REVIEW ON CELLULAR SIGNALING, GROWTH FACTORS, AND MECHANICAL STIMULUS IN NERVE REGENERATION

Ezegbe Chekwube Andrew1,3*image, Amarachi Grace Ezegbe2image

1Department of Pharmaceutical Technology and Industrial Pharmacy, University of Nigeria, Nsukka, Nigeria.

2Department of Home Science and Management, University of Nigeria, Nsukka, Nigeria.

3Nanoscience and Advanced Materials, Graduate Program (PPG-Nano), Federal University of ABC, Avenida dos Estados, Santo Andre, Sao Paulo, Brazil.

 

Abstract

 

The autonomous and peripheral constitute the entire nervous system. Peripheral nerve injury caused by trauma, accident and other associated factors always results in a huge loss of both the sensory and motor functions. The injured nerves can be successfully restored through the rebuilding of the functional axons. The complete recovery of PNI has not been optimized. Exogenous growth factor (GF) is a new therapeutic strategy that can be used in nerve regeneration. Growth factors mechanism of action is based on the ability to activate the signaling cascades via binding to the individual receptors in order to exert the multiple effects and restore the neuron and tissue regeneration. Although the GFs are limited by their short half-life and rapid deactivation. The use of nerve conduits has been able to reduce these limitations. The nerve conduits have been good biocompatibility and biofunctionality properties.

Keywords: Axons, growth factors, peripheral nerve injury, signaling cascade.

 

INTRODUCTION

 

Cellular signaling can be defined changes in cellular homeostasis, which causes cells to respond to different types of stimuli which could be in form of mechanical, electrical  and chemical transduction1.  Cell signaling is a process that enables a cell to interact with itself, other surrounding cells and the host environment1. Three major components are involved in cell signaling. They include the: signal, receptor and effector2. Signaling could occur in different forms viz the endocrine that involves long range communication, paracrine that involves short range, juxtracrine that involves the contact-dependent signaling and the autocrine. Growth factors are defined as polypeptides that can regulate the differentiation and proliferation of cells3. Growth factors that are soluble in nature can easily be incorporated directly into conduits in the nerve region. They play a crucial role in ensuring that numerous cell types that are involved in cell regeneration are supported4. Some of the commonly used growth factors include5:

 Nerve growth factor (NGF): It consist of three subunits: γ, β and α. Its main function is in the maintenance of mainly the basal forebrain cholinergic and sympathetic neurons6. The main action is based on its ability to bind the tyrosine kinase receptor (trkA) which enhances the choline acetyltransferase expression and its effect on neuron differentiation and maintenance7. Nerve growth factors can be increased at the injury site by insertion of Schwann cells into the nerve scaffolds6. The neurotrophic factor consists of peptides that are related both in central and peripheral nervous system7. Neurotrophins exist as noncovalent homodimers that are biologically active in nature8,9. Each molecule of the homodimer is made up of two pairs of antiparallel beta strands. Each of these beta strands is made up of highly flexible short loops10. The uniqueness of neurotropins is in their ability to bind to the receptors which include the tropomysin receptor kinase (TRK) and the tumor necrosis factor (TNF) alpha family of P75 receptor. The P45 receptor has similar affinity whenever it binds to neurotrophins, while the tropomysin receptor kinase is more specific in their binding. Nerve growth factors bind to trkA and BDNF, while NT-4/5 subsequently binds to trkB11.

Neuropoetic cytokines: They belong to the family of pleiotropic glycoprotein molecules that are active in biological activities, induction of immune responses, hematopoiesis regulation, control of cellular differentiation and wound healing induction12. The main signal mechanism for neuropoetic cytokine family is carried out through recruiting the common signal transduction receptor subunit13,14. Gp130 is not directly activated by neuropoetic cytokines, but they bind to specific ligand-binding subunits. IL-6 binds to the IL-6 receptor, LIF binds to the LIF receptor (LIFR) and the CNTF binds to the CNTF receptor (CNTFR). 

Brain derived neurotrophic factor (BDNF):  Brain and periphery have the highest concentration of this factor. Their major functions are in the promotion of the neuronal and synaptic growth, rapid maintenance of the cortex neurons and the basal forebrain. Its mechanism of action is based on their ability to bind to the trkB receptor and form the BDNF-trkB complex14.

Growth factors and their role in nerve regeneration

The NGFs belong to the peptide family. Their basic role is to ensure that the nerve fibers differentiate and survive at both the central and peripheral nervous systems15,16.

Neurotrophins are molecules that are made up of non-covalent homodimer beta chains17. They are separated from each other due to the composition of the binding sites. They play a major role in neurotrophic factors because they help the exons in growth cone during regeneration18.

Glial cell-lined derived neurotrophic factor (GDNF) 

They are made up of the GDNF, persephin (PSP), neurturin (NTN) and artemin (ART). The prominent member, GDNF helps in the motor neurons survival, while NTN assists in sympathetic neurons survival19. There are two major parts of receptors associated with GDNF. They are the GFRα1 subunit and C-ret subunit. The former serves as the binding site, while the later participates in signaling20-22.

