Osteogenic Effect And Mechanism Of Novel Nanostructured Lipid Carriers Containing Simvastatins For Bone Regeneration

In recent years, the technique of dental implant has become the main repair method for the patients with dentition defect or edentulous. But long-term missing teeth caused by caries or periodontitis could result in serious vertical and horizontal resorption of alveolar ridge, weight loss or even reached 50% of the original bone width; some trauma or cranial and maxillofacial tumors could lead to loss of teeth when accompanied with large alveolar crest defect; or patients with congenital malformations or genetic defects have inherent teeth and alveolar bone insufficiency; and in recent years, the increasing incidence rate and prevalence rate of population aging and intensified osteoporosis, leading to the decreased bone mineral density and the accelerated bone resorption. The volume of alveolar bone and the quality of bone are the key factors of the success of implant restoration. Therefore, how to solve the problem of bone defect, promote bone tissue regeneration, and restore the morphology and function of the jaw bone have become hot topics in recent years.Bone regeneration surgery up to 130 million in the globe annually, and treatment technologies from autologous bone graft, the same allogenic bone transplantation, xenogeneic bone tissue transplantation, new bone replacement grafts to bone tissue engineering technology have been studied and reported largely. But there are various problems with these treatment technologies, such as:complex technology, high cost, long cycle, biological safety, etc. Therefore, looking for a simple, inexpensive and safe way to promote bone regeneration is a direction in the field of bone regeneration. Some scholars have found that simvastatins, small molecule drugs, could induced autologous bone marrow mesenchymal stem cell differentiation into bone cells and promote the expression of BMP-2 osteogenic factor, therefore, the application of simvastatin in the field of bone tissue regeneration has become a focus in current research.Simvastatin (SV), a kind of medicine for reducing cholesterol and antilipemic agents commonly used in clinic is an inactive small molecule and a kind of A (HMG-COA) reductase inhibitor. After entering the human body, SV is converted into the active body by active enzyme in the liver cells and then inhibit the synthesis function of the alpha hydroxy acid. Since 1999 Mundy first found that statin drugs (simvastatin and lovastatin) can promote the expression of BMP-2 gene in bone cell from more than 3000 kinds of clinical drug, more and more focused researches on the osteogenetic effects of simvastatin have been done. The studies have shown that simvastatin has good osteogenesis, including promotion of BMSCs to migrate into the bone defect site, the differentiation of bone cells and angiogenesis and inhibition of osteoclast. But after oral administration of simvastatin, only less than 5% of the drug into the systemic circulation, which lead to lower effective drug concentration arrival at the position of the bone tissue. Besides, large dose application of SV would cause rhabdomyolysis and other complications. Thus, how to improve the availability of simvastatin and increase the local drug concentration in the acting cites are improtant problems to be solved.Drug delivery system is the combination of new nano technology and clinical drug optimization, which possess good therapeutic effect, reduce the adverse effect, achieve drug release and targeting, and could avoid drug premature diffusion and removal. Nano carrier system has been widely used to improve biological utilization degree of insoluble drugs, and there are various nano carrier forms, such as polymer nanoparticles, nano suspension, liposome, microemulsion, lipid nanoparticles, micelles. Lipid nanoparticles, consist of natural or synthetic lipids, can improve the solubility of insoluble drugs effectively, especially suitable for the biopharmaceutics classification system (BCS) in class II (low solubility, high permeability drugs) and class IV (low solubility, low permeability); can effectively increase the transmembrane ability; has strong selectivity to the target concentrated in the reticuloendothelial system, specifically targeted to liver and spleen; nanoparticles can also extend the residence time of the drug in the blood and be acted as the drug storage; non-toxic and non immunogenic, easy to be absorbed by the body; to avoid the use of toxic surface active agent and organic solvent; multiple route of administration function and simplisitic surface modification, and has good market prospects Etc. The second generation of lipid nanoparticles-nanostructured lipid carriers (nanostructured liquid carriers, NLC) is the mixture of liquid lipid (such as oleic acid, castor oil, medium chain fatty acid glycerol ester) and solid lipid. Due to the involvement of liquid lipid, NLC can significantly improve the drug loading ability and stability by disrupting the solid lipid originally ordered structure and reducing the prone to crystal transition and crystallization. Some researchers found that NLC loaded with simvastatin could obviously slow delayed drug release compared with pure simvastatin suspension. Studies have shown that the bioavailability of lipid nanoparticles loaded with SV was 2.55 times higher in the rat intestinal absorption than that of free SV.But the preparation of nano lipid carriers were adopted by solvent injection method or the method of ultrasonic emulsification which could introduce some organic solvents and remained in the final product. At the same time, it has not been reported about the osteogenic effect of SNs recently.Therefore, in order to find a better drug delivery system for improving the bioavailability of simvastatin, promoting the osteogenic effect of simvastatin, and provide a better solution and theoretical basis for