Introduction

Periodontitis is still considered as a globally prevalent disease [1]. The chronic presence of pathological factors may proceed to destruct the supporting periodontium of the teeth and lead to tooth loss. Early diagnosis of periodontitis prevents further structural damages to the periodontium, and it can be treated by removal of pathologic factors using scaling and root planning [2]. In the case of lost periodontal tissues, regeneration of the periodontium is considered as a challenging treatment. Numerous procedures and products have been developed and applied to regenerate lost periodontal tissue [3,4,5,6,7]. Such regenerative treatments are difficult and only effective in specific conditions with limited tissue reconstruction results, as the periodontium is a complex structure which possess various cell types [8].

Bone, as a connective tissue, preserves and supports organs and tissues within the body. It is also one of the important structures of the periodontal tissues surrounding teeth. Bone remodeling is a lifelong process to preserve bone structure and function. Some conditions like aging, trauma, obesity, congenital abnormalities, surgical removal of a mass within the bone, and cancer metastases to the bone, may interfere with the normal balance of bone remodeling and increase the demand for an efficient therapy to regenerate the bone tissue [9,10,11,12]. Autogenous and allogenous bone grafts are currently considered as a gold standard in bone regenerative therapies. However, numerous complications including, morbidity at graft harvesting site, limited harvesting sources, graft versus host disease (GVHD), need for secondary surgery, infection, and non-union formation are associated with these treatments [13,14,15,16,17]. Therefore, a new, safe, and efficient therapy is highly demanded to overcome the existing limitations. Bone remodeling involves various cells, such as bone cells (osteoblasts, osteoclasts, mechanosensitive osteocytes, and bone marrow stem cells), immune cells (T cells, dendritic cells, and monocytes), and articular cartilage cells [18]. Intercellular communication between cells is essential for bone remodeling [19]. This has directed recent studies towards investigating more suitable and efficient bone regenerative therapies especially when dealing with challenging defects that are beyond the spontaneously healing size.

Regenerative medicine is considered as a subdivision of translational medical science that focuses on identifying various approaches to efficiently replace or reestablish the normal structure and function of damaged tissues [20]. Stem cells have been considered as effective tools in regenerative medicine, with the potential to differentiate into various cell types, and having a wide range of applications including in tooth regeneration, wound healing, and treatment of various diseases [21, 22].

Oral tissues have been considered a suitable source of mesenchymal stem cells (MSCs), and the first dental derived stem cells were isolated from a dental pulp in 2000 [23]. Dental stem cells are regarded as an easily accessible and suitable source of stem cells with a well-known regenerative capacity. Dental derived stem cells include multiple types such as dental pulp mesenchymal stem cells (DP-MSCs), stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), and dental follicle progenitor cells (DFPCs) .There, still exists a search for finding more suitable stem cell origins in the oral cavity to be used in tissue regenerations and cell based therapies [24].

One of the secreted particles from MSCs is extracellular vesicle (EVs). EV is a term approved by International Society for Extracellular Vesicles (ISEV) for bilayer lipid membrane vesicles that are non-replicable, containing nucleic acids, proteins, lipids, and various signaling molecules [25]. Most eukaryotic cells secrete EVs, which have essential roles in intercellular communications. They carry active signals that can influence the activity of adjacent or distant recipient cells [26, 27]. It has been suggested that MSCs’ paracrine activity is controlled by growth factors and survival signals, as well as EVs. Current investigations have shown the beneficial contribution of MSC derived EVs in MSCs’ physiological functions [28]. Due to the challenges related to stem cell therapy, more recent studies have focused on other novel alternative regenerative methods such as cell free therapies on based paracrine signaling and use of such secreted particles to overcome these obstacles [29,30,31,32,33]. The investigation onstem cells and their mechanisms of action have revealed the important role of bioactive molecules of these cells and the media surrounding them [the conditioned media (CM)]. One of the most important secreted molecules that are released to the biological fluid or cell culture CM are EVs that show the same regenerative function as stem cells and can be considered as safe alternatives [34]. EVs’ valuable advantages over stem cell therapy are their relative ease of preservation and sterilization, and the capability of long-term storage without the risk of losing their properties. These cell-secreted particles provide broad bio-signaling functions for various targeted cell types.

