Biomaterials - Aligned electrospun nanofibers for peripheral nerve regeneration

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SURGGN05

BIOMATERIAL

Electrospun  Aligned    Nanofibers    for    

Peripheral  Nerve    Regeneration:    A    Review    

Ramanan  Bhashyam,    Yiangos    Psaras,    Cody    Scruggs,    Thanos    

Sofroniou  

    

    

Abstract     

Despite advances in the field of peripheral nerve regeneration, complete functional nerve repair still eludes our capabilities. Efficient regeneration and recovery not only presents a great clinical challenge due to the complexity of neural tissue, but also due to the error-prone mechanism of natural regeneration. With the current gold standard of autologous nerve grafting being limited by factors such as surgical expertise, donor tissue shortage and donor site morbidity, tissue engineering approaches are emerging as an attractive alternative. Electrospinning of aligned nanofibers has enjoyed being at the forefront of approaches, given the controllable architecture of the fibers and capability to mimic the physiological environment of the extracellular matrix. This approach is heavily dependent on the materials and parameters employed.

Although mechanically superior, synthetic polymers are flawed in their surface properties, while the reverse is true for naturally derived polymers. Additionally, alignment of nanofibers has shown favorable results towards functional regeneration of nerve tissue. This review will critically address the advantages and drawbacks of electrospun nanofibers; the clinical need, manufacturing process and currently used materials will be presented and the effect of fiber alignment will be evaluated using recent approaches.  

Electrospun  Aligned    Nanofibers    for    Peripheral    Nerve    Regeneration:    A    Review    

Table    of    Contents    

1.  Introduction (Ramanan Bhashyam)  .....................................................................................    3    

1.1 Peripheral nerve injury    ...................................................................................................................    3    

1.2 Structure of peripheral nerves  .......................................................................................................    4    

1.3 Biomechanics  ..................................................................................................................................    5    

1.4 Damage and repair of the peripheral nerve    ..................................................................................    7    

1.5 Tissue engineering and biomaterials for Peripheral Nerve regeneration  .................................    8    

2. Electrospinning (Yiangos Psaras)  .........................................................................................    9    

2.1 The electrospinning process  ..........................................................................................................    9    

2.2 Advantages and Disadvantages    ..................................................................................................    10    

2.3 Co-axial electrospinning  .............................................................................................................    11    

3. Electrospun nanofibers and material selection (Cody Scruggs)  .....................................    13    

3.1 Synthetic Polymers  ......................................................................................................................    13    

3.2 Natural Polymers (Yiangos Psaras)  ...........................................................................................    17    

3.3 Composite Polymers (Yiangos Psaras)  .....................................................................................    17    

3.3.1 Collagen    ..................................................................................................................................................    18    

3.3.2 Silk Fibroin    ............................................................................................................................................    19    

3.3.3 Chitosan    ..................................................................................................................................................    19    

3.3.4 Laminin    ...................................................................................................................................................    20    

4. Effects of aligned nanofibers on cell-scaffold interactions (Thanos Sofroniou)    ..........    22    

4.1 In vitro studies    ..............................................................................................................................    22    

4.2 In vivo studies    ...............................................................................................................................    25    

5. Conclusion (Ramanan Bhashyam)    .....................................................................................    29    

References  .................................................................................................................................    30    

    

Electrospun Aligned Nanofibers for Peripheral Nerve Regeneration: A Review

1.  Introduction

Treatment of peripheral nerve injuries and restoring the function of peripheral nerves  after  damage  still  remains  one  of  the  greater  clinical  challenges  due  to  the complex anatomy and functions of the nervous system. (1) Among several other types of peripheral  nervous  system  (PNS)  injuries,  complete  transection  of  the  nerve  with extensive gaps (>5mm) between neurons can lead to severe consequences and morbidity/mortality in patients unless there is a therapeutic intervention (2). With injuries exhibiting a wide range of causes, from road traffic accidents, stretch/traction injuries, wounding up to surgical malpractice, the need for alternative repair strategies is pressing. (3, 4)

1.1 Peripheral nerve injury

A recent report from Magellan Medical Technology Consultants determined the U.S. market for peripheral nerve repair to be 1.32 to 1.93 billion dollars per year. Concurrent with earlier reports (1, 3), upper extremity peripheral nerve damage was more frequent (84.4% out of 2,824 nerve repairs per year between the 25 surgeons consulted) than lower extremity damage. The average cost for the techniques and devices used for repair ranges between $780 up to $6,677.53 per case depending on the size of gap and method used. (5) The global nerve repair market is estimated to be around $4.5 billion as of 2013 with a compound annual growth rate (CAGR) of 11.5% and is presumed to reach

$7.5 billion by 2018, mainly due to forthcoming of novel techniques and devices. (6) Given the additional huge figures of 1 million peripheral nerve damage registered cases annually and approximately 700,000 PNS surgeries performed in the United States and Europe alone, the interest in new and improvement of existing repair strategies is more than validated. (7, 8)

1.2 Structure of peripheral nerves

Nerve fibers consisting of the axon and Schwann cells make up the functional unit of the peripheral nervous system (Table 1). (9) Axons can be up to 1m long and less than

1µm thin. With the cell bodies located in the spinal cord or dorsal root ganglia, the afferent (sensory) and efferent (motor) neurons of the PNS connect to the central nervous system (CNS) with sensory receptors, muscles and viscera. (9)

Table 1. Characteristics of peripheral nerve fibers. Values presented are from the minimum to the maximum. Fibers are further sub classified according to their electrophysiological nature (fiber contribution to compound action potential), fiber type (diameter, myelin thickness and conduction velocity), peripheral receptor and myelination. Adapted and modified from Topp et al. (2012). (9)

