Alveolar bone loss has long been a challenge in clinical dental implant therapy. Simvastatin (SV) has been demonstrated to exert excellent anabolic effects on bone. However, the successful use of SV to increase bone formation in vivo largely depends on the local concentration of SV at the site of action, and there have been continuing efforts to develop an appropriate delivery system. Specifically, nanostructured lipid carrier (NLC) systems have become a popular type of encapsulation carrier system. Therefore, SV-loaded NLCs (SNs) (179.4 nm in diameter) were fabricated in this study, and the osteogenic effect of the SNs was evaluated in a critical-sized rabbit calvarial defect. 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. Thus, this novel nanostructured carrier system could be a potential encapsulation carrier system for SV in bone regeneration applications.
ObjectivesThe present study aimed to clarify the role of the ERK1/2 pathway in simvastatin (SV)-loaded nanomicelles (SVNs)- and SV-mediated promotion of cell osteogenic differentiation and explore the molecular mechanisms by which SVNs exhibited a greater efficacy in promoting osteogenic differentiation than SV.Materials and methodsSVNs were synthesized using a dialysis method. MG63 cells were treated with 2.5, 0.25, and 0.025 μmol/L of the drug. The optimal drug dosage was determined by examining the proliferative activity and ALP activity of the MG63 cells. Subsequently, Western blot analysis was performed to analyze the levels of the phosphorylated ERK1/2 proteins in each experimental group at various time points. Finally, the inhibitor PD98059 was used to effectively inhibit the ERK1/2 pathway. The resulting changes in the proliferative activity of MG63 cells and the osteogenesis-related markers were analyzed.ResultsThe SVNs synthesized in the present study had a mean diameter of 27 nm. The encapsulation and drug-loading efficiencies were 52.03% ± 4.05% and 9.42% ± 0.66%, respectively. SVNs and SV exhibited optimum osteogenesis-promoting effects when the drugs were administered at a concentration of 0.25 μmol/L. The drug-induced activation of the ERK1/2 pathway reached a peak at 15 minutes after administration and then declined rapidly. From 24 hours to 7 days, SVNs and SV exerted an inhibitory effect on the ERK1/2 pathway rather than an activating effect. Throughout the whole experimental process, the regulatory effect of SVNs on the ERK1/2 pathway was significantly greater than that of SV. Inhibition of the ERK1/2 pathway by PD98059 markedly reduced the proliferative activity of the cells in all experimental groups. In addition, the ALP activity and the expression levels of the osterix (OSX) and osteocalcin (OC) proteins were drastically increased.ConclusionSVNs significantly increased the effect of SV-induced osteogenic differentiation by strongly inhibiting the ERK1/2 pathway.
Objectives. The present study intended to further verify that simvastatin-loaded nanomicelles (SVNs) enhanced the role of simvastatin (SV) in promoting osteoblast differentiation in vitro and to evaluate the effect of SVNs on bone defect repair in vivo. Methods. SVNs were synthesized by dialysis. MG63 cells were subjected to intervention with 0.25 μmol/l of SVNs and SV. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit and flow cytometry were used to determine cell proliferation activity, cell cycle distribution, and apoptosis. The osteoblastic differentiation of MG 63 cells was evaluated by measuring alkaline phosphatase (ALP) activity, ALP staining, and the expression levels of the osterix (Osx) and osteocalcin (OC) proteins. In addition, 0.5 mg of SVNs or SV was applied to the skull defect area of rabbits. Micro-CT, hematoxylin and eosin (HE) staining, and Masson’s trichrome staining were used for qualitative and quantitative evaluation of new bone in three dimensions and two dimensions. Results. The SVNs had a mean diameter of 38.97 nm. The encapsulation and drug-loading efficiencies were 54.57 ± 3.15 % and 10.91 ± 0.63 % , respectively. In vitro, SVNs and SV can inhibit the proliferation activity and promote osteogenic differentiation of MG63 cells by arresting MG63 cells at the G0/G1 phase without increasing the apoptosis rate. In vivo quantitative results showed that the bone mineral density (BMD), bone volume (BV)/total volume (TV) ratio, and trabecular number (Tb.N) in the gelatin sponge with SVNs (SVNs-GS) group and gelatin sponge with SV (SV-GS) group were 362.1%, 292.0%; 181.3%, 158.0%; and 215.2%, 181.8% of those in the blank control (BC) group, respectively. Histological results identified the new bone tissue in each group as irregular fibrous bone, and the arrangement of trabecular bone was disordered. There were significantly more osteoblasts and new capillaries around the trabecular bone in the SVNs-GS group and SV-GS group than in both the BC and drug-free nanomicelle (DFNs) groups. Both in vitro and in vivo, SVNs exhibited greater osteogenic efficacy than SV. Conclusion. SVNs significantly improved the osteogenic efficacy of SV.
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