Role of exosomes in nerve regeneration: Systematic review

INTRODUCTION

Neurological disorders are a leading cause of disability and death globally. In India, these disorders are significant contributors to the neurological disorder burden.^[1] Complete recovery from neurological disorders is dependent on neuroregeneration. Neuroregeneration is crucial for restoring function lost due to neurological diseases while aiming to repair or replace damaged neural tissue. Among emerging approaches, exosome-mediated therapies are notable for their potential in neuroregeneration and offering innovative ways to enhance recovery.^[2-4] Exosomes facilitate communication between cells and can deliver therapeutic molecules, promoting neuronal regeneration and functional recovery. Their ability to cross biological barriers, biocompatibility, and capacity to carry and protect therapeutic agents are key advantages.^[3]

With the evolving landscape of exosome research in neuroregeneration, there is a need for systematic reviews to show gaps in current knowledge and the potential for significant impact.^[4] Given the rapid advancements and diverse methodologies in the field, a systematic review is essential to consolidate evidence, identify effective strategies, and guide future research directions.

MATERIALS AND METHODS

Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standards were followed in the study design.^[5] The online tool was used to generate the PRISMA diagram [Figure 1].^[6]

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Figure 1:

Preferred Reporting Items for Systematic Reviews and Meta-Analyses flow chart.

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RESULTS

Sources of exosomes

Mesenchymal stem cells (MSCs) – either from bone marrow, adipose tissue, umbilical cord, or other tissues – were the most common source of exosomes, with 25 studies using MSC-derived exosomes. Schwann cells were the source of exosomes in ten studies. Induced pluripotent stem cells (iPSCs) were the source in two studies. Other cell types such as neurons, fibroblasts, endothelial cells, and platelets were each used in 1–2 studies. Nine studies did not specify or did not have access to the source of exosomes [Figure 2].

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Figure 2:

Exosomes in neuroregeneration.

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Transplantation approaches

Direct injection of exosomes into the injury site or localized tissues was used in 15 studies. Different injection sites included major pelvic ganglion, cavernous nerve, caudal vein, and wound sites. Loading exosomes into nerve conduits or grafts for transplantation was used in 12 studies. Exosomes were incorporated into various nerve conduits such as chitosan, collagen, and silk fibroin. Systemic intravenous injection was used in ten studies. No transplantation details were provided in nine studies.

Animal models

Rodents were the most common animal model, used in 67 studies. Rats were used in 58 studies, while mice were used in nine studies. Sprague-Dawley rats were the most popular rat strain, used in 44 studies. Wistar rats were used in eight studies. The sciatic nerve injury/defect model was the most common disease model, used in 55 studies. Other models such as cavernous nerve injury, cortical injury, and skin injury were each used in —one to two studies. Nine studies did not specify the animal model used.

Cells used

Schwann cells were the most used cell type, seen in 27 studies. They were often used for in vitro experiments and exosome collection. MSCs were used in 19 studies for exosome collection. Bone marrow and adipose tissue were common MSC sources. Dorsal root ganglion (DRG) neurons were used in five studies. Other cell types such as fibroblasts, macrophages, and endothelial cells were each used in —one to two studies.

Diverse study groups have been utilized to assess the therapeutic potential of exosome-mediated interventions across various models of nerve injury. The groups were delineated based on the type of nerve injury, the source of exosomes, and the regenerative strategies employed. Groups bridged with autografts, acellular nerve allografts, and iPSCs-derived exosomes to assess direct regenerative effects. Exosome treatment groups were compared with control groups to evaluate the efficacy of exosomal therapy.

Studies involving different concentrations of exosome preparations were done to establish dose-dependent effects on Schwann cell proliferation and nerve repair. Comparative analyses of exosome treatments derived from various cell types such as Schwann cells, adipose-derived stem cells, and bone marrow stromal cells were performed to determine the most efficacious source.

The studies evaluating exosomes for peripheral nerve regeneration in rodent models of sciatic nerve injury demonstrate significant improvements in several quantitative measures of nerve repair. Axonal regeneration was enhanced, with exosome treatments showing increased axon count, diameter, density, and area compared to controls at 8–12 weeks post-injury (P < 0.05).^[9] For example, one study found a two-fold increase in myelinated axon number after exosome treatment.^[10] These morphological improvements translated to better functional recovery, with sciatic functional index, compound muscle action potential, and tension recovery ratios being superior in exosome groups versus controls from 4 weeks onwards (P < 0.01).^[11] Muscle atrophy was also reduced by exosome therapy, with higher gastrocnemius muscle mass and larger muscle fiber diameter versus controls (P < 0.05).^[12]

Nanoparticle tracking analysis revealed exosome preparations in the 100–150 nm range, consistent with reported exosome sizes.^[13] Exosome uptake by Schwann cells and neurons was demonstrated by labeling techniques.^[14] Proliferation assays showed 1.5–2× higher Schwann cell proliferation after exosome treatment compared to controls by 7 days (P < 0.001). Flow cytometry quantified increased percentages of proliferating glial cells post-exosome administration.^[15] Quantitative polymerase chain reaction (qPCR) and RNA sequencing revealed exosome-mediated increases in neurotrophic, angiogenic, and myelination-related gene expression in target cells.^[16] Immunofluorescence staining demonstrated up to five-fold increases in new blood vessel formation after exosome therapy (P < 0.01). Axonal sprouting and outgrowth were also enhanced —two to three fold based on neurofilament staining.

