Page 151 - Physico-Chemical Niche Conditions for Bone Cells

a5 staining intensity per cell. n = 4 separate experiments, 30–40 cells/substrate/experiment (in total 150–200 cells/ experiment). (e) Relative mRNA levels of paxillin transcript variant a. n = 5 separate experiments. (f) Relative mRNA levels of paxillin transcript variant β. n = 5 separate experiments. (g) Relative integrin a5 gene expression. n = 5 separate experiments. **p < 0.001, *p < 0.05. PLL, poly-L- lysine coated glass; SLBs, supported lipid bilayers.
Osteogenic differentiation state after 17 hr of culture
To investigate the initial osteogenic gene expression response to RGD-functionalized SLBs, RUNX2, osteopontin (OPN), collagen type I (COL1a1), and alkaline phosphatase (ALP) gene expression was analyzed (Figure 6a). After 17 hr of culture, gene expression levels of OPN were 2.5-fold higher in cells cultured on SLBs with 0.2 μM RGD compared to those cultured on PLL-coated glass (Figure 6a; p < 0.05). There was a trend toward 1.5-fold higher mRNA expression levels of the early osteogenic marker RUNX2 in cells cultured on SLBs with 0.2 μM RGD compared to those cultured on PLL-coated glass (Figure 6a; n.s.). There were no differences regarding the expression of the late osteogenic marker COL1a1 between the different substrates (Figure 6a). ALP expression, also a late osteogenic marker, was too low to be determined (Figure 6a).
Osteogenic differentiation state after 1 week of culture
To further elucidate the effect of RGD-functionalized SLBs on osteogenic differentiation of pre- osteoblasts, gene expression of RUNX2, OPN, COL1a1, and ALP was also analyzed after 1 week of culture (Figure 6b). There was a 1.8-fold higher expression of COL1a1 in cells cultured on SLBs with 0.5 μM RGD than on PLL-coated glass (Figure 6b; p < 0.02). In cells cultured on SLBs without RGD and with 0.2 μM and 1.0 μM RGD, the mean expression of COL1a1 mRNA was 1.4-fold higher than in cells cultured on PLL (Figure 6b; n.s.). Compared to 17 hr, gene expression levels of OPN, COL1a1, and ALP were higher after 1 week, while RUNX2 expression was lower (Figure 6). This shows that after 1 week the expression of osteogenic genes in cells cultured on RGD-functionalized SLBs was comparable to the expression in cells cultured on PLL, if not higher.
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