La cécité cornéenne touche 12,7 millions personnes globalement. Il y a une pénurie des cornées de donneurs humains (CDH), et donc les tissues disponibles sont implanté préférentiellement dans les patients avec des troubles cornéens à faible risque comme le kératocône et la dystrophie endothéliale de Fuchs. Les patients qui ont un risque élevé d’inflammation, comme ceux avec des brûlures acides, alcalines et thermiques, des infections et des ulcères, ne reçoivent pas de greffes pour leurs maladies cornéennes. Les biomatériaux offrent une alternative aux CDH en permettant le développement de solutions de régénération cornéenne avec une longue durée de conservation, une thermostabilité pour un déploiement en zone rurale, et biocompatibilité chez les patients à haut risque. Les biomatériaux peuvent être développés sous forme d’implants cornéens solides à greffer dans des opacités cornéennes ou sous forme de liquides gélifiants injectables qui peuvent sceller des petites perforations cornéennes. Les implants cornéens solides conviennent aux chirurgiens ophtalmologiques, mais les produits de comblement liquides peuvent être utilisés par les prestataires de médecine d’urgence ou le personnel médical non spécialisé dans les zones où les chirurgiens ophtalmologistes ne sont pas disponibles. Cette thèse explore les formulations de biomatériaux pour les cornéens solides et gélifiants in situ, leurs performances en tant que dispositifs composites, l’ajout de la stérilisation terminale à la fabrication d’implants cornéens solides et le développement de futures protéines mimétiques du collagène pour la formulation d’hydrogel. Le premier objectif de cette thèse était de développer un implant cornéen solide adapté à l’implantation chez les patients cornéens à haut risque. Les implants cornéens peptide-mimant-le-collagène-polyéthylène glycol-phosphorylcholine (PMC-PEG-MPC) et les implants recombinants de collagène humain de type III-phosphorylcholine (RCHIII-MPC) ont réussi à régénérer les cornées de mini-porcs et de lapins, respectivement. La phosphorylcholine présente dans la formulation PMC-PEG-MPC a diminué l’inflammation et fourni une alternative cornéenne viable dans les brûlures alcalines à haut risque. Des nanoparticules d’argent coiffées de peptides étaient fabriquées avec succès à la surface d’un implant cornéen solide de collagène porcin de type I. Ces implants ont inhibé P. aeruginosa, S. aureus et S. epidermidis in vitro et empêché la formation de biofilm à l’interface air-liquide. Ces implants cornéens solides élargissent la gamme d’efficacité pour inclure les personnes souffrant de brûlures alcalines et d’infections. Finalement, on a validé une méthode de stérilisation terminale des implants cornéens solides. Le RCHIII-MPC a été stérilisé en phase terminale avec succès à l’aide d’une irradiation par faisceau d’électrons, offrant une future voie pour la stérilisation terminale des implants cornéens solides à base de biomatériaux. Le deuxième objectif était de concevoir un hydrogel qui se solidifierait in situ pour sceller les perforations cornéennes. Le PMC-PEG était combiné avec du fibrinogène pour former “LiQD Cornea”, le premier produit de comblement cornéen liquide à être chimiquement réticulé avec succès in situ pour sceller les perforations cornéennes et les plaies chirurgicales chez le lapin et les mini-porcs. Pour le troisième objectif, ce projet fournit également une méthodologie future pour la production de protéines mimétiques de collagène personnalisées pour les futures formulations d’hydrogel. Dans l’ensemble, le collagène et les biomatériaux inspirés du collagène se sont révélés être des greffes et des scellants cornéens prometteurs avec des voies viables de fabrication commerciale. / Corneal blindness and opacities affect 12.7 million people globally. There is a shortage of human donor corneas (HDCs), which are prioritized for patients with low risk corneal disorders like keratoconus and Fuch’s endothelial dystrophy. Patients with high-risk inflammatory conditions like acid, alklai and thermal burns, infections and ulcers are often unable to receive transplants to treat their corneal disorders. Biomaterials provide an alternative to HDCs by allowing the development of corneal regenerative solutions with a long-shelf life, thermostability for deployment in rural areas and biocompatibility in high-risk patients. Biomaterials can be developed as solid corneal implants to graft into large corneal opacities or as injectable in situ gelling liquids that can seal small corneal perforations. Solid corneal implants are suited for use by ophthalmic surgeons, but