DISCUSSION

The most common sources of exosomes were MSCs, Schwann cells, and iPSCs. The exosomes were most often locally injected into injury sites or loaded into nerve conduits/grafts for targeted delivery. There is a diversity of exosome sources and transplantation approaches being explored for neuroregeneration. Further studies are needed to compare the efficacy of different sources and delivery methods.

Rodent models, especially rats with sciatic nerve injury, were predominantly used to study neuroregeneration. The wide utilization of rodents in the studies mirrors the broader trends in biomedical research for nerve injury models. The dominant use of Sprague-Dawley rats and the sciatic nerve injury model underscores the preference for a standardized, well-characterized animal model that allows for reproducibility and comparison across studies.^[17,18] This selection reflects the ease of handling, cost-effectiveness, and extensive background knowledge available for these animals and injury models.

Schwann cells and MSCs were the main cell types studied for their exosome therapeutic effects. The predominance of MSC-derived exosomes in the studies aligns with the literature, suggesting that MSCs are a versatile and rich source of therapeutic exosomes due to their accessibility and regenerative properties.^[19,20] The inclusion of Schwann cells and iPSCs as sources also reflects their relevance in neuroregeneration, particularly given Schwann cells’ role in peripheral nerve repair and iPSCs’ pluripotency.^[21] The frequent use of Schwann cells in the studies underscores their pivotal role in peripheral nervous system regeneration, given their natural function in supporting axon regeneration after injury.^[22] This aligns with current research trends emphasizing Schwann cell-derived exosomes as key mediators of nerve repair mechanisms.^[23,24]

The utilization of MSCs from bone marrow and adipose tissue reflects their versatility and accessibility as sources for regenerative therapies, including their role in exosome production.^[25] MSC-derived exosomes have been shown to facilitate peripheral nerve regeneration, acting as vehicles for delivering regenerative factors to injury sites.

The reliance on rats and mice highlights the need for studies in larger animal models that better replicate human physiology. Compared to other systematic reviews, our study underscores the broad spectrum of exosome sources, which may enhance understanding of their mechanisms of action and therapeutic potential in neurodegenerative diseases.^[26]

The studies reviewed employed various delivery methods including local injections under the epineurium, systemic administration through caudal vein, and filling of nerve guide conduits. Direct injection into the injury site offers targeted therapy, potentially maximizing the local therapeutic effects of exosomes.^[19,20] The use of nerve conduits or grafts for exosome delivery suggests a novel approach to enhance nerve regeneration, possibly by providing a sustained release of exosomes at the injury site.^[21] Systemic intravenous injection indicates the exploration of a less invasive method, potentially offering a broader distribution of therapeutic effects.^[27]

Comparatively, these approaches mirror the diversity of strategies found in the existing literature on stem cell-derived exosomes for various regenerative purposes. However, the specific application to neuroregeneration emphasizes the innovative use of exosomes in neural repair and functional recovery.

Dosages ranged from microgram to particle number concentrations, with treatment frequencies spanning single doses to multiple administrations over weeks. The examination of dose-dependent effects of exosome preparations on Schwann cell proliferation and nerve repair, along with the comparative analyses of exosome treatments from different sources, underscores a critical aspect of regenerative medicine: The optimization of therapeutic strategies for enhanced efficacy. The findings suggest that not only the source of exosomes but also the concentrations at which they are applied play a significant role in their regenerative capabilities. This is consistent with studies highlighting the promotive effects of Schwann cell-derived exosomes on nerve regeneration,^[24,23] as well as the potential benefits of exosomes from adipose-derived stem cells and bone marrow stromal cells.^[25,28]

The implications of these results are profound, indicating that tailored exosome therapies based on specific cell sources and optimal dosing could significantly enhance outcomes in nerve repair. Thus, there are certain nuanced effects of exosome therapy, advocating for a more personalized approach to treatment.

Outcome assessments were conducted from hours to weeks post-treatment, with many studies noting significant improvements in structural and neurological function recovery. Investigations into the effect of exosome pre-treatment conditions, such as hypoxia or drug treatments, on their regenerative capacity were also done. Thus, there is scope for customization of the techniques.

Quantitative methods such as the Cell Counting Kit-8 assay and flow cytometry were used to measure cell proliferation rates and effects on Schwann cells after treatment with various exosome preparations. qPCR, high-throughput sequencing, and immunofluorescence staining were utilized to assess nerve regeneration efficacy and to quantify related protein and messenger RNA expressions. Tube formation assays and immunofluorescence staining provided quantitative measures of angiogenesis and axonal outgrowth, which are crucial for nerve regeneration.

