Engineering Extracellular Vesicles as Delivery Systems in Therapeutic Applications.
Advanced science (Weinheim, Baden-Wurttemberg, Germany)
Extracellular vesicles (EVs) are transport vesicles secreted by living cells and released into the extracellular environment. Recent studies have shown that EVs serve as "messengers" in intercellular and inter-organismal communication, in both normal and pathological processes. EVs, as natural nanocarriers, can deliver bioactivators in therapy with their endogenous transport properties. This review article describes the engineering EVs of sources, isolation method, cargo loading, boosting approach, and adjustable targeting of EVs. Furthermore, the review summarizes the recent progress made in EV-based delivery systems applications, including cancer, cardiovascular diseases, liver, kidney, nervous system diseases, and COVID-19 and emphasizes the obstacles and challenges of EV-based therapies and possible strategies.
Extracellular Vesicles in Breast Cancer: From Biology and Function to Clinical Diagnosis and Therapeutic Management.
International journal of molecular sciences
Breast cancer (BC) is the first worldwide most frequent cancer in both sexes and the most commonly diagnosed in females. Although BC mortality has been thoroughly declining over the past decades, there are still considerable differences between women diagnosed with early BC and when metastatic BC is diagnosed. BC treatment choice is widely dependent on precise histological and molecular characterization. However, recurrence or distant metastasis still occurs even with the most recent efficient therapies. Thus, a better understanding of the different factors underlying tumor escape is mainly mandatory. Among the leading candidates is the continuous interplay between tumor cells and their microenvironment, where extracellular vesicles play a significant role. Among extracellular vesicles, smaller ones, also called exosomes, can carry biomolecules, such as lipids, proteins, and nucleic acids, and generate signal transmission through an intercellular transfer of their content. This mechanism allows tumor cells to recruit and modify the adjacent and systemic microenvironment to support further invasion and dissemination. By reciprocity, stromal cells can also use exosomes to profoundly modify tumor cell behavior. This review intends to cover the most recent literature on the role of extracellular vesicle production in normal and cancerous breast tissues. Specific attention is paid to the use of extracellular vesicles for early BC diagnosis, follow-up, and prognosis because exosomes are actually under the spotlight of researchers as a high-potential source of liquid biopsies. Extracellular vesicles in BC treatment as new targets for therapy or efficient nanovectors to drive drug delivery are also summarized.
Review: Milk Small Extracellular Vesicles for Use in the Delivery of Therapeutics.
Small extracellular vesicles (sEVs, "exosomes") in milk have attracted considerable attention for use in delivering therapeutics to diseased tissues because of the following qualities. The production of milk sEVs is scalable, e.g., more than 10 sEVs may be obtained annually from a single cow. Milk EVs protect their cargo against degradation in the gastrointestinal tract and during industrial processing. Milk sEVs and their cargo are absorbed following oral administration and they cross barriers such as intestinal mucosa, placenta and the blood-brain barrier in humans, pigs, and mice. Milk sEVs do no alter variables of liver and kidney function, or hematology, and do not elicit immune responses in humans, rats, and mice. Protocols are available for loading milk sEVs with therapeutic cargo, and a cell line is available for assessing effects of milk sEV modifications on drug delivery. Future research will need to assess and optimize sEV shelf-life and storage and effects of milk sEV modifications on the delivery of therapeutic cargo to diseased tissues.
Efficacy of extracellular vesicles of different cell origins in traumatic brain injury: A systematic review and network meta-analysis.
Frontiers in neuroscience
Background:There was still no effective treatment for traumatic brain injury (TBI). Recently, many preclinical studies had shown promising efficacy of extracellular vesicles (EVs) from various cell sources. Our aim was to compare which cell-derived EVs were most effective in treating TBI through a network meta-analysis. Methods:We searched four databases and screened various cell-derived EVs for use in preclinical studies of TBI treatment. A systematic review and network meta-analysis were conducted for two outcome indicators, modified Neurological Severity Score (mNSS) and Morris Water Maze (MWM), and they were ranked by the surface under the cumulative ranking curves (SUCRA). Bias risk assessment was performed with SYRCLE. R software (version 4.1.3, Boston, MA, USA) was used for data analysis. Results:A total of 20 studies were included in this study, involving 383 animals. Astrocyte-derived extracellular vesicles (AEVs) ranked first in response to mNSS at day 1 (SUCRA: 0.26%), day 3 (SUCRA: 16.32%), and day 7 (SUCRA: 9.64%) post-TBI. Extracellular vesicles derived from mesenchymal stem cells (MSCEVs) were most effective in mNSS assessment on day 14 (SUCRA: 21.94%) and day 28 (SUCRA: 6.26%), as well as MWM's escape latency (SUCRA: 6.16%) and time spent in the target quadrant (SUCRA: 86.52%). The result of mNSS analysis on day 21 showed that neural stem cell-derived extracellular vesicles (NSCEVs) had the best curative effect (SUCRA: 6.76%). Conclusion:AEVs may be the best choice to improve early mNSS recovery after TBI. The efficacy of MSCEVs may be the best in the late mNSS and MWM after TBI. Systematic review registration:https://www.crd.york.ac.uk/prospero/, identifier CRD42023377350.
Hyper-inflammation of astrocytes in patients of major depressive disorder: Evidence from serum astrocyte-derived extracellular vesicles.
Brain, behavior, and immunity
Astrocyte-derived extracellular vesicles (ADEs) allow the in vivo probing of the inflammatory status of astrocytes practical. Serum sample and ADEs were used to test the inflammatory hypothesis in 70 patients with major depressive disorder (MDD) and 70 matched healthy controls (HCs). In serum, tumor necrosis factor α (TNF-α) and interleukin (IL)-17A were significantly increased, where as IL-12p70 was significantly reduced in the MDD patients compared with HCs. In ADEs, all inflammatory markers (Interferon-γ, IL-12p70, IL-1β, IL-2, IL-4, IL-6, TNF-α, and IL-17A) except IL-10 were significantly increased in the MDD patients, the Hedge's g values of elevated inflammatory markers varied from 0.48 to 1.07. However, there were no differences of all inflammatory markers whether in serum or ADEs between MDD-drug free and medicated subgroups. The association of inflammatory biomarkers between ADEs and serum did not reach statistically significance after multi-comparison correction neither in the HCs nor MDD patients. The spearman coefficients between inflammatory factors and clinical characteristics in the MDD patients, such as onset age, disease course, current episode duration, and severity of depression, were nonsignificant after multi-comparison correction. In the receiver operating characteristic curves analysis, the corrected partial area under the curve (pAUC) of each inflammatory markers in ADEs ranged from 0.522 to 0.696, and the combination of these inflammatory factors achieved a high pAUC (>0.9). Our findings support the inflammatory glial hypothesis of depression, and suggests that in human ADEs could be a useful tool to probe the in vivo astrocyte status.