liquid fillers can be used by emergency medicine providers or non-specialized medical personnel in areas where ophthalmic surgeons are not available. This thesis explores biomaterials formulations for solid and in situ gelling corneal biomaterials, their performance as composite devices, the addition of terminal sterilization to the manufacture of solid corneal implants, and the development of future collagen mimetic proteins for hydrogel formulations. The first objective of this thesis was to develop a solid corneal implant suitable for implantation in high-risk corneal patients. Collagen-like-peptide-polyethylene glycolphosphorylcholine (CLP-PEG-MPC) corneal implants and recombinant human collagen type III-phosphorylcholine implants were successful in regenerating the corneas of mini-pigs and rabbits, respectively. The phosphorylcholine present in the CLP-PEG-MPC formulation decreased inflammation and provided a viable corneal alternative in high-risk alkali burns. Peptide-capped nanoparticles were successfully fabricated on the surface of a porcine and S. epidermidis in vitro and prevented biofilm formation at the air-liquid interface. These solid corneal implants expand the range of efficacy to include individuals with alkali burns and infections. This thesis validated a method of terminal sterilization for solid corneal implants. RHCIII-MPC was successfully terminally sterilized using electron-beam irradiation, providing a future avenue for terminal sterilization of biomaterials-based solid corneal implants. The second objective was to design a hydrogel that will solidify in situ to seal corneal perforations. CLP-PEG was combined with fibrinogen to form LiQD Cornea, the first liquid corneal filler to be successfully chemically crosslinked in situ to seal corneal perforations and surgical wounds in rabbit and mini-pigs. For the third objective, this project also provides future methodology for the production of custom collagen mimetic proteins for future hydrogel formulations. Overall, collagen and collagen-inspired biomaterials were demonstrated to be promising corneal grafts and sealants with viable pathways to commercial manufacture.

Stem cell treatments for spinal cord injury (SCI) are effective in pre-clinical animal model research but not yet for humans. Two promising stem cell repair strategies involve (1) endogenous neural stem/progenitor cells (NSPCs) and (2) induced pluripotent stem cells (iPSCs). Delineating species differences in spinal cord NSPC biology is essential to inform human SCI endogenous regeneration and repair. Understanding the phenotypic differences between iPSC-derived NSPCs and primary spinal cord NSPCs would also improve the clinical application of iPSC-derived NSPC therapy in human SCI. To directly compare the molecular and functional attributes of spinal cord NSPCs between humans and animal models of SCI, we designed an in vitro model that allows the characterization of primary human, pig, and rat NSPCs under identical conditions. We found an enrichment of transcription factors in NSPCs of either species that may underlie their differentiation and proliferation potentials. Specifically, human NSPCs are neurogenic, whereas pig and rat NSPCs are gliogenic. Also, the proliferation rate of human and pig NSPCs is less than rat NSPCs. Subsequently, we expanded our in vitro model to examine the responses of NSPCs to inflammation and regenerative factors. Surprisingly, inflammation had induced glial scarring mechanisms from pig and rat NSPCs but potentiated neurogenesis of human NSPCs. We also found species-specific responses to regenerative factors that depend on the type of factor used, concentration, and duration of treatment. To assess the extent that iPSC-derived NSPCs phenocopy primary spinal cord NSPCs, we created iPSC-derived NSPCs with a previously reported brain or spinal cord phenotype and directly compared them with isogenic primary NSPCs. We found that iPSC-derived NSPCs exhibit an earlier developmental stage and a greater proliferation rate. We also found that primary NSPCs possess a unique differentiation potential and regional polarity along the rostral-caudal and dorsoventral axes. In summary, we discovered that species differences in NSPC biology exist between human and animal primary spinal cord NSPCs and that iPSC-derived NSPCs do not recapitulate the transcriptional nor functional attributes of primary spinal cord NSPCs. This thesis highlights the translational gap between pre-clinical research and the clinical application of stem cell treatments that target endogenous NSPCs or transplant iPSC-derived NSPCs.

