This thesis is focused on the development and evaluation of different hybrid scaffolds for the treatment of injuries in cartilage or bone. These hybrid materials were three-dimensional polycaprolactone macroporous scaffolds obtained through freeze extraction and particle leaching combined method and modified with hyaluronic acid or mineral particles. In order to facilitate the description of the obtained results, the thesis is divided in two sections dedicated to bone and cartilage tissue engineering respectively. In the case of bone tissue engineering we addressed the treatment of disorders associated with the spine that require spinal immobilization. This Thesis proposes the development of a synthetic macroporous support for intervertebral fusion as an alternative to commercial bone substitutes. Macroporous scaffolds were developed with bare polycaprolactone or its blends with polylactic acid in order to increase its mechanical properties and degradation rate. Furthermore, the scaffolds obtained were reinforced with hydroxyapatite or Bioglass®45S5 to improve their mechanical properties and turn them in bioactive scaffolds. The supports were characterized physicochemically and biologically to determine if they met the requirements of the project. Finally, materials were tested in vivo in a bone critical size defect preformed in a rabbit model against a commercial support. Cartilage engineering has been extensively studied in the last years due to the inherent limited self repair ability of this tissue. The second part of the thesis was focused in developing a construct composed by in vitro differentiated chondrocyte like cells in a hybrid scaffold for cartilage tissue engineering. Polycaprolactone hybrid substrates coated with hyaluronic acid scaffold were developed obtaining a substrate with positive influence over the development of chondrocyte phenotype in culture and able to protect the cells from excessive mechanical loading in the joint. Cell-scaffolds constructs were obtained combining hybrid scaffolds with mesenchymal stem cells and differentiating them to chondrocytes using chondrogenic culture medium combined with hypoxia, mechanical stimulus or co-culture. Finally the cellularized scaffolds were mechanically, biochemically and histologically characterized to determine the production of extracellular matrix and expression of chondrogenic markers. / Ródenas Rochina, J. (2015). Scaffold surface modifications and culture conditions as key parameters to develop cartilage and bone tissue engineering implants [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48526 / TESIS

abstract: Glioblastoma Multiforme (GBM) is a grade IV astrocytoma and the most aggressive form of cancer that begins within the brain. The two-year average survival rate of GBM in the United States of America is 25%, and it has a higher incidence in individuals within the ages of 45 - 60 years. GBM Tumor formation can either begin as normal brain cells or develop from an existing low-grade astrocytoma and are housed by the perivascular niche in the brain microenvironment. This niche allows for the persistence of a population of cells known as glioma stem cells (GSC) by supplying optimum growth conditions that build chemoresistance and cause recurrence of the tumor within two to five years of treatment. It has therefore become imperative to understand the role of the perivascular niche on GSCs through in vitro modelling in order to improve the efficiency of therapeutic treatment and increase the survival rate of patients with GBM. In this study, a unique three dimensional (3D) microfluidic platform that permitted the study of intercellular interactions between three different cell types in the perivascular niche of the brain was developed and utilized for the first time. Specifically, human endothelial cells were embedded in a fibrin matrix and introduced into the vascular layer of the microfluidic platform. After spontaneous formation of a vascular layer, Normal Human Astrocytes and Patient derived GSC were embedded in a Matrigel® matrix and incorporated in the stroma and tumor regions of the microfluidic device respectively. Using the established platform, migration, proliferation and stemness of GSCs studies were conducted. The findings obtained indicate that astrocytes in the perivascular niche significantly increase the migratory and proliferative properties of GSCs in the tumor microenvironment, consistent with previous in vivo findings. The novel GBM tumor microenvironment developed herein, could be utilized for further in-depth cellular and molecular level studies to dissect the influence of individual factors within the tumor niche on GSCs biology, and could serve as a model for developing targeted therapies. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2020

