Exosomes as a Promising Therapeutic Strategy for Peripheral Nerve Injury.

Peripheral nerve injury has a high incidence and often leads to severe losses of sensory and motor functions in the afflicted limb. Autologous nerve grafts are widely accepted as the gold standard for peripheral nerve repair, but the presence of inherent drawbacks dramatically reduces their usability. Numerous tissue engineering nerve grafts are developed as alternatives to autologous nerve grafts, and a variety of cells and neurotrophic factors are introduced into these grafts for improvement. However, they are still difficult to obtain satisfactory clinical results. Peripheral nerve regeneration following injury remains a significant challenge for researchers and clinicians. Exosomes are extracellular membranous nanovesicles that are secreted by most cells. As the key players of intercellular communication, exosomes play a fundamental role in the physiological and pathological processes of the nervous system. Accumulating evidence has suggested that exosomes can exert neurotherapeutic effects via mediating axonal regrowth, Schwann cell activation, vascular regeneration, and inflammatory regulation. Exosomes are emerging as a promising approach for treating peripheral nerve injury. Furthermore, they also provide possibilities for enhancing the repairing capacity of various nerve grafts. This review primarily highlights the regenerative effects of exosomes on peripheral nerve injury. The exosomes from distinct sources reported so far in the literature are summarized to understand their roles in the process of nerve repair. Moreover, the challenges that must be addressed in their clinical transformation are outlined as well. This review also provides further insight into the potential application of exosomes for peripheral nerve repair. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.

INTRODUCTION

Peripheral nerve injury (PNI) is the primary type of traumatic damage in the nervous system, and it affects over one million people worldwide annually [1]. These injuries frequently result in life-long disability, which drastically reduces patients’ quality of life [2]. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) possesses the intrinsic capacity to regenerate to a certain extent subsequent toPNI [3, 4]. End-to-end anastomosis is the preferred surgical technique for peripheral nerve repair [5]. When the extent of the nerve defect is greater and tensionless primary neurorrhaphy cannot be performed, the integrity of the nerve needs to be restored by nerve grafts [6]. Autologous nerve grafts (ANGs) have been accepted as the current “gold standard” treatment for PNI [7]. However, the clinical application of ANGs still has many critical drawbacks, such as limited availability of donor nerves, donor site morbidity, painful neuroma formation, additional surgery times, and mismatch in diameter or length [8, 9]. Furthermore, less than 50% of patients who underwent the transplantation of ANGs achieved effective functional recovery [10]. Numerous tissue engineering nerve grafts composed of various synthetic or natural biomaterials have been developed as substitutes for ANGs [11]. However, most of these substitutes fail to obtain satisfactory clinical outcomes, which may be associate with lack of regenerative cells, loss of neurotrophic factors, and destruction of extracellular matrix [12, 13]. Although much progress has been achieved in the pathophysiology of nerve injury and regeneration, peripheral nerve repair remains a significant clinical challenge [14]. It is necessary to explore a novel factor to create an ideal regenerative microenvironment and further improve therapeutic effects on PNI.

Exosomes are membranous nano-sized vesicles secreted into extracellular space by nearly all cell types, including mesenchymal stem cells (MSCs), immunocytes, neurons, cancerous cells, epithelial cells, osteocytes, and myocytes [15, 16]. They can mediate intercellular communication via delivering bioactive cargo, including lipids, proteins, and nucleic acids, to recipient cells [17]. Exosomes are present in different body fluids, such as blood, saliva, urine, amniotic fluid, and cerebrospinal fluid [18, 19]. They can act as modulators to manipulate the biological behaviors of target cells at distant or nearby areas, which is crucial for the maintenance of homeostasis in multicellular organisms [20]. The physiological properties of the nervous system are based on connection, integration, and signal exchange between various nerve cells [21]. Exosomes, as the key players of intercellular communication, play a fundamental role in the development, maintenance, function, and regeneration of the nervous system [22].

