Fig. 6: Scaffold preconditioning and transwells significantly improved the recellularization effiecinecy.
Representative hematoxylin and eosin-stained sections of recellularized constructs from each culturing condition after 14 days in vitro (A–C; scale bar = 100 μm). Cells were mainly located around the injection site when standard culturing conditions (RCSC) were used and there was limited migration (A). However, the different recellularization strategies (transwells, RCTW) and matrix metalloproteinase enzyme preconditioning in combination with TW (RCMMP+TW) clearly improved the cell density and the cellular distribution throughout the scaffolding structure (B, C). Surface cell density was visualized by scanning electron microscope and showed a dense coverage, independent of strategy used (D–F; inserts in C and F show the scaffold preconditioned with MMPs without cells; DCMMP; scale bar = 20 μm). Sheep fetal stem cells kept their pluripotency during recellularization independent of scaffold type, evident by the continuation of CD166, estrogen receptor-β (ER-β), vimentin (VIM), α-smooth muscle actin (SMA), and Ki67 (proliferation) expression (G–I; scale bars=100μm). When the recellularization efficiency was quantified and compared between groups and recellularization strategy, it became obvious that preconditioning with MMPs and using transwells were advantageous already after 3 days in vitro (J–L), but a substantial improvement was seen after 14 days (M–O) in protocol 1 (P1), protocol 2 (P2), and protocols 3 (P3), respectively. Graphs show box plot with each sample value, median ± IQR, and range. Significant levels; *p < 0.05; **p < 0.01; ***p < 0.001, and ****p < 0.0001.
