Growth Factors and Supporting Cells of Nerve Conduits for Peripheral Nerve Regeneration

Yang XIANG,Zhi-Wu CHEN,Jun-Shui ZHENG,Zhuan YANG,Guang-Hao LIN,Peng WEI

1Department of Plasticand reconstructive surgery,Ningbo First Hospital,Ningbo City,Zhejiang Province,315010,China;

2Ningbo university,Ningbo City,Zhejiang Province,315010,China.

ABSTRACT Peripheral nerve injury is a common disease that endangers human health.There is a variety of methods to repair peripheral nerve injury,the current “gold standard” approach is autograft,however it still faces many disadvantages.A new choice is the use of artificial nerve conduits,which are tubular structures and are designed to bridge nerve gaps.In order to bridge longer nerve gaps and gain ideal nerve regeneration effects,multiple technologies have been developed to the design of nerve conduits,such as selecting sutible materials,supplementing growth factors,transplanting supporting cells and so on.This review mainly introduce current progess in growth factors supplementation and supporting cells transplantation technology of nerve conduits.

KEY WORDS peripheral nerve injury,nerve conduits,growth factors,supporting cells

Peripheral nerve injury（PNI）is a common disease causing obvious physical disabilities,which due to trauma,congenital defects or surgical procedures.Peripheral nerve injury repair account for about 2.8%of all trauma surgeries[1].Currently,there is a variety of microsurgery methods to repair peripheral nerve injury,including direct tension-free neurorrhaphy,autograft/allograft transplantation.Direct tension-free neurorrhaphy.Despite developments in microsurgery,the motor recovery from peripheral nerve injury which obtain satisfactory results are unsatisfactory.For nerve defects more than 5mm in length,the graftings are the better choice[2].The current“golden standard” approach is autograft,however,still faces several disadvantages such as a need for second surgery,donor site morbidity,donor site size and shape mismatch and the possibility of neuroma formation[3].Another nerve graftings approach is allograft,which could overcome the shortcoming of autograft,but patients who receive allograft would undergo immunosuppression to avoid rejection[4].In efforts to address the shortcomings of these nerve grafting therapies,the primary alternative is the use of artificial nerve scaffolds.

The common type of nerve scaffolds are biological and synthetic nerve conduits,which are tubular structures designed to bridge nerve gaps.The nerve conduits creat a micro-environment for nerve regeneration between the nerve gaps,the nerve regenerative process in the microenvironment can be divided into five phases[5]:(1)the liquid filling phase:the first day after transplantation,the two broken ends of the nerve secreted fluids rich in neurotrophic factors and non-cellular matrix precursors,which accumulated in the conduit and formed a fluid filling regeneration chamber;(2)the matrix bridge formation phase:during the following week,the noncellular matrix precursors formed a longitudinal framework,also known as a "fiber cable," acted as a bridge between nerve breaks;(3)the cells migration phase:at two weeks after transplantation,a mixture of schwann cells,fibroblasts,and endothelial cells were secreted between nerve broken ends,these cells migrated along"fiber cables" and converged to form a matrix bridge,also known as the glial bands of Bungner;(4)the axons growth phase:at three weeks after transplantation,the regenerated axons grew along the matrix bridge and gradually reached the targets;(5)the myelin generation phase:about four weeks after transplantation,the regenerated axons reached the targets and formed myelin sheath with schwann cells.

The development of nerve conduits went through a long period.In 1882,the first”nerve conduit”consist of bone,which was actually hollow bone tube,was applied to bridge a 30mm nerve gap in a dog[6].With the developments of techniques,various nerve conduits had been manufactured and been approved in clinical application.For example,a study group developed collagen based biodegradable conduits,then they were installed in 12 mm (average)gaps in 19 patients and follow-up till 20 months,and the results reported satisfactory restoration of nerve functions as assessed by an independent observer[7].Another study installed Poly(DL-lactide-εcaprolactone)composed NGC into patients with forearm,elbow,wrist,palm and finger nerve injury sites,and the resulet revealed some advantages in the regain of sensory function,however,it also posed some serious disadvantages such as formation of neuroma in one out of 23 patients[8].Compared with traditional nerve repair methods,the nerve conduits have many advantages:reducing neuroma formation,avoiding donor site disfunctions,contributing to the concentration of growth factors and so on[9].However,their ability to repair large nerve gaps are still remained unknow.Fortunately,recent advances in materials science,nanotechnology and cell biology have promoted the biomimetic technologies of nerve conduit:including material,fabrication technique,architecture,and surface properties[10].These aspects are important for nerve regeneration and have been discussed for many times,the other technologies such as growth factor supplementation and cell transplantation have attracted low attention,but may be a future choice for integrating scaffold technologies.This review highlights the growth factors supplementation and cells transplantation technology in this field(Figure1).

