Accepted Manuscript

Biomaterials and cells for neural tissue engineering: Current

choices

Prerana Sensharma, G. Madhumathi, Rahul D. Jayant, Amit K.

Jaiswal

PII: S0928-4931(16)32866-1
DOI: doi: 10.1016/j.msec.2017.03.264
Reference: MSC 7776
To appear in: Materials Science & Engineering C
Received date: 29 December 2016
Accepted date: 28 March 2017

Please cite this article as: Prerana Sensharma, G. Madhumathi, Rahul D. Jayant, Amit K.
Jaiswal , Biomaterials and cells for neural tissue engineering: Current choices. The address
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 ACCEPTED MANUSCRIPT

Biomaterials and Cells for Neural Tissue Engineering: Current Choices

Prerana Sensharma1, §, Madhumathi G.1, §, Rahul D. Jayant3 and Amit K. Jaiswal2*

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1
School of Biosciences and Technology, VIT University, Vellore, 632014, Tamilnadu, India
2
Centre for Biomaterials, Cellular and Molecular Theranostics, VIT University, Vellore, 632014,

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Tamilnadu, India
3
Center for Personalized Nanomedicine, Institute of Neuro-Immune Pharmacology, Department
of Immunology, Herbert Wertheim College of Medicine, Florida International University (FIU),

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Miami, FL-33199, USA
§
Authors have equal contribution
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*
Corresponding Author

Amit K Jaiswal, Ph.D.

Associate Professor
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Email: amitj@vit.ac.in
Phone No (O): +91-9789280874
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Abstract

The treatment of nerve injuries has taken a new dimension with the development of tissue

engineering techniques. Prior to tissue engineering, suturing and surgery were the only options

for effective treatment. With the advent of tissue engineering, it is now possible to design a

scaffold that matches the exact biological and mechanical properties of the tissue. This has led to

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substantial reduction in the complications posed by surgeries and suturing to the patients. New

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synthetic and natural polymers are being applied to test their efficiency in generating an ideal

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scaffold. Along with these, cells and growth factors are also being incorporated to increase the

efficiency of a scaffold. Efforts are being made to devise a scaffold that is biodegradable,

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biocompatible, conducting and immunologically inert. The ultimate goal is to exactly mimic the
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extracellular matrix in our body, and to elicit a combination of biochemical, topographical and

electrical cues via various polymers, cells and growth factors, using which nerve regeneration
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can efficiently occur.

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Keywords: Nerve tissue engineering, Synthetic and natural polymers, Biomaterials, Hydrogels,

Nanofibers.
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1. Introduction
[1]
Nerve injuries affect millions of people worldwide. These injuries often affect youth of

employable age and leave them with a permanent disability of cognitive, motor or psychotic

nature. They should deal with a reduced quality of life as well as several debilitating social and

economic burdens. [2, 3] Peripheral nerve injuries affect 13-23 patients per 100,000 as reported in

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2015. They pose one of the major problems at trauma facilities. Etiologies associated with

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peripheral nerve injuries include penetrating injury, crush, traction, ischemia, and less common

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mechanisms such as thermal, electric shock, radiation, percussion, and vibration. Injured nerves

of the peripheral nervous system (PNS) have the ability to regenerate. However, spontaneous

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regeneration of the nerve, in the absence of any therapeutic intervention does not result in a good
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[3]
functional outcome. Despite early diagnosis surgical intervention does not result in the

recovery of functions as it was pre-injury. Traditional epineural neurorrhaphy promotes

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regeneration by direct contact, and can result in the formation of neuromas. Autologous grafts
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have high standards, but they still have limitations as the nerves are short aged, supply is limited.

They also present the possibility of causing morbidity, neuroma formation at site of harvest,
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[4]
scarring and sensory loss. Allogenic grafts could also be used but they would produce an
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immune response which will disable the cells that cure the injury. [5] The central nervous system

(CNS) consists of the brain, spinal cord and retina. It is limited in terms of its spontaneous
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regenerative capacity, limiting the possible treatment strategies. The etiologies of CNS injuries

are apoptotic and necrotic death of neurons (including photoreceptors), astrocytes and

oligodendrocytes, axonal injury, demyelination, excitotoxicity, ischemia, oxidative damage, and

inflammation. [6] Traumatic Brain Injury (TBI) can present symptoms ranging from headaches to

paralysis. However, the current treatment approaches focus on preventing any further damage,

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[2]
rather than facilitating further regeneration. Spinal cord injuries (SCI) occur majorly in traffic

accidents and due to elderly patients falling. They result in paraplegia and quadriplegia, which
[7]
cannot be effectively treated so far. Moreover, diseases of the CNS are complex, and can

cause decrease in cognitive, motor and sensory functions (as in the cases of Parkinson’s disease,

Alzheimer’s and Multiple Sclerosis) and loss of vision due to retinal defects (Retinitis

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Pigmentosa and Age related Macular degeneration). Pharmacological intervention is restricted to

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simply delaying the progression of disease. There are additional limitations as well. The site of

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cell transplantation has an inhospitable environment. Furthermore, limited drugs and biologics

can successfully cross the blood brain barrier thereby eliminating oral and intravenous drug

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administration methods (Figure 1). [6] Thus, we find the need for better approaches towards the
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treatment of nerve injuries. Tissue engineering centric approaches will enable regeneration,

repair and replacement of tissue at the site of injury. Consequently, functionality will be restored,
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even for complex CNS injuries like TBI. Treatment strategies combining cell transplantation,
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molecule delivery and biomaterial scaffold constructions are considered the greatest hope for

possible regeneration and functional recovery in SCIs. [8] Tissue engineering is achieved through
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the fabrication of a scaffold, which mimics all the properties of the tissue which should be
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repaired to favor cell penetration and tissue regeneration in three dimensions. Tissue engineering

comprises three main components namely, the biomaterial used, the cells and the biomechanical
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and/or mechanical stimuli (Figure 2).

Many different types of scaffolds have been studied for neural tissue engineering, namely

nanofibrous scaffolds, natural and synthetic scaffolds. The nanofibrous scaffolds have an
[9]
increasing application in tissue engineering. Many cell types like MSCs, iPSCs, ESCs cord

blood cells and majorly, Schwann cells are used. Various fabrication techniques like phase

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separation and electrospinning are used (Figure 3). Some of the growth factors used in case of

neural tissue engineering include NGF (Nerve growth factor), NT-3 (Neurotrophin-3) and BDNF

(Brain derived neurotrophic factor). Several products are commercially available for neural

tissue engineering. Neuragen®, NeuraWrap™, NeuroMatrix™, NeuroFlex™ are some FDA

approved collagen based nerve conduits available in the market. NeuroTube® is a nerve conduit

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made of Polyglycolic acid, while Neurolac™ is composed of Poly (D,L-lactide-co-ε-

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caprolactone) and Salutunnel™ is made of Polyvinyl alcohol. Neuragen® was the first

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commercially available, FDA-approved nerve conduit. These conduits have been reported

effective for regeneration of the nerve. In the United States alone they result in the expenditure

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of 150 billion dollars in the annual healthcare dollar. Over 200,000 repair procedures are
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[10]
performed in the United States annually. Thus, neural tissue engineering offers potentially

great solutions on the healthcare front, as well as great economic prospects in the market.
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2. Ideal properties of a scaffold

The scaffold used should be analogous to the natural ECM of the tissue and should support 3D
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cell cultures. To design an appropriate scaffold to repair the damage in a tissue we need to know
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the physical, chemical and mechanical properties of that tissue (Figure 4). The following

properties given below depend on one key parameter, which is the choice of biomaterial for
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scaffold fabrication. Depending on the requirements of the scaffold, the biomaterial is suitably

selected. [14]

2.1 Biocompatibility

Biocompatibility is a property of prime importance as it facilitates cell adhesion, proper

[14]
functionality of cells and migration and proliferation of cells on the scaffold. Surface

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modification of the scaffold can be done using bioactive molecules to make biomimetic

materials. Bioactive molecules like long chains of ECM proteins including fibronectin, laminin,

vitronectin and short peptide sequences are coated on the biomaterials. The surface modification

favors cell adhesion and cell proliferation. Bulk modification of the biomaterial is more

beneficial than surface modification. In bulk modification, the cell signaling peptides are

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integrated into the biomaterials and recognition sites are present both in the bulk and on the

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surface. Cell adhesion property depends upon surface properties like wettability and charge

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[15]
density. Along with biocompatibility, toxicity profiles also play a crucial role in cell

adhesion, growth and proliferation on the scaffold. [16]

2.2 Biodegradable
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One of the major advantages of synthesizing biodegradable scaffolds is that they eliminate the

need for surgical removal of the scaffold and they are absorbed by the surrounding tissues in the
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body. In case of neural tissue engineering, controlled biodegradable scaffolds are preferred as
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the scaffold is meant to support the growth of nerve cells and then be degraded by the body as
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subsequent repair takes place. A biodegradable scaffold will also facilitate the neighboring cells

to produce their own extracellular matrix (ECM). It should be taken care of that the by-products
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[14]
of biodegradation should be non-toxic as well and that they are easily dispose-off. The

cytotoxic effects could also enable neuroma formation. Biodegradability should favor the
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elimination of chronic inflammation. [17]

2.3 Porosity and Pore size

An ideal scaffold should possess the appropriate shape and porosity required to mimic its natural

tissue. High porosity and a pore size sufficient to aid in cell seeding, vascularization and

diffusion of growth factors and nutrients into the scaffold and surrounding tissues is necessary.

