Dual delivery of an angiogenic and an osteogenic growth factor for bone regeneration in a critical size defect model

Abstract

This study investigated the effects of dual delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) for bone regeneration in a rat cranial critical size defect. Four groups of scaffolds were generated with VEGF (12 microg), BMP-2 (2 mug), both VEGF (12 microg) and BMP-2 (2 microg), or no growth factor released from gelatin microparticles incorporated within the scaffold pores. These scaffolds were implanted within an 8 mm rat cranial critical size defect (n=8-9 for each group). At 4 and 12 weeks, implants were retrieved and evaluated by microcomputed tomography (microCT) and histological scoring analysis. Additionally, 4 week animals were perfused with a radiopaque material to visualize and quantify blood vessel formation. Histological analysis revealed that for all groups at 4 weeks, a majority of the porous scaffold volume was filled with vascularized fibrous tissue; however, bone formation appeared most abundant in the dual release group at this time. At 12 weeks, both dual release and BMP-2 groups showed large amounts of bone formation within the scaffold pores and along the outer surfaces of the scaffold; osteoid secretion and mineralization were apparent, and new bone was often in close or direct contact with the scaffold interface. MicroCT results showed no significant difference among groups for blood vessel formation at 4 weeks (<4% blood vessel volume); however, the dual release group showed significantly higher bone formation (16.1+/-9.2% bone volume) than other groups at this time. At 12 weeks, dual release and BMP-2 groups exhibited significantly higher bone formation (39.7+/-14.1% and 37.4+/-18.8% bone volume, respectively) than either the VEGF group or blank scaffolds (6.3+/-4.8% and 7.8+/-7.1% bone volume, respectively). This work indicates a synergistic effect of the dual delivery of VEGF and BMP-2 on bone formation at 4 weeks and suggests an interplay between these growth factors for early bone regeneration. For the doses investigated, the results show that the addition of VEGF does not affect the amount of bone formation achieved by BMP-2 at 12 weeks; however, they also indicate that delivery of both growth factors may enhance bone bridging and union of the critical size defect compared to delivery of BMP-2 alone.

Figure 1. Histological sections of composite scaffolds at 4 weeks

Fibrous tissue infiltration was observed in A) BLANK and B) VEGF groups, while some bone formation was observed in C) BMP-2 and D) DUAL groups. In B), the black arrow points to Microfil within a blood vessel, while the white arrows point to unperfused blood vessels. P: PPF scaffold; B: new bone. Bar represents 200 μm for all panels.

Figure 2. Histological sections of composite scaffolds at 12 weeks

In the A) BLANK and B) VEGF groups, the ingrowth tissue was mostly fibrous with minimal bone formation. However, in the C) BMP-2 and D) DUAL groups, there was significant bone formation in both the pores and along the scaffold surfaces, generally observed on the dural side (panel E, BMP-2 group). Intramembranous ossification (panel F, BMP-2 group) was characterized by osteoid secretion and mineralization (white arrows). P: PPF scaffold; B: new bone. Bar represents 200 μm for all panels.

Figure 3. Results of histological scoring

Scores for A) the hard tissue response at the bone-scaffold interface, B) the tissue response within the scaffold pores, and C) the amount of bone formation within the defect at 4 weeks (□) and 12 weeks (■) for the four groups examined in this study (BLANK, VEGF, BMP-2, and DUAL). Error bars represent standard deviation for n = 8–9. (*) denotes statistical significance at p<0.05. (#) denotes statistical significance at p<0.05 compared to the BMP-2 group at 12 weeks.

Figure 4. MicroCT images (maximum intensity projections) of cranial defects at 4 and 12 weeks

Panels A–D represent BLANK, VEGF, BMP-2, and DUAL groups at 4 weeks prior to decalcification; both blood vessels and bone were visible. Panels E–H represent BLANK, VEGF, BMP-2, and DUAL groups at 12 weeks; no blood vessels were visible because Microfil perfusion was not done at this time period. Bar represents 200 μm for all panels.

Figure 5. Results of scoring for bony bridging and union within the defect

Scores for bony bridging and union for the four groups (BLANK, VEGF, BMP-2, and DUAL) examined at 4 weeks (□) and 12 weeks (■). Error bars represent standard deviation for n = 8–9. (*) denotes statistical significance (p<0.05) between groups.

Figure 6. Results of microCT quantification of % blood vessel volume within the defect at 4 weeks

Values for % blood vessel volume are given for the four groups (BLANK, VEGF, BMP-2, and DUAL) examined as well as the EMPTY group as quantified by microCT. Error bars represent standard deviation for n = 8–9.

Figure 7. Results of microCT quantification of % bone volume within the defect at 4 and 12 weeks

Values are given for % bone volume of the four groups (BLANK, VEGF, BMP-2, and DUAL) examined as well as the EMPTY group as quantified by microCT. Error bars represent standard deviation for n = 8–9. At 4 weeks (□), the DUAL group was significantly different (p<0.05) than all other groups. At 12 weeks (■), both DUAL and BMP-2 groups were significantly different (p<0.05) from VEGF and BLANK groups.