Figure 1.
Maximal spreading length of fibroblasts and pore wall to wall distance within StarPEG-heparin cryogel.
Maximal spreading length dcell,spread of the different human dermal fibroblasts are represented by white bars and wall to wall distance dpore,min of the pore analyzed in the scaffold by dark grey bars. The overlapping area is displayed in light grey. Gaussian fit was performed for maximum cell spreading (continuous line) and pore diameter (dashed line) distribution. The data was obtained by combining all the imaging performed on the starPEG-heparin cryogel (Npores >100) and cells contained in pores (Ncells >200).”
Figure 2.
Filling of scaffold pores by fibroblasts for three pore size categories and three time points.
Confocal microscopy for visualization of actin cytoskeleton (green) and cell nuclei (blue) to illustrate the filling of scaffold pores by human fibroblasts at day 1, 3, and 7 for the three pore size categories (scale bar equals 100 µm). Data shown corresponds to the maximum projection images of the 3D image stacks recorded inside scaffolds. During the imaging, scaffold pores could be identified due to background staining of the scaffold material and are highlighted by dashed lines.
Figure 3.
Quantification of cell number during pore filling.
Evolution of cell position (cell nuclei) as a function of time and pore size for human fibroblasts (mean values of npores = 4 for each category, SEM = standard error of the mean) (a), the derived ratio between the number of cells that are located within the pores (#cellsin_pore), and the total number of cells (#cellstotal) in the pore (b).
Figure 4.
Remaining open cross-sectional area as a function of the number of cells.
The ratio between the space to be filled (i.e. remaining open cross-sectional area) and the initial cross-sectional area of the pores was plotted as a function of the number of cells within that pore for the different categories. Experimental data is shown as circles for the different categories. Simulation data is displayed as lines with different shadings from black to light grey for different variation ranges for the covered area (R = 1: new cells positioned at locations of largest covered area per cell; R = 0: random position of new cells on the remaining inner pore surface). Simulation was based on a 2D model. Experimental data points were extracted from z-stack maximum projection for 2D analysis. Lines represent the average of ten simulations. Simulation stopped when the remaining inner diameter of the pore was smaller than the cell size. Within each graph the cell organization for a remaining area between 0.5 and 0.6 of the initial pore area according to the simulation is given. Dashed lines represent pore closure extracted from experimental data. Due to the porosity of the fibronectin network the remaining open area did not reach a zero value.
Figure 5.
Fibronectin organization for three pore size categories and three time points.
Confocal microscopy for visualization of fibronectin network (red) to illustrate the filling of scaffold pores until day 1, 3, and 7 and the three pore size categories (scale bar equals 100 µm). Data shown corresponds to the maximum projection images of the 3D image stacks recorded inside scaffolds. During the imaging, scaffold pores were observed due to some background staining and where delimited here by dashed lines.
Figure 6.
Distribution of fibronectin and actin inside scaffold pores.
Actin (green) and fibronectin (red) intensity distribution from the pore center to the pore wall as extracted from histological images (Fig. 6a, npores = 4 for each category). From the maximum projection of the z-stack and the contoured pore, the Pearson coefficient for green and red channel colocalization was calculated. The whole intensity data range for each channel (0–255) was considered here (Fig. 6b, n = 5 for each category).
Figure 7.
Schematic representation of pore filling as a function of time.
Cells are represented by tensioned chords in green and fibronectin is in red. Initially cells are spreading on the pore wall and fibronectin is being produced but not visible as a connected network yet by the cells (top). The pore is subsequently filled by cells with time according to the model and produce fibronectin fibers being unfolded due to cell forces (middle). Finally the pore closes completely and at the same time fibronectin is further compacted towards the center (bottom).