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[16]
Ideally, 90 % porosity and pore size of 100µm-500µm is standard for yielding good results.

These factors contribute to the architecture of the scaffold. It is crucial to scaffolds that they have

interconnected pores to facilitate cell penetration and diffusion of nutrients to cells and ECM

present in the scaffold. Diffusing out of waste products also depends on the porous

interconnected structure. But while pores need to be large enough to accommodate diffusion of

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cells, nutrients and waste products, it should also be small enough to generate a highly specific

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surface area which results in efficient binding of cells to scaffold due to minimal ligand density.

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[14]

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2.4 Mechanical properties

The scaffold should have mechanical properties identical to that of the tissue at the implantation
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site. As this is not always feasible, materials with mechanical properties that can protect the cells
[16]
from compressive or tensile forces without disturbing the biomechanical cues are used.
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Polymeric nanofibers are highly advantageous with respect to this aspect. They have been shown
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to display unique mechanical properties, like the tensile modulus, tensile strength and shear
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modulus. These parameters have been found to increase with subsequent decrease in the

diameter of the fiber. Although the explanation behind this phenomenon has not been clearly
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elucidated yet, it has been postulated that there is an increase in macromolecular chain alignment

as the fiber diameter is decreased, resulting in a higher crystallinity of the nanofiber. Such unique
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mechanical properties facilitate the modulation of cell behavior and provide strength to resist the

forces exerted by cytoskeletal elements on the scaffold. Hence, optimal mechanical properties

and porous scaffold architecture together play a crucial role in facilitating cell infiltration and

vascularization which largely determine the making of a good scaffold. [14]

2.5 Provide multiple cues

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An ideal neural scaffold should be able to provide any one or a combination of cues including

mechanical, biochemical, topographical and electrical cues. These cues help the scaffold to

mimic the native extracellular matrix of the tissue in vivo, facilitates neurite outgrowth by

providing better contact guidance and promotes and enhances cell adhesion, proliferation,

migration and viability. [18] The mimicking of ECM will aid the surrounding cells to secrete their

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own ECM, which binds the cells to tissues and thus elicits signals that facilitate cell development

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[16]
and morphogenesis. Electrical stimulation has been found to be especially successful in the

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case of neural tissue engineering. Neuronal repair is highly unique due to the complexity of the

nervous system. Electrical cues have been found to enhance the nerve regeneration process and

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this has led to the increased usage of electrically conducive polymers like PPY and PANI in
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neural tissue engineering. [19]
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3. Scaffolds made from Natural Polymers

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The extracellular matrix has numerous functions in the body. It provides structural and
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mechanical support to the tissues, facilitates migration of cells, holds the cells together,

facilitates communication in cells so that daily cellular activities and wound healing can be
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[17, 20]
performed uninterrupted. Natural polymers has advantage as scaffold design as they

closely mimic the macromolecules that cells interact with in vivo. The frequently used natural
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polymers for scaffold design in neural tissue engineering are collagen, gelatin, hyaluronic acid,

chitosan, chitin, elastin, and alginate (Table 1).

3.1 Collagen

Collagen is one of the most widely studied constituents of the ECM due to its presence in all the

connective tissues in the body. There are 28 types of collagen that have been identified, all

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characterized by a triple helical structure. Collagen molecules are composed of three alpha

chains that assemble together. The amino acid sequence is characterized as Gly-X-Y-, wherein

glycine is essential at every third position to enable the tight packaging structure of collagen. The

presence of 4-hydroxyproline, formed during post-translational modification is a marker for

[21, 22]
collagen. Collagen is an eligible biomaterial for scaffolds pertaining to almost all types of

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tissues—it is used in bone tissue engineering, for skin, cornea potentially for the development of

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[22, 23, 24]
heart valves and other cardiovascular diseases, and so on. The versatility of collagen is

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attributed to the widespread distribution of collagen in the body. Some other advantageous

features of collage include low antigenicity, low inflammatory and cytotoxic response,

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biocompatibility, good water uptake capabilities, availability of several methods for isolation
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from various sources and the ability to tailor mechanical and cross linking properties as per need.
[20, 25]
Collagen 1 is suitable for implantation within the body because there are very few people
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who have humoral immunity against it. Moreover, a simple serological test can determine
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[22]
whether the use of this biomaterial will elicit an allergic response in the patient. A self-

organizing collagen guiding conduit was developed by Phillips et al, which is a Schwann cell
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containing conduit to be used at the site of injury in the Peripheral Nervous System. The implant
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was reported to show neurite extension from a dissociated dorsal root ganglia, and showed a

greater regeneration over all. [26] Collagen-based scaffolds are fabricated through electrospinning,
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among other methods. Collagen does not have good mechanical and structural stability upon

uptake of water. To prevent this, collagen can be used along with other natural and synthetic

polymers to modify properties like mechanical strength, permeability rate, compressive modulus,

cell number; cell metabolic activity etc. Different collagen concentrations bestow different
[22, 25]
properties to the scaffold. For instance, to increase the strength of a collagen based,

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scaffold, its cross-linking with glutaraldehyde vapors, formaldehyde and epoxy compounds was

done and the results obtained were that the scaffold mimicked the tensile strength of many

commercially available products, like Beschitin™ and Resolute™. However, there are greater
[20]
chances of cytotoxity and immune response using this. Studies have reported that greater

similarity of the nanofibrous collagen scaffold to the nerve tissue facilitates successful

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regeneration of neural tissue and thus makes an excellent scaffold for nerve regeneration.

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Collagen scaffolds have been made as composites along with several synthetic scaffolds to

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enhance the mechanical strength of the scaffold. In one study, Poly (L-lactic) co-poly (3-

caprolactone)-collagen nanofibrous scaffold was synthesized and bone marrow derived

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mesenchymal stem cells were incorporated in the scaffold. Results indicated that the
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differentiated MSCs on the PLCL/Coll scaffold had neuronal morphology with multipolar

elongations and expressed neurofilament and nestin proteins, which confirm neuronal induction.
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[27]
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3.2 Hyaluronic acid (And its derivatives)

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Hyaluronic acid (HA, also Hyaluranon) is a linear, non-branched non-sulfated

glycosaminoglycan (GAG) and is composed of repeating disaccharides (β-1, 4-D-glucuronic acid

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(known as uronic acid) and β-1, 3-N -acetyl- -glucosamide). It has sites for cell adhesion and is
[28]
non-immunogenic. Hyaluronic acid has found many applications in tissue engineering,
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especially for its use as a hydrogel scaffold. HA is one of the main components of hydrogels, as

it imparts the property of biodegradability to hydrogels made of non-biodegradable components

like Poly-Ethylene Glycol (PEG). [29] It plays a role pertaining to osteoarthritis, treatment, as an

aid in eye surgery, and for wound regeneration. It is used in soft tissue replacement surgically

and has several diagnostic applications. It serves as a diagnostic marker for cancer, rheumatoid

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[30,31]
arthritis, several liver pathologies as well as early organ rejection. Also, it has been

explored a s a medium for drug delivery via several routes like nasal, oral, pulmonary,

ophthalmic, topical, and parenteral. The possible targeting of cancer cells using HA has also

been explored. [32, 33, 34] The several advantages of using HA for scaffold building are its excellent

biocompatibility, high water content, capacity to degrade into safe products, limited

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immunogenicity, viscoelastic properties suitable for several tissue types and the ability to bind to

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specific cell surface receptors (CD44, RHAMM, and ICAM-1, etc.), thereby influencing several

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cellular processes. Thus, it is a plausible option for influencing
[28, 31, 33]
processes like wound healing, metastasis, etc. HA is especially advantageous for

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designing scaffolds for neural tissue regeneration due to its high abundance in the neural system
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(especially the Central Nervous System), making it an extremely biocompatible choice.