Nerve repair following PNI is involved in a series of highly interactive mechanisms, including axonal regrowth, Schwann cell (SC)activation, vascular regeneration, and inflammatory regulation [23, 24]. Recent studies indicated that exosomes could participate in these critical processes and exert neuroprotective effects [25, 26]. Exosomes are emerging as a promising approach for treating PNI.In this review, we discuss the current knowledge about the biogenesis and function of exosomes as well as their promoting effects on peripheral nerve regeneration. The exosomes from distinct sources reported so far in the literature are summarized to understand their roles in the process of nerve repair. Moreover, the challenges that must be addressed in their clinical transformation are outlined as well. This review also provides further insight into the potential application of exosomes for peripheral nerve repair.

BIOGENESIS AND FUNCTION OF EXOSOMES

Extracellular vesicles (EVs) were first discovered by Pan and Johnson during the maturation of sheep reticulocytes in 1983 [27]. EVs are defined as heterogeneous entities of phospholipid bilayer membrane-bound vesicles without any means of replication [28, 29]. EVs can be classified as apoptotic bodies, microvesicles, and exosomes based on their size, lipid composition, marker proteins, and mechanisms of discharge and formation [30]. Apoptotic bodies (0.5–3μm in diameter) are formed by random blebbing of the plasma membrane during cell apoptosis. Microvesicles (0.1-1μm in diameter) are secreted through direct plasma membrane budding. Unlike apoptotic bodies and microvesicles, exosomes are the smallest membranous vesicles (30–100 nm in diameter) that are generated by the endosomal pathway (Fig. ) [31, 32]. Briefly, the plasma membrane undergoes inward budding and forms early endosomes under the stimuli of various physical and chemical factors. Subsequently, as endosomes mature, intraluminal vesicles (ILVs) are generated by further invagination of the late endosome membrane, resulting in the formation of multivesicular bodies (MVBs). This process completes in two distinct pathways, including endosomal sorting complexes required for transport (ESCRT)-dependent and ESCRT-independent, which provide essential mechanisms for sorting and encapsulating specific molecules into ILVs [33]. Finally, MVBs may fuse with lysosomes for degradation. Alternatively, MVBs may fuse with the plasma membrane to release ILVs into extracellular space as exosomes [34]. Exosomes can interact with the recipient cells through extracellular receptor binding, fusion to the plasma membrane, or endocytosis-mediated internalization [35].

Exosomes have a “cup-shaped” morphology under observation by transmission electron microscopy, and their buoyant density is 1.13–1.19 g/cm2 [36, 37]. Several methods have been developed to isolate exosomes from cell culture medium and body fluids, including ultracentrifugation, density gradient separation, immunoaffinity capture, size exclusion chromatography, and flow cytometry [38]. Ultracentrifugation and density gradient separation are the most widely used isolating techniques [39]. However, these conventional methods have shortcomings of long isolating time, large sample demand, and integrity damage [40-42].

Exosomes can regulate the physiological status of recipient cells by delivering lipids, proteins, and nucleic acids while circulating in the extracellular environment [43]. These bioactive molecules contained in exosomes are heterogeneous, which varies depending on the origin and physiological or pathological conditions of parent cells [44]. Lipids and proteins are the main components of exosome membranes. Numerous lipids have been identified to form the lipid bilayer of exosomes, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, lysobisphosphatidic acid, ceramide, cholesterol, and sphingomyelin [45]. These lipid compounds are associated with the transmission of signal, the integrity of the structure, and the fusion of plasma membrane, which may affect the communication function of exosomes [46]. Furthermore, exosomes are enriched in a variety of function-specific proteins [47, 48]. GTPases, annexins, and flotillin play a crucial role in membrane transport and fusion. Heat shock proteins (Hsp 70 and Hsp 90) can modulate intracellular assembly and trafficking. Tetraspanins (CD9, CD63, CD81, and CD82) can mediate membrane fusion, cell migration, cell signaling, and intercellular adhesion. ALG-2-interacting protein X and tumor susceptibility gene (TSG) 101 are involved in MBV biogenesis. Among them, CD9, CD63, CD81, CD82, flotillin, and TSG 101 are generally recognized as biomarkers of exosomes. Besides these membrane-related proteins, a large number of bioactive proteins have also been identified in the cargo of exosomes, which can participate in the modulation of biological processes of target cells [49]. In addition, exosomes contain a wide range of genetic material, including messenger RNA (mRNA), micro RNA (miRNA), long noncoding RNA (lncRNA), and DNA [50]. The mRNA can be translated into the recipient cells, and the miRNA and lncRNA are involved in target gene modulation [51]. The function of DNA has not been fully elucidated yet. Among these molecules, miRNAs carried by exosomes have attracted much attention, due to their regulatory effects in gene expression. They can serve as important factors in intercellular interaction [52].

