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Biomaterials and cells for neural tissue engineering: Current choices
Accepted Manuscript Biomaterials and cells for neural tissue engineering: Current choices
Prerana Sensharma, G. Madhumathi, Rahul D. Jayant, Amit K. Jaiswal PII: DOI: Reference:
S0928-4931(16)32866-1 doi: 10.1016/j.msec.2017.03.264 MSC 7776
To appear in:
Materials Science & Engineering C
Received date: Accepted date:
29 December 2016 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 for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.03.264
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ACCEPTED MANUSCRIPT
Biomaterials and Cells for Neural Tissue Engineering: Current Choices
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Prerana Sensharma1, §, Madhumathi G.1, §, Rahul D. Jayant3 and Amit K. Jaiswal2*
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School of Biosciences and Technology, VIT University, Vellore, 632014, Tamilnadu, India Centre for Biomaterials, Cellular and Molecular Theranostics, VIT University, Vellore, 632014, Tamilnadu, India 3 Center for Personalized Nanomedicine, Institute of Neuro-Immune Pharmacology, Department of Immunology, Herbert Wertheim College of Medicine, Florida International University (FIU), Miami, FL-33199, USA
*
Corresponding Author
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Amit K Jaiswal, Ph.D. Associate Professor Email: [email protected] Phone No (O): +91-9789280874
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Authors have equal contribution
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ACCEPTED MANUSCRIPT 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
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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 scaffold. Along with these, cells and growth factors are also being incorporated to increase the
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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 extracellular matrix in our body, and to elicit a combination of biochemical, topographical and
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electrical cues via various polymers, cells and growth factors, using which nerve regeneration
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can efficiently occur.
Keywords: Nerve tissue engineering, Synthetic and natural polymers, Biomaterials, Hydrogels,
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Nanofibers.
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ACCEPTED MANUSCRIPT 1. Introduction Nerve injuries affect millions of people worldwide.
[1]
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
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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 mechanisms such as thermal, electric shock, radiation, percussion, and vibration. Injured nerves
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of the peripheral nervous system (PNS) have the ability to regenerate. However, spontaneous
functional outcome.
[3]
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regeneration of the nerve, in the absence of any therapeutic intervention does not result in a good Despite early diagnosis surgical intervention does not result in the
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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 have high standards, but they still have limitations as the nerves are short aged, supply is limited.
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They also present the possibility of causing morbidity, neuroma formation at site of harvest, [4]
Allogenic grafts could also be used but they would produce an
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scarring and sensory loss.
immune response which will disable the cells that cure the injury. [5] The central nervous system
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(CNS) consists of the brain, spinal cord and retina. It is limited in terms of its spontaneous 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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ACCEPTED MANUSCRIPT rather than facilitating further regeneration.
[2]
Spinal cord injuries (SCI) occur majorly in traffic
accidents and due to elderly patients falling. They result in paraplegia and quadriplegia, which cannot be effectively treated so far.
[7]
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,
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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 cell transplantation has an inhospitable environment. Furthermore, limited drugs and biologics
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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 treatment of nerve injuries. Tissue engineering centric approaches will enable regeneration,
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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, molecule delivery and biomaterial scaffold constructions are considered the greatest hope for
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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 repaired to favor cell penetration and tissue regeneration in three dimensions. Tissue engineering
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comprises three main components namely, the biomaterial used, the cells and the biomechanical 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 increasing application in tissue engineering.
[9]
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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ACCEPTED MANUSCRIPT 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
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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 commercially available, FDA-approved nerve conduit. These conduits have been reported
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effective for regeneration of the nerve. In the United States alone they result in the expenditure
performed in the United States annually.
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of 150 billion dollars in the annual healthcare dollar. Over 200,000 repair procedures are [10]
Thus, neural tissue engineering offers potentially
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great solutions on the healthcare front, as well as great economic prospects in the market.
2. Ideal properties of a scaffold
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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 the physical, chemical and mechanical properties of that tissue (Figure 4). The following
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properties given below depend on one key parameter, which is the choice of biomaterial for 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 functionality of cells and migration and proliferation of cells on the scaffold.
[14]
Surface
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ACCEPTED MANUSCRIPT 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
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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
density.
[15]
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surface. Cell adhesion property depends upon surface properties like wettability and charge Along with biocompatibility, toxicity profiles also play a crucial role in cell
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adhesion, growth and proliferation on the scaffold. [16]
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2.2 Biodegradable
One of the major advantages of synthesizing biodegradable scaffolds is that they eliminate the
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In case of neural tissue engineering, controlled biodegradable scaffolds are preferred as
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body.
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need for surgical removal of the scaffold and they are absorbed by the surrounding tissues in the
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the scaffold is meant to support the growth of nerve cells and then be degraded by the body as subsequent repair takes place. A biodegradable scaffold will also facilitate the neighboring cells
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to produce their own extracellular matrix (ECM). It should be taken care of that the by-products of biodegradation should be non-toxic as well and that they are easily dispose-off.
[14]
The
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cytotoxic effects could also enable neuroma formation. Biodegradability should favor the 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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ACCEPTED MANUSCRIPT Ideally, 90 % porosity and pore size of 100µm-500µm is standard for yielding good results.
[16]
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
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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]
2.4 Mechanical properties
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The scaffold should have mechanical properties identical to that of the tissue at the implantation site. As this is not always feasible, materials with mechanical properties that can protect the cells
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from compressive or tensile forces without disturbing the biomechanical cues are used.
[16]
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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 modulus. These parameters have been found to increase with subsequent decrease in the
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diameter of the fiber. Although the explanation behind this phenomenon has not been clearly elucidated yet, it has been postulated that there is an increase in macromolecular chain alignment
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as the fiber diameter is decreased, resulting in a higher crystallinity of the nanofiber. Such unique 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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ACCEPTED MANUSCRIPT 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,
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migration and viability. [18] The mimicking of ECM will aid the surrounding cells to secrete their
[16]
Electrical stimulation has been found to be especially successful in the
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and morphogenesis.
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own ECM, which binds the cells to tissues and thus elicits signals that facilitate cell development
case of neural tissue engineering. Neuronal repair is highly unique due to the complexity of the
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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 mechanical support to the tissues, facilitates migration of cells, holds the cells together,
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facilitates communication in cells so that daily cellular activities and wound healing can be performed uninterrupted.
[17, 20]
Natural polymers has advantage as scaffold design as they
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closely mimic the macromolecules that cells interact with in vivo. The frequently used natural 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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ACCEPTED MANUSCRIPT 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 is an eligible biomaterial for scaffolds pertaining to almost all types of
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collagen.
[22, 23, 24]
The versatility of collagen is
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heart valves and other cardiovascular diseases, and so on.
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tissues—it is used in bone tissue engineering, for skin, cornea potentially for the development of
attributed to the widespread distribution of collagen in the body. Some other advantageous
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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 from various sources and the ability to tailor mechanical and cross linking properties as per need. Collagen 1 is suitable for implantation within the body because there are very few people
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[20, 25]
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who have humoral immunity against it. Moreover, a simple serological test can determine whether the use of this biomaterial will elicit an allergic response in the patient.
[22]
A self-
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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 was reported to show neurite extension from a dissociated dorsal root ganglia, and showed a
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greater regeneration over all. [26] Collagen-based scaffolds are fabricated through electrospinning, 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 properties to the scaffold.
[22, 25]
For instance, to increase the strength of a collagen based,
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ACCEPTED MANUSCRIPT 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 chances of cytotoxity and immune response using this.
[20]
Studies have reported that greater
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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 enhance the mechanical strength of the scaffold. In one study, Poly (L-lactic) co-poly (3-
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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 differentiated MSCs on the PLCL/Coll scaffold had neuronal morphology with multipolar
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elongations and expressed neurofilament and nestin proteins, which confirm neuronal induction.
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[27]
Hyaluronic
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3.2 Hyaluronic acid (And its derivatives) acid
(HA,
also
Hyaluranon)
is
a
linear,
non-branched
non-sulfated
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glycosaminoglycan (GAG) and is composed of repeating disaccharides (β-1, 4-D-glucuronic acid (known as uronic acid) and β-1, 3-N -acetyl- -glucosamide). It has sites for cell adhesion and is [28]
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non-immunogenic.
Hyaluronic acid has found many applications in tissue engineering,
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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ACCEPTED MANUSCRIPT arthritis, several liver pathologies as well as early organ rejection.
[30,31]
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
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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 cellular processes. Thus, it is a plausible option for influencing [28, 31, 33]
HA is especially advantageous for
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processes like wound healing, metastasis, etc.
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designing scaffolds for neural tissue regeneration due to its high abundance in the neural system (especially the Central Nervous System), making it an extremely biocompatible choice.
