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Results 22 Fig. 6:

Results 22 Fig. 6: Alizarin Red staining quantification of osteogenically induced hMSCs after 21 days. There were no significant differences between normoxic and hypoxic osteogenic differentiation (p = 0.3819). Both controls showed nearly no Alizarin Red content and were significantly lower than osteogenically induced cells (p ≤ 0.001). n=2, r=3, Mean & SD. 3.1.2. Adipogenic differentiation Differentiation into the adipogenic lineage was visualized by Oil Red O staining of hMSCs after 21 days of induction. There was a clearly notable morphologic change between the differentiated cells (Fig. 7A/B) und the undifferentiated control group (Fig. 7C/D). Adipogenic induced cells were flattened, whereas undifferentiated cells showed a normal spindle-shape like morphology. Red stained lipid deposition was evidently visible in both culture conditions. In the macroscopic evaluation the normoxic group (Fig. 7A) showed less vacuoles compared to the hypoxic group (Fig. 7B). No vacuoles were visible in both control groups (Fig. 7C/D).

Results 23 Fig. 7: Exemplary microscopy images of Oil Red O stained hMSCs following adipogenic differentiation after 21 days in vitro. New vacuoles were formed by cell monolayer in normoxic (A) and hypoxic (B) culture conditions. Hypoxic cultured cells showed slightly more vacuoles compared to normoxic cultured cells. Undifferentiated cells were negative for Oil Red O staining in both cell culture conditions (C: Normoxia, D: Hypoxia). Bar: 200 µm. For lipid quantification of the adipogenic differentiation, vacuoles were labelled with 7-diethylamino-3,4-benzophenoxzine and fluorescence intensity was measured at 572 nm in relative fluorescence units (RFU). The results are presented in figure 8. Hypoxic cultured cells showed a two-times higher RFU (1,697.0 ± 534.7 RFU) equated with adipogenic differentiated cells in 21% oxygen (849.8 ± 320.7 RFU). Both controls showed nearly no RFU compared to the induced cells (Normoxia - 212.5 ± 18.5 RFU, Hypoxia - 178.0 ± 35.6 RFU). Statistical analysis revealed significant differences between the normoxic and the hypoxic group (p=0.0104). Control groups with normal cell culture media revealed also a significant difference compared to induced cells (Normoxia: p=0.0046, Hypoxia: p=0.001).

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