattform für die Untersuchung des Salmonelleninfektionsprozesses in einem menschlichen Kontext dar. Interessanterweise variierten die intrazellulären Salmonellenpopulationen während des Infektionsprozesses in ihrer Bakterienzahl, was auf eine erhöhte intrazelluläre Proliferation zurückgeführt werden konnte und somit ein heterogenes Verhalten der Salmonellen in einzelnen Zellen demonstriert. Darüber hinaus wurde durch die Anwendung von Einzel-Zell-Transkriptom-Analysen die Hochregulierung der Genexpression von Olfactomedin-4 (OLFM4) nachgewiesen; OLFM4 ist ein Protein mit verschiedenen Funktionen, darunter Prozesse der Zellimmunität sowie proliferierende Signalwege, und es wird häufig als Darmstammzellmarker verwendet. Diese OLFM4-Hochregulierung war zeitabhängig, auf mit Salmonella infizierten Zellen beschränkt und schien mit der intrazellulären Bakterienmasse zuzunehmen. Bei der Untersuchung der OLFM4-Regulationsmechanismen konnte eine nuclear factor κB-induzierte Hochregulierung ausgeschlossen werden, während die Hemmung der Notch-Signalübertragung zu einem Rückgang der OLFM4-Gen- und Proteinexpression führte. Darüber hinaus führte die Hemmung von Notch zu einer verminderten Bildung von filamentösen Salmonella. Insgesamt konnte durch die Verwendung des hier eingeführten primären Epithelgewebemodells ein heterogenes intrazelluläres bakterielles Verhalten beobachtet und eine bisher übersehene Wirtszellantwort - die Expression von OLFM4 durch einzelne infizierte Zellen - bei einem der am besten untersuchten enterischen Pathogene identifiziert werden. Dies beweist die Anwendbarkeit des vorgestellten Gewebemodells in der enterischen Infektionsforschung sowie die Bedeutung neuer Ansätze zur Entschlüsselung von Wirt-Pathogen-Interaktionen mit höherer Relevanz für den Wirt.

The inability to accurately reproduce the complexities of the in vivo tumor microenvironment with reductionist-based two-dimensional in vitro cell culture models has been a notable deterrent in identifying therapeutic agents that reliably translate to in vivo animal and human clinical trials. In an effort to address this, a growing number of three-dimensional (3D) in vitro tumor models capable of mimicking specific tumorigenic processes have emerged within the last decade. This concept stems from the understanding that cells cultured within 3D in vitro matrices have the ability to acquire phenotypes representative of the in vivo microenvironment. The objective of this project was to apply a tissue engineering approach towards developing a 3D in vitro tumor angiogenesis model. Initially, different scaffolds were investigated for supporting 3D tumor growth, including bacterial cellulose, electrospun polycaprolactone/collagen I, and highly porous electrospun poly(L-lactic acid). However, cancer cells cultured on these scaffolds demonstrated poor adhesion, sufficient adhesion with poor infiltration, and increased but still inadequate infiltration, respectively. Collagen I hydrogels were chosen as an appropriate scaffold for facilitating 3D in vitro tumor growth for two reasons -- cell-mediated degradation and immediate 3D cell growth. It was hypothesized that cancer cells cultured within collagen I hydrogels could be encouraged to recapitulate key characteristics of in vivo tumor progression. MDA-MB-231 human breast cancer cells were shown to experience hypoxia and undergo necrosis in response to limitations in oxygen diffusion and competition for nutrients. Upregulation of hypoxia-inducible factor-1" resulted in a significant increase in vascular endothelial growth factor gene expression. To capitalize on this endogenous angiogenic potential, microvascular endothelial cells were cultured on the surface of the designated "bioengineered tumors." It was hypothesized that paracrine signaling between tumor and endothelial cells co-cultured within this system would be sufficient for inducing an angiogenic response in the absence of exogenous pro-angiogenic growth factors. Endothelial cells in the co-culture group were shown to invasively sprout into the underlying collagen matrix, forming a capillary-like tubule network. This project culminated with the establishment of an improved in vitro tumor model that can be used as a tool for accurate evaluation and refinement of cancer therapies. / Ph. D.