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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 porous structure, with interconnected pores that allow for the transportation of nutrition,
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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 around the scar area. A dominant disadvantage associated with the use of HA is that cells do not
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adhere to its surface. Thus, to overcome this, it is blended with other biomaterials which will 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 promoted differentiation of Neural Stem Cells (NSCs) for neural regeneration in vitro.
[35]
Another disadvantage of HA is its water solubility, which makes it necessary to add a crosslinking agent to convert it into injectable form.
[36]
In one study, HA hydrogel that could be
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ACCEPTED MANUSCRIPT 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]
In another study, HA strands were cross linked using glutaraldehyde and coated
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hydrogel.
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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 graft. [38]
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3.3 HYAFF
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HYAFF is an HA derivative obtained by esterification of HA with benzyl ester. Some favorable properties of HYAFF are its biocompatibility, complete degradability, solubility in DMSO,
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stable on hydrolysis, strong interaction with polar molecules and its ability to promote cell [39]
Moreover, HYAFF can be used to design
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adhesion and proliferation of various cell types.
films, non-woven fabrics, gauzes, sponges, tubes and microsphere, thus broadening its
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applications. It is used for tissue repair, controlled drug release, nerve regeneration, delivery of [40]
Experiments have reported HYAFF based tube scaffolds
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growth factors, wound healing, etc.
are good substrates for nerve cell cultures and explants. It was reported that cells from rat sciatic
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nerves showed adhesion to the scaffold, followed by proliferation and colonization of the scaffold. Its properties are ideal for Peripheral Nervous System Tissue Regeneration.
[41]
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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ACCEPTED MANUSCRIPT 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,
43]
The advantages of alginate as a scaffold are its high biocompatibility, high
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[36,
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alginate hydrogels are formed by making them interact with divalent cations in aqueous solution.
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biodegradability, non-antigenicity and chelating property. The several types of scaffold that can be formed using alginate are summarized in a review by Sun & Tan et al. A study was conducted
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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 the repair of injured nervous tissue, where the repair is facilitated by the secretion of
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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 as a bioresourceable conduit supplemented with Schwann cells to replace nerve grafts. Enhanced
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neurite growth, viability and proliferation were observed.
[45]
Alginate conduits seeded with
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Schwann cells have also been fabricated along with the incorporation of fibronectin. Results indicated that regeneration rate, viability of the cells and axonal growth were enhanced using this
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conduit. [46]
3.5 Chitosan
Chitin is naturally found in the cell walls of crustaceans such as insects, crabs, shrimps as well as the cell wall of bacilli and fungi.
[25, 47]
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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ACCEPTED MANUSCRIPT 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
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of several fungi, yeast and bacteria and non-immunogenicity. Due to the absence of any protein
[49]
Chitin incorporated with other
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proved to be a good choice for neural tissue regeneration.
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or lipid in chitosan structure, no antibody can be developed against it. [48] Chitosan has also been
materials show an affinity for nerve cells. Chitin based nerve chambers can be used for effective
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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, growth and migration of Schwann Cells. This is very important because Schwann cells release
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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. Moreover, it has the mechanical strength necessary for effective neural regeneration. Chitosan
Chitosan scaffold pre-seeded with Schwann cells was synthesized as a bio-artificial nerve
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[50]
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conduits resulted in the sprouting of myelinated axons and successful recovery of nerve function.
graft for peripheral nerve regeneration. Good biocompatibility of the Schwann cells and chitosan
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fibers was observed. [51] In another study, chitosan conduit seeded with bone marrow stromal cell (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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ACCEPTED MANUSCRIPT 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]
To overcome the disadvantages posed by these grafts, synthetic
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of immune rejection.
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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 polymers, polymeric scaffolds are proving to very efficient scaffold materials.
[53, 54]
Polymeric
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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 mechanical properties can be easily engineered in these polymeric scaffolds, suitable to the
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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 the graft, leading to immune rejection of the graft. This property can also be easily tailored in
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synthetic polymers. Some widely used synthetic polymers for the fabrication of scaffolds include
sebacate,
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Poly -L-lactic acid (PLLA), Polycaprolactone (PCL), Polyhydroxybutyrate (PHB), Polyglycerol Poly-D,L-lactide-co-glycolic
acid
(PLGA),
Poly-L-lactate
(PLA),
Poly-3-
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hydroxybutyrate (PHB), Polyamide, Polydioxanone, Poly-e-caprolactone-co-ethyl ethylene phosphate (PCLEEP), Poly-D.L-lactide-co-caprolactone (PDLLA), Polyvinyl alcohol (PVA), Poly acrylonitrile-co-methylacrylate (PAN-MA) and copolymer of methyl methacrylate (MMA) and acrylic acid (AA) (PMMAAA).
[55]
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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ACCEPTED MANUSCRIPT 4.1 Polycaprolactone (PCL) PCL is a biodegradable and biocompatible polymer, which is cost efficient, possesses high elasticity, low toxicity, good mechanical properties and a slow degradation profile.
[56]
It has
been widely applied in tissue engineering including bone and neural tissue engineering. PCL
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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 free method, template synthesis was used where PCL fibers were synthesized using Alumina
PCL has been used in conjugation with natural polymers or coated with cells to improve its
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[54]
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nanoporous membrane. NaOH was then used to dissolve the template and yield PCL nanowires.
biocompatibility and cell adhesion properties. This will confer mechanical strength and
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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 allow the regeneration of nerve fibers in a contact-oriented fashion which increases the growth of [58]
In one study, PCL fibers have been extruded and embedded in poly-2-
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the fibers.
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hydroxyethyl methacrylate (HEMA) gel. The sonication of the PCL/HEMA composite was dissolved in acetone solvent leaching out PCL and leaving behind HEMA gel with oriented
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longitudinal channels embedded in it. The channel diameter can be controlled by altering the 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 (multipotent) stem cells to improve the effects on nerve regeneration.
[60]
A PCL scaffold seeded
with human mesenchymal stem cells was shown to induce adipogenic, chondrogenic and
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ACCEPTED MANUSCRIPT osteogenic lineages.
[16]
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
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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 [61]
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differentiated into Schwann cells and then integrated into the myelin sheath around axons.
PCL has also been used directly in conjugation with Schwann cells. Aligned PCL fibers
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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 nanofibrous PCL/gelatin scaffolds fabricated by electrospinning were seeded with Schwann cells
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to assist the direction of growth of regenerating axons in peripheral nerves. [63]
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4.2 Poly-L-lactic acid (PLLA)
PLLA is a biodegradable and biocompatible synthetic polymer. Nano-structured PLLA scaffolds
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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, varied distribution of pore size from 50-350 nm. It contains ester linkages in the polymer
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backbone which lead to bio-functionalization of the polymer with various biomolecules by 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 differentiation into different neurogenic lineages by culturing in differential media.
[14]
It has
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ACCEPTED MANUSCRIPT 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 aligned fibers yielded the same results with respect to NSC differentiation.
[64]
3-D PLLA nano-
structured porous scaffolds have been fabricated using liquid-liquid phase separation methods.
[65]
Several studies have proven that PLLA scaffolds
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differentiation and neurite outgrowth.
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The fabricated scaffold seeded with NSCs showed significant improvement in NSC
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efficiently support NSC differentiation and neurite outgrowth, thereby making them suitable to applications in neural tissue engineering. PLLA conduits incorporated with allogenic Schwann
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cells were studied to venture into the synthesis of bioactive nerve conduits which can potentially [66]
A heparin/collagen
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replace autologous nerve grafts and peripheral nerve regeneration.
encapsulating nerve growth factor multilayer was coated upon a PLLA scaffold using layer-by-
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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 superior benefits to Schwann cell proliferation and its cytoskeleton, PC12 differentiation and
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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) PLGA is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA). It is amorphous
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biodegradable polyester and is widely used for fabrication of scaffolds in neural tissue 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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ACCEPTED MANUSCRIPT 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
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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 pharmaceutical, medical engineering and various other industries. In one study, PLGA
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nanofibers were generated using electrospinning technique and NSCs were seeded onto the
outgrowth.
[14]
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scaffold. Results indicated high efficiency of cell adhesion, nerve cell differentiation and neurite In another study, the PLGA scaffold was conjugated with PEG (Polyethylene
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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 (SCI). The scaffold induced continuity of the spinal cord and led to a significant reduction in
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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 been shown to protect some important axonal cytoskeleton proteins to promote repair in SCI.
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Thus, the composite scaffold exhibited greater iNSC adhesion, cell growth and proliferation.
[68]
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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ACCEPTED MANUSCRIPT 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 muscle, cardiac muscle and Schwann cells.