Osteoblast derived extracellular vesicles induced by dexamethasone: A novel biomimetic tool for enhancing osteogenesis .
Frontiers in bioengineering and biotechnology
Extracellular vesicles (EVs) are newly appreciated communicators involved in intercellular crosstalk, and have emerged as a promising biomimetic tool for bone tissue regeneration, overcoming many of the limitations associated with cell-based therapies. However, the significance of osteoblast-derived extracellular vesicles on osteogenesis has not been fully established. In this present study, we aim to investigate the therapeutic potential of extracellular vesicles secreted from consecutive 14 days of dexamethasone-stimulated osteoblasts (OB-EV) to act as a biomimetic tool for regulating osteogenesis, and to elucidate the underlying mechanisms. OB-EV treated groups are compared to the clinically used osteo-inductor of BMP-2 as control. Our findings revealed that OB-EV have a typical bilayer membrane nanostructure of, with an average diameter of 178 ± 21 nm, and that fluorescently labeled OB-EV were engulfed by osteoblasts in a time-dependent manner. The proliferation, attachment, and viability capacities of OB-EV-treated osteoblasts were significantly improved when compared to untreated cells, with the highest proliferative rate observed in the OB-EV + BMP-2 group. Notably, combinations of OB-EV and BMP-2 markedly promoted osteogenic differentiation by positively upregulating osteogenesis-related gene expression levels of , , , , , and relative to BMP-2 or OB-EV treatment alone. Mineralization assays also showed greater pro-osteogenic potency after combined applications of OB-EV and BMP-2, as evidenced by a notable increase in mineralized nodules (calcium deposition) revealed by Alkaline Phosphatase (ALP), Alizarin Red Alizarin Red staining (ARS), and von Kossa staining. Therefore, our findings shed light on the potential of OB-EV as a new therapeutic option for enhancing osteogenesis.
PBMC-derived extracellular vesicles in a smoking-related inflammatory disease model.
European journal of immunology
Extracellular vesicles (EVs) function as mediators of intercellular communication and as such influence the recipient cell function. EVs derived from immune cells can carry out many of the same functions as their parental cells, as they carry costimulatory molecules, antigens, and antigen-MHC complexes. As a result, there is a strong interest in understanding the composition and origin of immune cell-derived EVs in order to understand their role in the pathogenesis of diseases. This study aimed to optimize methodologies to study immune cell-derived EVs. Peripheral blood mononuclear cell-derived small EVs were isolated and observed using conventional transmission electron microscopy and sized by nanoparticle tracking analysis. They were then enumerated and profiled using imaging flow cytometry and were further characterized using a flow cytometric multiplex bead assay. These techniques were then applied to our current research, namely smoking-related inflammatory disease. We present here a comprehensive approach to analyze PBMC-derived small EVs in smoking-related inflammatory disease following the Minimal Information for Studies of Extracellular Vesicle 2018 guidelines.
Role of adipocyte-derived extracellular vesicles during the progression of liver inflammation to hepatocellular carcinoma.
Journal of cellular physiology
Extracellular vesicles are membrane-bound cargos that vary in size and are stably transported through various bodily fluids. Extracellular vesicles communicate information between the cells and organs. Extracellular vesicles from the diseased cells alter cellular responses of the recipient cells contributing to disease progression. In obesity, adipocytes become hypertrophic and the extracellular vesicles from these dysfunctional adipocytes showed altered cargo contents instigating pathophysiological response leading to chronic liver diseases. In this review, the role of adipocyte-derived extracellular vesicles on the progression of liver inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma are extensively discussed. Newer approaches are crucial to take advantage of extracellular vesicles and their content as biomarkers to diagnose initial liver inflammation before reaching to an irreversible liver failure stage.
Extracellular vesicles in carcinoma microenvironment.
Biochemical Society transactions
Cancer is a significant cause of death, precluding increasing life expectancy worldwide. That is a multifactorial disease initiated by intrinsic or extrinsic factors that induce cell differentiation into cancer cells. However, cancer development, progression, and metastasis are not controlled only by cancer cells. The entire environment around these cells, named tumor microenvironment (TME), influences tumor development and spread. The tumor microenvironment is formed by cancer cells and heterogenous nonmalignant cells integrated with a complex extracellular matrix. The main cellular components of the TME are cancer-associated fibroblasts (CAFs), T lymphocytes, B cells, tumor-associated macrophages (TAMs), dendritic cells (DC), natural killer (NK) cells, tumor-associated neutrophils (TANs), Stem Cells, Endothelial Cells and their soluble secreted extracellular vesicles (EVs) that modulate cancer cells to establish and disseminate. This review provides a recent insight into the role of EVs secreted from different populations of the TME associated with the initiation and progression of carcinoma.
Extracellular vesicles and Alzheimer's disease in the novel era of Precision Medicine: implications for disease progression, diagnosis and treatment.
Extracellular vesicles (EVs), secreted membranous nano-sized particles, are critical intercellular messengers participating in nervous system homeostasis, while recent evidence implicates EVs in Alzheimer's disease (AD) pathogenesis. Specifically, small EVs have been shown to spread toxic proteins, induce neuronal loss, and contribute to neuroinflammation and AD progression. On the other hand, EVs can reduce amyloid-beta deposition and transfer neuroprotective substances between cells, mitigating disease mechanisms. In addition to their roles in AD pathogenesis, EVs also exhibit great potential for the diagnosis and treatment of other brain disorders, representing an advantageous tool for Precision Medicine. Herein, we summarize the contribution of small EVs to AD-related mechanisms and disease progression, as well as their potential as diagnostic and therapeutic agents for AD.
Extracellular vesicles derived from lung cancer cells promote the progression of lung cancer by delivering miR-151a-5p.
