P7. Moldable allograft bone gel infused with spine-derived exosomes triggers osteogenic induction

Abstract

BACKGROUND CONTEXT Exosomes are nanoscale vesicles that function as critical mediators of cell-to-cell communication via transportation of molecular cargo from a source cell to a target cell. It has previously been shown that the differentiation fate of primitive cells, such as mesenchymal stem cells (MSCs), can be modified toward an osteogenic path by the uptake of exosomes from defined cell types. Due to these properties, exosomes derived from a bone source such as spine seem an appropriate tool to guide the osteogenic differentiation of progenitor cells. PURPOSE A moldable bone gel was developed to serve as an osteoconductive support in filling bone voids. The effects of processing bone into a gelatinous material inhibits its innate osteoinductive properties. In an effort to restore the osteoinductive capacity of the bone gel product, spine-derived exosomes were added to the gel composition to restore bone graft material osteoinductivity and enhance handling. STUDY DESIGN/SETTING Purified exosomes were characterized by flow cytometry using coating latex beads with the nanoparticles and subsequently labeling these beads with known exosome markers, CD63, CD81 and CD9. Scanning Electron Microscopy (SEM) was performed to verify the size and morphology of the exosomes. Exosome concentration was determined using EXOCET Exosome Quantitation Kit (System Biosciences). PATIENT SAMPLE Exosomes were obtained from qualified cadaveric human spines by ultrafiltration and subsequent differential ultracentrifugation (Beckman Optima XE-90 Ultracentrifuge equipped with a SW32 rotor) of the clarified supernatant. OUTCOME MEASURES Scanning electron microscopy (SEM) was performed to verify the size and morphology of the exosomes. Exosome concentration was determined using EXOCET Exosome Quantitation Kit (System Biosciences). Protein concentration was determined using a Qubit 4.0 Fluorometer (Thermo Fisher), from which the purity of the exosome sample was determined by calculating exosome concentration per microgram of protein. A sensitive, quantitative method to assess the bone forming potential of C2C12 myoblast cell line was used. The expression of alkaline phosphatase, a widely accepted bone marker, was measured following treatment of C2C12 cells with spine-derived exosomes alone or in combination with bone gel using polycarbonate membrane transwells. METHODS Treatment with 50ng of BMP-4 was used as a positive control. Alkaline phosphatase expression was normalized to total protein content, which was measured with Pierce BCA Protein Assay Kit (Thermo Fisher). The osteoinductive (OI) index was calculated by using the following formula: (OI test sample result – OI negative control result)/OI negative control result/protein concentration. An index over 20% of negative baseline was considered as osteoinductive. RESULTS Spine-derived exosomes positively expressed the exosome flow cytometry markers tested. Specifically, they expressed 99 ± % of CD81, 85 ±14% of CD63 and 64 ± 35% of CD9. SEM imaging revealed most of the exosomes were approximately 100 nm in size, consistent with the expected physiological size range of exosomes (30 – 150nm). The mean concentration of the spine-derived exosomes obtained was 1.22 ± 1.09 × 1010 exosomes/mL of supernatant. The mean number of exosomes per microgram of protein was 3.31 ± 2.33 × 108 indicating relatively high purity. Osteoinductive testing was performed using different concentrations of exosomes either alone or in combination with bone gel. The OI index of treatment of C2C12 cells with BMP-4 or 2 × 108, 1 × 109, 2 × 109, 5 × 109 or 1 × 1010 exosomes alone was 28.5, 1.0, 3.7, 7.4, 11.8 and 27.6 respectively. The OI index of treatment with 2 × 108, 1 × 109, 2 × 109, 5 × 109 or 1 × 1010 exosomes, with each dose combined with 0.25cc of bone gel, was 0.9, 4.5, 6.2, 9.3 and 18.5 respectively. These results revealed a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression. All doses were 20% above negative baseline indicating an osteoinductive effect at doses ranging from 2 × 108 to 1 × 1010 exosomes alone or with bone gel. All data is expressed as mean ± SEM from 3 separate experiments. Statistical analysis was performed using Student's t-test or one-way ANOVA followed by Bonferroni's post hoc test if multiple group comparisons were performed. CONCLUSIONS In this preliminary study, in vitro osteoinductive effect of spine-derived exosomes alone or infused in bone gel on C2C12 cells was demonstrated. Although different concentrations of exosomes were tested, a single concentration (50ng) of BMP-4 was used as a positive control. Future studies will evaluate in vivo activity in a bone defect rat model. There are specific and key miRNA transcripts involved in the observed osteoinductive regulation to better understand the molecular mechanism of exosome-delivered therapy in the context of bone regeneration. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.