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Table 1 Characteristics of studies conducted on the application of EVs in bone regeneration

From: Extracellular vesicles in bone and periodontal regeneration: current and potential therapeutic applications

Indirect stimulator type Study mode Active EV cargo molecules Key function/targeted genes References
BMMSC-EVs In vitro miR-3084-3p, miR-680, miR-677-3p and miR-5100 RUNX2 and ALP up regulation, as well as matrix mineralization enhancement/activated Wnt/β-catenin [111]
hMSCs-EVs In vitro and in vivo (athymic nude mice) VEGF and BMP2 Induced osteogenic differentiation of naive MSCs and matrix mineralization, increased phosphorylated proteins/up regulation of RUNX2 and Osterix [112]
Dendritic cells-EVs In vitro Triggered osteogenic differentiation in MSCs/increased expression of Runx2 and ALP activity [113]
hASCs-EVs In vitro and in vivo (mouse calvarial defect model) miR-34a, let-7a, miR-218 Enhanced bone regeneration/increased expression of RUNX2, ALP, COL1A1 [114]
ASCs-EVs In vitro Wnt-3a protein Induced osteogenic differentiation of HOBs/increased expression of RUNX2, collagen I, osteopontin, and bone sialoprotein [115]
LPS-activated human monocytes-EVs and CM In vitro Promoted osteogenic gene expression in MSCs/increased expression of RUNX2 and BMP2 [116]
Mc3t3-EVs In vitro Phosphorylated eIF2a Promoted osteoblastic differentiation [117]
Cavin-1-PC3-EVs In vitro and in vivo(NOD/SCID mice) Proteins and miR-148a Induced osteoclast differentiation and osteoblast proliferation [118]
NOS-1 cells-EVs In vitro Ca, P, and Zn concentrations Increased mineral deposition mediated by the activation of ALP and calcium-binding proteins [119]
hBMSC-EVs In vitro and in vivo (SD rat calvarial defect model) miR-196a, miR-27a and miR-206 Promoted osteogenic function/increased expression of ALP, OCN, OPN and RUNX2 [99]
Multiple myeloma (MM) cells-EVs In vitro AKT pathway Modulation of osteoclasts function and differentiation/increased expression of TRAP, MMP9, and CTSK [120]
Osteoclast-EVs In vivo (mouse marrow, a model for the in vivo bone microenvironment) RANK Regulation of osteoclastogenesis/regulation of 1,25(OH)2 D3-stimulated osteoclast formation [121]
pMSCs-EVs In vitro Actin cytoskeleton, growth hormone, clathrin mediated endocytosis, and VEGF Stimulated microvascular endothelial cells migration in a concentration and oxygen-dependent manner/promoted vascular network formation [110]
hiPS-MSC-EVs In vitro and in vivo (rat model of calvarial bone defects) PI3K/Akt signaling pathway Enhanced proliferation and migration ability of hBMSCs and osteogenic differentiation/up-regulation of PDGFA, FGF1/2, FGFR1, COL1A1/2, BCL2L1/down-regulation of PTEN and GSK3β/activation of PI3K/Akt signaling pathway [122]
Rat bone marrow-derived MSCs-EVs In vitro and in vivo (nude mice) Enhanced bone regeneration and vessel formation/increased CD-31 positive cells [123]
BMP2/macrophage-EVs In vitro VEGF and BMP2 Increased osteoblastic differentiation and activated autophagy during osteogenic differentiation/enhanced expression of ALP, OPN, IBSP, Runx2, OCN, Col-I, and BMP signaling pathway [124]
hBMMSCs-CM In vitro and in vivo (rat calvarial bone defect model) IGF-1 and VEGF Enhancement of bone formation and MSC migration into the defect/enhanced expression of OCN and the Runx2 genes [125]
hBMMSCs-CM In vitro and in vivo (rat calvarial bone defect model) IGF-1, VEGF, TGF-B1, and HGF Increased migration and expression of osteogenic-related genes like ALP, OCN, and RUNX2 [92]
