Dose effect of dual delivery of vascular endothelial growth factor and bone morphogenetic protein-2 on bone regeneration in a rat critical-size defect model

Abstract

The dose effect of dual delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) on bone regeneration was investigated in a rat cranial critical-size defect (CSD). It was hypothesized that decreasing amounts of BMP-2 would result in a dose-dependent decrease in bone formation, and that this reduction in bone formation could be reversed by adding increasing amounts of VEGF. In vitro release kinetics of VEGF or BMP-2 were examined over 28 days. Next, scaffolds were implanted within a rat cranial CSD containing different combinations of both BMP-2 and VEGF. At 12 weeks, samples were analyzed using microcomputed tomography and histology. In vitro, VEGF and BMP-2 exhibited burst release in the first 24 h followed by a significant decrease in release rate over 27 days. Overall, BMP-2 had a more sustained release versus VEGF. An in vivo dose-dependent decrease in percentage of bone fill (PBF) was observed for BMP-2. The addition of VEGF was unable to reverse this decrease in PBF, although improvements in the number of bridged defects did occur in some groups. This suggests that for this particular model simultaneous release of BMP-2 and VEGF does not increase bone formation over BMP-2 alone at 12 weeks.

FIG. 1.

Pore interconnectivity of porous PPF scaffolds, as measured by microcomputed tomography (micro-CT). The sudden increase in the percentage inaccessible volume between a 192 and 224 μm size cut-off indicates an average pore interconnect size within that range.

FIG. 2.

Profile of the in vitro release of VEGF in collagenase buffer. Average percent cumulative VEGF release from GMPs only versus porous PPF scaffolds incorporating GMPs (composite). Error bars represent means ± standard deviation for n = 3.

FIG. 3.

Profile of the in vitro release of BMP-2 in collagenase buffer. Average percent cumulative BMP-2 release from GMPs only versus porous PPF scaffolds incorporating GMPs (composite). Error bars represent means ± standard deviation for n = 3.

FIG. 4.

Percent bone formation within the defect at 12 weeks as measured by micro-CT. Values are given for % bone formation of the groups performed by Patel et al. (Groups 0B/0V, 0B/12V, 2B/0V, and 2B/12V) and this current study (Groups 1B/0V, 1B/6V, 1B/12V, 0.5B/0V, 0.5B/6V, and 0.5B/12V). Error bars represent means ± standard deviation for n =  8–9. At 12 weeks, Groups 2B/0V and 2B/12V are statistically significantly different (p < 0.05) from all other groups, as denoted by asterisks (*).

FIG. 5.

Examples of micro-CT generated maximum intensity projections of rat cranial defects at 12 weeks. (A) Group 1B/0V, Sample #7: bone score = 3, 28.5% bone fill. (B) Group 1B/6V, Sample #10: bone score = 4, 40.0% bone fill. (C) Group 1B/12V, Sample #22: bone score = 3, 10.9% bone fill. (D) Group 0.5B/0V, Sample #31: bone score = 2, 3.1% bone fill. (E) Group 0.5B/6V, Sample #41: bone score = 3, 15.1% bone fill. (F) Group 0.5B/12V, Sample #49: bone score = 4, 35.0% bone fill. Bars represent 2 mm.

FIG. 6.

Average bone score within the defect at 12 weeks as measured by blinded observers assessing maximum intensity projections of micro-CT datasets. Values are given for bone score of the groups performed by Patel et al. (Groups 0B/0V, 0B/12V, 2B/0V, and 2B/12V) and this current study (Groups 1B/0V, 1B/6V, 1B/12V, 0.5B/0V, 0.5B/6V, and 0.5B/12V). Error bars represent means ± standard deviation for n =  8–9. Statistically significant differences (p < 0.05) are denoted by asterisks (*).

FIG. 7.

Representative histological sections of samples from (A) Group 1B/0V, (B) Group 1B/6V, (C) Group 1B/12V, (D) Group 0.5B/0V, (E) Group 0.5B/6V, and (F) Group 0.5B/12V. Note the pores in all groups are mainly filled with fibrous tissue, with a minimal amount of inflammation or bone formation. P, PPF scaffold; B, new bone. Bar represents 200 μm for all panels.

FIG. 8.

Average score for (A) the hard tissue response at the scaffold–bone interface, and (B) the tissue response within the scaffold pores within the defect at 12 weeks as measured by blinded observers assessing histological sections. Values are given for bone score of the groups performed by Patel et al. (Groups 0B/0V, 0B/12V, 2B/0V, and 2B/12V) and this current study (Groups 1B/0V, 1B/6V, 1B/12V, 0.5B/0V, 0.5B/6V, and 0.5B/12V). Note for Figure 8B that a significant difference also exists between Groups 1B/0V and 0.5B/0V. Error bars represent means ± standard deviation for n = 8–9. Statistically significant differences (p < 0.05) are denoted by asterisks (*).