In the nervous system, exosomes can be secreted by many cells, including neural stem cells, neurons, astrocytes, microglia, oligodendrocytes, SCs, and endothelial cells [53]. Exosomes emerge as a prominent form of intercellular communication, which has pivotal effects on mediating the physiological and pathological processes of the nervous system [54, 55]. Many studies have revealed the role of exosomes in nerve remodeling, nerve protection, neuronal development, and synaptic plasticity [56, 57]. Especially in the CNS, exosomes represent a promising strategy in the diagnosis and therapy of several neurological disorders, such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and brain injury [58]. Exosomes can provide information regarding the cells in the CNS, which contributes to determining the status of disease progression. Hence exosomes might serve as specific biomarkers for the diagnosis of CNS disorders [59]. Exosomes are also suggested as a therapeutic delivery system. They can pass through the blood-brain barrier and deliver their cargo to specific target cells, thereby exerting neurotherapeutic effects [60]. Furthermore, accumulating evidence shows that exosomes have important roles in promoting nerve repair in the PNS [61, 62]. Therefore, the potential of exosomes in the treatment of PNI becomes a subject of increasing interest.

THE EFFECTS OF EXOSOMES ON AXONAL REGROWTH

Axonal regrowth is a subject of intense investigation as it is the essential process of functional recovery following nerve injury. For the reconstruction of target tissue innervation, axons must extend across injury sites to reconnect with distal nerves. Regenerating axons develop distal tip expansions called growth cones that can sense growth and guidance cues [63]. Growth cones extend membranous protrusions called filopodia and lamellipodia that interact with the surrounding milieu [64]. The growth cone has a specialized sensory-motility structure characterized by its distinctive distribution of actin, microtubule, and neurofilament cytoskeletal proteins, which is closely associated with mediating axonal regrowth [65]. In the process of peripheral nerve repair, regenerating axons express various adhesion molecules that can promote SCs alignment and migration [66]. At the early stages of regeneration, axons can act as guiding substrates for SC migration in the proximal nerve stump [67]. Furthermore, axons are crucial for the maintenance of SC function. Dedifferentiation of SCs, which triggers several repair processes, is dependent on axonal contact [68]. When repair SCs lacked axonal contact for a long time, their regenerative capability diminished and the cell number decreased [69, 70]. Considering the limited period of successful nerve regeneration, the acceleration of axonal regrowth is particularly essential for peripheral nerve repair following injury.

