Extracellular vesicles as therapeutic tools in regenerative dentistry

Abstract

Dental and maxillofacial diseases are always accompanied by complicated hard and soft tissue defects, involving bone, teeth, blood vessels and nerves, which are difficult to repair and severely affect the life quality of patients. Recently, extracellular vesicles (EVs) secreted by all types of cells and extracted from body fluids have gained more attention as potential solutions for tissue regeneration due to their special physiological characteristics and intrinsic signaling molecules. Compared to stem cells, EVs present lower immunogenicity and tumorigenicity, cause fewer ethical problems, and have higher stability. Thus, EV therapy may have a broad clinical application in regenerative dentistry. Herein, we reviewed the currently available literature regarding the functional roles of EVs in oral and maxillofacial tissue regeneration, including in maxilla and mandible bone, periodontal tissues, temporomandibular joint cartilage, dental hard tissues, peripheral nerves and soft tissues. We also summarized the underlying mechanisms of actions of EVs and their delivery strategies for dental tissue regeneration. This review would provide helpful guidelines and valuable insights into the emerging potential of EVs in future research and clinical applications in regenerative dentistry.

Introduction

The high prevalence of damage or loss in dental and maxillofacial tissue has garnered global attention, as it greatly impacts the quality of life of patients and imposes a substantial financial burden on society [1]. Exogenous transplantation or the use of maxillofacial prostheses are currently the most common treatments for repairing dental and maxillofacial tissue [2]. However, these therapies only serve to halt disease progression and are unable to fully restore the normal physiological structure and function [3]. As a result, there is a pressing need for new treatments that can achieve genuine regeneration of dental and maxillofacial tissues.

Regenerative medicine has emerged as a promising approach to replace repaired tissue to restore normal biological functions and reduce the reliance on transplantation [4]. In particular, mesenchymal stem cells (MSCs) have shown significant potential in this field by animal and clinical studies [5]. MSCs possess remarkable abilities for self-renewal, multilineage differentiation, and robust immunomodulation, making them pivotal players in tissue regeneration [6]. However, their use has been restricted in the clinic due to concerns regarding uncontrollability and potential transformation risks, underscoring the need for alternative cell-free therapies [7]. Recent findings have shed light on the fact that MSCs primarily exert their effects through the secretion of cytokines or membranous vesicles. These secreted substances regulate the microenvironment surrounding damaged tissues and orchestrate subsequent regeneration processes via paracrine signaling [8].

Extracellular vesicles (EVs) were recently revealed as the primary component of paracrine signals of cells [9]. They constitute a heterogeneous group of cell secretomes and are secreted by almost all cell types. Based on the size, EVs can be classified into three subtypes: microvesicles, exosomes, and apoptotic bodies [10]. The International Society for Extracellular Vesicles has collectively termed these subtypes as “EVs” [11]. Compared to cells, EVs are non-replicable and exhibit lower immunogenicity and improved biocompatibility [12]. They play a crucial role in promoting the proliferation and differentiation of targeted cells and regulating the entire process of tissue regeneration [13, 14]. Previous studies have demonstrated that EVs can aid their parent cells in performing physiological functions [15]. Subsequent investigations have further explored the functions and underlying mechanisms of EVs [16], and shown that EVs can activate specific signaling pathways to facilitate cellular communication through their unique contents, including proteins, nucleic acids, and signaling peptides [17].

Compared to other organs, the oral cavity has direct communication with the external environment, which provides a favorable condition for the implantation of EVs. This also avoids the issue of EVs traversing the circulatory system, thereby reducing any residual or cumulative effects in non-treated areas. Consequently, the application of exogenous EVs in dental regenerative medicine has been extensively studied and has shown promising treatment effects [18] . Therefore, EVs, with their non-mutating and non-duplicating characteristics, are considered promising tools for dental tissue regeneration [19].

Fig. 1

Flow chart of literature search for EVs in regenerative dentistry

In this review, we examine the current literature (Fig. 1), regarding the functional roles of EVs in oral and maxillofacial tissue regeneration, including their impact on maxilla and mandible bone, periodontal tissues, temporomandibular joint cartilage, dental hard tissues, peripheral nerves, and soft tissues. Additionally, we summarize the underlying mode of actions of EVs (Fig. 2) and discuss their delivery strategies in the applications of regenerative dentistry. The existing challenges and the prospect of the future for EVs in dentistry regeneration are also discussed. This literature search was conducted in three databases (PubMed, Scopus and Google Scholar). English publications were searched using the keywords ((extracellular vesicles) OR (EVs) OR (exosomes) AND (dentistry regeneration) OR (dental tissue regeneration) OR (oral tissue regeneration)). After reviewing the titles and abstracts, 60 selected publications with full texts were selected for detailed analysis. 3 research articles were duplicated in the part of mechanism of EV-mediate dentistry regeneration.