Nerve type	Fiber          diameter (min. – max.)	Conduction        velocity (min. – max.)
Motor	0.2 – 20 mm	0.5 – 120 m/sec
Muscle   &   joint   proprioception (sensory)	6 – 20 mm	35- 120 m/sec
Cutaneous & deep mechanoreception (sensory)	0.2 – 12 mm	0.5 – 75 m/sec

Three layers of connective tissue reinforce the peripheral nerve fiber: the endoneurium (innermost), perineurium and epineurium (outermost) (Fig.1). (9, 10) Each layer varies in its components slightly; generally a mix of different collagen types alongside macrophages, fibroblasts and mast cells is observed. The perineurium is key in conferring mechanical strength to the nerve fiber by a concentric arrangement of its cells, interconnected by tight junctions. Epineurium consists of dense, irregular connective tissue and adipose tissue and surrounds the nerve trunk. (9-11)

Figure 1: Connective tissue layers of the peripheral nerve. As it can be seen, the epineurium is the outermost layer bundling fascicles into the nerve trunk; the perineurium is the protective layer of each fascicle. Fascicles are bundles of single nerve fibers, which in turn are covered by the endoneurium, a layer composed of type I & II collagen fibers and endoneurial fluid, which, similarly to cerebrospinal fluid, acts as a cushion. Adapted and modified from Tillett et al. (2004). (14)

1.3 Biomechanics

A significant aspect of peripheral nerves is their biomechanics, which are critical when developing repair strategies. Any artificial replacement of the nerves has to match the native physical and physiological properties of the nerve fiber; hence an increased understanding in native cellular properties will translate into better repair. The biomechanics allow the nerve fiber to respond to stimuli from the central nervous system in an adequate manner without injury (Table 2). Viscoelasticity and excursion are the key biomechanical features of peripheral nerve fibers. (9) While viscoelasticity permits the nerve to alter its length (strain) upon exposure to force applied (stress, which can be

tensile, compressive, shear or a combination), excursion is the longitudinal or transverse

(sometimes both) gliding of the nerve into surrounding tissue. (9-13)

Table  2.  Key  biomechanical  features  of  the  peripheral  nerve  and  their  use  to withstand the tensile, shear and compressive forces created by limb movements.

Biomechanical feature	Application
Viscoelasticity	Length alteration upon tensile stress
Excursion	Gliding  into  surrounding  tissue  to  resist shear forces
Resistance to pressure/compression increases	Preventing nerve damage upon movement

A typical load-elongation curve of peripheral nerves reveals that only minimal tension is required for the nerve fiber to adapt and elongate accordingly, after which it displays a markedly linear region with increasing load. This region demonstrates the stiffness (resistance of nerve to deformation) of peripheral nerves. After a certain amount of load the nerve becomes permanently deformed and enters the plastic zone following which it reaches its maximal mechanical capability before surrendering to mechanical failure and damage to the tissue occurs, with studies suggesting the perineurial layer to be the most susceptible. (12-14)

In addition, the ability of the peripheral nervous system to withstand pressure increases from surrounding tissue, such as the median nerve being exposed to carpal tunnel pressure rises in different wrist positions (11, 15), demonstrates the complex native biomechanical properties of peripheral nerve fibers. It has to be noted that biomechanical characterization of human peripheral nerves resulting in exact values of all these aforementioned parameters has yet to be achieved, with current results being both inconsistent and incomplete.

1.4 Damage and repair of the peripheral nerve

Unlike the CNS, the PNS has the capability of regenerating itself following traumatic injuries. Injuries resulting in transected nerve fibers trigger the process of Wallerian degeneration, where non-neuronal cells such as macrophages invade the tissue and assist the process of nerve regeneration. (2, 16) Morphological changes in the axon such  as  swelling  of  the  cell  body,  nucleus  displacement  to  the  periphery  and chromatolysis occur alongside macrophage invasion. Together with Schwann cells and various trophic factors such as nerve growth factor (NGF) and insulin-like growth factor (IGF-1), nerve regeneration is initiated and new axons sprout from the proximal end of the injured neuron. (16-18) The complete process is more complex than illustrated here, and since the information would be beyond the scope of this article, the reader is referred to other reviews describing peripheral nerve regeneration in more detail. (4,16-19)

Axon regeneration occurs at around 2-5 mm/day; subsequently, major injuries may take prolonged periods of time to heal. (4, 20) With studies proving that time to re- innervation being key in successful recovery and the likelihood of natural regeneration causing neuromas and scar tissue, clinical intervention is required to improve outcome. (19-21) Depending on the size of the gap and gravity of defect, sutures for minimal or no loss of nerve tissue and autologous nerve grafts for larger defects remain the current gold standard. (4, 22, 23) Since these methods are limited by factors such as surgical expertise, tissue availability, infections, scarring and donor site morbidity, the perfect nerve repair yet remains out of our reach and beyond our current capabilities.

1.5 Tissue engineering and biomaterials for Peripheral Nerve regeneration

Given the absence of adequate repair, tissue-engineering strategies using artificial grafts or biomaterials, are emerging as the hopeful new solutions. Tissue engineering allows the use of a scaffold seeded with cells and growth factors (among other biological factors) to act as a substitute for the injured nerve and has the main advantage that the material can be tailored to the patient’s needs. (24)

For peripheral nerve injuries, neural scaffolds ideally should aid in controlled growth of newly forming axons from the proximal to the distal end of the site of injury, provide mechanical support (matching the tensile strength of native nerve), permit nutrient, gas, and waste exchange and diffusion of neurotrophic factors, prevent scar tissue formation and create a favorable microenvironment for nerve regeneration. (4, 25-

28) In addition, an ideal nerve conduit should have appropriate mechanical and physical characteristics depending on the nature of the defect treated, be non-immunogenic and biodegradable (its degradation rate should match nerve regeneration rate). (4, 29, 30) Emerging nanotechnology approaches have been considering these factors to design and produce the ideal neural scaffold.

