| BackgroundAbnormal bone regeneration can cause the change of bone structure. Congenital malformation, trauma, cancer, osteoporosis, fracture, sinus lift, and osseointegration involve bone regeneration. It is very important for clinical practice to study the process of bone regeneration. Bone regeneration is a part of bone metabolism. Bone metabolism includes both bone formation and bone absorption, normally they are in dynamic balances. When bone absorption accelerates or bone formation reduces, osteoporosis will occur eventually. Epidemiological survey showed that in the world more than one hundred million people suffered from osteoporosis and the incidence of osteoporosis among women over 60 years old was 25%-50%.In USA, about 1.5 million people suffered from fracture caused by osteoporosis every year and most of them were women. In our country, with the aging of the population, osteoporosis has become a serious problem, and the incidence is increasing. Previous studies mainly focused on stopping bone absorption and preventing the loss of bone. However, it is no useful for the situation in which a great amount of bone has been lost. Therefore, most of the researches mainly concerned bone formation at present. The methods of new bone formation include autogenous bone graft, bone substitutes, guided bone regeneration, genetic engineering, and cell factor addition. In general, they have some shortcomings of technical complexity, high cost, long period and biological unsafety.Simvastatin, an HMG-COA reductase inhibitor and a new drug for hypercholesterolemia treatment in recent 20 years, is used to control the cholesterol level in practice and to prevent cardiovascular disease. Through animal experiments in 1999, Mundy found that among 3 million natual compounds, simvastatin was the only unique medicine which could promote bone metabolism. Simvastatin can enhance the activities of BMP-2 promoter and ALP accordingly promotes the formation of the mineralized nodules and bone. Thunyakitoisal PD found that simvastatin could decrease bone absorption by restraining the expression of MMP-9 of osteoblasts. Simvastatin have many advantages such as cheap, simple, convenient to get and so on.Increasing attention has been paid on the application of simvastatin to promote bone formation. However, the clinical application of simvastatin is confined because of general low bioavailability due to poor solubility.Amphiphilic block polymer consists of hydrophilic and oleophylic monomers. Solubilizing the insoluble drugs has become the hot topic of pharmaceutics in recent years. PLA has satisfactory biocompatibility and biodegradability. The intermediate metabolite of PLA is lactic acid, the end products are carbon dioxide and water.The end products do not accumulate in body. FDA authenticated PLA as a control release drug delivery, still it has the disadvantages like poor hydrophilicity, low degradation rate, difficult to control the degradation cycle. PEG has good hydrophilicity and biocompatibility, when the degree of polymerization is below 5000, it can be excreted from kidney and would not accumulate in body. The FDA has approved its safety and it can avoid the reaction with immunoglobulin, escape the captures of RES, liver, spleen, kidney, etc. PEG has a long blood circulation time. MePEG-PLA, one hydrophilic and oleophylic segmented copolymer, is one of the transmission system nanometer drug and has good biocompatibility and degradability. It can degrade into nontoxic PEG and PLA, and be excreted from kidney. When the concentration in water is more than critical micelle concentration, it will self-assemble, form the nanometer nuclear shell that nuclear produced from lyophobic part, nuclear composed of hydrophilic part. The nuclear packs the hydrophilic drug, while nuclear stops the interference from outside and prevents adsorption of immune globulin, avoid the catch of RES. So it can circulate in blood for a long time. MePEG-PLA has smaller size that it can pass physiology protective screen. It has special distribution in vivo that it can achieve passive targeting and it has the merit of surface modification easily to realize active targeting drug delivery dose.This thesis explored the method of producing simvastatins-loaded MePEG-PLA nanoparticles and studied the effects of simvastatins-loaded MePEG-PLA nanoparticles on the proliferation and differentiation of osteoblasts. The aim is to enhance the effect of simvastatins on the differentiation of osteoblasts by finding a more suitable drug-carrier. Objective1 To study and select the method of preparing simvastatins-loaded MePEG-PLA nanoparticles.2 To study the effects of simvastatins-loaded MePEG-PLA nanoparticles on the proliferation and differentiation of osteoblasts. Methods1) The critical micelle concentration (CMC) of MePEG-PLA was characterized by pyrene. MePEG-PLA-NPs were successfully prepared by the modified