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Development of innovative bioengineering approaches for the regeneration of dental enamel



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Dental enamel is the hard tissue that covers the tooth crown that is incapable of endogenous regeneration, once damaged, due to the loss of enamel secreting cells (i.e., ameloblast) following the tooth eruption. The current treatment options, including artificial filling materials, are of temporary solutions. This greatly raises the need of approaches to regenerate enamel, which, however, remains unachievable nowadays. To address this emerging need, this thesis aimed to develop novel bioengineering approaches for the regeneration of enamel tissue. The specific objectives are to develop (1) lipid-based gene delivery systems for Tbx1 encoding genes to dental epithelial cell line, HAT-7 cells, (2) an ionically cross-linkable bioink composed of alginate and carboxymethyl chitosan for bioprinting of HAT-7 cells, (3) a covalently cross-linkable hydrogel consisting of oxidized alginate and carboxymethyl chitosan with injectability and self-healing properties as a cell carrier to incorporate HAT-7 cells, for enamel formation in vitro. The first objective was to prepare and characterize a non-viral gene delivery system for the transfer of plasmid DNA encoding Tbx1. First, we prepared and characterized lipid-based nanoparticles that were based on two different cationic lipids: glycyl-lysine-substituted gemini surfactants with the 16-carbon tail and 1,2-dioleoyl-3-trimethylammonium-propane with three varying the ratios of cationic lipid nitrogen to pDNA phosphate (N/P ratios: 2.5, 5 and 10). The physicochemical properties and biological activities of these nanoparticles were examined in terms of particle size and zeta potential, morphology, DNA protection, cytotoxicity, and gene expression, with the results illustrating that the gemini surfactant-based nanaoparticles with the N/P ratio of 5 is the optimal formulation among those examined. Then, HAT-7 was transfected with the optimal formulation and cultured in 2D and 3D, photo-cross linkable gelatin methacrylate hydrogels, culture systems; followed by the assessment of ameloblast differentiation and enamel formation by evaluating the expression of ameloblast markers and by using mineralization assays. Results showed that the HAT-7 cells transfected with Tbx1 gene were able to robustly express secretory and maturation ameloblast markers, have higher calcium deposition, and form more extensive mineralized nodules compared to control cells. The second objective was to develop a bioink consisting of alginate and carboxymethyl chitosan for the fabrication of 3D HAT-7 cell-laden constructs by using three-dimensional extrusion bioprinting for enamel tissue engineering applications. Alginate and carboxymethyl chitosan were mixed at three different mixture ratios (2-4, 3-3- and 4-2) and printed as porous scaffolds while being ionically crosslinked in calcium chloride bath (100 mM). Then, the physico-mechanical and biological properties of the 3D printed scaffolds were characterized in terms of structural features, swelling and degradation behavior, mechanical properties, cell viability, cell morphology, and mineralization. Results indicated that alginate 4%-carboxymethyl chitosan 2% hydrogels showed higher viscosity, slower degradation rate, lower swelling ability and higher compressive mechanical properties. HAT-7 cells showed a high percentage of viability, maintained their morphology and secreted alkaline phosphatase. The third objective was to develop injectable self-healing hydrogels based on oxidized alginate and carboxymethyl chitosan for the use as a carrier of the dental epithelial stem cells for enamel regeneration applications. First, alginate was modified through oxidation reaction and chemically characterized. Then, oxidized alginate and carboxymethyl chitosan were mixed at three varying weight ratios (4:1, 3:1, 2:1) and in situ crosslinking occurred through Schiff base reactions, which was confirmed by chemical characterization. The physico-chemical and biological properties of the hydrogels were assessed in terms of gelation time, swelling ratio, structural, injectability, self-healing, antibacterial properties, cell morphology and viability, mineral deposition. The results indicated that hydrogels with the higher weight ratio of oxidized alginate had faster gelation time and lower swelling ability. Hydrogels formed highly porous structures and showed injectability and self-healing abilities, as well as antibacterial properties against two cariogenic bacteria. HAT-7 cells were able to maintain a high cell viability, without being affected by injection pressure, and could maintain their morphology, deposit minerals and secrete alkaline phosphatase. Taken together, this thesis presents the development of methods for bio-enamel formation in vitro based on gene therapy and tissue engineering principles. The combination of optimized lipid-based system for T-box1 gene delivery and the developed hydrogel-based scaffolds may provide a foundation to develop the optimal conditions for enamel regeneration in vitro and in vivo.



dental enamel, ameloblast differentiation, gene delivery, tissue engineering, 3D printing, injectable hydrogels



Doctor of Philosophy (Ph.D.)


Biomedical Engineering


Biomedical Engineering


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