Preparation of Coralline Hydroxyapatite Implant with Recombinant Human Bone Morphogenetic Protein-2-Loaded Chitosan Nanospheres and Its Osteogenic Efficacy.

OBJECTIVE: Spinal fusion is one of the most common surgical interventions for spine reconstruction. Despite the efforts to promote osteogenesis after spinal fusion, osteogenesis after spinal fusion remains a clinical challenge and new methods are still needed. The bone morphogenetic protein-2 (BMP-2) is a widely reported factor that can facilitate the osteogenesis in spinal fusion. In previous research, we found that the delivery of chitosan nanospheres could promote the effects of BMP-2 on osteogenic activity. The coralline hydroxyapatite (CHA) is one of the most frequently used implants in bone fusion. However, up to now no study has focused on the osteogenic efficacy of the CHA composite with recombinant human BMP-2 (rhBMP-2)-loaded chitosan nanospheres. This study aimed to investigate the effects of the CHA implant with rhBMP-2-loaded chitosan nanospheres on osteogenesis in spinal fusion. METHODS: The rhBMP-2-loaded microspheres and CHA composite (rhBMP-2 microspheres/CHA) were prepared and were used for implantation of the rats. All SD rats were divided into four groups: the rhBMP-2 microspheres/CHA composite group (containing 0.5 mg rhBMP-2), the rhBMP-2-loaded CHA (rhBMP-2/CHA) composite group (containing 0.5 mg rhBMP-2), the blank CHA group, and the negative control group. The microsphere morphology was scanned and analyzed using a scanning electron microscope. Micro-computed tomography examination and three-dimensional reconstruction were performed 4 weeks after the surgery. Hematoxylin and eosin staining was conducted for histological analysis. Both alkaline phosphatase (ALP) and calcium content were measured. RESULTS: The rhBMP-2-loaded CHA (rhBMP-2/CHA) composite was successfully prepared. Spherical regularity and a smooth and unwrinkled surface of the spheres were observed in all chitosan (CS)/rhBMP-2 microspheres. No side effects, infections, or abnormal behaviors were found in the animals. After 4 weeks of surgery, obvious new bone formation and bone fusion could be observed around the implant in both the rhBMP-2 microspheres/CHA composite group and the rhBMP-2/CHA composite group. No ectopic osteogenesis was found in the vertebral canal or other muscle tissues. After 4 weeks of implantation, in both the rhBMP-2 microspheres/CHA composite group and the rhBMP-2/CHA composite group, osteoid tissues could be found, and bone cells, bone marrow, and trabecular bone turned into mature sclerotin, obvious bone tissue formation could be also seen. Both ALP activity and calcium content in the rhBMP-2 microspheres/CHA composite group (6.52 ± 0.50 kat/g and 17.54 ± 2.49 µg/mg) were significantly higher than in all other groups. CONCLUSION: The composite with rhBMP-2-loaded CS nanospheres could enhance osteogenic efficacy and increase the ALP activity and calcium content. These results might provide a novel method for osteogenesis in spinal fusion and offer new insight into the role of BMP-2 in osteogenesis.

Effect of recombinant human bone morphogenetic protein delivered by chitosan microspheres on ectopic osteogenesis in rats.

In the present study, the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered by chitosan (CS) microspheres on ectopic osteogenesis was investigated in a rat model. rhBMP-2-loaded CS microspheres and blank CS microspheres were prepared. A total of 24 male Sprague Dawley rats were divided into 4 groups with 6 rats in each group: The CS/rhBMP-2 group, the rhBMP-2 group, in which rhBMP-2 was directly implanted (rhBMP-2 dose in either group, 1 mg), the CS blank group and the control group. X-ray was performed at 4 weeks after ectopic osteogenesis surgery and micro-computed tomography (CT) examination was scheduled at 1, 2, 3 and 4 weeks after the surgery to determine ectopic osteogenesis in the different groups. Histological analysis, and determination of alkaline phosphatase (ALP) activity and calcium content were also performed. The mean diameter of the osteoid tissues was 1.1±0.3 cm (range, 0.8-1.4 cm) in the CS/rhBMP-2 group, which was significantly bigger than that in the rhBMP-2 group (0.3±0.1 cm; range, 0.1-0.4 cm) at 4 weeks after the surgery. X-ray analysis and micro-CT scan indicated that the area of high-density tissues and the radionuclide intensity, as well as bone volume in the 3-dimensional reconstruction were greatest in the CS/rhBMP-2 group, followed by those in the rhBMP-2 group. All parameters, including bone mineral density, tissue mineral density, tissue mineral content and bone volume fraction, were significantly higher in the CS/rhBMP-2 group at 3 and 4 weeks after the surgery, compared with those in the rhBMP-2 group. The histological analysis, ALP activity analysis and determination of calcium content revealed that the CS/rhBMP-2 system had the greatest ability to induce osteoblast differentiation. In conclusion, the CS/rhBMP-2 microsphere delivery system significantly enhanced the induction and promotion effects of rhBMP-2 regarding ectopic osteogenesis. The present study enhances the basic data available for future application of the CS/rhBMP-2 microspheres delivery system and provides a deeper understanding of the role of BMP-2 in bone regeneration.

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

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.