Biomaterials Coursework - Yiangos, Thanos, Ram, Cody

Electrospun aligned nanofibers for nerve regeneration

Yiangos, Thanos, Ram, Cody

Contents

1.      Introduction

·         Discovery (clinical background and challenges of nerve regeneration)

·         Principle of Electrospinning (+schematic representation)

·         Introduction to nanofibers and their potential uses (+diagram)

·         Introduction to use of electrospun nanofibers in nerve regeneration

2.      Electrospinning nanofibrous scaffold for tissue engineering

·         Principle of Electrospinning (+schematic representation)

·         Controlled fibre alignment of electrospinning

3.      Electrospun  nanofibers for nerve regeneration

·         Types of polymers used for nanofiber production (and their pro’s/con’s)

·         Structure and properties of  electrospun nanofibers

·         Anisotropic properties of alignment nanofibres

·          

·         Comparison between random and aligned nanofibers (+ table for representation)

4.      Anisotropic effects of aligned nanofiber on Cell-scaffold interactions

·         In vitro

·         In vivo

·         Advantages of nanofibers over other methods for nerve regeneration (e.g. autografts)

5.      Conclusion Remarks

·         Future Outlook

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DRAFT - 47 hours to submit

Electrospun nano fibers for peripheral nerve regeneration

Electrospun aligned nanofibres for peripheral nerve injury, an Introduction

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 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)

Peripheral nerve injury statistics

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 CAGR (compound annual growth rate) 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)

 

Structure, biomechanics and damage of peripheral nerves

Nerve fibres consisting of the axon and Schwann cells make up the functional unit of the peripheral nervous system. (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 CNS with sensory receptors, muscles and viscera. (9)

Three layers of connective tissue reinforce the peripheral nerve fibre: the endoneurium (innermost), perineurium and epineurium (outermost). (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 fibre 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)

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 fibre; hence an increased understanding in native cellular properties will translate into better repair. The biomechanics allow the nerve fibre to respond to stimuli from the central nervous system in an adequate manner without injury. Viscoelasticity and excursion are the key biomechanical features of peripheral nerve fibres. (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)

A typical load-elongation curve of peripheral nerves reveals that only minimal tension is required for the nerve fibre 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)(13)(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 fibres.

Damage and repair of the peripheral nerve

Unlike the CNS, the peripheral nervous system has the capability of regenerating itself following traumatic injuries. Injuries resulting in transected nerve fibres 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 NGF and IGF-1, nerve regeneration is initiated and new axons sprout from the proximal end of the injured neuron. (16)(17)(18) Obviously, the entire 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 reinnervation 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.

Tissue engineering and biomaterials for PN regeneration

Given the absence of adequate repair, tissue-engineering strategies using artificial grafts, or biomaterials, are emerging as the hope on the horizon. 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 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 stump, provide mechanical support (matching the tensile strength of native nerve), permit nutrient/gas/waste exchange and diffusion of neurotrophic factors, prevent scar tissue formation and create a favourable microenvironment for nerve regeneration. (4)(25)-(28) In addition an ideal nerve conduit should have appropriate mechanical characteristics depending on the nature of the defect treated, be biocompatible and biodegradable (degradation rate should match nerve regeneration rate). (4)(29)(30) Emerging nanotechnology approaches have been considering these and additional 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 nanofibres with an organized and controllable architecture, mimicking the 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 nanofibres for peripheral nerve regeneration; biomaterial properties and the effect of fibre alignment will be evaluated and various materials will be critically assessed to find out the most suitable candidate.

The electrospinning process

Conventional tissue engineering processes follow a top-down methodology whereby a scaffold is tailored after the particular peculiarities of the tissue under investigation, followed by a cell seeding stage. Common approaches tend to attempt to produce biodegradable materials for the production of a scaffold that becomes progressively degraded and replaced with newly produced extracellular matrix deposited by the cells as these grow, proliferate and differentiate into the desired lineages. This aims to produce grafts that completely and ideally resemble and repair native tissue.

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 [3], its adaptation for applications in the biomedical field was introduced in 2001 by Li et al. [4]. The process uses three main components; a power source of high voltage, a syringe pump and a collector, whose particular parameters are vital in controlling and optimizing the scaffold [1].