clinical bone tissue regeneration, we fabricated nanostructured lipid carrier loaded with simvastatin and studied its osteogenic effect and mechanism in vitro and in vivo. In this study, the proposed new micro jet technology (Microfluidization), which could avoid organic solvent used and the operation is simple, was applied for preparing nanostructured lipid carrier carrying simvastatin. To verify the osteogenic effect and the related osteogenic mechanism of nanostructured lipid carriers carrying simvastatin, MG-63 cells were used in this study. And to further verify the osteogenic properties and mechanism of bone formation of nanostructured lipid carriers containing simvastatin, the critical bone rabbit skull defect will be established in this research.However, there is no report about nanostructured lipid carrier contained simvastatin for bone regeneration. Thus, we hypothesized that compared to the same content simple simvastatin nanostructured lipid carriers containing simvastatin has good in vitro release ability and it is more effective osteogenic effect of nanostructured lipid carriers loaded with simvastatins than free simvastatin in vitro and in vivo for bone regeneration.Objectives;(1) To find a better drug delivery system for improving the bioavailability of simvastatin;(2) To verify the more effective osteogenic effect of nanostructured lipid carriers loaded with simvastatins than free simvastatin in vitro and in vivo for bone regeneration.Methods:Part Ⅰ:synthesis, detection and optimization of the nanostructured lipid carriers loaded with Simvastatin1. Preparation and optimization of blank NLCWe prepared SV-loaded NLCs (SNs) using a microfluidization method. We chose oleic acid (OA) as the liquid lipid, stearic acid (SA) as the solid lipid and poloxamer 188 (P188) as the surfactant. The effects of the concentrations of OA (wt%,0.10 to 0.40) and P188 (w/v%,0 to 0.40) and the number of circulations in the microfluidizer (times,1 to 4) on the particle size, PI and zeta potential were then investigated and optimized using the BBD.2. Characteristics of the nanostructured lipid carriers loaded with SimvastatinBased on the optimized results for the response surfaces, we prepared SNs with three theoretical DLCs (5%,10% and 20%) (SV/NLCs, wt%). And particle size analysis, morphology, encapsulation efficiency and package sealing rate measurement and the in vitro release test were detected in this study. Among them, particle size, polydispersity index and the zeta potential were measured by Malvern laser particle size analyzer; morphology was observed by transmission electron microscopy; drug package encapsulation efficiency and drug loading rate were detected by high performance liquid phase chromatograph; in vitro release experiment by dialysis method.Part II:The in vitro osteogenic effect of nanostructured lipid carriers containing simvastatins for bone regeneration1. Cell culture of MG-63 cellThe MG-63 cells strain was recovered and were cultured under standard culture conditions (37℃ and 5% CO2) according to the manufacturer’s recommendations. The culture medium was refreshed every other day. daily observation, record their growth, proliferation.2. Effects of nanostructured lipid carriers containing simvastatins on osteoblast proliferation and differentiation.Experimental groups:SNs at doses ranging from 2.5 x 10-6 to 2.5 x 10-9 M; the equivalent concentrations of free SV served as the positive controls, and a blank served as the negative control. The effect of simvastatin loaded nanostructured lipid carriers on cell proliferation (MTS method), ALP activity, cells mineralization ability in vitro (Alizarin red staining) and the expression of OC protein and OC mRNA in MG-63 cells were detected.Part III:The in vivo osteogenic effect of nanostructured lipid carriers containing simvastatins for bone regeneration1. Establishment of animal model and specimen preparationTwenty New Zealand White rabbits (15-16 weeks,2.8-3.3 kg) were used in this experiment. The experimental protocol was approved by the Institutional Committee for Animal Care at Southern Medical University. All surgical procedures were performed under sterile conditions. A perpendicular incision was made, and a mucoperiosteal flap was raised. Four critical bone defects (8 mm in diameter) were then created and randomly divided into the following 4 groups:Group 1:No augmentation (blank control, BC); Group 2:Bone substitute (BS); Group 3:Bone substitute with 0.5 mg of SV (SVBS); and Group 4:Bone substitute with 2.5 mg of nanoparticles (0.5 mg SV) (SNBS). The animals were sacrificed 4 weeks after surgery. To label the bone, the fluorochromes calcein was then subcutaneously injected at a concentration of 10 mg/ml (10 mg/kg body weight) 3 and 4 days before euthanizing the animals. The specimens were removed en bloc and soaked in 4% buffered formaldehyde.2. Histomorphometric analysis(1) After one half of the specimens were completely fixed, they were demineralized with 14% EDTA for 4 weeks. They were then dehydrated in a graded ethanol (70-100%) series, cleared with xylene and embedded in paraffin. All the samples were sectioned into 4-μm-thick serial sections for hematoxylin and eosin (HE) or Masson’s trichrome staining, the ALP activity and an immunohistochemical analysis for osteocalcin (OC); all samples were stained in triplicate. The triplicate sections were separately evaluated by two blind observers with a digital microscope and a digital camera. The results were evaluated using image-analysis software (Image-Pro Plus 6.0).