Fig. 2

EVs parent cells source, EVs source, delivery strategies, functions and mechanisms of EVs in dental tissue regeneration. Abbreviations: EVs, extracellular vesicles; GMSC-EVs, gingival mesenchymal stem cell-derived extracellular vesicles; DPSC-EVs, dental pulp stem cell-derived extracellular vesicles; PDLSC-EVs, periodontal ligament stem cell-derived extracellular vesicles; DFC-EVs, dental follicle cell-derived extracellular vesicles; SCAP-EVs, stem cells from apical papilla-derived extracellular vesicles; SHED-EVs, human exfoliated deciduous teeth stem cell-derived extracellular vesicles; Macrophage-EVs, macrophage derived extracellular vesicles; ADMSC-EVs, adipose mesenchymal stem cell-derived extracellular vesicles; BMMSC-EVs, bone marrow mesenchymal stem cell-derived extracellular vesicles

Origin of EVs and their roles

EVs are nanoscale membrane vesicles and secreted by nearly all types of cells [20]. They are formed through the inward budding of multivesicular bodies that originate from late endosomal membrane invagination. These vesicles are subsequently released into the extracellular microenvironment by fusing with the plasma membrane [21]. During the process of EV formation, specific proteins, lipids, and nucleic acids are selectively recruited and encapsulated, granting EVs the ability to mediate paracrine crosstalk [22]. These proteins include adapter protein ALIX, endosome-related protein TSG101, and the transmembrane proteins CD9, CD63, and CD81 [23, 24]. The lipid bilayer of EVs typically comprises cholesterol, sphingomyelin, and phosphatidylserine, which significantly contribute to EV formation and their interaction with target cells [25, 26].

EVs interact with target cells through three main mechanisms, offering various avenues for studying signal pathways and therapeutic targets in different diseases [27]. Firstly, EVs engage in information transmission through receptor-ligand interactions, even without direct cell contact [15]. Secondly, EVs enhance cell adhesion properties by binding to the target cell membranes [28]. Lastly, EVs can fuse with the target cell membrane, delivering their contents into the cytoplasm and exerting biological effects [29]. The specific mechanisms of interaction depend on the composition and properties of EVs, as well as the characteristics of their parent cells [30]. Once released into the microenvironment, EVs transport their bioactive cargoes to specific cells, triggering a cascade of signaling pathways. The majority of EV components consist of proteins and nucleic acids, including DNA, mRNA, miRNA, tRNA, and non-coding RNA. While miRNA has been the focus of significant EV research due to its functional roles, more recent studies have indicated that proteins in EVs, rather than miRNA, play more critical roles in cell-cell communication.

Dental tissue-derived EVs

The oral cavity constitutes a multifaceted environment encompassing diverse tissues, including jaws, periodontium, gingiva, teeth, oral mucosa, and glands. Saliva and gingival crevicular fluid create the fluid milieu within the oral cavity. In addition, various coatings envelop these tissues, each harboring an array of bacteria, collectively forming the bacterial biofilm [31]. All cells from these tissues and bacteria can secret EVs to participate in the dental tissue development. More importantly, these EVs shape the ecological environment of the oral cavity and oral environment in turn affect the stability and bioactivity of these EVs. However, although all EVs play certain roles in the dental tissue development, EVs from stem cells derived from different dental tissues are mostly studied and utilized for dental tissue regeneration due to their multi-lineage differentiation and reproductive activity. These stem cells include dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), dental follicle progenitor cells (DFCs), gingival mesenchymal stem cells (GMSCs), stem cells from the apical papilla (SCAPs), alveolar bone-derived mesenchymal stem cells (ABMSCs) and stem cells from exfoliated deciduous teeth (SHEDs) (Fig. 3). They were utilized in different oral tissue regeneration according to their specific regenerative characteristics.

Notably, EVs sourced from DPSCs (DPSC-EVs) have garnered significant attention in the field of dentistry regeneration owing to their remarkable osteo/odonto-inductive capabilities [32, 33]. Furthermore, DPSC-EVs have exhibited enhanced anti-necrotic, immunomodulatory, and anti-apoptotic properties compared to EVs derived from bone marrow mesenchymal stem cells (BMMSC-EVs) [34]. On the other hand, EVs originating from PDLSCs (PDLSC-EVs) have been demonstrated to upregulate the expression of CD31 and VEGFA to promote angiogenesis. Additionally, they fortify osteogenesis through the regulation of insulin, AMPK, and MAPK signaling pathways, while also modulating the Th17/Treg balance to bolster anti-inflammatory capabilities [35,36,37,38]. GMSC-EVs and ABMSC-EVs have also emerged as significant contributors to bone regeneration. They exhibit anti-osteoclastogenic activity and convey miR-1260 to inhibit inflammatory bone loss [39, 40]. Furthermore, when combined with a small intestinal submucosa-extracellular matrix, GMSC-EVs facilitate tongue lingual papillae repair and promote the recovery of taste buds [41]. Moreover, EVs derived from SCAPs (SCAP-EVs) hold great promise for dentistry regeneration. They enhance dentinogenesis of BMMSCs and are considered potential candidates for dentin-pulp regeneration [35]. Meanwhile, EVs sourced from SHEDs (SHED-EVs) effectively mitigate inflammation in temporomandibular joint diseases.