Experimental cell research
Extracellular vesicles (EVs) participate in the occurrence and development of lung cancer. MiR-151a-5p has been reported to be highly expressed in the tumor tissues of lung cancer. The aim of this work was to investigate whether EVs can affect lung cancer progression by delivering miR-151a-5p. In this work, we found that miR-151a-5p was highly expressed in serum of lung cancer patients. Up-regulation of miR-151a-5p and down-regulation of Nedd4-binding partner-1 (N4BP1) were observed in lung cancer cell lines. The expression of miR-151a-5p was also increased in H520-derived EVs. H520-derived EVs promoted cell proliferation, inhibited apoptosis of H520 cells by delivering miR-151a-5p. EVs containing miR-151a-5p repressed E-cadherin expression and elevated N-cadherin and Vimentin expression to impede epithelial-mesenchymal transition (EMT) of H520 cells. Additionally, the interaction between miR-151a-5p and N4BP1 was verified by luciferase reporter assay, showing that miR-151a-5p interacted with N4BP1. MiR-151a-5p repressed N4BP1 expression by interacting with N4BP1. EVs containing miR-151a-5p promoted malignant phenotypes of H520 cells by targeting N4BP1. Finally, a tumor-bearing mouse model was constructed by inoculation of H520 cells with miR-151a-5p overexpression or knockdown. Overexpression of miR-151a-5p accelerated tumor growth of lung cancer in vivo, and repressed N4BP1 expression in the tumor tissues. Knockdown of miR-151a-5p caused opposite results. In conclusion, this work demonstrated that lung cancer cell-derived EVs secreted miR-151a-5p to promote cell proliferation, and inhibit apoptosis and EMT of lung cancer cells by targeting N4BP1, thereby accelerating lung cancer progression. Thus, this study suggests that EVs-derived miR-151a-5p may be a potential target for lung cancer treatment.
The CRISPR/Cas9 System Delivered by Extracellular Vesicles.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems can precisely manipulate DNA sequences to change the characteristics of cells and organs, which has potential in the mechanistic research on genes and the treatment of diseases. However, clinical applications are restricted by the lack of safe, targeted and effective delivery vectors. Extracellular vesicles (EVs) are an attractive delivery platform for CRISPR/Cas9. Compared with viral and other vectors, EVs present several advantages, including safety, protection, capacity, penetrating ability, targeting ability and potential for modification. Consequently, EVs are profitably used to deliver the CRISPR/Cas9 in vivo. In this review, the advantages and disadvantages of the delivery form and vectors of the CRISPR/Cas9 are concluded. The favorable traits of EVs as vectors, such as the innate characteristics, physiological and pathological functions, safety and targeting ability of EVs, are summarized. Furthermore, in terms of the delivery of the CRISPR/Cas9 by EVs, EV sources and isolation strategies, the delivery form and loading methods of the CRISPR/Cas9 and applications have been concluded and discussed. Finally, this review provides future directions of EVs as vectors of the CRISPR/Cas9 system in clinical applications, such as the safety, capacity, consistent quality, yield and targeting ability of EVs.
The roles of extracellular vesicles in major depressive disorder.
Frontiers in psychiatry
Major depressive disorder (MDD) is a serious mental disease characterized by depressed mood, loss of interest and suicidal ideation. Its rising prevalence has rendered MDD one of the largest contributors to the global disease burden. However, its pathophysiological mechanism is still unclear, and reliable biomarkers are lacking. Extracellular vesicles (EVs) are widely considered important mediators of intercellular communication, playing an important role in many physiological and pathological processes. Most preclinical studies focus on the related proteins and microRNAs in EVs, which can regulate energy metabolism, neurogenesis, neuro-inflammation and other pathophysiological processes in the development of MDD. The purpose of this review is to describe the current research progress of EVs in MDD and highlight their potential roles as biomarkers, therapeutic indicators and drug delivery carriers for the treatment of MDD.
Large tumour-derived extracellular vesicles as prognostic indicators of metastatic cancer patient survival.
British journal of cancer
Extracellular vesicles (EVs) are released by all cells and produced at particularly high levels by many cancer cells, often inducing pro-tumorigenic effects. Since these cancer EVs carry tumour proteins and RNAs, they can potentially be used at biomarkers. The heterogeneity of surface markers and cargos carried by EVs, however, presents some challenges to developing such approaches. Nanou et al.  found that automated counting of large tumour-derived EVs (tdEVs) performed at least as effectively as counting circulating tumour-derived cells (CTCs) and with higher sensitivity, in distinguishing the survival of patients with castration-resistant prostate cancer (CRPC), metastatic colorectal cancer (mCRC) and metastatic breast cancer (MBC), but not for non-small cell lung cancer (NSCLC). Subsequent work has suggested that these tdEVs may also be used to assess tumour subtype and that the number of large EVs produced by endothelial cells can also be increased in cancer patients. While by itself, the tdEV imaging approach used by Nanou et al.  is not specific enough to predict the survival of individual patients, in combination with other EV-associated assays, this test, perhaps enhanced through the inclusion of other tumour antigens, could prove invaluable in predicting cancer survival and other outcomes in the clinic.
An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue.
Journal of visualized experiments : JoVE
Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase Ι) with tissue, filtration through a 70 µm cell strainer, differential ultracentrifugation, and filtration through a 0.22 µm membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.
Extracellular vesicles in osteoarthritis of peripheral joint and temporomandibular joint.
Frontiers in endocrinology
Osteoarthritis (OA) is a disabling disease with significant morbidity worldwide. OA attacks the large synovial joint, including the peripheral joints and temporomandibular joint (TMJ). As a representative of peripheral joint OA, knee OA shares similar symptoms with TMJ OA. However, these two joints also display differences based on their distinct development, anatomy, and physiology. Extracellular vesicles (EVs) are phospholipid bilayer nanoparticles, including exosomes, microvesicles, and apoptotic bodies. EVs contain proteins, lipids, DNA, micro-RNA, and mRNA that regulate tissue homeostasis and cell-to-cell communication, which play an essential role in the progression and treatment of OA. They are likely to partake in mechanical response, extracellular matrix degradation, and inflammatory regulation during OA. More evidence has shown that synovial fluid and synovium-derived EVs may serve as OA biomarkers. More importantly, mesenchymal stem cell-derived EV shows a therapeutic effect on OA. However, the different function of EVs in these two joints is largely unknown based on their distinct biological characteristic. Here, we reviewed the effects of EVs in OA progression and compared the difference between the knee joint and TMJ, and summarized their potential therapeutic role in the treatment of OA.