The syringe pump contains a solution of polymer dissolved in a solvent and held in the pump by surface tension. On application of high voltage, charge repulsion within the polymer solution above a threshold level specific to the polymer solution and capillary diameter overcomes surface tension. At this point, the polymer solution is ejected from the syringe in what is called a Taylor cone to form a charged jet. Upon ejection, the solvent evaporates almost completely, leaving behind a fibrous jet of polymer, which is collected on the collector [1, 9]. 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 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 [22]. The diameter of fibers can be controlled by varying the molecular weight of the polymer, the electric field, the distance between the syringe and the mandrel, the rate of flow and the concentration of the solution [26].  The procedure is diagrammatically represented in figure 1.

Fig. 1 The electrospinning process.

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

           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 [8,9]. Other major disadvantages presented in electrospinning include the inability to produce homogeneous porosity [1].

           Conversely, the extensive use of electrospinning is indicative of its advantages. The method is quick and simple and produces scaffolds generally characterized by high surface area to volume ratio, which facilitates cell attachment and enhances mechanical properties relative to the biomaterials used [principles of TE book]. Importantly, electrospinning is extremely versatile in terms of incorporating more than one polymer as blends or mixtures including micro- and nanoparticles or growth factors for their controlled release [10, 11]. 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 [12]. Scaffolds can be produced using most available biomaterials since the main requirement is their dissolution in a solvent. Electrospun fibers can range in diameter from micrometers to nanometers, which closely resemble the structure of native extracellular matrix [principles of TE book], providing subsequently seeded cells with biomechanical cues that guide migration, differentiation and cell function. In the scope of this review, it must be mentioned that synergistic action between biomaterial properties and micro- or nano- architecture has been presented to have favorable results in cell behavior [7].  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 [7], has favorable effects on cell differentiation, as does the replication of native architecture by producing aligned electrospun fibers. 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 [7].

Electrospun nanofibers for nerve regeneration

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. (1) Although there are many manufacturing options, electrospun nanofibrous scaffolds have emerged as the leading candidate for PNS regeneration for several reasons.

First, the electrospinning technique allows the production of nanofibers that accurately replicate the scale and mechanical properties of fibers found in the native neural ECM. (2) Second, electrospinning can be used on a variety of materials whether they are synthetic or natural in origin, with the option to incorporate various growth factors and other bio-functional moieties. Lastly, the electro spinning procedure is not only cheap, but also easy to execute. (3) Although a single electro spun 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. (REFS?) These materials can be categorized as synthetic, natural or composites of synthetic and natural materials.   

Synthetic Polymers

Synthetic materials are attractive candidates for neural scaffolds due to their availability, ease of manufacturing and low variability between batches. (4) 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. Synthetic composite materials also exist and have been studied in nerve regeneration. An example of such a synthetic composite is the PGLA-PCL blend developed by Subramaninan et al (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. 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. (6) 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 (7). A recent example of one such study was undertaken by Koppes et al (2011). This group synthesized aligned PLLA nano fibers conjugated with laminin proteins. Results showed that alignment of nano fibers increase neutrite extension from dorsal root ganglion compared to randomly oriented PLLA nano fibers. Koppes group also showed that a synergistic effect could be achieved by seeding aligned PLLA nano fibers and applying a small  electrical stimulation to the scaffold. 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 figure 2.

Natural Polymer

           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, alginates 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-bath variation.

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; for example, they are all biodegradable and hydrophilic, allowing for cell attachment [20].

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 FDA approval [6]. The material presents hydrophilicity [36] 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 [13]. As collagen is biologically derived, it presents the disadvantage of being potentially immunogenic [6], however, its major disadvantage lies with its weak mechanical properties [24]. A further disadvantage hurdles specifically the production of electrospun scaffolds using pure collagen, as the fibrous morphology of scaffolds, deteriorates over time [23]. 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 [25]. Properties are dependent on the composition of the blend; however, the surface chemistry of collagen confers tunable hydrophilicity [7]. 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/ carpolactone) ((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 [7]. In their work, Seyer et al., have found that peripheral nerve collagen is composed of 81% type I and 19% type III collagen [13]. 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.