[16]
In one study, non-linear elastic biomaterial
scaffold was fabricated using a PGS/PLLA composite. The fabrication was done using core and
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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)
PHB is biodegradable aliphatic polyester synthesized by several bacteria using renewable carbon
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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 as a biomaterial and is FDA approved. A study reported that PHB is a potential neural protective
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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-3hydroxy butyrate-co-3-hydroxyvalate (PHBV) scaffold has been found to provide better support
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for adhesion and proliferation of osteoblasts and fibroblasts. In one study, PHB films were 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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ACCEPTED MANUSCRIPT 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
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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 natural polymers to enhance the overall physiochemical and biological properties of the
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fabricated scaffold. In one study, PVA fibres were blended with chitosan and the scaffold was fabricated using electrospinning. The results generated showed that the scaffold had balanced
4.7 Conductive polymers
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properties apt for neural cell regeneration. [72]
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Neural tissue engineering requires simultaneous biochemical, topographic and electrical cues for complete and efficient adhesion, differentiation and proliferation of cells. To enhance
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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 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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ACCEPTED MANUSCRIPT 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
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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 tissue engineering, but this could be overcoming by making composites of PPY.
[73]
Hence, it is
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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 biocompatibility and cell adhesion properties. It is also non-toxic, non-allergic, non-mutagenic
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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 the scaffold induced better neuronal growth and differentiation than PLGA scaffolds without [14]
In another study, PPY were polymerized on electrospun PLA and PCL
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PPY coating.
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templates generating conductive core-sheath nanofibers. The results indicated that PPY deposited on PCL was much smoother than that of the PLA fibers. This occurred due to the
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presence of a methyl group on PLA side chain, making it less hydrophilic in nature than PCL. 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 cell adhesion and growth.
[73]
In one study, the effect of electrical stimulation on Schwann cells
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ACCEPTED MANUSCRIPT 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
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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) PANI and PEDOT are versatile conducting polymers. In one study, it was shown that
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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 polymer with a biological matrix facilitates easier fabrication of conductive scaffolds. In another
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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 synthesized and then added to a solution of collagen, after which the cell suspensions were made.
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The 3-D conductive collagen gels exhibited good cyto-compatibilty and increased neurite [75]
An electrospun conductive
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outgrowth as compared to non-conductive collagen gels.
nanofiber scaffold was fabricated using PANI with PCL/Gelatin. This scaffold was then seeded
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with nerve stem cells and electrical stimulation was applied. Results indicated enhanced 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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ACCEPTED MANUSCRIPT 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 like replacing carbon atoms from tube wall using boron or nitrogen.
[14]
They exhibit
characteristic qualities like superior conductivity, remarkable stiffness and high aspect ratio.
[78]
One study demonstrated that
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structural stability of scaffolds when incorporated.
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They also have an innate ability to absorb strain and induce conductivity. It maintains the
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polyethyleneimine (PEI) deposited with multi-walled CNTs (MWCNTs) exhibited increased neurite growth and elongation in all directions. [14] Another study used carbon nanotube fibers
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(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 indicated that it generated directed nerve growth and overall increased differentiation and [76]
CNT network patterns have been used for selective growth and structural
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proliferation.
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polarization-controlled neuronal differentiation of human NSCs into neurons. The CNT patterns were found to provide synergistic cues, optimal nanotopography and biocompatibility for
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differentiation of hNSCs in physiological solution. Thus, CNTs have been proven to be a highly [79]
CNT networks on solid substrates have also
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suitable scaffold for controlling hNSC growth.
been used in the directed growth and differentiation of hMSCs. It has been reported that the
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hMSCs could recognize the arrangement of individual CNTs in the CNT network, which allowed directional growth of hMSCs following the direction of alignment of the CNTs. These hMSCs were found to exhibit enhanced proliferation and osteogenic differentiation.
[80]
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 elongation and enhanced differentiation of NSCs into mature neuronal cells.
[81]
Successful
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ACCEPTED MANUSCRIPT 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
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outgrowth and expression of neural markers were found to be similar as cells differentiated on
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poly-L-ornithine (PLO), which is a common growth substratum for neural stem cells.
[82]
hESCs
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have also been seeded on CNTs and proved to be successful candidates for neuronal differentiation. A poly (acrylic acid) grafted CNT thin film was fabricated and compared with
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PLO surfaces. This scaffold exhibited enhanced neuron differentiation and cell adhesion. [83]
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5. Nerve conduits
Nerve injuries can be treated using two approaches- designing a graft or a conduit. In a nerve
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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 5). This is a preferable alternative to nerve suturing, grafts, etc. because it does not elicit immune
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responses. Also, the nerve functionality recovery is not very good in any of these techniques. [84] Nerve conduits allow for guided nerve regeneration, and further prevent aberrant regeneration of the nerve. It is applicable for nerve injuries over short distances (4 cm).
[85]
They mimic a
microenvironment favorable for the regeneration of axon, with the incorporation of cells, ECM, neurotrophic factors as required.
[84, 85. 86]
They also enrich the neurotropic factors within the
ECM, thus providing a suitable micro environment for nerve regeneration.
[84]
Nerve conduits
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ACCEPTED MANUSCRIPT 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.
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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) and bi-component (BC) fibrous conduits were designed using PCL and gelatin solutions. Gelatin
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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 neuronal growth and differentiation as compared to MC conduits. [89]
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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 determines the degree of nutrient exchange between the external environment and the lumen. It
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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 scarring or hindrance to the process. The process of vascularization needs to be preceded by
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oxygen and nutrient delivery to the conduit lumen. Also, permeability is essential for the determination of any support cell’s viability.
[84, 91]
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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ACCEPTED MANUSCRIPT fibers, addition of salt and sugar crystals that can later be leached, injection molding solvent evaporation, gas foaming and phase separation.
[84, 93]
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,
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where the proximal and distal end of the stump are not planar in nature) are damaged, as well as
[91]
However, it must be noted that flexibility
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the growing tissues as well as the surroundings.
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the axon undergoing regeneration. [84, 90] A scaffold which is too rigid can cause compression of
must not compromise upon the mechanical strength of the conduit. Lesser mechanical strength
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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 nerve conduit can cause hindrance for the growing axon, compressing it as well as the
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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 copolymer ratio (which in turn influences the degradation profile as well.)
[53, 84, 90]
An optimal
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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 from the proximal to the distal stump. Then, it should start degrading. However, too rapid
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degradation causes inflammation and swelling. When it is too slow, there is compression. [84, 90] 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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ACCEPTED MANUSCRIPT 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
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regeneration. The membrane was coated with poly-L-lysine (PLL) to enhance cell attachment
[94]
A fully biodegradable conduit for peripheral nerve repair has also been
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regeneration.
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and growth on the membrane. The conduit proved to be extremely efficient for enhancing nerve
fabricated using synthetic polymers. A PLGA foam conduit with longitudinally aligned was
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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 regeneration and precisely guided neural regeneration was observed.
[95]
A biological chitin
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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 spinal cord hemisection injury, and created a local microenvironment that promoted the growth,
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migration and differentiation of the BMSCs. Furthermore, the conduit promoted axon growth
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and nerve regeneration, effectively reducing scar invasion. [96] Veins are also used as conduits materials, and are a good option for tubulization process
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due to their easy availability, biodegradability, inert and non-compressing nature. Tubulization is 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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ACCEPTED MANUSCRIPT 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 epineurium, more axonal regeneration, less inflammatory cells.
[98]
Bio-3D printing technology
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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 further by virtue of the several advantages it offers: complete control over the desired shape, size
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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 muscle, myocardium. [99,100]
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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 Schwann cells using a poly(lactic-co-glycolic acid) nerve conduit for regeneration of sciatic
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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 factor, platelet derived growth factor, transforming growth factor-β, vascular endothelial growth
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factor, brain-derived neurotrophic factor, nerve growth factor).It has shown immense potential as 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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ACCEPTED MANUSCRIPT 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
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synthesized from synthetic as well as natural biomaterials. The commercially available products
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have been enlisted in Table 3.
6. Cells Used for Neural Tissue Engineering
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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 then re-implanted at the site of injury/ regeneration in the patient. It is an ideal choice from an
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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 potentially require the resection of a large part of the patient’s skins, thereby causing pain and
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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 immunogenicity of the allogenic cells. This has found limited success. However, one of the
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mostly widely used allogenic cell-based grafts is Apligraft™. The third usable cell source is 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 been conducted in the past.
[103]
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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ACCEPTED MANUSCRIPT 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
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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
injury takes place and secretes N-cadherin, neurotrophins, gamma integrin’s, Neural Cell
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Adhesion molecules (N-CAM) as well as collagen and lamin in for ECM production.