Iron-loaded extracellular vesicles: angel or demon?
Free radical research
Extracellular vesicles (EVs) are identified as a non-classical way to mediate iron efflux except ferroportin. Interestingly, recent studies indicated that EVs pathway is a novel way involved in iron efflux. Mitochondria-derived vesicles (MDVs) are the potential mediator to load mitochondrial iron into EVs. Additionally, iron-replete cells resist excess iron-induced damage by secreting iron-loaded EVs, and the uptake of these EVs induces oxidative damage in the recipient cell. Importantly, iron-loaded EVs play a key role in aberrant iron distribution, which drives the progress of diseases like nonalcoholic fatty liver disease (NAFLD) and neurodegenerative diseases. Herein, we summarize extant research on intracellular iron export with an emphasis on EVs and put our eyes on the relationship between iron-loaded EVs with both parent and target cells. Iron-loaded EVs will be an important avenue for later research on their vital role in iron redistribution.
Emerging Roles of Neuronal Extracellular Vesicles at the Synapse.
The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry
Extracellular vesicles (EVs) are secreted from most, if not all, cell types and are implicated in short- and long-distance signaling throughout the body. EVs are also secreted from neurons and represent an emergent neuronal communication platform. Understanding the functional implications of EV signaling to recipient neurons and glia requires understanding the cell biology involved in EV biogenesis, cargo loading, secretion, uptake, and signal transduction in the recipient cell. Here we review these major questions of EV biology while highlighting recent new insights and examples within the nervous system, such as modulating synaptic function or morphogenesis in recipient neurons.
Tumor tissue derived extracellular vesicles promote diabetic wound healing.
Journal of diabetes and its complications
The diabetic wound nowadays remains a major public health challenge, which is characterized by overproduced reactive oxygen species (ROS). However, the current therapy for diabetic wounds is limited for reliable data in the general application. The growth of tumors has been revealed to share parallels with wound healing. Extracellular vesicles (EVs) derived from breast cancer have been reported to promote cell proliferation, migration and angiogenesis. The tumor tissue-derived EVs (tTi-EVs) of breast cancer performance a feature inheritance from original tissue and might accelerate the diabetic wound healing. We wonder whether the tumor-derived EVs are able to accelerate diabetic wound healing. In this study, tTi-EVs were extracted from breast cancer tissue via ultracentrifugation and size exclusion. Subsequently, tTi-EVs reversed the HO-induced inhibition of fibroblast proliferation and migration. Moreover, tTi-EVs significantly accelerated wound closure, collagen deposition and neovascularization, and finally promoted wound healing in diabetic mice. The tTi-EVs also reduced the level of oxidative stress in vitro and in vivo. Besides, the biosafety of tTi-EVs were preliminarily confirmed by blood tests and morphological analysis of major organs. Collectively, the present study proves that tTi-EVs can suppress oxidative stress and facilitate diabetic wound healing, which puts forward a novel function of tTi-EVs and provides potential treatment for diabetic wounds.
Extracellular Vesicles in Mental Disorders: A State-of-art Review.
International journal of biological sciences
Extracellular vesicles (EVs) are nanoscale particles with various physiological functions including mediating cellular communication in the central nervous system (CNS), which indicates a linkage between these particles and mental disorders such as schizophrenia, bipolar disorder, major depressive disorder, etc. To date, known characteristics of mental disorders are mainly neuroinflammation and dysfunctions of homeostasis in the CNS, and EVs are proven to be able to regulate these pathological processes. In addition, studies have found that some cargo of EVs, especially miRNAs, were significantly up- or down-regulated in patients with mental disorders. For many years, interest has been generated in exploring new diagnostic and therapeutic methods for mental disorders, but scale assessment and routine drug intervention are still the first-line applications so far. Therefore, underlying the downstream functions of EVs and their cargo may help uncover the pathogenetic mechanisms of mental disorders as well as provide novel biomarkers and therapeutic candidates. This review aims to address the connection between EVs and mental disorders, and discuss the current strategies that focus on EVs-related psychiatric detection and therapy.
Concise review: Current understanding of extracellular vesicles to treat neuropathic pain.
Frontiers in aging neuroscience
Extracellular vesicles (EVs) including exosomes are vesicular vesicles with phospholipid bilayer implicated in many cellular interactions and have the ability to transfer multiple types of cargo to cells. It has been found that EVs can package various molecules including proteins and nucleic acids (DNA, mRNA, and noncoding RNA). The discovery of EVs as carriers of proteins and various forms of RNA, such as microRNAs (miRNA) and long noncoding RNAs (lncRNA), has raised great interest in the field of drug delivery. Despite the underlying mechanisms of neuropathic pain being unclear, it has been shown that uncontrolled glial cell activation and the neuroinflammation response to noxious stimulation are important in the emergence and maintenance of neuropathic pain. Many studies have demonstrated a role for noncoding RNAs in the pathogenesis of neuropathic pain and EVs may offer possibilities as carriers of noncoding RNAs for potential in neuropathic pain treatment. In this article, the origins and clinical application of EVs and the mechanism of neuropathic pain development are briefly introduced. Furthermore, we demonstrate the therapeutic roles of EVs in neuropathic pain and that this involve vesicular regulation of glial cell activation and neuroinflammation.
Role of MSC-derived small extracellular vesicles in tissue repair and regeneration.
Frontiers in cell and developmental biology
Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and repair, secreting vesicles to the extracellular environment. Isolated exosomes were shown to affect angiogenesis, immunomodulation and tissue regeneration. Numerous efforts have been dedicated to describe the mechanism of action of these extracellular vesicles (EVs) and guarantee their safety, since the final aim is their therapeutic application in the clinic. The major advantage of applying MSC-derived EVs is their low or inexistent immunogenicity, prompting their use as drug delivery or therapeutic agents, as well as wound healing, different cancer types, and inflammatory processes in the neurological and cardiovascular systems. MSC-derived EVs display no vascular obstruction effects or apparent adverse effects. Their nano-size ensures their passage through the blood-brain barrier, demonstrating no cytotoxic or immunogenic effects. Several tests have been conducted with EVs obtained from different sources to understand their biology, molecular content, signaling pathways, and mechanisms of action. Application of EVs to human therapies has recently become a reality, with clinical trials being conducted to treat Alzheimer's disease, retina degeneration, and COVID-19 patients. Herein, we describe and compare the different extracellular vesicles isolation methods and therapeutic applications regarding the tissue repair and regeneration process, presenting the latest clinical trial reports.
