Exosomal miRNA have been shown to regulate many myogenic and osteogenic pathways involved in injury repair and healing. It is also known that rehabilitation and exercise can improve muscle mass and bone growth. The mechanisms by which this occurs in vivo are well studied, but the impact exosomes and their associated miRNA cargo have is unclear. With this knowledge and question in mind, we hypothesized that C2C12 myoblasts subjected to in vitro mechanical stimulus (“exercise”) would exhibit improved exosome production and differentially expressed miRNA cargo when compared to their static (“unexercised”) counterparts. C2C12 myoblasts were cultured using the FlexCell FX-5000TT bioreactor. Two exercise regimens were programmed: 1) low intensity regimen (LIR) (0–15% strain at 0.5 Hz for 24 hours) 2) high intensity interval regimen (HIIR) (12–22% strain at 1 Hz for 10 minutes followed by 50 minutes of rest repeated for 24 hours). Unexercised (static) cells were cultured in parallel. Exosomes were isolated using the Invitrogen Total Exosome Isolation Reagent. The Pierce BCA Protein Assay, System Bioscience's ExoELISA-ULTRA CD81 Kit and, SBI's ExoFlow-ONE EV labeling kit were used to confirm and quantify exosome number and protein concentration. The SBI Exo-NGS service was used to perform miRNA sequencing on isolated exosomes.Introduction and Objective
Materials and Methods
A single, locally-delivered injection of a human placental product containing multipotent stromal cells reduced severity of infection in an immunosuppressed murine osteomyelitis model and eliminated infection in 25% of animals compared with 0% of controls without the use of antibiotics. Implant–associated osteomyelitis is a serious orthopaedic condition and is particularly difficult to treat in immunosuppressed individuals. Despite great advancement in the field of biomaterials and pharmaceuticals, emerging patterns of antibiotic resistance, complex biofilm production and penetration of therapeutic concentrations of effective antibiotics into bone continue to represent unmet clinical challenges. The promise of adult multipotent stromal cells (MSCs) for tissue regeneration has been of intense interest in recent years. Among their many potential therapeutic uses, MSCs have also been shown to have direct antimicrobial properties. The objective of this study was to evaluate the efficacy of a locally–delivered human placental-based tissue product containing multipotent stromal cells (hAmSC) to reduce the severity of implant-associated Staphylococcus aureus osteomyelitis in an immunosuppressed murine model. We hypothesised that athymic mice with implant-associated osteomyelitis would have diminished infection following treatment with hAmSC as evidenced by decreased bioluminescence intensity and lower histologic scores for infection and bacterial load when compared to saline-treated controls.Summary Statement
Introduction