One of the major upcoming techniques is electrospinning. Electrospinning allows the production of natural, synthetic or semi-synthetic micro- or nanofibers with an organized, mimicking the extracellular matrix (ECM) and allowing cell adhesion, proliferation and ultimately nerve regeneration. Additionally, since incorporation of various biomolecules into these scaffolds is possible, electrospinning seems to be the future technique of choice for neural scaffold production. (31-34)

This article will review the use of electrospinning of nanofibers for peripheral nerve regeneration; biomaterial properties and the effect of fiber alignment will be evaluated and various materials will be critically assessed.

2. Electrospinning

2.1 The electrospinning process

Electrospinning has been widely used in the past decades as a rapid method of fabricating scaffolds with favorable features, which are presented later in this review. Initially  described  and  patented  in  1930  by  Anton  Formhals  as  a  method  for  the production of threads, its adaptation for applications in the biomedical field was introduced in 2002 by Li et al. (36, 37). The process uses four main components: a high voltage power source, a syringe pump, a syringe needle and a collector, whose particular parameters are vital in controlling and optimizing the scaffold. (35)

The syringe pump contains a solution of polymer dissolved in a solvent and held in the needle by surface tension. On application of high voltage, charge repulsion within the polymer solution above a threshold level specific to the polymer solution and needle capillary diameter overcomes surface tension. At this point, the polymer solution is ejected from the syringe in what is called a Taylor cone, formed due to the interaction of electrostatic forces within the solution, surface tension and charge, to form a charged jet, which follows a straight trajectory before spiraling due to its charge. (68) Upon ejection, the solvent evaporates almost completely, leaving behind a fibrous jet of polymer, which is collected on the collector. (35, 41) For the production of randomly arranged fibers a flat collector is used, whereas in order to collect aligned fibers a rotating mandrel is used. The procedure is diagrammatically represented in figure 2A. The speed at which the mandrel is rotating determines the degree of alignment of the fibers. As alignment itself is a function of the biomaterial being electrospun, this is usually subject to optimization in studies. Previous work presented that Collagen type I can be collected as random fibers at speeds corresponding to 0.16 m/s whereas at speeds of 1.4 m/s, fibers were aligned to the axis of rotation. (52)

Varying the distance between the syringe and the collecting mandrel can control the diameter of fibers. (56) Molecular weight of the polymer, the electric field and the conductivity of the solution have major effects as they affect the charge within the

solution. Conductivity of the solution has a pronounced effect, and is utilized in such ways as the addition of ionic salts to increase conductivity, as fibers of smaller diameter are produced with increasing conductivity. (69, 71) The concentration of the polymer solution is in itself a determinant of fiber diameter, as research findings present that solutions of higher concentration give fibers of larger diameter. (70, 71) It should also be noted that the feeding rate in the polymer, i.e. the rate at which the syringe pump ejects polymer solution has also been shown to affect fiber diameter, producing thicker fibers as the feeding rate increases. (71)

Upon its production, the aligned scaffold usually undergoes a drying stage of varying  durations  dependent  on  the  biomaterial  used  as  well  as  its  solvent  and  its intended application.

2.2 Advantages and Disadvantages

Electrospinning carries several inherent disadvantages, the major one being the inability to produce scaffolds of any complexity beyond that of either aligned or random sheets or simple masses of material. It is however possible to introduce complexity upon further manipulation of the product in simple ways such as rolling of flat sheets to form hollow of solid cylindrical tubes. Furthermore, although hierarchical structures cannot be directly produced, similar methods can combine several different electrospun products to introduce a varying degree of complexity. It should be noted however that the process cannot achieve such complexity as can be produced with alternative methods such as 3D printing. In the production of aligned electrospun scaffolds for the purposes of neural tissue engineering, it is common to rely on simple structures such as aligned hollow tubes formed prior to, or after, cell seeding. (40, 41)  Other major disadvantages presented in electrospinning include the inability to produce homogeneous porosity. (35)

Conversely, the extensive use of electrospinning is indicative of its advantages. The method is quick and simple and produces scaffolds generally characterized by high porosity and high surface area to volume ratio, which facilitates cell migration and attachment and enhances mechanical properties relative to the biomaterials used. (75)

Importantly, electrospinning is extremely versatile in terms of incorporating more than one polymer as composites or mixtures including micro- and nanoparticles or growth factors for their controlled release. (42, 43) The ability to spin blends can also be exploited to tailor the mechanical properties of scaffolds to those of native tissue in order to mimic the native mechanical environment as a means to increase the mechanical biocompatibility of grafts as well as direct cell behavior. Additionally, constructs are subject to a broad range of further processes for their functionalization. (44) Scaffolds can be produced using most available biomaterials since the main requirement is their dissolution in a solvent. Electrospun fibers, which range in diameters from micro- to nanometers, closely resemble the structure of the native ECM and provide the cells, which are seeded on the scaffold subsequently, with biomechanical cues that guide migration, differentiation and cell function. (75) In the scope of this review, it must be mentioned that synergistic action between biomaterial properties and micro- or nanoarchitecture has been presented to have favorable results in cell behavior. (39) Indeed, on investigation, it has been demonstrated that mimicking native chemical cues via the incorporation of natural biomaterials for peripheral nerve tissue engineering, namely a mixture of Collagen type I and type III, has favorable effects  on  cell  differentiation,  as  does  the  replication  of  native  architecture  by producing aligned electrospun fibers. (39) Combining both features however has an enhanced effect, giving increased proliferation and cellular phenotype when compared to the results of mimicking chemical cues and native architecture separately. (39)

2.3 Co-axial electrospinning

Co-axial electrospinning is a modified technique whereby two dissimilar polymer solutions are electrospun simultaneously to produce fibers as a core surrounded by a sheath. To achieve this, a smaller capillary delivering core polymer mixture is inserted inside the capillary, which ejects the sheath polymer mixture, and both mixtures can be controlled individually, as can be seen in 2B. (68) On application of the electrical field, the sheath polymer is ejected to form a Taylor’s cone and the stress generated applies one or both pulling forces on the core solution, known as “viscous dragging” and “contact

friction”, forcing the core solution to be ejected simultaneously. (72) This results in the ejection of a mixture of the two polymers, where one forms the core to the other.