Research progress on the role of extracellular vesicles derived from aging cells in osteoporosis.
The occurrence and development of many diseases are highly associated with the aging of the body. Among them, osteoporosis (OP) is a common age-related disease that tends to occur in the elderly population and is highly related to the aging factors in the body. In the process of aging transmission, the senescence-related secretory phenotype (SASP) can convey the information about aging through the paracrine pathway and endocrine mechanism through the extracellular vesicles (EVs) connected to SASP. EVs can be used as a way of conduction to join the connection between micro-environmental aging and age-related illnesses. EVs are double-layer membranous vesicles separated or secreted from the cell membrane, which mainly include microvesicles (MVs) and exosomes. Vesicular bodies secreted by this exocrine form carry a variety of cell-derived related substances (including a variety of proteins, lipids, DNA, mRNA, miRNAs, etc). These substances are mainly concentrated in human body fluids, especially can be transported to all parts of the body with the blood circulation system, and participate in the interactions between cells. Osteoporosis is closely associated with aging and aging cells, suggesting EVs were active in this pathological process. In this article, the basic mechanisms of aging cells in the occurrence and progression of osteoporosis through EVs will be discussed, to explore the connection between aging and osteoporosis, thereby providing a new perspective on the occurrence and development as well as prevention and treatment of osteoporosis.
Epithelial senescence in idiopathic pulmonary fibrosis is propagated by small extracellular vesicles.
BACKGROUND:Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that affects 3 million people worldwide. Senescence and small extracellular vesicles (sEVs) have been implicated in the pathogenesis of IPF, although how sEVs promote disease remains unclear. Here, we profile sEVs from bronchial epithelial cells and determine small RNA (smRNA) content. METHODS:Conditioned media was collected and sEVs were isolated from normal human bronchial epithelial cells (NHBEs) and IPF-diseased human bronchial epithelial cells (DHBEs). RESULTS:Increased sEV release from DHBEs compared to NHBEs (n = 4; p < 0.05) was detected by nanoparticle tracking analysis. NHBEs co-cultured with DHBE-derived sEVs for 72 h expressed higher levels of SA-β-Gal and γH2AX protein, p16 and p21 RNA and increased secretion of IL6 and IL8 proteins (all n = 6-8; p < 0.05). sEVs were also co-cultured with healthy air-liquid interface (ALI) cultures and similar results were observed, with increases in p21 and p16 gene expression and IL6 and IL8 (basal and apical) secretion (n = 6; p < 0.05). Transepithelial electrical resistance (TEER) measurements, a reflection of epithelial barrier integrity, were decreased upon the addition of DHBE-derived sEVs (n = 6; p < 0.05). smRNA-sequencing identified nineteen significantly differentially expressed miRNA in DHBE-derived sEVs compared to NHBE-derived sEVs, with candidate miRNAs validated by qPCR (all n = 5; p < 0.05). Four of these miRNAs were upregulated in NHBEs co-cultured with DHBE-derived sEVs and three in healthy ALI cultures co-cultured with DHBE-derived sEVs (n = 3-4; p < 0.05). CONCLUSIONS:This data demonstrates that DHBE-derived sEVs transfer senescence to neighbouring healthy cells, promoting the disease state in IPF.
Glycoproteomic Analysis of Urinary Extracellular Vesicles for Biomarkers of Hepatocellular Carcinoma.
Molecules (Basel, Switzerland)
Hepatocellular carcinoma (HCC) accounts for the most common form of primary liver cancer cases and constitutes a major health problem worldwide. The diagnosis of HCC is still challenging due to the low sensitivity and specificity of the serum α-fetoprotein (AFP) diagnostic method. Extracellular vesicles (EVs) are heterogeneous populations of phospholipid bilayer-enclosed vesicles that can be found in many biological fluids, and have great potential as circulating biomarkers for biomarker discovery and disease diagnosis. Protein glycosylation plays crucial roles in many biological processes and aberrant glycosylation is a hallmark of cancer. Herein, we performed a comprehensive glycoproteomic profiling of urinary EVs at the intact N-glycopeptide level to screen potential biomarkers for the diagnosis of HCC. With the control of the spectrum-level false discovery rate ≤1%, 756 intact N-glycopeptides with 154 N-glycosites, 158 peptide backbones, and 107 N-glycoproteins were identified. Out of 756 intact N-glycopeptides, 344 differentially expressed intact N-glycopeptides (DEGPs) were identified, corresponding to 308 upregulated and 36 downregulated N-glycopeptides, respectively. Compared to normal control (NC), the glycoproteins LG3BP, PIGR and KNG1 are upregulated in HCC-derived EVs, while ASPP2 is downregulated. The findings demonstrated that specific site-specific glycoforms in these glycoproteins from urinary EVs could be potential and efficient non-invasive candidate biomarkers for HCC diagnosis.
Immune Cells Release MicroRNA-155 Enriched Extracellular Vesicles That Promote HIV-1 Infection.
The hallmark of HIV-1 infection is the rapid dysregulation of immune functions. Recent investigations for biomarkers of such dysregulation in people living with HIV (PLWH) reveal a strong correlation between viral rebound and immune activation with an increased abundance of extracellular vesicles (EVs) enriched with microRNA-155. We propose that the activation of peripheral blood mononuclear cells (PBMCs) leads to an increased miR-155 expression and production of miR-155-rich extracellular vesicles (miR-155-rich EVs), which can exacerbate HIV-1 infection by promoting viral replication. PBMCs were incubated with either HIV-1 (NL4.3Balenv), a TLR-7/8 agonist, or TNF. EVs were harvested from the cell culture supernatant by differential centrifugation, and RT-qPCR quantified miR-155 in cells and derived EVs. The effect of miR-155-rich EVs on replication of HIV-1 in incubated PBMCs was then measured by viral RNA and DNA quantification. HIV-1, TLR7/8 agonist, and TNF each induced the release of miR-155-rich EVs by PBMCs. These miR-155-rich EVs increased viral replication in PBMCs infected in vitro. Infection with HIV-1 and inflammation promote the production of miR-155-rich EVs, enhancing viral replication. Such autocrine loops, therefore, could influence the course of HIV-1 infection by promoting viral replication.