SCs are the key components that modulate neuronal function and maintain their homeostasis under diverse conditions [71]. Exosomes derived from SCs, as an important mediator of SCs-to-axons communication, can regulate the regenerative processes of axons. In the study of Lopez-Verrilli et al. [72], they have found that the exosomes from dedifferentiated SCs were specifically internalized by axons. These exosomes can facilitate the survival of DRG neurons and increase axonal regrowth in vitro. Moreover, the regenerative capability of injured sciatic nerves can be significantly enhanced. They further illustrated that these SC-derived exosomes contribute to maintaining a pro-regenerating phenotype of growth cones and inhibiting the activity of the GTPase RhoA that is involved in growth cone collapse and axon retraction. Lopez-Leal et al. [73] have demonstrated that the shift of SC reprogramming into a repair phenotype that enhances neurite growth involved a modification in their miRNA cargo of exosomes. Increased expression of miRNA-21 is responsible for the pro-regenerative capacity of repair SC exosomes, which can promote axonal regeneration via PTEN downregulation and PI3-kinase activation in neurons. In addition to SC-derived exosomes, exosomes from MSCs have shown the ability to facilitate axonal regeneration. Bucan et al. [74] have demonstrated that adipose MSC-derived exosomes increased neurite outgrowth of DRG neurons in vitro and enhanced regeneration after crush injury of the sciatic nerve in vivo, which is mainly correlated with the presence of multiple neurotrophic factors in these exosomes. The study of Zhao et al. [75] has shown that bone marrow MSC-derived exosomes significantly improved neurite growth and increased axon length via the mechanism that may involve miRNA-mediated regulation of regeneration-related genes. Their in vivo results have revealed that there is a specific relationship between the bone marrow MSC-derived exosome doses and their effects on nerve regeneration. It is necessary to explore the appropriate dose of exosomes to achieve the beneficial effects of exosomes in nerve repair. The study of Tassew et al. [76] has found that fibroblast-derived exosomes facilitated robust neurite elongation of cultured cortical neurons, DRGs, and mouse retinal ganglion cells in an inhibitory milieu by promoting Wnt10b recruitment to lipid rafts and consequential activation of mTOR. Moreover, gingival MSC-derived exosomes have been demonstrated to promote DRG axonal regrowth [77]. Overall, these findings indicated that exosomes can play a crucial role in regenerative signaling transfer and constitute an imperative mechanism for axonal regrowth, which offers benefits for peripheral nerve repair.

THE EFFECTS OF EXOSOMES ON SCHWANN CELL ACTIVATION

SCs are the principal glial cells of peripheral nerves [78]. They establish myelin sheaths around axons, which contributes to maintaining the integrity of axons and increasing signal transmitting velocity [79]. The plasticity of SCs gives the repair capability following nerve damage to the PNS in contrast with the CNS [80, 81]. After PNI, SCs release from the degenerating nerve, dedifferentiate into progenitor-like repair cells, and go under phenotypic changes [82]. These specialized cells, together with macrophages, can eliminate Wallerian degeneration products and myelin debris to create a permissive environment for axonal regrowth [83]. In addition, repair SCs proliferate and extend longitudinally to form a layer of columnar organization beneath the basal lamina called bands of Büngner, which provides pathways for newborn axons [84]. Meanwhile, various cytokines, growth factors, neurotrophins, and extracellular matrix molecules are synthesized and secreted from SCs to facilitate the survival of neurons [85]. Following the contact with newborn axons, SCs will undergo redifferentiation for remyelination to accomplish the process of peripheral nerve repair [86].

Recent studies have documented that exosomes can promote peripheral nerve regeneration via supporting and regulating the biological behaviors of SCs. Chen et al. [87] demonstrated that after being internalized by SCs, adipose MSC-derived exosomes can enhance the proliferation, migration, myelination, and secretion of SCs by increasing the expression of corresponding genes in vitro. Their in vivo experiment showed that the exosome-treated group that optimized functionality of SCs achieved superior axonal regrowth, remyelination, and muscle restoration compared to the vehicle control group. Yin et al. [88] have revealed that adipose MSC-derived exosomes can inhibit the autophagy of SCs via miRNA-26b mediated downregulation of Kpna2 expression, which improves the regeneration of myelin sheaths in the repair process of PNI. Moreover, Liu et al. [89] have shown that adipose MSC-derived exosomes can inhibit the apoptotic process and improve the proliferation of SCs via upregulating the anti‐apoptotic Bcl‐2 mRNA expression and downregulating the pro-apoptotic Bax mRNA expression, which play a neuroprotective role in injured peripheral nerves. The study of Haertinger et al. [90] has shown that adipose MSC-derived EVs can facilitate the proliferation of SCs in a time- and dose-dependent manner. They further analyzed the way these EVs exert their promotional effects on SCs. The results revealed that adipose MSC-derived EVs entered SCs via an endocytosis-mediated internalization pathway rather than binding or fusing with the plasma membrane of SCs. These EVs were mainly localized around the nucleus and released their cargo through fusion with the endosomal membrane. miRNA contained within these EVs is probably the primary effector molecule, which can impact gene expression of SCs through the post-transcriptional mechanism in response to nerve injury. Moreover, it has been demonstrated that there exists mRNA of neurotrophic factors in these adipose MSC-derived EVs [90]. In addition to adipose MSC-derived exosomes, exosomes from other MSCs were also reported to have supportive effects on SCs. Rao et al. [77] demonstrated that the gingival MSC-derived exosomes could significantly promote SCs proliferation and the nerve conduits with these exosomes achieved superior remyelination in repairing 10 mm peripheral nerve defects in rats. Overall, these studies indicated that exosomes can exert their neuroprotective effects by acting on SCs, which underlies the further improvement of therapeutic effects on PNI.