Figure 1 The proximal end and distal end of peripheral nerve.Examples of molecular therapies and cellular therapies include growth factors,schwann cells and stem cells.

GROWTH FACTORS

Nerve growth factors,which play an important and complex role in regulating a large number of nerve and non-nerve cells phenotypic changes,could enhance nerve regeneration during peripheral nerve recovery,thus it’s vital to mimic their release.Although endogenous growth factors secreted by nerve cells in the distal nerve stem can support axons regeneration,but the production of endogenous growth factors decline over time,consequently the sustained supply of growth factors can’t be maintained.As a result,exogenous growth factors are necessary for axons regeneration.

Various of Growth factors and their applications

A number of growth factors have been employed in nerve regeneration,these growth factors can be mainly concluded:nerve growth factor(NGF),brain-derived neurotrophic factor(BDNF),neurotrophins-3(NT-3),glial cell line-derived neurotrophic factor(GDNF),plateletderived growth factor(PDGF)and so on[11].Portions of these growth factors have been applied into promoting multi-modal neurotrophic functions by targeting different biological pathways,including pathways needed for nerve regeneraiton.NGF is the major factors of growth factors,hence widely used both in vitro and in vivo.For example,Xia and Lv created a nanofibrous scaffold loaded with NGF and vascular endothelial growth factor.Compare with VEGF which only released within the first days,NGF could be continuously released for up to 1 month[12].BDNF,which is synthesized by Schwann cells,motor neurons,and a specific sub-group of DRG neurons,also contribute to the nerve regeneration.After nerve injury or complete transection,BDNF mRNA expression increase obviously[13].NT-3 belongs to neurotrophins,and has overlapping neurotrophic activity with NGF.According to some studies,NT-3,whose target cell was sensory neurons,had bioactive effect of neurogenesis and gliogenesis[14].Similarly,studies found that neurotrophin-4 (NT-4)might contribute to nerve regeneration[15].GDNF takes part in degenerative diseases and encourages survival of damaged midbrain dopaminergic neurons[16],study also reported that GDNF has potent effects on neuronal survival and repair of injured nerves[17].Another study found that plateletderived growth factor(PDGF)increased the number of nerve fibers in peri-implant tissues at early stage during a SD rats experiment,thus proving PDGF could promote nerve regeneration in peri-implant tissues at early stage[18].

Controlling growth factors release

Controlling growth factors release is vital for efficient nerve regeneraton,studies have proven that continuous release of growth factors promoting better nerve regeneration than rapid short-term release[19].As a result,several mechanisms promoting sustained release of growth factors have been designed to achieve this goal :growth factors are absorbed to the surface of the scaffold;growth factors are added into the scaffold materials during the fabrication of a scaffold;microspheres loaded with growth factors are trapped into the scaffold;growth factors are covalently immobilizated onto the scafold;an osmotic minipump or injection device is installed[20].For example,some studies integrated various growth factors (such as NGF,VEGF)[21]into the luminal wall and achieved ideal outcomes.Lackington found that guidance conduits with optimized NGF- and GDNFloaded microparticles had enhanced function ,and they could promote regeneration a 15 mm sciatic nerve defect in rats[22].Fadia investigated a biodegradable poly(caprolactone)(PCL)conduit with embedded doublewalled polymeric microspheres encapsulating glial cell line-derived neurotrophic factor (GDNF)capable of providing a sustained release of GDNF for >50 days in a 5-centimeter nerve defect in a rhesus macaque model,and the results demonstrated that the experiment group not only had similar functional recovery but also exhibited a statistically greater average area occupied by individual Schwann cells at the distal nerve compared to autograft[23].Some scientists have found that NGF loaded chitosan-PLGA double-walled microspheres have a potential clinical application in peripheral nerve regeneration after injury[24].Some experiments have demonstrated better nerve regeneration in rats inserted with GDNFloaded poly(ε-caprolactone-co-ethyl ethylene phosphate)(PCL-EEP)nanofibers than with plain PCLEEP fibers alone[25],due to the diffusion of growth factors from the scaffold surface and the release of growth factors during scaffold degrades.Shintani Kosuke investigated nerve conduits coated with dual controlled release of stromal cell-derived factor-1 (SDF-1)and basic fibroblast growth factor (bFGF)for peripheral nerve regeneration,and animal experiment results demonstrated their promoting peripheral nerve regeneration function[26].