In Canada, one in five people suffers from arthritis, of which the most common type is osteoarthritis (OA). OA is a group of joint diseases that cause pain and loss of range of motion and for which there is currently no cure. OA can be caused by numerous factors such as aging, genetics, environmental elements, and abnormal joint biomechanics (e.g., injury, obesity). These diseases are degenerative and lead to the progressive breakdown of joint cartilage, as well as changes in the underlying bone and other tissues of the joint over a period of years to decades. Articular cartilage incorporates a single type of resident cells, termed chondrocyte cells. These cells are entrapped within a dense extracellular matrix that limits their ability to proliferate and migrate to a site of injury, while the absence of blood vessels in the cartilage, amongst other factors, hinders the ability of progenitor cells to reach the site of injury, contributing to a limited capacity for intrinsic regeneration of the damaged tissue following an injury. As such, efforts to develop tissue engineering strategies that combine a biomaterial with bioactive signals to induce cells with the chondrogenic potential to regenerate tissue have been pursued actively. In this thesis, we investigate the potential of one such cartilage tissue engineering approach, whereby chondrocytes are encapsulated with alginate hydrogels incorporating inorganic polyphosphate (polyP), a promising chondrogenic signal. The driving hypothesis of the work was that polyanionic polyP would crosslink within the alginate hydrogel meshwork by ionic bonds with the multivalent cations used to form the hydrogel. Initial efforts focussed on optimizing the sterile chondrocyte encapsulation protocol for alginate beads, chondrocyte culture conditions to reduce proliferation – a response that is associated with dedifferentiation and a pathological state – and protocol for the incorporation of polyP in alginate bead when using calcium as a cationic crosslinker. We observed that polyP release from the calcium-alginate bead exhibited an important burst release to nearly 80% of the initial polyP loading within 24 hours of incubation in the culture medium. Increasing the alginate concentration led to approximately a 2.5-fold increase in polyP retention following the burst release. Subsequent incubation showed a more controlled release for at least 1 week. Efforts to reduce hydrogel swelling and increase its stability by substituting Ca2+ by Sr2+ as a crosslinker did not reduce the release rate during the burst release phase, nor did it increase the polyP retention following this initial stage. Other divalent cations including Mg2+ and Co2+, and pre-loading the polyP-alginate solution with a small concentration of Ca2+ did not impact the release profile either. Chondrocytes encapsulated in calcium- and strontium-alginate beads showed decreased DNA content and increased sulfated glycosaminoglycan accumulation at 1 week when polyP was incorporated in the beads compared to controls without polyP; however, this effect was lost at longer time points. These results suggest that this new material may find applications as a vehicle for the short-term delivery of polyP in joints and other tissues. Further efforts to improve the polyP release profile from alginate beads lead to promising results with the use of polyethylenimine (PEI) as a cationic tethering molecule between polyP and alginate. This thesis aims to generate novel biomaterials that can be used to stimulate cartilage tissue regeneration and to eventually develop a treatment strategy for OA. The work presented here will serve as a basis for continued efforts to ensure the prolonged retention of exogenous polyP into the joint.

Le chitosane présente des propriétés biologiques (biocompatibilité, biorésorbabilité, bioactivité) idéalement adaptées à des applications en ingénierie tissulaire. Dans cette étude partenariale (Programme ANR TECSAN 2010 ChitoArt), nous avons travaillé à l'élaboration d'hydrogels physiques de chitosane à propriétés physico-chimiques et biologiques variées et contrôlées, sans utilisation d'agents de réticulation externes. Ces hydrogels sont envisagés sous forme de tube mono ou pluri-membranaires pour une utilisation en tant que substituts vasculaires de petit diamètre (<6mm). En effet, l'ingénierie vasculaire présente, encore de nos jours, de nombreuses limitations lorsqu'il est question de vaisseaux de petits calibres. Notre démarche consiste en la modulation des paramètres structuraux (degré d'acétylation, masse molaire) et environnementaux (concentration du bain de gélification, du collodion) intervenants dans le procédé d'élaboration des hydrogels pour atteindre les critères physiques, biologiques et mécaniques compatibles avec cette application. L'étude morphologique des hydrogels par Cryo-Microscopie Électronique à Balayage (Cryo-MEB), via une méthode de préparation originale a permis une meilleure compréhension de l'organisation micro-structurale et multi-échelle des hydrogels de chitosane. Cette approche fondamentale a été couplée à une évaluation in vivo des propriétés biologiques des hydrogels ainsi qu'a des caractérisations mécaniques des substituts vasculaires. En particulier, l'évaluation de la suturabilité de nos substituts a mené au développement d'une formulation donnant lieu à des hydrogels physiques de chitosane suturables ayant fait l'objet d'un dépôt de brevet (N° de dépôt FR1363099). Le contrôle et la modulation des paramètres d'élaboration des hydrogels ont permis l'obtention de substituts vasculaire cellularisables et respectant les exigences (suture, compliance, résistance à l'éclatement) concernant leur implantation in vivo / Chitosan presents biological properties (biocompatibility, bioresorbability, bioactivity) ideally suited for tissue engineering. In this partnership study (ANR TECSAN 2010 ChitoArt program), we worked at the elaboration of physical chitosan hydrogels presenting various and controlled physicochemical and biological properties, without any external crosslinkers. These hydrogels are envisioned under mono- or poly-membranous tubes for small diameter vascular substitutes (<6mm) purposes. Indeed, vascular engineering presents, even today, numerous limitations for small calibre vessels. Our strategy consists in the modulation of both structural (degree of acetylation, molar mass) and environmental (neutralization bath and collodion composition and concentration) parameters involved in hydrogels elaboration process in order to reach physical, biological and mechanical requirements suitable for this application. The study of hydrogels morphology by Cryo-Scanning Electron Microscopy (Cryo-SEM), using an original sample preparation method led to a better comprehension of chitosan hydrogels fine structure and multi-scale organization. This fundamental approach was conducted through the in vivo biological evaluation of hydrogels but also to mechanical characterizations of vascular substitutes. In particular, our substitutes were evaluated in term of suture retention resulting in the development of a formulation that led to suturable physical chitosan hydrogels, which were protected by a patent (Deposit number: FR1363099). Hydrogels elaboration parameters control and modulation have resulted in the development of colonisable vascular substitutes matching their in vivo implantation requirements (suture retention, compliance, burst pressure)

Tissue engineering, with the ultimate goal of engineering artificial tissues or organs to replace malfunctioning or diseased ones inside the human body, provides a substitute for organ transplantation. Driven by the growing, tremendous gap between the demand for and the supply of donated organs, tissue engineering has been advancing rapidly. There has been great success in engineering artificial organs such as skin, bone, cartilage and bladders because they have simple geometry, low cell oxygen consumption rates and little requirements for blood vessels. However, difficulties have been experienced with engineering thick, complex tissues or organs, such as hearts, livers or kidneys, primarily due to the lack of an efficient media exchange system for delivering nutrients and oxygen and removing waste. Very few types of cells can tolerate being more than 200 μm away from a blood vessel because of the limited oxygen diffusion rate. Without a vasculature system, three-dimensional (3D) engineered thick tissues or organs cannot get sufficient nutrients, gas exchange or waste removal, so nonhomogeneous cell distribution and limited cell activities result. Systems must be developed to transport nutrients, growth factors and oxygen to cells while extracting metabolic waste products such as lactic acid, carbon dioxide and hydrogen ions so the cells can grow, proliferate and make extracellular matrix (ECM), forming large-scale tissues and organs. However, available biomanufacturing technologies encounter difficulties in manufacturing and integrating vasculature networks into engineered constructs. This work proposed a novel 3D bioprinting technology that offers great potential for integration into thick tissue engineering. The presented system offered several advantages, including that it was perfusable, it could print conduits with smooth, uniform and well-defined walls and good biocompatibility, it had no post-fabrication procedure, and it enabled direct bioprinting of complex media exchange networks.

Although millions of individuals worldwide are affected by blinding retinal degenerative diseases, most have very few options for treatment and no hope for vision restoration. Induced pluripotent stem cell (iPSC) replacement therapies represent a promising treatment option, but their effectiveness is limited by an overall lack of physical support for injected cells. Stem cell scaffolds can be used to provide this support by serving as an attachment platform for cells before, during, and after implantation. Thus, the design of polymer scaffolds with appropriate biochemistry, mechanical properties, and morphology is a critical step toward developing feasible stem cell therapies for blinding eye diseases. In this work, we aim to design a regenerative scaffold for the retina and determine the interplay among these three key design parameters. First, the feasibility of using a synthetic scaffold to grow and differentiate iPSCs to neural progenitor cells is demonstrated. The porous and degradable poly(lactic-co-glycolic acid) scaffolds employed were able to support a greater density of differentiating iPSCS than traditional tissue culture plastic. Additionally, the power of chitosan, a naturally occurring polymer, to overcome the toxic effects of copper nanoparticles is described. For two different cell types, various doses, and several time points, chitosan coated copper nanoparticles were significantly less toxic than non-coated particles. The mechanical properties of the human retina and the effects of aging and disease were also estimated using measurements of compressive modulus in animal models. In order to reach a range similar to native tissue, polymer mechanical properties were controlled using cross-linking density and surfactant templating. The influence of morphology was studied by inducing polymer structure changes via surfactant templating. Morphology significantly influenced water uptake and compressive modulus for both cross-linked poly(ethylene glycol) (PEG) and cross-linked chitosan hydrogels. Surfactant templating did not negatively affect the biocompatibility of PEG hydrogels and slightly improved the ability of chitosan hydrogels to support the growth and differentiation of iPSCs. Overall we have demonstrated the ability to tune polymer structure, mechanical properties, and biochemistry. These results add to the growing body of research aimed to understand and control cell/material interactions for biomaterial optimization.