THE EFFECTS OF EXOSOMES ON VASCULAR REGENERATION

The vascular system plays a fundamental role in maintaining the microenvironment homeostasis of the nervous system via the supply of blood, oxygen, and other nutrients, which is crucial for the development, maturation, and regeneration of the PNS [91]. It has been demonstrated that there is a close interlinkage between vascularization and nerve repair following injury [92]. In addition to the nutrient support, blood vessels can serve as tracks for SCs to migrate along, and vascular endothelial cells can secrete bioactive molecules that are conducive to neurite elongation [93]. Moreover, sufficient vascularization needs to be achieved to avoid central ischemia within nerve grafts, especially for long nerve defects with a large diameter [94]. However, vascular regeneration is still one of the great challenges in peripheral nerve repair. Nerve conduits and decellularized grafts that are substitutes of ANGs require a longer time to obtain formation and inosculation of microvessels, which may result in cell damage, intraneural fibrosis, oxidative stress, and other pathological processes [95, 96].

Tissue repair mechanisms entail significant endothelial cell regeneration and blood flow reestablishment in damaged and ischemic tissues. Recently, many studies have confirmed that exosomes serve as a key regulator in vascular regeneration. The influence of exosomes on revascularization in tissue repair has been intensively investigated. The study of Zhang et al. [97] has shown that umbilical cord MSC-derived exosomes enhanced vascular regeneration by delivering Wnt4 to activate Wnt/β-catenin in endothelial cells, which provides a meaningful mechanism for tissue regeneration. Furthermore, exosomes from adipose MSCs and bone marrow MSCs have been reported to exert pro-angiogenic effects in wound healing [98, 99]. Accumulating evidence has indicated that exosomes can mediate vascular regeneration to modulate nerve repair following injury. Zhang et al. [100] have demonstrated that exosomes from multipotent MSCs significantly increased the number of newly generated endothelial cells in the lesion boundary zone and achieved more newly formed vessels, which effectively improved the recovery of motor function in rats after traumatic brain injury. Yang et al. [101] have found that adipose MSC-derived exosomes can promote the mobility and revascularization of brain microvascular endothelial cells after oxygen-glucose deprivation via elevating the expression of miR-181b-5p and inhibiting the expression of its target transient receptor potential melastatin 7, which could offer advantages for ischemic stroke recovery. Exosome treatment-induced revascularization may promote functional recovery by enhancing neurite regrowth and synaptogenesis in the brain after stroke. Moreover, bone marrow MSC-derived exosomes can ameliorate ischemic brain injury by accelerating revascularization of endothelial cells and hindering neuron apoptosis through the delivery of miRNA-29b-3p that can activate the PTEN-mediated Akt signaling pathway [102]. Except for the above-mentioned exosomes derived from MSCs, circulating endothelial progenitor cell-derived exosomes have been identified to reduce brain cell apoptosis and improve sensorimotor function by enhancing revascularization and neurogenesis through the miR-126/PI3k signal pathway, which serves as a mechanism for the beneficial effects of exercise interventions on ischemic injury [103]. These studies illustrate that vascular regeneration mediated by exosomes is conducive to tissue regeneration in the nervous system, which provides a potential therapeutic strategy for PNI. Further investigations need to be conducted to determine the pro-angiogenic effects of exosomes in the treatment of PNI.

