Table 3:
Regenerative Outcomes in Preclinical EV Studies.
| Reference | Cell Source of EV | Test Method of Application | Species of Target Cell or Tissue | Outcome |
|---|---|---|---|---|
| Xia et al., 2019[99] | BM-MSCs | in vitro | Rat | 1. EV treatment attenuated NPC apoptosis after H2O2 exposure |
| 2. EV treatment restored iNOS, IL6, MMP3, MMP13, COL2A1, CASP1, IL1b, TXNIP, NLRP3, and SOX9 to untreated control levels | ||||
| 3. EV treatment increased the number of mitochondria and reduced mitochondrial dysfunction | ||||
| in vivo | 1. Intradiscal injection of EVs at 1μg/μL slowed the decrease in disc height index through 8 weeks compared to injury group | |||
| 2. Intradiscal delivery of EVs slowed the progression of IVDD through 8 weeks assessed by histological scoring | ||||
| 3. Intradiscal delivery of EVs restored MMP13 and COL2A1 equivalent to uninjured control through 8 weeks | ||||
| Lu et al., 2017[100] | BM-MSC | in vitro | Human | 1. EV treatment increased proliferation rate over 12-day period |
| 2. EV treatment increased ACAN, COL2A1, SOX9, and TIMP1 over 21 days in culture | ||||
| 3. EV treatment decreased MMP1 and MMP3 over 21 days in culture | ||||
| NPC | in vitro | 1. Migration activity increased with an increase in NPC-EV concentration | ||
| 2. NPC-EV treatment increased MSC ACAN, SOX9, COL2A1, HIF1a, CA12, and KRT19 expression | ||||
| 3. Changes in MSC expression were greater after EV treatment than indirect co-culture model with NPCs | ||||
| Bach et al., 2017[101] | NCs | in vitro | Canine | 1. EV treatment increased GAG and GAG/DNA in chondrocyte-like cell aggregates |
| 2. EV treatment increased GAG and collagen content in culture medium | ||||
| 3. Increase in EV treatment concentration increased DNA content, GAG content, and GAG/DNA ratio in a 7-day culture period | ||||
| 4. Significant positive correlation between total number of EVs used to treat chondrocyte-like cell aggregates and GAG content and GAG/DNA ratio | ||||
| Human | 1. EV treatment increased DNA, GAG and GAG/DNA in chondrocyte-like cell aggregates | |||
| 2. EV treatment increased GAG and collagen content in culture medium | ||||
| Lan et al., 2019[102] | NPCs | in vitro | Rat | 1. EV treatment increased ACAN, SOX9, COL2A1 expression in hBM-MSCs |
| 2. Knock down of Notch1 in MSCs resulted in higher upregulation of ACAN, SOX9, COL2A1 after EV treatment than controls | ||||
| Qi et al., 2019[103] | UC-MSCs | in vitro | Human | 1. EV treatment protected NPMSCs from high glucose induced injury |
| Cheng et al., 2018[104] | BM-MSC | in vitro | Rat | 1. Lower apoptosis rate for NPCs in EV treatment group when compared to untreated controls after application of TNFα |
| 2. miR-21 delivery via EVs inhibited TNFα-induced NPC apoptosis by targeting PTEN in the PI3K-Akt pathway | ||||
| in vivo | 1. Intradiscal injection of EVs alleviated TNFα induced NPC apoptosis in vivo | |||
| 2. No difference in Pfirmann grade between uninjured control and EVs treated IVDs | ||||
| 3. EV-treated IVDs appeared histologically similar to uninjured control IVDs through H&E staining | ||||
| Yuan et al., 2019[105] | CEPCs | in vitro | Rat | 1. Treatment with apoptotic bodies (Abs) promoted mineralization and upregulation of ALP, RUNX2, OCN, and COL1A1 in endplate chondrocytes |
| 2. Abs treatment promoted PPi metabolism modifications in endplate chondrocytes with an increase in Pi and decrease in PPi | ||||
| 3. Abs treatment decreased levels of ENPP1 and ANK expression, but increased TNAP expression | ||||
| 4. Treatment with H2O2 significantly increased the generation of Abs due to oxidative stress | ||||
| Moen et al., 2017[106] | NPCs | in vivo | Rat | 1. Application of miR-223-3p onto dorsal nerve roots decreased C-fiber responses (indirect application of NPC-EVs) |
| 2. miR-223 upregulated in NPC-EVs when the NP tissue is exposed to dorsal nerve roots | ||||
| Bach et al., 2016[107] | NCs | in vitro | Bovine | 1. The effects of porcine NCCM-P factors were negligible on bovine CLCs |
| Canine | 1. Canine NCCM pelletable factors increased the canine CLC GAG, GAG/DNA and COL2 content compared with controls | |||
| 2. Canine NCCM pelletable factors decreased VEGF and increased KRT19 expression | ||||