Effect of EVs in regenerative dentistry

Compared to tissues such as liver, skin, and muscle, oral tissues are generally constantly exposed to microorganisms from food, drink, and the oral microbiome and have limited blood supply during the regeneration process. This greatly affect their ability to heal efficiently and makes them more vulnerable to infections and inflammation. Numerous studies have highlighted that EVs derived from various cells, particularly stem cells, exhibit beneficial effects such as pro-regenerative, pro-vascularization, anti-inflammatory, and anti-apoptotic properties, irrespective of their distinctiveness from different sources [42]. These minuscule vesicles have exhibited the capacity to regenerate bone, dental tissues, and cartilage, rendering them promising therapeutic agents in the field of dental tissue regeneration [43] (Table 1)(Fig. 4).

Fig. 3

EVs from different dental tissue-derived stem cells used for dental tissue regeneration. EVs from different dental tissue-derived stem cells are mostly studied and utilized for dental tissue regeneration due to their multi-lineage differentiation and reproductive activity. EVs from different dental tissue-derived stem cells are mostly studied and utilized for dental tissue regeneration due to their multi-lineage differentiation and reproductive activity. Dental follicle progenitor cells are sourced from the connective tissue surrounding the developing tooth germ. Stem cells from the apical papilla are obtained from the apical papilla of incompletely developed teeth. Gingival mesenchymal stem cells are found within the gingiva. Stem cells from exfoliated deciduous teeth are harvested from the dental pulp of exfoliated primary teeth. Alveolar bone-derived mesenchymal stem cells can be extracted from the alveolar bone. Dental pulp stem cells are isolated from the dental pulp of permanent teeth. Periodontal ligament stem cells are sourced from the periodontal ligament of permanent teeth

Table 1 Effect of different EVs on dental tissue regeneration

Maxillofacial tissue regeneration

Maxillofacial diseases, such as congenital cleft palate, functional mandibular reconstruction, and conditions like odontogenic osteomyelitis or tumors, necessitate precise repair and functional restoration of the affected areas [44]. Noteworthy studies have demonstrated the efficacy of EVs in addressing these challenges [45]. For instance, in a model of bisphosphonate-related osteonecrosis of the jaw (BRONJ), the introduction of EVs derived from adipose mesenchymal stem cells (ADMSC-EVs) through tail vein injection in rats led to the formation of new jawbone and improvements in bone structure parameters [46]. BMMSC-EVs showcased preventive properties against the spread of chronic inflammation associated with aging cells. They further promoted osteogenesis and angiogenesis, effectively averting the occurrence of BRONJ [47]. DPSC-EVs implanted in a rat mandibular bone defect area also exhibited heightened jawbone density and facilitated the formation of new jawbone [48].

In addition to hard tissue regeneration, EVs have demonstrated promise in maxillofacial soft tissue regeneration. GMSC-EVs, when combined with small intestinal submucosa extracellular matrix, were implanted in a rat critical-sized tongue defect site, resulting in the regeneration of epithelial cells and the restoration of taste buds and lingual papilla [41]. Moreover, EVs derived from hair follicle epidermal neural crest stem cells, in conjunction with acellular nerve allografts, were employed to bridge facial nerve defects. This intervention led to thicker myelination and robust remyelination [49]. Additionally, SCAP-EVs enhanced angiogenesis and vascularization in a rat hard palate mucosa defect model [50].

Periodontal regeneration

The periodontium, encompassing the gingiva, periodontal ligament, and alveolar bone, serves as the structural support for teeth [51]. Periodontitis, a widespread global issue, is characterized by the progressive deterioration of the periodontium and inflammation [52]. The ultimate objective of periodontal tissue regeneration is to foster the development of new periodontal bone, complete with fresh periodontal ligaments, and the reattachment of the gingiva [53].

Numerous studies have unveiled the remarkable regenerative potential of EVs in periodontal tissue regeneration. For example, BMMSC-EVs have demonstrated the ability to stimulate alveolar bone formation and repair periodontal ligaments in models of periodontitis [54, 55]. Moreover, EVs released by M2 macrophages have proven effective in preventing alveolar bone loss [56]. The injection of EVs from ADMSCs into rat periodontal pockets has resulted in the formation of cellular periodontal tissue perpendicular to the cementum and alveolar bone [57, 58]. Moreover, EVs derived from dendritic cells have exhibited potential in treating degenerative alveolar bone diseases by promoting the coverage of soft tissues over the alveolar bone [59].

Emerging evidence suggests that EVs derived from dental tissues also contribute significantly to periodontal tissue regeneration. For instance, DPSC-EVs and SCAP-EVs have been reported to inhibit alveolar bone loss [60,61,62,63]. EVs derived from dental follicle progenitor cells (DFC-EVs) have been shown to enhance the formation of denser alveolar bone with increased trabecular thickness compared to control groups [64, 65]. Gingival mesenchymal stem cell-derived EVs (GMSC-EVs), PDLSC-EVs and SHED-EVs have proven efficient in repairing alveolar bone defects, accompanied by the development of new blood vessels [66,67,68,69].

