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ABSTRAKT:Isolering och karaktärisering av mesenkymala stamceller från periodontalligamentet hos friskatänderSYFTE: Att isolera och odla celler från periodontalligamentet samt karaktärisera dem sommesenkymala stamceller.MATERIAL OCH METOD: Friska premolarer gjordes tillgängliga vid ortodontiskaextraktioner. Den mellersta 1/3 av periodontalligamentet skrapades varpå en enzymatiskmetod användes för isolering av individuella celler. Resulterande celler odlades understandardiserade metoder. Karaktärisering av celler skedde genom flödescymetri med 2 olikapaneler av cellyta markörer; en för etablerat positiva uttryck och en för kända negativauttryck hos mesenkymala stamceller. Möjlighet av celler att differentieras in vitro tilladipocyter och osteocyter testades genom tillförsel av specifika substanser till odlingsmediet.RESULTAT: Celler från 11 av 13 tänder isolerades och odlades framgångsrikt adherenta tillodlingsytan i upp till 8 generationer. Celluttryck av de positiva markörerna CD73, CD90 samtCD44 bekräftades genom flödescymetri. Inget uttryck observerades för den negativa panelenCD45, CD34, CD11b, CD19 eller HLA class II. Uttrycket av CD105 kunde inte fastställas pgaofullständigt data. Försök till differentiering av celler till adipocyter och osteocyter visade påfenotypiska förändringar efter 21 dagar.SLUTSATS: Den här studien har bidragit till framgångsrik isolering och delvis karaktäriseringav mesenkymala stamceller från periodontalligamentet hos friska tänder. En icke-invasivmetod av detta slag, resulterande i tillgång till denna cellpopulation utgör ett lovande verktygför framtida studier med goda möjligheter till ytterligare kunskap applicerbart till kliniskasituationer inom tandvården. / ABSTRACT:Isolation and Characterization of Mesenchymal Stem Cells from the Periodontal Ligament ofHealthy TeethAIM: To isolate and culture viable cells from the periodontal ligament and confirming theiridentity as mesenchymal stem cells.METHODS AND MATERIALS: Healthy premolars were collected at the time oforthodontic extractions. The middle 1/3 of the periodontal ligament was scraped andsubsequent cell isolation was performed using an enzymatic method; yielding single cellisolates. Cells were cultured and maintained under standard culture conditions. Cellcharacterization was performed by flow cytometry using two sets of cell surface markers; oneknown to be present and one known to be absent in mesenchymal stem cells. Ability of thecells for in vitro differentiation into adipogenic and osteogenic lineages was tested usingspecifically formulated media supplements.RESULTS: Cells were successfully isolated from 11 of 13 teeth and were maintained asadherent cultures for up to 8 generations. Cellular expression of positive markers; CD73, CD90and CD44 were confirmed by flow cytometry. For the negative marker panel, expression ofCD45, CD34, CD11b, CD19 and HLA class II were not detectable. The expression of CD105was inconclusive. As determined by phenotypic changes, cells appeared to have undergoneadipogenic and osteocytic differentiation at 21 days.CONCLUSION: This study has resulted in successful isolation and partial characterization ofmesenchymal stem cells from the periodontal ligament of healthy teeth. Non-invasive accessto these cells, provides an excellent tool for future studies, potentially leading to beneficialknowledge transferable to the dental clinical situation.

<p>The work presented in this thesis was focused on design and construction of novel cell-loaded microcapsules by incorporation of bioactive molecules (proteins or peptides) for potential application in hemophilia B treatment. The objective of this study was to improve the viability and functionality of the encapsulated cells by creating biomimetic microenvironments for cells that more closely mimic their physiological extracellular matrix (ECM) environment.</p> <p>Three cell-adhesive molecules were used in this work: fibrinogen and fibronectin, two abundant proteins present in ECM, and arginine-glycine-aspartic acid (RGD) tri-peptide, the minimal essential cell adhesion peptide sequence and the most widely studied peptide for cell adhesion. Alginate, the most commonly used biomaterial used for cell encapsulation, was combined with either of these molecules to create biomimetic microcapsules. Non-modified alginate (control) and modified alginate matrices were used to encapsulate the factor IX (FIX) secreting cells for protein delivery. In this work, FIX-engineered cord blood-derived human mesenchymal stem cells CB MSCs were used as a cell source for FIX delivery.</p> <p>Our data suggested that fibrinogen-alginate, fibronectin-alginate and RGD-alginate microcapsules improved the viability of encapsulated MSC and are applicable in cell therapy technologies. However, fibrinogen-alginate and fibronectin-alginate microcapsules more significantly enhanced the proliferation and protein secretion from the encapsulated cells and may have potential for FIX delivery for hemophilia B and other inherited or acquired protein deficiencies. RGD-alginate microcapsules can v potentially be used for other tissue engineering applications with the aim of enhanced viability and attachment of the enclosed cells. Differentiation studies showed the osteogenic (but not chondrogenic or adipogenic) differentiation capability of FIX-engineered CB MSCs and their efficient FIX secretion while encapsulated in fibrinogen-alginate and fibronectin-alginate microcapsules.</p> / Doctor of Philosophy (PhD)

This study presents a viable strategy using fmoc-protected peptides hydrogels, to encapsulate and stretch mesenchymal stem cells (MSC). To explore the peptide hydrogel potential, a custom mechanical stretching device with polydimethylsiloxane (PDMS) chambers were used to stretch MSCs encapsulated in Fmoc hydrogels. We investigated the impact of fmoc- FF prepared in dimethyl sulfoxide (DMSO), 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and deionizied water in the self-assembly, and mechanical properties of the gels. The peptide hydrogel is formed through molecular self-assembly of peptide sequence into β-sheets that are connected with the π-π aromatic stacking of F-F groups. The hydrogels provided a stiff, hydrated gel with round nanofiber morphology representing an elastic modulus of 174-266 KPa. MSCs cultured on peptide hydrogels undergo viability, morphology, and alignment evaluations using MTT, live/dead, and phalloidin (F-actin) staining. The F-actins of 3D- cultured MSCs in Fmoc-FF/HFP, and Fmoc-FF/DMSO followed by mechanical stretching showed elongated morphology with defined microfilament fibers compared to the round and spherical F-actin shape of the control cells. Peptide gels with 5mM concentration preserved 100% viability of MSC. Results reveals the feasibility and conditions for successful cell encapsulation and alignment within peptide hydrogels. Encapsulation of MSC in peptide nanofiber followed by a stretching process present a promising tissue engineering platform. By enhancing our understanding of MSC-peptide hydrogel interactions, this research con- tributes to the development of biomaterials tailored for regenerative medicine.

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.