Fig 1: Expression of BMP2 alone was superior to the combined delivery of BMP2 and VEGFA in formation of radiopaque bone like structures in a critical-sized rat calvarial defect.Illustration of the location of scaffold implantation sites in rat calvaria and implantation of scaffolds seeded with ad-BMP2, ad-BMP2 + VEGFA, ad-GFP or untransduced rBMSC in rat calvarial bone defects (A and B). At 8 weeks, microCT examination of calvarial defects in the control groups showed few radiopaque bone-like structures (C and D). In contrast, in ad-BMP2 and ad-BMP2 + VEGFA scaffold explants at 8 weeks, abundant radiopaque bone-like areas were observed inside the defect (E and F). Quantification of radiopaque bone like structures in the defects at 2 weeks (K) demonstrated a slightly higher amount of bone formation in ad-BMP2 group, whereas at 8 weeks (L) significantly more bone-like structures were found in ad-BMP2 than in the untransduced, ad-GFP or in the ad-BMP2 + VEGFA group. ANOVA test with Bonferroni post hoc analysis was performed for statistical analysis in (K and L). The error bars represent SEM. ***, p < 0.000; **, p = 0.001 to 0.01;*, p = 0.01 to 0.05; ns, non-significant.
Fig 2: Adenoviral vector mediated delivery of BMP2 alone or in combination with VEGFA induced formation of capillary like structures in a critical sized rat calvarial bone defect.(A–D) Representative images of Masson's trichrome stained slides at 8 weeks showing higher numbers of blood capillaries/vessels in ad-BMP2 and ad-BMP2 + VEGFA explants than in the untransduced and ad-GFP explants. Black arrows indicate the pink/red stained capillaries/blood vessels containing brown/orange stained erythrocytes (inset D, arrowheads indicating erythrocytes). (E) Quantification of blood capillaries/vessels demonstrated approximately twice the number in ad-BMP2 and ad-BMP2 + VEGFA, compared with the untransduced and ad-GFP explants. However, no significant difference in the number of blood capillaries/vessels was found between the ad-BMP2 and ad-BMP2 + VEGFA groups. ANOVA test with Bonferroni post hoc analysis was performed for statistical analysis in (E). Error bars represent SEM. **, p = 0.001 to 0.01;*, p = 0.01 to 0.05; ns, non-significant.
Fig 3: Expression of BMP2 alone was superior to the combined delivery of BMP2 and VEGFA in formation of vital bony structures in a critical-sized rat calvarial defect.(A–H) Representative images of H & E/Masson's trichrome stained FFPE sections of scaffold explants from different experimental groups. Apart from the borders of the calvarial defects, formation of bony structures was not obvious in the untransduced and ad-GFP groups both at 2 (A and B, H & E stained slides) and at 8 weeks (E and F, Masson's trichrome stained slides). In both ad-BMP2 (C) and ad-BMP2 + VEGFA (D) groups, formation of bony structures (black arrows) at the borders of the defect (black dotted lines), peripherally and within the scaffold explants was observed as early as 2 weeks. Extensive bone formation (black arrows) with bony trabeculae extending throughout the thickness of the scaffolds was found at 8 weeks in ad-BMP2 (G) and ad-BMP2 + VEGFA (H) groups. Examination of the bony structure at 2 weeks revealed numerous osteocyte-like cells (black arrowheads, inset C and D).
Fig 4: Adenoviral vector mediated expression of BMP2 alone and in combination with VEGFA led to up-regulation of osteogenic molecules in vitro.Significantly higher mRNA levels of Alpl and Runx2 were found in ad-BMP2 and ad-BMP2 + VEGFA groups at both time points (A–D). The error bars represent SEM of 3 biological replicates (n = 3) done in 3 technical replicates. (E) Representative images and quantification (F and G) demonstrating higher alkaline phosphatase activity in ad-BMP2 and ad-BMP2 + VEGFA groups in monolayer culture, compared with the ad-GFP group at 3 and 14 days. Experiments were repeated at least three times. ANOVA test with Bonferroni post hoc analysis was performed for statistical analysis in A–D, F and G. ***, p < 0.001; **, p = 0.001–0.01; *, p = 0.01–0.05; ns, non-significant. (H) Significantly higher amount of calcium deposition was found in ad-BMP2 and ad-BMP2 + VEGFA groups both in monolayer culture and in poly(LLA-co-CL) scaffold, compared with the ad-GFP group on 21 days. rBMSC grown in osteogenic medium was used as a positive control. The experiments were repeated at least three times.
Fig 5: VEGFA and BMP2 adenoviral expression vectors respectively increased expression of both human and rat specific mRNA levels in rBMSC.Significant up-regulation of human and rat specific BMP2 mRNA (A, C, E and G) and secreted BMP2 protein (I and J) levels were found at day 3 and 14 in both ad-BMP2 and ad-BMP2 + ad-VEGFA groups, compared with the ad-GFP group. Similarly, significant up-regulation of human and rat specific VEGFA mRNA (B, D, F and H) was found in ad-BMP2 + VEGFA group, compared with the ad-GFP and ad-BMP2 groups at both time points. mRNA expression levels of Gapdh were used to normalize the data. (I and J) Higher levels of secreted rat specific BMP2 protein were found in the culture supernatant of ad-BMP2 and ad-BMP2 + VEGFA groups by using ELISA assay at 3 and 14 days. The error bars in (A–H) represent SEM of 3 biological replicates (n = 3) done in 3 technical replicates. The error bars in (I–J) represent SEM of 3 biological replicates (n = 3) done in duplicates. ANOVA test with Bonferroni post hoc analysis was used for statistical analysis in (A–J). ***, p < 0.001; **, p = 0.001–0.01; *, p = 0.01–0.05; ns, non-significant.
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