Given that periodontal diseases often involve inflammation, evaluating the function of EVs under inflammatory conditions is essential [70]. Research has demonstrated that EVs released from GMSCs treated with TNF-α effectively prevent periodontal bone resorption [39]. Furthermore, studies have shown that lipopolysaccharide (LPS)-preconditioned DFC-EVs promote the proliferation of PDLSCs and macrophages [71]. Similarly, LPS-preconditioned DFC-EVs have been found to be beneficial for the formation of integrated periodontal tissue in PDLSCs compared to healthy DFC-EVs [72].

Dental pulp regeneration

The dental pulp, the sole soft tissue within a tooth, resides within the pulp cavity, encircled by dentin. It comprises connective tissue, blood vessels, and nerves, rendering it vascularized and innervated [73]. Consequently, endodontic regeneration is a multifaceted process encompassing not only dental pulp regeneration and dentin-pulp complex formation but also pulp revascularization and neurological recovery [74].

Numerous research studies have shed light on the role of EVs in fostering dentin-pulp regeneration. EVs derived from various sources, such as SHEDs, DPSCs, SCAPs, Hertwig’s epithelial root sheath cells, and Schwann cells, have been subcutaneously implanted into mice, resulting in the promotion of dentin-pulp regeneration [35, 75,76,77,78]. Rats treated with LPS-preconditioned DPSC-EVs exhibited the formation of dental pulp-like tissue replete with new blood vessels in a model where dental pulp had been removed [79]. In another study, collagen containing SCAPs were placed at the root tip, and the cavity was filled with EVs derived from dental pulp tissue/stem cells, leading to the regeneration of dense pulp-like tissue and predentin-like tissue [80]. Intriguingly, Li et al. reported that apoptotic bodies, typically regarded as indicators of cellular end-of-life, spurred the formation of dental pulp-like tissue replete with abundant blood vessels [81]. As mentioned earlier, neurological recovery is also vital for dental pulp regeneration, with research highlighting the potential effects of EVs on neuroregeneration, thereby underscoring the promise of EVs in pulp regeneration [82].

Dental hard tissue regeneration and mineralization

Dental hard tissues encompass enamel, dentin, and cementum. Enamel is primarily composed of hydroxyapatite crystals, whereas dentin and cementum are a combination of hydroxyapatite and organic matrix [83]. Research has shown that EVs play a role in the formation and mineralization of dental hard tissues [84, 85].

In a rat pulpotomy model, DPSC-EVs prompted the creation of dentin tubes and reparative dentin bridges [86]. Another study illustrated how DPSC-EVs, in conjunction with dentin matrix, heightened the proliferation, migration, and odontogenesis of dental pulp cells, thus contributing to the continuous formation of reparative dentin [87]. Zhao et al. reported that EVs originating from macrophages with different polarization phenotypes had distinct effects on cementoblast mineralization. Specifically, EVs derived from M2 macrophages fostered cementum mineralization and curtailed root resorption [88]. Jiang et al. posited that EVs facilitated communication between epithelial and mesenchymal cells. Epithelial cell-derived EVs were found to stimulate mesenchymal cells to produce dentin sialoprotein (DSP) and partake in mineralization, while mesenchymal cell-derived EVs induced epithelial cells to generate ameloblastin and amelogenin [89]. Moreover, it was proposed that intracellular ameloblast secretory EVs played a role in enamel mineralization [84].

Temporomandibular cartilage regeneration

The temporomandibular joint (TMJ) is an intricate joint, comprising the mandibular condyle and the articular surfaces of the temporal bone, both covered with dense articular cartilage [90]. Temporomandibular joint osteoarthrosis (TMJOA) is a degenerative disease characterized by an imbalance between the synthesis and degradation of the condylar matrix mediated by chondrocytes [91]. This imbalance leads to the breakdown of the condylar matrix, resulting in joint disorganization, biomechanical alterations, disruption of the microenvironmental homeostasis around cartilage cells, and inflammation [92]. Because the TMJ cavity is an enclosed joint space with well-defined boundaries, this enclosed space provides a contained environment for injected substances, preventing their immediate dispersion into the surrounding tissues. Therefore, it has great potential to employ therapeutic EVs for the treatment of TMJOA.

Shen et al. firstly demonstrated that extracellular vesicles derived from BMMSCs under hypoxic conditions can enhance the proliferation, migration, and anabolic capacity of chondrocytes in vitro. Moreover, they exhibited pro-chondrogenic potential in vivo [93]. Similarly, SHED-EVs have shown the capability to down-regulate the expression of proinflammatory factors and matrix metalloproteinases, indicating their potential to mitigate inflammation in the temporomandibular joint and prevent further cartilage damage [94]. Other than dental tissue derived EVs, EVs derived from human embryonic mesenchymal stem cells have been observed to enhance chondrogenesis, leading to the formation of new hyaline cartilage closely resembling healthy tissue in a rat model of TMJOA [95].

