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Identifier 000452005
Title 3D mechanical metamaterial scaffolds for tissue engineering applications
Alternative Title Μηχανικά υλικά τριών διαστάσεων ως ικριώματα για εφαρμογές στη μηχανική ιστών
Author Φλαμουράκης, Γεώργιος
Thesis advisor Βελόνια, Κέλλυ
Reviewer Φαρσάρη, Μαρία
Ρανέλλα Ανθή
Abstract The aim of this thesis is to utilize the unlimited versatility of light-based additive manufacturing alongside with the formulation of photoresins in order to produce different mechanical metamaterial structures that will be used as scaffolds for the study of cellular responses of different cell lines. Furthermore, this study investigates in great depth the osteogenic differentiation potential of those environments in Mesenchymal Stem Cell differentiation. For the purpose of the study, two main mechanical metamaterial scaffolds were fabricated by Multiphoton Polymerization on flat glass substrates. More precisely, i) Auxetic scaffolds that display negative Poisons’ Ratio were fabricatedand characterized, and ii) tetrakaidekahedron lattice (popularly known as Kelvin foam) that shows great stiffness while being extremely lightweight. Both metamaterials were fabricated using the hybrid organic-inorganic photresin SZ2080TM. For the fabrication of the fluorescent free version of those scaffolds, a novel approach was followed where the photoinitiator of the material was replaced by a dye that was later used as a post fabrication treatment of the polymeric structure in order to quench the highlevels of fluorescence. Moreover, several optimizations needed to take place as the scaffolds needed to have a large size in order to host a great number of cells to make accurate measurements. Initially, an organic-inorganic photo resin was used to fabricate simple structure such as boxes and cylinders that were used in a nano indenter system to characterize its’ mechanical properties. The results that we acquired showed that the polymerized material shows great hardness and stiffness which is proportional to the laser power of the system. Afterwards, using 3D software we created two different types of Auxetic scaffolds based on the bowtie geometry. Those scaffolds were simulated under axial force and the negative poison ratio was proved theoretically and experimentally. Following the simulations, several optimization steps were made to the fabrication procedure in order to 3D print high resolution-large scaffolds that then used for cell culturing. We proved that even the material itself is very hard with a Hardness of around 2GPa, due to the special geometry of the unit cells cell were able to bend and deform it easily without breaking it.The second part of this thesis involves the development and optimization of a variant of SZ2080TMthat has low autofluorescence. For that instance, Sudan Black B (SBB) dye was used as a replacement of the photoinitiator that the resin in synthesized by default with, and as a post fabrication treatment. We showed that SBB can initiate photo-polymerization with MPL technique as we created complicated metamaterial scaffolds, and that the newly fabricated structures show 99% lower autofluorescence. Then, cytotoxicity assays were performed with MSCs, and confocal microscopy was used to visualize the cells in those scaffolds. As a result, this material is excellent for sell culture as no cytotoxicity was observed and confocal microscopy could be ultimately used for quantification of proteins in the cells.The third part involves advanced biology experiments in an effort to study how auxetic mechanical environment affects the osteogenic differentiation of Mesenchymal Stem Cells (MSCs), as in recent years the effect of mechanical properties in that cell line has been thoroughly studied. More precisely, the two scaffolds mentioned before, were fabricated through Multiphoton Lithography (MPL) on the same glass substrate and MSCs were cultured on them for different time periods from 2 days to 28 days in the presence and absence of osteogenic differentiation medium. Then, the samples were fixed and observed with Scanning electron microscopy (SEM), and Confocal microscopy. Moreover, we developed a novel protocol in order to prepare the sample for Polymerase chain reaction (PCR) of the cells cultured on the scaffolds as this was a very challenging procedure due to the small size of the fabricated scaffolds. Lastly, we quantified two important proteins for mechanotrunsdaction and differentiation: the Yes associated protein 1 (YAP) and Runt-related Transcription factor 2 (Runx2). Mechanotransduction, by definition, isthe mechanism by which cells translate mechanical stimuli into cellular responses to a variety of mechanical loadsand characteristics such as stiffness or Hardness. We showed that YAP protein is translocated to the nucleus in a higher ratio on Kelvin foam, where it remains in the cytoplasm on the Auxetic. Lastly, Runx2 shows an increase in the case of Auxetic scaffold, hinting the reverse relationship thatthose two proteins have. Analysis of those very complicated 3D environments was conducted with ImageJ through a novel custom pipeline developed especially for the need of this study. Lastly, a new approach for the fabrication of large scaffolds wasfollowed by using a stereolithographic approach with a commercially available 3D printer that uses photosensitive resin and a UV lamp to 3D print the structures. We showed that with this approach it is possible to create large mechanical metamaterial scaffolds from complicated STL files and use them with several different cell lines where no cytotoxicity was observed. In that way, the main drawback of the limited size is surpassed as a great number of cells can be cultured, and the scaffolds are easily manipulated.
Language English
Subject Multiphoton polymerization
Αυξητικά υλικά
Διφωτονικός πολυμερισμός
Μεταυλικά
Issue date 2022-11-23
Collection   School/Department--School of Sciences and Engineering--Department of Materials Science and Technology--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/e/2/8/metadata-dlib-1668765161-162936-22257.tkl Bookmark and Share
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