Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects

Abstract

We previously showed that the repair of bone defects is regulated by neural and vascular signals. In the present study, we examined the effect of topically applied β-nerve growth factor (β-NGF) on neurogenesis and angiogenesis in critical-sized bone defects filled with collagen bone substitute. We created two symmetrical defects, 2.5 mm in diameter, on either side of the parietal bone of the skull, and filled them with bone substitute. Subcutaneously implanted osmotic pumps were used to infuse 10 μg β-NGF in PBS (β-NGF + PBS) into the right-hand side defect, and PBS into the left (control) defect, over the 7 days following surgery. Immunohistochemical staining and hematoxylin-eosin staining were carried out at 3, 7, 14, 21 and 28 days postoperatively. On day 7, expression of β III-tubulin was lower on the β-NGF + PBS side than on the control side, and that of neurofilament 160 was greater. On day 14, β III-tubulin and protein gene product 9.5 were greater on the β-NGF + PBS side than on the control side. Vascular endothelial growth factor expression was greater on the experimental side than the control side at 7 days, and vascular endothelial growth factor receptor 2 expression was elevated on days 14 and 21, but lower than control levels on day 28. However, no difference in the number of blood vessels was observed between sides. Our results indicate that topical application of β-NGF promoted neurogenesis, and may modulate angiogenesis by promoting nerve regeneration in collagen bone substitute-filled defects.

Keywords: angiogenesis; collagen; nerve regeneration; neural regeneration; protein gene product 9.5; vascular endothelial growth factor; β III-tubulin; β-nerve growth factor.

Figure 1

Creation and repair of bone defects. (A) Trephine drill; (B) incision; (C) exposed cranium; (D) two critical-sized bone defects in the parietal bone; (E) placement of osmotic pumps into subcutaneous pockets; (F) defects filled with bone substitute; (G) fixation of pumps; (H) cementation of spacers; (I) sutured incision.

Figure 2

Prepared (empty) osmotic pump used for drug infusion into the bone defect.

Figure 3

Effect of topical application of β-NGF neurogenesis in collagen bone substitute-filled defects. (A–C) Immunohistochemistry for β III-tubulin (A), NF160 (B), and PGP9.5 (C) at bone defect site (× 400; positive immunoreactivity appears brown). (D) Quantitation of β III-tubulin, PGP9.5 and NF160 immunoreactivity in collagen bone substitute-filled defects. All data are expressed as the mean ± SD (n = 6). *P < 0.05, vs. PBS side (paired t-test). β-NGF: β-Nerve growth factor; PGP9.5: protein gene product 9.5; NF160: neurofilament 160; AOD: average optical density; d: days.

Figure 4

Effect of topical application of β-NGF on angiogenesis in collagen bone substitute-filled defects. (A) Blood vessels in the bone defect during repair (hematoxylin-eosin staining, × 400). (B) Number of blood vessels at the bone defect site increased on both sides until days 7–14, and diminished thereafter. All data are expressed as the mean ± SD (n = 6). β-NGF: β-Nerve growth factor; d: days.

Figure 5

Effect of topical application of β-NGF on VEGF and VEGFR-2 immunoreactivity in collagen bone substitute-filled defects. (A, B) Immunohistochemistry for VEGF (A) and VEGFR-2 (B) at the bone defect site (× 400; positive immunoreactivity appears brown). (C) Quantitation of VEGF and VEGFR-2 immunoreactivity in collagen bone substitute-filled bone defects. All data are expressed as the mean ± SD (n = 6). *P < 0.05, vs. PBS side (paired t-test). β-NGF: β-Nerve growth factor; VEGF: vascular endothelial growth factor; VEGFR-2: VEGF receptor 2; AOD: average optical density; d: days.