Mechanisms of EV-mediate dentistry regeneration

Fig. 4

The therapeutic effects of extracellular vesicles on different dental tissue regeneration. a EVs derived from LPS-preconditioned DFCs loden on hydrogel applied in the treatment of periodontitis by repairing lost alveolar bone and promoting periodontal tissue regeneration. This figure is adapted and is freely accessible from reference [72], Licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). b EVs derived from BMMSCs prevent BRONJ by preventing the spread of chronic inflammation and promoting angiogenesis and osteogenesis. This figure is adapted and is freely accessible from reference [46], c TDM and EVs isolated from DPSCs promote reparative dentin formation. This figure is adapted and is freely accessible from reference [87], Licensed by Sage Publications and Copyright Clearance Center. d EVs derived from osteoclasts promote bone regeneration. This figure is adapted and is freely accessible from reference [14], Reprinted under the terms of the Creative Commons CC-BY license. Abbreviations: AB, alveolar bone; PL, periodontal ligament; D, cementum; ZOL, zoledronic acid; TDM, dentin matrix; D, dentin; P, pulp tissue; DB, dental bridge; BV./TV., bone volume/total volume; OCs-col, osteoclasts on collagen; OC-EVs-col, EVs derived from osteoclasts on collagen

Understanding these mechanisms is essential for harnessing the full regenerative potential of EVs. The mechanisms underlying extracellular vesicle-mediated dentistry regeneration are a complex and dynamic interplay of cellular and molecular processes. They facilitate key aspects of regeneration, including but not limited to osteogenesis, odontogenesis, mineralization of dental hard tissues, angiogenesis, immunomodulation (Table 2; Figs. 5 and 6). Through their ability to transfer these bioactive molecules, EVs modulate various signaling pathways, gene expression, and cellular behaviors, ultimately contributing to the repair and regeneration of dental tissues.

Fig. 5

The effective components and functions of EVs for dental tissue regeneration. EVs are released upon the fusion of multivesicular bodies with plasma menbranes. They aid in dental tissue regeneration by promoting odontogenic differentiation, osteogenesis differentaition, dental hard tissue mineralization, angiogenesis and regulating immunomodultion through different cargos, including but not limited to protein, MicroRNA, and mRNA

Table 2 Underlying mechanisms of EVs in the dental tissue regeneration

EVs increase odontogenic differentiation

Odontogenic differentiation constitutes a pivotal process in tooth development, and emerging research affirms the role of extracellular vesicles in inducing odontogenic differentiation and upregulating the expression of dental-related markers such as dentin sialophosphoprotein (DSPP) and dental matrix protein (DMP) [35, 96]. Of note, DPSC-EVs have been observed to undergo cellular endocytosis in a dose-dependent manner, consequently activating the p38/MAPK pathway and intensifying odontogenic differentiation [76]. Moreover, DPSC-EVs have demonstrated their potential to transport nuclear factor I/C (NFIC), a pivotal transcription factor central to tooth development. This transport, in turn, promotes the proliferation, migration, and dentinogenesis of SCAPs [97]. Studies utilizing miRNA sequencing have unveiled alterations in miRNA profiles following the uptake of DPSC-EVs, underscoring the role of EVs in orchestrating odontogenic differentiation through the TGFβ1/Smads signaling pathway [98]. In addition, miR-758-5p transported by DPSC-EVs under inflammatory conditions has the capacity to stimulate BMP signaling to ultimately govern odontogenic differentiation [99]. Meanwhile, EVs originating from Hertwig’s Epithelial Root Sheath Cells have been shown to activate the Wnt/β-catenin pathway, thereby establishing a conducive microenvironment for odontogenic differentiation by fostering the connection between epithelial cells and mesenchymal cells [78].

EVs promote osteogenic differentiation

Osteogenic differentiation plays a pivotal role in bone formation, including craniofacial and alveolar bone remodeling and repair [100]. Several studies have diligently explored the potential roles of EVs in osteogenesis. Reports indicate that PDLSC-EVs contributed to the alveolar bone regeneration by mitigating the overactivation of the Wnt signaling pathway and suppressing NF-κB activity of osteoprogenitor cells [68, 101]. Meanwhile, DFC-EVs have been shown to activate the MAPK pathway, aiding in the repair of alveolar bone defects [99, 100]. In contrast to DFC-EVs, SHED-EVs regulate osteogenesis through the AMPK pathway [69]. SHED-EVs transport mitochondrial transcription factor A mRNA, thereby instigating mitochondrial aerobic metabolism and consequently augmenting bone regeneration [102].

DPSC-EVs, GMSC-EVs, and SCAP-EVs are shown to promote the osteogenic differentiation of stem cells via the miRNAs they carry [39, 63, 99, 101]. For instance, miR-181b-5p found within osteocyte-derived EVs facilitates the osteogenic differentiation of PDLSCs through the PTEN/AKT pathway [103]. Conversely, miR-129-5p within plasma secretory EVs inhibits jawbone osteogenesis via the FZD4/β-catenin pathway [104]. Except for miRNA, mRNA and proteins in EVs can also contribute to dental bone regeneration by upregulating the osteogenic differentiations of stem cells. EVs derived from M2 macrophages transport IL-10 mRNA, activating the cellular IL-10/IL-10R pathway directly, thereby promoting osteogenesis and preserving bone homeostasis [56]. ADMSC-EVs expedite alveolar bone repair by transmitting calcitonin gene-related peptide (CGRP), a significant neuropeptide expressed during bone repair [58]. Meanwhile, umbilical cord mesenchymal stem cell-derived EVs (UMSC-EVs) were reported to enhance the osteoblastic differentiation capability of PDLSCs via the P13K/AKT pathway [105].

