Increasing emphasis has been placed on the use of renewable resources, on decreased reliance on petroleum in order to better utilize global energy needs. Biological structures available in nature have been a constant inspiration to the design and fabrication of the new line of functional biomaterials whose unique phenomena can be exploited in novel applications. In tissue engineering for example, a natural biomimetic material with close resemblance to the profile features existed in a native extracellular matrix could provide a temporary functional platform to regulate and control cellular interactions at a molecular level and to subsequently direct a tissue regeneration. However, the lack of rigidity of natural materials typically limits their mass production. One promising approach to address this shortcoming is to introduce a biomimetic composite material reinforced by high purity nanofibers found in nature. As an attractive reinforcing filler phase, cellulose nanowhiskers (CNWs) offer exceptional properties such as high aspect ratio, large interface area, and significant mechanical performance. As such, CNWs could integrate a viable nanofibrous porous candidate, resulting in superior structural diversity and functional versatility. Inspired by the fascinating properties of cellulose and its derivatives, we have designed two bio-inspired nanocomposite materials reinforced with CNWs in this work. The successful grafting of CNWs within the host matrix and their tendency to interconnect with one another through strong hydrogen bonding gave rise to the formation of a three-dimensional rigid percolating network, fact which imparted considerable mechanical strength and thermal stability to the entire structure with only a small amount of filler content, i.e. 3 wt.%. Also, the biocompatibility of the nanocomposite was probed by in-vitro incubation of human-bone-marrow-derived mesenchymal stem cells (MSCs), which resulted in the invasion and proliferation of MSCs around the nanocomposite at day 8 of culture. The green functional biomaterial with its unique features in this work could open new perspectives in the self-assembly of nanobiomaterial for tissue-engineered scaffolding, while it could make the design of the next generation of fully green functional biomaterial a reality.

Three-dimensional collagen/chitosan scaffolds fabricated by freeze-drying technique in 96-well polystyrene and PDMS plates were optimized during this study. Surface tension is, by and large, one of the most limiting factors in fabricating freeze-dried scaffolds in small format well plates. Traditionally, bowl-shaped top surfaces of collagen/chitosan scaffolds were common in polystyrene 96-well plate; whereas for PDMS 96-well plate, dome-shaped surfaces were formed. These surface tension phenomena are not desirable in cell studies especially during initial cell seeding. A combination of surface treatment and change of freeze-drying regime were developed to mitigate the surface tension problem in PS and PDMS 96-well plates respectively. Collagen/chitosan scaffolds of varying concentration and composition were experimented in both polystyrene and PDMS 96-well plates. Thin water film treatment with UV cross-linking was successfully used to eliminate meniscus in PS well plates; pre-cooling, on the other hand, was utilised to treat scaffold solutions in PDMS well plates. The resultant matrices all had flat top surfaces and average thickness of 1 mm. As expected, scaffolds with lower overall polymer concentration or, from a compositional perspective, scaffolds with high chitosan content generally had larger pores. Microscopic observation by multi-photon microscope was performed and chemical analyses were conducted to characterize the surface-treated scaffolds. In addition, scaffolds were tested in vitro using DLD-1 cells, hMSCs and fibroblasts for their biological performance. The purpose of this study was to address the problem of using small format culture wells for the fabrication of freeze-dried collagen-based scaffolds for studies of cell growth in 3D culture and in microfluidic perfusion bioreactors.

The field of tissue engineering has grown immensely since its inception in the late 1980s. However, currently commercialized tissue engineered products are simple in structure. This is due to a pre-clinical bottleneck in which complex tissues are unable to be fabricated. 3D bioprinting has become a versatile tool in engineering complex tissues and offers a solution to this bottleneck. Characterizing the mechanical properties of engineered tissue constructs provides powerful insight into the viability of engineered tissues for their desired application. Current methods of mechanical characterization of soft hydrogel materials used in tissue engineering destroy the sample and ignore the effect of 3D bioprinting on the overall mechanical properties of a construct. Herein, this work reports on the novel use of a non-destructive method of viscoelastic analysis to demonstrate the influence of 3D bioprinting strategy on mechanical properties of hydrogel tissue scaffolds. 3D bioprinting is demonstrated as a versatile tool with the ability to control mechanical and physical properties. Structure-function relationships are developed for common 3D bioprinting parameters such as printed fiber size, printed scaffold pattern, and bioink formulation. Further studies include effective real-time monitoring of crosslinking, and mechanical characterization of multi-material scaffolds. We envision this method of characterization opening a new wave of understanding and strategy in tissue engineering. / Graduate