Acetylsalicylic acid (ASA), aspirin, is a renowned NSAID that its role in the process of bone metabolism has recently come to light. However, the influence of ASA on the odontogenesis of human dental pulp cells (HDPCs) remains elusive. In search of materials that would synergize the healing potential of the dental pulp, this study aimed to investigate the role of ASA on the odontogenesis of HDPCs in vitro and the influence of ASA on TGF-ß1 liberation from dentin. HDPCs were cultured in a culture medium with different concentrations of ASA: 25, 50, 75, 100, 200 μg/mL and 0 μg/mL as a control. The mitochondria activity of HDPCs was assessed using an MTT assay. Crystal violet staining and triton were used to evaluate cell proliferation rates. ALP activity was measured with the fluorometric assay. Expressions of DSP and RUNX2 were determined with ELISA. DSP and RUNX2 mRNA levels were measured with RT‐qPCR. Alizarin red staining was conducted to evaluate the mineralized nodule formation. Dentin slices were submerged in PBS (negative control), 17% EDTA (positive control), and ASA before collecting the solution for TGF-ß1quantification by ELISA. The data were analyzed by t tests and ANOVA followed by the Tukey post hoc tests. P values < 0.05 were considered statistically significant. The results showed that 25-50 μg/mL ASA promoted mitochondria activity of HDPCs at 72h (P<0.05) and yielded significantly higher proliferation rates of HDPCs than the control at 14d and 21d (P<0.001). All concentrations of ASA promoted odontogenic differentiation of HDPCs by enhancing the mineralization and the levels of DSP, RUNX2, and their mRNA expression in a dose-dependent manner (P<0.05). Also, ASA yielded significantly higher TGF-ß1 liberation after conditioning dentin for 5min (P<0.001) and 10min (P<0.05). In conclusion, the data suggest that ASA promotes the odontogenic potential of HDPCs and TGF-ß1 liberation from dentin in vitro and might be incorporated into the novel pulp capping materials for dental tissue regeneration.

At present there is an extraordinary need to overcome barriers in regards to discovering novel and enhanced biomaterials for various tissue engineering applications. The need for durable orthopaedic implants is on the rise to limit issues such as revision surgery. A promising pathway to enhance fixation is to accelerate the onset and rate of early cellular adhesion and bone growth through nanoscale surface topography at the implant surface. The main aim of this research project was to investigate cellular response to altered physical and mechanical characteristics of materials suitable for orthopaedic applications. Four injection moulded polymeric substrates were produced, each with varied compositional and topographical characteristics. The four materials fabricated are Polyether-ether-ketone (PEEK), PEEK with 30% glass fibre (GL/PEEK) composite, PEEK and GL/PEEK with grooved topography. SEM and AFM analysis was used to investigate the groove dimensions and surface roughness of all samples followed by mechanical testing using a nano indenter to detect the Young’s modulus, stiffness and hardness of all four substrates. These tests were performed to determine which material has similar characteristics to cortical bone. These tests were followed by wettability and surface energy testing. Cell-substrate adhesion was examined using a cell viability assay to identify if there is a significant difference (p<0.05) between the percentage of viable cells on all four PEEK based materials. Imaging of MG-63 osteosarcoma cells using immunohistochemistry staining kits was conducted to observe the relationship between cell length and surface topography followed by a comparison between HaCaT (skin) cells and MG-63 (bone) cells. Following experimental testing mechanical variations between PEEK and GL/PEEK were identified alongside physical characterization differences. The grooved topography increased the surface roughness of PEEK and GL/PEEK in comparison to the planar surface. After 72 hours a correlation between the increased surface roughness and the percentage of viable MG-63 cells could be identified. When assessing the effect surface topography has on the water contact angles and surface energy, all four substrates showed no correlation. However, the grooved topography did increase the water contact angle and reduced the surface energy of PEEK in comparison to planar PEEK. Images of the four substrates after cell culture observed the grooved topography to affect the cellular orientation of both MG-63 and HaCaT cells. Polycaprolactone (PCL) scaffolds with a concentration of 1, 3, and 5% triclosan (an antimicrobial and antifungal agent) were fabricated using electrospinning. In addition to PCL + Triclosan scaffolds PCL with a concentration of 1% silver (an antimicrobial agent that can reduce the risk of infection) and 1, 3, and 5% triclosan were also electrospun. The pore size and fibre diameters of the scaffolds were investigated using SEM and Image J software followed by wettability and surface energy testing. MG-63 cells were cultured on all PCL scaffolds to study cellular viability percentage after 24 and 72 hours. The findings obtained showed the physical characteristics of PCL scaffolds to affect cellular viability of MG-63 cells. The output from these findings aim to provide data at a proof of concept level in understanding the relationship between the mechanical and physical characteristics of biomaterials and cellular behaviour.