| 3. At least 4 d of freezing at −70 °C did not influence the biological activity of canine Canine NCCM pelletable factors on canine CLC micro-aggregates compared to non-frozen controls | ||||
| 4. Protein aggregates and EVs exerted a moderate concentration-dependent anabolic effect, but only on canine CLCs | ||||
| Bari et al., 2018[108] | ASCs | in vitro | Human | 1. Exosomes were less abundant than microvesicles in lyo-secretome |
| 2. Lyo-secretome was not hematolytic at any of the tested concentrations | ||||
| 3. Cell metabolic activity remained at least ≥60% when treated with lyo-secretome | ||||
| 4. Lyo-secretome became cytotoxic to NPCs at a concentration of over 50 mg/mL | ||||
| 5. Lyo-secretome (5–50 mg/mL) protected NPCs from the oxidative stress damages induced by H2O2 | ||||
| Liao et al., 2019[109] | BM-MSCs | in vitro | Human | 1. EVs led to protective effect by reducing ER stress-induced apoptosis |
| 2. EVs regulated UPR activation in response to AGEs-induced ER stress in human NPCs | ||||
| 3. EVs protected against ER stress-related apoptosis partly through the AKT and ERK activation in human NPCs | ||||
| in vivo | Rat | 1. EVs inhibited the activation of AGEs-induced ER stress-related cell apoptosis and slowed the progression of IVDD | ||
| Chen et al., 2020[110] | NPCs | in vitro | Rat | 1. Senescent NPC EVs showed an increase in the relative expression of P21 and P53 |
| 2. Senescent NPC-EV treatment led to a lower growth rate, fewer colony forming units, and higher SA-β-gal positivity in healthy NPCs | ||||
| 3. Senescent NPC-EV treatment led to more G1 phase cells and fewer S phase cells compared to the control group | ||||
| 4. siRNA transfection of EV treated NPCs led to a decrease in P21 and P53 expression, higher growth rate, and lower SA-β-gal positivity | ||||
| Hingert et al., 2020[111] | BM-MSCs | in vitro | Human | 1. EV treatment increased cell proliferation and decreased cellular apoptosis in degenerated disc cells |
| 2. EV-treated disc cell pellets demonstrated 3X greater ECM production compared to control disc cell pellets | ||||
| 3. EV treatment suppressed secretion of MMP-1 in disc cells | ||||
| Hu et al., 2020[112] | NPCs | N/A | N/A | 1. Rapamycin and bafilomycin A1 led to induction of NPC autophagy and EV secretion in an autophagy-dependent manner |
| 2. siRNA against ATG5 induced accumulation of ILVs and decrease in isolated EVs | ||||
| 3. Knockdown of RhoC and ROCK2 with siRNA inhibited secretion of EVs | ||||
| Li et al., 2020[113] | BM-MSCs | in vitro | Human | 1. Proliferation activity, collagen II, and aggrecan expression decreased in NPCs cultured at pH 5.9 – 6.7 |
| 2. Caspase-3 and MMP-13 expression increased in NPCs cultured at pH 5.9 – 6.7 | ||||
| 3. EV treatment led to an upregulation of collagen II and aggrecan, and a downregulation of matrix-degrading enzymes | ||||
| Li et al., 2020[114] | BM-MSCs | in vitro | Human | 1. EVs suppressed IL1β-induced inflammation and apoptosis of AF cells by suppressing autophagy |
| 2. EVs supported AF cell viability after IL1β treatment | ||||
| 3. EVs inhibited AF cell autophagy by activating the PI3K/AKT/mTOR signaling pathway | ||||
| Luo et al., 2021[115] | CESCs | in vitro | Rat | 1. Treatment with healthy CESC-EVs inhibited apoptosis compared to degenerated CEP stem cell-derived EVs |
| 2. Healthy CESC-EVs inhibited apoptosis of NPCs by activating the PI3K/AKT pathways | ||||
| in vivo | 1. Healthy CESC-EVs alleviated IVDD via activation of PI3K/AKT pathways | |||
| Song et al., 2020[116] | NPCs | in vitro | Human | 1. circRNA_0000253 was highly upregulated in degenerative NPC-EVs |
| 2. circRNA_0000253 promoted an IVDD phenotype by adsorbing miRNA-141-5p and downregulating SIRT1 in vitro | ||||
| in vivo | Rat | 1. circRNA_0000253 accelerated IVDD by adsorbing miRNA-141-5p and downregulating SIRT1 in vivo | ||
| Sun et al., 2020[117] | NCs | in vitro | Human | 1. 0.5MPa-conditioned EVs inhibit endothelial cell angiogenesis through miR-140-5p and regulate Wnt/β-catenin signaling |
| 2. NP EV-derived miR-140-5p is negatively associated with angiogenesis in clinical samples | ||||
| in vivo | Mouse | 1. 0.5MPa-conditioned EV treatment reduced vascularization in degenerated IVDs | ||
| Sun et al., 2021[118] | AFCs | in vitro | Human | 1. HUVECs phagocytose AFC-EVs |