Small non-coding RNA landscape of extracellular vesicles from a post-traumatic model of equine osteoarthritis.
Frontiers in veterinary science
Extracellular vesicles comprise an as yet inadequately investigated intercellular communication pathway in the field of early osteoarthritis. We hypothesised that the small non-coding RNA expression pattern in synovial fluid and plasma would change during progression of experimental osteoarthritis. In this study, we conducted small RNA sequencing to provide a comprehensive overview of the temporal expression profiles of small non-coding transcripts carried by extracellular vesicles derived from plasma and synovial fluid for the first time in a posttraumatic model of equine osteoarthritis. Additionally, we characterised synovial fluid and plasma-derived extracellular vesicles with respect to quantity, size, and surface markers. The different temporal expressions of seven microRNAs in plasma and synovial fluid-derived extracellular vesicles, eca-miR-451, eca-miR-25, eca-miR-215, eca-miR-92a, eca-miR-let-7c, eca-miR-486-5p, and eca-miR-23a, and four snoRNAs, U3, snord15, snord46, and snord58, represent potential biomarkers for early osteoarthritis. Bioinformatics analysis of the differentially expressed microRNAs in synovial fluid highlighted that in early osteoarthritis these related to the inhibition of cell cycle, cell cycle progression, DNA damage and cell proliferation as well as increased cell viability and differentiation of stem cells. Plasma and synovial fluid-derived extracellular vesicle small non-coding signatures have been established for the first time in a temporal model of osteoarthritis. These could serve as novel biomarkers for evaluation of osteoarthritis progression or act as potential therapeutic targets.
Extracellular vesicles in COVID-19 prognosis, treatment, and vaccination: an update.
Applied microbiology and biotechnology
The lethality of the COVID 19 pandemic became the trigger for one of the most meteoric races on record in the search for strategies of disease control. Those include development of rapid and sensitive diagnostic methods, therapies to treat severe cases, and development of anti-SARS-CoV-2 vaccines, the latter responsible for the current relative control of the disease. However, the commercially available vaccines are still far from conferring protection against acquiring the infection, so the development of more efficient vaccines that can cut the transmission of the variants of concerns that currently predominate and those that will emerge is a prevailing need. On the other hand, considering that COVID 19 is here to stay, the development of new diagnosis and treatment strategies is also desirable. In this sense, there has recently been a great interest in taking advantage of the benefits offered by extracellular vesicles (EVs), membrane structures of nanoscale size that carry information between cells participating in this manner in many physiological homeostatic and pathological processes. The interest has been focused on the fact that EVs are relatively easy to obtain and manipulate, allowing the design of natural nanocarriers that deliver molecules of interest, as well as the information about the pathogens, which can be exploited for the aforementioned purposes. Studies have shown that infection with SARS-CoV-2 induces the release of EVs from different sources, including platelets, and that their increase in blood, as well as some of their markers, could be used as a prognosis of disease severity. Likewise, EVs from different sources are being used as the ideal carriers for delivering active molecules and drugs to treat the disease, as well as vaccine antigens. In this review, we describe the progress that has been made in these three years of pandemic regarding the use of EVs for diagnosis, treatment, and vaccination against SARS-CoV-2 infection. KEY POINTS: • Covid-19 still requires more effective and specific treatments and vaccines. • The use of extracellular vesicles is emerging as an option with multiple advantages. • Association of EVs with COVID 19 and engineered EVs for its control are presented.
Tumour-derived small extracellular vesicles contribute to the tumour progression through reshaping the systemic immune macroenvironment.
British journal of cancer
BACKGROUND:Tumour-derived small extracellular vesicles (sEVs) play a crucial role in cancer immunomodulation. In addition to tumour immune microenvironment, the peripheral immune system also contributes significantly to cancer progression and is essential for anticancer immunity. However, a comprehensive definition of which and how peripheral immune lineages are regulated by tumour-derived sEVs during cancer development remains incomplete. METHODS:In this study, we used mass cytometry with extensive antibody panels to comprehensively construct the systemic immune landscape in response to tumour development and tumour-derived sEVs. RESULTS:Systemic immunity was dramatically altered by tumour growth and tumour-derived sEVs. Tumour-derived sEVs significantly and extensively affected immune cell population composition as well as intracellular pathways, resulting in an immunosuppressive peripheral and tumour immune microenvironment, characterised by increased myeloid-derived suppressor cells and decreased Ly6CCD8 T cells. These sEVs largely promoted hematopoietic recovery and accelerate the differentiation towards myeloid-derived suppressor cells. The knockdown of Rab27a reduced sEV secretion from tumour cells and delayed tumour growth and metastasis in vivo. CONCLUSIONS:These results highlight that tumour-derived sEVs function as a bridge between peripheral immunity regulation and the tumour microenvironment, and contribute to cancer progression through altering the composition and function of the global immune macroenvironment.
Circulating brain-derived extracellular vesicles expressing neuroinflammatory markers are associated with HIV-related neurocognitive impairment.
Frontiers in immunology
Background:Neurocognitive impairment remains prevalent in people with HIV (PWH) despite long term virological suppression by antiretroviral therapy (ART) regimens. Systemic and neuro-inflammatory processes are suggested to contribute to the complex pathology leading to cognitive impairment in this population, yet the underlying mechanisms remain unresolved. Extracellular vesicles (EVs) play a central role in intracellular communication and have emerged as key modulators of immunological and inflammatory responses. In this report, we examined the impact of EVs in PWH experiencing cognitive deficits to determine their relevance in HIV associated neuropathology. Methods:EV phenotypes were measured in plasma samples from 108 PWH with either cognitive impairment (CI, n=92) or normal cognition (NC, n=16) by flow cytometry. Matched cerebrospinal fluid (CSF)-derived EVs were similarly profiled from a subgroup of 84 individuals who underwent a lumbar puncture. Peripheral blood mononuclear cells were assayed by flow cytometry to measure monocyte frequencies in a subset of 32 individuals. Results:Plasma-EVs expressing CD14, CD16, CD192, C195, and GFAP were significantly higher in HIV-infected individuals with cognitive impairment compared to individuals with normal cognition. Increased CSF-EVs expressing GFAP and CD200 were found in the cognitive impairment group compared to the normal cognition group. Frequencies of patrolling monocytes correlated with plasma-EVs expressing CD14, CD66b, MCSF, MAP2, and GFAP. Frequencies of CD195 expression on monocytes correlated positively with plasma-EVs expressing CD41a, CD62P, and CD63. Expression of CD163 on monocytes correlated positively with CSF-EVs expressing GFAP and CD200. Finally, the expression of CD192 on total monocytes correlated with CSF-EVs expressing CD200, CD62P, and CD63. Conclusions:EVs expressing monocyte activation and neuronal markers associated with HIV associated cognitive impairment, suggesting that distinct EV subsets may serve as novel biomarkers of neuronal injury in HIV infection. Further circulating platelet EV levels were linked to monocyte activation indicating a potential novel interaction in the pathogenesis of HIV-related cognitive impairment.
