Sustained dual release of placental growth factor-2 and bone morphogenic protein-2 from heparin-based nanocomplexes for direct osteogenesis

Abstract

Objective

To compare the direct osteogenic effect between placental growth factor-2 (PlGF-2) and bone morphogenic protein-2 (BMP-2).

Methods

Three groups of PlGF-2/BMP-2-loaded heparin–N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) nanocomplexes were prepared: those with 0.5 μg PlGF-2; with 1.0 μg BMP-2; and with 0.5 μg PlGF-2 combined with 1.0 μg BMP-2. The loading efficiencies and release profiles of these growth factors (GFs) in this nanocomplex system were quantified using enzyme-linked immunosorbent assay, their biological activities were evaluated using cell counting kit-8, cell morphology, and cell number counting assays, and their osteogenic activities were quantified using alkaline phosphatase and Alizarin Red S staining assays.

Results

The loading efficiencies were more than 99% for the nanocomplexes loaded with just PlGF-2 and for those loaded with both PlGF-2 and BMP-2. For the nanocomplex loaded with just BMP-2, the loading efficiency was more than 97%. About 83%–84% of PlGF-2 and 89%–91% of BMP-2 were stably retained on the nanocomplexes for at least 21 days. In in vitro biological assays, PlGF-2 exhibited osteogenic effects comparable to those of BMP-2 despite its dose in the experiments being lower than that of BMP-2. Moreover, the results implied that heparin-based nanocomplexes encapsulating two GFs have enhanced potential in the enhancement of osteoblast function.

Conclusion

PlGF-2-loaded heparin–HTCC nanocomplexes may constitute a promising system for bone regeneration. Moreover, the dual delivery of PlGF-2 and BMP-2 appears to have greater potential in bone tissue regeneration than the delivery of either GFs alone.

Keywords:

• placental growth factor-2
• bone morphogenic protein-2
• heparin
• nanocomplexes
• osteogenesis

Supplementary material

Figure S1 TEM images of heparin–HTCC nanocomplexes before and after PlGF-2/BMP-2/dual-GF loading.

Notes: (A) Blank heparin-nanocomplexes; (B) BMP-2-loaded heparin-nanocomplexes; (C) PlGF-2-loaded heparin-nanocomplexes (scale bar =200 nm).

Abbreviations: TEM, transmission electron microscopy; HTCC, N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride; PlGF-2, placental growth factor-2; BMP-2, bone morphogenetic protein; GF, growth factor.

Acknowledgments

This study was supported by the Natural Science Foundation of Guangdong Province, People’s Republic of China (S2013010015805), as well as Guangdong Science and Technology Projects (2014A010105025). We thank all the research staff members at the Department of Oral Implantol-ogy, Guanghua School of Stomatology and the Institute of Polymer Science, Sun Yat-sen University.

Disclosure

The authors report no conflicts of interest in this work.