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 [18] and presents enhanced mechanical properties due to eggshell-like microstructure [16]. Additionally, it promotes a regenerative response [16] is non-immunogenic [17], water permeable [18], and has a degradation profile which is controllable by the method of recrystallization. As a result, silk fibroin is a material that has been used for the production of vascular grafts [14] and cartilage repair [15] purposes amongst its other applications.

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, its has been presented that coating with fibronectin, gelatin or collagen enhance cell attachment and growth [17].

Alternative biomaterials include chitosan, a fully or partially deacetylated derivative of chitin [28,30]. 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 [37]. Chitosan is hydrophilic [29] and has antibacterial properties [31], an advantageous feature when considering its application as an implantable material for nerve tissue engineering.

The application of pure chitosan is heavily deterred due to its properties. Brittleness and rigidity arise due to strong intermolecular and intramolecular hydrogen bonding [37]. Low mechanical strength and the inability to maintain a predetermined shape on implantation under physiological conditions [32] are also significant downsides. Specifically, chitosan is normally unsuited for electrospinning due to its insolubility in most common solvents [37] and due to high repulsion between charged ionic groups in the polymer backbone under high electric fields [38]. 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 [28], tubular membrane constructs being used for rat sciatic nerve repair [32] and membranes achieving nerve regeneration and functional recovery in rat models [33].

Laminin is a basement membrane protein, which has a fibrous morphology in vivo [35], highlighting its relevance with the electrospinning method. It facilitates cell attachment, growth and migration [35]. 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 [35].

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 favors cell attachment and drives neurite outgrowth [34, 35]. 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 [34]. Haven’t been able to find the physical/chemical properties of laminin

For the subsequent research studies the following tasks will be done ASAP: 1. editing and cleanup, 2. removal of unnecessary text, 3. formal english (vocabulary / grammar), 4. cohesive flow of writing to show interrelation and meaning/significance of studies, 5. proper citing of references. Other than these, any other comment or feedback related (i.e. the content, approach, etc.) is welcomed.

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In vitro studies of aligned electrospun fibers for PNs regeneration

Aligned electrospun fibers can direct neurite outgrowth and are promising for nerve regeneration applications. Geometrical parameters such as fiber alignment, diameter and density influence nerve regeneration. Aligned electrospun nanofibers PLLA of varying diameters on NSCs and Schwann cells (SCs) from chick Dorsal Root Ganglion. Neurite outgrowth and SC migration were guided along the aligned fibers. Neurite length was 42% and 36% shorter than those on the intermediate and larger fiber substrates respectively. SCs migration did not correlate with neurite extension always. Topography alone, sometimes, is sufficient to guide neuritis without the leading support of SCs. Intermediate fibers (750nm) promoted neurite extension independent of SC migration and large fibers (1300nm) promoted neurite extension and SC migration. The packing density of fibers may also influence neurite extension and Schwann cell migration. Therefore the fiber diameter and space between fibers should be considered when constructing electrospun nanofibrous scaffolds . (65. Wang 2010)

Neural stem cells treated with retinoic acid (rNSCs). rNSCs on laminin-coated electrospun polyethersulfone (PES) fiber mesh. Average diameter (280nm, 750nm, 1500nm). rNSCs 40% increase in oligodendrocyte differentiation on 280nm and 20% increase in neuronal differentiation on 750nm compared to polystyrene surface culture. Cells stretched on other fibers at 280nm but along single fiber on larger fibers. Higher level of proliferation, migration and cell spreading and lower degree of cell aggregation observed with gradually lower fiber-diameter. rNSCs higher proliferation on laminin coated 2D surface compared to fiber-meshes in serum-free-medium with FGF-2 (20ng/ml). This shows in fiber topography plays vital role in differentiation and proliferation of rNSCs in culture. Topographical cues with biochemical signals can regulate the lineage specification of stem cells. (63..2008.Christopherson)

