Characteristics And In Vivo Osteogenic Effect Of Simvastatin-containing MPEG-PLA Nanoparticles On Bone Regeneration

Background:One of the tremendous challenges in dental implantation is the treatment of patients with severely resorbed alveolar bone. Such resorption can reduce bone height and width, which makes dental implant placement in the appropriate sites unavailable. Over the years, multiple materials and techniques have been developed to solve this problem, such as augmentation of the sinus in the posterior maxilla, guided bone regeneration, split bone technique, autogenous bone blocks, and bone marrow derived mesenchymal stem cells (BMMSCs). But there were still a lot of disadvantages in preliminary study in material and techneque, for example,complex surgical procedures, a second surgical aera for alternative bone graft, expensive cost, etc.Our group has also studied nano-hydroxyapatite/coralline (nHA/coral) blocks that were pre-vascularized by coating them with vascular endothelial growth factor (VEGF) in a canine mandibular critical-size defect model in beagles. Recent studies have been shown that several small-molecule drugs have been explored as indirect alternative routes to promote osteogenesis. Simvastatin (SV),as small molecule drug mainly used clinically to decrease serum cholesterol levels, has been shown to enhance bone osteogenesis by enhancing the expression of BMP-2, mobilizing MSCs migration to bone defects, promoting angiogenesis, enhancing the expression of osteogenic factors in newly formed bone tissue and inhibiting osteoclast activity. However, SV is an inactive lactone drug and is poorly absorbed into the systemic circulation in less than 5% of the ingested dosage after oral administration. It has become a hotspot to search for a drug carrier to increase the water solubility of SV and to promote the osteogenesis of SV in the future.It is one of aims of this study to find a drug carrier which increase water solubility of SV and promote the osteogenesis of SV.Recent investigations have been shown that nanoscale polymeric micelles was characterised by their unique core-shell architecture and outstanding,which has possible biodegradability and high drug-loading efficiency and pleiotropic surface functionalization and especially the ability of promoting cell adhesion and targeting.Polymeric nanoparticles have been highlighted as advanced drug delivery systems in cancer therapy and in other fields,many preclinical and clinical studies of small molecular drugs based on biodegradable polymer nanoparticles have been performed.Monomethoxy poly(ethylene glycol)-poly(lactic acid) (MPEG-PLA) diblock copolymers, copolymerized polyethylene glycol (PEG) and poly(D,L-lactide) (PLA) have served as highly biocompatible subjects in drug delivery systems,MPEG-PLA copolymers possess a unique core-shell architecture (a hydrophobic inner core (PLA) and a hydrophilic outer shell (PEG)) and outstanding characteristics similar to those of other nanoscale polymeric micelles, such as biodegradability, controlled drug release, high drug-loading efficiency, high stability and prolonged circulation after systemic administration or local application. Osteogenesis of SMP in vitro has been confirmed in some research. However, few reports of the internal osteogenic effects of SV-loaded MPEG-PLA NPs are available, and the effects of these nanoparticles in a critical-sized rabbit calvarial defect have not yet been determined. According to the research progress at home and abroad, we propose the mpeg-pla nanoparticles as SV drug carrier, there may be due to changing the SV hydrophobic to hydrophilic and have sustained release characteristics, thereby increasing the SV local concentration effect; and because of the chemical and physical properties of nanoparticles have the potential to increase the SV osteogenesis effect and with animal experiments confirmed this hypothesis.Objective:We will construct SV-loaded MPEG-PLA NPs (SMP) using a modified-SESD method and invesgate its characteristics,and the other one aim of this investigation is that we administer SMP to the standard skull bone defects models of rabbit to explore osteogenic effect of SMP on bone regeneration in vivo.Methods:1. Preparation of blank or SV-loaded MPEG-PLA NPs by a modified-SESD method.The distribution of NP sizes, the polydispersity index (PI) and zeta position of the SMPs were measured by dynamic laser light scattering; The morphology of the nanoparticles was investigated using transmission electron microscopy (TEM); A fixed amount of lyophilized SMPs was completely dissolved in acetonitrile at a constant volume 10 ml and measured via ultraviolet spectrophotometry using an ultraviolet spectrophotometer to check the encapsulation ratio and drug-loading efficiency of SV encapsulated into the MPEG-PLA NPs; The physical states of drug and MPEG-PLA in nanoparticles were investigated by fourier transform infra-red spectroscopy (FT-IR) and differential scanning calorimetry (DSC).2.16 New Zealand white healthy male rabbits (24 weeks,3.0-3.5 kg) were used in this experiment. Preparation of the standard skull bone defects (diameters of 8mm and depth of 1mm) models of rabbit was performed. The four defects were randomly divided into the following groups