Moreover, HA plays a role in wound healing. Several strategies for successful axonal
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regeneration using HA exist. HA also reduces glial scar formation. HA Hydrogel has a very
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porous structure, with interconnected pores that allow for the transportation of nutrition,

penetration of cells, blood vessels and nerves. Use of HA scaffold in vivo has reported less glial
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scarring, smaller gliosis thickness and lesser glial fibrillary acidic protein (GFAP) positive cells
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around the scar area. A dominant disadvantage associated with the use of HA is that cells do not

adhere to its surface. Thus, to overcome this, it is blended with other biomaterials which will
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enhance the binding of cells and consequentially increase neural tissue regeneration. An example

is collagen-HA scaffold, which had desired mechanical properties for CNS regeneration and
[35]
promoted differentiation of Neural Stem Cells (NSCs) for neural regeneration in vitro.

Another disadvantage of HA is its water solubility, which makes it necessary to add a cross-
[36]
linking agent to convert it into injectable form. In one study, HA hydrogel that could be

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degraded by cell released enzymes was fabricated and seeded with mouse mesenchymal stem

cells. The HA hydrogel was modified to contain acrylate groups and cross linked using matrix

metalloproteinase (MMP) degradable cross linkers. Results indicated that faster MSC

proliferation and migration occurred in the presence of RGD and MMP degradation sites in the
[37]
hydrogel. In another study, HA strands were cross linked using glutaraldehyde and coated

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with polylysine. This was then seeded with Schwann cells. Results indicated higher attachment

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of cells, water insolubility of HA and lesser biodegradability, hence making it a potent nerve

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graft. [38]

3.3 HYAFF

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HYAFF is an HA derivative obtained by esterification of HA with benzyl ester. Some favorable
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properties of HYAFF are its biocompatibility, complete degradability, solubility in DMSO,

stable on hydrolysis, strong interaction with polar molecules and its ability to promote cell
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[39]
adhesion and proliferation of various cell types. Moreover, HYAFF can be used to design
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films, non-woven fabrics, gauzes, sponges, tubes and microsphere, thus broadening its

applications. It is used for tissue repair, controlled drug release, nerve regeneration, delivery of
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[40]
growth factors, wound healing, etc. Experiments have reported HYAFF based tube scaffolds
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are good substrates for nerve cell cultures and explants. It was reported that cells from rat sciatic

nerves showed adhesion to the scaffold, followed by proliferation and colonization of the
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[41]
scaffold. Its properties are ideal for Peripheral Nervous System Tissue Regeneration. The

ability of HYAFF to support adhesion and proliferation of Schwann cells and endothelial cells

obtained from peripheral nerve has been studied. Results indicated that HYAFF is a good choice

of biomaterial for peripheral nerve cell culture and nerve explants. [42]

3.4 Alginate

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It is a naturally derived, linear polysaccharide obtained from brown algae and bacteria. It is

composed of (1–4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid monomers (G).

Alginate is pH dependent anionic and can thus interact with positively charged proteoglycans

and other molecules, a property which can be utilized for delivery of cationic drugs. Also,

alginate hydrogels are formed by making them interact with divalent cations in aqueous solution.

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[36, 43]
The advantages of alginate as a scaffold are its high biocompatibility, high

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biodegradability, non-antigenicity and chelating property. The several types of scaffold that can

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be formed using alginate are summarized in a review by Sun & Tan et al. A study was conducted

to evaluate the effects of alginate concentration on neurotropic factor release. The study inferred

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that NSC-seeded alginate beads with a high G, non-PLL-coated composition may be useful in
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the repair of injured nervous tissue, where the repair is facilitated by the secretion of

neuroprotective factors, while the alginate beads coated with PLL and a high M concentration
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were fragile and susceptible to maximum breakage. [43, 44] Alginate hydrogel (AH) has been used
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as a bioresourceable conduit supplemented with Schwann cells to replace nerve grafts. Enhanced
[45]
neurite growth, viability and proliferation were observed. Alginate conduits seeded with
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Schwann cells have also been fabricated along with the incorporation of fibronectin. Results
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indicated that regeneration rate, viability of the cells and axonal growth were enhanced using this

conduit. [46]
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3.5 Chitosan

Chitin is naturally found in the cell walls of crustaceans such as insects, crabs, shrimps as well as
[25, 47]
the cell wall of bacilli and fungi. Chitosan is obtained by the alkaline deacetylation of

chitin. The degree of deacetylation of chitin is process-dependent. The degree of deacetylation

along with the molecular weight affects several properties like solubility, mechanical strength

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and degradation. The evaluation of chitosan in terms of mechanical properties, growth factor

delivery, etc. concluded that it is an excellent choice for construction of scaffold. [47] It has found

several applications like in bone tissue engineering, skin tissue engineering, etc. Several

advantages of chitosan are its biocompatibility, biodegradability, non-toxic, inhibition of growth

of several fungi, yeast and bacteria and non-immunogenicity. Due to the absence of any protein

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or lipid in chitosan structure, no antibody can be developed against it. [48] Chitosan has also been

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[49]
proved to be a good choice for neural tissue regeneration. Chitin incorporated with other

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materials show an affinity for nerve cells. Chitin based nerve chambers can be used for effective

nerve regeneration over long gaps as well as functional recovery. A dog sciatic nerve gap of 50

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mm was repaired using chitosan-based scaffold. Chitosan based scaffolds promote the adhesion,
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growth and migration of Schwann Cells. This is very important because Schwann cells release

neurotropic factors, express neuron-specific ligands and guide neurite outgrowth, thus playing an
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irreplaceable role in nerve tissue regeneration. Schwann cells also secrete and deposit ECM.
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Moreover, it has the mechanical strength necessary for effective neural regeneration. Chitosan

conduits resulted in the sprouting of myelinated axons and successful recovery of nerve function.
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[50]
Chitosan scaffold pre-seeded with Schwann cells was synthesized as a bio-artificial nerve
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graft for peripheral nerve regeneration. Good biocompatibility of the Schwann cells and chitosan

fibers was observed. [51] In another study, chitosan conduit seeded with bone marrow stromal cell
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(BMSC) derived Schwann cells was fabricated. Results indicated the profound efficacy of the

conduit in treating critical peripheral nerve defects by supporting nerve conduction, myelin

regeneration and development of myelinated axons. [52]

4. Scaffolds made from Synthetic materials

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Although the gold standard for the treatment of nerve injuries which are greater than 5mm in size

is said to be autologous nerve grafts, they have several shortcomings, including limited

availability of graft tissue, donor site morbidity, neuroma formation, nerve site mismatch, and

sometimes possible lack of functional recovery. Allogenic grafts pose the obvious disadvantage
[49, 16]
of immune rejection. To overcome the disadvantages posed by these grafts, synthetic

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scaffolds are being devised that exactly mimic the biological and mechanical properties of the

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cells and ECM in vivo. Due to the extreme versatility of the composition and formation of

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[53, 54]
polymers, polymeric scaffolds are proving to very efficient scaffold materials. Polymeric

biomaterial scaffolds can be used for peripheral and central nerve injuries, both in vivo and in

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vitro. Properties like biodegradability, non-inflammatory, non-toxicity, porosity and other
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mechanical properties can be easily engineered in these polymeric scaffolds, suitable to the

characteristics of the in vivo tissue where the graft should be implanted. [18] The hydrophilicity of
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the synthetic polymer also plays a crucial role, as hydrophobicity elicits monocyte adhesion to
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the graft, leading to immune rejection of the graft. This property can also be easily tailored in

synthetic polymers. Some widely used synthetic polymers for the fabrication of scaffolds include
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Poly -L-lactic acid (PLLA), Polycaprolactone (PCL), Polyhydroxybutyrate (PHB), Polyglycerol

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sebacate, Poly-D,L-lactide-co-glycolic acid (PLGA), Poly-L-lactate (PLA), Poly-3-

hydroxybutyrate (PHB), Polyamide, Polydioxanone, Poly-e-caprolactone-co-ethyl ethylene

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phosphate (PCLEEP), Poly-D.L-lactide-co-caprolactone (PDLLA), Polyvinyl alcohol (PVA),

Poly acrylonitrile-co-methylacrylate (PAN-MA) and copolymer of methyl methacrylate (MMA)

[55]
and acrylic acid (AA) (PMMAAA). Conductive polymers like Polypyrrole (PPY) and

polyanaline (PANI) are also gaining popularity in nerve tissue engineering by their ability to

conduct electrical signals (Table 2).

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4.1 Polycaprolactone (PCL)

PCL is a biodegradable and biocompatible polymer, which is cost efficient, possesses high
[56]
elasticity, low toxicity, good mechanical properties and a slow degradation profile. It has

been widely applied in tissue engineering including bone and neural tissue engineering. PCL

fibers have most commonly been generated by the method of electrospinning. [57] But it has been

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experimentally observed that fabrication or bioactive molecule encapsulation using organic

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solvents may engender cytotoxic effects when the graft is being implanted in the body. A solvent

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free method, template synthesis was used where PCL fibers were synthesized using Alumina

nanoporous membrane. NaOH was then used to dissolve the template and yield PCL nanowires.