A thin layer of calcified cartilage at the native cartilage-to-bone junction facilitates integration between deep zone articular cartilage and subchondral bone, while maintaining the integrity of the two distinct tissue regions. Regeneration of this interface remains a significant clinical challenge for long-term and functional cartilage repair. The strategy for osteochondral interface formation discussed in this thesis focuses on the design and optimization of a biomimetic scaffold for stable calcified cartilage formation. The ideal interface scaffold supports chondrocyte biosynthesis and the formation of calcified cartilage with physiologically-relevant mechanical properties. Furthermore, the interface scaffold allows for osteointegration and the maintenance of the calcified cartilage matrix. It is hypothesized that ceramic presence and zonal chondrocyte interactions regulate cell biosynthesis and mineralization, and these cell-matrix and cell-cell interactions are essential for calcified cartilage formation and maintenance. Biomimetic design parameters for an interface scaffold were determined by characterizing the native interface in terms of mineral and matrix distribution. A composite hydrogel-hydroxyapatite scaffold was then designed to support formation of a functional calcified cartilage matrix. The hydrogel phase maintains the chondrocyte phenotype and allows for incorporation of ceramic particles, while the biomimetic ceramic phase is osteointegrative and decreases the need for cell-mediated mineralization. This scaffold was optimized <italic>in vitro</italic> based on hydrogel type, chondrocyte population, and ceramic particle size. The collective findings from these cell-ceramic interaction studies determined that hypertrophic chondrocytes, cultured in the presence of micron-sized hydroxyapatite particles, exhibit enhanced hypertrophy and matrix deposition. Scaffold ceramic dose and seeding density were also optimized for promoting calcified cartilage formation <italic>in vitro</italic>. In order to implement the scaffold for integrative cartilage repair, a scaffold was designed to regenerate both uncalcified and calcified cartilage on a bilayered hydrogel scaffold. Furthermore, a polymer-ceramic nanofiber component was added to augment the original design for <italic>in vivo</italic> implementation. The hydrogel-nanofiber composite scaffold was evaluated <italic>in vivo</italic> and found to support mineralization and osteointegration within the bone region while preventing endochondral ossification within the repair tissue. Finally, inspired by the stratified organization of zonal chondrocyte populations above the calcified cartilage interface, the layered hydrogel model was used to determine the role of zonal chondrocyte organization on calcified cartilage stability. This thesis collectively explores cell-ceramic and cell-cell interactions, and their ramifications for calcified cartilage formation and maintenance. Specifically, ceramic presence promotes the deposition of a calcified cartilage matrix by hypertrophic chondrocytes in a dose-dependent manner, and furthermore, communication between surface zone and deep zone chondrocyte populations suppresses mineralization within articular cartilage above the calcified cartilage interface. It is anticipated that the scaffold design strategy developed in this thesis can also be applied to the regeneration of other complex interfaces where there are transitions from soft-to-hard tissue.

In recent studies it was reported that the D-amino acid containing peptide HYD1 was used in the treatment of necrotic cell death in multiple myeloma cell lines and showed promising biological activity and in vivo activity. It was meaningful to explore strategies for increasing the therapeutic efficacy of HYD1, a linear peptide. These efforts led to the development of MT1-101 (cyclized peptidomimetics), a lead compound that showed increased in vitro activity and in vivo activity. MTI-101 was found to bind the cell adhesion molecule CD44 and induce programmed necrosis in myeloma cell lines. It was important to improve on the binding efficiency of the MTI-101 to this target and explore more cost effective ways to synthesize this peptide. This lead to developing Cyclic beta-hairpin-like peptoid scaffolds, which introduced diverse families of random peptoid-body libraries that will be screened to find small stable scaffolds that compete with and can replace antibodies as cell-surface targeting reagents. The synthesis of peptoids on solid-support can be more cost effective and a large library can be developed using a diverse library of primary amines. This initiated this thesis project to develop a generalized scheme for the synthesis of TRP-like primary amine peptoid side chains used in the cyclic beta-hairpin - Like scaffolds.

