| BackgroundBone defects are clinical complications that surgeons often faced with, which are mostly caused by trauma, infection and tumor resection. Infected nonunion are often caused by trauma and improper handling of serious emergency, leading to bone defects, which make the treatment more difficult. Muscle atrophy, osteoporosis, joint stiffness are most common complications of this disorder. Other complications including pain and physical dysfunction bring not only great physical and psychological burden on patients, but also the heavy financial burden.Due to the poor outcome of treatment in post-traumatic bone defects, amputation was the ultimate treatment of such diseases in the past. Cancellous bone grafting for treatment of bone defects is a major breakthrough. Bone transporting and skeletal traction is mainly used for 2-10cm bone defects, but complications including delayed fracture healing and a long treating time still exist.Free bone flap is used for defects of 5-12cm, but the common clinical complicationsare graft instability and postoperative re-fracture. Bone substitutes are another hot areas which are still under research, but still can not meet the need of post-traumatic bone defect. Above all, although the traditional methodcan promote healing of bone defects in some extent, there still exist the difficulty in management, longer treatment period required repeated surgery and other complications, which will limit the wide spread of the above methods.Bone Tissue engineering is a major breakthrough in solving the problem of grafting materials. There is no core technology succeeding in translating engineering bone into autologous bone by now. Therefore, it is necessary to find a new method to promote bone formation. BMP-2 is widely used in clinical treatment because it can effectively promote healing of fractures; But the high need, short supply, difficulty to prepare itself and the high priceseverely limit its widespread clinical application. As a major clinical lipid regulating drugs, simvastatin was found thatcan promote the expression of BMP-2 in osteoblasts, contributing to the healing of fractures. However, the application of free simvastatin is limited because the water-soluble rate is low and the drug is easy metabolized in the liver. Moreover, the side effect of the large dose of clinical applications and the cytotoxic problems produced by large dose oflocal injection still exist.Since the mechanism of RNAi was discovered, the technology of RNAi has been widely used in silencing a variety of target genes, which has broad prospects in the future. RNAi technology was used in this article to downregulate the expression of noggin genes in order to remove its inhibition of BMP-2. Therefore, this will promote the osteogenesis of MC3T3-E1 cells. As small molecules,the delivery of siRNA into cells also must be addressed at this stage.viral vectors were often used in the past, which showed a high efficiency of cell transfection but presented issues of biosafety. siRNA molecules were not very stable in vivo circulation. Moreover, they were easily swallowed by macrophages and degraded by nuclease. They are difficult to pass through the cell membrane which are with the negative charge because the large molecular weight and the same negative charge in itself and they can not paly the role of target gene silencing Therefore, a new and effective way must be found to promote its effect.This paper proposed a delivery technology of multi-functional nanocomplexes, to explore a clinically applicable, safety and economical way to the treatment of osteogenesis. First, according to the mechanism that the simvastatin-induced VEGF promotes vascularization and then induce the expression of BMPs, the application of simvastatin will promote osteogenesis; Secondly, RNA interference (RNAi) reduced the noggin, which is the antagonistic protein of BMPs. Therefore, simvastatin, in conjunction with noggin siRNA, will promote osteogenesis of MC3T3-E1 cells in two fields of gene transcription and protein interaction. However, the direct use of siRNA and simvastatin present problems of low treatment efficiency and easy degradation. So they are both hardly transmitted to the area of bone formation. Thirdly, the use of multifunctional nanocomplexes will transmitnoggin siRNA and simvastatin simultaneouslyto the targeted area to play the synergies for better promoting osteogenesis. It will also improvethe administration efficiency of simvastatin and reduce the dose of simvastatin to avoid the potential side effects.Therefore, we have designed a pH-sensitive triblock copolymermPEG-bPEI-PAsp (DIP-BA), which can be used for joint transmission of simvastatin and noggin siRNA. Hence, it will play the dual effect of drug and gene therapy. Simvastatin will induce the expression of BMP-2after acting on osteoblasts.Noggin, as specific antagonists of BMP-2, will antagonize osteogenesis process. The use of noggin siRNA can downregulate the elevated levels of noggininduced by BMP-2.The Synergy of drug and RNA interference therapeutics will enhance the effect in treating bone defects.In this paper, we investigate potential applications of the pH-sensitive triblock copolymer in the treatment of bone defects in vitro.PurposeThe purpose of this study was to design a pH-sensitive triblock copolymer and to explore its structure and traits,to study its ability in loading simvastatin and noggin siRNA, the toxic effects of pH-sensitive triblock copolymer without loading simvastatin in MC3T3-E1 cells; the proliferation of MC3T3-E1 cells by free simvastatin and copolymer