Mouse ESCs induced to neural progenitors by adding retinoic-acid to embryoid body culture for 4 days. ESCs induced to differentiate into specific neural lineages such as neurons oligodendrocytes astrocytes when seeded on electrospun nanofibrous scaffolds. PCL nanofibers were being used and the aligned PCL nanofibers directed the neurite.outgrowth. (61.2008.Xie)

Electrospun polyvinyl-alcohol-(PVA)/chitosan nanofibrous scaffolds synthesized with large pore sizes. PVA fibers blended with some.chitosan. Porosity measured at various depths by image analysis method. Scaffold evaluated structure physicochemical biodegradability swelling. PC12 nerve cells used found to have most balanced properties for nerve cells. Chitosan to PVA scaffolds enhances viability and proliferation of nerve.cells which increases the biocompatibility of the scaffolds with just a small percentage (number?) of chitosan to PVA scaffolds. PVA/chitosan is more promising than PVA scaffolds. (59.2011. Alhosseini)

Synthetic nerve guidance conduits(NGCs). Tissue engineering scaffold within conduit must be similar to the linear microenvironment of healthy nerve. Aligned poly(lactic-co-glycolic acid)/bioactive polyanhydride fibrous substrates were fabricated with diameters 600-/+200nm. SEM shows high-level alignment. Schwann cells and dissociated rat dorsal root ganglia elongated and proliferated parallel to oriented electrospun fibers with significantly lower Schwann cell process length and neurite outgrowth compared to random nanofibers (*). Aligned polyanhydride mat promising supplement scaffold for interior of degradable polymer NGC. Bioctive SAA (salicylic acid based polyanhydride fibers).  (54.2011)

While known that aligned fibers influence neurite outgrowth and schwann cell migration, mechanisms unclear. Thin films of aligned poly-acrylonitrile methylacrylate (PAN-MA) fibers or solvent casted smooth. PAN-MA films to study the role of differential protein adsorption on topography dependent neural cell responses. Aligned nanofiber films showed enhanced adsorption of fibronectin compared to smooth films. Fibronectin plays important role in modulating Schwann cell migration and neurite outgrowth from DRG cultures based on function blocking antibodies against cell adhesion motifs. AFM showed aligned PAN-MA fibers influenced fibronectin distribution and promoted aligned fibronectin network formation compared to smooth PAN-Ma films. In presence of topographical cues, Schwann cell generated fibronectin matrix was also organized in a topographically sensitive manner. Concluding that fibronectin adsorption mediated the ability of topographical cues to influence Schwann cell migration and neurite outgrowth, important insight for approaches to scaffold designs for peripheral nerve gaps. Fibronectin presentation, conformation and organization contributes heavily to enhanced Schwann cell migration and neurite.outgrowth on fiber-based films compared to smooth films of same composition. To develop scaffolds that match or exceed autografts deeper understanding of mechanisms which scaffold properties affect nerve regeneration. (53.2011.Mukhatyar)

Neurite outgrowth on electrospun nanofibers with uniaxial alignment. Effects of varying fiber density, surface coating and supporting substrate. Neurite outgrowth in the direction of nanofiber alignment although resembles native structure of nerve tissue is more complicated than just guiding them along them. DRG as model system to study interactions between neurites and uniaxially aligned nanofibers. Study showed that neurites can grow parallel or perpendicular to the aligned nanofibers depending on these factors: density of nanofibers, protein deposited on the surfaces (surface chemistry) of the nanofibers and surface properties of the substrate on which the nanofibers were supported. Myosin II inhibition on the nanofiber guided growth of neurites by adding blebbistatin to the culture medium. Myosin II could play role in perpendicular contact guidance but not in parallel contact guidance of neurite outgrowth. These finding will offer new insights to the design of nanofiber based scaffolds for nerve repair and provide new guidelines for construction of neuronal network architecture (neural circuits). (48.2014.Xie.)

