Cells in multicellular organisms are surrounded by a complex three-dimensional macromolecular extracellular matrix (ECM). This matrix, traditionally thought to uniquely serve a structural function providing support and strength to cells within tissues, is increasingly being recognized to have pleiotropic effects in neurogenesis and regeneration processes such as neocortex folding, stem cell niche maintenance, peripheral nerve regeneration, axonal growth, and many more. ECM mediates these processes via cell-ECM interactions which provide the cells with a wealth of signals including biophysical and mechanical cues in a spatiotemporal manner. Owing to the importance of the surrounding microenvironment, modern neural tissue engineering strategies have focused on the development of engineered biomaterials capable of finely instructing the neuronal response according to their physicochemical characteristics. Neurons and neural stem cells are in fact sensitive to their mechanical and topographical environment, and cell–substrate binding contributes to this sensitivity by activating specific signaling pathways for basic cell function. In addition, the advances in nanotechnology have opened the possibility of introducing decorative nano-motifs that interact with cells at the molecular level. Successful strategies in tissue engineering are driven by not only advances in the synthesis of highly instructive biomaterials but also greatly depend on the right selection of cell sources. As a matter of fact, advances in neural tissue engineering have been strongly hampered by the poor availability of cell sources, considering that primary neurons are the only type of cells that do not proliferate. The discovery of induced pluripotent stem cells (iPSCs) has addressed many of the cell-related limitations in neural tissue engineering, offering the possibility to consistently produce a wide range of neural cell lines. Advances in cell biology have led to the development of iPSCs-derived brain spheroid, which surely represent the most promising tools for several neural tissue engineering applications ranging from in vitro modelling of neurodegenerative diseases (i.e., Parkinson's, Huntington's and Alzheimer's), biomaterials testing and drug screening platforms. The overarching goal of my doctoral work was to engineer biomaterials with instructive physicochemical properties to elicit beneficial cellular responses that are suitable for different neural tissue engineering applications such as nerve regeneration and 3D in vitro modelling. In the first study (Chapter 2), I evaluated the compounded effects of surface stiffness and micro-topography on dorsal root ganglion and human bone-marrow mesenchymal stem cells behavior. To this end, arrays of parallel microchannels of different geometries were introduced on the surface of chitosan films by electrophoretic replica deposition. In addition, a novel chemical crosslinking with citric acid was performed to both enhance the long-term stability of the chitosan films and fine-tune the surface stiffness for the investigation of its role in cell behavior. In the second study (Chapter 3), I developed a novel nanocomposite consisting of a collagen hydrogel decorated with glycine-derived carbon nanodots (Gly-CNDs). After a comprehensive physicochemical characterization of the resulting nanocomposite, I evaluated the effects exerted on neuronal differentiation and electrophysiological maturation of mouse iPSCs-derived brain spheroid. In the third study (Chapter 4), I optimized an alignable collagen-based hydrogel characterized by anisotropically oriented fibers with potential applications in both peripheral and central nervous system repair. I established a protocol that encompasses the introduction in the collagen solution of biodegradable laminin-functionalized magnetic microbeads and the time-controlled application of an external magnetic field. The regenerative potential of the hydrogel was unveiled using mouse iPSCs-derived neural stem cells.

Chemically cross-linked hydrogels (chemical gels) offer a number of enhanced properties over their physical counterparts, particularly in biomedical applications such as drug delivery, tissue engineering, and cell encapsulation. Conventional chemical gels are generally too elastic to be introduced into the body without requiring surgical implantation, making them challenging to use in a clinical context. In response, this thesis is focused on developing injectable analogues of conventional hydrogel-based biomaterials as well as advanced, engineered injectable hydrogels, enabling the facile use of these hydrogels in biomedical applications. Cross-linking is achieved using hydrazone chemistry, in which one precursor is functionalized with aldehyde groups and the other is functionalized with hydrazide groups. Following coextrusion of the reactive precursors, a stable hydrogel network spontaneously forms within seconds. By employing this chemistry as a standard in all of this work, a number of injectable hydrogel systems with well-defined properties (including swelling, drug loading and release, optical properties, gel formation and degradation kinetics, response to the temperature of the surrounding environment, and tissue response) have been generated that can be tuned by rationally varying the charge content in the precursor polymers, the number of cross-linking functional groups used, the reactivity of the electrophilic cross-linking units, and the length and number of hydrophobic affinity domains present within the gels. This work therefore presents a series of independent methods for customizing hydrogels so that they may be adapted to a number of different biomedical applications. / Dissertation / Doctor of Philosophy (PhD)

