| BackgroundWith rapid development of modern transportation, cases of bone defect caused by various trauma are increasing. Moreover, bone tumor, infection and deformity also become the main reason of bone defect. All these need artificial bone or bone substitute to overcome the problem. Nowadays, the most common method of bone defect treatment is autogenous bone implantation, but the donor's limitation and related complications restrict its application and increase the suffering of patients. On the other hand, allogeneic bone has great antigenicity, espeasially when it was used in large bone defect, severe immune rejection will result in surgery failure. Further, allogenic bone has limited resource and potential problem of disease spreading and medical ethics, which restrict its clinical application. Therefore, in order to overcome the shortcomings of autogenous and allogenic bone in clinical application, the artificial bone material replacing the nature bone become the urgent demands increasingly.Bone tissue engineering have become a hot issue in the orthopedic research these days. Tissue engineering is the use of a combination of cells, engineering and materials methods, and suitable biochemical and physio-chemical factors to improve or replace biological functions. It was proposed by American National Scienee Foundation (NSF) in 1987 for the first time. Then, Crane put forword the scientific significance, research methods, current status and prospects of bone tissue engineering systematically, which aroused general concern. In 1996, International Union of Materials Research Societies pointed out two strategies of bone tissue engineering:(1) the scaffold and cytokines were assembled in vitro and then implanted into the body, to induce new bone formation; (2) implant osteoblasts-loaded scaffold into the body.Research on bone growth factors have been one of the most active subject in the field of bone tissue engineering. In respect that exogenous growth factor can significantly enhance cell proliferation and differentiation, delay cell aging and stimulate tissue repair and regeneration, it has become a focus of tissue engineering research. However, directly application of growth factors in body results in inactivation due to rapid dilution and metabolism or enzyme digestion. In recent years, researches on active growth factor released carrier transfer from traditional carrier to microsphere carrier, which prolongs the release time of growth factor. With the development of controlled-release drug delivery and targeted drug delivery technology, sustained release of growth factors is being increasingly widespread researched. Therefore, nanospheres are introduced, which will accelerate the development and utilization of exogenous growth factors and become an important research interest of tissue engineering. According to the current researches, bone morphogenetic proteins (BMP) are bone growth factors with the most positive osteogenic activity. US Food and Drug Administration Home (FDA) authorized the application of recombinant human bone morphogenetic protein-2 (rhBMP-2) for spinal fusion in July 2002. However, exogenous rhBMP-2 is not stable and has short half-life in vivo and poor biological membrane permeability. Thereby, the application of microsphere release system to release peptide is one of the ways to solve the problem.Polymer drug carries can be divided into two categories:synthetic polymer carrier (polylactic acid, polycaprolactone, polyacrylate, etc.) and natural polymers carrier (gelatin, cellulose, chitosan, etc.). However, synthetic polymer carriers have poor biocompatible, and sometimes contain trace amounts of impurities resulting in cytotoxicity, such as organic tin catalyst added in production process of polylactic acid. As a natural polymer without those drawbacks, chitosan is produced commercially by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans (crabs, shrimp, etc.) and cell walls of fungi. It is not only biocompatible and biodegradable with nontoxic degradation products, but also have anti-bacterial, bleeding, inhibition of cancer metastasis. Therefore, chitosan microspheres have obvious advantages as drug carriers than the microspheres made of other materials. (1) Surface of chitosan microspheres has abundant functional groups, which can absorb or entrap different characteristic drugs; (2) Chitosan