INTRODUCTION: Mesenchymal stromal cells (MSC) combined with biphasic calcium phosphate biomaterial (BCP) is a promising clinical strategy to repair and regenerate lost bones . Further, MSC derived extracellular vesicles (MSC:EV) are established factors of paracrine inter-cellular communication with various cell types, including immune cells, which impacts their regenerative potential  . However, scarce studies have explored the immune modulation behavior of EV isolated from MSC+BCP constructs (MSC+BCP:EV). The aim of this study was to isolate and characterize MSC+BCP:EV, including from inflammatory primed cells (MSCp+BCP:EV).Isolated EVs from all MSC groups were exposed to primary human macrophages to determine change in macrophage maturation and polarization states.
METHODS: EV isolation was done by using size exclusion chromatography (SEC) columns (PURE-EV, HansaBioMed Life Sciences Ltd., Estonia). After confirming the particle size in eluted EV fractions via dynamic light scattering (DLS) and protein amount by BCA, fraction no. 8-15 were selected for further analysis. EVs were characterized by TEM and flow cytometry. A human cytokine 27-plex assay was used with a Bio-Plex® 200 System (both from Bio-Rad Laboratories) to measure the cytokine content of EVs. Macrophages were obtained from differentiation of primary blood derived monocytes, which were isolated from donor buffy coats via magnetically activated cell sorting (MACS, Miltenyi Biotec GmBH).
RESULTS: Size and morphologies of EVs from different MSC system were found comparable. Further, MSC+BCP:EV showed less proinflammatory, whereas MSCp+BCP:EV showed more immunomodulatory and angiogenic cytokine profile compared to MSC:EV. Functional macrophage analysis revealed increased potential of MSCp+BCP:EV to induce unpolarized/naive macrophages (M0) into an anti-inflammatory phenotype (M2), as compared to EVs from an unprimed construct (MSC + BCP:EV) and MSC alone (MSC:EV). Further, it was found that MSCp + BCP:EVs also have an enhanced potential for bi-directional macrophage polarization switching (i.e., from pro- to anti-inflammatory state and vice-versa).
CONCLUSIONS: This study established methods to isolate and characterize EVs from a MSC and biomaterial constructs. We showed that both priming and biomaterials have differential effect on EV-cytokines and hence immunomodulation by EVs. EVs derived from primed MSC + BCP constructs showed enhanced potential to modulate both naïve (M0) and pro-inflammatory (M1) human macrophage subsets towards an anti-inflammatory (M2) type. Such properties were attributed to the higher levels of immunomodulatory cytokines present in the MSCp + BCP derived EVs. Thus, our study provides new insights into role of EVs in MSC-biomaterial induced bone regneration .
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