Controlled Release of Bone Morphogenetic Protein-2 Augments the Coupling of Angiogenesis and Osteogenesis for Accelerating Mandibular Defect Repair

Abstract

Reconstruction of a mandibular defect is challenging, with high expectations for both functional and esthetic results. Bone morphogenetic protein-2 (BMP-2) is an essential growth factor in osteogenesis, but the efficacy of the BMP-2-based strategy on the bone regeneration of mandibular defects has not been well-investigated. In addition, the underlying mechanisms of BMP-2 that drives the bone formation in mandibular defects remain to be clarified. Here, we utilized BMP-2-loaded hydrogel to augment bone formation in a critical-size mandibular defect model in rats. We found that implantation of BMP-2-loaded hydrogel significantly promoted intramembranous ossification within the defect. The region with new bone triggered by BMP-2 harbored abundant CD31+ endomucin+ type H vessels and associated osterix (Osx)+ osteoprogenitor cells. Intriguingly, the new bone comprised large numbers of skeletal stem cells (SSCs) (CD51+ CD200+) and their multi-potent descendants (CD51+ CD105+), which were mainly distributed adjacent to the invaded blood vessels, after implantation of the BMP-2-loaded hydrogel. Meanwhile, BMP-2 further elevated the fraction of CD51+ CD105+ SSC descendants. Overall, the evidence indicates that BMP-2 may recapitulate a close interaction between functional vessels and SSCs. We conclude that BMP-2 augmented coupling of angiogenesis and osteogenesis in a novel and indispensable way to improve bone regeneration in mandibular defects, and warrants clinical investigation and application.

Keywords: angiogenesis; bone morphogenetic protein-2; hydrogel; mandibular defect; osteogenesis.

Figure 1

Fabrication and characterization of BMP-2-loaded hydrogel. (A) Schematic chart showing the synthesis of BMP-2-loaded GelMA hydrogel. (B,C) Quantitative analysis of the degradation rate and swelling ratio of GelMA and BMP-2-loaded GelMA hydrogels. (D) Representative SEM image of the microstructure of GelMA and BMP-2 loaded GelMA hydrogels. Scale bar 25 μm.

Figure 2

Implantation of BMP-2-loaded hydrogel promoted bone regeneration in mandibular defects of rats. (A) Schematic chart of the study design. (B) Trichrome staining of mandibular bone. Scale bar, 250 μm. The black dashed line indicates the border of the new bone and old bone. NB: new bone, OB: old bone. (C) Quantitative analysis of the ratio of new bone to old bone (NB/OB). (D) Reconstructed 3D images of the defect in mandibular bone. Scale bar, 750 μm. (E) Quantitative analysis of bone volume/total tissue volume (BV/TV), trabecular number (Tb.N), bone mineral density (BMD), and trabecular thickness (Tb.Th). The p value was calculated by one-way ANOVA and Dunn’s multiple comparisons test.

Figure 3

BMP-2 enhanced angiogenesis and growth of type H vessels after implantation during the mandibular defect repair. (A) Immunofluorescent staining of type H vessels (CD31+ Emcn+) in the mandibular bone. Scale bar, 50 μm. The white arrow indicates the type H vessels. (B) Quantitative analysis of the area fraction of type H vessels in the new bone. The p value was calculated by one-way ANOVA and Dunn’s multiple comparisons test.

Figure 4

BMP-2 increased the number of osterix (Osx)+ osteoprogenitor cells coupled with vessels after implantation during the mandibular defect repair. (A) Immunofluorescent staining of Osx+ osteoprogenitor cells in the mandibular bone. Scale bar, 50 μm. The white arrow indicates the Osx+ osteoprogenitor cells. The hollow arrow indicates the vessels. (B) Quantitative analysis of the number of Osx+ osteoprogenitor cells in the new bone. The p value was calculated by one-way ANOVA and Dunn’s multiple comparisons test.

Figure 5

Implantation of BMP-2-loaded hydrogel facilitated the growth of CD51+ skeletal stem cells (SSCs) and their descendants during the bone regeneration of mandibular defects. (A) Immunofluorescent staining of CD51+ cells in the mandibular bone. Scale bar, 50 μm. The white arrow indicates the CD51+ SSCs and descendants. The hollow arrow indicates the vessels. (B) Quantitative analysis of the number of CD51+ cells in the new bone. The p value was calculated by one-way ANOVA and Dunn’s multiple comparisons test.

Figure 6

The fraction of CD51+ CD105+ SSCs descendants was elevated after implantation of BMP-2-loaded hydrogel in the newly regenerated bone. (A,C) Representative images and quantitative analysis of immunofluorescent staining of CD51+ CD200+ SSCs in the new bone. Scale bar, 50 μm. The white arrow indicates the CD51+ CD200+ SSCs. The hollow arrow indicates the vessels. (B,D) Representative images and quantitative analysis of immunofluorescent staining of CD51+ CD105+ SSCs descendants in the new bone. Scale bar, 50 μm. The white arrow indicates the CD51+ CD105+ SSCs descendants. The hollow arrow indicates the vessels. The p value was calculated by one-way ANOVA and Dunn’s multiple comparisons test.