TO THE EDITOR
We were fortunate to read an article on “Bone marrow mesenchymal stem cells in treatment of peripheral nerve injury” by Zou et al[1]. We highly acknowledge the findings of the authors, who comprehensively summarized the biological characteristics of bone marrow derived mesenchymal stem cells (BMSCs), outlined the effects and mechanisms of BMSCs in alleviating peripheral nerve injury (PNI). We are grateful to the authors for their commitment to BMSCs to alleviate nerve damage, which will help elucidate the related biological mechanisms of BMSCs to improve PNI, providing a solid foundation for basic and clinical research.
In recent years, numerous studies have confirmed the effectiveness of BMSCs in improving PNI[2-4]. For example, studies have shown that local or caudal intravenous injection of BMSCs significantly increased the amplitude of complex muscle action potential, the number and diameter of fibers, and increased the density, number and diameter of axons in PNI rats[2]. It also significantly reduces collagen deposition in nerve tissue, indicating that BMSCs have certain anti-inflammatory effects[2]. Similar studies found that compared with the control group, intramuscular injection of BMSCs significantly increased sciatic functional index, nerve conduction velocity, myelin thickness, and wet weight recovery rate of gastritis muscle[3], suggesting that BMSCs promoted nerve function recovery and neurogenesis in PNI rats. Similarly, BMSCs were co-cultured with peripheral nerve extracts from rats with sciatic nerve injury in vitro, and it was found that BMSCs differentiated into Schwann cells (SCs), astrocytes and neurons in vitro[4], which may be one of the important mechanisms by which BMSCs promote neurogenesis. Further studies have found that the mechanism of BMSCs promoting the repair of injured nerves can be mainly divided into the following three aspects: Nerve regeneration, axon regeneration and myelin reconstruction. These three processes occur simultaneously and interact with each other. First, BMSCs can differentiate into neurons and SCs, which extend pseudopods to wrap axons after contact with axons, thus promoting myelin reconstruction and axon regeneration. Secondly, BMSCs secrete neurotrophic factors and growth factors, and differentiate vascular endothelial cells to promote angiogenesis. The ability of vascular regeneration plays a key role in the repair process of injured nerves, not only providing sufficient oxygen and nutrition for nerve growth and rapidly discharging wastes, but also providing direction for the migration of SCs. This facilitates a microenvironment that promotes axon regeneration and nerve regeneration. In addition, BMSCs also express a variety of chemokines and cytokines to regulate inflammatory responses and restore the neural microenvironment[5].
Furthermore, mesenchymal stem cells (MSCs) have been widely used in the field of biological tissue engineering in recent years, which is beneficial to improve the regeneration and repair after PNI. Among them, neural tissue-specific extracellular matrix (ECM) is the microenvironment on which cells depend, containing a variety of growth factors, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) and neurotrophin 3 (NT-3), which are essential for nerve regeneration and are considered to be natural materials for promoting axon and nerve regeneration. Decellularized ECM (dECM) is composed of scaffold material, supporting cells, ECM and NGFs. It has been found that BMSCs-derived dECM significantly improved axonal and myelin regeneration, restored the functional recovery of PNI, created a microenvironment conducive to axonal growth, and successfully achieved the ultimate goal of nerve regeneration and repairing the function of nerve defects[6]. It is suggested that the advantage of ECM as a biomaterial is that it provides an excellent microenvironment for cell survival, value-added and differentiation, guides the phenotype of cell-specific differentiation and proliferation, and provides a specific physical structure for tissue and organ repair and regeneration. Similarly, acellular nerve allograft (ANA) well mimics the natural structure and microenvironment of the ECM, which is conducive to nerve regeneration. Li et al[7] found that ANA combined with BMSCs transplantation significantly up-regulated the levels of interleukin (IL)-10, down-regulated the levels of IL-1β and tumor necrosis factor-α in a sciatic nerve injury model rat, promoted the