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[54]
PCL has been used in conjugation with natural polymers or coated with cells to improve its
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biocompatibility and cell adhesion properties. This will confer mechanical strength and

biocompatibility to the scaffold. A tubular prosthesis of PCL in combination with aligned

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collagen was synthesized to direct aligned axonal growth of the nerve fibers. Aligned implants
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allow the regeneration of nerve fibers in a contact-oriented fashion which increases the growth of
[58]
the fibers. In one study, PCL fibers have been extruded and embedded in poly-2-
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hydroxyethyl methacrylate (HEMA) gel. The sonication of the PCL/HEMA composite was
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dissolved in acetone solvent leaching out PCL and leaving behind HEMA gel with oriented

longitudinal channels embedded in it. The channel diameter can be controlled by altering the
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concentration of PCL used in the composite. The fabrication of such gels using synthetic

polymers with oriented channels will provide support and contact guidance to regenerating

neuritis and axons. [59] PCL nanotubes have also been used in conjugation with Adipose-derived
[60]
(multipotent) stem cells to improve the effects on nerve regeneration. A PCL scaffold seeded

with human mesenchymal stem cells was shown to induce adipogenic, chondrogenic and

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[16]
osteogenic lineages. In another study, nonwoven aligned nanofibrous nerve conduits were

synthesized, composed of poly (L-lactide-co-caprolactone) and polypropylene glycol in a ratio of

70:30 by electrospinning seeded with neural crest stem cells (NCSC) obtained from ESCs and

iPSCs. NCSCs are multipotent in nature and can give rise to cells of mesodermal and ectodermal

lineages. Results indicated that NSCS-engrafted nerve conduits had faster regeneration potential

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of sciatic nerves. These cells also promoted axonal myelination. It was observed that NCSCs

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[61]
differentiated into Schwann cells and then integrated into the myelin sheath around axons.

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PCL has also been used directly in conjugation with Schwann cells. Aligned PCL fibers

fabricated by electrospinning seeded with human Schwann cells indicated enhanced peripheral

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nerve regeneration by promoting formation of bands of Bungner.[62] Aligned and random
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nanofibrous PCL/gelatin scaffolds fabricated by electrospinning were seeded with Schwann cells

to assist the direction of growth of regenerating axons in peripheral nerves. [63]

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4.2 Poly-L-lactic acid (PLLA)

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PLLA is a biodegradable and biocompatible synthetic polymer. Nano-structured PLLA scaffolds

are widely used in neural tissue engineering owing to its analogous structure to the natural ECM
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of nerve cells, including ultrafine continuous fibers, high surface-to-volume ratio, high porosity,
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varied distribution of pore size from 50-350 nm. It contains ester linkages in the polymer

backbone which lead to bio-functionalization of the polymer with various biomolecules by

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means of covalent conjugation. This polymer also poses several disadvantages including poor

biocompatibility, release of acidic products on degradation, poor process ability and premature

failure of mechanical features during degradation. In one study, PLLA conjugated with

mesenchymal stem cells was made into a nanofibrous scaffold. It was shown to induce
[14]
differentiation into different neurogenic lineages by culturing in differential media. It has

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been experimentally proven that electrospun aligned PLLA fibers are shown to support neurite

extension and axon regeneration better than electrospun random fibers, although random and
[64]
aligned fibers yielded the same results with respect to NSC differentiation. 3-D PLLA nano-

structured porous scaffolds have been fabricated using liquid-liquid phase separation methods.

The fabricated scaffold seeded with NSCs showed significant improvement in NSC

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[65]
differentiation and neurite outgrowth. Several studies have proven that PLLA scaffolds

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efficiently support NSC differentiation and neurite outgrowth, thereby making them suitable to

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applications in neural tissue engineering. PLLA conduits incorporated with allogenic Schwann

cells were studied to venture into the synthesis of bioactive nerve conduits which can potentially

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[66]
replace autologous nerve grafts and peripheral nerve regeneration. A heparin/collagen
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encapsulating nerve growth factor multilayer was coated upon a PLLA scaffold using layer-by-

layer self-assembly mechanism. The scaffold aimed to provide biomolecular as well as physical
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cues to the regenerating nerve. Sustained release of nerve growth factor for two weeks, as well as
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superior benefits to Schwann cell proliferation and its cytoskeleton, PC12 differentiation and

neurite growth was observed as compared to the aligned PLLA nanofibrous scaffolds. [67]
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4.3 Poly-D,L-lactic-co-glycolic acid (PLGA)

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PLGA is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA). It is amorphous

biodegradable polyester and is widely used for fabrication of scaffolds in neural tissue
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engineering due its biodegradability, non-toxicity and film forming ability. PLGA is said to have

similar properties to that of skeletal tissue. It has also been effectively employed as a scaffold

for regeneration of heart valves, blood vessels and cartilage. It poses a disadvantage, when

exposed to long term cyclic strain exposure; it undergoes plastic deformation and fails. [16] It also

releases acidic products on degradation, leading to a decrease in pH of the tissue which may

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result in aseptic inflammation. To overcome this, the PLA to PGA ratio can be controlled during

the synthesis of the polymer. Since PLA is more hydrophobic and crystalline in nature than

PGA, the degradation profile of PLGA can be altered and made slower by using higher

concentrations of PLA in the synthetic polymer. Another major disadvantage is the lack of

natural adhesion sites, as it is a synthetic polymer. This can be rectified by using techniques like

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hydrolysis, aminolysis, blending and covalent attachment of adhesive peptides. It has been

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approved by the FDA for biomedical applications. Its other areas of application include

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pharmaceutical, medical engineering and various other industries. In one study, PLGA

nanofibers were generated using electrospinning technique and NSCs were seeded onto the

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scaffold. Results indicated high efficiency of cell adhesion, nerve cell differentiation and neurite
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[14]
outgrowth. In another study, the PLGA scaffold was conjugated with PEG (Polyethylene

glycol) for improved mechanical strength and seeded with mouse embryonic fibroblasts
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reprogrammed into induced neural stem cells (iNSCs) for the treatment of spinal cord injury
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(SCI). The scaffold induced continuity of the spinal cord and led to a significant reduction in

cavity formation. PEG, which is composed of repetitive oxygen vinyl has higher hydrophilicity,
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and hence forms a great composite with PLGA by overcoming its shortcomings. PEG has also
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been shown to protect some important axonal cytoskeleton proteins to promote repair in SCI.
[68]
Thus, the composite scaffold exhibited greater iNSC adhesion, cell growth and proliferation.
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A PLGA scaffold was fabricated with the objective of inhibition of neurite growth. This was

achieved by silencing Nogo-66 receptor gene using small interfering RNA in BM-MSCs and

SCs. The strategy was seen as effective for spinal cord injury treatment. [69]

4.4 Polyglycerol sebacate (PGS)

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PGS is a chemically cross-linked elastomeric polymer and confers a high amount of toughness to

the scaffold. It can support several cells including fibroblasts, hepatocytes, endothelial, smooth
[16]
muscle, cardiac muscle and Schwann cells. In one study, non-linear elastic biomaterial

scaffold was fabricated using a PGS/PLLA composite. The fabrication was done using core and

shell electrospinning technique. The results indicated that elastomeric mesh aids the growth and

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proliferation of enteric neural crest progenitor cells (ENCs). The scaffold also exhibited soft,

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tissue like mechanical properties on studying the stress-strain curve. [70]

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4.5 Polyhydroxy butyrate (PHB)
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PHB is biodegradable aliphatic polyester synthesized by several bacteria using renewable carbon

sources. Since the monomeric component of PHB, 3-hydroxy butyric acid (3-HBA) is a stable
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analyte in human serum and microbial PHB is recognized by mammals, PHB is widely applied
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as a biomaterial and is FDA approved. A study reported that PHB is a potential neural protective

agent and acts by reducing the number of apoptotic glial cells in mice. It has high crystallinity,
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resulting in a brittle nature of scaffold and longer degradation time. A PHB blend with poly-3-
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hydroxy butyrate-co-3-hydroxyvalate (PHBV) scaffold has been found to provide better support

for adhesion and proliferation of osteoblasts and fibroblasts. In one study, PHB films were
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blended with poly-L-lactide-co-caprolactone (PLCL), which is a copolymer of PLA and PCL.