Regulating growth factors dose

Another method for promoting nerve regeneraton is regulating growth factors dose,dose is not only key for effective repair but also key for nonaberrant repair.The dose released into the tubular structure should be carefully controlled,because a low dose has limited clinical effects,and a high dose can cause aberrant axonal regeneration,poor functional recovery outcomes,high cost and failure to achieve the target[27].The growth factors embedded in the neural scaffolds in vivo are homogeneous and not able to simulate the growth factors gradient in cell environment ,because axons have a tendency to move toward higher concentrations of growth factors which is called growth cone chemotaxis[28],so the effect of directional guidance in the conduit is somewhat reduced[29].Growth factor gradients have been inserted into neural scaffolds to mimic the physiological environment,further promoting axons extending in search of their appropriate targets,often over long distances with the assistance of growth cones detecting and following molecular gradients[30].Bellamkonda investigated the heterogeneous distribution of nerve growth factors in the tubular structure ,which presented concentration gradient distribution,and the results suggested that the heterogeneous distribution had better regeneration effect on the 20mm gap of the sciatic nerve in rats than the uniform distribution[31].Sun et al.invented a new method which could produce growth factor gradients.In detail,they used encapsulated bone marrow MSCs (BMSCs)capable of producing high levels of BDNF to encapsulate conduit,which presented dissimilar cell attachment and distribution after 6 weeks in vivo,thus inducing Schwann cell transfer from the center to the distal end[32].Chang et al.developed a natural biodegradable multi-channeled scaffold composed by oriented electrospun nanofibers containing a neurotrophic gradient.For the gradient,the authors followed two strategies:the NGF was simply blended with gelatin and BDNF was encapsulated in nanoparticles further embedded in gelatin hydrogel.The gelatin scaffold was divided into five regions from A1-A5(low concentration to high concentration,from proximal to distal site).Their strategy promoted intense nerve regeneration in critical sciatic nerve defect in a rabbit model[33].

SUPPORTING CELLS

The success of peripheral nerve repair is largely dependent on the distal endogenous schwann cells.Dedifferentiated schwann cells have to migrate across the injury site to form the binger's zone,whereas schwann cells may undergo senescence at the injury site and accelerate senescence when crossing larger gaps.Studies have found that the repair effects are improved by adding schwann cells,stem cells to the empty conduits,as outlined below.