Novel techniques like nanoparticle tracking analysis were employed to assess exosome size and concentration, and established methods like the Western blot analyses were used to analyze protein expressions and the effects of treatments. The nanoparticle tracking analysis confirms the size consistency of exosome preparations, aligning with established parameters for exosomes, which facilitates their identification and characterization.^[23] The enhanced uptake of exosomes by Schwann cells and neurons, along with significant proliferation observed through various assays, underscores the effectiveness of exosome therapy in promoting cellular processes critical for nerve repair.^[24]

Moreover, the increase in neurotrophic, angiogenic, and myelination-related gene expression as revealed by qPCR and RNA sequencing indicates that exosome therapy not only supports the growth of nerve cells but also promotes the formation of new blood vessels and myelination, essential for the restoration of nerve function.^[29] This multi-faceted impact of exosomes, evidenced by up to five-fold increases in new blood vessel formation and enhanced axonal sprouting, suggests a robust mechanism through which exosomes contribute to nerve regeneration.

These results are in harmony with other systematic reviews that have highlighted the regenerative effects of exosomes in peripheral nerve injuries, indicating their role in enhancing neuronal survival, growth, and plasticity.^[30] Recommendations for future research include further elucidation of the molecular mechanisms underlying exosome-mediated regeneration and exploration of optimized dosing and administration routes to maximize therapeutic outcomes.

Sciatic nerve injury model studies demonstrated improved axonal growth, reinnervation, and motor function recovery, assessed through the sciatic functional index, nerve conduction velocity, and compound muscle action potential recovery indices. Measures included cross-sectional area, diameter, and number of myelinated axons, with evaluations conducted at 8 and 12 weeks postoperatively. This holds huge promise for neurological disease cure. The focus on the sciatic nerve injury model, used in a majority of the studies, highlights its importance in peripheral nerve repair research, given its relevance to human peripheral nerve injuries. However, the limited variety in animal models and injury types could pose a risk of narrow research outcomes that might not fully translate to human conditions.^[31] In comparison with other systematic reviews, our findings likely align with the emphasis on rodents for preclinical models in nerve repair.

The DRG neurons in our review highlight the importance of neuronal components in studying nerve regeneration, given their direct involvement in transmitting signals from the peripheral to the central nervous system.

Exosome treatments enhanced functional recovery and axon outgrowth, and reduced muscle atrophy, which were quantified through muscle strength tests, tension recovery ratios, and muscle fiber analysis. And thus, they establish a strong role for exosomes in neuroregenerative medicine. The results from studies evaluating exosomes for peripheral nerve regeneration in rodent models, particularly sciatic nerve injury, indicate a significant therapeutic potential of exosome treatments. These findings are consistent with current research that highlights the role of exosomes in promoting nerve regeneration and functional recovery after injury. For instance, studies have shown that exosomes derived from various cell sources can enhance axonal regeneration, improve functional recovery, and reduce muscle atrophy following peripheral nerve injury.^[2,32]

These improvements in axon count, diameter, density, area, and functional recovery metrics such as the sciatic functional index and compound muscle action potential underscore the efficacy of exosome treatments in nerve repair processes. The reduction in muscle atrophy further supports the multifaceted benefits of exosome therapy in the context of peripheral nerve injuries.

Comparatively, these results align with other systematic reviews, suggesting exosomes as a promising therapeutic strategy for peripheral nerve injuries, showcasing their regenerative effects across various models and species.^[33]

The study emphasizes the dose-dependent and source-specific effects of exosome treatments. Recommendations include the need for further research to optimize exosome therapy protocols, such as identifying the most efficacious exosome sources and concentrations. In addition, translational studies to evaluate the clinical applicability of these findings in human nerve regeneration would be pertinent. We advocate for more high-quality, low risk of bias studies to be conducted in this field to strengthen the evidence base going forward.

The studies support the efficacy of exosome therapy in rodent nerve injury models across morphological, functional, proliferative, and genetic measures. Translation to humans and large animals is now required to further validate these findings.

CONCLUSION

Evidence for the possibility of exosome-based treatments to promote nerve regeneration is included in this systematic review. In preclinical models of nerve injury, exosomes produced from different cell sources, including Schwann cells and mesenchymal stem cells, have diverse restorative effects. These outcomes include reduced muscle atrophy, improved axonal regeneration, remyelination, and functional recovery. The regulation of neurotrophic signaling and angiogenesis are the basic processes that underlie these advantages. But the majority of studies have a high risk of bias, which emphasizes the need for more thorough, well-thought-out research. Subsequent investigations ought to concentrate on enhancing exosome supplies, transport techniques, and dosage schedules. Furthermore, larger animal model research and potential clinical trials are essential to confirm the safety and effectiveness of exosome therapy for human neuron regeneration. Notwithstanding the drawbacks, exosomes have a promising future as a cutting-edge treatment strategy in neuroregenerative medicine, as evidenced by the consistently favorable results across a range of investigations.