hBMMSCs-CM In vitro and in vivo (rat calvarial bone defect model) VEGF Increased bone formation, and blood vessel formation [94]
hBMMSCs-CM In vivo (mouse H-DO model) MCP-1/-3 and IL-3/-6 Accelerated distraction osteogenesis accelerated new bone callus formation [126]
hBMMSCs-CM In vitro and in vivo (rat BRONJ models) IGF-1, VEGF, angiogenin, IL-6, and M-CSF Increased bone healing with complete soft tissue coverage/increased expression levels of the Runx2, OCN and VEGF-A [127]
Hypoxic hDPCs-CM In vitro and in vivo (mouse DO model) VEGF-A165 and Ang‐2 protein Increased α-SMA‐positive mature blood vessels and blood vessel formation/promoted new callus formation and upregulation of factor 8 gene [128]
hFMSCs-CM In vitro and in vivo (rat DO model) Upregulation of mRNA expression of ALP, OCN, OPN, Osx, and Runx2 [129]
hFMSCs-CM In vitro and in vivo (nude mice) Up-regulation of p21and down-regulation of bax and p53 Reduced SA-β-gal expression and activity/enhanced cell proliferation and osteogenic differentiation/activation of sirt1and foxo3a/induced expression of osteogenic genes, including Alp, Runx2 and Rex1/highly expression of COL1A1 and OPG [130]
hESC-MSCs-EVs In vivo (critical sized osteochondral defects in a rat model) Restored cartilage and subchondral bone after 12 weeks/high levels of GAG and type II collagen [131]
hBMMSCs-CM and EVS In vitro and in vivo (femur fracture model of CD9−/− mice) High expression of miR-4532, miR‐125b‐5p, and miR‐4516 in EVs. High levels of MCP‐1, MCP‐3, and SDF‐1 in CM Accelerated fracture healing/increased TRAP-positive cells and cells positive for the vascular marker αSM [132]
hiPSCs-MSC-EVs In vitro and in vivo (rat critical size bone defect model) Enhanced cell proliferation and ALP activity/up-regulated mRNA and protein expression of osteoblast-related genes/stimulated bone regeneration and angiogenesis [133]
uMSCs-EVs In vivo (rat stabilized femoral fracture model) - Enhanced angiogenesis and bone healing processes/enhanced osteogenic differentiation/increased VEGF and HIF-1α expression/enhanced proliferation/migration and tube formation of HUVECs [134]
DPSCs-EVs In vitro Enhanced DPSCs viability, migration and mineralization potential/a time-dependent increase of TGF-1 and a TEGDMA concentration-dependent increase of both TGF-1 and FGF-2 in CM [135]
hGMSCs-CM In vitro and in vivo (rat calvarial defect model) Induction of new bone formation and osseointegration through expressing or up-regulating genes involved in ossification or regulation of ossification [136]
hGMSCs-EVs In vitro and in vivo (rat calvarial defect model) Improved bone healing by showing better osteogenic properties and greater osteogenic inductivity/increased RUNX2 and BMP2/4 mRNA expression/up-regulation of osteoblast differentiation genes/inducing the regulation of adhesion molecules [137]
In vitro and in vivo (rat calvarial defect model) Up-regulation of osteogenic genes, such as TGFB1, MMP8, TUFT1, TFIP11, BMP2, and BMP4/induced bone regeneration/improved mineralization process and inducing extensive vascular network [138]
In vitro and in vivo (rat calvarial defect model) Enhanced osseous regeneration, vascularization, and osseointegration/increased levels of VEGF and VEGFR2 [98]
In vitro DSPP, BMP7, DDR2, USP9X, NCOA2, PEG10, LPA Presence of osteogenic lineage proteins/higher osteogenic differentiation and lower adipogenic differentiation [139]
Bone marrow interstitial fluid EVs In vitro and in vivo (mice femoral defect model) miR-96-5p, miR-182-5p, and miR-183-5p Suppress osteogenic differentiation, increase cell senescence/decreases Hmox1/suppresses BMSC proliferation and mineralization [140]