The production of fibers of specific diameters is governed in much the same way as  it  is  in  normal  electrospinning,  however,  for  the  successful  application  of  this technique,  several  factors  must  be  taken  into  consideration.  Chief  among  these  is matching the viscosities of both solutions, conductivity and solvent evaporation pressure. It is vital that the latter is considered, since high pressure on evaporation of the core solvent can cause a vacuum and have detrimental effects on the structure of the fibers. The interaction of solvents used for each polymer must also be taken into consideration, as any precipitation of polymer as a result of interacting with the second polymer’s solvent will cause errors in the technique. (74) Conversely, the miscibility of the two solvents is debated. The concentration of each solution, affects the diameter of the fibers produced in a dynamic way as there is a balance between the thickness of the core and the thickness of the sheath with respect to each other and the net diameter of the fiber. (73) As with all instances of producing blended polymer composites, co-axial electrospinning has the inherited advantages of tuning both bulk and surface material properties such as degradation rates and mechanical properties of hydrophilicity, as well as achieving controlled factor release. (68) For an in-depth review of co-axial electrospinning, the authors suggest the work of Moghe and Gupta. (68)

Co-axial electrospinning benefits from the same advantages and suffers the same disadvantages as conventional electrospinning. The increased complexity allows for the production of a blend with a unique structure to its fibers, and its respective advantages mentioned above, arising from the sheathing of core polymer. The introduction of complexity to the procedure affects the simplicity of its design. A a result, although the setup remains relatively unchanged, it becomes increasingly difficult to carry out the procedure due to the complexity of solvent interactions; specific solvents need to be matched. Additionally, the prolonged interaction between the core and sheath materials may induce a change to either, bringing about undesirable effects. (38)

3. Electrospun nanofibers and material selection

The design of scaffolds suitable for peripheral nerve regeneration is among the most challenging aspects of tissue engineering. Requirements for the ideal neural scaffold have been outlined earlier in the article and although a single electrospun material has yet to be discovered that replicates all the desired properties of native neural ECM, several polymeric materials have been studied and show promise as suitable options for PNS regeneration. These materials can be categorized as synthetic, natural or composites of synthetic and natural materials.

3.1 Synthetic Polymers

Synthetic materials are attractive candidates for neural scaffolds due to their availability, ease of manufacturing and low variability between batches. (76) Of the many synthetic polymers studied, polyesters such as poly lactic glycolic acid (PLGA) and poly L-lactic acid (PLLA) represent the most widely studied synthetic polymers for PNS regeneration. (77) As thermoplastics, these materials can be shaped fairly easy through methods such as molding and extrusion, but their biodegradable properties due to their linear aliphatic polyesters make them especially useful in nerve tissue engineering. The degradation process can occur through either hydrolysis or enzymatic action and depends upon the structure and composition of the material (78). By tailoring the size and composition of the scaffolds, unique degradation profiles can be attained to meet the demands  in  specific  nerve  injury  sites.  Besides  PLGA  and  PLLA,  other  synthetic materials such as PHBV and PCL have shown to be effective synthetic scaffolds (79-80) Poly 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) is a thermoplastic that is naturally produced by the bacteria.  PHBV is an attractive material since it is renewable and has properties similar to other conventional thermoplastics. This material, however, suffers from high cost of synthesis mostly due to bacterial culture requirements. (81) Despite this, limited success has been found by Molamma et al., who showed that when PHBV nanofibers were aligned and generated by electrospinning, PC12 cells grew more efficiently and orientated themselves along the direction of the fibers. (79)

Figure 2: A - Traditional electrospinning, B - Co-axial electrospinning.

To further make use of synthetic materials, composite materials have been created to achieve better overall material. An example of such a synthetic composite is the

PGLA-PCL blend developed by Subramanian et al. in 2012. This group showed that this material was capable of supporting Schwann cell growth and proliferation and showed that alignment of nanofibers resulted in higher axial tensile strength and increased

Schwann cell proliferation. (82) Though the biodegradability and ease of manufacturing associated with synthetic materials makes them attractive, they are often relatively hydrophobic which results in poor cell interaction. To address these issues, the surfaces of these materials are often modified by plasma deposition and subsequently coated with native ECM proteins such as laminin, fibronectin, RGD sequences and other cellular

adhesion proteins (83). A recent example of one such study was undertaken by Koppes et al. in 2014. This group synthesized aligned PLLA nanofibers conjugated with laminin proteins. Results showed that alignment of nanofibers increase neurite extension from dorsal root ganglion compared to randomly oriented PLLA nanofibers. Koppes group

also showed that a synergistic effect could be achieved by seeding aligned PLLA nanofibers and applying a small electrical stimulation to the scaffold. (83) These results, amongst others, are promising indications that wholly synthetic scaffolds may be a realistic option for Peripheral nerve regeneration. However, as the field is still in its infancy, a significant amount of research must be undertaken to ensure consistent results. A more condensed overview of synthetic scaffolds for peripheral nerve regeneration is presented in table 3.

