Loading BMP-2 on nanostructured hydroxyapatite microspheres for rapid bone regeneration

Abstract

Introduction: Tissue engineering is a promising strategy for bone regeneration in repairing massive bone defects. The surface morphology of implanted materials plays a key role in bone healing; these materials incorporate osteoinductive factors to improve the efficiency of bone regeneration.

Materials and methods: In the current study, nanostructured hydroxyapatite (nHAp) micro-spheres were prepared via a hydrothermal transformation method using calcium silicate (CS) microspheres as precursors; the CS microspheres were obtained by a spray-drying method. The nHAp microspheres constructed by the nano-whiskers significantly improved the ability of the microspheres to adsorb the bioactive protein (BMP-2) and reduce its initial burst release. To evaluate the in vivo bone regeneration of microspheres, both conventional hydroxyapatite (HAp) and nHAp microspheres were either loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) or not loaded with the protein; these microspheres were implanted in rat femoral bone defects for 4 and 8 weeks.

Results and discussion: The results of our three-dimensional (3D) micro-computed tomography (CT) and histomorphometric observations showed that the combination of the nano-structured surface and rhBMP-2 obviously improved osteogenesis compared to conventional HAp microspheres loaded with rhBMP-2. Our results suggest that the nHAp microspheres with a nanostructured surface adsorb rhBMP-2 for rapid bone formation; they therefore show the potential to act as carriers in bone tissue regeneration.

Keywords: BMP-2; bone regeneration; hydrothermal transformation; hydroxyapatite; nanostructure.

Figure 1

SEM morphologies of the HAp microspheres (A, B) and the nHAp microspheres obtained via hydrothermal treatment of the CS microspheres in Na₃PO₄ aqueous solution at 180°C for 24 h (C, D). Abbreviations: CS, calcium silicate; HAp, hydroxyapatite; nHAp, nanostructured Hap; SEM, scanning electron microscopy.

Figure 2

XRD patterns of the CS microspheres (A) and the nHAp microspheres obtained via hydrothermal treatment of the CS microspheres in Na₃PO₄ aqueous solution at 180°C for 24 h (B). Abbreviations: CS, calcium silicate; HAp, hydroxyapatite; nHAp, nanostructured hydroxyapatite; XRD, X-ray diffraction.

Figure 3

FTIR spectrum of the nHAp microspheres obtained via hydrothermal treatment of the CS microspheres in Na₃PO₄ aqueous solution at 180°C for 24 h. Abbreviations: CS, calcium silicate; FTIR, Fourier transform infrared; nHAp, nanostructured hydroxyapatite.

Figure 4

The amount of loaded rhBMP-2 on 1 g HAp and nHAp microspheres (A). Cumulative in vitro release curves of the HAp and nHAp microspheres over a period of 12 days (B). ^*Significant differences, P < 0.05. Abbreviations: HAp, hydroxyapatite; nHAp, nanostructured HAp; rhBMP-2, recombinant human bone morphogenetic protein-2.

Figure 5

3D micro-CT reconstruction of bone regeneration in the femoral bone defect from animals with the most new bone formation (A). The defect sites were analyzed to calculate the BMD (B) and the percentage of new BV relative to TV (BV/TV) (C) (n = 3 rats/batch). ^*Significant differences, P < 0.05. Abbreviations: BMP, bone morphogenetic protein; HAp, hydroxyapatite; nHAp, nanostructured HAp; BMD, bone mineral density; BV, bone volume; CT, computed tomography; 3D, three-dimensional; TV, tissue volume; w, weeks.

Figure 6

Histological images of newly formed bone in femoral bone defect at 4 and 8 weeks after operation (A). The percentage of new bone area assessed at 4 (B) and 8 weeks (C) after implantation by histomorphometric analysis. All the new bone area data were normalized to the percentage of maximum new bone area value. ^*Significant differences, P < 0.05; scale bar = 100 μm. ^**Significant differences, P < 0.01. Abbreviations: BMP-2, bone morphogenetic protein-2; HAp, hydroxyapatite; nHAp, nanostructured HAp; w, weeks.