Current degradable orthopedic fixation devices do not typically facilitate tissue integration during healing. Proposed here is a novel combination of processing methods to enhance the tissue integration capability of degradable thermoplastics used in temporary orthopedic fixation devices. The provision of open pores in devices used to affix reconstructed hard tissues would allow for local cells to infiltrate during the healing process. Any openly porous structure is inherently weakened in comparison to its monolithic peers (i.e. decreased relative bulk modulus), such that the matrix materials must be made more resilient in keep the device from becoming friable. These processing methods aim to improve degradable surgical fixation devices at multiple levels of design: both through the inclusion of porous morphology, processing changes, and additives to regain mechanical integrity. Biomimetic pores are added for cellular infiltration by dissolving a porogen’s interpenetrating polymer network. The addition of open pores significantly reduces the bulk stiffness. More uniform phase separation has led to better pores, but the objects still need more resilience. Carbon nanomaterials are used to improve on the mechanics and surface chemistry of the polymer matrix material, composites of polylactide/nanodiamond are produced through cryogenic milling and solid state polycondensation. The addition of minute amounts of functionalized nanodiamond has remedied the brittle failure of the material, by cryogenic milling and solid state polycondensation of poly((D,L)lactide-co-glycolide) and hydroxyl functionalized detonation nanodiamonds. This composite has also demonstrated increased cytocompatability with 7F2 osteoblasts, as analyzed by cellular adhesion through fluorescence microscopy and alamar blue assay. / Bioengineering

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)

Corneal diseases and insults can result in opacification of the cornea and ultimately lead to blindness. Treatment options for patients are limited due to limited donor availability and the fact that many patients are not eligible for certain treatments due to the nature of their condition. When conventional treatment options are not beneficial for a patient, artificial corneal replacement is necessary. Current artificial replacements induce epithelial downgrowth, where the remaining host corneal cells grow underneath the replacement ultimately leading to implant extrusion. Therefore, surface modification of these synthetic materials is necessary in order to allow proper epithelialization on the surface. This work focuses on the creation of a novel corneal scaffold consisting of poly(2-hydroxyethyl methacrylate) (pHEMA) which is surface modified by 3-(acrylamido)phenylboronic acid (APBA), a molecule known to have cell-binding properties through its ability to bind sugars found throughout the cell membrane. Surfaces were modified using two different polymerization techniques: conventional free radical polymerization (CFRP) and a controlled polymerization technique known as atom transfer radical polymerization (ATRP). It was hypothesized that ATRP would yield more uniform APBA brushes than the conventional method, and therefore create a more efficient cell-binding surface than the conventional method. Following each modification, the surface chemical composition of the materials was confirmed by ATR-FTIR, XPS and surface wettability measurements. Once prepared, NIH 3T3 mouse embryo fibroblasts were seeded onto the surfaces and cell viability was assessed through an MTT assay. The results revealed no cell viability on the APBA-modified surfaces, with surface hydrophobicity, grafting density and surface toxicity (for surfaces modified through ATRP) contributing to the lack of cell attachment. / Thesis / Master of Applied Science (MASc)

Ocular drug delivery to the posterior segment of the eye is extremely challenging. The delivery of the pharmaceuticals is made difficult by the numerous barriers that are present in the eye, as well as the isolated nature of the eye. The eye also consists of efficient drainage routes that eliminate the drug that has entered the eye successfully. Because of these reasons, drug delivery to the posterior segment of the eye is challenging and complicated. As a result, conventional eye drops are an inefficient way to deliver the pharmaceuticals to the eye as <5% of the administered dose is delivered to the anterior segment of the eye, and a negligible amount is delivered to the posterior tissues. The work presented in this thesis focuses on the design, synthesis, and characterization of the PLGA nanoparticles as a drug delivery vehicle to treat diseases associated with the posterior segment of the eye. The slow-release formulation was developed using PLGA nanoparticles and synthesized by the Double Emulsion Method (W1-O-W2). The PLGA nanoparticles were optimized by following various protocols and formulations to obtain the highest encapsulation efficacy and desired particle size range by changing the intensity of sonication, speed of ultracentrifugation, composition, and amount of the stabilizer and PLGA nanoparticles. The nanoparticles showed a 97% encapsulation efficiency with Bovine Serum Albumin (BSA) and a particle size of 201 nm. The slow-release formulation was further developed by immobilization of the particles in a thermogelling PNIPAAM scaffold. In vitro drug release results suggest that PNIPAAM containing PLGA nanoparticles produced in this work has the potential to be further developed and used as a drug delivery vehicle for the posterior segment of the eye. / Thesis / Master of Applied Science (MASc)

