Efficient delivery of recombinant human bone morphogenetic protein (rhBMP-2) with dextran sulfate-chitosan microspheres

Abstract

Bone morphogenetic protein-2 (BMP-2) serves an important role in the development of bone and cartilage. However, administration of BMP-2 protein alone by intravenous delivery is not very effective. Sustained delivery of stabilized BMP-2 by carriers has been proven necessary to improve the osteogenesis effect of BMP-2. The present study constructed a novel drug delivery system using dextran sulfate (DS)-chitosan (CS) microspheres and investigated the efficiency of the delivery system on recombinant human bone morphogenetic protein (rhBMP-2). The microsphere morphology, optimal ratio of DS/CS/rhBMP-2, and drug loading rate and entrapment efficiency of rhBMP-2 CS nanoparticles were determined. L929 cells were used to evaluate the cytotoxicity and effect of DS/CS/rhBMP-2 microspheres on cell proliferation. Differentiation study was conducted using bone marrow mesenchymal stem cells (BMSCs-C57) cells treated with DS/CS/rhBMP-2 microspheres or the control microspheres. The DS/CS/rhBMP-2 microspheres delivery system was successfully established. Subsequent complexation of rhBMP-2-bound DS with polycations afforded well defined microspheres with a diameter of ~250 nm. High protein entrapment efficiency (85.6%) and loading ratio (47.245) µg/mg were achieved. Release of rhBMP-2 from resultant microspheres persisted for over 20 days as determined by ELISA assay. The bioactivity of rhBMP-2 encapsulated in the CS/DS microsphere was observed to be well preserved as evidenced by the alkaline phosphatase activity assay and calcium nodule formation of BMSCs-C57 incubated with rhBMP-2-loaded microspheres. The results demonstrated that microspheres based on CS-DS polyion complexes were a highly efficient vehicle for delivery of rhBMP-2 protein. The present study may provide novel orientation for bone tissue engineering for repairing and regenerating bone defects.

Keywords: bone induction; chitosan; dextran sulfate; in vitro release; nanometer microsphere; preparation; recombinant human bone morphogenetic protein-2.

Figure 1.

(A) Scanning electron microscope images and (B) atomic force microscopy images of CS-DS/rhBMP-2 microspheres. CS, chitosan; DS, dextran sulfate; recombinant human bone morphogenetic protein 2.

Figure 2.

rhBMP-2 release profiles for CS-DS. Data are presented as the mean ± standard deviation (n=3). rhBMP-2, recombinant human bone morphogenetic protein 2. CS, chitosan; DS, dextran sulfate.

Figure 3.

Microscopic images of BMSCs-C57 in the (A) CS/DS/rhBMP-2, (B) rhBMP-2, (C) CS/DS (D) and control groups (magnification, ×200). CS, chitosan; DS, dextran sulfate; rhBMP-2, recombinant human bone morphogenetic protein 2.

Figure 4.

Trend graph of the ALP activity of bone marrow mesenchymal stem cells-C57 in each group. Data are presented as the mean ± standard deviation (n=4). A, CS/DS/rhBMP-2 microsphere group; B, rhBMP-2 group; C, CS/DS group; D, control group; ALP, alkaline phosphatase; CS, chitosan; DS, dextran sulfate; rhBMP-2, recombinant human bone morphogenetic protein 2; OD, optical density.

Figure 5.

Microscopic images of Alizarin Red staining displaying the formation of calcium nodules in BMSCs-C57 cells following osteogenic culturing for 14 days in (A) CS/DS/rhBMP-2 microsphere group, (B) rhBMP-2 group, (C) CS/DS group and (D) control group. Blue arrows indicate calcium nodules. Scale bars=2 µm. CS, chitosan; DS, dextran sulfate; rhBMP-2, recombinant human bone morphogenetic protein 2.

Figure 6.

Microscopic image of recovered sub-cultured L929 cells (magnification, ×200).

Figure 7.

Cytotoxicity influence on L929 cells in each group at different time points revealed by an MTT assay. Data are presented as the mean ± standard deviation. CS, chitosan; DS, dextran sulfate; rhBMP-2, recombinant human bone morphogenetic protein 2; OD, optical density.