Results 18 3. Results 3.1. Characterisation of hMSCs Dominici et. al. stated the minimal criteria for defining multipotent mesenchymal stem cells: First, cells must be plastic-adherent on normal polystyrene surfaces in normal culture conditions. Second, they can be differentiated into the osteogenic, adipogenic and chondrogenic mesenchymal lineage. Third, hMSCs must express CD105, CD73 and CD90 and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules 19 . 3.1.1. Osteogenic differentiation Osteogenic differentiation was induced for 21 days. After this period the osteogenic differentiation was quantified by Alizarin Red staining. Both, normoxic (Fig. 3A) and hypoxic (Fig. 3B) cultured cells differentiated into osteoblasts and produced a homogenous matrix mineralisation. By macroscopic appearance, the hypoxic group seemed to have a slightly denser extracellular matrix. For both culture conditions the standard media induced no osteogenic differentiation (Fig. 3: C - Normoxia, D - Hypoxia).
Results 19 Fig. 3: Representative Alizarin Red staining of osteogenic differentiated hMSCs. Cell monolayers in normoxic (A) as well as hypoxic (B) conditions showed a strong and homogenous matrix mineralisation. Both controls produced no calcified matrix, while normoxic cells appeared in the typical spindle shape morphology within monolayers (C). Whereas, the hypoxic group formed cell accumulation and cellular multilayers (D). Bar: 200 µm. Exemplary bright field confocal microscopy images for normoxic and hypoxic differentiated hMSCs (A & B) as well as both controls (C & D) are presented in figure 4. Both osteogenic differentiated groups showed a homogenous cellular monolayer with a dense ECM deposition. There were various calcium accumulations in the normoxic group (Fig. 4A, white arrows), whereas few of them were visible within the hypoxic group (Fig. 4B). Normoxic hMSCs in control media formed a normal confluent monolayer with clearly distinguishable cell nuclei (Fig. 4C). In contrast, 2% oxygen lead to cellular polylayers with higher non-mineralized ECM production (Fig. 4D). Corresponding topographical images are displayed in figure 5. Calcium accumulations, which were visible in bright field images, were higher than the global extracellular matrix depositions (Fig. 5A). Calcified matrix production can be quantified by calculation of the mean surface roughness. These values are shown in supplementary