This capability has attracted attention to use EVs for transferring particular messages to multiple heterogeneous cells involved in tissue regeneration therapies such as craniofacial bone and tissue regenerations. The current review aims to summarize the available evidence on EVs’ function and also their potential applications in bone and periodontal regeneration.

General characteristics of EVs: biogenesis, components, and composition

EVs have been previously classified into three main subtypes based on their cellular origin, size, or biogenesis. This includes (1) exosomes (30–150 nm) with an endocytic origin, (2) microvesicles (100–1000 nm) formed by budding of the plasma membrane, and (3) apoptotic bodies (500 nm–2 µm) derived from dying cells (Fig. 1) [35]. Based on new guidelines and the fact that determining the exact biogenesis pathway of EV is still considered difficult, use of a more general term of EV is recommended. Moreover, for identifying EV subtypes, use of more operational terms which refer to either their physical characteristics such as size, density, biochemical composition, descriptions of conditions or cell of origin is suggested [25, 36].

Fig. 1
figure 1

Mechanisms of maturation and secretion of extracellular vesicles

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Exosomes were first recognized in 1981 [37] and can be distinguished from other EVs by their protein and lipid composition. They can be secreted from almost all cell types and they can be found in body fluids (e.g., blood, breast milk, saliva, semen, and urine) [38, 39].

Exosomes are formed by the inward budding of endosomal membranes of multivesicular Endosome (MVE) and form intraluminal vesicles (ILV). These exosomes are released due to the fusion of the MVE with the plasma membrane [35].

Depending on their endosomal origin, EVs/exosomes may contain membrane transport and fusion proteins (Annexins, Rabs, flotillin), tetraspanins (CD9, CD63, CD81, CD82), heat shock proteins (Hsp70, Hsp 90), proteins associated with MVB formation, including Endosomal Sorting Complex Required for Transport (ESCRT) proteins, apoptosis-linked gene 2-interacting protein X (Alix), Tumor Susceptibility Gene 101 (TSG101), transmembrane receptors including MHC molecules and integrins as well as lipid-related proteins and phospholipases [40, 41]. They also contain cytosolic proteins such as cytoskeletal proteins (Actin, Tubulin, Profilin, Cofilin) and various metabolic enzymes (AChE, GAPDH and Pyruvate kinase) [42]. Therefore, it should be taken into consideration that different sources of exosomes may cause a variation in these markers’ expression. Commonly used markers of exosomes identification include tetraspanins, Alix, flotillin, TSG101, and Rab5b [27]. So far, more than 4400 different proteins in addition to the membrane proteins have been recognized as cargo for intercellular communication [43]. Moreover, exosomes contain specific raft-associated lipids such as cholesterol, ceramide, sphingolipids, and phosphoglycerides with long and saturated fatty-acyl chains [44,45,46]. The genomic molecules such as mRNA, miRNAs, and lncRNAs are mentioned as other exosome components associated with the regulation of gene expression. Exosome miRNA content is specific to the parental cell type and cell condition (e.g., inflammation and hypoxia) (Fig. 2) [47].

Fig. 2
figure 2

Components and potential applications of extracellular vesicles

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EVs are released in body fluids such as blood, semen and urine and may also be isolated from cell culture condition mediums [48,49,50].

Cell culture media are convenient sources of EVs that can result in a reproducible and high gain of EVs. Because of the high chance of EVs’ contamination in culture media that are hard to distinguish during the isolation process, alternative ways such as EVs-depleted FBS are considered to prevent the influence on the type, cargo, and amount of released EVs [51,52,53,54,55,56]. Numerous factors affect EVs’ secretion, including oxidative stress, hypoxia, and calcium ions [57]. These vesicles are distinguished by different sets of lipids, functionally active ribonucleic acids (e.g., mRNA, miRNA), and parental cell-derived cytosolic and membrane proteins [58,59,60]. EV-based therapies are relatively more convenient than cell-based therapeutics. However, identifying the EV separation, storage and retrieval methods which have been shown to significantly alters both the physical and biological properties of EVs, are challenging topics of research, and are yet being extensively studied to help pave the path for a better translation and clinical application of EVs [25, 48, 61, 62]. 

