Extracellular Vesicles Derived from Primed Mesenchymal Stromal Cells Loaded on Biphasic Calcium Phosphate Biomaterial Exhibit Enhanced Macrophage Polarization

Abstract

Mesenchymal stromal cells (MSC) loaded on biphasic calcium phosphate biomaterial (MSC + BCP) have been used as an advanced therapy medicinal product to treat complex maxillofacial bone defects in patients. Further, MSC-derived extracellular vesicles (EVs) are established vehicles of paracrine factors, supporting inter-cellular communication between MSC and other interacting cell types, such as monocytes/macrophages. However, the information about the immunomodulatory potential of EVs derived from MSC and biomaterial constructs (MSC + BCP:EV) and inflammatory primed constructs (MSCp + BCP:EV) are scarce. Hence, we isolated and characterized EVs from these different systems, and compared their cytokine contents with plastic-adherent MSC-derived EVs (MSC:EV). When EVs from all three MSC systems were added to the primary blood-derived macrophages in vitro, significantly higher numbers of M0 (naive) macrophages shifted to M2-like (anti-inflammatory) by MSCp + BCP:EV treatment. Further, this treatment led to enhanced switching of M1 polarized macrophages to M2 polarized, and conversely, M2 to M1, as evaluated by determining the M1/M2 ratios after treatment. The enhanced macrophage modulation by MSCp + BCP:EV was attributed to their higher immunomodulatory (TNFα, IL1β, IL5), angiogenic (VEGF), and chemokine-rich (RANTES, MCP1, MIP1β) cytokine cargo. In conclusion, we successfully isolated and characterized EVs from MSC + BCP constructs and demonstrated that, depending upon the tissue microenvironment, these EVs contribute towards modulating the macrophage-mediated inflammation and healing responses. The study offers new insights into the use of biomaterial-induced EVs for MSC secretome delivery, as a step towards future ‘cell-free’ bone regenerative therapies.

EV‐COMM: A database of interspecies and intercellular interactions mediated by extracellular vesicles

“…They can be categorised into different subtypes based on their size or biogenesis mode, that is, exosomes (30–150 nm), microvesicles (100–1000 nm) and apoptotic bodies (1000 nm–5 μm). In the absence of specific markers and overlapping sizes, the term “small EVs” (sEVs) applies to both exosomes and microvesicles up to 200 nm in size and “medium and/or large EVs” (m/lEVs) refers to >200 nm in size (Rana et al., 2022 ; Théry et al., 2018 ). Different species use various names for extracellular vesicles (EVs), including EVs‐like nanovesicles, exosome‐like extracellular vesicles and exosome‐like nanovesicles (ENVs).…”

Coupling BCP Ceramics with Micro‐Vibration Stimulation Field for Cascade Amplification from Immune Activation to Bone Regeneration

“…Moreover, compared with Plate‐CM one, SS‐CM group increased gene expression of osteogenic markers such as ColI and Bsp on day 3, Runx2 , Osx and Alp on day 7, which was in agreement with our previous study, indicating that BCP ceramics could induce macrophages toward M2 phenotype to release a mass of anti‐inflammatory cytokines and growth factors, which could reduce inflammation and provide a local inflammatory microenvironment suitable for osteogenesis. [ 54,55 ] VS‐CM group not only showed the highest ALP activity and osteogenic gene expression (i.e., Runx2 , ColI , Bmp2 , Alp , Bsp and Osx ) in vitro, but also significantly up‐regulated the expression of osteogenic genes in vivo, including the early osteogenic markers like Bmp‐2 and Alp , and the late osteogenic markers like Ocn and Opn . A nude mice subcutaneous implantation model found that VS‐CM group promoted the early bone formation, as evidenced by the accelerated formation of collagen‐rich osteoid‐like tissues and the highest expression of osteogenic marker (BMP‐2).…”

Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model

“…The protein concentration of EV samples was measured by Pierce™ BCA Protein assay kit (Thermo Fisher Scientific, USA), following the manufacturer’s instructions. EVs morphology was recorded by transmission electron microscopy (TEM), according to a previously described protocol [ 24 ]. The final EV preparations were examined using a Jeol JEM1400 transmission electron microscope (Jeol Ltd., Tokyo, Japan).…”