THE EFFECTS OF EXOSOMES ON INFLAMMATORY REGULATION

The inflammatory response is an essential pathological process of nerve repair, which has a major influence on the prognosis [104]. After PNI, SCs activate and secrete a variety of inflammatory cytokines and chemokines during Wallerian degeneration. Subsequently, macrophages are recruited to further accelerate the clearance of axon and myelin debris and initiate an inflammatory cascade [67, 105]. As the primary immune cell type in the repair stage after nerve injury, macrophages play a crucial role in coordinating a series of immune events, which is essential for successful nerve regeneration [106, 107]. Influenced by local microenvironment cues, macrophages have heterogeneous phenotypes, including classically-activated M1 cells and alternatively-activated M2 cells [108]. M1 macrophages can secrete inflammatory cytokines, such as IL-1β, IL-6, TNF-α, and IFN-γ, which may result in further tissue damage. M2 macrophages have an inhibitory role in the immune response by producing IL-4 and IL-10 [109, 110]. Meanwhile, they could exert neuroprotective effects by releasing various growth factors, which contribute to promoting vascular regeneration and functional recovery [7, 93, 111]. During the early stage of inflammation, macrophages mainly show the M1 pro-inflammatory phenotype and transform into M2 anti-inflammatory phenotype in the late stage, which can effectively control the inflammation development [26]. The inflammatory response helps to turn peripheral nerve tissue into a state that supports regeneration [112]. However, excess activation of neuroinflammation can aggravate the extent of nerve injury via secondary damage, which may lead to delayed or insufficient regeneration. In the repair process following PNI, the appropriate regulation of inflammatory response can inhibit neuron apoptosis and axon demyelination caused by neuroinflammation [113, 114]. In addition, it can also prevent the development of an acute inflammatory response to a chronic persistent state, which contributes to reducing the incidence of undesired symptoms, such as neuropathic pain [115]. Therefore, inflammation has been identified as a therapeutic target for PNI.

Many studies have suggested that exosomes can serve as modulators to manipulate immune responses in the nervous system [116]. MSC-derived exosomes, as the essential mediators responsible for paracrine effects, have been demonstrated to possess similar anti-inflammatory functions to parent cells, which can provide favorable conditions for nerve regeneration [117]. Sun et al. [110] have demonstrated that umbilical cord MSC-derived exosomes can promote the healing of spinal cord injury through alleviating the inflammation of the injury region. Their findings showed that these exosomes could alter the macrophage polarization from M1 to M2 phenotype, which is conducive to inhibiting the excess inflammatory response. Moreover, the in vivo studies have revealed that except for the transformation of macrophage phenotype, these exosomes can improve functional recovery after injury via downregulation of the pro-inflammatory cytokines, such as TNF-α, MIP-1α, IL-6, and IFN-γ. The study of Ni et al. [118] has found that bone marrow MSC-derived exosomes can exert neuroprotective effects by attenuating early neuroinflammation in mice with traumatic brain injury via regulating the polarization of microglia/macrophage. Moreover, in the PNS, bone marrow MSC-derived exosomes have been demonstrated to alleviate the inflammatory response, thereby facilitating neurovascular remodeling and functional recovery of diabetic peripheral neuropathy in diabetic mice [119]. In the study of Ma et al. [120], a rat model of sciatic nerve transection was utilized to analyze the effects of umbilical cord MSC-derived EVs on peripheral nerve repair. They found that these EVs can migrate to nerve defects, and then serve immunosuppression function by decreasing the level of pro-inflammatory cytokines IL-6 and IL-1β and increasing the level of anti-inflammatory cytokine IL-10 at the distal nerve stumps, which can offer advantages for peripheral nerve regeneration. Gingival MSC-derived exosomes also have been reported to participate in the inhibition of inflammation by regulating the secretion of IL-1 receptor antagonist, which improved the therapeutic effects of chitin conduits in rat sciatic nerve injury [77, 121]. In addition to MSC-derived exosomes, SC-derived exosomes have been shown to possess a regulatory role in the inflammatory phase of nerve injury, due to the presence of two proteins, αB-crystallin and galectin-1 [122]. Together, these studies indicated that exosome-mediated immunomodulatory effect contributes to rational control of neuroinflammation, which facilitates the establishment of beneficial microenvironment for peripheral nerve regeneration.