[105]
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Xenogeneic sources are used to provide acellular nerve grafts. These keep the ECM intact and elicit low immune response. These can be cultured with Schwann cells, which is a very effective
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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] Experimental data provided by Rodrigues et al shows that the immune response caused by the
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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] 6.2 Bone marrow derived Mesenchymal stem cells
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They are stromal cells which have the potential to differentiate into different cell lineages and 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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ACCEPTED MANUSCRIPT 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
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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, forskolin, recombinant human bFGF, and recombinant human platelet derived growth factor-AA,
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and recombinant human heregulin β-1 causes differentiation of Stem Cells to SC-like phenotype. Some features essential to use of these stem cells for clinical purposes are ease of accessibility,
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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 myelination. The presence of ECM proteins like collagen I, collagen IV, laminin, fibronectin and
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neurite guidance proteins further reinforce regeneration. Moreover, the expression of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), glial cell line- derived
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neurotrophic factor (GDNF), ciliary neurotrophic factor and neurotrophin-3 (NT-3) has been 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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ACCEPTED MANUSCRIPT 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]
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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 are potentially tumorigenic, immunogenic and their use could spark several ethical controversies. The application of Embryonic stem cell based therapies for several neurodegenerative
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[111]
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diseases like Parkinson’s disease, Alzheimer’s disease, as well as injuries have been reviewed in detail. [110] However, it has been reported that their application to neural tissue regeneration is
M
limited, and mesenchymal precursors have therefore been derived.
[111]
Neural stem cells are
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multipotent stem cells that give rise to different cell types within the neuronal lineage like astrocytes, oligodendrocytes, etc. Studies have reported that they play a regenerative role in
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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 functions. [117, 118] Apart from BM-MSC, mesenchymal stem cells can also be obtained from the
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adipose tissue, fetal tissue, hair follicle tissue and dental pulp. While it was initially established that mesenchymal stem cells could only differentiate to cells of the mesoderm, it is now established that they can differentiate into cell types outside the mesodermal lineage.
[122]
Experimental evidence of neural cell markers being expressed from isolated human mesenchymal stem cells has been reported.
[119]
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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ACCEPTED MANUSCRIPT 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
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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 injuries. Although autografts are the golden standards used, several other alternatives are being
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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 site of injury, the extent to which the tissue has been injured and the personal profile of the
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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 determine the method of treatment. Although synthetic materials are gaining popularity due to
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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. Conductive polymers have also led to a substantial increase in neurite growth and extension. The
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method of fabrication also plays a crucial role in determining the physical properties of the 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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ACCEPTED MANUSCRIPT 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
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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.
8. Acknowledgement: Amit Kumar Jaiswal gratefully acknowledges VIT University, Vellore
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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 other non-financial support from Institute of NeuroImmune Pharmacology (INIP) and Centre for
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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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equally.
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authors have given approval to the final version of the manuscript. §These authors contributed
Conflict of Interest: All authors declare no financial and any other kind of conflict.
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Figure 1. An overview of CNS and PNS Injuries. Types of nerve injuries and their possible 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 (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 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, flexibility, swelling and degradation rate.
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Figure 7. Types of cells used in Neural Tissue Engineering summarized.
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ACCEPTED MANUSCRIPT List of Tables Table 1. Natural biomaterials and cells in neural tissue engineering Natural Materials Disadvantage
Cells used
Collagen
Versatility, low antigenicity, inflammatory and cytotoxic response, biocompatibility, good water uptake capabilities, availability of several isolation methods, ability to tailor mechanical and cross linking properties.
Weak mechanical and structural stability upon uptake of water.
BM-MSCs and Schwann cells.
Hyaluronic acid
Good biocompatibility, high water content, safe degraded products, limited immunogenicity, viscoelastic properties and ability to influence wound healing, metastasis etc.
Non-adherence of cells and water solubility.
HYAFF
Biocompatibility, complete degradability, solubility in DMSO, stable on hydrolysis, strong interaction with polar molecules and ability to promote cell adhesion and proliferation.
High biocompatibility, high biodegradability, non-antigenicity and chelating property.
References [16, 20, 14, 27]
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Advantage
[28, 31, 34, 40, 36, 37, 38]
Release of acidic degradation products, poor processability and loss of mechanical properties very early during degradation.
Schwann cells.
[111, 112, 116. 117, 42]
Unstable mechanical properties and lack of the specific cellrecognition signals.
NSCsc) ad Schwann cells.
[118, 43, 44, 45, 46 ]
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Alginate
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Biocompatibility, biodegradability, nontoxicity, inhibition of growth of fungi, yeast and bacteria and nonimmunogenicity.
Some forms of chitosan may be toxic.
Schwann cells and BMSCd)derived Schwann cells.
[119, 48, 50, 51, 52]
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 PCL
Advantage Biodegradable, biocompatible, possesses high elasticity, low
Disadvantage Cytotoxic effects on using organic solvents.
Cells used Adiposederived multipotent SCs,
References [56, 14, 60, 61, 62, 63]
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toxicity, good mechanical properties and a slow degradation profile.
[14, 65, 66]
Biodegradable, ultrafine continuous fibers, high surfaceto-volume ratio, high porosity, varied distribution of pore size
Poor biocompatibility, release of acidic products on degradation, poor process ability and premature failure of mechanical features during degradation.
MSCs, NSCsc) and Schwann cells.
PLGA
Biodegradability, non-toxicity and film forming ability.
Plastic deformation and failure on exposure to long term strain, releases acidic products on degradation.
NSCs, NPCsd) and Mouse embryonic fibroblasts.
[14, 16, 129, 68]
PHB
Potential neural protective agent, high crystallinity, longer degradation time, and provides support for cell adhesion and proliferation (of osteoblasts, fibroblasts).
Poor biocompatibility.
OECse), MSCs, NSCs and hMSCs.
[70, 121, 126, 123]
PVA
Non-toxic, hydrophilic and high mechanical strength.
Poor biocompatibility.
Dorsal root ganglia.
[72, 124, 125]
PPY
Exhibits rigidity, good biocompatibility and cell adhesion properties, non-toxic, non-allergic, nonmutagenic and nonhaemolytic.
Insoluble, nonbiodegradable and poor process stability.
Schwann cells.
[75, 76, 126, 127]
PANI
Versatility, conductive, good biocompatibility and increased neurite outgrowth.
Inability to degrade and chronic inflammation.
hMSCs and NSCs.
[19, 75, 76, 125, 126]
PEDOT
Versatility, conductive, good biocompatibility and
Inability to degrade and chronic inflammation.
hMSCs and NSCs.
[19, 75, 77, 128]
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hNSCs, hMSCs, Mouse embryonic NSCs and hESCsf).
[129, 130, 131, 78, 79, 80, 81, 82, 83]
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Superior conductivity, remarkable stiffness, high aspect ratio, maintains structural stability of scaffolds, biocompatibility, optimal nanotopography and induces conductivity.
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hMSC – human Mesenchymal stem cells, b)NCSC – Neural crest stem cells, c)NSC – Neural stem cells, d)NPC – 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 Biomaterial
Polyglycolic acid (PGA)
Degradation
Diameter
Length
Characteristics
Nerve Conduits
3 months
2.3 mm
4 cm
Woven, flexible, externally corrugated tube that can bridge nerve gaps between 8 mm and 3 cm.
NeuroMatrixTM Type I Collagen
4-8 months
2-6 mm
2.5 cm
Semi-permeable, clinically effective and tensionless repair without associated morbidity.
NeuraGenTM
36-48 months
1.5-7 mm
2-3 cm
When hydrated, it is an easy to handle, soft, pliable, non-friable,
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Neurotube®
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Type I Collagen
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4-8 months
2-6 mm
2.5 cm
Flexible, fully resorbable and semipermeable and kink resistant up to 60o.
Axoguard® Nerve Connector
Types I, III, IV and VI Collagen (Porcine)
3 months
1.5-7 mm
10 mm
Only porcine submucosal ECM aid for tensionless repair of severed nerve ends with less than a 5 mm gap.
NeuroLac®
Poly(DLlactide Єcaprolactone); PCL
16 months
1.5-10 mm
3 cm
SaluTunnelTM Nerve Protector
Salubria®
Nonbiodegradable
2-10 mm
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NeuroFlexTM
A transparent, synthetic, bioresorbable conduit that allows for efficient nerve stump positioning. Flexible, tubular sheath with longitudinal slit that allows for easy placement at site of injury and provides a protective environment.
2, 5 and 10 mm
6.35 cm
Flexible, tubular sheath that provides a protective environment.
3 months
2, 5 and 7 mm
5 cm
A biocompatible nerve cuff with sufficient tensile strength, suture retention strength and ability to withstand compressive forces.
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Nerve Cuffs
Axoguard® Nerve Protector
Types I, III, IV and VI Collagen (Porcine)
Nonbiodegradable
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Salubria® (Polyvinyl alcohol, PVA and waterbased)
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SaluBridgeTM
NeuraWrapTM
Type I Collagen
36-48 months
3-10 mm
2-4 cm
An absorbable collagen implant with longitudinal slit that allows for easy placement and protects the neural environment.
NeuroMendTM
Type I Collagen
4-8 months
4-12 mm
2.5-5 cm
Resorbable collagen matrix with longitudinal slit. When hydrated, it is an easy
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Fig. Biomaterials and cells used in neural tissue engineering
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ACCEPTED MANUSCRIPT HIGHLIGHTS
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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 injuries.