EVs facilitate dental hard tissue mineralization

The mineralization process of dental hard tissue is a multifaceted phenomenon characterized by intricate interactions among various organic compounds [106]. Within this context, EVs serve as reservoirs of numerous factors that contribute to the formation of hydroxyapatite crystals and calcium phosphate [107]. Nevertheless, the precise mechanism through which EVs mediate mineralization remains the subject of debate and ongoing research. For instance, Chaudhary et al. demonstrated that EVs derived from the 17IIA11 cell line transport factors that induce enamel mineralization through the activation of the Erk1/2 pathway [108]. Another investigation proposed that miR-135a in EVs promote the reciprocal interaction between epithelial and mesenchymal cells, thereby activating the Wnt/β-catenin signaling pathway and facilitating the production of dentin matrix proteins [89]. Furthermore, researchers have postulated that ameloblast secretory EVs engage in interactions with orphan phosphatase 1 (PHOSPHO1) and play a role in amelogenesis [84]. Similarly, EVs have been found to participate in the transport of Dentin Phosphophoryn (DPP) to the extracellular matrix, thereby contributing to the mineralization process [109].

EVs accelerate angiogenesis

Blood vessels play a pivotal role in delivering vital bioactive elements, encompassing growth factors, nutrients, and progenitor cells, to sites of regeneration, thereby contributing significantly to the maintenance of homeostasis [110]. Accumulating evidence suggests that EVs exhibit the capacity to expedite angiogenesis in the context of dental regeneration [111, 112]. This facilitation primarily hinges on the transfer of microRNA (miRNA) payloads encapsulated within EVs. For instance, PDLSC-EVs augmented the vascularization of dental periodontal ligaments through the transmission of vascular endothelial growth factor (VEGF) via miR-17-5p [111]. Similarly, SHED-EVs transferred miR-26a to initiate the TGF-β/Smad signaling pathway, thereby fostering angiogenesis [75]. Another investigation illustrated that SHED-EVs regulated angiogenesis via the activation of the AMPK signal pathway [69]. Moreover, SHED-EVs were reported to enhance angiogenesis even under hypoxic conditions, accomplished by the transfer of let-7f-5p and miR-210-3p, which respectively modulate the AGO1/VEGF and miR-210-3p/ephrinA3 signaling pathways [113].

In addition to miRNAs, proteins transported by EVs significantly contribute to the expediting of angiogenesis. SCAP-EVs mediated the action of Cdc42, thereby promoting vascularization and aiding in the repair of craniofacial soft tissue [50]. DPSC-EVs regulated the activation of angiogenesis by modulating the translation elongation factor Tu via the transcription factor EB (TFEB)-autophagy pathway [81]. Additionally, DPSC-EVs demonstrated the capacity to promote angiogenesis and activate the p38 MAPK pathway, showcasing substantial angiogenic potential for pulp regeneration [114].

EVs regulate immunomodulation

Extracellular vesicles play a pivotal role in orchestrating immunoregulatory processes, with a significant contribution stemming from the encapsulated miRNAs. For instance, miR-100-5p within SHED-EVs [94], miR-935 in SCAP-EVs [63], and miR-25-3p found in salivary secretory EVs [37] have demonstrated the capacity to modulate immune responses. Notably, specific miRNAs encapsulated within EVs play immunomodulatory roles through different signaling pathways. For example, EVs derived from embryonic stem cells (EBC-EVs) have been shown to suppress inflammation through the activation of adenosine receptor-dependent AMPK and AKT/ERK signaling pathways [92]. The NF-κB transcription factor, known for its pivotal role in regulating inflammatory responses, is proved to be another key response element in the immunomodulation process of EVs [115]. Numerous studies have established that EVs exert influence over immune responses in the regenerative dentistry by modulating the NF-κB signaling pathway [61, 116,117,118]. In the realm of osteoimmunology, the RANKL (NF-κB ligand) and osteoprotegerin (OPG) system bear significance [71, 119]. EVs have demonstrated their ability to regulate the RANKL-RANK-OPG signaling within the context of osteoimmunology in dental bone regeneration [55]. Furthermore, the equilibrium between Th17 and Treg cells is revealed of importance in modulating inflammation to aid in the dental tissue regeneration [120]. EVs contribute significantly to this balance by virtue of various factors, including specific miRNAs like miR-1246 and miR-155-5p, as well as cytokines such as TGF-β and IL-10 [38, 59, 62].