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13586号 / 論医博第2306号 / 新制||医||1070(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 川口 義弥, 教授 波多野 悦朗, 教授 齋藤 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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60 % of school children have some form of untreated tooth decay or have suffered trauma to the front teeth which results in pulp damage. If left untreated, these teeth are susceptible to premature fracture/loss under daily stresses. In cases of adolescent tooth loss, teenagers cannot get dental implants until after the growth spurts; their only option is using removable dentures which lowers their quality of life. Conventional endodontic treatment (root canal treatment) is used in cases of pulp necrosis, but cannot be performed in immature permanent teeth due to major differences in tooth anatomy. Currently the American Dental Academy has approved a procedure called Regenerative Endodontic Treatment (RET) for such cases, but the outcomes are still unpredictable and the method is largely unreliable. One issue that we are trying to address in this work is the regeneration of the pulp-dentin complex (PDC), specifically the interface. Endeavors in regenerating either pulp or dentin have been successful individually, but the interface region is the anatomical and physiologic hallmark of the PDC and has not been addressed. We have proposed a biomimetic scaffold to facilitate early stage stratification of these different tissues and allow recapitulation of their interface. Tissue engineering principles and biomaterial processing techniques were used simultaneously to encourage dental pulp stem cells into mineralize selectively only on one side. This effectively allows the scaffold to serve as the interface region between the hard dentin and the soft vascular pulp. / Bioengineering

Mesenchymal stem cells (MSCs) have great potential to improve clinical outcomes for many inflammatory and degenerative diseases through delivery of exogenous MSCs via injection or cell-laden scaffolds and through mobilization and migration of endogenous MSCs to injury sites. MSC fate and function is determined by microenvironmental cues, specifically dimensionality, topography, and cell-cell interactions. MSC responses of migration and differentiation are the focus of this dissertation. Cell migration occurs in several physiological and pathological processes; migration mode and cell signaling are determined by the environment and type of confinement in three-dimensional (3D) models. Tendon injury is a common musculoskeletal disorder that occurs through cumulative damage to the extracellular matrix (ECM). Studies combining nanofibrous scaffolds and MSCs to determine an optimal topographical environment have promoted tenogenic differentiation under various conditions. We investigated cellular response of MSCs on specifically designed nanofiber matrices fabricated using a novel spinneret-based tunable engineered parameters production method (STEP). We designed suspended and aligned nanofiber scaffolds to study cellular morphology, tendon marker gene expression, and matrix deposition as determinants for tendon differentiation. The delivery and maintenance of MSCs at sites of inflammation or injury are major challenges in stem cell therapies. Enhancing stem cell homing could improve their therapeutic effects. Homing is a process that involves cell migration through the vasculature to target organs. This process is defined in leukocyte transendothelial migration (TEM); however, far less is known about MSC homing. We investigated two population subsets of MSCs in a Transwell system mimicking the vasculature; migrated cells that initiated transmigration on the endothelium and nonmigrated cells in the apical chamber that failed to transmigrate. Gene and protein expression changes were observed between these subsets and evidence suggests that multiple signaling pathways regulate TEM. The results of these experiments have demonstrated that microenvironmental cues are critical to understanding the cellular and molecular mechanisms of MSC response, specifically in homing and differentiation. This knowledge has identified scaffold parameters required to stimulate tenogenesis and signaling pathways controlling MSC homing. These findings will allow us to target key regulatory molecules and cell signaling pathways involved in MSC response towards development of regenerative therapies. / Ph. D.