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Prerana Sensharma, G. Madhumathi, Rahul D. Jayant, Amit K. Jaiswal PII: DOI: Reference:
S0928-4931(16)32866-1 doi: 10.1016/j.msec.2017.03.264 MSC 7776
To appear in:
Materials Science & Engineering C
Received date: Accepted date:
29 December 2016 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 for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.03.264
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ACCEPTED MANUSCRIPT
Biomaterials and Cells for Neural Tissue Engineering: Current Choices
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Prerana Sensharma1, §, Madhumathi G.1, §, Rahul D. Jayant3 and Amit K. Jaiswal2*
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School of Biosciences and Technology, VIT University, Vellore, 632014, Tamilnadu, India Centre for Biomaterials, Cellular and Molecular Theranostics, VIT University, Vellore, 632014, Tamilnadu, India 3 Center for Personalized Nanomedicine, Institute of Neuro-Immune Pharmacology, Department of Immunology, Herbert Wertheim College of Medicine, Florida International University (FIU), Miami, FL-33199, USA
*
Corresponding Author
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Amit K Jaiswal, Ph.D. Associate Professor Email: [email protected] Phone No (O): +91-9789280874
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Authors have equal contribution
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§
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ACCEPTED MANUSCRIPT 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
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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 scaffold. Along with these, cells and growth factors are also being incorporated to increase the
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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 extracellular matrix in our body, and to elicit a combination of biochemical, topographical and
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electrical cues via various polymers, cells and growth factors, using which nerve regeneration
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can efficiently occur.
Keywords: Nerve tissue engineering, Synthetic and natural polymers, Biomaterials, Hydrogels,
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Nanofibers.
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ACCEPTED MANUSCRIPT 1. Introduction Nerve injuries affect millions of people worldwide.
[1]
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
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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 mechanisms such as thermal, electric shock, radiation, percussion, and vibration. Injured nerves
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of the peripheral nervous system (PNS) have the ability to regenerate. However, spontaneous
functional outcome.
[3]
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regeneration of the nerve, in the absence of any therapeutic intervention does not result in a good Despite early diagnosis surgical intervention does not result in the
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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 have high standards, but they still have limitations as the nerves are short aged, supply is limited.
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They also present the possibility of causing morbidity, neuroma formation at site of harvest, [4]
Allogenic grafts could also be used but they would produce an
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scarring and sensory loss.
immune response which will disable the cells that cure the injury. [5] The central nervous system
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(CNS) consists of the brain, spinal cord and retina. It is limited in terms of its spontaneous 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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ACCEPTED MANUSCRIPT rather than facilitating further regeneration.
[2]
Spinal cord injuries (SCI) occur majorly in traffic
accidents and due to elderly patients falling. They result in paraplegia and quadriplegia, which cannot be effectively treated so far.
[7]
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,
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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 cell transplantation has an inhospitable environment. Furthermore, limited drugs and biologics
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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 treatment of nerve injuries. Tissue engineering centric approaches will enable regeneration,
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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, molecule delivery and biomaterial scaffold constructions are considered the greatest hope for
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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 repaired to favor cell penetration and tissue regeneration in three dimensions. Tissue engineering
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comprises three main components namely, the biomaterial used, the cells and the biomechanical 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 increasing application in tissue engineering.
[9]
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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ACCEPTED MANUSCRIPT 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
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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 commercially available, FDA-approved nerve conduit. These conduits have been reported
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effective for regeneration of the nerve. In the United States alone they result in the expenditure
performed in the United States annually.
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of 150 billion dollars in the annual healthcare dollar. Over 200,000 repair procedures are [10]
Thus, neural tissue engineering offers potentially
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great solutions on the healthcare front, as well as great economic prospects in the market.
2. Ideal properties of a scaffold
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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 the physical, chemical and mechanical properties of that tissue (Figure 4). The following
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properties given below depend on one key parameter, which is the choice of biomaterial for 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 functionality of cells and migration and proliferation of cells on the scaffold.
[14]
Surface
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ACCEPTED MANUSCRIPT 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
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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
density.
[15]
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surface. Cell adhesion property depends upon surface properties like wettability and charge Along with biocompatibility, toxicity profiles also play a crucial role in cell
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adhesion, growth and proliferation on the scaffold. [16]
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2.2 Biodegradable
One of the major advantages of synthesizing biodegradable scaffolds is that they eliminate the
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In case of neural tissue engineering, controlled biodegradable scaffolds are preferred as
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body.
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need for surgical removal of the scaffold and they are absorbed by the surrounding tissues in the
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the scaffold is meant to support the growth of nerve cells and then be degraded by the body as subsequent repair takes place. A biodegradable scaffold will also facilitate the neighboring cells
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to produce their own extracellular matrix (ECM). It should be taken care of that the by-products of biodegradation should be non-toxic as well and that they are easily dispose-off.
[14]
The
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cytotoxic effects could also enable neuroma formation. Biodegradability should favor the 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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ACCEPTED MANUSCRIPT Ideally, 90 % porosity and pore size of 100µm-500µm is standard for yielding good results.
[16]
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
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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]
2.4 Mechanical properties
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The scaffold should have mechanical properties identical to that of the tissue at the implantation site. As this is not always feasible, materials with mechanical properties that can protect the cells
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from compressive or tensile forces without disturbing the biomechanical cues are used.
[16]
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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 modulus. These parameters have been found to increase with subsequent decrease in the
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diameter of the fiber. Although the explanation behind this phenomenon has not been clearly elucidated yet, it has been postulated that there is an increase in macromolecular chain alignment
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as the fiber diameter is decreased, resulting in a higher crystallinity of the nanofiber. Such unique 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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ACCEPTED MANUSCRIPT 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,
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migration and viability. [18] The mimicking of ECM will aid the surrounding cells to secrete their
[16]
Electrical stimulation has been found to be especially successful in the
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and morphogenesis.
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own ECM, which binds the cells to tissues and thus elicits signals that facilitate cell development
case of neural tissue engineering. Neuronal repair is highly unique due to the complexity of the
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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 mechanical support to the tissues, facilitates migration of cells, holds the cells together,
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facilitates communication in cells so that daily cellular activities and wound healing can be performed uninterrupted.
[17, 20]
Natural polymers has advantage as scaffold design as they
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closely mimic the macromolecules that cells interact with in vivo. The frequently used natural 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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ACCEPTED MANUSCRIPT 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 is an eligible biomaterial for scaffolds pertaining to almost all types of
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collagen.
[22, 23, 24]
The versatility of collagen is
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heart valves and other cardiovascular diseases, and so on.
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tissues—it is used in bone tissue engineering, for skin, cornea potentially for the development of
attributed to the widespread distribution of collagen in the body. Some other advantageous
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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 from various sources and the ability to tailor mechanical and cross linking properties as per need. Collagen 1 is suitable for implantation within the body because there are very few people
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[20, 25]
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who have humoral immunity against it. Moreover, a simple serological test can determine whether the use of this biomaterial will elicit an allergic response in the patient.
[22]
A self-
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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 was reported to show neurite extension from a dissociated dorsal root ganglia, and showed a
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greater regeneration over all. [26] Collagen-based scaffolds are fabricated through electrospinning, 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 properties to the scaffold.
[22, 25]
For instance, to increase the strength of a collagen based,
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ACCEPTED MANUSCRIPT 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 chances of cytotoxity and immune response using this.
[20]
Studies have reported that greater
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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 enhance the mechanical strength of the scaffold. In one study, Poly (L-lactic) co-poly (3-
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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 differentiated MSCs on the PLCL/Coll scaffold had neuronal morphology with multipolar
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elongations and expressed neurofilament and nestin proteins, which confirm neuronal induction.
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[27]
Hyaluronic
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3.2 Hyaluronic acid (And its derivatives) acid
(HA,
also
Hyaluranon)
is
a
linear,
non-branched
non-sulfated
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glycosaminoglycan (GAG) and is composed of repeating disaccharides (β-1, 4-D-glucuronic acid (known as uronic acid) and β-1, 3-N -acetyl- -glucosamide). It has sites for cell adhesion and is [28]
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non-immunogenic.
Hyaluronic acid has found many applications in tissue engineering,
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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ACCEPTED MANUSCRIPT arthritis, several liver pathologies as well as early organ rejection.
[30,31]
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
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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 cellular processes. Thus, it is a plausible option for influencing [28, 31, 33]
HA is especially advantageous for
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processes like wound healing, metastasis, etc.
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designing scaffolds for neural tissue regeneration due to its high abundance in the neural system (especially the Central Nervous System), making it an extremely biocompatible choice.