which are SMP group, SV group, BO group and BC group.1.5 mg of nanoparticles containing 0.5 mg SV mixed with 5 mg BioOss(?) was administered to bone defects of models for SMP group;0.5 mg of SV mixed with 5 mg BioOss(?)for SV group; 5 mg BioOss(?) for BO group; blank control for BC group. The rabbits of each group were sacrificed 4 weeks after operation. No decalcified hard tissue grinding was used to observe fluorescent tags on bone graft areas by fluorescence microscope, rates of fluorescent tags on bone graft areas were measured.All samples were fixed in 4% formaldehyde for 24 h at room temperature. The new bone formation within the defect region was evaluated by CT and X-ray. The samples were cleared with xylene and embedded in paraffin. Next, 4-μm-thickness serial sections were performed and stained by hematoxylin and eosin (HE). all samples were stained by triplicate Masson’s trichrome. The expression of bone morphogenetic protein-2 (BMP-2) (osteoblast-specific factors and a marker of osteoblast differentiation) was measured by immunohistochemistryl. All of the experimental data were expressed as the means ± the standard deviation (SD). One-way ANOVA was used to analyze difference between groups. The homogeneity of the variance was evaluated using the Levene test.Results:In this study, monomethoxy poly(ethylene glycol)-poly(lactic acid) (MPEG-PLA, MP) was synthesized, and the osteogenic effect of SV-loaded MPEG-PLA (SMP) nanoparticles on critical-sized bone defect was evaluated in vivo. The average size of SMPs was 41.3±1.6 nm.There was a significant difference in the cumulative release outcomes of SMPs compared with the controlled release.The figure of nanoparticles was a fine spherical shape with smooth surfaces without aggregation or adhesion under Transmission Electron Microscope (TEM).The EE (39.12±1.65%) and DL (7.23±1.05%) of the SMPs were analyzed by the spectrum of FT-IR and DSC,which indicated that Most of the simvastatins was wrapped in the synthesis of SMP, only a small amount of drugs was adsorbed on the surface of nanoparticles.All of 16 New Zealand White rabbits survived postoperatively; without surgical infection, diets and activities was normal; after 4 weeks of operation. Comparision in average bone graft areas among four groups was as following:MP>SV>BS≥BC. There was a clear boundary between planted bone area and the host bone in each defect.The characteristics of X-ray of new bone regeneration of skull defects of rabbit models were as following:There were no obvious transmission shadows about mal-osteogenesis in bone graft areas of SMP groups; new bones was connected closely with natural bone; Density of new bone at all bone graft areas were not same; The bone density was more dense in SMP group and most less BC group.Green fluorescent belts at each graft area in undecalcified hard tissue was observed and shown that new bone was regenerated in four weeks after operation. The comparation of the amount of new bone regeneration at graft areas among four groups was as following:SMP group> SV group>BS group>BC group.The ratio of the new bone area in the SMP group (23.98 ±1.92%) was markedly higher than that in the other three groups (P<0.05). A significant difference was found in SV (18.90±1.23%) compared with BO (8.78±0.43%)(P<0.05). The ratio of new bone area in the BC group (8.29±1.83%) was notably lower than that in the SMP and SV groups (P<0.05),and no differences between the BC and BS groups(P= 0.617>0.05), but many osteoblasts were present around the bone substitute.In agreement with the histomorphometric results, Masson’s trichrome staining for collagen regeneration revealed significant collagen fibrous tissue and osteoblasts around the bone substitute in the defect area, especially in the SMP group. At high magnification, multiple dark blue-stained collagen fibers, the main component of the bone mineralization matrix, formed a thinly woven composition and were connected to form the trabecular structure. Meanwhile, multiple active osteoblasts, in which the cell nuclei dyed pink were observed around the collagen or bone substitutes.A number of BMP-2-stained cells were observed around the newly formed immature bone or bone substitute in the defect area in the SMP group, especially. The cell nuclei of the BMP-2-stained cells, dyed dark brown, showed multiple mitotic figures, such as shuttle shapes or multiple angle shapes, similar to the osteoblasts in the histological images. The results also revealed that bone formation was positively related to the number of BMP-2-stained cells in the defect area. Percentage of BMP-2-stained cells in the SMP and SV groups had relatively higher than that in the BC (P<0.05)and BS groups (P<0.05). Like the aforementioned new bone ratio, significant difference in the score of the immunoreactivity of BMP-2 between the SMP group and the SV group (P<0.05) and no differences between the BC and BS groups (P=0.553> 0.05).Conclusions:MP successfully enclosed simvastatin, The solubility of simvastatin was greatly enhanced. MPEG-PLA nanoparticles as drug delivery system can enhance the effects of simvastatins on osteoblasts proliferation and differentiation. SV-loaded MPs significantly improved the anabolic bone effects of SV by topical administration in vivo.