Table 3: Synthetic Materials or PNS

Synthetic polymer	Chemical structure	Material characteristics and mechanical properties of aligned fibers	Fiber diameter	Cell type	Experimental outcome
PCL (80) Poly (ε- caprolactone)	O H 2 O            C             C 5                n	Biodegradeable polyester semicrystalline polymer Relatively hydrophobic Tensile strength: Not reported Youngd modulus: Not reported	Aligned Fiber 1,030 ± 84 Random Fiber 934 ± 54	Dorsal Root Ganglia	DRG cells grew along the direction of aligned fibers, with enhanced growth in fibers containing elastin
PHBV (79) Poly ( 3- hydroxybutyrat e-co-3- hydroxyvalerat e)	CH3                  O                   CH2CH3         O      H2                                         H2                    O        C        C        C        O        C        C        C H                             H n	Bacterially produced thermoplastic Biodegradable Tensile Stress (MPa) 1.49 ±0.07 Youngs Modulus (MPa) 25.98 ± 2.31	Parallel Aligned 2,170 ± 370 nm Perpendicula r aligned 2,370 ± 560 nm	PC12 Nerve Cells	Cells on aligned nanofibers showed contact guidance and orientation of nerve cells along the direction of the Fibers, showing bipolar extension. Though collagen composites performed better.
PLLA (83) Poly Lactic acid	O                                O O        C        CH        O        C        CH CH3                                     CH3           n	Thermoplastic polymer Relatively hydrophobic Tensile Stress (MPa) Not reported Youngs Modulus (MPa) Not reported	Parallel Aligned 2,170± 370 nm Perpendicula r Aligned 2,370 ± 560 nm	Dorsal Root Ganglion	DRG cells grown on laminin coated parallel-aligned fibers proved to be able to grow further compared to cells grown on perpendicular fibers. Also, small electrical stimulation was shown to increase

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          16

					nuetrite growth and also increased the length cells could achieve on just parallel-fibers alone. (synergistic effect)
*PLGA-PCL (82) Poly (lactic-co- glycolic aid)	O                                   O O        CH2          C           O        CH2          C n	Brittle and degrades in bulk PCL overcome limitations Tensile strength (MPa) 0.7 ± 0.2 Young’s modulus (MPa) 7.5 ± 3.0* Elongation (%) 28.7 ± 6.0	Aligned 227 ± 60 nm	Schwann Cells	Schwann cells were shown to grow and proliferate significantly more on aligned nanofibers than random fibers. .

3.2 Natural Polymers

Natural polymers are characterized as polymeric materials that can be derived from  plants,  animals  or  human  tissues.  (8)  Examples  of  natural  polymers  include collagen, silk fibroin, chitosan, alginate and starch. These materials are readily bioactive and have low toxicity. However, these materials are not without drawbacks. Natural polymers are generally harder to source than synthetic polymers and can be difficult to process often having considerable batch-to-batch variation.

3.3 Composite Polymers

Although  synthetic  materials  tend  to  be  cheaper,  easier  to  obtain  and  have superior mechanical properties, they tend to have unfavorable hydrophobic properties. Additionally, despite their biodegradability, they are inferior in terms of mimicking the chemical properties of native extracellular matrix. To rectify this, research has focused on

the incorporation of natural biomaterials in the field. These materials have their own specific properties, but, being natural, share several important advantages such as being biodegradable and hydrophilic, allowing for cell attachment. (51)

3.3.1 Collagen

Perhaps the most commonly used natural biomaterial in nerve tissue engineering is collagen. Indeed, interest in its biomedical applications has resulted in collagen type I conduits gaining approval for the United States Food and Drugs Administration (FDA). (38) The material presents hydrophilicity and enhances cell adhesion, as well as offering the potential to satisfy required properties that have yet to be elucidated, since collagen is a major component of peripheral nerve matrix. (65, 45) As collagen is biologically disadvantage lies with its weak mechanical properties. (38, 54) A further disadvantage hurdles derived, it presents the disadvantage of being potentially immunogenic, however, its major specifically the production of electrospun scaffolds using pure collagen, as the fibrous morphology of scaffolds, deteriorates over time.(53) This feature which is particularly  detrimental  when  electrospinning  nanofibers  and  even  more  so  when aligning,  as  the  fibers  tend  to  fuse  to  produce  an  uneven  sheet  of  collagen.  This effectively destroys the main advantages of aligned electrospinning, nanotopography and large surface area.

As a result, collagen is incorporated in scaffolds as part of a composite in order to supplement its mechanical properties and to stabilize its morphology. (55) Properties are dependent on the composition of the blend; however, the surface chemistry of collagen confers tunable hydrophilicity. (39) Evidence for the favorable interaction of collagen with cells of neuronal lineage can be seen in the work of Kijeńska et al., where blends of poly (L-lactic acid/ caprolactone) (P(LLA/CL)) and collagens I and III were investigated. It was shown that blends of P(LLA/CL) with collagen I and III presented higher C17.2 cell proliferation, elongation and differentiation than pure P(LLA/CL) scaffolds. Additionally, blends containing both types of collagen presented increased neurite outgrowth than those containing only one type of collagen. (39) In their work, Seyer et al., have found that peripheral nerve collagen is composed of 81% type I and 19% type III collagen. (45) On the basis of this, it is presented that cellular interaction with a

nanoenvironment reminiscent of the normal niche has very prominent effects on the outcome of neural tissue growth.

3.3.2 Silk Fibroin

Silk fibroin is a biomaterials derived from silkworm silk. It presents a multitude of properties, some of which are unusual to natural biomaterials: it has high tensile strength and presents enhanced mechanical properties due to eggshell-like microstructure. (48, 50) Additionally, it promotes a regenerative response is non-immunogenic, water permeable, and has a degradation profile which is controllable by the method of recrystallization. (48-50) As a result, silk fibroin is a material that has been used for the production of vascular grafts and cartilage repair purposes amongst its other applications. (46, 47)

As with collagen, silk fibroin is more commonly used as part of a composite biomaterial as its pure form has been shown to be unfavorable for cellular attachment and growth. Instead, it has been presented that coating with fibronectin, gelatin or collagen enhance cell attachment and growth. (49)