CHALLENGES OF EXOSOME CLINICAL TRANSFORMATION

By analyzing the recent literature, it can be seen that exosomes have great potential for promoting nerve regeneration following PNI. In the future, exosome-based therapy is expected to be a feasible strategy for peripheral nerve repair. Nevertheless, there are some challenges remaining to be further addressed. First of all, the lack of stable and effective approaches to isolate and purify exosomes is the main drawback hampering the clinical application of exosomes. In recent years, to overcome the restrictions of traditional isolating methods, several commercially available kits have been developed mainly based on size-based precipitation technology, and they have been proven to be more efficient, reliable, and reproducible [123], providing the possibility for standardized enrichment of exosomes. Secondly, it should be noted that the cargo of exosomes is a mixture of various functional proteins and RNA molecules, which may result in the heterogeneity of exosomal functionality and the risk of adverse reactions. It is necessary to regulate exosomes in a way to specifically orient them toward the desired target, which contributes to achieving optimal therapeutic outcomes with few side effects. The surface proteins of exosomes, along with their molecular cargo can serve as biomarkers, making it possible to screen exosomes with different functional properties through immune techniques. Thirdly, the appropriate administration route of exosomes is a key issue to be explored in PNI treatment. The effectiveness of systemic administration routes in the clinical use of exosomes needs to be further assessed. In contrast, local administration of exosomes to the PNI site may be a feasible way to fully exert their neurotherapeutic capabilities. Studies have reported that the effect of nerve repair has been significantly improved by integrating exosomes into tissue engineering nerve grafts [77]. However, the mechanism underlying exosomal functionality in PNI repair remains to be investigated further. In addition, the effects of exosomes on health and diseases are diverse, which have not been thoroughly elucidated and controlled. Exosomes may also be involved in the replication and propagation of transmissible pathogens. These may bring potential safety risks to patients. It is essential to ensure the consistent mass production of exosomes, while the supervision of exosomal safety cannot be ignored. Overall, much work needs to be done to overcome the limitations in the clinical transformation of exosomes.

CONCLUSION AND PERSPECTIVES

Peripheral nerve repair following injury is a complicated process based on the interaction of regenerative units. Exosomes, as signaling vehicles with multifaceted functions, have been shown to play a vital role in peripheral nerve regeneration. They can promote neuron survival and axonal regrowth, enhance SCs migration and proliferation, facilitate vascular regeneration, and regulate the inflammatory response, indicating that exosomes have a great neurotherapeutic capacity (Fig. ). In recent years, although cell-based therapies have demonstrated efficacy in some clinical trials for peripheral nerve repair, there exist several inherent risks, such as nerve sacrifice, microvasculature occlusion, neoplastic transformation, and immune rejection. Moreover, the viability and potency of transplanted cells are difficult to maintain [124, 125]. These factors greatly limit the clinical application of cell-based therapies. Exosomes possess similar therapeutic effects and functional properties as parent cells. Also, they evade the issues allied with cell transplantation [117], which indicates that exosomes have massive advantages as cell-free therapies for PNI. Moreover, exosomes have the capacity to deliver their cargo to specific targets over a long distance, therefore exosomes are suggested as a therapeutic delivery system. Exosomes can be tailored by appropriate strategies to carry specific bioactive substances or drugs to achieve better nerve repair effects. In summary, exosomes represent a highly promising strategy for peripheral nerve regeneration and exhibit massive potential for clinical transformation. The safety of exosomes needs to be thoroughly investigated before they can be used clinically.