Articular cartilage lesions are often caused by joint trauma and can progress to osteoarthritis (OA) if left untreated. Cartilage tissue engineering is a promising approach for chondral lesion repair, involving the cultivation of cell-seeded scaffolds to generate neocartilage tissues recapitulating composition, structure, and function of native cartilage. Transforming growth factor beta (TGF-β) is widely utilized in cartilage tissue engineering for its ability to promote chondrogenesis and extracellular matrix (ECM) biosynthesis. Conventionally, TGF-β is supplemented in culture medium at supraphysiologic doses (10-100 ng/mL) during in vitro cultivation to regenerate neocartilage with native-matched sGAG content and mechanical properties. However, these doses are 10-1000-fold higher than the physiologic range, promoting undesirable tissue features that are detrimental to the functional behavior of hyaline cartilage. Additionally, TGF-β gradients from media supplementation can induce pronounced heterogeneities in ECM distribution, potentially compromising the survival of engineered cartilage under physiologic loading. The dissertation aims to enhance cartilage regeneration quality using bio-inspired latent TGF-β (LTGF-β) conjugated scaffolds. We hypothesize that LTGF-β scaffolds can achieve uniform delivery of moderated, near-physiologic doses of TGF-β through cell-mediated activation, inducing homogeneous and more hyaline cartilage-like tissue growth. We first evaluated the impact of physiologic TGF-β doses on tissue growth. To address issues related to TGF-β concentration gradients and tissue heterogeneities, we employed a reduced-size construct model. Our findings demonstrate that physiologic doses of TGF-β promote significant enhancements in tissue properties for reduced-size tissues, while also mitigating undesirable outcomes associated with excessive TGF-β. Subsequently, we developed bio-inspired LTGF-β-conjugated scaffolds to deliver physiologic doses of TGF-β. We established a quantification platform based on TGF-β autoinduction to accurately measure the bioactivity level of delivered TGF-β, confirming conjugated LTGF-β can be activated in physiologic range. Further, this quantification platform exhibits versatility for applications in native tissue studies and other TE platforms. Lastly, we determined that LTGF-β conjugation led to enhancements in tissue functional properties comparable to native tissue, while mitigating the abnormal features of neocartilage associated with TGF-β excesses. Moreover, LTGF-β conjugation significantly improves tissue spatial homogeneities in composition and mechanical properties, offering promising implications for enhancing clinical regeneration outcomes.

[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

<p dir="ltr">Approximately 2.39 million Americans suffer from inflammatory bowel disease (IBD), an autoimmune disorder that is characterized by chronic inflammation of the gastrointestinal (GI) tract. Current treatment options for IBD, which are limited, include oral medications, surgery, and supportive care. These therapeutics often times are not effective and are associated with high toxicity. Thus, there is a pressing clinical need for a therapy that can be delivered both locally and precisely, while also having an improvement in efficacy and lower toxicity.</p><p dir="ltr">This study introduces three novel microrobot designs fabricated using stereolithography (SLA) 3D printing, which aims to address the challenges seen in IBD treatment. The microrobots utilize a reservoir design to encapsulate the drug for an on-demand release, allowing for improved control and precision. The SLA microrobots were evaluated for cytotoxicity as well as drug release capabilities. We were able to demonstrate a local release of a protein on-demand at a biologically relevant temperature. The integration of microrobots in IBD therapy has the capability to significantly improve patient outcomes and quality of life, offering a more efficient and less toxic treatment approach.</p>

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.