3.3.3 Chitosan

Alternative biomaterials include chitosan, a fully or partially deacetylated derivative  of  chitin.  (57,59).  Originally,  chitin  is  collected  from  the  exoskeleton  of insects, shells of crustaceans, or from fungal cell walls. Its structural similarity to glycosaminoglycans of the extracellular matrix initially attracted interest as a biomaterial. (66) Chitosan is hydrophilic and has antibacterial properties, an advantageous feature when considering its application as an implantable material for nerve tissue engineering. (58, 60)

The application of pure chitosan is heavily deterred due to its properties. Brittleness and rigidity arise due to strong intermolecular and intramolecular hydrogen bonding. (66) Low mechanical strength and the inability to maintain a predetermined shape on implantation under physiological conditions are also significant downsides. (61) Specifically, chitosan is normally unsuited for electrospinning due to its insolubility in

most common solvents and due to high repulsion between charged ionic groups in the polymer backbone under high electric fields. (66, 67) As a result, chitosan follows the general trend of natural polymers to confer their advantages while mediating their disadvantages as parts of composite biomaterials. To date, chitosan has seen application and intense research in neural tissue engineering, with composite conduits achieving functional  recovery  and  hind  leg  mobility  in  canine  sciatic  nerve  repair,  tubular membrane constructs being used for rat sciatic nerve repair and membranes achieving nerve regeneration and functional recovery in rat models. (57, 61, 62)

3.3.4 Laminin

Laminin is a basement membrane protein, which has a fibrous morphology in vivo, highlighting its relevance with the electrospinning method. It facilitates cell attachment, growth and migration. (64) Unlike other proteins used in electrospinning, such as collagen, laminin nanofibers retain their structural features when wet; hence laminin does not require chemical crosslinking for its fixation, which could compromise bioactivity by altering the protein structure. (64) As with previous natural biomaterials, laminin has seen application as a blend. It has been shown multiple times, as can be evidenced by Neal et al. in a murine model, that incorporation of laminin in scaffolds favours cell attachment and drives neurite outgrowth. (63, 64) When pure synthetic scaffolds were compared to laminin-containing blend scaffolds for the regeneration of rat tibial defect, sensory function recovery was more pronounced in scaffolds that contained laminin. (63)

Table 4: Natural vs composite materials

Natural/composite polymer	Material characteristics and mechanical properties of aligned fibers	Fiber diameter	Cell type	Experimental outcome
Collagen (84)	Tensile Strength 4.8 ± 0.7 Ultimate strain % 13.7 ± 1.9	150nm	PIECs culture	collagen promoted cell attachment, correct morphology
Collagen/TPU (84) (8 wt.%/3 wt.%)	Tensile strength 4.53 ± 0.3 Ultimate strain % 142.8 ± 7.2	720nm	PIECs culture	coaxial electrospun nanofibers could provide better growth condition for cell proliferation
10%laminin/PCL (63)	Tensile strength <1 MPa Elastic Modulus <1.5 MPa	100nm	PC12 cells	laminin-PCL blend nanofibers maintain the bioactivity of pure laminin nanofibers, while Alignment of nanofibers increases the length of neurite extension

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4. Effects of aligned nanofibers on cell-scaffold interactions

By forming nanofibrous scaffolds through electrospinning and by manipulating several important parameters such as fiber orientation, fiber diameter, density of nanofibers, deposition of factors such as protein on the surface of the scaffold, the topographical and biochemical cues produced by the scaffold can be engineered towards peripheral nerve regeneration. Important parameters that alter these cues include the orientation  and  diameter  of  the  nanofibers,  the  density  of  the  fibers,  the  surface properties, surface morphology, porosity, degradation rate, mechanical properties and others.

4.1 In vitro studies

It has been known for more than a decade that altering the orientation of the fibers in a scaffold as well as the size of the diameters can significantly alter neurite outgrowth. (85) Yang et al. used PLLA to test the response of neuronal stem cells (NSCs) on aligned and  random  fibers.  Neurite  outgrowth  was  parallel  to  the  direction  of  the  aligned fibers.  The diameter of the fibers was also examined and the fibers with the smallest diameter produced a higher differentiation rate of NSCs than scaffolds made with larger fiber diameter.

Neurite outgrowth along the direction of the aligned fibers using PLLA was also observed in other studies. (86, 87) Xie and coworkers noticed neurite outgrowth in mouse embryonic stem cells (ESCs) along PCL electrospun nanofibers. The cells were also able to differentiate into specific neurons such as oligodendrocytes and astrocytes. (86) Wang et al. found that Schwann cell migration is also being directed through contact guidance along the nanofibers but was found to be independent of neurite outgrowth. (87) Surprisingly, smaller fiber diameters produced shorter neurite outgrowth. This result may have been affected by the density of the fibers in the scaffold, as it is known that reducing the fiber diameter increases fiber density.

Subramanian and coworkers showed that Schwann cells seeded on aligned PLGA nanofibers not only direct their migration along the fiber substrates but also increase their proliferation compared to random nanofibers. (88)

The mechanical properties of the aligned nanofibers differ from the random nanofibers in a way that mimic the native microenvironment of the ECM more closely, allowing for higher proliferation rates than random nanofibers. In addition, aligned nanofibers have better deformability than random nanofibers, which could permit better cell-scaffold interaction.

Another study investigated a PLGA-PCL co-blended nanofibrous scaffold. Schwann cells were cultured and were seen migrating along the aligned nanofibers, whereas on random nanofibers reduced proliferation was noticed. This was the result of better flexibility, slower degradation rate and enhanced cell-scaffold interaction, which is observed in the native microenvironment of the tissue allowing Schwann cells to adhere and proliferate on the scaffold. (89)

The alignment of the fibers produces elongation of neurites along the same direction as the fiber’s orientation that most often results in the formation of bipolar neurons. Random nanofibers, however, usually do not produce bipolar neurons but their elongation can span several directions. Two studies using poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) as substrate demonstrated multipolarity in seeded nerve cells. (90,  79)  Masaeli  and  coworkers  showed  Schwann  cell  alignment  on PHB/PHBV/Collagen nanofibers, which developed along the direction of the fibers whereas stem cells seeded on random nanofibers displayed multipolarity. It is worth noting that collagen enhanced proliferation and differentiation independent to the increased proliferation seen by the alignment of the nanofibers. (90) Prabhakaran et al. noticed similar effects of fiber orientation and collagen incorporation. (79) The diameter of the nanofibers decreased in both studies with the integration of PHBV into the scaffolds.

