Figure 5
Focal adhesion immunostaining and total collagen and elastin synthesis.
(a) Immunostaining images (scale bar: 100 μm) of RDFs show vinculin (green), phalloidin (red) and dapi (blue) on different surface modification type. The upper panel shows staining for phalloidin only to exhibit the first population of RDFs morphology on X, CHF 3 and NaOH, where cells were poorly spread. The lower panel shows that the second population of RDFs morphology on X, CHF 3 and NaOH, where cells were more spread, exhibited vinculin sites and organized actin cytoskeleton. Ar and O2 provided a homogenous spread of cells, enhanced actin stress fibers and vinculin expression throughout the polymer. (b) CHCl3 displayed a homogenous population of cells with enhanced actin filaments expression, but different vinculin density. Vinculin sites found in larger pores (5–20 μm) shown by the arrow heads expressed dense circular vinculin sites, while smaller pores (0.5–2.5 μm) displayed by the arrows expressed elongated vinculin staining. (c) Quantification of vinculin density per treatment. Statistics were done by One-way ANOVA with a post Tukey test (n = 8). (d) Quantification of collagen secreted per different rod showed statistically significant increase in production after surface modification for day 4 conditioned medium (CM), day 7 refresh medium (ref) and CM (∧ indicating exception to NaOH, # to CHF 3). CM cultures triggered a significant increase in comparison to refresh medium, (& indicating exception to CHF 3 and O2). CHCl3 secreted the highest collagen amount in comparison to all treatments (red star, or Φ indicating exception to O2). (e) A statistically significant increase of elastin secretion was seen on treated rods for day 4 conditioned medium (CM), day 7 refresh medium (ref) and CM (# indicating exception to CHF 3). CM cultures triggered a significant increase in comparison to refresh medium on day 4. At day 7 CM, CHCl3 provided the best secretion of elastin in comparison to all other surface modification. Data are shown as mean ± s.d. (n = 6). Blue symbols (*P < 0.05, ** P < 0.01, *** P < 0.001) indicate statistical significances in comparison to unmodified, red symbols indicate the best parameter from all the treatment types and black symbols evaluate statistical differences between the different treatments.