loading simvastatin. Then, we study the endocytosis and localization of MC3T3-E1 cells incubating with triblock copolymer. Finally, we study the osteogenesis promoted by simvastatin and noggin siRNA loading by nanocomplexes at the level of gene and protein.Methods1. Synthesizing nanopolymer mPEG-bPEI-PAsp(DIP-BA) by chemical methods and to test and characterize the nanopolymer by 1H-NMR test.2. Brookhaven Instruments exploreparticle sizes and zeta potentials of nanocomplexes (B-PEM/BCL-2) prepared at various N/P ratios (n=5);3. TEM images and figures of Time-cumulative release amount to test simvastatin release from nanocomplex(SIM-mPBP/Noggin siRNA or SIM-mPBP) at pH 7.4 or 5.0;4. Agarose gels explore the electrophoretic mobility of noggin siRNA after complexing with B-mPBP or D-mPBP at various N/P ratios;5. CCK8 method explorethe cytotoxicity of the nanocomplex at different concentration in MC3T3-E1 cells; CCK8 method explore the proliferation or cytotoxicity of free simvastatin at different concentration in MC3T3-E1 cells; CCK8 method explore the proliferation of nanocomplex at different concentration of simvastatin in MC3T3-E1 cells;6. Confocal laser scanning microscopy (CLSM) and flow cytometry test the intracellular absorption of nanocomplex;7. Real-time PCR analysis explore the efficacy of various concentration and various time(concentration of SIM=luM) of mPBP/SCR in inducing BMP-2/reducing noggin expression in MC3T3-E1 cells at mRNA level, eal-time PCR analysis explore the efficacy of various concentration of noggin siRNA in inducing BMP-2 /reducing noggin expression in MC3T3-E1 cells at mRNA level;8. Real-time PCR analysis explorethe efficacy of mPBP/siRNA(concentration of SIM=1,10Um; concentration ofnoggin siRNA=120nM)in inducing BMP-2 /reducing noggin expression in MC3T3-E1 cells at mRNA level. We also further confirm the result of real-time PCR analysis by western blotting, ALP and Alizarin red staining.Results1. We successfully synthesized nanopolymer and 1H-NMR spectras show that nanopolymer was successfully synthesized by chemical methods;2. Particle size and zeta potential tested by Brookhaven Instruments show that nanocomplexes (SIM-mPBP@siRNA) have smaller Particle size(40 nn) and weak zeta potential(+8mV) when N/P ratios is 10;3. TEM images and test of in vitro simvastatin release show that the particle size distribution is about 35 nm, simvastatin at a slow release status; when pH 5.0, thenanocomplexes become aggregation and enlargement, simvastatin at a fast release status;4. Electrophoretic mobility of siRNA in agarose gel after complexing with B-mPBP and D-mPBP at various N/P ratios show that siRNA is fully compound withoutmobility when N/P ratios is 10.Whether nanocomplexes load with simvastatin do not affect its ability of loading with siRNA;5. CCK8 method shows that even when the concentration of nanocomplexes is up to 430 ug/mL(N/P ratios is 10), survival rate of MC3T3-E1 cells is still as high as 90.5%, a part of the positive is neutralized when nanocomplexes composite siRNA, reducing their toxicity to MC3T3-E1 cells;6. Free simvastatin have no proliferation or toxicityon MC3T3-E1 cell at Different concentrations.nanocomplexes loading simvastatin have a certain effect on cell proliferation at the concentration of less than lOuM and inhibit cell proliferation at the concentration of 1 OuM7. Confocal laser scanning microscopy (CLSM) images and flow cytometry show nanocomplexescan be efficientlyendocytosed by MC3T3-E1 cells,the transfection efficiency is up to 86.4%. Simvastatin and siRNA slowly release when nanocomplexes were into the lysosome; Simvastatin and siRNA started to release at 10min,released more at 30min, most at 60min, completely releasedat 120min.8. Real-time PCR analysis show that the concentration of luM and the time of 48h were the best contributing factors. When the concentration of noggin siRNA was higher, the inhibition for noggin was better,120 nM is the bestconcentration for noggin inhibition.9. When the concentration of noggin siRNA is 120 nM, the best concentration of simvastatin for promoting osteogenesis is luM, which is confirmed by western blotting, ALP and Alizarin red staining.ConclusionThe purpose of this article is to explore the application of pH-sensitive triblock copolymer in the treatment of bone defects. We successfully synthesized PEG-g-bPEI-g-PAsp(DIP-BA) and test the potential and particle size of nanocomplexes by TEM images and Brookhaven Instruments.Our results show that we have successfully synthesizeda nanocomplex which has smaller particle size(about 40 nm) and pH-sensitive core,presenting uniform distribution.This nanocomplex can avoid quickly being ruled out by the kidneys and uptake by the RES. On the other hand, it also increased the water solution of simvastatin, prolonged blood circulating time of this drug, therefore, improved its therapeutic effect.The biological experimental results strongly show it will promote osteogenesis when simvastatin and noggin siRNA are simultaneously transmitted into MC3T3-E1 cells by pH-sensitive triblock copolymer. Simvastatin will promote the high rate of BMP-2 expression, which also induce the expression of noggin. The upregulation of noggin may be effectively reduced by noggin siRNA, thereby reducing the effect of inhibiting osteogenesis, the synergism of simvastatin and noggin siRNA will better promoted osteogenesis.The results further show that nanocomplex provide a good platform for the development of the treatment of bone defects and bone tissue engineering. |
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