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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 porous structure, with interconnected pores that allow for the transportation of nutrition,
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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 around the scar area. A dominant disadvantage associated with the use of HA is that cells do not
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adhere to its surface. Thus, to overcome this, it is blended with other biomaterials which will 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 promoted differentiation of Neural Stem Cells (NSCs) for neural regeneration in vitro.
[35]
Another disadvantage of HA is its water solubility, which makes it necessary to add a crosslinking agent to convert it into injectable form.
[36]
In one study, HA hydrogel that could be
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ACCEPTED MANUSCRIPT 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]
In another study, HA strands were cross linked using glutaraldehyde and coated
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hydrogel.
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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 graft. [38]
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3.3 HYAFF
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HYAFF is an HA derivative obtained by esterification of HA with benzyl ester. Some favorable properties of HYAFF are its biocompatibility, complete degradability, solubility in DMSO,
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stable on hydrolysis, strong interaction with polar molecules and its ability to promote cell [39]
Moreover, HYAFF can be used to design
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adhesion and proliferation of various cell types.
films, non-woven fabrics, gauzes, sponges, tubes and microsphere, thus broadening its
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applications. It is used for tissue repair, controlled drug release, nerve regeneration, delivery of [40]
Experiments have reported HYAFF based tube scaffolds
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growth factors, wound healing, etc.
are good substrates for nerve cell cultures and explants. It was reported that cells from rat sciatic
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nerves showed adhesion to the scaffold, followed by proliferation and colonization of the scaffold. Its properties are ideal for Peripheral Nervous System Tissue Regeneration.
[41]
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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ACCEPTED MANUSCRIPT 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,
43]
The advantages of alginate as a scaffold are its high biocompatibility, high
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[36,
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alginate hydrogels are formed by making them interact with divalent cations in aqueous solution.
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biodegradability, non-antigenicity and chelating property. The several types of scaffold that can be formed using alginate are summarized in a review by Sun & Tan et al. A study was conducted
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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 the repair of injured nervous tissue, where the repair is facilitated by the secretion of
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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 as a bioresourceable conduit supplemented with Schwann cells to replace nerve grafts. Enhanced
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neurite growth, viability and proliferation were observed.
[45]
Alginate conduits seeded with
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Schwann cells have also been fabricated along with the incorporation of fibronectin. Results indicated that regeneration rate, viability of the cells and axonal growth were enhanced using this
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conduit. [46]
3.5 Chitosan
Chitin is naturally found in the cell walls of crustaceans such as insects, crabs, shrimps as well as the cell wall of bacilli and fungi.
[25, 47]
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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ACCEPTED MANUSCRIPT 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
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of several fungi, yeast and bacteria and non-immunogenicity. Due to the absence of any protein
[49]
Chitin incorporated with other
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proved to be a good choice for neural tissue regeneration.
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or lipid in chitosan structure, no antibody can be developed against it. [48] Chitosan has also been
materials show an affinity for nerve cells. Chitin based nerve chambers can be used for effective
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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, growth and migration of Schwann Cells. This is very important because Schwann cells release
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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. Moreover, it has the mechanical strength necessary for effective neural regeneration. Chitosan
Chitosan scaffold pre-seeded with Schwann cells was synthesized as a bio-artificial nerve
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[50]
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conduits resulted in the sprouting of myelinated axons and successful recovery of nerve function.
graft for peripheral nerve regeneration. Good biocompatibility of the Schwann cells and chitosan
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fibers was observed. [51] In another study, chitosan conduit seeded with bone marrow stromal cell (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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ACCEPTED MANUSCRIPT 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]
To overcome the disadvantages posed by these grafts, synthetic
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of immune rejection.
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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 polymers, polymeric scaffolds are proving to very efficient scaffold materials.
[53, 54]
Polymeric
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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 mechanical properties can be easily engineered in these polymeric scaffolds, suitable to the
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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 the graft, leading to immune rejection of the graft. This property can also be easily tailored in
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synthetic polymers. Some widely used synthetic polymers for the fabrication of scaffolds include
sebacate,
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Poly -L-lactic acid (PLLA), Polycaprolactone (PCL), Polyhydroxybutyrate (PHB), Polyglycerol Poly-D,L-lactide-co-glycolic
acid
(PLGA),
Poly-L-lactate
(PLA),
Poly-3-
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hydroxybutyrate (PHB), Polyamide, Polydioxanone, Poly-e-caprolactone-co-ethyl ethylene phosphate (PCLEEP), Poly-D.L-lactide-co-caprolactone (PDLLA), Polyvinyl alcohol (PVA), Poly acrylonitrile-co-methylacrylate (PAN-MA) and copolymer of methyl methacrylate (MMA) and acrylic acid (AA) (PMMAAA).
[55]
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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ACCEPTED MANUSCRIPT 4.1 Polycaprolactone (PCL) PCL is a biodegradable and biocompatible polymer, which is cost efficient, possesses high elasticity, low toxicity, good mechanical properties and a slow degradation profile.
[56]
It has
been widely applied in tissue engineering including bone and neural tissue engineering. PCL
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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 free method, template synthesis was used where PCL fibers were synthesized using Alumina
PCL has been used in conjugation with natural polymers or coated with cells to improve its
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[54]
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nanoporous membrane. NaOH was then used to dissolve the template and yield PCL nanowires.
biocompatibility and cell adhesion properties. This will confer mechanical strength and
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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 allow the regeneration of nerve fibers in a contact-oriented fashion which increases the growth of [58]
In one study, PCL fibers have been extruded and embedded in poly-2-
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the fibers.
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hydroxyethyl methacrylate (HEMA) gel. The sonication of the PCL/HEMA composite was dissolved in acetone solvent leaching out PCL and leaving behind HEMA gel with oriented
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longitudinal channels embedded in it. The channel diameter can be controlled by altering the 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 (multipotent) stem cells to improve the effects on nerve regeneration.
[60]
A PCL scaffold seeded
with human mesenchymal stem cells was shown to induce adipogenic, chondrogenic and
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ACCEPTED MANUSCRIPT osteogenic lineages.
[16]
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
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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 [61]
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differentiated into Schwann cells and then integrated into the myelin sheath around axons.
PCL has also been used directly in conjugation with Schwann cells. Aligned PCL fibers
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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 nanofibrous PCL/gelatin scaffolds fabricated by electrospinning were seeded with Schwann cells
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to assist the direction of growth of regenerating axons in peripheral nerves. [63]
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4.2 Poly-L-lactic acid (PLLA)
PLLA is a biodegradable and biocompatible synthetic polymer. Nano-structured PLLA scaffolds
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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, varied distribution of pore size from 50-350 nm. It contains ester linkages in the polymer
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backbone which lead to bio-functionalization of the polymer with various biomolecules by 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 differentiation into different neurogenic lineages by culturing in differential media.
[14]
It has
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ACCEPTED MANUSCRIPT 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 aligned fibers yielded the same results with respect to NSC differentiation.
[64]
3-D PLLA nano-
structured porous scaffolds have been fabricated using liquid-liquid phase separation methods.
[65]
Several studies have proven that PLLA scaffolds
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differentiation and neurite outgrowth.
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The fabricated scaffold seeded with NSCs showed significant improvement in NSC
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efficiently support NSC differentiation and neurite outgrowth, thereby making them suitable to applications in neural tissue engineering. PLLA conduits incorporated with allogenic Schwann
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cells were studied to venture into the synthesis of bioactive nerve conduits which can potentially [66]
A heparin/collagen
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replace autologous nerve grafts and peripheral nerve regeneration.
encapsulating nerve growth factor multilayer was coated upon a PLLA scaffold using layer-by-
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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 superior benefits to Schwann cell proliferation and its cytoskeleton, PC12 differentiation and
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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) PLGA is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA). It is amorphous
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biodegradable polyester and is widely used for fabrication of scaffolds in neural tissue 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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ACCEPTED MANUSCRIPT 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
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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 pharmaceutical, medical engineering and various other industries. In one study, PLGA
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nanofibers were generated using electrospinning technique and NSCs were seeded onto the
outgrowth.
[14]
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scaffold. Results indicated high efficiency of cell adhesion, nerve cell differentiation and neurite In another study, the PLGA scaffold was conjugated with PEG (Polyethylene
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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 (SCI). The scaffold induced continuity of the spinal cord and led to a significant reduction in
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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 been shown to protect some important axonal cytoskeleton proteins to promote repair in SCI.
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Thus, the composite scaffold exhibited greater iNSC adhesion, cell growth and proliferation.
[68]
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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ACCEPTED MANUSCRIPT 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 muscle, cardiac muscle and Schwann cells.
