PMC

SMCs expressing constitutively active V14-RhoA were cultured in PEG-fibrinogen constructs of varying stiffness for 7 days and subsequently stained for F-actin (A, red) and vinculin (B, red). The co-expressed GFP is shown in green in both A and B. White arrows denote evidence of vinculin-rich focal adhesion-like structures in SMCs expressing V14-RhoA. C.) Representative Western blot for vinculin of day 7 cell lysates from both V14-RhoA expressing and wild-type SMCs (From left to right, C = 448 Pa, non-transduced control, followed by V14-RhoA samples increasing in compressive modulus from left to right). Quantification of Western blots with * denoting statistical significance relative to the day 7, 448 Pa condition (N=6 separate experiments; error bars denote SEM).

SMCs were cultured within PEG-fibrinogen hydrogels of varying stiffness (448 Pa=red, 1008=green, 2306=blue, and 5408=black) and their DNA content quantified via a Hoechst dye. Constructs containing wild-type SMCs (solid lines) or V14-RhoA expressing SMCs (dashed lines) were assayed at 1, 3, 5, 7, 10, and 14 days post-seeding. Data represent mean ± SEM with N=2 gels for every experiment and N≥4 separate experiments at every condition. The * denotes statistical significance relative to the day 1 count at the same stiffness condition. The # denotes statistical significance between wild-type and V14-RhoA cells at any given condition (P≤0.05).

SMCs cultured within PEG-fibrinogen hydrogels of different stiffnesses were fixed and stained for vinculin (red) and F-actin (green). A.) SMCs did not fully spread nor show evidence of F-actin bundling after 1 day in culture. B.) After 7 days, SMCs were fully spread and showed some F-actin bundling. C.) F-actin bundling was qualitatively higher in the stiffer matrices after 14 days in culture, shown by the high magnification images (top row). However, at all time points and stiffness conditions, there was a notable absence of classic, punctate focal adhesion structures, denoted by vinculin staining, as is normally seen on 2-D substrates. Scale bars in top rows = 10 µm, bottom rows = 50 µm.

SMCs were cultured within PEG-fibrinogen hydrogels of varying stiffness for either 7 or 14 days and subsequently analyzed for their differentiated state via both Western blotting and immunofluorescent staining. Quantitative Western blot analysis of two markers specific to SMC differentiation, α-actin (A) and calponin (B) revealed that their expression was regulated by the stiffness of the surrounding 3-D matrix, but only in conjunction with constitutively active RhoA. Quantitative values shown are relative to the expression at day 7 for 448 Pa without V14-RhoA transduction. The * denotes statistical significance from the 448 Pa non-transduced condition. C.) After 7 days of culture in 3-D PEG-fibrinogen, wild-type and V14-RhoA SMCs were fixed and immunofluorescently stained for α-actin and calponin and visualized by confocal microscopy. In the top row, α-actin=green and calponin=red, (wild-type SMCs); middle row, α-actin=red (V14-RhoA SMCs); bottom row, calponin=red (V14-RhoA SMCs). Scale bar = 10 µm.

A.) A schematic representation of PEG-fibrinogen hydrogels and their synthesis is shown. 1.) The full heterotrimeric fibrinogen protein contains plasmin-sensitive sequences (black arrows) and disulfide bonds (green arrow). 2.) Synthesis of PEG-fibrinogen gels involves dissociating the heterotrimer into its representative α, β, and γ chains (one representative chain is shown). These chains are then PEGylated via a Michael-type addition reaction, forming thiol linkages between diacrylated PEG and cysteine residues in the fibrinogen monomers. 3.) Upon exposure to UV-light in the presence of a photoiniator, the remaining unreacted acrylate end groups on PEGDA are induced to react together, or with additional exogenous PEGDA (up to 2% by weight in this study), to form a cross-linked hydrogel network. B.) Average concentrations of fibrinogen and acrylated PEG for PEG-fibrinogen hydrogels used throughout all experiments are tabulated. Fibrinogen concentration was determined using a Pierce BCA protein assay, and PEGDA concentration determined by lyophilizing and weighing a known volume of product. C.) The compressive moduli of PEG-fibrinogen hydrogels increase from 448 Pa to 5804 Pa by increasing the amount of exogenous cross-linkable PEGDA from 0–2% by weight. Compressive moduli were determined by compressing each sample five consecutive times using an MTS Synergie 100 and extracting values from the linear region of the stress-strain curve (0–4% total strain). The * denotes statistical significance relative to the 0% condition (P<0.005). D.) Representative scanning electron micrographs of fixed, flash-frozen, freeze-dried, and carbon-sputtered acellular PEG-fibrinogen hydrogels imaged on a Zeiss Ultra 55 SEM are shown. Images A and B are of a 448 Pa gel, and C and D are of a 5408 Pa gel. Scale bars in the top row of images are 10 µm. Scale bars in the bottom row are 1 µm. E.) SMCs cultured in PEG-fibrinogen hydrogels for 1, 7, or 14 days were subsequently stained using a LIVE/DEAD Viability kit from Molecular Probes. Data represent quantification of several images for each condition and time point. There were no statistical differences between the data for any of the PEG-fibrinogen hydrogels. Viability assays in PEG-RGDS constructs (adapted from []) and were performed in parallel as controls.