PLCL has been used in several applications including controlled-release drug delivery systems,

monofilament surgical sutures and absorbable nerve guides. It is an FDA approved polymer for

biomedical applications. This scaffold was reported to support cardiovascular, cartilage and

nerve regeneration. The composite scaffold possesses better physical and mechanical properties

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and promotes enhanced cell adhesion, differentiation and proliferation. The study used olfactory

ensheathing cells (OECs), which are an important class of glial cells promoting nerve

regeneration of olfactory system, which in turn plays a crucial role in neural growth of CNS and

PNS. The scaffold was shown to display a much favorable fiber diameter and increased

hydrophilicity, which led to better proliferation of OECs. [71]

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4.6 Polyvinyl alcohol (PVA)

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PVA is a non-toxic, hydrophilic and biocompatible polymer. Although it has high mechanical

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strength, its biocompatibility is not satisfactory. Hence, composites of PVA are made with

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natural polymers to enhance the overall physiochemical and biological properties of the

fabricated scaffold. In one study, PVA fibres were blended with chitosan and the scaffold was
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fabricated using electrospinning. The results generated showed that the scaffold had balanced

properties apt for neural cell regeneration. [72]

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4.7 Conductive polymers

Neural tissue engineering requires simultaneous biochemical, topographic and electrical cues for
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complete and efficient adhesion, differentiation and proliferation of cells. To enhance

topographic cues, aligned fiber scaffolds are being generated by electrospinning that favors
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neurite extension unidirectional. [73] Biochemical cues are enhanced using composite scaffolds of
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synthetic and natural polymers, or by embedding cells like Schwann cells, stem cells or growth

factors on the surface of the scaffold. Since neurons are involved in electrical signal

transmission, electrical cues play a crucial role in directing the development of axons and neurite

growth. Conductive polymers are those with electrons present in their backbone, which enables

them to conduct electricity. Skeletal and nerve cells respond to electrical stimulation. Some

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common conductive polymers used are Polypyrrole (PPY), Poly-3, 4-ethylenedioxythiphene

(PEDOT), Polyaniline (PANI) and carbon nanotubes.

4.7.1 Polypyrrole (PPY)

PPY is a conductive polyacetylene derivative that is used in drug delivery systems, nerve

regeneration and biosensor coatings for neural probes. Its characteristic features include exhibits

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rigidity, insolubility and poor process stability which occur due to the conjugation in the

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molecular backbone of PPY. Its non-biodegradability poses a major challenge to its use in neural

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[73]
tissue engineering, but this could be overcoming by making composites of PPY. Hence, it is

usually conjugated with other synthetic or natural polymers to obtain a stable, biocompatible and

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biodegradable scaffold. PPY is more suited for peripheral nerve injury regeneration, has good
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biocompatibility and cell adhesion properties. It is also non-toxic, non-allergic, non-mutagenic

and non-haemolytic, making it ideal for biomedical applications. In one study, PPY conductive
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meshes were formed on random and aligned electrospun PLGA fibers. The results indicated that
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the scaffold induced better neuronal growth and differentiation than PLGA scaffolds without
[14]
PPY coating. In another study, PPY were polymerized on electrospun PLA and PCL
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templates generating conductive core-sheath nanofibers. The results indicated that PPY
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deposited on PCL was much smoother than that of the PLA fibers. This occurred due to the

presence of a methyl group on PLA side chain, making it less hydrophilic in nature than PCL.
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Increased neurite extension was observed due to electrical stimulation. Also, it was greater on

PCL/PPY scaffolds owing to the smooth texture, as neurons have smooth surfaces due to the

myelin sheath present on them. The efficiency of electrical stimulation is said to be caused due to

an increased adsorption of ECM glycoproteins onto the surface of PPY, which results in better
[73]
cell adhesion and growth. In one study, the effect of electrical stimulation on Schwann cells

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was studied using Polypyrrole in conjugation with chitosan. Results indicated that the

PPY/Chitosan scaffold supported cell adhesion, spreading and proliferation. In the presence of

electrical stimulation, enhanced expression of nerve growth factor (NGF) and brain-derived

neurotrophic factor (BDNF) was observed. Hence, nerve regeneration can be significantly

enhanced using electrical stimulation on conductive scaffolds by means of increased

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neurotrophin secretion. [74]

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4.7.2 Polyaniline (PANI) and Poly-3,4-ethylenedioxythiophene (PEDOT)

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PANI and PEDOT are versatile conducting polymers. In one study, it was shown that

differentiation of human mesenchymal stem cells (MSCs) was increased primarily due to the use

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of hybrid macroporous hydrogels made of PANI and PEG diacrylate. Dispersing the conductive
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polymer with a biological matrix facilitates easier fabrication of conductive scaffolds. In another

study, the characteristics of scaffold deposited with PEDOT were also studied, and it was
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observed that they increase axon growth in nerve conduits. PANI and PEDOT were chemically
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synthesized and then added to a solution of collagen, after which the cell suspensions were made.

The 3-D conductive collagen gels exhibited good cyto-compatibilty and increased neurite
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[75]
outgrowth as compared to non-conductive collagen gels. An electrospun conductive
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nanofiber scaffold was fabricated using PANI with PCL/Gelatin. This scaffold was then seeded

with nerve stem cells and electrical stimulation was applied. Results indicated enhanced
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attachment, proliferation and neurite outgrowth of NSCs. [76] PEDOT has been cross linked with

the polymer Polystyrene sulfonate (PSS) to obtain a conjugated polymer scaffold. Human neural

stem cells were then seeded onto this scaffold. On electrical stimulation, elongation and

differentiation of NSCs into neurons and formation of longer neurites was observed. [77]

4.7.3 Carbon Nanotubes

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They are a type of conducting polymers that need to be functionalized to aid neural signal

transport, dendrite adhesion and elongation. They can be functionalized by substitution reactions
[14]
like replacing carbon atoms from tube wall using boron or nitrogen. They exhibit

characteristic qualities like superior conductivity, remarkable stiffness and high aspect ratio.

They also have an innate ability to absorb strain and induce conductivity. It maintains the

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[78]
structural stability of scaffolds when incorporated. One study demonstrated that

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polyethyleneimine (PEI) deposited with multi-walled CNTs (MWCNTs) exhibited increased

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neurite growth and elongation in all directions. [14] Another study used carbon nanotube fibers

(CNF) made with the natural polymer agarose. The agarose/CNF composite is conductive, non-

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toxic and functionalization can facilitate improved cell adhesion and proliferation. Results
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indicated that it generated directed nerve growth and overall increased differentiation and
[76]
proliferation. CNT network patterns have been used for selective growth and structural
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polarization-controlled neuronal differentiation of human NSCs into neurons. The CNT patterns
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were found to provide synergistic cues, optimal nanotopography and biocompatibility for

differentiation of hNSCs in physiological solution. Thus, CNTs have been proven to be a highly
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[79]
suitable scaffold for controlling hNSC growth. CNT networks on solid substrates have also
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been used in the directed growth and differentiation of hMSCs. It has been reported that the

hMSCs could recognize the arrangement of individual CNTs in the CNT network, which allowed
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directional growth of hMSCs following the direction of alignment of the CNTs. These hMSCs
[80]
were found to exhibit enhanced proliferation and osteogenic differentiation. Another form of

CNT has been studied, where a CNT rope substrate was developed and seeded with neural stem

cells. On electrical stimulation, it was observed that it had a positive impact on promoting neurite
[81]
elongation and enhanced differentiation of NSCs into mature neuronal cells. Successful

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single walled carbon nanotube (SWNT) scaffolds have also been demonstrated. Layer- by- layer

(LBL) assembled SWNT- polyelectrolyte multilayer thin films seeded with mouse embryonic

neural stem cells from cortex have been found to successfully differentiate into neurons,

astrocytes and oligodendrocytes with clear formation of neurites. The biocompatibility, neurite

outgrowth and expression of neural markers were found to be similar as cells differentiated on

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[82]
poly-L-ornithine (PLO), which is a common growth substratum for neural stem cells. hESCs

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have also been seeded on CNTs and proved to be successful candidates for neuronal

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differentiation. A poly (acrylic acid) grafted CNT thin film was fabricated and compared with

PLO surfaces. This scaffold exhibited enhanced neuron differentiation and cell adhesion. [83]

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5. Nerve conduits
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Nerve injuries can be treated using two approaches- designing a graft or a conduit. In a nerve

conduit, proximal and distal nerve stumps are inserted into the two ends of the conduit, and
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axons start regenerating from the proximal end and selectively grow into the distal ends (Figure
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5). This is a preferable alternative to nerve suturing, grafts, etc. because it does not elicit immune

responses. Also, the nerve functionality recovery is not very good in any of these techniques. [84]
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Nerve conduits allow for guided nerve regeneration, and further prevent aberrant regeneration of
[85]
the nerve. It is applicable for nerve injuries over short distances (4 cm). They mimic a

microenvironment favorable for the regeneration of axon, with the incorporation of cells, ECM,
[84, 85. 86]
neurotrophic factors as required. They also enrich the neurotropic factors within the
[84]
ECM, thus providing a suitable micro environment for nerve regeneration. Nerve conduits

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can be ECM taken from the cells of the donor and then decellularized, to prevent immune

reaction or also manufactured artificially. The manufactured nerve conduits can be synthetic,

biological or hybrid conduits, which are biosynthetic. A wide range of synthetic materials like

polyesters, polyurethanes and polyols can be used in the synthesis of synthetic nerve conduits.

