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Introduction 8 induction

Introduction 8 induction of migration and differentiation of preosteoclasts 56,57 . This induction is driven by the preosteoclasts differentiation factor receptor activator of nuclear factor kappa-B ligand (RANKL). The crosstalk of these cells and therewith the active programmed cell deaths during bone remodelling makes quiescence progenitor cells necessary. Interestingly, there are two different progenitor cells for osteoblasts (hMSCs) and osteoclasts (HSCs) respectively, whereupon these stem cells linger in close proximity within specific niches until activation. 1.2.4. Bone stem cell niche The stem cell niche is defined as a microenvironment where multipotent stem cells reside surrounded by highly specified cells and extracellular matrix. The interaction of chemical and/or physical signals (cytokines, interleukins, cell-cell contacts and cellmatrix contacts) can either keep the cell’s quiescence or induce their activation in respect to cell migration and differentiation by a complex cascade 58 . These cell-matrix contacts are mainly mediated by integrin anchoring junctions which are transmembranic hemidesmosomes. Integrins consist of several α- (18 types) and β- (8 types) subunits which end up in a broad combinatorial diversity. By this connection of matrix, cellular surface and intermediate filaments, integrins can influence the shape, rigidity and activate signalling cascades 59 . Up to now, stem cell niches were described for various tissues such as hair follicle, intestine and bone marrow 60,61 . Currently, few data is available about the specific stem cell niche for hMSCs. It is believed that hematopoetic stem cells (HSCs) residue in close proximity to hMSCs. The niche of HSCs has been extensively investigated for over 32 years. Currently it serves as a model for the microenvironment niche of MSCs 62 . There a two distinguished niches for HSCs, a perivascular niche near osteoblasts in a quiescence state and a vascular niche in an activated state. Both niches provide structural and trophic support, topographic information and appropriate physiological signals to regulate stem cell function. Osteoblastic cells regulate the fate of HSCs by the activation of various signal pathways, such as Wnt or Notch 63 . Interestingly, primitive hematopoietic stem and progenitor cells promote the osteogenic differentiation of MSCs trough cell-cell interaction and paracrine signalling 64 . This might be another indication that HSCs and MSCs are in close proximity in vivo. Additionally, recent studies suggested the localisation of hMSCs in a perivascular spot and in neighbourhood of pericytes, within bone marrow 65,66 with an oxygen level below 4% 67 .

Introduction 9 1.3. Aim of the study Up to now, most biological analysis of hMSCs were performed under normoxic cell culture conditions. Due to the extending evidence that hMSCs are located in vivo in a low oxygen perivascular niche with oxygen levels less than 4%, the currently used 21% oxygen in cell culture is in fact hyperoxic compared to the physiological concentrations. We, therefore, aimed at identifying differences in hMSC characteristics between the oxygen levels of 2% O2 (called hypoxia) and 21% O2 (called normoxia). Main questions: I. What is the influence of different oxygen levels on long-term proliferation of hMSCs? II. Can morphological differences and alterations in the distribution of subpopulations over the period of cell culture until senescence between the two oxygen levels be observed? III. What is the effect of different extracellular matrix proteins - ECM (collagen I, fibronectin and laminin) in combination with different oxygen levels on cellular volume? IV. Are there changes in cell migration on different ECM proteins between different oxygen levels? V. Does the oxygen level affect integrin expression?