Extracellular vesicles 101

Extracellular vesicles (EVs) are lipid-bound, nanoscale vesicles released by cells and are known to facilitate intercellular communication. The three main EV subtypes are 1) exosomes: ranging from 30 to 150 nm in diameter and are formed by the endosomal route; 2) microvesicles: diameters from 100 nm up to 1 µm and bud off directly from the plasma membrane and 3) apoptotic bodies: diameter from 50 nm up to 5000 nm and derived from the apoptotic cell disassembly (Doyle and Wang, 2019). Due to the lack of specific markers for exosomes, the preferred nomenclature is EVs or small-derived EVs. Exosomes are typically characterised by their size and highly enriched expression of proteins such as tetraspanins (CD9, CD63, and CD81), flotillin-1, integrins, Hsp70, TSG101, and Alix. EVs are seen as a new tool for regenerative research which was initially discovered from the beneficial effect of MSC conditioned media due to paracrine signaling (Gnecchi et al., 2008). MSC EVs are the most commonly tested EVs for regenerative medicine and have shown promising results for their role in regeneration of different injured tissues eg. liver fibrosis, lung disorders, osteoarthritis, colitis, myocardial injury, spinal cord injury and retinal injury (Ghafouri-Fard et al., 2021). The source of MSCs used for EV isolation includes bone marrow, umbilical cord tissue, adipose tissue, oral, human induced pluripotent stem cells and synovial tissue. Studies have also shown that blood-derived EVs have regenerative potential for wound healing, myocardial injury, hindlimb ischemia and hepatic fibrosis (summarised in (de Boer and Davies, 2021)). The purity of all EV isolations must be taken into account particularly with the issue of lipoprotein contamination (de Boer et al., 2021).

MSC-EVs have shown promise for treatment of bone injuries in preclinical studies. A recent systematic review of 12 reports (1947 to 2020; meta-analysis) found MSC-EVs exhibited statistically significant bone healing for bone volume/total volume (22.2%) and new bone formation (26.1%) compared to controls (Kirkham et al., 2021). MSC-EVs have been found to be effective for bone regeneration despite the animal model used with various bone defects and diseases, eg. calvaria defects, fractures, distraction osteogenesis, osteoporosis, osteonecrosis, and irradiation-induced bone loss (systematic review of 23 papers with sample size of 690 rats or mice and 38 rabbits; up to 2020) (Tan et al., 2020). MSC-EVs also have therapeutic benefits for cartilage repair such as promoting repair and regeneration of osteochondral defects and alleviating osteoarthritis degeneration; seen by increased cellular proliferation and matrix deposition (systematic review of 13 studies with sample size of 378 rats or mice and 56 rabbits; up to 2019) (Tan et al., 2021).

Various hydrogels have been investigated to encapsulate EVs for diverse regenerate purposes which is required to prevent rapid loss of EVs (de Boer and Davies, 2021). A systematic review of MSC-EVs combined with scaffolds in bone regeneration (16 papers; 2012-2020) found overall positive effects in vivo on bone regeneration and mineralization, activation of the pathway for bone regeneration, induction of vascularization, and modulation of inflammation (Re et al., 2021). The in vitro data showed positive results for bone mineralization and proangiogenic ability. The types of scaffolds used were hydrogels (chitosan, collagen, PLA with PEI, hyaluronan), scaffolds (PLA, PCL or tricalcium phosphate), 3D-printed titanium alloy, decalcified bone matrix, and hydrogel pure matrix. In addition, EVs derived from endothelial cells (ECs) could also play a role in bone remodeling as studies have shown they are able to regulate the maturation process of osteoblasts and osteoclasts; however, as their mechanisms are not well understood more studies need to be performed on EC-derived EVs and their effect on bone tissue (Yin et al., 2021).

Aspects that still need to be elucidated include substantial heterogeneity between studies, possible bias reporting which clouds the accurate assessment of MSC-EVs therapeutic potency, suitable cell source, EV production protocol and concentration, translation into clinic, incomplete reporting and lack of standardization in outcome measurements.