Spinal cord injury and stroke are two devastating neurological events that lack effective clinical treatments. Recent neuroregenerative approaches involving the delivery of cells or drugs to the injured tissue have shown promise, but face critical challenges to clinical translation. Herein, hyaluronan-methylcellulose (HAMC) hydrogels were investigated as a versatile means of overcoming the challenges facing central nervous system cell and drug delivery. HAMC was shown to support the viability of encapsulated human umbilical tissue-derived cells, demonstrating utility as a scaffold for therapeutic cell delivery to the injured spinal cord. In a drug delivery context, release of the neuroregenerative drug cyclosporin A from the hydrogel was tunable over 2-28 days and the drug diffused to the stem cell niche in the brain and persisted for up to 24 days at a stable concentration when the HAMC-based system was implanted epi-cortically. HAMC is thus a promising tool for emerging neuroregenerative therapies.

Spinal cord injury and stroke are two devastating neurological events that lack effective clinical treatments. Recent neuroregenerative approaches involving the delivery of cells or drugs to the injured tissue have shown promise, but face critical challenges to clinical translation. Herein, hyaluronan-methylcellulose (HAMC) hydrogels were investigated as a versatile means of overcoming the challenges facing central nervous system cell and drug delivery. HAMC was shown to support the viability of encapsulated human umbilical tissue-derived cells, demonstrating utility as a scaffold for therapeutic cell delivery to the injured spinal cord. In a drug delivery context, release of the neuroregenerative drug cyclosporin A from the hydrogel was tunable over 2-28 days and the drug diffused to the stem cell niche in the brain and persisted for up to 24 days at a stable concentration when the HAMC-based system was implanted epi-cortically. HAMC is thus a promising tool for emerging neuroregenerative therapies.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13068号 / 論医博第2123号 / 新制||医||1019(附属図書館) / 33219 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 開 祐司, 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

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)

<p>Intervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to low back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Additionally, while cell delivery to the pathological IVD has significant therapeutic potential for enhancing NP regeneration, the development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the NP region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into biomaterial scaffolds for NP tissue engineering or cell delivery to the disc may be beneficial for promoting NP cell survival and phenotype. In this dissertation, laminin-111 (LM111) functionalized poly(ethylene glycol) (PEG) hydrogels were developed and evaluated as biomaterial scaffolds for cell-based NP regeneration. </p><p>Here, PEG-LM111 conjugates with functional acrylate groups for crosslinking were synthesized and characterized to allow for protein coupling to both photocrosslinkable and injectable PEG-based biomaterial scaffolds. PEG-LM111 conjugates synthesized using low ratios of PEG to LM111 were found support NP cell attachment and signaling in a manner similar to unmodified LM111. A single PEG-LM111 conjugate was conjugated to photocrosslinkable PEG-LM111 hydrogels, and studies were performed to evaluate the effects of hydrogel formulation on immature NP cell phenotype in vitro. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, softer LM111 presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111 presenting and PEG-only gels. When cells were encapsulated in 3D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (<1 kPa) PEG-LM111 hydrogels. </p><p>A novel, injectable PEG-LM111 hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. PEG-LM111 conjugates were crosslinked via a Michael-type addition reaction upon the addition of PEG-octoacrylate and PEG-dithiol. Injectable PEG-LM111 hydrogel gelation time, mechanical properties, and ability to retain delivered cells in the IVD space were evaluated. Gelation occurred in approximately 20 minutes without an initiator, with dynamic shear moduli in the range of 0.9 - 1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 hydrogel carrier, as compared to cells in liquid suspension. </p><p>The studies presented in this dissertation demonstrate that soft, LM111 functionalized hydrogels may promote or maintain the expression of specific markers and cell-cell interactions characteristic of an immature NP cell phenotype. Furthermore, these findings suggest that this novel, injectable laminin-functionalized biomaterial may be an easy to use and biocompatible carrier for delivering cells to the IVD.</p> / Dissertation