microspheres have good adhesion which are suitable for oral, nasal, gastrointestinal and other mucosal administration, especially has unique advantages of antigen and adjuvant delivery; (3) Chitosan microsphere has rich polysaccharide chains which can be recognized by specific cells (or tissue), resulted in sustained release of drugs; (4) Chitosan can influence cell function of F-actin and open cell channels, helping to improve penetration of drugs into the cells instantly. Therefore, since the 90s of 20th century, chitosan microspheres carrier of drugs has become a hot track in the new formulations research. Jong reported that TGF-β1 loaded chitosan microspheres prepared by emulsification crosslinking method, were embedded into porous collagen-chitosan skeleton with cartilage cells. After 3-days culture, its amount of mucopolysaccharide and cartilage cells was significantly higher than that of TGF-β1 solution group, indicated that TGF-β1 loaded chitosan microspheres stimulated the proliferation of cartilage cells. In summary, the research on various types of chitosan microspheres and nanoparticles as drug release and targeting carrier, has launched a wide range all over the world.Recently, the research on bone growth factors loaded chitosan microspheres mainly focus on its property, release pattern, induction and differentiation effect of cells in vitro. However, chitosan microspheres are micron-sized, and the studies on bone growth factors loaded chitosan nanospheres is few. Furthermore, the studies on osteogenic activity of drug-loaded nanospheres compounded to the artificial bone are much fewer. Therefore, this paper has a certain innovation and significance.Objective1. To prepare the rhBMP-2 loaded chitosan nanospheres and study its physical, chemical and pharmacological properties, including loading rate, entrapment efficiency and release kinetics.2. To evaluate the biocompatibility of rhBMP-2 loaded chitosan nanospheres, which further lay the foundation of bone formation activity research.3. To analyze the differentiation from rat bone mesenchymal stem cells to osteroblasts induced by rhBMP-2 loaded chitosan nanospheres, and to assess its osteogenic activity in vitro.4. To evaluate the ectopic bone formation activity of rhBMP2-loaded chitosan nanospheres in vivo.5. To prepare the compound of rhBMP-2 loaded chitosan nanospheres and coralline hydroxyapatite and study its bone inductive effect.Methods1. RhBMP2-loaded chitosan nanospheres was prepared by ionic crosslinking method with TPP as a crosslinking agent. Then the morphological properties and particle size distribution were evaluated by transmission electron microscopy (TEM) and particle size analyzer. Its loading rate, entrapment efficiency and release kinetics was analyzed by ELISA.2. Mouse fibroblast L929 was thawed and cultured with DMEM high glucose medium (containing 10% fetal bovine serum). The cytotoxicity test of rhBMP-2 loaded chitosan nanospheres comparing chitosan nanospheres with no rhBMP-2 was carried out referring to GB/T 16175-2008:Biological Evaluation Test Methods For Medical Organic Silicon Materials.3. Rat bone mesenchymal stem cells (BMSC) were cultured with DMEM low glucose medium (containing 10% fetal bovine serum). The rhBMP-2 loaded chitosan nanospheres were added to BMSC to co-culture, with the same dose of rhBMP-2 as control group. Then the proliferation and differentiation of BMSC induce by rhBMP-2 loaded chitosan nanospheres were analyzed by MTT method and ALP activity assay.4. Ectopic bone formation test in Sprague Dawley (SD) rat was implemented to evaluate the ectopic bone formation activity of rhBMP-2 loaded chitosan nanospheres. RhBMP-2 loaded chitosan nanospheres and the same dose of rhBMP-2 as control group were implanted intramuscularly respectively. Then, gross observation, X-ray examination, histological observation, ALP activity assay and calcium content test were performed after 4 weeks.5. RhBMP-2 loaded chitosan nanospheres were loaded into the coralline hydroxyapatite (CHA) by vacuum freeze-drying method. The rat posterolateral intertransverse process spinal fusion model was established, and rhBMP-2 chitosan nanospheres loaded CHA was implanted between L4～L5 transverse process. Finally, fusion rate was detected by Micro CT scan, three-dimensional reconstruction and palpation, and its bone formation activity was assessed by ALP,Ca2+ and histological analysis.Results1. The rhBMP-2 loaded chitosan nanospheres were prepared successfully. It was spherical in shape with a smooth surface and well distributed. The average particle diameter was 230 nm, the entrapment efficiency was (66.867±4.575)% and the loading rate was 33.437±2.290μg/mg. Degradation rate of rhBMP-2 loaded chitosan nanospheres was (57.567±2.759)% after 45 days. Its release of rhBMP-2 was biphasic kinetics, while the initial phase was the burst release phase and the latter phase was sustained release phase, which could sustainedly release rhBMP-2 up to 30 days. The degradation rate at 30 days was (90.133±3.564)%.2. Cytotoxicity test on mouse fibroblast cells L929 showed that the cytotoxicity grades of different dilution of extraction of rhBMP-2 loaded chitosan nanospheres group and chitosan nanospheres group were 0 or 1, indicated both had no cytotoxicity.3. Cytotoxicity test and ALP activity assay on rat bone mesenchymal stem cells demonstrated that rhBMP-2 loaded chitosan nanospheres and rhBMP-2 in the same dose significantly stimulated the proliferation and differentiation of BMSC. However, rhBMP-2 loaded chitosan nanospheres had greater activity than rhBMP-2 (P<0.05).4. Results of ectopic bone formation test indicated that rhBMP-2 loaded chitosan nanospheres and rhBMP-2 both had significant ectopic bone formation activity after 4 weeks, while the blank group and negative group had no ectopic bone formation activity. However, both the ALP and Ca2+ contents of rhBMP-2 loaded chitosan nanospheres group were larger than those of rhBMP-2 group with significant difference (P＜0.05), which indicated that the former had better ectopic bone formation activity.5. The rat posterolateral intertransverse process spinal fusion model was successfully established.The fusion rate of rhBMP-2 chitosan nanospheres-loaded CHA group and rhBMP-2 loaded CHA group were 100%, but both the ALP and Ca2+ contents of rhBMP-2 chitosan nanospheres-loaded CHA group were larger than those of rhBMP-2 loaded CHA group with significant difference (P<0.05),CHA group and negative control group did not observe the fusion.Conclusion1. Chitosan nanospheres prepared by ionic crosslinking method could be used as the efficient carrier of rhBMP-2, with the advantage of simple process, smooth surface, good stability, favorable drug loading rate, encapsulation rate and sustained drug release and degradation rate. Therefore, it provides a new approach and new ideas for further application of rhBMP-2 loaded chitosan nanoparticles to repair bone defects in bone tissue engineering.2. GB/T 16175-2008:Biological Evaluation Test Methods For Medical Organic Silicon Materials is the criterion for cytotoxicity test of organic materials and polymers which are used to implant into humane body, and the MTT method is the gold standard of cytotoxicity test in vitro. Both point out that the results are reliable. Results showed that the cytotoxicity grades of rhBMP-2 loaded chitosan nanospheres were 0 or 1, indicated that it had potential good biocompatibility in vivo.3. RhBMP-2 loaded chitosan nanospheres could significantly stimulated the proliferation and differentiation of BMSC when it was co-cultured with BMSC, which indicated that rhBMP-2 loaded chitosan nanospheres had good osteoblast-induced activity in vitro and could be used as a carrier of bone growth factors to continue further studies in vivo osteogenic activity. 4. Results of intramuscular implantation test proved that rhBMP-2 loaded chitosan nanospheres and rhBMP-2 both had significant ectopic bone formation activity while the blank group and negative group had no ectopic bone formation activity. However, both the ALP and Ca2+ contents of rhBMP-2 loaded chitosan nanospheres group were larger than those of rhBMP-2 group with significant difference. These showed a good sustained drug release of chitosan nanospheres in vivo, which resulted in better ectopic bone formation activity.5. Results of spinal fusion test in rat showed that rhBMP-2 chitosan nanospheres loaded CHA had perfect fusion effect, indicated that rhBMP-2 loaded chitosan nanospheres compounded with CHA decelerate the drug release rate and improved osteogenic activity. These further laid a solid experimental and theoretical basis of large animal research and clinical application.
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