For biological nerve conduits, polyesters, proteins and polysaccharides can be used. Composites

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of biological and synthetic materials are used for hybrid nerve conduits. [87, 88] Nerve conduits can

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be designed in different ways, like tubular, fibrous and matrix type. In one study, mono (MC)

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and bi-component (BC) fibrous conduits were designed using PCL and gelatin solutions. Gelatin

stabilizes the electrospun PCL fibers and provides cell adhesion properties. The growth of dorsal

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root ganglia (DRG) neurons was studied. It was observed that BC conduits exhibited better
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neuronal growth and differentiation as compared to MC conduits. [89]

Properties like permeability, flexibility, swelling and degradation rate determine the
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efficiency and quality of nerve conduits (Figure 6). The permeability of the nerve conduit
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determines the degree of nutrient exchange between the external environment and the lumen. It

also influences the selective inclusion or exclusion of growth factors or nutrients in the
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regeneration process, as well as excluding the invasion of undesirable cells that can cause
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scarring or hindrance to the process. The process of vascularization needs to be preceded by

oxygen and nutrient delivery to the conduit lumen. Also, permeability is essential for the
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[84, 91]
determination of any support cell’s viability. The permeability must also be adequate for

the disposal of waste material, while ensuring that none of the secreted neurotrophic factors are

excreted out. [89] This is dependent upon the size of the pores within the conduit, and an optimum

molecular weight cut off 50,000 has been determined. [92] There are several methods to make the

nerve conduits permeable—cutting holes into the walls, rolling of meshes, electrospinning of

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fibers, addition of salt and sugar crystals that can later be leached, injection molding solvent
[84, 93]
evaporation, gas foaming and phase separation. The concept of asymmetric porosity is a

very effective method to minimize scar tissue formation and maximize nutrient delivery. [53, 92]

The flexibility ensures that none of the surrounding tissues (especially in case of a joint,

where the proximal and distal end of the stump are not planar in nature) are damaged, as well as

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the axon undergoing regeneration. [84, 90] A scaffold which is too rigid can cause compression of

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[91]
the growing tissues as well as the surroundings. However, it must be noted that flexibility

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must not compromise upon the mechanical strength of the conduit. Lesser mechanical strength

can cause tearing, kinking or breakage. Thus, it is important that an optimal polymer or

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copolymer ratio be ascertained through experimental studies. [90] Undesirable swelling within the
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nerve conduit can cause hindrance for the growing axon, compressing it as well as the

surrounding tissues. Swelling can be caused by the accumulation of degradation products due to
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the conduit degradation. Swelling can be controlled by varying the conduit dimensions and its
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[53, 84, 90]

copolymer ratio (which in turn influences the degradation profile as well.) An optimal

degradation profile is needed for nerve regeneration to occur without hindrance. Biodegradability
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should ideally follow a trend wherein it remains intact through the process of nerve regeneration
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from the proximal to the distal stump. Then, it should start degrading. However, too rapid

degradation causes inflammation and swelling. When it is too slow, there is compression. [84, 90]
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Thus, nerve conduits are very efficient in treating nerve injuries. Their efficiency is increased by

incorporating different types of support cells, using fibers as channels for guidance and using

bioactive agents like growth factors. The only limitation is the length of the injury that can be

treated with a conduit

5.1 Materials for Conduit Fabrication

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Both synthetic and biological materials can be used for the fabrication of conduits. The scaffold

can be degradable or non-degradable. With nerve conduits, degradable scaffolds with optimum

degradation rates are desired. One example of a biological material based conduit is a Schwann

cell seeded chitosan based nerve guidance conduit that was fabricated for improving nerve

regeneration. The membrane was coated with poly-L-lysine (PLL) to enhance cell attachment

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and growth on the membrane. The conduit proved to be extremely efficient for enhancing nerve

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[94]
regeneration. A fully biodegradable conduit for peripheral nerve repair has also been

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fabricated using synthetic polymers. A PLGA foam conduit with longitudinally aligned was

fabricated and seeded with Schwann cells. The unique channeled architecture also allowed the

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incorporation of neurotrophic factors into the conduit in a controlled fashion. Substantial axonal
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[95]
regeneration and precisely guided neural regeneration was observed. A biological chitin

conduit was fabricated for treatment of spinal cord injuries. The conduit was biodegradable and
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seeded with BM-MSCs. It caused the spinal cord to retain its tubiform shape 14 weeks after
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spinal cord hemisection injury, and created a local microenvironment that promoted the growth,

migration and differentiation of the BMSCs. Furthermore, the conduit promoted axon growth
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and nerve regeneration, effectively reducing scar invasion. [96]

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Veins are also used as conduits materials, and are a good option for tubulization process

due to their easy availability, biodegradability, inert and non-compressing nature. Tubulization is
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the process of using a tubular structure to wrap the site of nerve repair, with or without additional

regeneration promoting factors. The medial layer and adventitia of the vein is rich in collagen

and laminin, which effectively promote nerve regeneration. The composition of tissue in vein

bears similarities to that of the nerve. Veins can act as a successful barrier for preventing scar

tissue formation. They possess the permeability to allow diffusion of nutrients, growth factors,

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etc. Additionally, they are a low-cost, relatively abundant option which does not require

additional injuries as severe as those in nerve grafts. [97] Vein conduits were successfully used for

sciatic nerve repair in rats. Favorable observations were: less epineural scarring, thinner
[98]
epineurium, more axonal regeneration, less inflammatory cells. Bio-3D printing technology

has been employed to fabricate scaffold-free conduits using human fibroblasts. Successful

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regeneration was observed using the conduit in rat sciatic nerve model, along with complete

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biodegradation at 8 weeks post-surgery. This method of conduit fabrication should be studied

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further by virtue of the several advantages it offers: complete control over the desired shape, size

and strength of the structure, absence of any foreign materials that could cause allergy,

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immunogenic reaction or infections. It has been used for bone, cartilage, bladder, skeletal
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muscle, myocardium. [99,100]

Platelet rich plasma (PRP) therapy has been demonstrated as an effective method for
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regeneration of nerve. Autologous platelet rich plasma gel was used as a seeding matrix for
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Schwann cells using a poly(lactic-co-glycolic acid) nerve conduit for regeneration of sciatic

nerve defects in rabbits. The use of this therapy is justified by the presence of several blood
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proteins (like fibrinogen) as well as growth factors in platelet rich plasma (Insulin-like growth
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factor, platelet derived growth factor, transforming growth factor-β, vascular endothelial growth

factor, brain-derived neurotrophic factor, nerve growth factor).It has shown immense potential as
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a neurogenic, neuroprotective and neuroinflammatory therapeutic modulator system. The

biomolecules delivered by PRP modulate macrophage polarization, early inflammation, stem

cell-like myelinating SC activation, as well as the active resolution of inflammation, fibrogenesis

and angiogenesis. These are processes crucial to full recovery of the nerve. Post activation a

three-dimensional fibrin gel is formed. In the case of Schwann cells and mesenchymal stem cells,

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significantly superior adherence of the cells, consistent maintenance of the differentiated state as

well as more uniform distribution of the cells has been observed when fibrin is used as a matrix

and not simply supplied external.[101,102] The Food and Drug Administration (FDA) has approved

several nerve guidance conduits and protective wraps for commercial use (Table 3). These are

synthesized from synthetic as well as natural biomaterials. The commercially available products

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have been enlisted in Table 3.

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6. Cells Used for Neural Tissue Engineering

There are three types of cell sources that can be used for tissue engineering, namely, autologous,

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allogenic and xenogeneic. Autologous cells are harvested from the patient, cultured ex vivo and
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then re-implanted at the site of injury/ regeneration in the patient. It is an ideal choice from an

immunological perspective, as the cells used from one’s own body does not elicit any immune
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responses. However, this approach has its own limitations as the harvesting of organs could
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potentially require the resection of a large part of the patient’s skins, thereby causing pain and

discomfort. The paucity of autologous cells that can be harvested is also a limitation. The second
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cell source is allogeneic, which requires immunosuppression or other strategies to reduce the
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immunogenicity of the allogenic cells. This has found limited success. However, one of the

mostly widely used allogenic cell-based grafts is Apligraft™. The third usable cell source is
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referred to as Xenogeneic, where cross-species transplantation takes place. Again, overcoming

the possibility of immune response poses a major hurdle, along with cross-species pathogen

infectivity. However, successful pigs to human transplants for the treatment of diabetes have
[103]
been conducted in the past. The cells used specifically in neural tissue regeneration include

Schwann cells, bone marrow derived Mesenchymal stem cells and other stem cell sources like

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Embryonic Stem cells, Nerve stem cells, Mesenchymal and Induced Pluripotent stem cells

(Figure 7).[104]

6.1 Schwann cells

Schwann cells produce neurotrophic factors and cause neurite outgrowth in the cells which helps

in the repair of peripheral nerve injuries. Schwann cells play an important role in the process of

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axonal outgrowth. This is preceded by Wallerian degeneration and re-myelination and are

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integral parts of the PNS’s natural wound healing process. Schwann cells get activated when an

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injury takes place and secretes N-cadherin, neurotrophins, gamma integrin’s, Neural Cell
[105]
Adhesion molecules (N-CAM) as well as collagen and lamin in for ECM production.

