Effects of bone tissue engineering triad components on vascularization process: comparative gene expression and histological evaluation in an ectopic bone-forming model

Figures & data

Table 1. Experimental groups of mice used in this study.

Experimental group	Implanted material	Number of treated mice	Number of time points for implant extraction	Number of sacrificed mice per time point
EPB	1 × 10^4 ECs (ASCs at the 12th day of in vitro endothelial differentiation) in 18 µL of DMEM and 2 µL of PRP as a liquid component, and 10 mg (∼0.02 cm^3) of BMM	20	4 (1, 2, 4, 8 weeks after implantations)	5
PB	18 µL of DMEM and 2 µL of PRP as a liquid component, and 10 mg of BMM	20	4 (1,2,4, 8 weeks after implantations)	5
B (control group)	20 µL of DMEM as a liquid component, and 10 mg of BMM	20	4 (1, 2, 4, 8 weeks after implantations)	5

Table 2. Primers used for qPCR.

Gene name and abbreviation	QuantiTect primer assay
β-actin (Actb)	Mm_Actb_2_SG, QT01136772
Early growth response-1 (Egr-1)	Mm_Egr1_1_SG, QT00265846
Vascular endothelial growth factor receptor 1 (Flt-1)	Mm_Flt1_1_SG, QT00096292
Vascular cell adhesion molecule-1 (Vcam-1)	Mm_Vcam1_1_SG, QT00128793
von Willebrand factor (Vwf)	Mm_Vwf_1_SG, QT00116795
Osteopontin (Spp1)	Mm_Spp1_1_SG, QT00157724

Figure 1. CD44 (A and B) and VCAM-1 (C and D) immunoexpression in ASCs one day after isolation (A, C and E) and one day after passage 3 (P03) (B, D and F). Negative controls (E and F).

Figure 2. VCAM-1 immunoexpression in ASCs induced into ECs 7 (A), 12 (B) and 15 days (C) after P03 and in non-induced ASCs: 7 (D), 12 (E) and 15 days (F) after P03. Negative controls: 7 (G), 12 (H) and 15 (I) days after ASCs induction.

Figure 3. Egr-1 (A) and Flt-1 (B) relative expression in EPB, PB and B implants at 1-, 2-, 4- and 8-week (w) time points.

Figure 4. Vcam-1 (A) and Vwf (B) relative expression in EPB, PB and B implants at 1-, 2-, 4- and 8-week (w) time points.

Figure 5. VCAM-1 immunoexpression (ellipse) at the 2-week time point (A–C) and at the 8-week time point (D–F). EPB (A and D), PB (B and E) and B (C and F) implants. Bio-Oss (star); blood vessels (arrowhead).

Figure 6. Negative controls for immunohistochemistry: at the 2-week time point (A–C) and the 8-week time point (D–F). EPB (A and D), PB (B and E) and B (C and F) implants.

Figure 7. Toluidine blue staining: at the 2-week time point (A–C) and at the 8-week time point (D–F). EPB (A and D), PB (B and E) and B implants (C and F). Bio-Oss (star), osteoblast-like cells (long, thin arrow), multinucleated giant cells (short, thick arrow), osteoid-like tissue (ellipse).

Figure 8. Relative expression of Spp1 in EPB, PB and B implants at 1-, 2-, 4- and 8-week (w) time points.

Figure 9. Osteopontin immunoexpression (ellipse) at the 2-week time point (A–C) and at the 8-week time point (D–F). EPB (A and D), PB (B and E) and B implants (C and F). Bio-Oss (star), osteoblast-like cells (long, thin arrow), multinucleated giant cells (short, thick arrow).