Interacting cellular systems with nano-interfaces has shown great promise in promoting differentiation, regeneration, and stimulation. Functionalized nanostructures can serve as topological cues to mimic the extracellular matrix network to support cellular growth. Nanostructures can also generate signals, such as thermal, electrical, and mechanical stimulus, to trigger cellular stimulation. At this stage, the main challenges of applying nanostructures with biological systems are: (1) how to mimic the hierarchical structure of the ECM network in a 3D format and (2) how to improve the efficiency of the nanostructures while decreasing its invasiveness. To enable functional neuron regeneration after injuries, we have developed a 2D nanoladder scaffold, composed of micron size fibers and nanoscale protrusions, to mimic the ECM in the spinal cord. We have demonstrated that directional guidance during neuronal regeneration is critical for functional reconnection. We further transferred the nanoladder pattern onto biocompatible silk films. We established a self-folding strategy to fabricate 3D silk rolls, which is an even closer system to mimic the ECM of the spinal cord. As demonstrated by in vitro and in vivo experiments, such a scaffold can serve as a grafting bridge to guide axonal regeneration to desired targets for functional reconnection after spinal cord injuries. Benefited from the robust self-folding techniques, silk rolls can also be used for heterogeneous cell culture, providing a potential therapeutic approach for multiple tissue regeneration directions, such as bones, muscles, and tendons. For achieving neurostimulation, we have developed photoacoustic nanotransducers (PANs), which generate ultrasound upon excitation of NIR II nanosecond laser light. By surface functionalize PAN to bind to neurons, we have achieved an optoacoustic neuron stimulation process with a high spatial and temporal resolution, proved by in-vitro and in-vivo experiments. Such an application can enable non-invasive, optogenetics free and MRI compatible neurostimulation, which provides a new direction of gene-transfection free neuromodulation. Collectively, in this thesis, we have developed two systems to promote functional regeneration after injuries and stimulate neurons in a minimally invasive manner. By integrating those two functions, a potential new generation of the bioengineered scaffold can be investigated to enable functional and programmable control during the regeneration process.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13265号 / 論医博第2179号 / 新制||医||1038(附属図書館) / (主査)教授 別所 和久, 教授 安達 泰治, 教授 戸口田 淳也 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22349号 / 医博第4590号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 椛島 健治, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Blood vessels mimics (BVMs) are tissue-engineered blood vessels used to test vascular devices in an environment that mimics some simple anatomical factors of native blood vessels. It is important to accurately and consistently assess tissue-engineered blood vessels, although there is currently a lack of standardization in Cal Poly’s Tissue Engineering Lab and in the entirety of the field. The goal of this thesis was to develop and optimize imaging and image quantification techniques for tissue-engineered blood vessels. The first aim of this thesis optimized and compared imaging and assessment techniques for electrospun scaffolds. Images from different SEMs were compared to determine the benefits and drawbacks of each microscope. Several materials were also imaged using these microscopes to characterize polymers at the microscopic scale and to compare the quality of images from different SEMs. The second aim of this thesis validated and implemented a MATLAB-based automatic fiber diameter measurement tool. Fiber measurements were obtained from a manual ImageJ method, a semi-automatic DiameterJ method, and a new automatic MATLAB method and compared to evaluate accuracy and user variability of the MATLAB tool. The results of this aim validated the accuracy of the MATLAB tool and showed that it resulted in lower user variability as compared to other fiber diameter measurement methods. The third aim of this thesis developed imaging techniques for novel silicone BVMs at each stage of development. Evaluation techniques to quantify cell adhesion and coverage on silicone BVMs using SEM, widefield fluorescent imaging, and immunochemistry were developed. After refining those methods, they were applied and adapted to silicone BVMs with deployed devices. BBI, H&E, and PECAM-1 staining were all found to be effective assessment methods for silicone BVMs. Overall, the work described in this thesis increased the consistency, standardization, and accuracy of scaffold and BVM assessment in Cal Poly’s Tissue Engineering Lab.