In recent years, however, more studies are investigating semi-synthetic materials than  purely  synthetic  ones.  This  is  because  it  has  been  realized  that  adding  natural

materials onto the scaffold is a superior method in mimicking the native microenvironment. In fact, creating a scaffold that mimics the ECM of the peripheral nervous system is the main challenge in tissue engineering.  (79)

Kijenska and coworkers added collagen I and collagen type III on a synthetic blend P(LLA-CL). C17.2 type neural stem cells (NSCs) were cultured and seeded on both the biocomposite scaffold P(LLA-CL)/Collagen I/Collagen III and the P(LLA-CL) which acted as a control. In the biocomposite scaffold there was a 22% increase in cell proliferation compared to the synthetic-blend scaffold indicating the favorable topographical  and  biochemical  cues  produced  with  introduction  of  collagen  in  the scaffold. The morphology of the NSCs was better in aligned nanofibers compared to random nanofibers for both materials. (39)

In another study by Christopherson et al., laminin was coated on the synthetic polyethersulfone (PES) and was used as a fiber substrate for NSCs to investigate fiber diameter. The diameter of the nanofibers was made to be small (250nm), intermediate (750nm) and large (1500nm). The neurites were elongated on top of several nanofibers in the small diameter group but elongated in only one nanofiber in the larger diameters. Despite this, the cells seeded on small diameters did not aggregate and showed increased proliferation and migration compared to the other larger diameters. The neuronal differentiation of NSCs to oligodendrocytes was 20% higher in the small diameter group of nanofibers compared to its larger diameter counterparts. (92)

Gelatin has been explored in a few studies, where it has been added to synthetic materials to form semi-synthetic blends and currently is showing promising results. Mobarakeh and co-workers developed random and aligned PCL/Gel scaffolds where gelatin was incorporated in different concentrations yielding two different PCL/Gel blends. PCL/Gel 50:50 and PCL/Gel 70:30 formulations relating to the percentage of their concentration were developed. PCL/Gel 70:30 accounted for the highest differentiation rate and greater proliferation of NSCs compared to both the PCL/Gel (50:50 and the pure PCL. The mechanical properties of PCL/Gel 70:30 were found to be similar to that of nerve tissue. (93) Another study created a scaffold blending PLLA and Gelatin  (PLLA/Gel)  forming  a  non-woven  material  that  had  degradation  rates  and

mechanical properties that were similar to peripheral nerves. Retinoic acid was incorporated in the scaffold, which enhanced the differentiation of NSCs into motor neuronal lineages and the development and elongation of neurites. (94) A natural blend consisting of gelatin and hyaluronic acid was investigated by Liou et al.. The relative concentration of hyaluronic acid was made to vary across the different blends and although no significant difference was observed in the attachment and proliferation of Schwann cells being cultured, the expression of several genes and proteins associated with normal Schwann cell function such as Nrg1 and P0 was significantly higher in the HA-Gel blend compared to Gel alone. (95)

Strong anisotropy is favorable in nerve regeneration. (96) Additionally, the tensile strength of nanofibrous scaffolds can affect the regenerative capacity of nerve tissue as demonstrated by Zhang and coworkers. (97) By characterizing the effects of nanofibrous scaffolds made of silk fibroin when combined with the synthetic blend P(LLA-CL) to form SF/P(LLA-CL) 25:75, it was found that the aligned SF/P(LLA-CL) yielded a significantly increased Schwann cell (SC) proliferation compared to the aligned P(LLA- CL) and both groups of random nanofibers. The migration of SCs was along the aligned nanofibers as expected. The aligned nanofiber was demonstrated to have a high tensile strength in parallel of the direction of the nanofibers, which can be an important factor in SC migration and neurite outgrowth.

4.2 In vivo studies

In the regeneration of peripheral nerve, the development of a suitable nerve guidance conduit (NGC) is essential. NGCs can be made to have different fiber orientations on the exterior and interior so that different topographical cues will affect the cells  to  alter  cell  differentiation,  elongation,  migration  and  proliferation  differently. Griffin et al. investigated the effect of Schwann cells on salicylic acid based polyanhydride fibers blended with PLGA. The neurite outgrowth and SC migration along the  direction  of  the  aligned  nanofibers  was  very  promising.  (98)  As  a  result, polyanhydride  fibers  can  be  added  on  nanofibrous  scaffolds  to  provide  a  controlled release system for which SAA is known to have anti-inflammatory effects. (99) This could minimize the immune response associated with PLGA.

Another biocompatible material is chitosan, examined by Alhosseini and coworkers. Polyvinyl-alcohol (PVA) fibers were blended with chitosan in a way that a highly porous scaffold was generated. PC12 nerve cells were seeded on the scaffold and were shown to have higher viability and proliferation than the PVA fibers alone. It’s worth noting that only a small increase in the percentage of chitosan on the scaffold resulted in a significant increase in the biocompatibility of the overall scaffold. (100)

NGCs are of great interest in bridging nerve gaps because of their high surface to volume ratio, flexibility and highly porous structure. (101) A synthetic blend composed of PLGA and PCL (PLGA/PCL) was studied in vivo by formulating an NGC to bridge a