BMSCs-EVs In vitro and in vivo(intravenous injection in mice) miR221, miR451a, miR654-3p, miR106b-3p, miR155-5p and miR210-5p Reversal of growth inhibition, DNA damage and apoptosis on exposure/stimulation of normal murine marrow stem cells to proliferate [141]
Osteoblast-derived EVs In vitro RANKL Stimulation of RANKL–RANK signaling/facilitation of osteoclast formation [142]
MC3T3-E1-EVs (mouse preosteoblast cell line) In vitro and in vivo (mice subcutaneous back defect model) TRIP-1 Increased matrix mineralization during differentiation/initiation of the calcium phosphate nucleation process/increased expression of Runx2 and alkaline phosphatase [143]
Osteoclast derived EVs In vitro and in vivo (mice femoral defect model) miR-214-3p Inhibition of osteoblast activity/reduction in bone formation [144]
Human synovial fluid EVs In vitro miR-26a-5p, miR-146a-5p, miR-328-5p, and miR-4654 Regulation of EVs miRNA cargo by estrogen signaling/increased catabolic activity and inflammatory genes/decreased anabolic genes [145]
Murine-derived BMMSCs-EVs In vitro and in vivo (mice subcutaneous defect model) miR-151-5p Promotion of osteogenic differentiation/reduction of adipogenic differentiation/increased mineralized nodule formation/up-regulation of the Runx2, ALP, and OCN/increased in vivo bone formation/inhibition of IL4Rα expression/down-regulating mTOR pathway activation [146]
hBMSCs-EVs In vitro miR-199b, miR-218, miR148a, miR-135b, and miR-221 Modulatory effect on RNA degradation, mRNA surveillance pathway, Wnt signaling pathway, and RNA transport/potential effect on pathogenesis of osteogenic dysfunction [147]
hMSCs-EVs In vitro miR-31-3p/5p and miR-10b-5p Increased ALP activity compared with the initial time point/induction of osteogenic differentiation in a stage-dependent manner/enrichment of the pathways endocytosis (hsa04144), regulation of actin cytoskeleton (hsa04810), spliceosome (hsa03040), RNA transport (hsa03013), mRNA surveillance pathway (hsa03015) and protein digestion and absorption (hsa04974) [148]
hASCs-EVs In vitro and in vivo (rat calvarial defect model) miR-375 Improved osteogenic differentiation/inhibition of IGFBP3 [149]
Supernatants of rat bone marrow EPCs derived EVs In vitro and in vivo (rat unilateral tibial DO model) miR-126 Accelerated bone regeneration/enhanced mechanical properties/higher vessel density/enhanced proliferation, migration, and angiogenic capacity/down-regulation of SPRED1 and activated Raf/ERK signaling [150]
hBMSCs-EVs In vitro and in vivo (rat calvarial defect model) Activation of AKT/mTOR pathway/stimulation of angiogenesis in HUVECs/enhanced bone regeneration and angiogenesis [151]
EPCs-EVs In vitro Inhibition of osteoblastic differentiation of BMSCs/inhibition of osteogenic genes expression/increased BMSCs proliferation [152]
MSCs-CM In vitro and in vivo (rat calvarial defect model) Enhanced cellular migration/promoted bone regeneration and angiogenesis during early stages [95]
SHED-EVs In vitro Wnt3a and BMP2 Promoted osteogenic differentiation/higher ALP activity/up-regulated osteogenic gene expression (RUNX2, OPN and OCN) [153]
In vitro and in vivo (rat femoral defect model) Collagen Type I, fibronectin, decorin, and biglycan Enhanced proliferation, migration and osteogenic differentiation of (BM-MSCs)/accelerated bone formation [154]
BMSCs-EVs In vitro and in vivo (mice femoral defect model) miR-26a Enhanced expression of Ocn, Runx2 and Alpl/increased ALP activity/promoted matrix mineralization/enhance osteoblastic differentiation/enhanced bone mass/accelerated bone healing [155]