EVs are involved in several biological interactions, such as intercellular communication, transportation of proteins and nucleic acids, tumorigenesis, and metabolism. They may also be used in diagnostic and therapeutic applications in various diseases, as host immune response modulators, and prions carriers [60, 63]. EVs membrane proteins may interact with cell surface and result in intercellular signaling. The mentioned process is done when a vesicles fuses with the target cell membrane via EVs surface proteins such as Alix or TSG101, and tetraspanins such as CD9, CD63, CD81, and CD82 [64, 65]. Also, internalization into a recipient cell may deliver cargo such as proteins and RNA that are active inside the recipient cell [66].

EVs have also been considered as therapeutic nano delivery systems as they have low immunogenicity, a long half-life in circulation, and are capable of penetrating through the brain-blood barrier [67,68,176]. Tumor-derived EVs, by contrast, inhibit macrophage maturation associated with TGF-β [177]. Generally, modulation of innate or adaptive immunity by EVs is a potential target for clinical therapeutics in bone regeneration.

Periodontal regeneration

Routine periodontal treatments can successfully reduce the number of pathogens in a periodontal defect; however, a predictable treatment procedure for reconstructing the lost structures has not been found yet [1]. Some evidence support that MSC-derived EVs can be useful in promoting periodontal ligaments regeneration. As noted, the secretomes of MSCs are known to be responsible for their regenerative effects, containing proteins, lipids, nucleic acid, and trophic factors as growth factors, chemokines, cytokines, hormones, and EVs. Therefore, many studies have started using EVs or their CM as cell-free techniques in periodontal regeneration. Different sources and delivery routes have been used for this purpose (Table 2).

Table 2 Characteristics of studies conducted on the application of EVs for periodontal regeneration
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Most studies on periodontal regeneration have utilized MSCs CM reporting positive outcomes. Kawai et al. [181] have used Human bone marrow MSCs-CM and reported that it may lead to the enhancement of periodontal tissue regeneration by stimulating angiogenesis and even the mobilization of endogenous MSCs.

Among different sources of MSCs, periodontal ligament stem cells (PDLSCs) are the most commonly studied and potentially considered the most suitable source for periodontal regeneration [183, 184]. They are easily accessible and capable of secreting mineralized structures and can be the best choice for periodontal regeneration due their similar origin. PDLSCs carried by hydroxyapatite/tricalcium phosphate (HA/TCP) were shown to be able to form a cementum/PDL-like structure in vivo [183]. Transplantation of PDLSC-CM has been investigated in some studies and demonstrated considerable new PDL attachment and bone defect regeneration. Nagata et al. investigated the regenerative potential of conditioned mediums (CMs) acquired from cultured periodontal ligament stem cells (PDLSCs) on regenerating periodontal defects models in rats. Their results suggested improved periodontal regeneration and reported a suppression of the inflammatory response caused by TNF-α production as a result of this treatment method [93]. Qiu et al. explored the periodontal tissue regeneration by conditioned media from gingival mesenchymal stem cells (GMSCs) or PDLSCs in rat periodontal defects. Their results showed that similar to PDLSC-CM, GMSC-CM transplantation significantly promoted periodontal defect regeneration in rats. They expressed that this resulted from the regulation of inflammation by MSC-CM and also the facilitation of osteogenic differentiation of bone progenitor cells in the wound region.

EVs isolated from adipose-derived stem cells were used by Mohammed et al. as supplementary treatment to the non-surgical periodontal therapy. They divided 50 rats with ligature-induced periodontitis into four groups, including control, SRP, ADSCs, and exosome group. EVs were locally injected through the pockets by using a plastic syringe after doing a scaling and root planning. They evaluated the progress at different intervals, and the EVs treated group showed the best result with a significantly higher area percentage of newly formed tissues [179].

