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Abbreviations 62 MRI

Abbreviations 62 MRI Magnetic resonance imaging NCP Non-collagenous protein PBS Phosphate-buffered saline PDT Population doubling time PFA Paraformaldehyde p-FAK Phosphorylated focal adhesion kinase PLL Poly-L-lysine PLLG Polylactide-co-glycolide p-SCR Phosphorylated tyrosine kinase SCR RANKL Receptor activator of nuclear factor kappa-B ligand RFU Relative fluorescence unit RNA Ribonucleic acid ROS Reactive oxygen species RS Rapidly self-renewing RT Room temperature RT-PCR Real-time polymerase chain reaction SEM Scanning electron microscopy SS Spindle-shaped TE Tissue Engineering TERT Telomerase reverse transcriptase TGF-β Transforming growth factor-β VEGF Vascular endothelial growth factor

List of figures 63 List of figures Fig. 1: Schematic overview of the multilineage potential of human mesenchymal stem cells 21 . ............ 3 Fig. 2: Schematic illustration of bone hierarchy from macroscopic to nanoscale level 35 . ....................... 6 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. ....................................... 19 Fig. 4: Bright field confocal microscopy images of osteogenic differentiated hMSCs under normoxic (A) and hypoxic (B) conditions. Standard cell culture media served as a control (C & D). White arrows indicate condensed calcium depositions. Bar: 50µm. .................................................. 20 Fig. 5: Topographical confocal white light microscopy images of osteogenic differentiated hMSCs. Calcium depositions within the differentiated groups were higher than mean ECM height (A: normoxia, B: hypoxia). Control groups had constantly less ECM height. White arrows indicate condensed calcium depositions. Bar: 50µm............................................................................. 21 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. ..................................... 22 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..................... 23 Fig. 8: Lipid content of adipogenic differentiated hMSCs was measured 21 days after adipogenic induction. Results are shown in relative fluorescence units (RFU). Hypoxic cultured cells showed a significant, two-times higher lipid content compared to normoxic cultured cells (p=0.0104). Undifferentiated cells showed significantly less RFU compared to induced groups (Normoxia: p=0.0046, Hypoxia: P=0.001). n=2, r=3, Mean & SD............................................ 24 Fig. 9: Exemplary cluster of differentiation (CD) expression acquired by flowcytometry analysis for normoxic and hypoxic cultured hMSCs. Isotype controls are shown in red and CD markers are blue. There was no difference between each group. A: Cells in both culture conditions were positive for CD73, CD90 and CD105, B: Cells in both culture conditions were negative for CD14, CD19, CD45 and HLA-DR (A & B, normoxia: upper row, hypoxia: lower row)............. 25 Fig. 10: Cumulative population doublings (cum PD) of normoxic and hypoxic cultured hMSCs. Up to day 12 cells showed a comparable proliferation. The following days, normoxic cultured cells decreased in proliferation and went into senescence after day 52 (red dashed line). Hypoxic cultured cells showed a nearly linear proliferation over the entire time period until they went into senescence after day 64 (blue dashed line)...................................................................... 26

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