Depot injectable biodegradable nanoparticles loaded with recombinant human bone morphogenetic protein-2: preparation, characterization, and in vivo evaluation

Abstract

Objective: The aim of this study is to utilize the biocompatibility characteristics of biodegradable polymers, viz, poly lactide-co-glycolide (PLGA) and polycaprolactone (PCL), to prepare sustained-release injectable nanoparticles (NPs) of bone morphogenetic protein-2 (BMP-2) for the repair of alveolar bone defects in rabbits. The influence of formulation parameters on the functional characteristics of the prepared NPs was studied to develop a new noninvasive injectable recombinant human BMP-2 (rhBMP-2) containing grafting material for the repair of alveolar bone clefts.

Materials and methods: BMP-2 NPs were prepared using a water-in-oil-in-water double-emulsion solvent evaporation/extraction method. The influence of molar ratio of PLGA to PCL on a suitable particle size, encapsulation efficiency, and sustained drug release was studied. Critical size alveolar defects were created in the maxilla of 24 New Zealand rabbits divided into three groups, one of them treated with 5 μg/kg of rhBMP-2 NP formulations.

Results: The results found that NPs formula prepared using blend of PLGA and PCL in 4:2 (w/w) ratio showed the best sustained-release pattern with lower initial burst, and showed up to 62.7% yield, 64.5% encapsulation efficiency, 127 nm size, and more than 90% in vitro release. So, this formula was selected for scanning electron microscope examination and in vivo evaluation. Histomorphometric analysis showed 78% trabecular bone fill, mostly mature bone in the defects treated with rhBMP-2 in NPs within 6 weeks.

Conclusion: The prepared NPs prolonged the release and the residence time of rhBMP-2 in rabbits, which led to the formation of adequate bone in critical size alveolar bone defects in 6 weeks. This noninvasive method has application for the primary restoration of alveolar bone defects.

Keywords: BMP-2; PCL; PLGA; alveolar defects; biodegradable polymers; nanoparticle.

Figure 1

SEM images of rhBMP-2 nanoparticles. Abbreviations: SEM, scanning electron microscopy; rhBMP-2, recombinant human bone morphogenetic protein-2; DDL, Dynamic data links.

Figure 2

Release of rhBMP-2 from nanoparticles prepared with various PLGA:PCL ratios. Abbreviations: rhBMP-2, recombinant human bone morphogenetic protein-2; PLGA, poly lactide-co-glycolide; PCL, polycaprolactone.

Figure 3

Plasma levels of BMP-2 after local injection of 5 μg/kg of BMP-2 isotonic saline into the created alveolar bone defects in rabbits (group A). Abbreviation: BMP-2, bone morphogenetic protein-2.

Figure 4

Photomicrograph of a bone defect in group A (H&E stain, at 4× magnification). Abbreviations: H&E, hematoxylin and eosin; px, pixels.

Figure 5

Plasma level of BMP-2 after injection of isotonic saline into the created defects in the rabbits (group B). Abbreviation: BMP-2, bone morphogenetic protein-2.

Figure 6

Photomicrographs of a bone defect in group B (slides stained with H&E stain, at 4 × magnification (A) and 10 × magnification (B)). Abbreviations: H&E, hematoxylin and eosin; px, pixels.

Figure 7

Plasma level of BMP-2 after injection of 5 μg/kg of rhBMP-2 nanoparticles into the surgically created alveolar bone defects in the rabbits (group C). Abbreviations: BMP-2, bone morphogenetic protein-2; rhBMP-2, recombinant human BMP-2.

Figure 8

Photomicrographs of a bone defect in group C (slides stained with H&E stain, at 10 × magnification (A), 20 × magnification (B), and 40 × magnification (C)). Abbreviation: H&E, hematoxylin and eosin.