Vascularization and osteogenesis in ectopically implanted bone tissue-engineered constructs with endothelial and osteogenic differentiated adipose-derived stem cells

Figure 1 Relative endothelial-related gene expression in uninduced adipose-derived stem cells. Relative endothelial-related gene expression in uninduced adipose-derived stem cells at the 12th day after the third passage (P03-12d cells) compared to uninduced adipose-derived stem cells obtained 1 d after the third passage (P03-1d cells; calibrator sample). Upregulation of expression of all examined endothelial-related genes (Vwf, Egr1, Flt1 and Vcam1) was found in P03-12d cells. Vwf: Von Willebrand factor; Egr1: Early growth response 1; Flt1: Vascular endothelial growth factor receptor 1; Vcam1: Vascular cell adhesion molecule 1.

Figure 2 Dynamics and patterns of relative gene expression levels in ectopic osteogenic implants. Relative gene expression levels in BC (implants containing only BMM carrier), BPUI (implants containing uninduced adipose-derived stem cells) and BPEO (implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells) implants extracted at one (1w), two (2w), four (4w) and eight (8w) weeks after implantation (observation points) were presented comparing the expression of the same genes in the calibrator sample (P03-1d cells combined with PRP). A: Von Willebrand factor; B: Early growth response 1; C: Vascular endothelial growth factor receptor 1; D: Vascular cell adhesion molecule 1; E: Osteopontin. Number of sacrificed mice per experimental period (1w, 2w, 4w, 8w) per group was six, giving six samples per group [n (BC) = 6, n (BPUI) = 6, n (BPEO) = 6] for relative gene expression analysis. For each examined gene expression, results were presented as mean value ± standard deviation. Error bars represent standard deviation. ^aP < 0.05 BC vs BPUI within the same observation point. ^bP < 0.05 BC vs BPEO within the same observation point. ^cP < 0.05 BPUI vs BPEO within the same observation point. Vwf: Von Willebrand factor; Egr1: Early growth response 1; Flt1: Vascular endothelial growth factor receptor 1; Vcam1: Vascular cell adhesion molecule 1; Spp1: Osteopontin; BC: Implants containing only bone mineral matrix carrier; BPUI: Implants containing uninduced adipose-derived stem cells, platelet-rich plasma and bone mineral matrix carrier; BPEO: Implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and bone mineral matrix carrier.

Figure 3 Representative hematoxylin and eosin sections of ectopic osteogenic implants. Representative hematoxylin and eosin sections of: A: Two-week-old BC [implants containing only bone mineral matrix (BMM) carrier]; B: Two-week-old BPUI (implants containing uninduced adipose-derived stem cells, platelet-rich plasma and BMM carrier); C: Two-week-old BPEO (implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and BMM carrier); D: Eight-week-old BC implants; E: Eight-week-old BPUI implants; F and G: Eight-week-old BPEO implants. All images acquired at bright field. Scale bars show 50 μm (A-F) and 100 μm (G). BMM granules (orange star), cellularity (black circle), blood vessels (orange arrows), osteoblast-like cells (orange arrowhead), multinucleated giant cells (black arrowhead), osteon-like structures (rounded rectangle), hemorrhagic fields (orange circle), area of resorbed granules (ellipse), tissue regression between BMM granules (orange ellipse).

Figure 4 Representative Masson’s trichrome sections of ectopic osteogenic implants. Representative Masson’s trichrome sections of: A: Two-week-old BC [implants containing only bone mineral matrix (BMM) carrier]; B: Two-week-old BPUI (implants containing uninduced adipose-derived stem cells, platelet-rich plasma and BMM carrier); C: Two-week-old BPEO (implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and BMM carrier); D: Eight-week-old BC implants; E: Eight-week-old BPUI implants; F: Eight-week-old BPEO implants. All images acquired at bright field. Scale bar shows 50 μm; BMM granules (orange star), cellularity (black circle), blood vessels (orange arrows), osteoblast-like cells (orange arrowhead), multinucleated giant cells (black arrowhead), hemorrhagic fields (orange circle), weave-like collagen fibers (curved double-arrow connector), regular collagen fibers (elbow double-arrow connector).

Figure 5 Histomorphometric analysis of ectopic osteogenic implants. Percentage of vascularization was determined in: A: Two-week-old and B: Eight-week-old BC [implants containing only bone mineral matrix (BMM) carrier], BPUI (implants containing uninduced adipose-derived stem cells, platelet-rich plasma and BMM carrier) and BPEO (implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and BMM carrier). Four samples (n = 4) per each group per experimental period [n (BC) = 4, n (BPUI) = 4, n (BPEO) = 4] were taken for this analysis. For each group for both experimental periods, results were presented as mean values ± standard deviation. Error bars represent standard deviation. ^aP < 0.01 BC vs BPUI. ^bP < 0.01 BC vs BPEO. ^cP < 0.05 BPUI vs BPEO. ^dP < 0.05 BC and BPEO. BC: Implants containing only bone mineral matrix carrier; BPUI: Implants containing uninduced adipose-derived stem cells, platelet-rich plasma and bone mineral matrix carrier; BPEO: Implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and bone mineral matrix carrier.