| 2. Degenerated AFC-EVs promoted cell migration and upregulation of IL-6, TNF-α, MMP-3, MMP-13, and VEGF, while non-degenerated AF cell-derived EVs demonstrated inverse effects | ||||
| Tang et al., 2021[119] | NPCs | in vitro | Human | 1. Bulk electroporation of cells with FOXF1 led to FOXF1 plasmids in designer EVs and demonstrated efficient cell uptake |
| PMEFs | in vivo | Mouse | 1. Injection of FOXF1-loaded EVs into IVDs showed significant upregulation of FOXF1 and Brachyury compared to controls | |
| Wen et al., 2021[120] | BM-MSCs | in vitro | Mouse | 1. EV treatment led to an increase in COL2 and ACAN staining intensity and decrease in SA-β and TUNEL positive NPCs |
| 2. A reduction in EV-derived miR-199a led to an impaired protective effect of EVs on NPCs | ||||
| 3. EV-derived miR-199a promotes repair by targeting GREM1 and downregulating TGFβ pathway | ||||
| in vivo | 1. EV treatment led to increased levels of miR-199a and decreased levels of MMP3-, MMP6-, TIMP1-, and TUNEL-positive cells | |||
| Xiang et al., 2020[121] | USCs | in vitro | Human | 1. EV treatment led to a decrease in GRP78, GRP94, Caspase 3, and Caspase 12 expression under stress-induced conditions |
| 2. EVs inhibit excessive activation of unfolded protein response under stress-induced conditions | ||||
| 3. EVs regulate stress by activating AKT and ERK signaling pathways in NPCs under stress-induced conditions | ||||
| in vivo | Rat | 1. EVs inhibited ER stress-associated cell apoptosis and decelerated IVDD progression in vivo | ||
| Xie et al., 2020[122] | MSCs | in vitro | Rat | 1. EVs inhibited apoptosis and TBHP-induced CEP calcification |
| 2. Downregulation of miR-31-5p impaired EV protective effects | ||||
| 3. miR-31-5p negatively regulated ATF6-related ER stress and inhibited CEP apoptosis and calcification | ||||
| in vivo | 1. Sub-endplate injection of EVs ameliorate IVDD hallmarks | |||
| 2. Downregulation of EV-derived miR-31-5p inhibited EV protective effects in vivo | ||||
| Yuan et al., 2020[123] | PLMSCs | in vitro | Human | 1. EV-derived AntagomiR-4450 ameliorates NPC damage by promoting proliferation and migration |
| 2. EV-derived AntagomiR-4450 decreased MMP13, IL6, IL1β, CASP3 expression, and increased COL2 and ACAN expression | ||||
| in vivo | Mouse | 1. EV-derived AntagomiR-4450 attenuated IVDD damage by repressing miR-4450 and increasing ZNF121 expression | ||
| 2. EV-derived AntagomiR-4450 ameliorated gait abnormality | ||||
| Zhang et al., 2020[124] | MSCs | in vitro | Mouse | 1. EV treatment inhibited pyroptosis by suppressing the NLRP3 pathway |
| 2. EV treatment inhibited LPS-induced pyroptosis in NPCs | ||||
| 3. EV-derived miR-410 suppressed LPS-induced pyroptosis in NPCs | ||||
| in vivo | 1. EV treatment and miR-410 treatment alleviated IVDD severity | |||
| Zhang et al., 2020[125] | NPCs | in vitro | Rat | 1. Rapamycin treatment led to an increase in miR-27a in NPCs and their EVs |
| 2. EV-derived miR-27a alleviated IL1β-induced ECM degradation by downregulating MMP13 in NPCs | ||||
| Zhu et al., 2020[126] | BM-MSCs | in vitro | Mouse | 1. EV treatment attenuated NPC apoptosis by reducing inflammatory cytokine secretion and activating MAPK pathway |
| 2. EV-derived miR-142-3p targets mixed MLK3 and inhibits NPC apoptosis and promotes MAPK signaling | ||||
| 3. MLK3 overexpression abolished EV effects on inflammation, NPC apoptosis, and MAPK signaling activation | ||||
| Zhu et al., 2020[127] | BM-MSCs | in vitro | Rat | 1. EV treatment led to inhibition of apoptosis, ECM catabolism, and fibrosis in TNFα-treated NPCs |
| 2. miR-532-5p was abundant in TNFα-treated MSC-derived EVs and was less abundant in apoptotic NPCs | ||||
| 3. RASSF5 is an empirically validated target of miR-532-5p |
MSC = Mesenchymal Stem Cell; BM-MSC = Bone Marrow-derived Mesenchymal Stem Cell; NPC = Nucleus Pulposus Cell; NC = Notochordal Cell; AFC = Annulus Fibrosus Cell; UC-MSC = Umbilical Cord-derived Mesenchymal Stem Cell; ASC = Adipose-derived Mesenchymal Stromal Cell; CEPC = Cartilage Endplate Chondrocyte; CESC = Cartilage Endplate Stem Cell; PMEF = Primary Mouse Embryonic Fibroblast; USC = Urine-derived Stem Cell; PLMSC = Placental Mesenchymal Stem Cell; HUVEC = Human Umbilical Vein Endothelial Cell.