Engineering 2021, eight,12 ofFigure 6. Characterization on the isolated WJMSCs. Morphological traits of WJMSCs (A1 3). Differentiation of WJMSCs towards “osteogenic”, “adipogenic” and “chondrogenic” lineages (A4 six). The thriving differentiation of WJMSCs into “osteocytes”, “adipocytes” and “chondrocytes” was verified utilizing the histological stains Alizarin Red S, Oil Red O and Alcian Blue, respectively. CFUs assay of WJMSCs at P1 to P3 (A7 9). In vitro angiogenesis assay functionality. Photos showing the developed network have been acquired after 1, four and eight h (A10 12). Immunophenotyping analysis of WJMSCs P3 (B). Determination of total quantity (C) and viability of WJMSCs at P1 3 (D). The images A1 six and A10 12 were acquired with CD32a Protein C-6His original magnification ten and scale bars one hundred .Figure 7. Histological analysis of hUAs (A ). Overview of repopulated hUAs of groups A and B (B,C). Decellularized hUA served because the control group (A). Repopulated hUA of group A (B). WJMSCs P3 in group A have been located only to the tunica adventitia. Repopulated hUA of group B (C). Around the contrary, WJMSCs P3 in group B migrated successfully towards the inner vascular wall. Pictures represented with original magnification 2.5and scale bars 500 . Pictures inside the black squares represented with original magnification 10 scale bars one hundred .To further confirm the proliferative activity on the WJMSCs P3 inside the repopulated hUAs, immunohistochemistry against Ki67 was performed (Figure 9). The expression of Ki67 was confirmed in each groups. Nonetheless, a greater distribution of Ki67 was observed in group B, further confirming the H E staining outcomes.Bioengineering 2021, eight,13 ofFigure eight. Histological evaluation of repopulated vascular EIF4EBP1 Protein web grafts with WJMSCs P3, located in the tunica adventitia. Decellularized hUAs stained with H E (A,G,M). Repopulated hUAs of group A (C,I,O) and group B (E,K,Q) stained with H E. Immunohistochemistry against Ki67 in decellularized hUAs (B,H,N), and repopulated hUAs of group A (D,J,P) and group B (F,L,R). Images (A ) presented with original magnification 10 scale bars 100 . Photos (G ) presented with original magnification 20 scale bars 50 . Photos (M ) presented with original magnification 40 scale bars 25 .Figure 9. Histological analysis of repopulated vascular grafts with WJMSCs P3, positioned inside the tunica intima. Decellularized hUAs stained with H E (A,G,M). Repopulated hUAs of group A (C,I,O) and group B (E,K,Q) stained with H E. Immunohistochemistry against Ki67 in decellularized hUAs (B,H,N), and repopulated hUAs of group A (D,J,P) and group B (F,L,R). Photos (A ) presented with original magnification 10 scale bars 100 . Pictures (G ) presented with original magnification 20 scale bars 50 . Images (M ) presented with original magnification 40 scale bars 25 .The immunofluorescence final results indicated the expression of MAP kinase in each groups (Figure ten and Figure S3). Even so, the greater distribution and expression of MAP kinase were observed in group B in comparison to group A (Figure ten). The MFI of the MAP kinase expression in TA and TI in repopulated hUAs of group A and group B was 13.9 2.1 and 1.1 0.three, and 45.6 7.1 and 45.3 5.1, respectively. Accordingly, for the DAPI stain, the MFI in repopulated hUAs of groups A and B have been 7.six 1.1 and 39.8 two.five, and 43.4 four.4 and 41.five 4.three, respectively. The statistically significant differences wereBioengineering 2021, 8,14 ofobserved within the study groups, concerning either the MAP kinase expression (p 0.01) or the DAPI.