Tendon function is essential for quality of life, yet the pathogenesis and healing of tendinopathy remains poorly understood compared to other musculoskeletal disorders. The aim of regenerative medicine is to replace traditional tissue and organ transplantation by harnessing the developmental potential of stem cells to restore structure and function to damaged tissues. The recently discovered interdependency of cell phenotype and biophysical environment has created a paradigm shift in cell biology. This dissertation introduces a dynamic in vitro model for tendon function, dysfunction and development, engineered to characterize the mechanobiological relationships dictating stem cell fate decisions so that they may be therapeutically exploited for tendon healing. Cells respond to mechanical deformation via a complex set of behaviors involving force-sensitive membrane receptor activity, changes in cytoskeletal contractility and transcriptional regulation. Effective ex vivo model systems are needed to emulate the native environment of a tissue and to translate cell-matrix forces with high fidelity. A naturally-derived decellularized tendon scaffold (DTS) was invented to serve as a biomimetic tissue culture platform, preserving the structure and function of native extracellular matrix. DTS in concert with a newly designed dynamic mechanical strain system comprises a tendon bioreactor that is able to emulate the three-dimensional topography, extracellular matrix proteins, and mechanical strain that cells would experience in vivo. Mesenchymal stem cells seeded on decellularized tendon scaffolds subject to cyclic mechanical deformation developed strain-dependent alterations in phenotype and measurably improved tissue mechanical properties. The relative tenogenic efficacies of adult stem cells derived from bone marrow, adipose and tendon were then compared in this system, revealing characteristics suggesting tendon-derived mesenchymal stem cells are predisposed to differentiate toward tendon better than other cell sources in this model. The results of the described experiments have demonstrated that adult mesenchymal stem cells are responsive to mechanical stimulation and, while exhibiting heterogeneity based on donor tissue, are broadly capable of tenocytic differentiation and tissue neogenesis in response to specific ultrastructural and biomechanical cues. This knowledge of cellular mechanotransduction has direct clinical implications for how we treat, rehabilitate and engineer tendon after injury. / Ph. D.

Nanoparticles can be used as building blocks of materials. Properties of such materials depend on the organization of the constituent particles. Thus, control over particle organization enables control over material properties. However, robust and scalable methods for arranging nanoparticles are still lacking. This dissertation explores the use of an externally applied magnetic field to organize magnetic nanoparticles into microstructures of desired shape. It extends to proofs of concept towards applications in material design and tissue engineering. First, external control over dipolar self-assembly of magnetic nanoparticles (MNPs) in a liquid dispersion is investigated experimentally. Scaling laws are derived to explain experimental observations, correlating process control variables to microstructure morphology. Implications of morphology on magnetic properties of such structures are then explored computationally. Specifically, a method is proposed wherein superparamangetic nanoparticles, having no residual magnetization, can be organized into anisotropic structures with remanence. Another application explores the use of magnetic forces in organizing human cells into three-dimensional (3D) structures of desired shape and size. When magnetized cells are held in place for several days, they are seen to form inter-cellular contacts and organize themselves into tight clusters. This provides a method for 3D tissue culture without the use of artificial scaffolding materials. Finally, a method to pattern heterogeneities in the stiffness of an elastomer is developed. This makes use of selective inhibition of the catalyst of crosslinking reactions by magnetite nanoparticles. The last chapter discusses future possibilities. / Ph. D.

High levels of ionizing radiation are known to cause degradation and/or cross-linking in polymers. Lower levels of ionizing radiation, such as x-rays, are commonly used in the treatment of cancers. Material characterization has not been fully explored for polymeric materials exposed to therapeutic radiation levels. This study investigated the effects of therapeutic radiation on three porous scaffolds: polycaprolactone (PCL), polyurethane (PU) and gelatin. Porous scaffolds were fabricated using solvent casting and/or salt leaching techniques. Scaffolds were placed in phosphate buffered saline (PBS) and exposed to a typical cancer radiotherapy schedule. A total dose of 50 Gy was broken into 25 dosages over a three-month period. PBS was collected over time and tested for polymer degradation through high performance liquid chromatography (HPLC) and bicinchoninic acid (BCA) protein assay. Scaffolds were characterized by changes in microstructure using Scanning Electron Microscopy (SEM), and crystallization using Differential Scanning Calorimetry (DSC). Additionally, gelatin ε-amine content was analyzed using Trinitrobenzene Sulfonic Acid Assay (TNBSA). Gelatin scaffolds immersed in PBS for three months without radiation served as a control. Each scaffold responded differently to radiation. PCL showed no change in molecular weight or microstructure. However, the degree of crystallinity decreased 32% from the non-irradiated control. PU displayed both changes in microstructure and a decrease in crystallinity (85.15%). Gelatin scaffolds responded the most dramatically to radiotherapy. Samples were observed to swell, yet maintain shape after exposure. As gelatin was considered a tissue equivalent, further studies on tissues are needed to better understand the effects of radiotherapy. / Master of Science