polarization of M1-type macrophages to M2-type, and significantly increased the number of regulatory T-cells, suggesting that the engineered therapy exhibited a more significant immunomodulatory effect and contributed to the maintenance of immune homeostatic properties in vivo. Similarly, the combination therapy significantly increased gene expression of BDNF, NGF, VEGF, NT-3, and transforming growth factor-β, showing significantly greater pro-vascular regenerative effects; it also increased myelin basic protein (MBP), recombinant protein zero expression, suggesting that ANA binding to BMSCs promoted myelin regeneration. In conclusion, the above studies demonstrated that the bioengineering therapy significantly modulated the inflammatory response, angiogenesis and myelinogenesis in vivo. In addition, recent studies have found that chitin is an ideal biomaterial for constructing nerve conduits because of its antimicrobial activity, biocompatibility, nontoxicity, and biodegradability. Polydopamine is an adhesive widely used for its high hydrophilicity, long-lasting adhesion ability, and excellent biochemical properties. It was found that BMSCs-derived exosomes (BMSCs-Exos) loaded into polydopamine-modified chitin conduits significantly increased the Exos content in SCs, also increased the levels of BDNF, NGF, VEGF and ciliary neurotrophic factor in SCs and the SCs’ proliferative capacity, increased Jun and sex determining region box 2 and myelin-related genes (MBP and early growth response gene 2) expression, and increased the average length of dorsal root ganglion (DRG) neuromasts and the numbers of axons at the distal ends of the regenerating nerve bundles and laminated myelin sheaths of regenerated nerve fibers[8], suggesting that this combination therapy activates and maintains the reparative phenotype of SCs and releases neurotrophic factors, thus providing a better microenvironment for nerve regeneration to provide a better microenvironment to repair and alleviate PNI.
Importantly, the reason why MSCs are considered an attractive therapeutic strategy for the treatment of PNI is due to the powerful paracrine capacity of MSCs. As a major component of paracrine action in MSCs, Exos are considered to be a core mediator involved in PNI, which transports a variety of NTs to receptor cells, thereby promoting vascularization and nerve regeneration[9,10]. Zhao et al[11] injected BMSCs-Exos into the gastrocnemius to explore the effect of Exos on sciatic nerve regeneration, and found that Exos could be retrograde transferred into the DRG neurons via blood and nerve fibers both in vivo and in vitro, and could be well internalized and ingested by the neurons. The authors next analyzed in depth the effect of BMSCs-Exos on nerve growth and whether this effect was related to microRNAs (miRNAs), the results showed that the ability of Exos to promote neurite growth was significantly reduced after miRNA knockdown, suggesting that BMSCs-Exos promoted the nerve regeneration of cultured primary DRG neurons through a miRNA-dependent mechanism. Further analysis showed that Exos significantly increased the average number of myelinated nerve fibers, diameter of myelinated nerve fibers and latency of thermal pain and sciatic nerve motor functional index, suggested that Exos promoted regeneration and functional recovery of damaged peripheral nerves. Accordingly, BMSCs-Exos are expected to be a novel nanoparticular drug carrier for peripheral nerve regeneration.
As a short non-coding RNA, miRNA plays an important role in the pathogenesis of PNI. It was found that the release of miR-21, miR-142-3p, miR-146b, miR-203-3p and miR-221 increased in rat DRG neurons 7 days after PNI. In particular, miR-221 was also elevated in the serum of PNI model rats from day 7 to day 28, indicating that miRNAs may be involved in the pathophysiological progression of PNI[12]. Wen et al[13] further analyzed and found that the miR-221-3p overexpression promoted the proliferation and migration of SCs, and accelerated the growth of neurites. MiR-338-3p overexpression promotes myelination of SCs. MiR-338-3p/miR-221-3p overexpression significantly improved sciatic functional index of rats, conduction velocities of compound muscle action potential and muscle recovery rate, it is suggested that miR-338-3p/miR-221-3p enhance functional recovery after sciatic nerve transection. In conclusion, the above studies have shown that miR-221-3p and miR-338-3p promote nerve regeneration and functional recovery.