10mm  nerve  gap  in  the  sciatic  nerve  of  a  rat.  (102)  The  PLGA/PCL  blend  was synthesized to form random and aligned fiber orientations with varied fiber diameters. Both the aligned and random fibers in the tubes resulted in reconnection of the sciatic nerve and re-innervation of the target muscles, while no reconnection or re-innervation was observed in the control group without treatment. Contact guidance by topographical cues elongated the axons along the direction of the aligned fibers. This particularly favorable feature is noted when compared to the randomly orientated fibers that resulted in poor elongation of the axons. PLGA/PCL fibers of nanoscale diameter resulted in greater cell attachment than on microscale fibers because the cells have more surface area to adhere and therefore higher cell density per unit of space. PLGA was also investigated for a sciatic nerve defect by Wang et al. where incorporation of NGFs on the NGC produced significantly better results than the PLGA formulation alone, after 12 weeks of implantation. (103) The PLGA/NGF NGC showed similar results with the autograft treatment group. Perhaps slightly more promising was the fabrication of PLGA with silk- fibroin (SF) by Li et al. The PLGA/SF formulation was again comparable to autografts but silk fibroin (SF) incorporation can help mimic the ECM, encourage cell adhesion and alter the mechanical properties and microarchitecture of NGCs.   (104 )The enhanced regenerative effects of SF were also observed by Wang et al. which blended SF with aligned  P(LLA-CL)  nanofibers.  The  SF/P(LLA-CL)  conduits  produced  significantly better recovery than P(LLA-CL) alone.   (105) More studies are required for the silk fibroin incorporation to NGCs in vivo studies.

Apart from PLGA, PCL has also received attention for in vivo experiments. (65,

96)  Yu  and  coworkers  constructed  PCL/Collagen  blends,  which  formed  an  NGC  to bridge an 8mm sciatic nerve gap in rats. Although muscle re-innervation was observed, the regeneration of the nerves was too slow and premature 4 months post-implantation. (65) Nevertheless, the high surface area of the PCL/Collagen conduit makes this material a promising blend for future investigation.

More recently, a study done in 2014 by Beigi et al. added stem cells from human exfoliated deciduous teeth (SHED) on the NGC, which was implanted in the sciatic nerve of a rat. It was found that the addition of SHED on the conduit enhanced functional and sensory recovery significantly 16 weeks post implantation compared to the conduit without SHED. This particular cell seeding approach with these specific cells (SHED) most probably accounts for its improved recovery, as these cells are known to express neurotrophic factors that enhance nerve proliferation. (106) A better understanding of cell biology through research of related scientific fields such as cell biology and stem cell biology can assist the advancement of cell seeding on scaffolds and their interaction with different cells in a way that cell behavior can be directed as planned.

A different approach to cell seeding is the incorporation of protein factors as discussed before. Kaklinoki and coworkers added the laminin-derived peptide AG73 to a synthetic  polymer  blend  consisting  of  PLLA  and  oligo(D-lactic  acid)  (ODLA)  and showed that after 6 months of bridging a 10mm nerve gap of a rat, there was a significant improvement in the recovery of the nerve with the semisynthetic nerve conduit P(LLA)/ODLA-AG73 compared to the pure PLLA fabrication. (58)

Most of the NGC formulations comprise of at least one synthetic material but Wang and coworkers investigated the formation of a mesh tube synthesized only by chitosan. They showed that the orientated tubes produced larger number of axons and greater axon diameter in the rat’s sciatic nerve. Functional recovery of the defected nerve was also observed after 20 weeks post-implantation. The aligned chitosan nanofibers were comparable to the isograft and significantly better than the random nanofibrous mesh tube both in terms of axon diameter in addition to number of axons as well as functional recovery.  (107)

Perhaps one of the best strategies in the construction of NGCs is the incorporation of various neurotrophic factors, natural filler materials like collagen and fibroin in small quantities and cells on NGCs (108). It is expected that the best NGCs will encompass this approach as it can greatly improve the recovery of peripheral nerves. More in vitro and in vivo studies nonetheless are required to understand the effects of peripheral nerves upon the complexity of these factors.

An NGC maybe preferred over the current standard treatment even if it is not more  effective,  as  such  an  improvement  would  eliminate  most  of  the  morbidity associated with autograft treatments. Therefore, although NGCs with spectacular regenerative capabilities would be ideal for peripheral nerve regeneration, a small but significant advancement in the development of appropriate NGCs in the near future could greatly improve the treatment of peripheral nerves.

5. Conclusion

With the natural repair of peripheral nerves being a flawed mechanism and the shortcomings of the current clinical gold standard (autologous repair), tissue engineering approaches using biomaterials, in particular electrospinning, rise to the occasion. Electrospun nanofibers have shown the potential to bypass the limitations of autologous repair since they not only support the structural integrity of cells, but also permit incorporation of biomolecular and topographical cues (in particular alignment) to guide cellular  attachment  and  proliferation.  Ease-of-manufacture  and  cost-effectiveness  are other factors speaking in favor of this method.

Challenges to this approach remain two-fold: (1) our understanding of the peripheral nervous system, in particular the biomechanical properties, is yet incomplete; (2) the ideal scaffold configurations to replicate all the desired properties regarding peripheral nerve regeneration has yet to be discovered.

Synthetic polymers may have outstanding mechanical properties; nonetheless they fall short of the requirements in surface chemistry and cellular interactions. Natural materials on the other hand, be they derived from human, animal, or insect, are deposited as tissues grow and mature, thus sacrificing their mechanical properties for the more important function of cellular interactions. It therefore has to be recognized that neither synthetic nor natural materials possess the exact required peculiarities to achieve ideal peripheral nerve regeneration individually.

Currently, the blending of natural and synthetic polymers mediates the problem by providing a middle ground solution when attempting to regenerate nervous tissue. As the desired properties are predominantly dependent on the location and size of the defect under consideration, the specific mechanical and physical properties must be tuned for each scenario. The chemistry of the native tissue also needs to be considered to bring an enhanced effect. To the authors’ knowledge and to date, no attempt has been made to do so with the use of a blend of several biomaterials and it is in this that our future prospects should lay.

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