Bridging larger nerve gaps between proximal and distal ends needs exogenous tubular constructs with uniaxially aligned topographical cues to promote the axonal regrowth due to the lack of fibrin cable formation. Aligned and random PLGA-PCL (blend) nanofibers with diameter 230 -/+ 60nm have been electrospun. DSC is the technique that was used to confirm homogeneity of blending PLGA-PCL. Alignment of fibers is quantified by calculating relative angle of each fiber. Tensile strength, porosity, contact angle and biodegradation of uniaxial PLGA-PCL nanofibers measured and compared with corresponding random fibers. Pore size, Young’s modulus and degradation of aligned scaffold significantly less than random fibers (p<0.05). Schwann cell adhesion guided along direction of aligned nanofibers. In vitro cell adhesion and proliferation of Schwann cells on aligned nanofibers evaluated and compared with random nanofibers. Significantly higher number of cells on aligned scaffolds compared to random fibers. Study shows aligned nanofibers better flexibility, lesser pore size with adequate porosity, slower degradation rate, topographical cues, better cell-scaffold interaction, contact guidance and proliferation than random fibers. Results show alignment influences Schwann cells’ adhesion and proliferation. So, axially aligned nanofibers may mimic the fibrin cable architecture, making it ideal scaffold for axonal growth. (37.2012.Subramanian)

Schwann cell growth and myelin sheath regeneration using Poly(Hydroxyalkanoate) composite scaffold. Scaffolds with cells is a combination approach. Electrospun scaffold was done by blending poly(3-hydroxybutyrate) (PHB) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in different compositions in order to investigate myelin membrane surrounding nerve axons. Increasing PHBV decreased diameter of nanofibers based on SEM. Blend composition, fiber alignment and collagen integration on Schwann cells function studied. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited bipolar morphology that oriented along the fiber direction while SCs grown on random fibers showed multipolarity. Collagen in nanofibers increased SCs proliferation on day 14, GDNF gene expression day 7 and NGF secretion day 6. Aligned PHB/PHBV potential scaffolds and collagen type 1 improves cell differentiation. Aligned PHB/PHBV nf scaffolds showed higher SCs proliferation after 14 days compared to random nf. Collagen incorporation to the blend increased SCs proliferation as well as neurotrophin secretion and GDNF expression. (36.2013. Masaeli)

2D random and 3D longitudinally oriented nanofibers of poly(lactide-co-glycolide) (PLGA) studied surface morphology, mechanical properties, porosity, degradation and wettability and shown that the orientation of the fibers plays a major role in these properties. Average diameter of random PLGA was 197-/+72nm and aligned PLGA was 187 -/+ 121nm. Pore size of aligned was significantly lower than random nanofibers but percentage porosity of both scaffolds was comparable. Random PLGA degraded faster than aligned PLGA. Tensile strength and young’s modulus of random PLGA significantly higher than aligned PLGA. Schwann cells cultured in vitro show that aligned fibers assist the direction of Schwann cells and increased proliferation than random fibers. Aligned nanofibers have better deformability, slow degradation, comparable porosity and orientation cues compared to random nanofibers. (35.2011.Subramanian)

Aligned and random 25:75 wt% silk fibroin/poly[(l-lactic acid)-co-(epsilon-caprolactone)] SF/P(LLA-CL) nanofibrous scaffolds where the mechanical properties of aligned scaffolds present a strong anisotropy with much higher tensile strength in parallel than in perpendicular direction. Schwann cell viability studies show that aligned scaffolds significantly promote cell growth and the direction of SC elongation is parallel to the direction of fibers for aligned scaffolds. (34.2013.Zhang)

Polymer blending is one of the most effective methods for providing new, desirable biocomposites for tissue engineering applications. Random and aligned Poly(epsilon-caprolactone)/gelatin PCL/gelatin biocomposite nanofibrous scaffolds with different PCL/gelatin ratios: 50:50 and 70:30. Chemical and mechanical properties were measured. PCL/gelatin 70:30 nanofiber showed the most balanced properties meeting all the necessary specifications for nerve tissue and was used in vitro with nerve stem cells (C17.2 cells). 70:30 enhanced nerve differentiation and proliferation compared to PCL scaffolds and enhanced neurite outgrowth. Neurite outgrowth and nerve cell elongation was parallel to the direction of fibers. (29.2008.Mobarakeh)