Delivery strategy of EVs

Fig. 6

The mode of action of extracellular vesicles in promoting different dental tissue regeneration. a EVs derived from DPSCs specifically activate endogenous EC autophagy by transferring TUFM, thereby causing angiogenesis. The acceleration of vascular reconstruction promotes dental pulp regeneration. This figure is adapted and is freely accessible from reference [81], Licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). b EVs derived from DPSCs under an inflammatory microenvironment participate in the regulating of odontogenic and osteogenic differentiation by miR-758-5p/LMBR1/BMP2/4 axis. This figure is adapted and is freely accessible from reference [99], Licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). c EVs derived from GMSCs under inflammation microenvironment enhance M2-type macrophage polarization and prevent periodontal bone loss. This figure is adapted and is freely accessible from reference [39], Licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). Abbreviations: EC, endothelial cells; hDPSC, human dental pulp stem cells; TUFM, Tu translation elongation factor, mitochondrial; TFEB, transcription factor EB; VEGF, vascular endothelial growth factor; ANG2, angiotensin 2; hDPSC-apoVs, apoptotic vesicles from human dental pulp stem cells; BMP, bone morphogenetic protein; LMBR1, limb development membrane protein 1; TNF-α, tumor necrosis factor α; DPSC-EV, EVs from dental pulp stem cells; iDPSC-EVs, EVs from dental pulp stem cells under inflammatory environment; PDLSC, periodontal ligament stem cells

The delivery of extracellular vesicles represents a critical aspect of their utilization in various therapeutic contexts. Effective delivery approaches for EVs are essential to harness their regenerative and therapeutic potential. Various strategies have been developed to facilitate the precise and targeted delivery of EVs to specific tissues or cells, ranging from direct injection to more sophisticated engineered delivery systems. In the context of regenerative dentistry, delivering EVs to oral tissues has their own particularity due to the special oral anatomical and physiological characteristics. To be specific, the oral cavity is rich in saliva containing enzymes and chemicals and oral tissues are subject to constant mechanical stress due to activities such as chewing and speaking. All these factors might affect the stability and function of extracellular vesicles which have to be taken into consideration in the application EVs in regenerative dentistry.

EVs delivery by injection

Due to the nano-size of EVs and well-established clinical procedure of intravenous injection, EVs have been initially and widely utilized through intravenous injection in various biomedical and therapeutic applications. In the field of regenerative dentistry, intravenous injection of EVs is proved to be feasible and effective. For example, in a BRONJ rat model, intravenously administered EVs were found to effectively modulate genes associated with osteogenesis and inflammation in the maxilla to promote bone regeneration [116]. However, EVs delivered systemically can become diluted in the bloodstream, which may reduce their concentration at the oral sites, potentially reducing the specificity of the treatment. Given the special oral anatomical structure, there is a preference for employing extracellular vesicles through direct injection into the target site in the context of regenerative dentistry regeneration. Research studies have provided evidence of the effectiveness of locally injected EVs in promoting the formation of new epidermal tissue and enhancing vascularization, as demonstrated in a mouse model of palatal gingiva wound healing [50]. Moreover, in an experimental model of alveolar bone loss, locally injected EVs exhibited slower clearance and demonstrated higher affinity compared to systemic injection [59]. However, it is crucial to acknowledge a significant limitation associated with the use of injected EVs, namely the stability and retention of EVs post-administration, particularly in the oral cavity. The special oral environment and the salivary flow can result in a notable loss of EVs, which significantly affect the therapeutic effectiveness and potential applications of injected EVs for the treatment of dental diseases.

EVs delivery by carriers

The direct injection of EVs into target sites in the oral cavity presents challenges related to their stability and retention in vivo. In contrast, the utilization of carrier-based delivery systems, such as hydrogels and ceramics, offers notable advantages, primarily concerning controlled release and prolonged retention duration. This carrier-based EV delivery exhibits significant potential for augmenting the therapeutic effectiveness of EVs in the field of dental regenerative medicine [121, 122]. Given the superior biocompatibility and tunability, hydrogel materials are widely used to deliver cells and EVs via minimally invasive procedures for tissue regeneration. Collagen, a natural hydrogel derived from most tissues, stands out as a popular choice for delivering EVs in the realm of regenerative dentistry. When incorporated into collagen hydrogels, EVs have demonstrated their capacity to enhance osteogenesis, odontogenesis, and the regeneration of bone and dentin-like tissues across a spectrum of oral disease conditions [35, 48, 54, 55, 72]. Given that hydrogels have a texture similar to soft tissue, it is of great advantage that apply collagen hydrogels in the regeneration of dental pulp. Several studies have demonstrated that collagen hydrogels help promote angiogenesis, thus speeding up the rate of dental pulp regeneration [76,77,78, 80]. Gelatin, a collagen derivative, also exhibits controlled-release properties for EVs, amplifying their effectiveness in promoting dentin formation [97]. Furthermore, alternative hydrogels like chitosan and alginate hydrogel have found utility as carriers for EVs in dental regeneration endeavors [55, 61, 67, 101]. Additionally, synthetic polymers such as PLGA/pDA, PLGA, and PEG-PLGA-PEG, engineered with precision to control their physical and chemical attributes, offer a means of achieving more predictable and sustained EV release for dental tissue regeneration [58, 66, 86]. However, it’s worth noting that the sustained release of EVs primarily relies on the physical encapsulation provided by these hydrogels, which typically spans several days. Consequently, there is a growing interest in developing advanced materials capable of enabling long-term EV delivery in the context of regenerative dentistry, given the chronic nature of most oral diseases.

