Dual delivery of BMP-2 and bFGF from a new nano-composite scaffold, loaded with vascular stents for large-size mandibular defect regeneration

Abstract

The aim of this study was to investigate the feasibility and advantages of the dual delivery of bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) from nano-composite scaffolds (PLGA/PCL/nHA) loaded with vascular stents (PLCL/Col/nHA) for large bone defect regeneration in rabbit mandibles. Thirty-six large bone defects were repaired in rabbits using engineering bone composed of allogeneic bone marrow mesenchymal stem cells (BMSCs), bFGF, BMP-2 and scaffolds composed of PLGA/PCL/nHA loaded with PLCL/Col/nHA. The experiments were divided into six groups: BMSCs/bFGF/BMP-2/scaffold, BMSCs/BMP-2/scaffold, BMSCs/bFGF/scaffold, BMSCs/scaffold, scaffold alone and no treatment. Sodium alginate hydrogel was used as the carrier for BMP-2 and bFGF and its features, including gelling, degradation and controlled release properties, was detected by the determination of gelation and degradation time coupled with a controlled release study of bovine serum albumin (BSA). AlamarBlue assay and alkaline phosphatase (ALP) activity were used to evaluate the proliferation and osteogenic differentiation of BMSCs in different groups. X-ray and histological examinations of the samples were performed after 4 and 12 weeks post-implantation to clarify new bone formation in the mandible defects. The results verified that the use of sodium alginate hydrogel as a controlled release carrier has good sustained release ability, and the combined application of bFGF and BMP-2 could significantly promote the proliferation and osteogenic differentiation of BMSCs (p < 0.05 or p < 0.01). In addition, X-ray and histological examinations of the samples exhibited that the dual release group had significantly higher bone formation than the other groups. The above results indicate that the delivery of both growth factors could enhance new bone formation and vascularization compared with delivery of BMP-2 or bFGF alone, and may supply a promising way of repairing large bone defects in bone tissue engineering.

Figure 1

In vitro degradation of sodium alginate hydrogel formed with 3% sodium alginate aqueous solution and 5% aqueous calcium chloride solution in phosphate buffer solution (PBS) for 25 days at 37 °C.

Figure 2

Cumulative release rate of bovine serum albumin (BSA) per time point from sodium alginate hydrogel.

Figure 3

Four groups of cell proliferation were analyzed by alamarBlue assay: C + S, BMSCs/scaffold; C + S + bFGF, BMSCs/bFGF/scaffold; C + S + BMP-2, BMSCs/BMP-2/scaffold; C + S + bFGF + BMP-2, BMSCs/bFGF/BMP-2/scaffold.

Figure 4

Changes in alkaline phosphatase (ALP) activity of BMSCs in all four groups on culture days 1, 3, 5, 7 and 14. * A value of p < 0.05 and ** A value of p < 0.01 were considered statistically significant.

Figure 5

Cumulative release rate of bFGF and BMP-2 per time point from sodium alginate hydrogel and nano-composite scaffolds (PLGA/PCL/nHA) loaded with vascular stents (PLCL/Col/nHA).

Figure 6

Tissue slices of different mandibular implants at weeks 4 and 12 stained with hematoxylin and eosin (H&E). a(a₁) Scaffold alone; b(b₁) BMSCs/scaffold; c(c₁) BMSCs/bFGF/scaffold; d(d₁) BMSCs/BMP-2/scaffold; e(e₁) BMSCs/bFGF/BMP-2/scaffold. B, bone; S, scaffold; O, osteoid matrix; NB, new bone; BV, blood vessel.

Figure 7

Tissue slices of different mandibular implants at weeks 4 and 12 stained with Masson staining. a(a₁) Scaffold alone; b(b₁) BMSCs/scaffold; c(c₁) BMSCs/bFGF/scaffold; d(d₁) BMSCs/BMP-2/scaffold; e(e₁) BMSCs/bFGF/BMP-2/scaffold; F, collagen fibers.

Figure 8

X-ray radiographs of large-sized mandibular defects repaired using different implants at week 12 post-implantation. (a) Scaffold alone; (b) BMSCs/scaffold; (c) BMSCs/bFGF/scaffold; (d) BMSCs/BMP-2/scaffold; (e) BMSCs/bFGF/BMP-2/scaffold.

Figure 9

The analysis of new bone density of large-sized mandibular defects repaired using different implants shown at week 12 post-implantation. (Mean ± SD; n = 3; * p < 0.05).