Schwann cells

How do schwann cells contribute their functions in vivo?It is generally believed that they play a role by increasing electrical impulse conductivity through the axons myelin sheath,by releasing biological activity signals to provide neurotrophic support to motor and sensory axons,and by acting on the production and maintenance of basal membrane[34].The purpose of adding schwann cells(SCs)into tubular structures is to improve the repair effects by supplementing aging schwann cells at the site of injury.SCs can be harvested from different type of sources(such as allogeneic,syngeneic,autologous,celllines,orstemcells)[35].It has been considered that autograft is the gold standard of peripheral nerve regeneration and autologous Schwann cells are the gold standard of cellular-based therapies[36].The advantages of autologous schwann cells can be mainly concluded that they avoid inducing an immune response after transplantation,prior to other exogenous cells.May F et al.used mice to perform bilateral excision of cavernous nerve segments,then cavernous nerves were reconstructed using unseeded silicon tubes,nerve autografts and silicon tubes seeded with either Glial-cellline-derived (GDNF)-overexpressing or green fluorescent protein (GFP)-expressing Schwann cells (SCs),and the results proved that Schwann-cell-seeded scaffolds combined with neurotrophic factors are superior to unseeded tubes and autologous nerve grafts[37].Another clinical study have hypothesized that autologous human schwann cells(ahSC)could improve axons function.In the study,participants were paraplegics with neurologically complete,trauma-induced spinal lesions.Autologous SCs were cultured in vitro from a sural nerve harvested from each participant and injected into the epicenter of the spinal lesion,after one year,there was no evidence of additional spinal cord damage,mass lesion,or syrinx formation.The application of allogenic SCs is limited due to complexities associated with host immunological rejection and immunosuppressive drugs.A less commonly used cells is genetically modified schwann cells,which overexpress a pro-regenerative cytokines or neurotrophic factors,so that their phenotype is quite different from that of autologous schwann cells.For example,in a recent study,schwann cells in vitro were isolated and infected with lentivirus vector,in order to guide the glial nerve growth factor overexpressed[1],then these cells were added into transplants to repair 14 mm of rat sciatic nerve defects.The results showed that early recovery of nerve was ideal,however constant ovexpression went against regeneration eventually,leading to abnormal repair[38].Gene modification has promising prospects,but further research on more transient gene expression sequences to regulate the release of overexpressed cytokines is needed,in order to overcome some current limitations.The use of Schwann cells still faces a few disadvantages such as the effort of collection,the time-consuming expansion in culture and a inappropriate immunogenicity,thus requiring further immune suppression strategies to be considered[39].Because of these drawbacks,the attention shifted to the use of undifferentiated stem cells.

Stem cells

In addition to growth factors,successful nerve regeneration requires interaction between neuronal and non-neuronal supporting cells in the extracellular matrix.Stem cells have great potential to promote neural regeneration,and have the potential to differentiate into supporting cells to promote neural regeneration.There are many stem cell types,such as embryonic stem cells,which can be called true pluripotent cells because of their potential to differentiate into any cell types.Adult stem cells,also known as somatic stem cells,are pluripotent and can differentiate into specific lines of cells.Not only stem cells can differentiate into glial fibroacidic protein positive schwann cells and support myelin sheath[40],but also they can differentiate into fibroblastlike cells that produce both extracellular matrix proteins and neurotrophic factors[41].Clinical use of stem cells is sill under restrictions,for example,it is regulated and monitored by the food and drug administration (FDA)in the United States.In Europe,the European drug agency calls cell-related therapies as "advanced medical products",although the European agency supports stem cell research,the infrastructure which allows cell-related therapies to be transferred to the clinical use is still under development[42].The following will disscuss the major classification of stem cells.

Neural stem cells

Neural stem cells(NSCs)have also been used for peripheral nerve regeneration.NSCs,which

Isolated from the adult striatum,have the remarkable ability to divide,proliferate,and experience multilineage differentiation in vitro[43].NSCs could differentiate into schwann cells,and they were first found having the ability in vitro experiments.In detail,they were added into collagen tubular structures to repair 15 mm gap of rat sciatic nerve ,and the result showed that they improved regeneration significantly compared with control group[44].In another study,the neural stem cells also improved the ability of nerve conduits in rabbit models[45].In conclusion,these studies illustrated the potential of neural stem cells to improve nerve regeneration.However,at present the technology is not available to extract neural stem cells from the organization of human brain in clincal practise,so the use of these cells is limited to the xenogenous source,which is likely to cause an immune response.Due to the need of immunosuppression,it raises the overall cost of treatment.Lee et al.found that the addition of IL12p80 together with NSCs in NGCs improved motor function recovery,promoted nerve regeneration and increased the diameter of newly regenerated nerve up to 4.5 fold with mouse sciatic nerve defects[46].

