Mesenchymal Stem Cell-Derived Extracellular Vesicles in Bone-Related Diseases: Intercellular Communication Messengers and Therapeutic Engineering Protagonists

Abstract

Extracellular vesicles (EVs) can deliver various bioactive molecules among cells, making them promising diagnostic and therapeutic alternatives in diseases. Mesenchymal stem cell-derived EVs (MSC-EVs) have shown therapeutic potential similar to MSCs but with drawbacks such as lower yield, reduced biological activities, off-target effects, and shorter half-lives. Improving strategies utilizing biotechniques to pretreat MSCs and enhance the properties of released EVs, as well as modifying MSC-EVs to enhance targeting abilities and achieve controlled release, shows potential for overcoming application limitations and enhancing therapeutic effects in treating bone-related diseases. This review focuses on recent advances in functionalizing MSC-EVs to treat bone-related diseases. Firstly, we underscore the significance of MSC-EVs in facilitating crosstalk between cells within the skeletal environment. Secondly, we highlight strategies of functional-modified EVs for treating bone-related diseases. We explore the pretreatment of stem cells using various biotechniques to enhance the properties of resulting EVs, as well as diverse approaches to modify MSC-EVs for targeted delivery and controlled release. Finally, we address the challenges and opportunities for further research on MSC-EVs in bone-related diseases.

Keywords: bone-related diseases; extracellular vesicles; functional modification; mesenchymal stem cells.

Figure 1

Schematic summary of the cross-talk functions of EVs and EV-based treatment strategies for bone-related diseases. EVs serve as an important communicator between cells and organs related to the skeletal system and regulate cellular function. The strategies of functional-modified EVs for bone-related disease treatment can be summarized into two aspects. First, parental cells can be pretreated with different techniques to improve the properties of EVs. Second, surface modification of EVs and the following combination with different carrier systems enables the targeted delivery and sustained release of EVs, which can optimize and broaden the application of EVs.

Figure 2

EVs stimulated by cytokines may regulate macrophages phenotype. (A) Transmission Electron Microscope (TEM) images of inflammation-stimulated ADSC- derived sEV (IAE) and normal ADSC-derived sEV (AE). Scale bar = 100 nm. (B) Schematic diagram revealed that sEVs were connected to the scaffolds through TGase: glutamine residues (purple), lysine residues (gray), TGase (green), and sEVs (yellow). (C) The release curves of sEVs showed that scaffolds with TGase exhibited complete release up to day 7. (D) Immunofluorescence staining showed that the proportion of M2 macrophages in the entire macrophage population was significantly upregulated by IAE; M1 marker (CD86, red), M2 marker (CD206, green), and nuclei (DAPI, blue). Scale bar = 100 µm. (E) Immunohistochemical staining of collagen I and II of temporomandibular joint sections and magnified defect sites at week 8 posttreatment indicated a good regenerative effect in IAE group. Black boxes indicate magnified microscopic fields of view. Upper panel scale bar, 1 mm; lower panel scale bar, 200 µm. Reprinted from Liu Y, Zhang Z, Wang B, et al. Inflammation-stimulated MSC-derived small extracellular vesicle miR-27b-3p regulates macrophages by targeting CSF-1 to promote temporomandibular joint condylar regeneration. Small. 2022;18(16):1. © 2022 Wiley-VCH GmbH.

Figure 3

Engineered exosomes derived from BMSCs preconditioned with magnetic stimulation. (A) Scheme of the fabrication of three types of exosomes: BMSC-Exos, BMSC-Fe₃O₄-Exos and BMSC-Fe₃O₄-SMF-Exos. BMSC-Fe₃O₄-SMF-Exos were obtained via magnetic stimulation using Fe₃O₄ nanoparticles and a static magnetic field (SMF). (B) Morphology of exosomes was observed by TEM; the red arrows indicate exosomes. (C) Fe₃O₄ nanoparticles and SMF increased the production of exosomes in BMSCs. *p<0.05, ***p<0.001. Representative fluorescent images of the osteogenic marker Runx2 (D) and the angiogenic marker CD31 (E) revealed that magnetic stimulation potentiated osteogenesis and angiogenesis in calvarial defects 12 weeks post-treatment; white arrows mark the newly formed vessel. Reprinted from Wu D, Chang X, Tian J, et al. Bone mesenchymal stem cells stimulation by magnetic nanoparticles and a static magnetic field: release of exosomal miR-1260a improves osteogenesis and angiogenesis. J Nanobiotechnology. 2021;19(1):209. Creative Commons.

Figure 4

Surface modification of EVs via micelle-mediated physical modification and chemical modification. (A) Schematic illustration of DS-EXOs as a cell-free therapeutic system for rheumatoid arthritis (RA). (B) Mechanism by which the DS-EXOs reprogram macrophages. The DS-EXOs reach joints inflamed by RA owing to their ability to target activated macrophages after systemic administration. (C) TEM images of EXOs and DS-EXOs. (D) Ex vivo organ distribution images revealed that the number of Cy5.5-labeled DS-EXOs at the inflamed sites was significantly higher than that of Cy5.5-labeled bare EXOs. (E) Representative images of the collagen-induced arthritis (CIA) mice treated with PBS, bare EXOs and DS-EXOs of different concentrations. Reprinted from You DG, Lim GT, Kwon S, et al. Metabolically engineered stem cell-derived exosomes to regulate macrophage heterogeneity in rheumatoid arthritis. Sci Adv. 2021;7(23):eabe0083. Creative Commons.

Figure 5

Immobilizing exosomes within collagen hydrogels for spatially vascularization and host integration. (A) Scheme of the overall strategy for engineering Az-EVs via metabolic glycan labeling and the following Az-EV immobilization within DBCO-collagen. (B) TEM image showed the morphology of EVs. (C) Assessment of immobilization and chronic release of radiolabeled Az-EVs and control non-Az-EVs in DBCO-collagen and unmodified collagen gels (n = 3). (D) Gross appearance of collagen gel explants 7 days post-implantation. (E) Histological analysis of host vascular ingrowth via endothelial CD31 staining (n = 7). (F) Histological analysis of M1 macrophage staining (CCR7) (n = 7). Adapted from Xing Y, Yerneni SS, Wang W, Taylor RE, Campbell PG, Ren X. Engineering pro-angiogenic biomaterials via chemoselective extracellular vesicle immobilization. Biomaterials. 2022;281:121357. Creative Commons.