On the other hand, ceramics are frequently used to deliver EVs for hard tissue regeneration in oral diseases due to their mechanical properties such as hardness and corrosion resistance and chemical components that is calcium and phosphate ions. Hydroxyapatite (HA), which has been available on the market for clinical therapy since the 1970s, is considered the most conventional ceramics for regenerating dental hard tissues [123]. For instance, in an ectopic dentin regeneration model, the group utilizing EVs with HA exhibited significant formation of dentin-like tissue [71]. Another noteworthy ceramic material is beta-tricalcium phosphate (β-TCP) and it is known for its biodegradability. It promotes rapid bone tissue regeneration when used in conjunction with EVs in a periodontitis model. This structure facilitates angiogenesis and actively contributes to the formation of bone tissue [68, 69].

It is of note that a burst release frequently occurs when loading EVs in ceramic materials because EVs are mostly adsorbed on to the ceramic surfaces through hydrophilic action. The combination of exosome and hybrid scaffolds might exert better regenerative effects than organic or inorganic materials alone, which needs further investigations in the regenerative dentistry.

Limitations and future perspectives

While extracellular vesicles have demonstrated significant progress in advancing dentistry regeneration [124,125,126,127], their widespread implementation in clinical trials is contingent upon addressing several limitations and challenges. First, EV composition exhibits dependency on various factors, including the cell type, donor age, state, and the microenvironment in which parent cells reside, all of which influence their functional roles [128]. For instance, the immune profiles of mesenchymal stem cell-derived EVs (MSC-EVs) have been substantiated age-dependent variations [129]. Furthermore, EVs derived from samples of differing ages exhibit disparate effects during the osteogenesis process and display varying degrees of efficacy in bone repair [130]. Consequently, it is imperative to investigate EVs from diverse contextual sources to unravel the underlying mechanisms that intricately govern their therapeutic efficacy.

The other challenge that hampers the potential utilization of extracellular vesicles for dentistry regeneration is a dependable method for isolating and purification of EVs from cells or bodily fluids. Current isolation methods encompass ultracentrifugation, size-exclusion chromatography, asymmetrical flow field-flow fractionation, and immunoaffinity-based techniques [131]. Comparative analyses of these methods have revealed variations in EV particle yield and purity [132]. For instance, one study compared the purification of serum-EVs using ultracentrifugation and Total Exosome Isolation reagent, with the latter displaying superior purity based on miRNA profiles [133]. Meanwhile, the conditions of the liquid for EV isolation, such as viscosity, preservation environment, and treatment methods, also influence EV purification outcomes. Nonetheless, a comprehensive comparison and optimization strategies for EV purification remains relatively unexplored. Moreover, these methods often incur high costs while yielding limited quantities of vesicles, thereby impeding their clinical applicability. Therefore, more advanced techniques for efficient and standardized EV isolation and purification are required for the future clinical application of EVs in regenerative dentistry. A relevant issue in this challenge is the standardization of EV usage, especially the determination of optimal dosages or concentrations. Notably, studies have reported a wide range of EV concentration/dosage across different investigations (Table 1). A general trend in the literature suggests that higher EV doses tend to yield relatively better tissue regeneration outcomes [134, 135], yet none of the studies provide definitive guidance regarding the optimal EV concentration for their respective animal models. To ensure consistency and efficacy, it is imperative to establish good manufacturing practices (isolation and purification) and comprehensive standards and guidelines for the clinical application of EVs [136].

The management of chronic dental diseases necessitates continuous engagement of extracellular vesicles. However, the sustained presence of EVs and their therapeutic effects at injury sites over extended periods remains a challenge. To address this, the development of a proficient delivery system for EVs offers distinct advantages in augmenting their therapeutic efficacy when integrated with modified scaffolds [137]. Consequently, forthcoming research endeavors may develop novel EV-loaded scaffolds, encompassing controlled release profiles, in vivo degradation characteristics, and loading efficiency. For instance, injectable microspheres with sustained release kinetics of EVs have been devised for addressing irregular tissue defects and for periodontitis [138]. More investigation on material-EV interaction would aid in optimizing the adaptability and plasticity of such scaffolds to ensure their effectiveness to deliver EVs.

Conclusion

Extracellular vesicles have emerged as pivotal elements in cellular interactions and hold the potential to revolutionize regenerative dentistry by facilitating tissue regeneration, encompassing the maxillofacial, periodontal, dental, and temporomandibular cartilage regions. These vesicles, sourced from diverse origins, make substantial contributions to regenerative dentistry through various mechanisms, including the promotion of odontogenesis, osteogenesis, dental hard tissue mineralization, angiogenesis, and modulation of the immune response. Leveraging diverse delivery strategies has allowed for more effective utilization of EVs, enhancing their regenerative efficacy in the field of dental tissue regeneration. Nonetheless, the successful clinical translation of EV-based therapies hinges upon addressing several critical challenges. These include the optimization of EV yield, the establishment of a standardized definition for EVs, and the development of novel EV delivery strategies.