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

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]

hPDLSCs-EVs

hPDLSCs-PEI-EVs

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]

hPDLSCs-CM

hPDLSCs-EVs

hPDLSCs-PEI-EVs

In vitro and in vivo (rat calvarial defect model)

–

Enhanced osseous regeneration, vascularization, and osseointegration/increased levels of VEGF and VEGFR2

[98]

DMSCs-EVs

BMSCs-EVs

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]

SMCs-CM

PCs-CM

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, Tb⋅Th and Tb⋅N, and enhanced angiogenesis and expression of VEGFR2 and CD31

Enhanced proliferation, migration and tube-forming capacities/activated PI3K/Akt signaling pathway

[163]

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, Tb⋅Th and Tb⋅N, and decreased Tb⋅Sp/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]