Simvastatin-Loaded Nanomicelles Enhance the Osteogenic Effect of Simvastatin

Abstract

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 $54.57\pm 3.15\%$ and $10.91\pm 0.63\%$ , 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.