Embryonic stem cells

Embryonic stem cells(ESCs)are self-replicating pluripotent stem cells derived from early human embryos,which limits their availability,but their diversity ensures that human embryonic stem cell types can be used for research.ESCs are able to differentiate into schwann-like cells,which express schwann cell phenotypes,and have been shown to be associated with axons migration[47].During a study,ESCs were injected into an epinuclear conduit,which was connected to the sciatic nerve in rats,and the results confirmed that 64% of the normal axons crossed the 1cm gap,while only 7% of the autologous nerve grafts group were counted.More than 1/3 of the ESCs differentiated into schwann cells and survived for at least 3 months[48].ESCs could also differentiate into NSCs.During a study,NSCs differentiated from ESCs were seeded into a biodegradable nerve conduit,then the conduit was transplanted a rat sciatic nerve injury model.A robust regeneration front was observed across the entire width of the conduit seeded with the differentiated neural crest cells.Moreover,the up-regulation of several regeneration-related genes was observed within the dorsal root ganglion and spinal cord segments harvested from transplanted animals[49].However,ethical issues have hindered the use of embryonic stem cells.Although embryonic stem cells do not develop tumors as they differentiate into spinal stem cells,they tend to form teratomas and induce immune rejection[50].

Bone Marrow Stromal Cells

Bone Marrow Stromal Cells(BMSCs),which have pluripotency,are another kind of stem cells.The most obvious advantage of them is that they can be isolated from human tissues,mainly from the bone marrow of ischium and sternum,so that minimizing the risk of an immune response.These cells have the potential to differentiate into tissues other than mesoderm,for example,they have successfully differentiated into the Schwann cell phenotype with beta-mercaptoethanol and retinic acid,further combined with tubular structures,and the results have proved that they can improve the repair capacity of the sciatic nerve in rats[51].Zheng et al.performed a experiment,during which rat BMSCs were genetically modified with recombinant lentiviruses to construct TrkA-overexpressing BMSCs.They were then seeded in acellular nerve allografts to bridge 10-mm rat sciatic nerve defect.Eight weeks after surgery,the analyses demonstrated improved axon growth,as well as significantly higher expression of myelin basic protein and superior results of myelinated fiber density,axon diameter and myelin sheaths thickness,revealing a superior outcome in terms of nerve regeneration[52].Lankford et al.found that intravenous delivered BMSCs exosomes tend to migrate into the injury site,where they exert their beneficial effects,thus promoting the recovery of contusion injury of the spinal cord in rats[53].

Adipose-Derived Stem Cells

Adipose-Derived Stem Cells(ADSCs)can also be used for nerve repair,with the advantage of being able to isolate 5000 cells from 1g of adipose tissue,500 times the number of stem cells available from the same amount of bone marrow[54].Thus,in addition to being available from autologous tissue,it is also more available than other cell types.Schilling et al.performed a study.During the study undifferentiated ADSCS injected directly in the muscles connected to the damaged nerve were found to have increased presence of IL-10 and Ki67,which helped in delaying the onset of muscular atrophy[55].The result proved that undifferentiated ADSCs have the ability to promote nerve regeneration.Ching et al found that differentiated ADSCS could also enhance nerve regeneration function[56].

Fetal tissue derived stem cells

A number of stem cells similar to mesenchymal stem cells have been isolated from fetal tissues,including wool and chorionic membranes,amniotic fluid,umbilical cord tissue,and blood[57].These tissue sources have a distinct advantage,as they are collected from the fetal age approximately 9 months near the fertilization ,and have less time to accumulate cell mutations and genetic irregularity which are common in adult stem cells[58].The youth of these cells also ensures their extremely robust regenerative activity,and surpass the fat-derived stem cells[58]. Stem cells derived from fetal tissues such as umbilical cord tissue called human umbilicalcord mesenchymal stem cells (HUCMSCs)can secrete 14 different neurotrophic factors related to enhance peripheral nerve regeneration[59].As a result,HUCMSCs paracrine effects probably lead to the efficacy of those cells in the treatment of nerve injuries[60].

Induced pluripotent stem cells

Induced pluripotent stem cells(IPSCs)are a kind of somatic cells that have been genetically modified to express stem cell-like phenotypes in morphology and growth behavior.Induced pluripotent stem cells,which induced from Somatic cells,avoid immune rejection and can be obtained from the body with lower levels of dysfunction.The stem cells induction process requires cells reprogramming,which is accomplished through reverse transcription by transcription factors,and the process always takes weeks.In vivo experiments in rats,Ikeda demonstrated that induced pluripotent stem cells and fibroblast growth factors added into neural conduits had synergistic effects on nerve regeneration,whereas autogenous nerve groups had the best effects[61].

