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Discussion 40 gen

Discussion 40 gen concentrations below 2% 75-82 . In contrast to our study, the majority of these studies, first expanded cells under normoxic conditions and only differentiated without hypoxic preconditioning was performed under reduced oxygen concentrations. On the contrary, Volkmer and colleagues could show that hypoxic preconditioning for three days could restore the osteogenic potential of hMSCs and lentiviral immortalised hMSCs 83 . Currently, there are controversial reports of osteogenic potential and HIF-1α interplay of MSCs in lowered oxygen concentrations. Tamama K et. al. described a strong HIF reliance in the first seven days, but simultaneously pointed out a possible stage dependency during osteogenic differentiation 84,85 . These discrepancies show that more knowledge of the molecular mechanisms is inevitable. Adipogenic differentiation Differentiation into the adipogenic lineage revealed a significant higher and bigger vacuole formation in hypoxic conditions. Along visual appearance, Oil Red O quantification showed a two-fold higher lipid content for hypoxic cultured cells. Up to now there is also no consensus about the prospect of success for adipogenic differentiation. It could be shown, that murine adipose derived stem cells showed a higher adipose lineage potential when they were differentiated in 21% oxygen saturation. Additionally, hypoxic preconditioning prior adipogenic differentiation in normoxic conditions significantly increased vacuole formation and expression of the adipogenic linked genes PPARγ, LPL and FABP4 compared to a complete normoxic setup 86 . Several other studies indicated more adipogenic differentiation potential under normoxic cell culture conditions 75,87-90 or more differentiation potential within lower oxygen concentrations 91-93 . Considering, that the local oxygen concentration within fat is around 7% 94 , an increased potential of adipogenic differentiation in hypoxic cell culture conditions is likely. Surface marker profile Along the positive results for differentiation into the osteogenic or adipogenic lineage, the surface protein profile was assessed according to Dominici et. al. 19 . The positive markers CD73, CD90 and CD105 were strongly expressed in both culture conditions (< 95%). On the other hand, non-mesenchymal markers (CD14, CD19, CD45 and HLA-DR) showed weak fluorescence intensity (> 2%). Holzwarth et. al. showed com-

Discussion 41 parable results for hMSCs in 1% oxygen 75 . Interestingly, initial impurity of the main population with cells from non-mesenchymal origin decrease over increasing passage numbers 95 . Furthermore, human embryonic stem cells (hESCs) showed a different expression of CD44, CD105 and PDGFRα when cultured for three weeks in normoxic or hypoxic conditions 96 . Additionally, multipotent mesenchymal stromal cells from Wharton’s jelly slightly lost the positive expression of CD73 within Hypoxia 97 . Continuative studies must show, if the loss of the positive markers also occur in MSC populations in vitro. The comparability of these studies is limited due to multiple differences, like oxygen concentration, media height, time of preconditioning prior to the experiments, like direct splitting of obtained cells or subsequent splitting after a normoxic cell expansion phase, sealed/unsealed cell culture hoods or donor viability in each different study. In relation to stem cell characterisation, cells in our study showed an osteogenic and adipogenic differentiation potential and had a suitable surface marker profile. After proving the stem cell character of the obtained cells, further experiments investigating cell characteristics for both culture conditions were performed. Differences of cell characteristics Proliferation and metabolic differences To realize the concept of Tissue Engineering, it is essential to obtain high cell numbers in a short period of time. Consequently, it is important to culture the cells under the ideal culture conditions in order to obtain a high rate of proliferation. For the first 12 days of cell culture no difference for the cumulative population doublings could be observed between normoxic and hypoxic cultured cells. Hypoxic cells maintained a constant proliferation capacity for a total time of 64 days in culture, while cells in 21% oxygen decreased their cumulative cell doubling rate and went into senescence after 52 days in culture. Before going into senescence, the cum PD was doubled from 15 (Normoxia) to 30 (Hypoxia). HMSCs cultured within 2% O2 showed a consistent population doubling time over the complete culture period, whereas cells in 21% O2 demonstrated a highly volatile doubling time. The cellular metabolic activity revealed

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