In this study, the authors fabricated electrospun gelatin and hyaluronan-gelatin (HA-Gel) composite nanofibers to deliver suitable growth environment for Schwann cells. The fiber diameters of Gel, 0.5 HA-Gel, 1 HA-Gel, 1.5 HA-Gel were 130-/+30nm, 294-/+87nm, 362-/+129nm and 224-/+54nm respectively. RT4-D6P2T rat Schwann cells were cultured in vitro. Cell attachment and proliferation rates were not significantly different on these matrices. Schwann cells with HA-Gel showed better organized F-actin than SCs with Gel. Also, the expression level of several genes and proteins including Nrgl and P0 were significantly higher on HA-Gel than on Gel. These markers indicate the favourable metabolic changes that occur in Schwann cells in HA-Gel which is important in understanding peripheral nerve regeneration. (22.2013.Liou)

Biggest challenges in peripheral nerve in tissue engineering is to make artificial nerve graft that mimics well the ECM and assists in nerve regeneration. Nanotopography and orientation of the fibers in scaffolds affects significantly the nerve cell morphology and outgrowth and alignment of fibers affects guidance of cells (aligned fibers provide contact guidance of the cells). P(LLA-CL)/Collagen I/Collagen III biocomposite scaffolds on NSCs C17.2.  P (LLA-CL)/collagen I/collagen III is compared to P (LLA-CL) scaffolds and aligned fibers were compared to random nanofibrous scaffolds. Aligned P(LLA-CL)/Collagen I/Collagen III with diameter ~250nm and tensile strength of 11.59-/+1.68MPa, showed 22% increase in cell proliferation compared with aligned pure P(LLA-CL) scaffolds. The semi-synthetic compositions showed better cell proliferation compared to the purely synthetic and aligned fibers were superior to random fibers. Aligned P(LLA-CL)/Collagen I/Collagen III showed both higher proliferation and improved cellular phenotype of NSCs which indicated that it was the most suitable. Therefore, the collagen blended with this polymer increases cell proliferation on scaffold. (20.2012.Kijenska)

Both random and aligned nanofibers of PHBV and PHBV/Collagen have been fabricated through electrospinning with average diameters of 438nm and 231nm respectively. Nerve cells (PC12) have been cultured and seen how biocompatible they are and their neurite extension assessed by immunostaining techniques. Cell proliferation of PHBV/ColI50:50 was 40.01% higher than pure PHBV nanofibers. PHBV/ColI75:25 had 5.48% higher proliferation of nerve cells than pure PHBV. Aligned nanofibers provided contact guidance and directed the orientation of the nerve cells along the direction of the fibers and produced elongated cell morphologies exhibiting bipolar neurite extensions as required for nerve regeneration. Aligned biocomposite nanofiber was more promising than random nanofibrous scaffold for nerve regeneration. Nerve cells showed multipolar phenotype on the random nanofibers. (19.2013.Prabhakaran)

More than a decade old study but significant its findings stand relevant up to today when PLLA was electrospun to aligned and random nanofibrous and microfibrous scaffolds on NSCs. The study clearly showed that aligned nanofibers were favorable  [poly (L-lactic acid) (PLLA). Aligned nanofibers showed NSC elongation and neurite outgrowth matching the direction of the aligned fibers and no effect did fiber diameter have on cell orientation and neurite outgrowth. Also NSC differentiation rate was higher for nanofibers than for microfibers but unrelated to fiber alignment. (15.2004.Yang)

Another study focus on the molecular aspects of the aligned electrospun nanofibers on NSCs. The study aimed to differentiate NSCs into motor neurons with PLLA/Gel non-woven material which has degradation rates and mechanical properties similar to peripheral nerve tissue. Retinoic Acid (RA) and purmorphamine were incorporated into the scaffold and were released in a control manner differentiating the seeded NSCs into β-III-tubulin, HB-9, Islet-1 and choactase-positive motor neurons. The non-woven material assisted in the differentiation into motor neuronal lineages and encourage neurite outgrowth. (13.2013.Binan)