Figure 6 Immunoexpression of vascular endothelial growth factor receptor 2 in representative sections of ectopic osteogenic implants. A: Two-week-old BC [implants containing only bone mineral matrix (BMM) carrier]; B: Two-week-old BPUI (implants containing uninduced adipose-derived stem cells, platelet-rich plasma and BMM carrier); C: Two-week-old BPEO (implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and BMM carrier); D: Eight-week-old BC implants; E: Eight-week-old BPUI implants; F: Eight-week-old BPEO implants, G: Vascular endothelial growth factor receptor 2 immunohistochemistry optical density score in implants extracted two (2w) and eight (8w) weeks after implantation (observation points). All images acquired at bright field. Scale bar shows 20 μm. Immunoreactivity is visualized as brown color and represents immunoexpression of the applied antibody. Vascular endothelial growth factor receptor-2 positive cell (black circle); Vascular endothelial growth factor receptor-2 negative cell (orange circle). ^aP < 0.01 BC 2w vs BPUI 2w. ^bP < 0.001 BC 8w vs BPEO 8w. ^cP < 0.001 BPUI 2w vs BPEO 2w. ^dP < 0.001 BPUI 8w vs BPEO 8w. VEGFR-2: Vascular endothelial growth factor receptor 2; BC: Implants containing only bone mineral matrix carrier; BPUI: Implants containing uninduced adipose-derived stem cells, platelet-rich plasma and bone mineral matrix carrier; BPEO: Implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and bone mineral matrix carrier; IHC: Immunohistochemistry; OD: Optical density.

Figure 7 Immunoexpression of CD31 in representative sections of ectopic osteogenic implants. A: Two-week-old BC [implants containing only bone mineral matrix (BMM) carrier]; B: Two-week-old BPUI (implants containing uninduced adipose-derived stem cells, platelet-rich plasma and BMM carrier); C: Two-week-old BPEO (implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and BMM carrier); D: Eight-week-old BC implants; E: Eight-week-old BPUI implants; F: Eight-week-old BPEO implants; G: CD31 immunohistochemistry optical density score in implants extracted two (2w) and eight (8w) weeks after implantation (observation points). All images acquired at bright field. Scale bar shows 50 μm. Immunoreactivity is visualized as brown color and represents immunoexpression of the applied antibody. CD31 positive cells (black circle), CD31 negative cells (orange circle). ^aP < 0.01; BC 8w vs BPUI 8w. ^bP < 0.001; BC 2w vs BPEO2w. ^cP < 0.05; BC 8w vs BPEO 8w. ^dP < 0.01; BPUI 2w vs BPEO 2w. ^eP < 0.001; BPUI 8w vs BPEO 8w. BC: Implants containing only bone mineral matrix carrier; BPUI: Implants containing uninduced adipose-derived stem cells, platelet-rich plasma and bone mineral matrix carrier; BPEO: Implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and bone mineral matrix carrier; IHC: Immunohistochemistry; OD: Optical density.

Figure 8 Immunoexpression of osteocalcin in representative sections of ectopic osteogenic implants. A: Two-week-old BC [implants containing only bone mineral matrix (BMM) carrier]; B: Two-week-old BPUI (implants containing uninduced adipose-derived stem cells, platelet-rich plasma and BMM carrier]; C: Two-week-old BPEO (implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and BMM carrier); D: Eight-week-old BC implants; E: Eight-week-old BPUI implants; F: Eight-week-old BPEO implants; G: Osteocalcin immunohistochemistry optical density score in implants extracted two (2w) and eight (8w) weeks after implantation (observation points). All images acquired at bright field. Scale bar shows 20 μm. Immunoreactivity is visualized as brown color and represents immunoexpression of the applied antibody; osteocalcin-positive fields (orange ellipse). ^aP < 0.01; BC 2w vs BPUI2w. ^bP < 0.001; BC 8w vs BPUI 8w. ^cP < 0.001; BC 8w vs BPEO 8w. ^dP < 0.05; BPUI 2w vs BPEO 2w. ^eP < 0.001; BPUI 8w vs BPEO 8w. ^fP < 0.01; BC 2w vs BC8w. ^gP < 0.001; BPUI 2w vs BPUI 8w. BC: Implants containing only bone mineral matrix carrier; BPUI: Implants containing uninduced adipose-derived stem cells, platelet-rich plasma and bone mineral matrix carrier; BPEO: Implants containing simultaneously applied endothelial and osteogenic differentiated adipose-derived stem cells, platelet-rich plasma and bone mineral matrix carrier; IHC: Immunohistochemistry; OD: Optical density.