Figure 6

Angiogenesis regulated by cell bricks prevented central necrosis and ossification of constructs. Evaluation of angiogenesis in B-CB-P grafts at the fourth week and 12th week. (A, C) Histological images reveal infiltration of capillaries at the fourth week and 12th week, predominantly distributed in BMSC regions, as confirmed by CD31 immunofluorescent staining (B, D). (E) Comparison of the number of blood vessels between the fourth week and 12th week as described previously; the value represents the cumulative number of all slides examined (n = 8). The fourth-week grafts allowed more blood vessel formation than those in the 12th week. (F) Immunostaining of vascular endothelial growth factor (VEGF) revealed decreasing expression of VEGF in BMSC regions through 4 and 12 weeks in the B-CB-P group, but increasing expression in the B-C-P group. Positive control, native cancellous bone; negative control, native auricular cartilage; bar = 50 μm, magnification × 100. (G) Real-time RT-PCR revealed the expression of VEGF in the B-CB-P and B-C-P groups, which were all normalized to rabbit auricular cartilage. *Statistically significant difference from rabbit auricular cartilage (P <0.05). #Statistically significant difference from the B-CB-P group (P <0.05). ▲Statistically significant difference of each group at two time points. B-C-P, BMSCs–chondrocytes–PRP; B-CB-P, BMSCs–cell bricks–PRP; B-P, BMSCs–PRP; BMSC, bone marrow-derived mesenchymal stem cell; HE, hematoxylin and eosin; HFP, high power field; PRP, platelet-rich plasma.