| Background:Bone defect and bone non-union are the clinical common diseases, how to repair bone defect and promote bone healing is also an important content in the research of bone tissue engineering. Scaffolds, cells and factors are the three main elements of bone tissue engineering research. In recent years, the studies of bone growth factors were increasing, BMP-2 is one of the most important bone growth factors, the function of promoting bone repair has been confirmed by related researches. BMP-2’s level is very little in the body, expression of recombinant gene has realized the mass production of rh BMP-2, makes it possible for clinical application, but rh BMP-2’s half-life is very short and degraded quickly in the body, if rh BMP-2’s usage was increased a lot,it would bring a series of related complications, and its clinical application was seriously limited. Microspheres slow-release technology is one of the ways to solve this problem, after rh BMP-2 and microspheres were mixed together, not only can which protect the factor and extend it’s action time in the body, but also can reduce the complications and the cost of treatment at the same time. Therefore, the purpose of this research is to prepare a nano-carrier for rh BMP-2’s clinical application, makes it could be more widely used to cure bone diseases. Objective:1. To prepare recombinant human bone morphogenetic protein-2 loaded gelatin nanoparticle(rh BMP-2-GN), and to test its physicochemical properties and drug release characteristics. 2. To evaluate the biological influence of rh BMP-2-GN on BMSCs. 3. To offer the proper nano-carriers for the clinical application of rh BMP-2, and to provide the necessary basis and more reliable solutions for next experiments in animals. Methods:1. The preparation of rh BMP-2-GN and detections:(1) To prepare blank gelatin nanoparticles.(2) Scanning electron microscopy(SEM) was used to observe the surface morphology of gelatin nanoparticles and calculate its average particle size. Transmission electron microscopy(TEM) was used to observe the internal structure of gelatin nanoparticles. After absorbing water, Particle Size Analyzer was used to detect the average particle size and dispersity of GN, and its swelling ratio was calculated.(3) Recombinant human bone morphogenetic protein-2 and gelatin nanoparticles were mixed together to prepare rh BMP-2-GN, Enzyme-linked immunosorbent assay(ELISA) was used to measure the amount of rh BMP-2 which was loaded, its entrapment efficiency and drug loading were also calculated.(4) Rh BMP-2-GN was dispersed in the PBS and the cumulative percentage of sustained-release was caiculated.2. The biological effect of rh BMP-2-GN on bone marrow derived mesenchymal stem cells(BMSCs):(1) CCK-8 test was used to detect rh BMP-2-GN’s influence on the early proliferation of BMSCs.(2) Alkaline phosphatase(ALP) staining was used to detect rh BMP-2-GN’s influence on BMSCs’ s early differentiation.(3) Alizarin red S calcium nodules staining was used to detect rh BMP2-GN’s influence on BMSCs’ s terminal differentiation. Results:1. The preparation and detection of rh BMP-2-GN:(1) Blank gelatin nanoparticles were successfully prepared in two step desolvation method.(2) Physical properties of gelatin nanoparticles: Gelatin nanoparticles’ s surface was smooth, spherical structured, had a lot of small channels within, dispersed uniformly, gelatin nanoparticles of dry powder had an average particle size of 171.49 nm and the average particle size was 313.01 nm after swelling in water, the swelling rate of the gelatin nanoparticles was 1.83.(3) Rh BMP-2-GN was successfully prepared, its entrapment efficiency and drug loading was 98.13 ± 0.131% and 58.89± 0.079 ng/mg respectively.(4) Drug release time of rh BMP-2-GN was at least one month in vitro.2. The biological effect of rh BMP-2-GN on BMSCs:(1) The cell proliferation of BMSCs was better in rh BMP-2-GN group than the control group and the GN group on the 4th and 7th days(P < 0.05)(2) ALP staining of of BMSCs in the rh BMP-2-GN well was deeper than the control well and the GN well on 4th and 7th days, the difference was vey apparent.(3) Rh BMP-2-GN well have more calcium nodules than the control well and the GN well on the 28 th day,the difference was also very significant. Conclusions:1. " Two step desolvation " method was successfully used to prepare blank-gelatin nanoparticles, rh BMP-2-GN had a high entrapment efficiency and drug loading, and a good sustained-release effect in vitro.2. Rh BMP-2 was gradually released through the degradation of GN, the influence of which was prolonged, significantly promoted the early proliferation of BMSCs and BMSCs’ s differentiation to osteoblasts... |
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