[16]
In one study, non-linear elastic biomaterial
scaffold was fabricated using a PGS/PLLA composite. The fabrication was done using core and
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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)
PHB is biodegradable aliphatic polyester synthesized by several bacteria using renewable carbon
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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 as a biomaterial and is FDA approved. A study reported that PHB is a potential neural protective
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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-3hydroxy butyrate-co-3-hydroxyvalate (PHBV) scaffold has been found to provide better support
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for adhesion and proliferation of osteoblasts and fibroblasts. In one study, PHB films were 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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ACCEPTED MANUSCRIPT 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
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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 natural polymers to enhance the overall physiochemical and biological properties of the
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fabricated scaffold. In one study, PVA fibres were blended with chitosan and the scaffold was fabricated using electrospinning. The results generated showed that the scaffold had balanced
4.7 Conductive polymers
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properties apt for neural cell regeneration. [72]
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Neural tissue engineering requires simultaneous biochemical, topographic and electrical cues for complete and efficient adhesion, differentiation and proliferation of cells. To enhance
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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 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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ACCEPTED MANUSCRIPT 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
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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 tissue engineering, but this could be overcoming by making composites of PPY.
[73]
Hence, it is
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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 biocompatibility and cell adhesion properties. It is also non-toxic, non-allergic, non-mutagenic
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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 the scaffold induced better neuronal growth and differentiation than PLGA scaffolds without [14]
In another study, PPY were polymerized on electrospun PLA and PCL
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PPY coating.
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templates generating conductive core-sheath nanofibers. The results indicated that PPY deposited on PCL was much smoother than that of the PLA fibers. This occurred due to the
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presence of a methyl group on PLA side chain, making it less hydrophilic in nature than PCL. 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 cell adhesion and growth.
[73]
In one study, the effect of electrical stimulation on Schwann cells
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ACCEPTED MANUSCRIPT 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
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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) PANI and PEDOT are versatile conducting polymers. In one study, it was shown that
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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 polymer with a biological matrix facilitates easier fabrication of conductive scaffolds. In another
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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 synthesized and then added to a solution of collagen, after which the cell suspensions were made.
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The 3-D conductive collagen gels exhibited good cyto-compatibilty and increased neurite [75]
An electrospun conductive
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outgrowth as compared to non-conductive collagen gels.
nanofiber scaffold was fabricated using PANI with PCL/Gelatin. This scaffold was then seeded
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with nerve stem cells and electrical stimulation was applied. Results indicated enhanced 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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ACCEPTED MANUSCRIPT 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 like replacing carbon atoms from tube wall using boron or nitrogen.
[14]
They exhibit
characteristic qualities like superior conductivity, remarkable stiffness and high aspect ratio.
[78]
One study demonstrated that
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structural stability of scaffolds when incorporated.
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They also have an innate ability to absorb strain and induce conductivity. It maintains the
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polyethyleneimine (PEI) deposited with multi-walled CNTs (MWCNTs) exhibited increased neurite growth and elongation in all directions. [14] Another study used carbon nanotube fibers
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(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 indicated that it generated directed nerve growth and overall increased differentiation and [76]
CNT network patterns have been used for selective growth and structural
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proliferation.
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polarization-controlled neuronal differentiation of human NSCs into neurons. The CNT patterns were found to provide synergistic cues, optimal nanotopography and biocompatibility for
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differentiation of hNSCs in physiological solution. Thus, CNTs have been proven to be a highly [79]
CNT networks on solid substrates have also
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suitable scaffold for controlling hNSC growth.
been used in the directed growth and differentiation of hMSCs. It has been reported that the
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hMSCs could recognize the arrangement of individual CNTs in the CNT network, which allowed directional growth of hMSCs following the direction of alignment of the CNTs. These hMSCs were found to exhibit enhanced proliferation and osteogenic differentiation.
[80]
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 elongation and enhanced differentiation of NSCs into mature neuronal cells.
[81]
Successful
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ACCEPTED MANUSCRIPT 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
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outgrowth and expression of neural markers were found to be similar as cells differentiated on
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poly-L-ornithine (PLO), which is a common growth substratum for neural stem cells.
[82]
hESCs
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have also been seeded on CNTs and proved to be successful candidates for neuronal differentiation. A poly (acrylic acid) grafted CNT thin film was fabricated and compared with
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PLO surfaces. This scaffold exhibited enhanced neuron differentiation and cell adhesion. [83]
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5. Nerve conduits
Nerve injuries can be treated using two approaches- designing a graft or a conduit. In a nerve
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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 5). This is a preferable alternative to nerve suturing, grafts, etc. because it does not elicit immune
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responses. Also, the nerve functionality recovery is not very good in any of these techniques. [84] Nerve conduits allow for guided nerve regeneration, and further prevent aberrant regeneration of the nerve. It is applicable for nerve injuries over short distances (4 cm).
[85]
They mimic a
microenvironment favorable for the regeneration of axon, with the incorporation of cells, ECM, neurotrophic factors as required.
[84, 85. 86]
They also enrich the neurotropic factors within the
ECM, thus providing a suitable micro environment for nerve regeneration.
[84]
Nerve conduits
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ACCEPTED MANUSCRIPT 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.
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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) and bi-component (BC) fibrous conduits were designed using PCL and gelatin solutions. Gelatin
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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 neuronal growth and differentiation as compared to MC conduits. [89]
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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 determines the degree of nutrient exchange between the external environment and the lumen. It
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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 scarring or hindrance to the process. The process of vascularization needs to be preceded by
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oxygen and nutrient delivery to the conduit lumen. Also, permeability is essential for the determination of any support cell’s viability.
[84, 91]
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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ACCEPTED MANUSCRIPT fibers, addition of salt and sugar crystals that can later be leached, injection molding solvent evaporation, gas foaming and phase separation.
[84, 93]
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,
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where the proximal and distal end of the stump are not planar in nature) are damaged, as well as
[91]
However, it must be noted that flexibility
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the growing tissues as well as the surroundings.
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the axon undergoing regeneration. [84, 90] A scaffold which is too rigid can cause compression of
must not compromise upon the mechanical strength of the conduit. Lesser mechanical strength
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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 nerve conduit can cause hindrance for the growing axon, compressing it as well as the
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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 copolymer ratio (which in turn influences the degradation profile as well.)
[53, 84, 90]
An optimal
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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 from the proximal to the distal stump. Then, it should start degrading. However, too rapid
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degradation causes inflammation and swelling. When it is too slow, there is compression. [84, 90] 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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ACCEPTED MANUSCRIPT 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
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regeneration. The membrane was coated with poly-L-lysine (PLL) to enhance cell attachment
[94]
A fully biodegradable conduit for peripheral nerve repair has also been
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regeneration.
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and growth on the membrane. The conduit proved to be extremely efficient for enhancing nerve
fabricated using synthetic polymers. A PLGA foam conduit with longitudinally aligned was
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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 regeneration and precisely guided neural regeneration was observed.
[95]
A biological chitin
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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 spinal cord hemisection injury, and created a local microenvironment that promoted the growth,
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migration and differentiation of the BMSCs. Furthermore, the conduit promoted axon growth
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and nerve regeneration, effectively reducing scar invasion. [96] Veins are also used as conduits materials, and are a good option for tubulization process
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due to their easy availability, biodegradability, inert and non-compressing nature. Tubulization is 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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ACCEPTED MANUSCRIPT 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 epineurium, more axonal regeneration, less inflammatory cells.
[98]
Bio-3D printing technology
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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 further by virtue of the several advantages it offers: complete control over the desired shape, size
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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 muscle, myocardium. [99,100]
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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 Schwann cells using a poly(lactic-co-glycolic acid) nerve conduit for regeneration of sciatic
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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 factor, platelet derived growth factor, transforming growth factor-β, vascular endothelial growth
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factor, brain-derived neurotrophic factor, nerve growth factor).It has shown immense potential as 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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ACCEPTED MANUSCRIPT 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
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synthesized from synthetic as well as natural biomaterials. The commercially available products
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have been enlisted in Table 3.
6. Cells Used for Neural Tissue Engineering
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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 then re-implanted at the site of injury/ regeneration in the patient. It is an ideal choice from an
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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 potentially require the resection of a large part of the patient’s skins, thereby causing pain and
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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 immunogenicity of the allogenic cells. This has found limited success. However, one of the
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mostly widely used allogenic cell-based grafts is Apligraft™. The third usable cell source is 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 been conducted in the past.
[103]
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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ACCEPTED MANUSCRIPT 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
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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
injury takes place and secretes N-cadherin, neurotrophins, gamma integrin’s, Neural Cell
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Adhesion molecules (N-CAM) as well as collagen and lamin in for ECM production.