Interactions between neurotrophic factors

There are differences that exist for both GDNF family and neuropoietic cytokines23-25. Damage to the axon leads to significant increase of BDNF mRNA within 8 hrs26, while in a healthy neuron, BDNF is under expressed, thus within the 7th day of injury, the BDNF level returns to normal. Following external damage, trkB mRNA increases on the second day, while on the 7th day, it reaches the peak. The content and localization of the axonal damage are two major factors that affect the neuropoietic cytokine receptors27. After damage to the axon, changes occur at the cellular and molecular level, which are characterized by phagocytic processes28.  Whenever an injury occurs at the axonal end, the expression of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) increases in the distal part, while the expression of NT-3 and NT-4 neurotrophin reduces29. In an intact nerve, the level of NGF mRNA is very low, while in a damaged axon, it increases to 10 times in the distal part within the first 12 hrs. After 72 hrs post-injury, it decreases back to its normal level and remains like that for about three weeks30-35. In a damaged axon, the BDNF mRNA increases at the distal part, although the increase is slow when compared to that of NGF mRNA. Although GDNF has been detected in healthy nerve, in a damaged axon, it usually peaks in distal part after 7th day and remains like that for atleast two weeks36.

Mechanical stimulus (mechanisms, biomaterials, types of stimulus and results)

Ultrasound: Ultrasound can serve two major functions: as a diagnostic and as a therapeutic tool. The mechanical energy generated by the ultrasound helps to stimulate tissue regeneration37. Ultrasound wave can come in either continuous or pulsed. The low intensity pulsed ultrasound is preferable due to the fact that it involves low intensity of mechanical wave in a pulsatile manner, which results in reduction of heat generation38. The ultrasound stimulation that regulates intracellular signaling mechanism induction of fibroblasts by mechanical force leads to enhancement of collagen production and also provision of a structural support for axonal repair38.

Extracorporeal shock wave (ESW)

The difference between extracorporeal shock wave (ESW) and ultrasound is that ESW applies a higher mechanical pressure that is about one thousand (1,000) times compared to that of ultrasound39. ESW has a lot of therapeutic applications, among them is in the repair of peripheral nerve injury.

Types of extracorporeal shock wave

  1. Focused extracorporeal shock wave (FESW)
  2. Radial extracorporeal shock wave (RESW)

Focused extracorporeal shock wave is applied in deep treatment areas that can reach up to 12 cm, while radial extracorporeal shock wave is applied to a depth of about 3-4 cm40. A mechanical stimulus is generated by the extracorporeal shock wave that provokes two major physical effects which include mechanotransduction and cavitation. In peripheral nerve repair, mechanotransduction plays a major role by affecting the development of the gene regulation of the myelin cells, differentiation of Schwann cell and the regeneration of axons41.

Peripheral nerve injury repair biomaterials  

In tissue engineering, any biomaterial used in nerve conduit production must possess some basic characteristics properties which include: bio-compatibility, biodegradability, permeability, bio-chemical properties, flexibility and resistance to collapse and tension42. The biocompatibility property of a biomaterial is further subdivided into 343,44.

i). Blood compatibility: This infomr about the ability of the biomaterial not to initiate hemolysis or coagulation in the human body.

ii). Histocompatibility: The surrounding tissues should be free from any side effects.

iii). Mechanical compatibility: The properties presented by the biomaterial must be similar to that of the host tissue. Permeability is another important parameter that should be possessed by a conduit biomaterial. This is because it enhances cell viability and also promotes the exchange of gas, nutrients and waste materials45. According to Funakoshi et al., a direct relationship exists between the conduit permeability and pore size. To facilitate nerve growth and repair, nerve conduits with large pores are preferable. In nerve regeneration, a semi-permeable conduit is more preferable when compared to both low permeable and impermeable conduits46. The nerve guide diameter has a lot of influence on the nerve regeneration outcome. This is because the injured nerve has to match the nerve guide diameter47. Conduit wall thickness also has a major role to play in axonal growth. According to Naveilhan et al., conduit walls that have more than 0.8 mm thick reduces axonal growth which affects the permeability and porosity reduction which are important factors to consider in nerve regeneration47. Another important feature that affects nerve regeneration outcome is the wall thickness48.

Natural based biomaterials

In nerve regeneration, many natural-based biomaterials have been used. They include polysaccharides such as: hyaluronic acid, alginate, chitin and chitosan. Proteins such as: collagen, gelatin, fibrin and keratin49.