In vivo studies of aligned electrospun fibers for PNs regeneration

[[ *probably remove this study, unnecessary.   More than a decade study. Nerve GF(poly(phosphoester)) within NGCs. NGF containing polymeric microspheres fabricated from a biodegradable poly(phosphoester) (PPE) polymer were loaded into silicone or PPE conduits to provide for prolonged, site specific delivery of NGF. Conduits used for 10mm gap in rat sciatic nerve model. 3 months post-transplantation, morphological analysis revealed higher values of fiber diameter, fiber population and fiber density and lower G-ratio at the distal end of regenerated nerve cables collected from NGF microsphere-loaded silicone conduits, compared with control conduits loaded with either saline alone, BSA microspheres or NGF protein without microencapsulation. Beneficial effects noted on Fiber diameter, G-ratio and fiber density observed in permeable PPE NGCs. Results confirm long-term promoting effect of exogenous NGF on morphological regeneration of PNs. This approach (microsphere protein release system incorporated into NGCs) has great promise.  (50.2003.Xu)]]

Chitosan nanofiber mesh tube on schwann cell alignment and rat sciatic nerve defect maybe a promising substitute for autogenous nerve graft. Oriented chitosan nanofiber tube showed better results than non-oriented both for schwann cells and sciatic nerve. (64.2008.Wang)

NGFs into aligned core shell nanofibers by coaxial electrospinning and created aligned fibrous nerve guidance conduits (NGCs). PLGA/NGF NGC on 13 mm rat sciatic nerve defect. In 12 weeks post implantation, PLGA/NGF NGC showed significantly better results than the PLGA NGC group but no significant difference was observed compared to the autograft group. In the PLGA/NGF group, more nerve fibers regenerated and the regenerated nerves were more mature than PLGA group. (62.Wang-2010)

PLLA (biodegradable but not bioactive) with oligo(D-lactic.acid)(ODLA) nerve conduit with AG73 peptide (*). Transplanted at 10mm gap of rat sciatic nerve. Six months, electrophysiological evaluation showed better functional reinnervation than silicone tube or unmodified PLLA conduit. Degradation ratio of the PLLA/ODLA-AG73 conduit should be optimised to suppress unwanted reinnervation with surrounding muscles. (58.2011.Kaklinoki)

Collagen/poly(ε -caprolactone) nerve-conduit with tailored degradation rate. Collagen/PCL promoted schwann cell adhesion, elongation and proliferation. In vivo successful regeneration through 8mm sciatic nerve gap in adult rats achieving similar electrophysiological and muscle reinnervation results as autografts. Regenerated nerve fibers still in pre-mature stage 4 months postoperatively, the implanted collagen/PCL-nerve conduits helped more axons regenerating through the conduit lumen and gradually degraded at great similar speed of nerve regeneration rate. Collagen/PCL porous nerve conduit high surface area (*). Future studies shall incorporate growth factors and cells, cell electrospinning and adding electrical materials as well. (55.2011.Yu)

Electrospinning was used to fabricate nerve conduits from poly (l-lactide-coglycolide) -silk.fibroin) PLGA-silk fibroin nanofibers. 10mm defects in sciatic nerves of Sprague-dawley rats. The nerve conduits were transplanted into the defect area of the nerve and six weeks after operation; morphological and functional assessment showed nerve conduits from PLGA-silk fibroin grafts promoted the regeneration of peripheral nerves. Similar to nerve autograts and promising alternative to it. PLGA-SF NCs can mimic ECM architecture and promote cell attachment. Benefits over autografts include: fabrication different dimensions degradation rate, mechanical properties, microarchitectures and not being subject to limited supply or permanent loss of nerve function and morbidity at donor site and need for multiple surgeries. Histological assessment & TEM studies showed similar PLGA-SF NCs with autografts. However, overall results were not better than those using autologous nerve grafts in situ so more studies are needed for this type of materials to develop artificial nerve bridging.substitute for nerve grafts. (52.2012.Li.)