Dermal derived precursor cells

Dermal derived precursor cells are a kind of multipotent cells isolated from the dermal papilla of the hair and beard follicles.These cells have the ability to differentiate into mesodermal and neuronal cells,including neurons,schwann cells,and smooth muscle cells[62].Dermal derived precursor cells shared many key characteristics with another dermal pluripotent cell type(nerve-ridge stem cells),and further study showed that dermal derived precursor cells from the face were derived from nerve-ridge[63].Because of their superficial location,these cells are readily available in the adult stem cell group in regenerative medicine.

Hair follicles derived stem cells

In addition to dermal-derived progenitors,there is another group of pluripotent adult stem cells located in the skin,adjacent to hair follicles.Hair follicle-derived stem cells are located at the exudated site,in the superficial layer of the dermal papilla layer[64].This bulge acts as a reservoir of dermal stem cells on the surface,which have the ability to form new hair,skin,and sebaceous glands[65].Hair folliclederived stem cells can be identified by their expression of nestin,which is associated with nerve progenitor cells[66].Hair follicle-derived precursor cells have the ability to differentiate into nerve ridge factional cells such as schwann cells,neurons,melanocytes,smooth muscle cells,and chondrocytes[67].

CONCLUSIONS

Neural conduits have great prospects in the field of peripheral nerve regeneration,however,currently approved technologies are still under expectation,especially for the injury of large gaps.Various efforts have been made to improve current techniques,and better simulations of the anatomy and physiology of peripheral nerves have yielded remarkable results.Although autograft remains the gold standard,bionics in the area of catheter design have improved the regenerative performance of neural stents.To fully understand the clinical potential of these technologies,multifunctional facilities combined with biomimetic materials,models,structures and surface modifications should be investigated in depth.Furthermore,new technologies in neural scaffolds combined with other regenerative strategies such as growth factor supplementation,stem cell transplantation,and cell-surface polysaccharide engineering will have additional synergies.Overall,recent technologies in the field of bionics present exciting possibilities for artificial neural scaffolds that may exceed the effectiveness of current clinical practice.

The field of peripheral nerve regeneration and tissue engineering benefits from advances in biomaterials,nerve catheter manufacturing,substrates,and growth factors.Despite these advances,the potential application of unrestricted autologous stem cells to peripheral nerve injury may represent the greatest breakthrough and the greatest challenge to regenerative medicine to date.Although research in this area presents many possibilities,each with advantages and limitations,suitable stem cell resources will be regulated by improvements in manufacturing,quality control and development regulation ultimately.As stem cell integration and other aspects of the peripheral neural field have improved,the combination and effectiveness of these artificial nerves will increase,with a looser limit on the length of neural defects that can be repaired without autologous transplantation.In this way,the combination and possible synergy result in improved results of nerve regeneration.

Rapid advances in the field of research have led to improvements in the ability of hollow nerve conduits repairing long gaps.Functional and morphological techniques have been used to evaluate the performance of nerve conduits in vivo.Studies have increasingly focused on describing the differences in presentation between tubular structures and autograft,thus making neural conduits clinically relevant.Several studies have reported the same effects of neural conduits in vivo in comparison with autograft,especially neural conduits added with biochemical factors,cells or neurotrophic factors[68].Despite the advances in stem cell technology,the ideal combination of neural conduits and cell types is still unknown.Researchers have identified two important strategies for the release of growth factors:controlled release and dose-regulated release.Although growth factor release kinetics and dosing can be standardized in vitro,transformation of the system in vivo,which often assessed by the regenerative activity of neurofactorloaded conduits,is difficult to monitor.Another problem is that the successful repair requires more than one cell type in order to improve cells activities,the cascade release of neurotrophic factors has overcomed this problem but on the other hand costly.Although the combination of growth factor bears watching,further researchs are still needed to explore their potential and cost benefit equilibrium.We believe that as the improvements in the technologies of nerve conduits design both structurely and biochemically,nerve conduits would replace autografts in the future.

ACKNOWLEDGEMENTS

Chinaprovince(2020376249,2019326481); the Natural Science Foundation of Ningbo(2013A6102647,2015A610207).