Interestingly, BMSCs-Exos, as novel nanomedicines, contain miRNAs, growth factors, anti-inflammatory molecules, neurotrophic factors, etc., all of which contribute to regeneration of blood vessels, axon regeneration, and neuronal survival at the site of nerve injury. In particular, miRNAs are thought to be the material basis for the neuroprotective effects of BMSCs-Exos. For example, one study found that BMSCs-Exos containing miR-17-92 significantly increased axon density, primary and secondary neuronal branching and spine density, directly induced the production of new neurons, decreased phosphatase and tensin homolog deleted on chromosome 10 levels, increased phosphorylation of protein kinase B, mammalian target of rapamycin and glycogen synthase kinase 3 beta[14]. It is suggested that BMSCs-Exos miR-17-92 enhances nerve regeneration and recovery of neurological function through activation of phosphatidylinositide 3-kinases/protein kinase B/mechanistic target of rapamycin/glycogen synthase kinase 3 beta genesis[14]. Another study showed that BMSCs-Exos were absorbed and internalized by astrocytes[15]. Moreover, BMSCs-Exos miR-124-3p decreased p38 mitogen activated protein kinase (MAPK) and increased glutamate transporter-1 (GLT-1) expression in astrocytes. The neuronal cell death was significantly reduced. On the contrary, after treatment with Exos miR-124-3p inhibitor, p38 MAPK expression was increased, GLT-1 expression was decreased, and neuronal cell death was increased. It is suggested that ExosmiR-124-3p alleviate glutamate-mediated excitatory toxicity, reduce nerve cell death, and play a neuroprotective role by regulating the expression of p38 MAPK and GLT-1 in astrocytes[15]. Similarly, BMSCs-Exos containing miR-23a and miR-125b enhanced angiogenesis and nerve regeneration, possibly through the toll-like receptor/nuclear factor kappa-B signaling pathway, that suppressed the inflammatory response of the organism[16]. In addition, Lu et al[17] found that MSCs-Exos were mainly enriched in neurons and microglia on both sides of the spinal cord, and most of them was endocytosed by neurons and microglia. Further analysis showed that Exos miR-26a-5p reduced the levels of IL-6β, IL-4, and tumor necrosis factor-α by targeting the wingless type mouse mammary tumor virus integration site family, member 5A in the spinal cord of mice. The protein and mRNA levels of receptor-like tyrosine kinase, calmodulin-dependent protein kinase 2 and nuclear factor of activated T cells 1 were increased. The activation of microglia was significantly inhibited. These results suggest that Exos miR-26a-5p may alleviate inflammatory pain induced by nerve injury through the wingless-type mouse mammary tumor virus integration site family, member 5A signaling pathway. Moreover, Exos miRNA is low in immunogenicity, risk of tumor formation, easy to prepare, store, and transport, and easy to administer at specific times. Therefore, Exos miRNAs have been shown to be promising therapeutic targets for a variety of neurological injury disorders, especially PNI. However, Exos still face significant challenges in moving from basic trials to clinical applications. First of all, Exos production is small, and expanding its output is the primary solution to be solved in the future. Second, there is a lack of uniform standards for Exos isolation and purification procedures, which will affect the isolation, purification and mass production of Exos, thereby hindering its clinical implementation. Moreover, the safety and quality of Exos products warrant further investigation. Then, in the case that the basic experiment has proved that Exos has consistency and safety and efficacy, large sample size and multi-center clinical trials are still needed to verify.
In recent years, there have also been some researches to combine bioengineering and Exos efficiently, which is conducive to the establishment of a microenvironment for nerve regeneration as well as taking advantage of the good safety profile of nanomaterials[5,7,8,18]. The good electrophysiological properties of electroconductive hydrogel (ECH), which is almost the same as that of endogenous neural tissues, has been widely used for nerve regeneration after PNI. Yang et al[18] found that ECH loaded with BMSCs-Exos (ECH-Exos) electroconductive nerve dressing in vitro promoted the attachment and migration of SCs, decreased inducible nitric oxide synthase and tumor necrosis factor-a, while increased the M2 phenotype secretion of anti-inflammatory markers such as arginase 1 and IL-10 protein and gene levels, and decreased p-ikappaBalpha, p-P65, suggesting that the ECH-Exos hydrogel may inhibit nuclear factor kappa-B signaling pathway to induce macrophage polarization from M1 to M2 phenotype. Similarly, ECH-Exos was found to increase regenerated axonal fibers and sciatic function index, enhancing axonal fiber regeneration at the lesion as well as recovery of motor function after PNI; increase in NF-200, MBP, and S-100β proteins, confirming that ECH-Exos hydrogel treatment promotes post-PNI axonal regeneration and remyelination after PNI; significantly increased levels of MAPK kinase (MEK), p-MEK, extracellular regulated protein kinases (ERK), and p-ERK proteins, suggesting that activation of the MEK/ERK pathway by ECH-Exos hydrogel is a key factor in the recovery of motor function after PNI. MEK/ERK pathway enhances myelinated axonal regeneration, which in turn promotes the functional recovery of damaged nerves. In conclusion, ECH-Exos holds great promise for nerve regeneration, functional recovery and pain relief in PNI patients. Thus, ECH-Exos may represent a promising therapeutic regimen for PNI with broad potential for alleviating neurological deficits. In addition, the recent discovery that organoids are tiny models of organs that can summarize the function of cells and tissues in time and space has had a major impact on the field of neurobiology, providing the possibility to study human PNI in a petri dish[19]. Moreover, organoid technology has been transformed from pure matrix gel to bio-mixed or hydrogels containing cellular nutrient solution, which can significantly promote the growth and differentiation of MSCs, and aims to simulate the biomechanical and biochemical properties of natural ECMs, thus laying a solid foundation for the creation of functional in vitro organoids, such as healthy sciatic nerve models. This proves the versatility and tunability of hydrogels[19].