spontaneous emulsification solvent diffusion method (modified-SESD), which was selected on the basis of particle size and polydispersity. MePEG-PLA concentration in organic solvent, the volume ratio of oil and water and the volume ratio of acetone and ethanol were used as factors to optimize the technology of preparation through the orthogonal test design. The nanoparticles were characterized by dynamic light scattering technique for size, size distribution and Zeta Potential, TEM for morphological properties, ultraviolet spectrophotometry method for drug encapsulation efficiency and drug loading efficiency.2) The physical states of drug and MePEG-PLA in nanoparticles were investigated by fourier transform infra-red spectroscopy (FT-IR) and differential scanning calorimetry (DSC).3) To study the effects of simvastatins-loaded MePEG-PLA nanoparticles on MG63 cells proliferation and differentiation, the experiment was divided into five groups:normal contol, simvastatin, blank MePEG-PLA nanoparticles, simvastatins-loaded MePEG-PLA nanoparticles, DMSO. The activities of proliferation, alkaline phosphatase (ALP) activities, BMP-2(Bone morphogenetic protein-2), ALP and osteocalcin (OC) gene expression of MG63 cells were detected with MTS, ALP kit, fluorescent real-time PCR, respectively. Results1) The critical micelle concentration (CMC) of the copolymer was determined to be 0.588mg/L. The particle size, polydispersity and Zeta Potential of simvastatins-loaded MePEG-PLA nanoparticles which prepared by modified-SESD method were around 28.7±3.6nm,0.242 and-8.06mV. The drug loading efficiency was (6.23±0.75)% and drug encapsulation efficiency was (37.78±1.31)%. The morphology of nanoparticles exhibited a fine spherical shape with smooth surfaces without aggregation or adhesion observed by TEM.2) The spectrum of FT-IR and DSC indicated that simvastatins were entrapped in nanoparticles and a little were adsorbed on the surface of nanoparticles. 3) The effects of simvastatins-loaded MePEG-PLA nanoparticles on MG63 cells proliferation and differentiation. MTS outcome:compared with control group, both blank MePEG-PLA nanoparticles group and DMSO group have no statistical significance (P>0.05), both simvastatins-loaded MePEG-PLA nanoparticles group and simvastatin group have statistical significance (P<0.05),there is statistical significance between simvastatins-loaded MePEG-PLA nanoparticles group and simvastatin group(P<0.05). both blank MePEG-PLA nanoparticles group and DMSO group have no effect on the proliferation of MG63 cells, the proliferation of MG63 cells were inhibited in both simvastatins-loaded MePEG-PLA nanoparticles group and simvastatin group and the proliferation of MG63 cells were inhibited more heavily in simvastatins-loaded MePEG-PLA nanoparticles group. The outcome of ALP activity:Compared with control group, both blank MePEG-PLA nanoparticles group and DMSO group have no statistical significance (P>0.05), both simvastatins-loaded MePEG-PLA nanoparticles group and simvastatin group have statistical significance (P<0.05), there is statistical significance between simvastatins-loaded MePEG-PLA nanoparticles group and simvastatin group (P<0.05). both blank MePEG-PLA nanoparticles group and DMSO group have no effect on the ALP activity of MG63 cells, the ALP activity of MG63 cells was enhanced in both simvastatins-loaded MePEG-PLA nanoparticles group and simvastatin group and the ALP activity of MG63 cells were enhanced more greatly in simvastatins-loaded MePEG-PLA nanoparticles group.The outcome of ALP,OC and BMP-2 mRNA expression:Compared with control group, both blank MePEG-PLA nanoparticles group and DMSO group have no statistical significance(P>0.05), both simvastatins-loaded MePEG-PLA nanoparticles group and simvastatin group have statistical significance (P<0.05), there is statistical significance between simvastatins-loaded MePEG-PLA nanoparticles group and simvastatin group(P<0.05). both blank MePEG-PLA nanoparticles group and DMSO group have no effect on the ALP, OC and BMP-2 mRNA expression of MG63 cells, the ALP, OC and BMP-2 mRNA expression of MG63 cells was enhanced in both simvastatins-loaded MePEG-PLA nanoparticles group and simvastatin group and the ALP, OC and BMP-2 mRNA expression of MG63 cells were enhanced more greatly in simvastatins-loaded MePEG-PLA nanoparticles group. Conclusion1) Simvastatins-loaded MePEG-PLA nanoparticles were easily prepared by modified-SESD method. The morphology of nanoparticles exhibited a fine spherical shape with smooth surfaces without aggregation or adhesion. The solubility of simvastatin was greatly enhanced.2) Simvastatin was entrapped in nanoparticles and a little was adsorbed on the surface of nanoparticles.3) Polymer nanoparticles as drug delivery system can improve the effects of simvastatins on osteoblasts proliferation and differentiation. |
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