PLAGA/TCP Scaffolds Loaded With RhBMP-2 Chitosan Microspheres For Treatment Of Bone Defects In Rabbits

Bone defect is one of the unsolved clinical problems. With thedevelopment of tissue engineering, bridging bone defects with tissue engineeredbone grafts showed great promise. However, the osteoinductive efficacy oftissue engineered grafts is relatively low, which has been a factor limiting theapplication of tissue engineered bone grafts. rhBMP-2 is one of the mostimportant cytokines which play a key role in osteogenesis and bone modeling.In the present study, rhBMP-2 was loaded into the chitosan microspheres, whichcan slowly release rhBMP-2 for a long period of time. The rhBMP-2 loadedchitosan microspheres were then incorporated into PLGA/TCP scaffolds toconstruct a novel artificial bone with osteoinductive property. The artificial bonewas expected to promote osteogenesis when bridging bone defects.1. Preparation of rhBMP-2 loaded chitosan microsphere and its propertiesObjective: To prepare and characterize a novel controlled release system forrhBMP-2 and to evaluate the feasibility of chitosan microsphere as carriers forcontrolled release of rhBMP-2.Methods: The rhBMP-2 loaded chitosanmicrosphere was prepared using an emulsion cross-linking method with genipin as the cross-linking agent. Scanning electron microscopy was used to evaluatethe morphological properties and particle size distribution. An ELISA assay wasadopted to determine the actual drug loading and in vitro drug release kinetics.ResultsResults: The rhBMP-2 loaded chitosan microspheres were spherical in shape,and showed a smooth surface and a high encapsulation efficiency (>85%). Fromthe in vitro release experiment, the rhBMP-2 was controlled release from thechitosan microsphere over 30 days. Conclusions: The rhBMP-2 loaded chitosanmicrosphere prepared by emulsion cross-linking method shows a goodcontrolled release property. Such a novel type of controlled release system hasthe potential of being applied for controlled cytokine delivery and the bonetissue engineering.2. rhBMP-2 Chitosan microspheres and osteogenic capacity of ForeignMinister capacitive detectionObjective: To investigate the biocompatibility of chitosan microsphere withcultured bone marrow stromal cells (BMSCs), and to evaluate the capability ofrhBMP-2 loaded microsphere in inducing the differentiation of BMSCs toosteoblasts. Methods: BMSCs were obtained from the male New Zealandrabbits with ages of 2 to 4 weeks. The third passage of BMSC was used and fourgroups were included in the present study. A group (blank control group):BMSCs were cultured with DMEM supplemented with 10% fetal bovine serum(FCS); B group: BMSCs were co-cultured with DMEM supplemented withchitosan microsphere in which nothing was incorporated; C group: BMSCs wereco-cultured with DMEM supplemented with rhBMP-2 (0.2μg/ml); D group:BMSCs were co-cultured with DMEM supplemented with rhBMP-2 loadedchitosan microsphere (0.2μg/ml). Three days after co-culture, the apoptosis rateof BMSCs in the A and B groups were detected using flow cytometry. Seven and 14 days after incubation, the cells in A, C and D groups were examined byALP and alizarin bordeaux staining. ResultsResults: The apoptosis rate of BMSCs in Aand B groups were in the similar range, indicating that the chitosanmicrospheres were non-toxic to BMSCs. The ALP was positive in both C and Dgroups, and the intensity of ALP was similar in C and D groups. The alizarinBordeaux was also positive in both C and D groups, and the number of calciumnodus in D group was significantly than that in C group.Conclusions: Thechitosan microspheres prepared by an emulsion cross-linking method showedgood compatability, and the degradation products were non-toxic to BMSCs.The rh-BMP2 loaded chitosan microspheres can release bioacitive rhBMP-2gradually, which can induce the differentiation of BMSCs to osteoblasts.3. PLGA/TCP scaffolds loaded with rhBMP-2 chitosan microspheres fortreatment of bone defects in rabbitsObjective: To investigate the feasibility and efficacy of using the PLGA/TCPscaffolds loaded with rhBMP-2 chitosan microspheres to treat bone defects inrabbits.Methods: The rhBMP-2 chitosan microspheres were prepared using anemulsion cross-linking method. An ELISA assay was adopted to determine theactual drug loading and in vitro drug release kinetics. The rhBMP-2 chitosanmicrospheres were incorporated into the PLGA/TCP scaffold. The composites ofrhBMP-2 microspheres and the PLGA/TCP were then used to bridge the bonedefects (diameter: 0.6 cm, depth: 1.0 cm) at the condyle of the left femur inrabbits. Altogether 45 rabbits were randomized into 5 groups in the present study.In group A (n=10) the defects were not treated. The PLGA scaffold was used ingroup B (n=10), the rhBMP-2/ PLGA scaffold in group C (n=10), and therhBMP-2 micorsphere / PLGA scaffold in group D (n=10). Group E (n=5) wasthe normal control. X-ray, Micro-CT and histological studies were conducted to identify new bone formation in groups A to D at 4 and 12 weeks aftersurgery.ResultsResults:The rhBMP-2 chitosan microspheres showed a highencapsulation efficiency (>85%) and the rhBMP-2 was released from thechitosan microspheres for over 30 days. At 4 weeks postoperatively, plenty newbone and mature trabecular bone were observed, and most of the PLGA/TCPscaffolds were degraded in group D. The ratio of new bone formation wassignificantly higher in group D (42.63%±8.52%) than in group C(23.95%±5.28%, p=0.034) and group B (2.63%±0.71%, p<0.050), and alsosignificantly higher in group C than in group B. At 12 weeks postoperatively, thePLGA/TCP scaffolds and chitosan microspheres were completely degraded, andthe bone defects were filled with mature bone in group D. In addition, the ratio ofnew bone formation was significantly higher in group D (74.25%±8.91%) than ingroup C (37.26%±6.45%, p=0.021) and group B (5.78%±1.21%, p<0.050).Micro-CT examination at 12 weeks showed that the indices of bone mineraldensity, bone volume fraction, trabecular thickness, trabecular number andtrabecular spacing in group D were significantly higher than in groups B and C(P<0. 0001). Such indices were not observed in group A. Conclusion: ThePLGA/TCP composites loaded with rhBMP-2 microspheres are effective instimulating new bone formation when used to bridge bone defects in rabbits,highlighting their potential application in clinical settings.