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Abstract II 1.5-fold

Abstract II 1.5-fold increased metabolic activity of MSCs in hypoxia compared to normoxic cells. The assessment of subpopulations revealed a heterogeneous distribution of rapidly self-renewing (RS), spindle-shaped (SS) and flattened (FC) cells in normoxic conditions over the complete culture period of 52 days, whereas hypoxic cell culture mainly consisted of RS and SS subpopulations before going into senescence on day 64. Nevertheless, both groups had the typical flattened cell appearance during senescence. Additionally, the cell volume measurements were carried out on different surfaces (polystyrene surface and collagen I, fibronectin and laminins coated glass slides) to obtain more information about cell morphology on bone extracellular matrix (ECM) proteins. Normoxic cells appeared slightly bigger on all surfaces, compared to cells under hypoxic cell culture conditions. Time-lapse microscopy analysis was performed to evaluate migratory differences. Results showed that hypoxic cultured cells had a significantly higher relative migration velocity and Eucledian distance for cells on polystyrene, as well as collagen I and laminin differently coated surfaces compared to the normoxic cultured cells. Furthermore, differences in integrin expression were evaluated using RT-PCR. In this regard, an upregulation of the subunits α1, α3, α6, αv, β1 and β3 in hypoxic conditions was observed, while α2 and β5 were higher expressed in normoxic cultured cells. No differences were determined for integrin α5 and α11. Taken together, the results indicate that MSCs are strongly sensitive to different oxygen levels in matters of differentiation capacity, metabolic activity, morphology, migration and integrin expression. Future studies will have to disclose the involved molecular pathways to obtain a better understanding of the influence of oxygen on hMSCs. This knowledge will be of permanent importance in order to find the best terms for hMSCs to heal large bone defects.

Contents III Contents Abstract..................................................................................................... I Contents ................................................................................................. III 1. Introduction........................................................................................ 1 1.1. Mesenchymal stem cells and their clinical application .....................................1 1.1.1. Major aspects of Tissue Engineering ........................................................2 1.1.2. Mesenchymal stem cells...........................................................................2 1.2. Bone biology ....................................................................................................4 1.2.1. Bone development ....................................................................................4 1.2.2. Bone anatomy on a macro- and nanostructual level .................................5 1.2.2.1. Mechanical characteristics of bone....................................................6 1.2.2.2. Biophysical characteristics of bone....................................................7 1.2.3. Bone biology .............................................................................................7 1.2.4. Bone stem cell niche.................................................................................8 1.3. Aim of the study ...............................................................................................9 2. Material and Methods ...................................................................... 10 2.1. Mesenchymal stem cell characterisation and cell culture...............................10 2.1.1. Mesenchymal stem cell extraction ..........................................................10 2.1.2. Cell culture conditions.............................................................................10 2.1.3. Osteogenic differentiation .......................................................................11 2.1.4. Adipogenic differentiation........................................................................12 2.1.5. Surface maker analysis...........................................................................12 2.2. Cell characteristics .........................................................................................13 2.2.1. Cumulative population doubling ..............................................................13 2.2.2. WST-1 metabolic assay ..........................................................................14 2.2.3. Morphological changes ...........................................................................14 2.2.4. Cell volume measurement ......................................................................15 2.2.5. Time-lapse analysis ................................................................................15 2.2.6. RT-PCR ..................................................................................................16 2.3. Statistical analysis..........................................................................................17 3. Results.............................................................................................. 18

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