In conclusion, regeneration and repair of PNI is a hot direction of basic and clinical research in recent years. The goal of peripheral nerve repair is to minimize recovery time and function. BMSCs have limited its application due to its shortcomings. For example, inadequate sources of BMSCs may be an urgent issue, and the optimal dose and route of administration and their relationship need to be explored. Secondly, BMSCs transplantation carries the risk of immune rejection, low survival rate and tumor formation, which makes effective treatment of PNI challenging. In addition, cross-sectional comparative studies of BMSCs treating different PNI transgenic mice are lacking, and the differences in their therapeutic effects remain unclear. Finally, genomics, proteomics and other technologies are urgently needed to explore the beneficial components of BMSCs in alleviating PNI and the detailed mechanism[9]. It is still unclear whether the beneficial components of BMSCS are equally effective in humans, which needs to be tested through large-scale clinical trials. In addition, advanced translational models are still needed in the future to bridge the gap between basic research and clinical practice and promote successful translational BMSCs. Similarly, the BMSC inevitably faces some ethical issues. First, should donors and recipients be informed when serious diseases are discovered during the reprogramming of donated cells? Second, will donors be charged for donating cells? How much do you charge? Then, stem cell therapy as an unproven therapy, in the trial phase of whether the treatment will affect the rights of patients. Finally, the connection between basic stem cell research and clinical research needs to be solved. As we continue to address these ethical and challenging challenges, we believe that BMSCs have a very promising application.
BMSCs-Exos are considered as a new mediator of intercellular communication in the peripheral nerve microenvironment, which has the advantages of low immunogenicity, low risk of tumor formation, and a good safety profile, and has a great potential[20]. Therefore, BMSCs-Exos are considered as a safe and attractive cell-free nanomedicine. Bioengineering therapies with good ex vivo and ex vivo biocompatibility and favorable safety profile increase the yield of Exos and improve their biological functions, which play an important role in promoting the recovery of damaged nerves. Engineered Exos exhibit better characteristics, such as stronger targeting, more active ingredients, and higher transport efficiency. However, the current research on bioengineering BMSCs-Exos in PNI may only be at the beginning, and many challenges remain. The first challenge is quality standardization, the lack of a standard Exos preparation and separation system, which seriously affects the neuroprotective role of Exos, hindering its clinical application. The second challenge is related to efficacy and safety. Research in the field of bioengineering nanomaterials is just in its infancy, and large-scale basic experiments and clinical trials are needed to confirm reliability. In addition, more research is needed to explore the precision with which biomaterials can be efficiently loaded into Exos, widespread production, and the minimization of adverse reactions. While most advanced research lacks data from clinical trials, bioengineering and nanomaterials offer a new avenue for precision medicine, we firmly believe that with the deepening of basic and clinical research in bioengineering and nanotechnology, it is expected to elucidate the key molecular biological mechanisms of BMSCs-Exos to promote nerve regeneration, search for potential regenerative targets, and then provide a scientific basis for the standardized treatment of PNI, which will bring attractive therapeutic perspectives for the rehabilitation of patients with neurological injuries.
