gms | German Medical Science

Objectives: The repair of large-sized bone defects that severely compromise musculoskeletal function is one of the major challenges for orthopedic surgeons. Extracellular vesicles (EVs)-based cell-free therapy has been of interest as a promising alternative tool for the restoration of bone defects. This study aims to explore if MSCs-derived EVs secreted upon sustainable simvastatin stimulation (denoted as BMSCEVsim) could inherit the osteogenic potential from simvastatin and might be exploited as an osteo-enhancer to stimulate osteogenesis.

Methods: First, isolated BMSCEVsim were characterized and identified via Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM) analysis. Next, the uptake of BMSCEVsim by BMSCs was confirmed by cellular internalization assays. The proliferation and migration capacity of BMSCs treated with varying dosages of BMSCEVsim were evaluated via Calcein AM staining, CCK-8, and scratch wound assays. Furthermore, the osteogenic differentiation of BMSCEVsim-treated BMSCs was examined by detecting biomineralization markers and BMP-2 immunofluorescence staining. The osteogenesis-related gene expression levels of type 1A collagen (Col1A1), alkaline phosphatase (ALPL), bone morphogenetic protein 2 (BMP-2), bone gamma-carboxyglutamic acid-containing protein (BGLAP) coding for osteocalcin, Secreted Phosphoprotein 1 (SPP1) coding for osteopontin, runt-related transcription factor 2 (RUNX2) were detected in BMSCEVsim-treated BMSCs using quantitative real-time PCR (qRT-PCR). The experiments were carried out considering the following experimental groups: Control (PBS), control (Simvastatin), BMSCEVssim (5 µg/mL), BMSCEVssim (10 µg/mL), and BMSCEVssim (20 µg/mL).

Results and conclusion: Cellular uptake assays indicated that fluorescently labeled BMSCEVssim were phagocytosed by BMSCs. Besides, in vitro proliferation and migration assays verified that the proliferation rate and migration rate in the BMSCEVssim group at 20 ug/mL was significantly higher than those in either the low concentration BMSCEVssim (5- and 10 ug/mL) or solely simvastatin treatment groups. Mineralization experiments revealed that a large amount of dispersed mineralized nodules were formed in BMSCs treated with various dosages of BMSCEVssim. Furthermore, the osteogenesis-related gene expression levels of Col1A1, ALPL, BMP-2, BGLAP, SPP1, and RUNX2 were significantly upregulated after stimulation with 20 ug/mL BMSCEVssim group in comparison to a plain simvastatin stimulation.

Simvastatin-stimulated BMSCEVssim could function as an osteo-enhancer given their apparent pro-osteogenic capacity. In addition, these EVs were able to promote in vitro proliferation and migration of BMSCs. Besides, the pro-osteogenic effect exhibited by BMSCEVssim was positively correlated with the increasing concentration of BMSCEVssim.