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

Abstract

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.

Keywords: Chitosan nanospheres; Coralline hydroxyapatite implant; Osteogenesis; Spinal fusion; rhBMP-2.

Fig 1

Characterization of chitosan (CS)/recombinant human bone morphogenetic protein‐2 (rhBMP‐2) microspheres. The CS/rhBMP‐2 microspheres under transmission electron microscopy (TEM). Spherical regularity and smooth and unwrinkled surface of the spheres were observed in all microspheres.

Fig 2

Micro‐computed tomography (CT) evaluation for fusion of the coralline hydroxyapatite (CHA) composite. CT and three‐dimensional reconstruction for different groups. The red cycle showed the fusion place of the implant. New bone formation and bone fusion could be observed around the implant in both the recombinant human bone morphogenetic protein‐2 (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 tissue ×5.

Fig 3

Histological assay. Hematoxylin and eosin staining for tissues around the implant in different groups. Osteoid tissues, bone cells, bone marrow, trabecular bone turning into mature sclerotin, and bone tissue formation could be seen in both the recombinant human bone morphogenetic protein‐2 (rhBMP‐2) microspheres/coralline hydroxyapatite (CHA) composite group and the rhBMP‐2/CHA composite group. No bone tissue formation was observed around the implants in the CHA blank group and the negative group.