[105]
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Xenogeneic sources are used to provide acellular nerve grafts. These keep the ECM intact and elicit low immune response. These can be cultured with Schwann cells, which is a very effective
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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] Experimental data provided by Rodrigues et al shows that the immune response caused by the
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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] 6.2 Bone marrow derived Mesenchymal stem cells
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They are stromal cells which have the potential to differentiate into different cell lineages and 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
31
ACCEPTED MANUSCRIPT 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
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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, forskolin, recombinant human bFGF, and recombinant human platelet derived growth factor-AA,
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and recombinant human heregulin β-1 causes differentiation of Stem Cells to SC-like phenotype. Some features essential to use of these stem cells for clinical purposes are ease of accessibility,
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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 myelination. The presence of ECM proteins like collagen I, collagen IV, laminin, fibronectin and
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neurite guidance proteins further reinforce regeneration. Moreover, the expression of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), glial cell line- derived
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neurotrophic factor (GDNF), ciliary neurotrophic factor and neurotrophin-3 (NT-3) has been 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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ACCEPTED MANUSCRIPT 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]
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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 are potentially tumorigenic, immunogenic and their use could spark several ethical controversies. The application of Embryonic stem cell based therapies for several neurodegenerative
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[111]
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diseases like Parkinson’s disease, Alzheimer’s disease, as well as injuries have been reviewed in detail. [110] However, it has been reported that their application to neural tissue regeneration is
M
limited, and mesenchymal precursors have therefore been derived.
[111]
Neural stem cells are
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multipotent stem cells that give rise to different cell types within the neuronal lineage like astrocytes, oligodendrocytes, etc. Studies have reported that they play a regenerative role in
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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 functions. [117, 118] Apart from BM-MSC, mesenchymal stem cells can also be obtained from the
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adipose tissue, fetal tissue, hair follicle tissue and dental pulp. While it was initially established that mesenchymal stem cells could only differentiate to cells of the mesoderm, it is now established that they can differentiate into cell types outside the mesodermal lineage.
[122]
Experimental evidence of neural cell markers being expressed from isolated human mesenchymal stem cells has been reported.
[119]
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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7. Conclusion
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We have discussed the various approaches that are currently being used to treat peripheral nerve injuries. Although autografts are the golden standards used, several other alternatives are being
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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 site of injury, the extent to which the tissue has been injured and the personal profile of the
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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 determine the method of treatment. Although synthetic materials are gaining popularity due to
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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. Conductive polymers have also led to a substantial increase in neurite growth and extension. The
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method of fabrication also plays a crucial role in determining the physical properties of the 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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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.
8. Acknowledgement: Amit Kumar Jaiswal gratefully acknowledges VIT University, Vellore
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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 other non-financial support from Institute of NeuroImmune Pharmacology (INIP) and Centre for
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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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equally.
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authors have given approval to the final version of the manuscript. §These authors contributed
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 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 (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 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, flexibility, swelling and degradation rate.
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Figure 7. Types of cells used in Neural Tissue Engineering summarized.
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ACCEPTED MANUSCRIPT List of Tables Table 1. Natural biomaterials and cells in neural tissue engineering Natural Materials Disadvantage
Cells used
Collagen
Versatility, low antigenicity, inflammatory and cytotoxic response, biocompatibility, good water uptake capabilities, availability of several isolation methods, ability to tailor mechanical and cross linking properties.
Weak mechanical and structural stability upon uptake of water.
BM-MSCs and Schwann cells.
Hyaluronic acid
Good biocompatibility, high water content, safe degraded products, limited immunogenicity, viscoelastic properties and ability to influence wound healing, metastasis etc.
Non-adherence of cells and water solubility.
HYAFF
Biocompatibility, complete degradability, solubility in DMSO, stable on hydrolysis, strong interaction with polar molecules and ability to promote cell adhesion and proliferation.
High biocompatibility, high biodegradability, non-antigenicity and chelating property.
References [16, 20, 14, 27]
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Advantage
[28, 31, 34, 40, 36, 37, 38]
Release of acidic degradation products, poor processability and loss of mechanical properties very early during degradation.
Schwann cells.
[111, 112, 116. 117, 42]
Unstable mechanical properties and lack of the specific cellrecognition signals.
NSCsc) ad Schwann cells.
[118, 43, 44, 45, 46 ]
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Alginate
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Biocompatibility, biodegradability, nontoxicity, inhibition of growth of fungi, yeast and bacteria and nonimmunogenicity.
Some forms of chitosan may be toxic.
Schwann cells and BMSCd)derived Schwann cells.
[119, 48, 50, 51, 52]
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 PCL
Advantage Biodegradable, biocompatible, possesses high elasticity, low
Disadvantage Cytotoxic effects on using organic solvents.
Cells used Adiposederived multipotent SCs,
References [56, 14, 60, 61, 62, 63]
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toxicity, good mechanical properties and a slow degradation profile.
[14, 65, 66]
Biodegradable, ultrafine continuous fibers, high surfaceto-volume ratio, high porosity, varied distribution of pore size
Poor biocompatibility, release of acidic products on degradation, poor process ability and premature failure of mechanical features during degradation.
MSCs, NSCsc) and Schwann cells.
PLGA
Biodegradability, non-toxicity and film forming ability.
Plastic deformation and failure on exposure to long term strain, releases acidic products on degradation.
NSCs, NPCsd) and Mouse embryonic fibroblasts.
[14, 16, 129, 68]
PHB
Potential neural protective agent, high crystallinity, longer degradation time, and provides support for cell adhesion and proliferation (of osteoblasts, fibroblasts).
Poor biocompatibility.
OECse), MSCs, NSCs and hMSCs.
[70, 121, 126, 123]
PVA
Non-toxic, hydrophilic and high mechanical strength.
Poor biocompatibility.
Dorsal root ganglia.
[72, 124, 125]
PPY
Exhibits rigidity, good biocompatibility and cell adhesion properties, non-toxic, non-allergic, nonmutagenic and nonhaemolytic.
Insoluble, nonbiodegradable and poor process stability.
Schwann cells.
[75, 76, 126, 127]
PANI
Versatility, conductive, good biocompatibility and increased neurite outgrowth.
Inability to degrade and chronic inflammation.
hMSCs and NSCs.
[19, 75, 76, 125, 126]
PEDOT
Versatility, conductive, good biocompatibility and
Inability to degrade and chronic inflammation.
hMSCs and NSCs.
[19, 75, 77, 128]
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PLLA
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hNSCs, hMSCs, Mouse embryonic NSCs and hESCsf).
[129, 130, 131, 78, 79, 80, 81, 82, 83]
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Superior conductivity, remarkable stiffness, high aspect ratio, maintains structural stability of scaffolds, biocompatibility, optimal nanotopography and induces conductivity.
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CNTs
hMSC – human Mesenchymal stem cells, b)NCSC – Neural crest stem cells, c)NSC – Neural stem cells, d)NPC – 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 Biomaterial
Polyglycolic acid (PGA)
Degradation
Diameter
Length
Characteristics
Nerve Conduits
3 months
2.3 mm
4 cm
Woven, flexible, externally corrugated tube that can bridge nerve gaps between 8 mm and 3 cm.
NeuroMatrixTM Type I Collagen
4-8 months
2-6 mm
2.5 cm
Semi-permeable, clinically effective and tensionless repair without associated morbidity.
NeuraGenTM
36-48 months
1.5-7 mm
2-3 cm
When hydrated, it is an easy to handle, soft, pliable, non-friable,
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Neurotube®
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Product
Type I Collagen
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4-8 months
2-6 mm
2.5 cm
Flexible, fully resorbable and semipermeable and kink resistant up to 60o.
Axoguard® Nerve Connector
Types I, III, IV and VI Collagen (Porcine)
3 months
1.5-7 mm
10 mm
Only porcine submucosal ECM aid for tensionless repair of severed nerve ends with less than a 5 mm gap.
NeuroLac®
Poly(DLlactide Єcaprolactone); PCL
16 months
1.5-10 mm
3 cm
SaluTunnelTM Nerve Protector
Salubria®
Nonbiodegradable
2-10 mm
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NeuroFlexTM
A transparent, synthetic, bioresorbable conduit that allows for efficient nerve stump positioning. Flexible, tubular sheath with longitudinal slit that allows for easy placement at site of injury and provides a protective environment.
2, 5 and 10 mm
6.35 cm
Flexible, tubular sheath that provides a protective environment.
3 months
2, 5 and 7 mm
5 cm
A biocompatible nerve cuff with sufficient tensile strength, suture retention strength and ability to withstand compressive forces.
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6.35 cm
Nerve Cuffs
Axoguard® Nerve Protector
Types I, III, IV and VI Collagen (Porcine)
Nonbiodegradable
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Salubria® (Polyvinyl alcohol, PVA and waterbased)
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SaluBridgeTM
NeuraWrapTM
Type I Collagen
36-48 months
3-10 mm
2-4 cm
An absorbable collagen implant with longitudinal slit that allows for easy placement and protects the neural environment.
NeuroMendTM
Type I Collagen
4-8 months
4-12 mm
2.5-5 cm
Resorbable collagen 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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Fig. Biomaterials and cells used in neural tissue engineering
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ACCEPTED MANUSCRIPT HIGHLIGHTS
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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 injuries.
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