(2) One half of the samples were dehydrated with a graded series of ethanol concentrations before being embedded in resin and sliced into 30-μm-thick sections. Fluorescence microscopy images of the calcified sections were then taken with a confocal microscope using the appropriate filters. The newly formed mineralized bone in the defect area was observed using ZEN 2009 Light Edition software based on the grey values. Finally the non-decalcified 30-μm-thick cuts were stained with both toluidine blue. And the newly formed mineralized bone were calculated with the Image-Pro Plus 6.0 software.Statistical analysisSPSS 19.0 statistical analysis software for data analysis. The data for normality test analysis, and the results with x±s if the data with normal distribution. Then the data were analyzed with single factor variance analysis (one way ANOVA) or ANOVA for factorial design (general linear model). At the same time, variance homogeneity test was tested by Levene’s test method, further pairwise comparison (Bonferroni test) was done if the variance is homogeneous; if variance not neat, the approximate f Welch test method and further multiple comparison (tamhane’T2 method). If the information does not accord with normal distribution, Kruskal-Wallis H nonparametric test were adopted and further Mann-Whitney U test for pairwise comparison. Standard test a=0.05.Results:(1) In this study, NLCs were successfully developed using a microfluidization method to generate an appropriate delivery system for loading SV. In addition, the effects of the composition of the NLCs were investigated and optimized using the BBD. The recommended optimal preparation conditions for nanoparticles using the BBD were shown to be as follows:the concentration of OA was 40%(wt%), the concentration of P188 was 0.4%(w/v%), and the number of circulations in the microfluidizer was 4. Based on the optimized results for the response surfaces, we prepared SNs with three theoretical DLCs (SV/NLCs, wt%). The mean diameter of the SNs was 174.7 nm, encapsulation efficiency (EE) was up to 94.1% and actual drug-loading capacity (DLC) reach up to 18.2. The in vitro drug release results for the nanoparticles with the three theoretical DLCs showed obvious and prolonged controlled release from the lipid carriers compared with free SV, approximately three times that of the corresponding free SV suspension.2. To test the hypothesis that the biologic effect of SNs would be greater than that of the equivalent concentrations of free SV, a cellular uptake study was performed, and the osteogenic effects were evaluated by cytology. We found that the uptake of SNs by MG-63 cells occurred via endocytosis and the fluorescence intensity directly increased with concentration and time at 37℃. The next cell proliferation test conducted in our study suggested that SNs and SV had positive effects at low concentrations and negative effects at a relatively high concentration. SN showed better effective effect on the proliferation of MG-63 cells not only in a dose-dependent manner but also in a time-dependent manner. The inhibitory effect of a high dose of SV could be reduced by the controlled and sustained release of SV from nanoparticle formulations. ALP activity was obviously up-regulated by the SNs, with an activity level far superior to that induced by the equivalent concentration of free SV. The formation and maturation of mineralized calcium nodules in the later period of osteoblast differentiation were also promoted by SNs, especially at 2.5 × 10-7 M and 2.5×10-8 M SV. Moreover, the expression of the OC gene and protein was dose-dependently and more strongly enhanced by the SNs, particularly in the SN group with an SV concentration of 2.5 ×10-7M.3. To investigate the in vivo stability, host reactions and the early bone regeneration capability of SNs, a critical-sized bone defect (CSD) model was created in rabbit calvarias in our present study. Our results revealed that the SNs significantly enhanced bone formation in vivo, as evaluated by hematoxylin and eosin (HE) staining, immunohistochemistry, and a fluorescence analysis. The SNs exhibited good stability and effectively induced osteogenesis to form mostly woven bone. The results of a histomorphometric analysis revealed that new bone covered only 4.13±0.71% of the defect area in the BC group, The new bone area ratio increased to 10.38±0.52% with the addition of 0.5 mg SV and further increased to 16.26±0.59% in the SN group, which contained 0.5 mg SV, indicating an abundance of newly formed bone in the defect area after 4 weeks without any inflammation in both test groups. Moreover, the histology and fluorescence microscopy images indicated that some irregular and asymmetric bone had formed at the defect edge in all groups at 4 weeks. Some samples had small bony islands in the central area of the bone defect, away from the defect edge. Furthermore, the Masson trichrome staining results demonstrated that the local application of SNs can significantly improve collagen regeneration and neovascularization in the bone defect at the early stages. Specifically, the expression of the OC protein was enhanced to a greater extent by the SNs, and the immunohistochemical analysis revealed that the formation of new bone positively correlated with the number of OC-stained cells, which was significantly higher in the SNBS group than in the SVBS group after 4 weeks (P<0.05).Conclusion:1. SNs with a higher EE and DLC were successfully prepared using a microfluidization method, at the same time, SNs possess prior physical and chemical properties. The in vitro cumulative profiles of SV release from the SNs demonstrated controlled release.2. The proliferation and differentiation effect of SNs was dose-dependent and was more effective on MG-63 cells compared with the equivalent free SV. The results confirmed that the nanostructured lipid carrier loading simvastatin can promote osteogenesis effect through the mechanism of promotion of osteoblast proliferation and differentiation similar to simvastatin, but the throughout medicine of the effect of nanostructured lipid carrier loading simvastatin on osteogenesis need further research.3. The local application of SNs contained with 0.5mg SV can significantly improve bone regeneration at the early stages of bone healing in a critical-sized bone defect (CSD) model.