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Xenogeneic sources are used to provide acellular nerve grafts. These keep the ECM intact and
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elicit low immune response. These can be cultured with Schwann cells, which is a very effective

approach to repair injury in the PNS. However, practically this approach is almost obsolete
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because SCs have limited resources, are difficult to purify and can cause immune reactions. [105]
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Experimental data provided by Rodrigues et al shows that the immune response caused by the

Schwann cells affected the reinnervation process. Moreover, the result provided by the
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autologous SCs was lower than that obtained by autograft. [110]

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6.2 Bone marrow derived Mesenchymal stem cells

They are stromal cells which have the potential to differentiate into different cell lineages and
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can thus be investigated as a plausible alternative to the use of SCs. This cell type overcomes

most of the drawbacks that the use of SCs presents: multi-directional differentiation potential,

and have many clinical advantages, such as the ease of accessibility, rapid proliferation in culture

and successful integration into the host tissue without producing an eminent immune response.

While studies have shown that transplanted SCs are more efficient than BM-MSCs for axonal

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regeneration, BM-MSCs can differentiate to Schwann-like Cells (SLCs) which could improve

the nerve regeneration significantly. [126] However BM-MSCs are said to have less differentiation

capacity and proliferation potential as compared to other types of MSCs, like derived from

Adipose and Dental pulp. [111] Experimental evidence showing the differentiation BM-MSCs into

neural cell types have been reported under in vitro conditions for rats. [112]

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6.3 Other sources

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Stem cells from other sources can be used for nerve tissue regeneration by differentiation into

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SC-like phenotype. Exposure to β-mercaptoethanol, all-trans retinoic acid, fetal bovine serum,

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forskolin, recombinant human bFGF, and recombinant human platelet derived growth factor-AA,

and recombinant human heregulin β-1 causes differentiation of Stem Cells to SC-like phenotype.
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Some features essential to use of these stem cells for clinical purposes are ease of accessibility,

ability to rapidly expand in culture, capable of in vivo survival and integration into host tissue
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and must be amenable to stable transfection and expression of exogenous genes. These then
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assemble to form Bands of Bungner followed by axonal regeneration and subsequent

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myelination. The presence of ECM proteins like collagen I, collagen IV, laminin, fibronectin and

neurite guidance proteins further reinforce regeneration. Moreover, the expression of nerve
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growth factor (NGF), brain derived neurotrophic factor (BDNF), glial cell line- derived

neurotrophic factor (GDNF), ciliary neurotrophic factor and neurotrophin-3 (NT-3) has been
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described in the case of differentiated stem cells. [108] Other sources for harvesting stem cells are

embryonic stem cells, nerve stem cells, mesenchymal stem cells and induced pluripotent stem

cells. [113] These must be differentiated into SLC polycells. [110, 111] SLCs derived from Human

umbilical cord blood mesenchymal cells (hUCBMSCs) have been investigated as the seed to

repair large sciatic nerve. These cells have been known to promote axonal regeneration and

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functional recovery at the site of transected sciatic nerve. Analysis has revealed that fourteen

important growth factors were secreted and acted via paracrine mechanisms to promote

regeneration. Furthermore, the ECM secreted by these cells was also investigated and found to

promote peripheral nerve repair. [114,115]

Embryonic stem cells are pluripotent stem cells harvested from the human blastocyst with

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immense proliferation capacity. The advantage of using these cells is that they are bereft of the

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impact of age and disease and cause no discomfort upon extraction. However, these stem cells

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are potentially tumorigenic, immunogenic and their use could spark several ethical controversies.
[111]
The application of Embryonic stem cell based therapies for several neurodegenerative

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diseases like Parkinson’s disease, Alzheimer’s disease, as well as injuries have been reviewed in
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detail. [110] However, it has been reported that their application to neural tissue regeneration is
[111]
limited, and mesenchymal precursors have therefore been derived. Neural stem cells are
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multipotent stem cells that give rise to different cell types within the neuronal lineage like
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astrocytes, oligodendrocytes, etc. Studies have reported that they play a regenerative role in

response to events like ischemic stroke, multiple sclerosis and other neurodegenerative
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occurrences. Thus, the plasticity of the neural stem cells can be manipulated to give regenerative
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functions. [117, 118] Apart from BM-MSC, mesenchymal stem cells can also be obtained from the

adipose tissue, fetal tissue, hair follicle tissue and dental pulp. While it was initially established
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that mesenchymal stem cells could only differentiate to cells of the mesoderm, it is now
[122]
established that they can differentiate into cell types outside the mesodermal lineage.

Experimental evidence of neural cell markers being expressed from isolated human
[119]
mesenchymal stem cells has been reported. Induced Pluripotent stem cells are generated

from somatic cells, such as fibroblasts, by upregulating the expression of specific genes (Oct3/4,

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Sox2, c-Myc, and Klf4) that activate the pluripotency genes in these normal somatic cells. This

can induce pluripotency in previously somatic cell type and use the patient’s own cells thereby

eliminating the risk of immune rejection. There are several studies reporting the differentiation of

iPSCs into neural cell types. [61, 110]

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7. Conclusion

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We have discussed the various approaches that are currently being used to treat peripheral nerve

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injuries. Although autografts are the golden standards used, several other alternatives are being

devised to overcome their disadvantages. It is important to note that the method used can be

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idealized on general basis. The type of graft or conduit to be designed majorly depends on the
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site of injury, the extent to which the tissue has been injured and the personal profile of the

patient, where age and possible allergies may also factor in. Hence, the best way to devise an
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ideal method for treatment of the injury would be to first diagnose the crucial factors that
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determine the method of treatment. Although synthetic materials are gaining popularity due to

their versatility of fabrication and mechanical strength, it is observed that synthetic polymers as
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composites with natural polymers are highly efficient in case of both grafts and conduits.
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Conductive polymers have also led to a substantial increase in neurite growth and extension. The

method of fabrication also plays a crucial role in determining the physical properties of the
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scaffold. Pore size, random or oriented alignment can be controlled by using varied polymer

concentration, and by using various templates for fabrication. Nanofibers are proving to be more

efficient than microfibers by effectively increasing cell adhesion, growth and proliferation. Cells

like Schwann cells and various stem cells can be seeded to improve the biological activity of the

scaffold. Cell adhesion molecules like integrin’s or cadherin’s have been incorporated into the

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scaffolds to improve the adhesive property of scaffold which will result in better cell growth and

differentiation. Growth factors like NGF, BDNF, GDNF and BFGF are also being incorporated

into the scaffold for improved biological activity. Thus, a combination of fabrication techniques

and materials used in synthesis of scaffold seem to elicit a better response and faster

regeneration. This kind of multi-dimensional approach to developing scaffolds should be applied

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to achieve an ideal scaffold for the subsequent treatment of nerve injuries.

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8. Acknowledgement: Amit Kumar Jaiswal gratefully acknowledges VIT University, Vellore

for the support through “Seed Grant for Research”. Rahul Dev Jayant would like to acknowledge

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Pilot Funding (Grant # 800008542) from Herbert Wertheim College of Medicine (HWCOM) and
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other non-financial support from Institute of NeuroImmune Pharmacology (INIP) and Centre for

Personalized NanoMedicine (CPNM), Department of Immunology, Florida Internal University

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(FIU), Miami, USA.

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Author Contributions: The manuscript was written through contributions of all authors. All
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authors have given approval to the final version of the manuscript. §These authors contributed
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equally.
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Conflict of Interest: All authors declare no financial and any other kind of conflict.

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List of Figures

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Figure 1. An overview of CNS and PNS Injuries. Types of nerve injuries and their possible
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effects: PNS injuries (at the left) are mainly categorized into three types—Neuropraxia (2.),
Axonotmesis (3.) and Neurotmesis (4.) Injuries to the CNS (right) occur in the brain
encompassing traumatic brain injuries (TBI) and neurodegenerative diseases and in the spine
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(SCIs). Figure adopted from references 11 and 12.