Alterations in the LRRK2-Rab pathway in urinary extracellular vesicles as Parkinson's disease and pharmacodynamic biomarkers.
NPJ Parkinson's disease
Expression or phosphorylation levels of leucine-rich repeat kinase 2 (LRRK2) and its Rab substrates have strong potential as disease or pharmacodynamic biomarkers. The main objective of this study is therefore to assess the LRRK2-Rab pathway for use as biomarkers in human, non-human primate (NHP) and rat urine. With urine collected from human subjects and animals, we applied an ultracentrifugation based fractionation protocol to isolate small urinary extracellular vesicles (uEVs). We used western blot with antibodies directed against total and phosphorylated LRRK2, Rab8, and Rab10 to measure these LRRK2 and Rab epitopes in uEVs. We confirm the presence of LRRK2 and Rab8/10 in human and NHP uEVs, including total LRRK2 as well as phospho-LRRK2, phospho-Rab8 and phospho-Rab10. We also confirm LRRK2 and Rab expression in rodent uEVs. We quantified LRRK2 and Rab epitopes in human cohorts and found in a first cohort that pS1292-LRRK2 levels were elevated in individuals carrying the LRRK2 G2019S mutation, without significant differences between healthy and PD groups, whether for LRRK2 G2019S carriers or not. In a second cohort, we found that PD was associated to increased Rab8 levels and decreased pS910-LRRK2 and pS935-LRRK2. In animals, acute treatment with LRRK2 kinase inhibitors led to decreased pT73-Rab10. The identification of changes in Rab8 and LRRK2 phosphorylation at S910 and S935 heterologous phosphosites in uEVs of PD patients and pT73-Rab10 in inhibitor-dosed animals further reinforces the potential of the LRRK2-Rab pathway as a source of PD and pharmacodynamic biomarkers in uEVs.
Extracellular Vesicles in Acute Kidney Injury.
Extracellular vesicles (EVs) are promising novel cellular communicators and biomarkers in acute kidney injury (AKI). These submicron vesicles derive from all cell types along the urinary tract and reflect molecular processes of their parent cells and physiological and pathological conditions in AKI. Several EV protein and RNA biomarker candidates have been identified. They have shown to differentiate AKI etiology and pinpoint to disease mechanisms. In fact, EV research has opened up a new frontier of biomarker discovery since some less abundant biomarkers are concentrated in EVs, which makes them more easily detectable. EVs are also functional and are involved in intra-nephron communication. Tubular-interstitial communication is current focus in EV research in AKI as it can help to understand maladaptive processes in AKI. EVs are also promising therapeutic tools and have been shown to be regenerative in many different models of AKI. Rigorous studies are needed to validate these findings, and more sensitive EV detection and characterization tools need to be developed to dissect EV biology in renal disease and AKI.
Real-Time Dissection of the Transportation and miRNA-Release Dynamics of Small Extracellular Vesicles.
Advanced science (Weinheim, Baden-Wurttemberg, Germany)
Extracellular vesicles (EVs) are cell-derived membrane-enclosed structures that deliver biomolecules for intercellular communication. Developing visualization methods to elucidate the spatiotemporal dynamics of EVs' behaviors will facilitate their understanding and translation. With a quantum dot (QD) labeling strategy, a single particle tracking (SPT) platform is proposed here for dissecting the dynamic behaviors of EVs. The interplays between tumor cell-derived small EVs (T-sEVs) and endothelial cells (ECs) are specifically investigated based on this platform. It is revealed that, following a clathrin-mediated endocytosis by ECs, T-sEVs are transported to the perinuclear region in a typical three-stage pattern. Importantly, T-sEVs frequently interact with and finally enter lysosomes, followed by quick release of their carried miRNAs. This study, for the first time, reports the entire process and detailed dynamics of T-sEV transportation and cargo-release in ECs, leading to better understanding of their proangiogenic functions. Additionally, the QD-based SPT technique will help uncover more secrets of sEV-mediated cell-cell communication.
MSC-derived extracellular vesicles as nanotherapeutics for promoting aged liver regeneration.
Journal of controlled release : official journal of the Controlled Release Society
Aging is one of the critical factors to impair liver regeneration leading to a high incidence of severe complications after hepatic surgery in the elderly population without any effective treatment for clinical administration. As cell-free nanotherapeutics, mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been demonstrated the therapeutic potentials on liver diseases. However, the effects of MSC-EVs on the proliferation of aged hepatocytes are largely unclear. In this study, we found MSCs could reduce the expression of senescence-associated markers in the liver and stimulate its regeneration in aged mice after receiving a two-thirds partial hepatectomy (PHx) through their secreted MSC-EVs. Using RNA-Seq and AAV9 vector, we mechanistically found that these effects of UC-MSC-EVs partially attributed to inducing Atg4B-related mitophagy. This effect repairs the mitochondrial status and functions of aged hepatocytes to promote their proliferation. And protein mass spectrum analysis uncovered that DEAD-Box Helicase 5 (DDX5) enriches in UC-MSC-EVs, which interacts with E2F1 to facilitate its nuclear translocation for activating the expression of Atg4B. Collectively, our data show that MSC-EVs act nanotherapeutic potentials in anti-senescence and promoting regeneration of aged liver by transferring DDX5 to regulate E2F1-Atg4B signaling pathway that induce mitophagy, which highlights the clinical application valuation of MSC-EVs for preventing severe complications in aged population receiving liver surgery.