Tissue engineering is a multidisciplinary subject aimed at producing the immunologically tolerant artificial tissues/organs to repair or replace damaged ones. In this field, tissue scaffold plays a key role to support cell growth and new tissue regeneration. For fabrication of tissue scaffolds with individual external geometry and predefined inner structure, rapid prototyping (RP) systems based on fluid dispensing techniques have proved to be very promising. The present research conducted a comprehensive study on the dispensing-based fabrication process. <p>First of all, the scaffold materials are characterized in terms of their biocompatibility and flow behaviour. The biocompatibility of biomaterials of PLLA, PCL, collagen, chitosan, and gelatine is evaluated in terms of supporting neuron cells adhesion and outgrowth. Chitosan solution (2% w/v) in acetic acid is shown to be the most promising among the examined biomaterials for the fabrication of nerve tissue scaffolds. Its non-Newtonian flow behaviour is identified by using a commercial rheometer. <p>In the fabrication process, the flow rate of biomaterials dispensed, the profile of strand cross-sections, and the scaffold porosity are very important and must be precisely controlled. A model is developed to represent the flow rate of biomaterials dispensed under the assumptions that the flow is incompressible, steady, laminar, and axisymmetric. Also, the profile and size of line strands at different layers and portions are modeled based on the Young-Laplace equation. Thus the dispensing-based fabrication process can be predicted in terms of the flow rate and the scaffold porosity. <p>The effects of operation conditions on the fabrication result are identified theoretically and experimentally. Simulation result shows that a higher driving pressure, a higher temperature, and a larger needle diameter will result in a larger size of the strand cross-sections and lower scaffold porosity. The change pattern, however, is nonlinear, which is affected by the fluid surface tension and non-Newtonian flow behaviour of scaffold biomaterials. <p>To verify the effectiveness of the developed models, experiments were carried out on a commercial dispensing system (C-720, Asymtek, USA). To avoid the possible error derived from the temperature difference between the dispensing system and the rheometer, a new method is presented to characterize the fluid properties used for model predictions. Experimental results illustrate that the developed models, combined with the new identification method, are very promising to predict the dispensing-based fabrication process.