The synthetic biodegradable polymer poly(lactide-co-glycolide) (PLGA) has been widely explored as substrate biomaterials for controlled drug delivery and tissue engineering. ECM component heparin and bone mineral hydroxyapatite (HA) are attractive biomaterials which can functionalize the PLGA surface to improve cell cell response and to bring in the dual growth factor delivery, because heparin and HA both can improve cell responses and bind with various proteins. To combine the osteoconductivity of HA and the controlled drug release of PLGA microspheres, HA coated PLGA microspheres were developed by a 3 hour rapid HA precipitation on the PLGA microsphere surface. Effects of various fabrication parameters on microsphere and HA coating morphology were evaluated. This core-shell composite worked as a dual drug delivery device and demonstrated better cell cell response than PLGA microspheres without HA coating. Three different methods, including osmogen, extractable porogen and gas-foaming porogen, were evaluated to fabricate porous microspheres as injectable cell scaffolds in the tissue engineering. The gas-foaming method produced covered porous PLGA microspheres, on which a skin layer covered all the surface pores. The skin layer was hydrolysed by NaOH to control the surface porosity. The modified open porous microspheres have large continued surface areas between pores, which provided more continued areas for cell adhesion. The porous microspheres with controllable surface porosity and large surface continuity between pores could be novel injectable cell scaffolds. Heparin was immobilized on the open porous PLGA microspheres by a facile layer-by-layer assemble to combine the advantages of porous structure and the protein binding from heparin. The heparin-coated porous microspheres promoted cell adhesion, spreading, proliferation and osteogenic differentiation. Growth factor-like protein lactoferrin was immobilized on the heparin coated porous microspheres, which further enhanced MG-63 proliferation and osteogenic differentiation. The heparin-coated porous microspheres are promising multi-functional devices for controlled drug delivery and injectable cell delivery.

Les siRNA se sont des agents actifs et spécifiques couramment utilisés en thérapie génique. L’intérêt d’utiliser des nanoparticules en tant que vecteurs des siRNA est leur capacité à protéger ces derniers de la dégradation par les nucléases et également de leur permettre d’être délivrés au niveau de leur cible thérapeutique. Afin d’appuyer cette théorie, ce travail s’est concentré sur un modèle cellulaire de sarcome d’Ewing, dans le but de mettre au point un nouveau système galénique pour le transport de siRNA formé des nanoparticules bifonctionnelles de diamants fluorescentes recouvertes par des polymères cationiques. Ces nano-vecteurs sont capables d’induire efficacement l’inhibition de l’expression de l’oncogène EWS-Fli1 dans les cellules en culture s’ils transportent des siRNA dirigés contre ce gène. Par ailleurs, les nanodiamants, grâce à leurs propriétés de fluorescence stable et intense, ont constitué des outils de détection permettant de suivre leurs voies de pénétration, leur biodistribution cellulaire, ainsi que la cinétique de libération des siRNA dans le cytoplasme. Enfin, un modèle de nanodiamants fonctionnalisés par le polyéthylenimine a été choisi pour la poursuite des travaux biologiques en raison de son efficacité de vectorisation. / SiRNA are powerful and commonly used agent for the specific inhibition of gene expression. They need to be vectorized by nanoparticles to facilitate cell penetration and their protection from degradation in biological media. At first, cationic nanodiamonds coated with cationic polymers were developed and were able to adsorb siRNA on their surface. Using antisense siRNA against the oncogene EWS-Fli1, nanodiamonds allowed to efficiently induce the inhibition of expression of the oncogene EWS-FLI1 in cultured Ewing sarcoma cells. As a second goal of this study, the fluorescence of red color center created in the nanodiamonds was used to follow their pathways, their cellular biodistribution and the kinetics of release of siRNA into the cytoplasm. In conclusion, nanodiamonds functionalized by polyethylenimine showed a better transfection efficiency and were chosen for further biological studies.

Heart failure accounts for more deaths in the United States than any other pathology. Unfortunately, repairing the heart after pathological injury has become an overwhelming task for physicians and researchers to overcome. Fortunately, cellular cardiomyoplasty has emerged as a promising solution for sufferers of heart failure. Such a therapy is limited in efficacy due to poor engraftment efficiencies, however. To address this issue, we have developed a tissue engineered vehicle for cell delivery. Use of a "cardiac patch" resulted in localized and efficient delivery of human mesenchymal stem cells (hMSC) to infarcted myocardium. Application of a cardiac patch also attenuated adverse remodeling. Additionally, the culture of stem/progenitor cells within three dimensional collagen constructs led to modulations in cell function, which did not promote enhanced angiogenesis in vitro or in vivo. Despite enhanced neovessel formation, hMSC patches were more beneficial at augmenting myocardial repair compared to directly injected hMSC. Lastly, although hMSC represent an effective cell source option for enhancing cardiac repair they require additional purification and expansion steps which inherently delay the turnover before treatment. Therefore, suitable cell alternative are being sought. Human embryonic stem cell derived mesenchymal (B4) cells display several phenotypic similarities to hMSC. B4 progenitor cells responded similarly to hMSC in 3D culture. In addition B4 progenitor cell patch application to infarcted myocardium resulted in similar indices of repair compared to hMSC. Thus, a tissue engineering approach represents an effective cell delivery strategy and induces modulations in cell function which may demonstrate pathological significance.