Diagnosis potential of subarachnoid hemorrhage using miRNA signatures isolated from plasma-derived extracellular vesicles.
Frontiers in pharmacology
The diagnosis and clinical management of aneurysmal subarachnoid hemorrhage (aSAH) is currently limited by the lack of accessible molecular biomarkers that reflect the pathophysiology of disease. We used microRNAs (miRNAs) as diagnostics to characterize plasma extracellular vesicles in aSAH. It is unclear whether they can diagnose and manage aSAH. Next-generation sequencing (NGS) was used to detect the miRNA profile of plasma extracellular vesicles (exosomes) in three patients with SAH and three healthy controls (HCs). We identified four differentially expressed miRNAs and validated the results using quantitative real-time polymerase chain reaction (RT-qPCR) with 113 aSAH patients, 40 HCs, 20 SAH model mice, and 20 sham mice. Exosomal miRNA NGS revealed that six circulating exosomal miRNAs were differentially expressed in patients with aSAH versus HCs and that the levels of four miRNAs (miR-369-3p, miR-410-3p, miR-193b-3p, and miR-486-3p) were differentially significant. After multivariate logistic regression analysis, only miR-369-3p, miR-486-3p, and miR-193b-3p enabled prediction of neurological outcomes. In a mouse model of SAH, greater expression of miR-193b-3p and miR-486-3p remained statistically significant relative to controls, whereas expression levels of miR-369-3p and miR-410-3p were lower. miRNA gene target prediction showed six genes associated with all four of these differentially expressed miRNAs. The circulating exosomes miR-369-3p, miR-410-3p, miR-193b-3p, and miR-486-3p may influence intercellular communication and have potential clinical utility as prognostic biomarkers for aSAH patients.
Hybrid extracellular vesicles for drug delivery.
Extracellular vesicles (EVs) are expected to serve as interesting drug delivery vectors as they may offer unique and new properties for drug delivery. Their natural origin, protein and nucleic acid composition, and intrinsic pleiotropic therapeutic effects could enable new possibilities in the field of drug delivery. Here, we aimed to review the methods used to produce Hybrid EVs, a recently emerged type of EV-based vector made from both EVs and synthetic vectors to exploit their respective properties. Hybrid EV/synthetic objects can be obtained by incubation, electrostatic interactions, polyethylene glycol (PEG)-mediated fusion, co-extrusion, freeze-thawing, or simple EV surface modification, leading to different types of objects. We also opted to review the properties of these vectors, and specifically compared them with those of other drug delivery vectors. It has to be noticed that only a limited number of study report loading metrics that allow cross article comparison. Based on this critical analysis, we attempted to draw the pith and marrow from these relatively difficult-to-compare studies and integrate them into the more general context of opportunities in drug delivery and drug development, with a particular focus on oncology.
microRNAs profiling of small extracellular vesicles from midbrain tissue of Parkinson's disease.
Frontiers in molecular neuroscience
Small extracellular vesicles (sEVs) are generated by all types of cells during physiological or pathological conditions. There is growing interest in tissue-derived small extracellular vesicles (tdsEVs) because they can be isolated from a single tissue source. Knowing the representation profile of microRNA (miRNA) in midbrain tissue-derived sEVs (bdsEVs) and their roles is imperative for understanding the pathological mechanism and improving the diagnosis and treatment of Parkinson's disease (PD). bdsEVs from a rat model of PD and a sham group were separated and purified using ultracentrifugation, size-exclusion chromatography (SEC), and ultrafiltration. Then, miRNA profiling of bdsEVs in both groups was performed using next-generation sequencing (NGS). The expression levels of 180 miRNAs exhibited significant differences between the two groups, including 114 upregulated and 66 downregulated genes in bdsEVs of PD rats compared with the sham group ( < 0.05). Targets of the differentially expressed miRNAs were predicted by miRanda and RNAhybrid, and their involvement in the signaling pathways and cellular function has been analyzed through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO). Furthermore, we explored the expression levels of miR-103-3p, miR-107-3p, miR-219a-2-3p, and miR-379-5p in bdsEVs, sEVs derived from plasma, and plasma of both groups of rats. Interestingly, the expression levels of miR-103-3p, miR-107-3p, miR-219a-2-3p, and miR-379-5p were elevated in bdsEVs and sEVs from plasma; in contrast, their expression levels were decreased in plasma of the rat model of PD. In summary, miRNAs may play a significant role in the onset and development of PD, and miRNAs need to be selected carefully as a research subject for exploring the pathological mechanism and the potential therapeutic targets and diagnostic markers of PD.
DPSC-Derived Extracellular Vesicles Promote Rat Jawbone Regeneration.
Journal of dental research
Repair and functional reconstruction of large jawbone defects remain one of the challenges in the field of head and neck surgery. The recent progress in tissue engineering technologies and stem cell biology has significantly promoted the development of regenerative reconstruction of jawbone defects. The multiple trophic activities of extracellular vesicles (EVs) produced by mesenchymal stem cells (MSCs) may play a critical role in their therapeutic effects. Accumulating evidence has shown the promise of dental pulp stem cells (DPSCs) in bone regeneration, but less is known about the regenerative effects of DPSC-EVs on jawbone defects. The purpose of this study is to explore the osteogenic effects of DPSC-EVs on jawbone marrow-derived MSCs (JB-MSCs) in vitro and their osteoinductive effects in a mandibular bone defect model in rats. Our results showed that JB-MSCs could efficiently uptake DPSC-EVs, which in turn significantly promoted the expression of osteogenic genes, such as runt-related transcription factor 2 (), alkaline phosphatase (), and osteocalcin (), as well as the osteogenic differentiation capability of JB-MSCs. Meanwhile, we found that the pro-osteogenic effect in vitro induced by DPSC-EVs was comparable to that induced by BMP-2 (bone morphogenetic protein 2), currently the only Food and Drug Administration-approved osteoinductive growth factor. In vivo, animals that were locally treated with DPSC-EVs laden with a commercially available collagen membrane exhibited a relatively fast wound closure and increased new bone density at the mandible defects. Our results provide evidence for the osteogenic and osteoinductive effects of DPSC-EVs on jawbone regeneration. Due to the accessibility, rapid proliferation, and osteogenic propensity of DPSCs, DPSC-EVs may represent a safe cell-free therapeutic approach for craniofacial bone regeneration.