Grandes defectos óseos constituyen un reto para el campo clínico, ya que no puede ser reparado por el propio organismo, sino que requieren la implantación de injertos de hueso adecuado. Para superar los inconvenientes de los injertos procedentes de fuentes autólogas o allogeneicas, la ingeniería de tejidos óseos pretende sustituir el tejido perdido utilizando el cultivo de células in vitro sobre biomateriales porosos. El cultivo de células en grandes andamios porosos ha demostrado ser difícil, que requiere bioreactores, que se utilizan para el cultivo de tejidos y el estudio del comportamiento de células en 3D de los andamios. De interés especial es el condicionamiento mecánico de los tejidos cultivados por bioreactor de la ingeniería del tejido óseo, que es capaz de aumentar el potencial osteogénico de los injertos sintéticos.En este trabajo, dos sistemas de bioreactores fueron desarrollados para permitir comprender las propiedades bioactivas de andamios de diferentes materiales y la mecanoregulación del comportamiento de células o tejidos. Un sistema de bioreactor de perfusión in vitro fue desarrollado para el sembrado y cultivo de células incorporadas en cilindros de un biomaterial poroso. Varios estudios para la determinación de los parámetros del sembrado de células aplicable se llevaron a cabo, así como experimentos de cultivo de células bajo flujo de fluido constante con una estimulación mecánica adicional por alternancia del flujo.Un sistema de cámara ósea fue desarrollado como un bioreactor in vivo. El sistema produjo un defecto óseo grande en tibias de perros y permitió la implantación repetida de grandes andamios porosos de materiales diferentes. El tejido creciendo en los andamios permite extraer conclusiones sobre las propiedades de osteoconductividad u osteinductividad de los andamios. Además, un dispositivo de compresión se ha desarrollado para aplicar cargas cíclicas en los andamios en vivo para estudiar el efecto de la estimulación mecánica en el desarrollo de los tejidos.Los estudios con el sistema de perfusión desarrollado han demostrado que el sembrado de células en grandes andamios porosos es posible, lo que se considera crucial para el cultivo celular. El largo tiempo de cultivo de células mostró la proliferación de las células madre mesenquimales hasta dos semanas. El patrón de estimulación utilizado en el estudio aumentó la expresión de la osteocalcina, lo que indica una mayor actividad de las células, pero la ausencia de expresión de RunX2 y colágeno I impidió la determinación concluyente de la diferenciación.El sistema desarrollado de la cámara ósea demostró su funcionalidad en el entorno quirúrgico durante los experimentos in vivo. Complicaciones durante los experimentos no permitieron la aplicación de las cargas cíclicas de los andamios implantados. La formación de hueso retrasada debido al defecto óseo creado y material de andamios restantes no permitieron conclusiones definitivas acerca de las propiedades del material del andamio. Sin embargo, el estudio proporciona datos para el desarrollo futuro del dispositivo y protocolo clínico.Los estudios realizados constituyen una novedad en respecto a la creación de bioreactores para el estudio de la andamios porosos sintéticos de grandes dimensiones in vitro e in vivo. Los sistemas desarrollados constituyen la base para otros estudios en mecanobiología de las células óseas y los tejidos. / Large bone defects constitute a challenge for the clinical field, because they cannot be repaired by the body itself, but require the implantation of suitable bone grafts. To overcome the drawbacks of grafts from autologous or allogous sources, modern bone tissue engineering aims to replace lost tissue by cultivating cells in vitro on porous biomaterials. The cell culture on large porous scaffolds has shown to be difficult, requiring bioreactors, which are used for tissue culture and the study of cell behaviour in 3D scaffolds. Of special interest is the mechanical conditioning of the cultured tissue for bioreactor-based bone tissue engineering, which is able to enhance the osteogenic potential of the synthetic grafts.In this work two bioreactor systems were developed to allow insight into bioactive properties of different scaffold materials and the mechanoregulation of cell or tissue behaviour. An in vitro perfusion bioreactor system was developed for the cell seeding and culture on porous biomaterial cylinders. Several studies for the determination of applicable cell seeding parameters were conducted, as well as experiments of cell culture under steady fluid flow with additional mechanical stimulation by alternating fluid flow. A bone chamber system was developed as an in vivo bioreactor. The system produced a large bone defect in dog tibia and allowed the repeated implantation of large porous scaffolds of different material compositions.The ingrowing tissue was observed to allow conclusions about osteoconductive or osteinductive properties of the scaffolds. Additionally a compression device was developed to apply cyclic loading on the scaffolds in vivo to study the effect of mechanical stimulation on tissue development.The studies with the developed in vitro perfusion bioreactor system have shown that it is possible to seed cells throughout large porous scaffolds, which is deemed crucial for the further cell culture. The long time cell culture showed the proliferation of mesenchymal stem cells up to two weeks. The stimulation pattern used in the study enhanced the expression of osteocalcin, indicating an enhanced cell activity, but the absence of RunX2 and collagen I expression rendered the determination of differentiation inconclusive.The developed bone chamber system proved to be functional in the surgical environment during the in vivo experiments. Occurring complications during the experiments did not allow the application of the cyclic loading of implanted scaffolds. Delayed bone formation due to created bone defect and remaining scaffold material did not allow final conclusions about the scaffold material properties. Nevertheless the study provides input for further development of the device and clinical protocol.The conducted studies constitute a novelty regarding the creation of bioreactors for the study of synthetic porous scaffolds of large dimensions in vitro and in vivo. The developed systems form the basis for further studies in mechanobiology of bone cells and tissue.

Tissue engineering is a multidisciplinary subject aimed at producing the immunologically tolerant artificial tissues/organs to repair or replace damaged ones. In this field, tissue scaffold plays a key role to support cell growth and new tissue regeneration. For fabrication of tissue scaffolds with individual external geometry and predefined inner structure, rapid prototyping (RP) systems based on fluid dispensing techniques have proved to be very promising. The present research conducted a comprehensive study on the dispensing-based fabrication process. <p>First of all, the scaffold materials are characterized in terms of their biocompatibility and flow behaviour. The biocompatibility of biomaterials of PLLA, PCL, collagen, chitosan, and gelatine is evaluated in terms of supporting neuron cells adhesion and outgrowth. Chitosan solution (2% w/v) in acetic acid is shown to be the most promising among the examined biomaterials for the fabrication of nerve tissue scaffolds. Its non-Newtonian flow behaviour is identified by using a commercial rheometer. <p>In the fabrication process, the flow rate of biomaterials dispensed, the profile of strand cross-sections, and the scaffold porosity are very important and must be precisely controlled. A model is developed to represent the flow rate of biomaterials dispensed under the assumptions that the flow is incompressible, steady, laminar, and axisymmetric. Also, the profile and size of line strands at different layers and portions are modeled based on the Young-Laplace equation. Thus the dispensing-based fabrication process can be predicted in terms of the flow rate and the scaffold porosity. <p>The effects of operation conditions on the fabrication result are identified theoretically and experimentally. Simulation result shows that a higher driving pressure, a higher temperature, and a larger needle diameter will result in a larger size of the strand cross-sections and lower scaffold porosity. The change pattern, however, is nonlinear, which is affected by the fluid surface tension and non-Newtonian flow behaviour of scaffold biomaterials. <p>To verify the effectiveness of the developed models, experiments were carried out on a commercial dispensing system (C-720, Asymtek, USA). To avoid the possible error derived from the temperature difference between the dispensing system and the rheometer, a new method is presented to characterize the fluid properties used for model predictions. Experimental results illustrate that the developed models, combined with the new identification method, are very promising to predict the dispensing-based fabrication process.