Despite being the most prevalent presentation of Age-Related Macular Degeneration (AMD), dry AMD (dAMD) lacks a therapeutic treatment. Retinal pigment epithelium (RPE) dysfunction preceding the onset of dAMD has inspired interest in regenerative medicine approaches seeking to replenish the RPE and preserve visual acuity. Cell delivery to the subretinal space however has been met with challenges surrounding ease of access and invasive surgical implantation. Two-dimensional scaffolds have made use of natural and polymeric materials to act as carriers for RPE cells and various progenitor lines. These substrates mitigate issues surrounding the handling of delicate cell sheets harvested for transplant. As well, they are often successful in preserving RPE phenotype, supporting growth, and can be fine tuned to possess morphologies comparable to native extracellular matrix (ECM). Despite aiming to act as replacement Bruch’s membrane on which RPE resides, two-dimensional substrates are often notably bulky and require traumatic surgery for implantation. As a result, the use of injectable methods of cell delivery has gained appeal. Bolus injections, despite improved methods of administration, are correlated with issues of inadequate cell localization. In response, three-dimensional hydrogel carriers for retinal applications aim to encapsulate cells, allowing for better cell distribution as these materials spread throughout the subretinal space. Increased viscosity of hydrogels as compared to saline injections, is hypothesized to improve cell loss and reduce aggregation. Of particular interest are in situ gelling systems, which undergo physical changes upon injection. Gelation upon delivery works to further assist in maintaining the cells within their target site. Purity and reproducibility concerns associated with the use of natural materials in the development of hydrogel cell carriers, have inspired the use of synthetic thermoresponsive poly-N-isopropylacrylamide (pNIPAAm). pNIPAAm undergoes a liquid to gel transition at a lower critical solution temperature (LCST) of 32°C. Copolymerization with various hydrophobic and hydrophilic groups can be used to adjust gel properties such as increasing or decreasing LCST, allowing for degradation, and improving water retention. In the work described herein, two NIPAAm-based thermoresponsive hydrogels intended for use as subretinal cell carriers are proposed. / Thesis / Master of Applied Science (MASc)

<p>Efficient delivery of therapeutic cell and pharmaceutical suspensions to the posterior segment of the eye remains an elusive goal. Delivery is made difficult by blood ocular barriers that separate the eye from systemic circulation, the compartmentalized structure of the eye that limits diffusion across the globe, and effective clearance mechanisms that result in short drug residence times. The work presented in this thesis focuses on the design, synthesis, evolution and refinement of novel biomaterial scaffolds ultimately intended to facilitate the minimally invasive delivery of therapeutic payloads into the posterior segment of the eye. The first generation materials presented in this work (Chapter 2) consist of linear chains of temperature-sensitive amine-terminated poly(N-isopropylacrylamide) (PNIPAAm) grafted onto the backbone of type I collagen. Second generation materials (Chapter 3) saw the inclusion of the lubricious polysaccharide, hyaluronic acid (HA), and replacement of the bulky collagen backbone, which was observed to impede scaffold gelation, with small cell adhesive RGD peptide sequences. The introduction of degradability was the emphasis of third generation copolymers (Chapter 4) and was achieved through copolymerization with dimethyl-γ-butyrolactone acrylate (DBA). The DBA lactone side group was found to undergo a hydrolysis dependent ring opening, which raises copolymer LCST above physiologic temperature, triggering the gelled scaffold to solubilize and be excreted from the body via renal filtration without the liberation of any degradation by-products. Degradation was found to occur slowly, which is favourable for long-term release scaffolds intended to decrease the frequency of injections required to maintain therapeutically relevant concentrations within the vitreous. Finally, the design of a fourth generation material is discussed (Chapter 5), in which optical transparency is achieved through copolymerization of third generation materials with polyethylene glycol (PEG) monomers of varying molecular weight. Synthesis, design and characterization of the various copolymers is described herein.</p> / Doctor of Philosophy (PhD)