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Figure 2. Overview of Neural Tissue Engineering approach. Two approaches: Conventional

(Types of grafts) and Tissue engineering approach. Types of stimuli, cells and biomaterials are
elaborated.
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Figure 3. Schematic diagram showing the apparatus for electrospinning: Electrospinning is a

method for scaffold fabrication. The above mentioned parameters can be varied to make
scaffolds with different properties.
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Figure 4. The ideal properties of scaffolds used in Neural Tissue Engineering depend on the
right choice of biomaterial and its corresponding chemical, physical and mechanical properties.
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Figure 5. Schematic figure depicting different constituent elements of a nerve conduit. The
regeneration occurs from proximal to distal stump. The conduit itself can be of three types based
on degradation profile desired. Several elements can be added (shown at the left) to promote
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regeneration. It is effective for severed axon having stumps with less than or equal to 3 cm gap.
Figure has been adopted from references 107 and 109.
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Figure 6. The properties that determine the idealness of a scaffold include its permeability,
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flexibility, swelling and degradation rate.
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Figure 7. Types of cells used in Neural Tissue Engineering summarized.
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List of Tables

Table 1. Natural biomaterials and cells in neural tissue engineering

Natural Materials

Material Advantage Disadvantage Cells used References

[16, 20, 14, 27]
Collagen Versatility, low Weak mechanical and BM-MSCs

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antigenicity, structural stability upon and Schwann
inflammatory and uptake of water. cells.

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cytotoxic response,
biocompatibility, good

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water uptake
capabilities,
availability of several
isolation methods,

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ability to tailor
mechanical and cross
linking properties.
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[28, 31, 34, 40, 36, 37, 38]
Hyaluronic acid Good Non-adherence of cells Mouse MSCs
biocompatibility, high and water solubility. and Schwann
water content, safe cells.
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degraded products,
limited
immunogenicity,
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viscoelastic properties
and ability to influence
wound healing,
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metastasis etc.
[111, 112, 116. 117, 42]
HYAFF Biocompatibility, Release of acidic Schwann
complete degradation products, cells.
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degradability, poor processability and

solubility in DMSO, loss of mechanical
stable on hydrolysis, properties very early
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strong interaction with during degradation.

polar molecules and
ability to promote cell
adhesion and
proliferation.

Alginate High biocompatibility, Unstable mechanical NSCsc) ad [118, 43, 44, 45, 46 ]

high biodegradability, properties and lack of Schwann

non-antigenicity and the specific cell- cells.
chelating property. recognition signals.

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[119, 48, 50, 51, 52]

Chitosan Biocompatibility, Some forms of chitosan Schwann
biodegradability, non- may be toxic. cells and
toxicity, inhibition of BMSCd)-
growth of fungi, yeast derived
and bacteria and non- Schwann
immunogenicity. cells.
a)
BM – Bone marrow derived, b)MSCs – Mesenchymal stem cells, c)NSCs – Neural stem cells, d)BMSC – Bone
marrow stem cells

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Table 2. Synthetic biomaterials and cells in neural tissue engineering

Synthetic Materials

Material Advantage Disadvantage Cells used References

[56, 14, 60, 61, 62, 63]
PCL Biodegradable, Cytotoxic effects on using Adipose-
biocompatible, organic solvents. derived
possesses high multipotent
elasticity, low SCs,

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toxicity, good hMSCsa),

mechanical NCSCsb) and
properties and a slow Schwann
degradation profile. cells.
[14, 65, 66]
PLLA Biodegradable, Poor biocompatibility, MSCs,
ultrafine continuous release of acidic products NSCsc) and
fibers, high surface- on degradation, poor Schwann
to-volume ratio, high process ability and cells.
porosity, varied premature failure of
distribution of pore mechanical features

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size during degradation.

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[14, 16, 129, 68]
PLGA Biodegradability, Plastic deformation and NSCs,
non-toxicity and film failure on exposure to NPCsd) and

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forming ability. long term strain, releases Mouse
acidic products on embryonic
degradation. fibroblasts.

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PHB Potential neural Poor biocompatibility. OECse), [70, 121, 126, 123]

protective agent, MSCs, NSCs

high crystallinity, and hMSCs.
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longer degradation
time, and provides
support for cell
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adhesion and
proliferation (of
osteoblasts,
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fibroblasts).
[72, 124, 125]
PVA Non-toxic, Poor biocompatibility. Dorsal root
hydrophilic and high ganglia.
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mechanical strength.
[75, 76, 126, 127]
PPY Exhibits rigidity, Insoluble, non- Schwann
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good biodegradable and poor cells.

biocompatibility and process stability.
cell adhesion
properties, non-toxic,
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non-allergic, non-
mutagenic and non-
haemolytic.
[19, 75, 76, 125, 126]
PANI Versatility, Inability to degrade and hMSCs and
conductive, good chronic inflammation. NSCs.
biocompatibility and
increased neurite
outgrowth.
[19, 75, 77, 128]
PEDOT Versatility, Inability to degrade and hMSCs and
conductive, good chronic inflammation. NSCs.
biocompatibility and

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increased neurite
outgrowth.
[129, 130, 131, 78, 79, 80,
CNTs Superior Cytotoxicity and non- hNSCs,
81, 82, 83]
conductivity, biodegradability. hMSCs,
remarkable stiffness, Mouse
high aspect ratio, embryonic
maintains structural NSCs and
stability of scaffolds, hESCsf).
biocompatibility,
optimal

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nanotopography and

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induces conductivity.
a)
hMSC – human Mesenchymal stem cells, b)NCSC – Neural crest stem cells, c)NSC – Neural stem cells, d)NPC –

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Neural progenitor cells, e)OEC – Olfactory progenitor cells, f)hESC – human Embryonic stem cells.

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Table 3. FDA approved nerve conduits and cuffs

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Product Biomaterial Degradation Diameter Length Characteristics

Nerve Conduits
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Neurotube® Polyglycolic 3 months 2.3 mm 4 cm Woven, flexible,

acid (PGA) externally corrugated
tube that can bridge
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nerve gaps between 8

mm and 3 cm.

NeuroMatrixTM Type I 4-8 months 2-6 mm 2.5 cm Semi-permeable,

Collagen clinically effective and
tensionless repair
without associated
morbidity.

NeuraGenTM Type I 36-48 months 1.5-7 mm 2-3 cm When hydrated, it is an

Collagen easy to handle, soft,
pliable, non-friable,

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porous collagen tube.

NeuroFlexTM Type I 4-8 months 2-6 mm 2.5 cm Flexible, fully

Collagen resorbable and semi-
permeable and kink
resistant up to 60o.

Axoguard® Types I, III, IV 3 months 1.5-7 mm 10 mm Only porcine

Nerve and VI submucosal ECM aid
Connector Collagen for tensionless repair of
(Porcine) severed nerve ends with

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less than a 5 mm gap.

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NeuroLac® Poly(DL- 16 months 1.5-10 mm 3 cm A transparent,
lactide Є- synthetic, bioresorbable

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caprolactone); conduit that allows for
PCL efficient nerve stump
positioning.

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SaluTunnelTM Salubria® Non- 2-10 mm 6.35 cm Flexible, tubular sheath
Nerve biodegradable with longitudinal slit
Protector that allows for easy
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placement at site of
injury and provides a
protective environment.
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Nerve Cuffs

SaluBridgeTM Salubria® Non- 2, 5 and 6.35 cm Flexible, tubular sheath

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(Polyvinyl biodegradable 10 mm that provides a

alcohol, PVA protective environment.
and water-
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based)

Axoguard® Types I, III, IV 3 months 2, 5 and 7 5 cm A biocompatible nerve

Nerve and VI mm cuff with sufficient
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Protector Collagen tensile strength, suture

(Porcine) retention strength and
ability to withstand
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compressive forces.

NeuraWrapTM Type I 36-48 months 3-10 mm 2-4 cm An absorbable collagen

Collagen implant with
longitudinal slit that
allows for easy
placement and protects
the neural environment.

NeuroMendTM Type I 4-8 months 4-12 mm 2.5-5 Resorbable collagen

Collagen cm matrix with
longitudinal slit. When
hydrated, it is an easy

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to handle, soft, pliable,

non-friable and porous.

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Graphical Abstract
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Fig. Biomaterials and cells used in neural tissue engineering

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HIGHLIGHTS

1. Neural tissue engineering is an alternative to using grafts for nerve injuries.

2. Biomaterials, Cells and Stimuli are the three main components of Neural tissue engineering.
3. Biomaterials used for the fabrication of scaffolds can be natural or synthetic or composites of
both.
4. Different cell types and growth factors have been incorporated in scaffolds.
5. Commercially fabricated nerve conduits and cuffs are available for different types of nerve

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injuries.

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