Page 144 - Physico-Chemical Niche Conditions for Bone Cells

RGD-functionalized supported lipid bilayers modulate pre-osteoblasts
intensity and phosphopaxillin cluster number and size were measured using ImageJ as described [29].
Gene expression analysis
After 17 hr or 1 week of culture, cells were lysed using TRIreagent (Invitrogen; Fisher Scientific). Total RNA was extracted using RNAqueous-micro kit (Invitrogen) according to the manufacturers' protocol and measured using nanodrop 2000 (Thermo scientific). Complementary DNA (cDNA) was synthesized using 100 ng RNA for cells cultured for 17 hr, and 200 ng RNA for cells cultured for 1 week in 20 μL reaction mixture using Superscript VILO MasterMix (Invitrogen). For each target gene 5 μL of 10 ´ diluted cDNA was amplified in duplicate using Fast SYBRTM Green Master Mix (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA) on a StepOne Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific). Target proliferation marker genes Ki67 and CCND1 and osteogenic marker genes RUNX2, OPN, COL1a1, and ALP were analyzed. Gene expression levels were calculated relative to the housekeeping gene HPRT1 using the delta Ct method. Primer sequences are listed in Table 1.
Statistics
Data shown are mean ± SEM. Statistical analysis was performed using IBM SPSS version 25 (SPSS Inc., La Jolla, CA). Differences were considered significant if p <0.05. Data on cell density, focal adhesion, and gene expression obtained from cultures on different substrates were compared using one-way ANOVA with Bonferroni post hoc tests, Kruskall–Wallis test with pairwise comparisons, or ANCOVA. Data on cell morphology (Figure 4) were not normally distributed and tested per category using mixed model ANOVA with category as within- subjects factor, substrate as between-subjects factor, and percentage of cells as the dependent variable. A significant interaction effect (category*substrate) was considered as an indication that at least one substrate differed from at least one of the other substrates. When a significant interaction effect was observed, differences were further investigated by repeating the mixed model ANOVA with PLL excluded, which always resulted in a nonsignificant interaction effect, indicating that the distribution of the measurement in cells on PLL was different from that of the measurement on other substrates. To investigate whether the percentage of cells was different between substrates in a certain category, one-way ANOVA with Bonferroni post hoc tests (or Kruskall–Wallis tests with pairwise comparisons if parametric methods were not appropriate) was performed for every category. Mean ± SEM was calculated and significant differences tested using Kruskall–Wallis test with pairwise comparisons, since data were not normally distributed.
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