Artificial nanofibers nerve guidance conduits are of interest in bridging nerve gaps and associated peripheral nerve regeneration because of high surface area, flexibility and porous structure. Electrospun poly(epsilon-caprolactone)/gelatin PCL/Gel nanofiber mats were fabricated, rolled around a Cu wire and fixed by medical grade adhesive to obtain tubular shaped biograft. This was in order to bridge 10mm sciatic nerve gap in rat models. SCs from human exfoliated deciduous tooth (SHED) were transplanted to the site of nerve injury through the nanofibrous nerve guides. Nerve gap was grafted using 1. nanofiber nerve guide, 2. Nanofiber nerve guide seeded with SHED, 3. Suturing, and untreated nerve gap was negative control. Cell culture carried for SHED-nanofiber interaction and its viability within the nerve guides after 2 and 16 weeks of implantation. Walking track analysis, plantar test, electrophysiology and immunohistochemistry performed to evaluate functional recovery of nerve regeneration. Vascularization was studied by H&E staining. Results showed SHED seeded on nanofibers NG can survive and promote axonal regeneration in rat sciatic nerves, whereby the biocompatible PCL/Gel nerve guide with cells can support axonal regeneration and is promising tissue engineering graft for PN regeneration. [[(they did nerve guides with pcl/gel nanofibers tested its effects with and without SHED on that sciatic nerve. Controls were sutured sciatic nerve and untreated sc.n. NG with SHED was superior to the one without as for nerve regrowth, functional and sensory recovery and histological assessment.]] This could be due to the expression of neurotrophic factors related to neural crest origin of these cells. (40.2014.Beigi)

Micro- nanofiber tubes for sciatic nerve transection regeneration. Electrospun tubes composed of biodegradable polymers (PLGA/PCL blend) with no coating or drug loading for 10 mm nerve gap regeneration of sciatic nerve in a rat. Four months after surgery, sciatic nerves did not reconnect in the control group (lesioned animals without the treatment) whereas in most animals of the experimental group the electrospun tubes induced nervous regeneration and functional reconnection of the two severed sciatic nerve tracts. Myelination and collagen IV deposition have been found together with the regenerated fibers. Reinnervation has also occured of the target muscles in the majority of the treated animals. Guidance conduits can be loaded with various fillers like collagen, fibrin or self-assembling peptide gels or loaded with neurotrophic factors and seeded with cells. Nervous projections were mostly aligned with the longitudinal conduit axis. The fibrous micro-structure provides mechanical stability to soft tissues while nanostructure adds more substrate surface for cell attachment (higher cell density per unit of space).  (23.2008.Panseri) *

Aligned silk fibroin (SF) blended with poly(l-lactic acid-co-ε-caprolactone) (P(LLA-CL)) and aligned P(LLA-CL) without silk fibroin reeled into aligned nerve guidance conduits. SF/P(LLA-CL) NGC was used to bridge a 10mm defect in the sciatic nerve of rats which was assessed 4 and 8 weeks post-transplantation. The SF/P(LLA-CL) NGC was significantly better compared to the P(LLA-CL) in all the results. (6.2010.Wang)

Conclusion

As has been remarked in wisdom by the Greek philosopher Aristotle, perfection and excellence is achieved in appropriate measure and moderation. The outstanding qualities of synthetic polymers in the way of mechanical capability, although to be commended and utilized, fall short of the requirements in surface chemistry and cellular interaction in the attempt to replicate and repair that which evolution has honed over the passing of millennia. Much as every achievement does on the extreme end of the scale, natural materials, be they derived from the human, animal, or insect, are deposited as tissues grow and mature, thus sacrifice their own mechanical properties for their more important function of interacting with the cell. It is to be recognized, that in the same way that all excellence is achieved in moderation, neither synthetic nor natural materials possess the exact required peculiarities to bring about peripheral nerve regeneration individually. Hence, in the way of moderation, and in the biomaterial aspect of this review, it is presented by the authors that blending of natural and synthetic polymers is the best possible choice to be made when attempting to engineer nerve tissue. As the desired properties are dependent on the location at which we attempt to treat, it stands to reason that specific mechanical properties must be tuned for each scenario. It should in no way be left unstated that the individual beneficial effects of natural biomaterials must also supplement each other in order to bring about an enhanced effect, as the attempt to engineer nerve tissue should be an attempt to mimic the chemistry of native tissue as mush as its physical make up. 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 lie.

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