Figure 4: Alkali-treatment of Ti enhances ECM deposition, while co-culture of fibroblasts with blood clots induces ECM remodelling.
(a–f) Immunofluorescent micrographs of native (a–c) and alkali-treated (d–f) Ti either exposed to blood (a,d), blood and subsequent seeded fibroblasts (b,e) or fibroblasts only (c,f), 24 h after seeding fibroblasts. Samples were immunostained for cell nuclei (DAPI) (blue), fibronectin (Fn) (green) and fibrin (red). One field of view of each condition (using the same magnification) from donor 4 is presented as XY (top) and XZ side view maximum intensity projections (middle) and as surface rendered 3D representations (bottom) at identical magnification. (g,h) Quantification of extracellular matrix components Fn (g) and fibrin (h) on native or alkali-treated Ti exposed to blood (white bars), blood & fibroblasts (grey bars) or fibroblasts (dark grey bars) 24 h after fibroblast seeding. Total ECM volumes per field of view were quantified from confocal micrograph z-stacks. Reported values in boxplots represent mean (middle square), median (central line), 25th to 75th percentile (box) and standard deviation (whisker) of 7 fields of view of duplicate Ti surfaces per condition repeated for 5 donors. Statistically significant differences between conditions are indicated by (***) for p < 0.001. Alkali-treatment of Ti significantly increased surface-adhering Fn and fibrin ECM compared to native Ti. Fibroblasts were shown to assemble new Fn matrix, either cultured on bare Ti or on blood clots. Remodelling of blood clots through fibroblasts was observed by trends in reduction of Fn and fibrin compared to blood only and fibroblasts residing in pits within blood clots. For timeline see Fig. 2a.
