Results 38 showed practically no difference. Polar circles indicate the Eucledian distance and radial lines indicate the movement angle. 3.2.6. Integrin profile An insight into the cell-matrix contacts of both hMSCs was realised by an integrin expression screening on mRNA level for the integrin α-subunits 1, 2, 3, 5, 6, 11 and v, as well as the β-subunits 1, 3 and 5. All samples were normalised against GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Figure 20 shows an overview of all screened integrins. The integrin α-subunits 1, 3, 6 und v and the β-subunits 1 and 3 were higher expressed in hypoxic hMSCs then in normoxia. α2 and β5 were higher expressed in normoxic cells. There was no difference in the expression levels of in- tegrin α5 and α11. Fig. 20: Integrin expression differences on polystyrene of both culture settings were assessed by polymerase chain reaction (PCR). Normoxic PCRs are red labelled, whereas hypoxic PCRs are blue labelled. PCR was normalized with GAPDH. Hypoxic cells express the α-subunits 1, 3, 6 and v on a higher level than normoxic cultured cells. α2 is higher and α5 and α11 respectively is equally expressed in normoxic cells compared to the hypoxic group. β1 and β3 subunits, despite β5, are also higher expressed within hypoxic hMSCs.
Discussion 39 4. Discussion Since the first discovery of a positive effect on long-term in vitro cell survival of diploid cells under low oxygen concentration by Packer and Fuehr 1977 71 , researchers worldwide currently specify the environmental conditions within specific cell and stem cell niches. Ambient oxygen concentration of 21%, which is regularly used for cell culture conditions, is much higher than in most human tissues (2-9%) 72 . Consequently, it does not seem to reflect physiologic conditions of the stem cell niche. Due to the fact, that direct oxygen measurement inside the stem cells’ niches have not been realized up to now, computational models try to estimate oxygen levels with implication of cell types, oxygen consumption and numbers of cell layers. These studies indicate even lower oxygen levels within bone marrow (1-7%) compared to normal organs 73,74 . In the presented study, differentiation potential, growth characteristics, morphological and metabolic differences from primarily obtained femoral human mesenchymal stem cells were evaluated in 21% O2 (normoxic) and 2% O2 (hypoxic) conditions. Furthermore, differences in integrin expression caused by different oxygen levels were screened using RT-PCR. Additionally, cell migration on bone specific proteins was assessed under the two different oxygen concentrations. Stem cell characterisation In the presented study, hMSCs were induced into the osteogenic or adipogenic lineage for 21 days in normoxic and hypoxic conditions following calcium or fat content quantification combined with microscopy to prove multilineage potential. Furthermore, surface marker expression was measure by flow cytometry in accordance to the minimal criteria by Dominici et. al. 19 . Osteogenic differentiation Our results showed only minor differences for the Alizarin Red staining. The subsequent quantification did not reveal any significant difference of calcium deposition between 2% and 21% oxygen. Three-dimensional analysis, using confocal white light microscopy, indicated a more agglomerated calcium deposition in normoxic conditions. Numerous studies evaluating the osteogenic differentiation of hMSC under different oxygen levels, consistently reported a decreased osteogenic potential in oxy-