SMSCs-EVs In vitro and in vivo (rat model of GC-induced ONFH) Enhanced proliferation and anti-apoptotic responses of BMSCs/inhibition of the decreased osteogenic response caused by MPS injection [156]
BMSCs-EVs In vitro and in vivo (rat model of unilateral tibial DO) Increased the ALP activity and calcium mineral deposition/upregulation of ALP, RUNX2, and OCN/mediated through the SDF-1/CXCR4 axis [157]
hWJ-MSCs-EVs In vitro and in vivo (rat model of GIONFH) miR-21 Anti-apoptotic and proliferative effects on MLO-Y4 osteocytes mediated by the PTEN-AKT signaling pathway [158]
BMSCs-EVs In vitro and in vivo (rabbit SANFH model) Increased OCN and ALP expression/accelerated trabecula bone regeneration and enhanced proliferation, migration and tube formation of HUVECs through HIF-1α pathway [159]
hUC-MSC-EVs In vitro and in vivo (SNFH rats Enhanced callus formation, reduced apoptotic cells, and elevated expressions of CD31, BMP-2, and VEGF [160]
BMSC-J-EVs In vitro and in vivo (critical-sized mouse calvarial defect model) Induced osteogenic differentiation with significant increase in ALP and alizarin red staining/upregulation of osteogenic marker (ALP, OSX and RUNX2) expression [161]
BMSCs-EVs In vitro and in vivo (ONFH rabbit model) microRNA-122-5p Attenuated ONFH development by down-regulating SPRY2 via the RTK/Ras/mitogen-activated protein kinase (MAPK) signaling pathway [162]
iPS-MSC-Exo In vitro and in vivo (steroid-induced rat osteonecrosis model) Reduced osteonecrosis, improved bone structure parameters including increased BV/TV, bone surface area over bone volume, TbTh and TbN, and enhanced angiogenesis and expression of VEGFR2 and CD31
Enhanced proliferation, migration and tube-forming capacities/activated PI3K/Akt signaling pathway
hUC-MSCs-EVs In vitro and in vivo (mouse femoral fracture model) miR-126 Promoted proliferation, migration, and tube formation of HUVECs/enhanced angiogenesis through a SPRED1/Ras/Erk pathway [164]
hBM-MSCs-EVs In vitro and in vivo (rat model of ONFH) miR-224‐3p Enhanced angiogenesis by promoting proliferation, migration, and tube formation of HUVECs/ameliorated osteonecrosis, with increased bone viability/increased protein levels of FIP200 and VEGF [165]
BM-MSCs-EVs In vitro and in vivo (rat femoral fracture model) miRNA-128-3p Increased newly formed callus, BV/TV, and gene expression levels of RUNX2, ALP, and Col I/enhanced osteogenic differentiation through Smad5 inhibition [166]
hUC-MSCs-EVs In vitroand in vivo (rat model of disuse osteoporosis) miR-1263 Improvements in bone histology and structural parameters including increased BV/TV, TbTh and TbN, and decreased TbSp/reduced apoptosis of bone marrow MSCs with decreased Mob1 and increased YAP expression [167]
BMMSC-EVs In vitroand in vivo (rat model of femoral nonunion) Accelerated bone healing with more new bone formation, higher radiographic score and BV/TV/improved proliferation and migration of y HUVECs and MC3T3-E1 cells/increased angiogenesis-related markers (CD31, VEGF, and HIF-1α)/upregulation of osteogenesis markers (OPN and OGN, BMP-2, Smad1, and RUNX2)/overall improved fracture healing score [168]
hUC-MSCs-EVs In vivo (rat femoral fracture model) Significant fracture healing with upregulated expression levels of β-catenin and Wnt3a and osteogenic marker genes including Col I, OPN, and RUNX2 [169]
BM-MSCs-EVs In vitroand in vivo (rat irradiation bone loss model) Reduced oxidative stress, accelerated DNA damage repair, and reduced proliferation inhibition and cell senescence-associate protein expression/activated Wnt/β-catenin pathway [170]