Many important biomaterials are composed of multiple layers of networked fibers. A prime example is in the field of tissue engineering, in which damaged or diseased native tissues are replaced by artificial tissues that are grown on fibrous polymer networks. For load bearing tissues, it is critical that the mechanical behavior of the engineered tissue be similar to the behavior of the native tissue that it will replace. In the case of soft tissues such as heart valves, the macroscale mechanical behavior is highly anisotropic and nonlinear. This behavior is a result of complex deformations of the collagen and elastin fibers that form the extracellular matrix (ECM). The microstructure of engineered tissues must be properly designed to reproduce this unique macroscopic behavior. While there is a growing interest in modeling and simulation of the mechanical response of this class of biomaterials, a theoretical foundation for such simulations has yet to be firmly established. This work introduces a method for modeling materials that have a layered, fibrous network microstructure. Methods for characterizing the complex network geometry are first established. Then an algorithm is developed for generating realistic network geometry that is a good representation of electrospun tissue scaffolds, which serve as the primary synthetic structure on which engineered tissues are grown. The level of fidelity to the real geometry is a significant improvement on previous representations. This improvement is important, since the scaffold geometry has a strong influence over the macroscopic mechanical behavior of the tissue, cell proliferation and attachment, nutrient and waste flows, and extracellular matrix (ECM) generation. Because of the importance of scaffolds in tissue formation and function, this work focuses on characterizing scaffold network geometry and elucidating the impact of geometry on macroscale mechanics. Simulation plays an important role in developing a detailed understanding of scaffold mechanics. In this work, Cosserat rod theory is used to model individual fibers, which are connected to form a network that is treated as a representative volume element (RVE) of the material. The continuum theory is the basis for a finite element discretization. The nonlinear equations are solved using Newton's method in a parallel implementation that is capable of accurately capturing the large, three-dimensional fiber rotations and large fiber stretches that result from the large macroscopic deformations experienced by these biomaterials in their natural environment. Comparisons of simulation results with existing analytical models of soft tissues show that these models can predict the behavior of scaffold networks with reasonable accuracy, despite the significant differences between soft tissue and scaffold network microstructural geometry. The simulations also reveal how macroscale loading is related to the microscale fiber deformations and the load distribution among the fibers. The effects of different characteristics of the microstructural geometry on macroscopic behavior are explored, and the implications for the design of scaffolds that produce the desired macroscopic behavior are discussed. Overall, the improved modeling of electrospun scaffolds presented in this work is an important step toward designing more functional engineered tissues.

Tissue engineering of the annulus fibrosus (AF) is challenging due to its complex lamellar structure. Polyurethane scaffolds have shown promise in AF tissue engineering. The current study examines whether matrix protein coatings (collagen type I, fibronectin, or vitronectin) would enhance cell attachment and promote cell and collagen orientation that more closely mimics native AF. The results demonstrate that the greatest cell attachment occurred with fibronectin (Fn)-coated scaffolds. Cells on Fn-coated scaffolds were also aligned parallel to scaffold fibers, a process that involved α5β1 integrin, determined by integrin-specific blocking antibodies. The inhibition of this integrin reduced AF cell spreading and alignment and the changes in cell shape were regulated by the actin cytoskeleton, demonstrated using cytochalasin D inhibitor. Cells on Fn-coated scaffolds formed fibrillar Fn, synthesized significantly more collagen, and showed alignment of type I collagen that more closely mimics native AF therefore facilitating the development of the tissue in vitro.