Chew et al. have also applied EVs derived from hMSCs in regeneration of surgically created periodontal defect in a rat model. They reported that single implantation of collagen sponges has the potential to elevate periodontal regeneration by increasing bone construction and enhancing functional PDL length. They suggested that this could be related to adenosine receptor activation of AKT and ERK signaling pathways, promoting proliferation and migration of PDL cells [178]. MSC-derived EVs have also documented to be able to enhance the repair of osteochondral defects through a reduction in proinflammatory cytokines and an increase in regenerative M2 macrophage counts [185]. Since the activation of pathways that cause bone loss requires an adequate amount of inflammatory factors concentration, the anti-inflammatory characteristic of MSC-derived EVs gained noticeable attention for periodontal regeneration applications [186]. The available evidence indicates that EV based therapies are of valuable therapeutic approaches and have the potential of increasing the success of periodontal regenerative therapies [182]. Further research is still necessary in order to find ideal and standardized sources of EVs, their effective concentration, frequency of treatment and suitable scaffolds or delivery routes used for efficient regeneration of the complex structure of periodontal tissue and further develop their therapeutic applications in this field.

Future landscapes of EVs applications

Many studies have focused on biomedical EVs applications. Based on their physical functions, EVs of particular cell types have been used as therapeutic mediators in immune therapy, drug delivery, vaccination trials, and regenerative medicine. For example, Xu et al. [187] highlighted the utility of EVs for the development of cancer diagnostics and therapeutics. Mianehsaz et al. [188] reviewed the evidence for EVs from MSCs as a new cell-free therapy method osteoarthritis and joint damage. Synthetic EVs, tunable EVs, and EVs mimetics, as well as EVs designed to overexpress or knockdown signaling pathways associated with pathological conditions, are considered as the next generation of EVs-based products to be studied and developed which may have potential applications in oral and craniofacial diseases [101].

Adequate standards for EVs isolation, manipulation, and characterization need to be defined to reach future progression in the clinical application of EVs. Factors such as the distribution, cargoes, and the purification protocols can manipulate EVs effects [63]. Since EV cargoes depend significantly on their origins, it is essential to profile EVs before clinical applications [189]. There are two commonly used purification procedures, which exploit either repeated ultracentrifugation or ultrafiltration. These techniques provide only a low EVs yield and take a relatively long time. For instance, 5 × 106 myeloma cells can deliver only 5–6 µg of EVs [190, 191]. Achieving good manufacturing practices requires development in EVs isolation techniques. Therefore, developments in EVs studies highly rely on finding novel methods to efficiently isolate them.

It should be noted that delivering EVs in therapeutic dosage to target cells, particularly via systemic injection, may not always be as simple as it looks. Riau et al. proposed the possibility of using the encapsulated form of EVs with biodegradable or highly porous hydrogels. Approaches to encapsulate nanoparticles, like EVs, and instances of possible materials for sustained delivery of the EVs from the stem cells are also main areas of focuses in several studies [192].

After injection, EVs are distributed mostly in the bone, lung, spleen, liver, and kidney. Therefore, it is necessary to assess the clearance and final dosage in the organs [193]. It is still imprecise how to end the biological effects of EVs when the satisfactory outcome is accomplished. Moreover, the half time of EVs applied should be considered to be long enough for achieving the therapeutic aim. Thus, examining EVs in pre-clinical models before moving on to the clinical phase is crucial for their correct translational applications.

Conclusion and prospects

Several recent studies have been exploring the application of EVs in regenerative medicine. According to these studies, EVs have the potential of regulating immune microenvironment, promoting vascularization, facilitating osteoblasts activity, proliferation and mineralization. Significant development has been made to explore EVs biology, structure, and contents as well as understanding the exact mechanisms by which EVs may alter target cells functions. It is well established that the source of stem cells and their culture conditions affect the functional properties of the secreted EVs. EV-based therapies are considered as novel free-cell therapy approach which is easier to handle and reduces the risk of tumorigenesis, host rejection, and infections associated with direct cell therapy. The available research indicates a great potential for EV application for improvement in success and predictability of bone and periodontal tissue regeneration therapies.

However, we are still facing challenges for ideal clinical application of EVs and further investigations are needed to achieve a protocol for efficient engineering of these nano-bioparticles to maintain exact composition and structure of isolated EVs.