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Discussion 44 abridgment

Discussion 44 abridgment of the wide variety of cellular movement dynamics. For two decades, Horwitz and colleagues are researching on the mechanism of cell migration 128-132 and defined a sequence of actions with the coordination of force generation, cell adhesion and membrane recycling for cellular migration. The initial cell polarisation is followed by membrane extension (lamellipodia) and interim in the formation and stabilisation of new focal adhesions 59,133 . In the presented study, cell velocity and Euclidean distance (shortest distance from start to end point) of hMSCs were evaluated. Since the surface is of paramount importance in respect to cell migration the four previously described surface coatings were evaluated for both cell culture conditions. Hypoxic cells showed a 1.6-fold increased cell velocity on polystyrene surface. Coating with the most prominent ECM proteins of bone - collagen I, fibronectin and laminin – showed a tremendous effect for MSCs within hypoxia. Hypoxic cells had a 1.4-times higher velocity on collagen I and laminin compared to normoxic cells. Remarkably, such strong difference couldn’t be shown for fibronectin coating with just a 10% enhancement of cell velocity. Similar normoxic experiments with transmembrane assays showed also a significant motogenic effect of collagen I, vitronectin and fibronectin proteins (soluble as well insoluble form) for hMSCs 134 . Wang et. al. could show that hypoxic conditioned medium from epithelial cells has a positive effect on locomotion of marrow-derived endothelial progenitor cells 135 . In addition, MSCs demonstrate an strong cumulative cellular migration response to hypoxia and IL-6 towards tumour milieu 136 . In general, hypoxic environment has a strong influence onto tumour metastasis 137,138 . A critical point in our study might be the relatively high serum concentration of 5% within the media which might affect the protein coating and therewith cell velocity. Recent findings reported no influence of fetal calve serum onto human fetal skin fibroblasts migration 139 . However, to avoid discrepancies with literature, velocity was calculated relatively to normoxic controls and still showed strong oxygen dependence. Integrin expression Docheva et. al. wrote a broad-ranged review of biological adhesion mechanism of hMSCs 140 . Despite diverse results for each subunit, hMSCs are mainly sensitive for collagens, laminins, fibronectin and other bone-related ECM substitutes, like vitronectin, osteopontin or osteocalcin. We could identify differences in integrin expres- sion for the two culture conditions. In 21% O2, only α2 and β5 were higher expressed

Discussion 45 on mRNA level, whereas α1, α3, α6, αv, β1 and β3 were higher detectable within hy- poxic cultured cells. No differences were found for α5 and α11. A recent study indi- cated the integrin subunit α2, in interaction with mechanotransduction, plays a major role during osteogenic differentiation 141 . In addition, crosstalk between integrin α5 and IGF2/IGFBP2 (insulin-like growth factor 2/insulin-like growth factor binding protein) promotes differentiation into the osteogenic lineage in hMSCs 142,143 and is upregulated during osteogenic differentiation on PLLG substrates 144 . We hypothize, that this crosslink between integrins involved in osteogenic differentiation of bone progenitor cells and their higher or equal expression in normoxic conditions might be another hint that osteogenic differentiation occurs in higher oxygen levels than to the stem cell niche. Interestingly, it could be shown, that reactive oxygen species (ROS) significantly reduce integrin expression on protein level within MSCs 145 . Due to the fact, that hypoxia increases the intracellular ROS level, focal adhesion complexes should also be degraded in hypoxic conditions. Therewith it could be highlighted, that focal adhesion-related molecules, such as phosphorylated focal adhesion kinase (p-FAK) and phosphorylated-tyrosine kinase SRC (p-SCR) as well as the integrin subunits αv and β1 are significantly downregulated when incubated with H2O2 146 . Our results re- vealed an upregulation of various integrin subunits in a low oxygen concentration, and therefore a protective mechanism to avoid that degradation must be involved. Major hints for the regulation of mesenchymal stem cell integrin expression are provided from other cell types. HIF plays the pivotal role for integrin regulation in hypoxic conditions for integrin β1 upregulation in fibroblasts during hypoxic wound healing 147 , inflammatory adhesion of leukocytes to activated endothelia by integrin β2 but not β1 upregulation 148 or migration, invasion and adhesion of trophoblast stem cells 149 . In conclusion, continuative studies must reveal, if HIF is also responsible for integrin regulation in hypoxic cultured MSCs. Furthermore the protective mechanism of focal adhesion complexes in hypoxia must be evaluated.

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