Polysaccharides

i). Hyaluronic acid (HA): It consist of glycosaminoglycan moiety which is involved in cellular process regulation50. Some unique properties associated with hyaluronic acid include: biocompatibility, support of axonal growth and its non-adhesive nature51. Although some of the limitations associated with HA which are: fast degradation and low mechanical properties, it can still be used as a conduit internal filler mostly in hydrogel form. 

ii). Alginate: Alginate has a wild application in the biomedical field52. Chemical reactions is one major way that is used in the modification of alginate. When alginate is oxidized with sodium alginate, it gives rise to alginate dialdehyde53. One of the limitations associated with alginate use in promoting nerve regeneration is its weak mechanical resistance. The physiological loading conditions can be improved when alginate is combined with other polymers54. According to Pfister et al., he blended alginate with a biomaterial of natural origin-chitosan which gave rise to a support of nerve regeneration for short nerve gaps. Due to the hydrophilic nature of the chitosan, the blended mixture possessed a good permeability and adequate mechanical strength55. The techniques used in the manufacture of alginate include: magnetic templating, electrospinning, gas forming, emulsion freeze drying and 3D printing56,57. Alginate can also be used in nerve regeneration as a conduit internal filler that is applied growth factor delivery58.

iii). Chitin and chitosan: Chitin is a member of the glycosaminoglycan family with the presence of N-acetyl-D-glucosamine moiety. Chitin most abundant in nature is found in the exoskeleton of arthropods59. Chitin application spans the food industry, agriculture, pharmaceutics and medicine especially when used in its partial deacetylated form as chitosan60,61. There are some unique properties that make chitosan suitable to be used in peripheral nerve regeneration. They include its biocompatibility, ability to support axonal growth and tendency to reducing scar62. Although chitosan has low mechanical strength, it can be modified in order to improve its mechanical stability63. Other unique properties associated with chitosan include: its versatility and easy modification of the surface structure64. An investigation in the nerve regeneration in rat sciatic nerves 3 months after 10 mm nerve repair with chitosan conduits that had three different deacetylation degrees65.  The study indicated that there was no significant differences among the experimental groups at functional, biomolecular and morphological levels66. Reaxon® a chitosan nerve conduit was commercialized in 2015. It was able to bridge nerve gaps up to 26 mm due to some of its unique advantages such as transparency, flexibility and resistance to collapse67.

Proteins

Collagen: Collagen is used in nerve conduit repair73. According to Saltzman et al., a hollow conduit (10 mm) reported better results in rat nerve regeneration and muscle re-innervation than the polyglycolic acid (PGA) filed conduits of collagen. The limitations associated with the use of collagenase in nerve tissue repair is due to its low mechanical stress resistance and weak manipulability74. It is recommended that collagen should be combined with chitosan in order to increase its mechanical strength75.

Gelatin: The thermal denaturation of collagen results in the production of gelatin. Gelatins physical and mechanical properties could be easily altered by using various cross-linking agents76. One of the most common cross-linkers used was genipin, a natural substance with low cytotoxicity. According to Chen Y et al., he used a genipin cross-linked gelatin conduit to repair a rat sciatic nerve (10 mm) for 8 weeks. The result obtained after 8 weeks, showed that most of the regenerated axons were not myelinated76. Proanthocyanidin was another cross linker that was used to stabilize a gelatin conduit. According to Liu et al., it was used to repair a 10 mm nerve gap and the regeneration after 8 weeks, was assessed. The biocompatibility and degradation rate of the conduit was tested. The in vivo studies after 8 weeks showed that the conduit was well integrated into the surrounding tissues77. Another natural cross linker used was bis(vinylsulfomethyl). The result obtained after 8 weeks in a 10 mm rat sciatic nerve defect showed that it reduced gelatin swelling and improved its mechanical properties78.

Silk fibroin

Silk fibroin is used in biomedical applications. It has repeated amino acidic sequence, thus having a very good mechanical property. It can easily degrade82.

Keratin

It has some unique characteristics that makes it useful as a biomaterial. They include its biocompatibility, biodegradability, bioactivity and its hydrophilic surface. Although it has some limitations such as poor physical and mechanical properties, various cross-linking agents can be used to improve it86,87. Gupta and Najak deployed the use of keratin as a protein source for scaffold fabrication. The results obtained showed that they produced a keratin-alginate scaffold88.

Polyesters

Polyester is a biopolymer that is naturally biodegradable. The type commonly used in tissue engineering is polyhydroxyalkanoates (PHA). Some advantages associated with PHA include pH stability and biocompatibility. One of the limitations of its use is high cost, although it could be reduced to the barest minimum by the development of recombinant microorganisms88.

 

CONCLUSIONS

 

Overtime, there has been an advancement on the comprehension of peripherous nervous injury, although there is still room for improvement. With growing research on other growth factors, they hold a great promise as a tool for studying intracellular communication among cells.

 

ACKNOWLEDGEMENT

 

Authors are thankful for University of Nigeria, Nsukka, Nigeria to provide necessary facilities for this work.

 

AUTHOR’S CONTRIBUTION

 

Ezegbe CA: investigation, visualization, writing editing. Ezegbe AG: review, editing. Both authors checked and approved final version of the manuscript.

 

DATA AVAILABILITY

 

The accompanying author can provide the empirical data that were utilized to support the study's conclusions upon request.

 

CONFLICT OF INTEREST

 

None to declare.

 

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