Vat photopolymerization (VP) is an additive manufacturing (AM) technology that permits the fabrication of parts with complex geometries and feature sizes as small as a few microns. These attributes make VP an attractive option for the fabrication of scaffolds for tissue engineering. However, there are few printable materials with low cytotoxicity that encourage cellular adhesion. In addition, these resins are not readily available and must be synthesized. A novel resin based on 2-acrylamido-2-methyl-1-propanesulfonic acid (NaAMPS) and poly(ethylene glycol) diacrylate (PEGDA) was formulated and printed using VP. The mechanical properties, water content, and high fidelity of the scaffold indicated promise for use in tissue engineering applications. Murine fibroblasts were observed to successfully adhere and proliferate on the scaffolds. The growth, migration, and differentiation of a cell is known to dependent heavily on its microenvironment. In engineered constructs, much of this microenvironment is provided by the tissue scaffold. The physical environment results from the scaffold's geometrical features, including pore shape and size, porosity, and overall dimensions. Each of these parameters are known to affect cell viability and proliferation, but due to the difficulty of isolating each parameter when using scaffold fabrication techniques such as porogen leaching and gas foaming, conflicting results have been reported. Scaffolds with pore sizes ranging from 200 to 600 μm were fabricated and seeded with murine fibroblasts. Other geometric parameters (e.g., pore shape) remained consistent between scaffold designs. Inhomogeneous cell distributions and fewer total cells were observed in scaffolds with smaller pore sizes (200-400 μm). Scaffolds with larger pores had higher cell densities that were homogeneously distributed. These data suggest that tissue scaffolds intended to promote fibroblast proliferation should be designed to have pore at least 500 μm in diameter. Techniques developed for selective placement of dissimilar materials within a single VP scaffold enabled spatial control over cellular adhesion and proliferation. The multi-material scaffolds were fabricated using an unmodified and commercially available VP system. The material preferences of murine fibroblasts which resulted in their inhomogeneous distribution within multi-material scaffolds were confirmed with multiple resins and geometries. These results suggest that multi-material tissue scaffolds fabricated with VP could enable multiscale organization of cells and material into engineered constructs that would mimic the function of native tissue. / Doctor of Philosophy / Vat photopolymerization (VP) is a 3D printing (or additive manufacturing) technology that is capable of fabricating parts with complex geometries with very high resolution. These features make VP an attractive option for the fabrication of scaffolds that have applications in tissue engineering. However, there are few printable materials that are biocompatible and allow cells attachment. In addition, those that have been reported cannot be obtained commercially and their synthesis requires substantial resources and expertise. A novel resin composition formulated from commercially available components was developed, characterized, and printed. Scaffolds were printed with high fidelity. The scaffolds had mechanical properties and water contents that suggested they might be suitable for use in tissue engineering. Fibroblast cells were seeded on the scaffolds and successfully adhered and proliferated on the scaffolds. The growth, migration, and differentiation of cells is influenced by the environmental stimuli they experience. In engineered constructs, the scaffold provides many of stimuli. The geometrical features of scaffolds, including how porous they are, the size and shape of their pores, and their overall size are known to affect cell growth. However, scaffolds that have a variety of pore sizes but identical pore shapes, porosities, and other geometric parameters cannot be fabricated with techniques such as porogen leaching and gas foaming. This has resulted in conflicting reports of optimal pore sizes. In this work, several scaffolds with identical pore shapes and porosities but pore sizes ranging from 200 μm to 600 μm were designed and printed using VP. After seeding with cells, scaffolds with large pores (500-600 μm) had a large number of evenly distributed cells while smaller pores resulted in fewer cells that were unevenly distributed. These results suggest that larger pore sizes are most beneficial for culturing fibroblasts. Multi-material tissue scaffolds were fabricated with VP by selectively photocuring two materials into a single part. The scaffolds, which were printed on an unmodified and commercially available VP system, were seeded with cells. The cells were observed to have attached and grown in much larger numbers in certain regions of the scaffolds which corresponded to regions built from a particular resin. By selectively patterning more than one material in the scaffold, cells could be directed towards certain regions and away from others. The ability to control the location of cells suggests that these printing techniques could be used to organize cells and materials in complex ways reminiscent of native tissue. The organization of these cells might then allow the engineered construct to mimic the function of a native tissue.

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

The overall goal of this research project is to develop methods for producing a tissue engineered ligament. The envisioned tissue engineering strategy involves three steps: seeding bone marrow stromal cells (BMSCs) onto electrospun scaffolds, processing them into cords that allow cell infiltration, and conditioning them with uniaxial cyclic stretch. These steps were addressed in three complimentary studies to establish new methods to engineer a tissue with ligament-like cells depositing organized extracellular matrix (ECM). In the first study scaffold topographies were systematically varied to determine topographies that induce cells to orient and differentiate into ligament-like cells in static culture. Scaffolds — electrospun from poly (ester-urethane urea) (PEUUR) with different fiber diameters degrees of fiber alignments — were biocompatible and supported cell growth. Topographic cues guided cell alignment, and cell elongation increased with increasing fiber alignment. Finally, expression of the ligament-like markers collagen type I and decorin were enhanced on the smallest fiber diameters compared to larger diameters. In the second study BMSCs — seeded onto aligned electrospun PEUUR scaffolds — were cyclically stretched to determine the effect of dynamic mechanical stimulation on BMSC alignment and differentiation. BMSCs remained aligned parallel to the direction of fiber alignment and expressed ligament markers (e.g. collagen type I, decorin, scleraxis, and tenomodulin) on electrospun scaffolds after the application of stretch. However, the cyclic stretch regimen was not able to enhance expression of ECM components. In the third study techniques were developed to produce more clinically relevant constructs with improved cell infiltration. Specifically, a co-electrospun scaffold composed of two well integrated components was developed to create larger pores. The scaffold was also embedding in a photo-crosslinkable hydrogel to prevent the fibers from collapsing. These results demonstrate the feasibility of making a tissue engineered ligament by seeding BMSCs on an aligned, co-electrospun scaffold with submicron diameter fibers and then applying cyclic mechanical stretch. Future work will involve combining these three steps to achieve materials suitable for in vivo testing. / Ph. D.