Repair strategies for injured peripheral nerve: Review

Life Sciences 243 (2020) 117308

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Repair strategies for injured peripheral nerve: Review a,⁎

b

a

Chand Raza , Hasib Aamir Riaz , Rabia Anjum , Noor ul Ain Shakeel a b

T a

Department of Zoology, Government College University, Lahore 54000, Pakistan Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Surgical nerve repair Nerve vasculature Nerve guidance conduits Axon guidance

Compromised functional regains in about half of the patients following surgical nerve repair pose a serious socioeconomic burden to the society. Although surgical strategies such as end-to-end neurorrhaphy, nerve grafting and nerve transfer are widely applied in distal injuries leading to optimal recovery; however in proximal nerve defects functional outcomes remain unsatisfactory. Biomedical engineering approaches unite the efforts of the surgeons, engineers and biologists to develop regeneration facilitating structures such as extracellular matrix based supportive polymers and tubular nerve guidance channels. Such polymeric structures provide neurotrophic support from injured nerve stumps, retard the fibrous tissue infiltration and guide regenerating axons to appropriate targets. The development and application of nerve guidance conduits (NGCs) to treat nerve gap injuries offer clinically relevant and feasible solutions. Enhanced understanding of the nerve regeneration processes and advances in NGCs design, polymers and fabrication strategies have led to developing modern NGCs with superior regeneration-conducive capacities. Current review focuses on the advances in surgical and engineering approaches to treat peripheral nerve injuries. We suggest the incorporation of endothelial cell growth promoting cues and factors into the NGC interior for its possible enhancement effects on the axonal regeneration process that may result in substantial functional outcomes.

1. Introduction The ultimate goal of the peripheral nerve repair strategies is to achieve maximal functional regains and shortening the recovery duration. Billions ($150) of dollars are spent annually for the treatment and repair procedures of injured peripheral nerve in the United States [1]. Although mounting evidence suggests the substantial functional regains in mild and moderate nerve injuries through pharmacological, medicinal plant-derivatives and genetic manipulations [2–4], however the application of said strategies is limited in severe nerve trauma. Thus, surgical engineering and combined-therapy approaches are employed in severe nerve injuries to cope the grim situation [1,5–8]. Nerve injuries are classified based on the extent and severity of damage caused [9]. Neuropraxia is the mildest and 1st type of nerve injury and involves no damage to the nerve, except transient changes in myelin and altering the activities of the ion channel to block the nerve conduction. Axonotmesis is the 2nd type of nerve injury and involves the disruption of axonal components of the nerve. Discontinued axons in the distal stump need to be cleared through a process of Wallerian degeneration before the advancements of the regenerating nerve fibers. Neurotemesis is the 3rd and most severe type of nerve injury and involves the complete transection of the nerve trunk, recovery is nearly ⁎

impossible without surgical intervention [10]. Nerve fiber bundles (fascicles) constitute the nerve trunk, the failure to re-establish the correct fascicle alignment following transection injury is a critical defect leading to poor functional outcomes [11]. It necessitates the advances in microsurgical procedures for effective repair strategies. 2. Surgical repair Conventional direct suturing involves the end-to-end fascicular or epineural suturing, however, if the gap between nerve stumps is > 5 mm, then grafting or conduits are installed keeping in view the relevant considerations. Importantly, nerve transfer has become an effective and preferred clinical strategy for proximal upper limb nerve injuries [12]. 2.1. End-to-end neurorrhaphy After the removal of the necrotic tissues from nerve ends, the approximation and alignment of nerve fascicles and nerve vasculature is made and finally the epineurial sutures (for example 10–0 sutures) are applied to avoid malrotation of the transected nerve ends. Either separate sutures or the continuous sutures are applied to stitch the nerve

Corresponding author. E-mail address: [email protected] (C. Raza).

https://doi.org/10.1016/j.lfs.2020.117308 Received 26 October 2019; Received in revised form 9 January 2020; Accepted 13 January 2020 Available online 15 January 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.

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protected) distal stump of common peroneal nerve was cross sutured with freshly transected tibial nerve, higher numbers of the regenerated motor axons were observed [25]. It suggests the effective restoration can be enhanced through preventing the distal nerve atrophy and thus may enhance the functional recovery in proximal nerve injuries and may overcome the distance and time constraints. Nerve transfer in clinics is gaining increased attention as a time-effective strategy to repair the high ulnar nerve injuries. The ulnar nerve fascicle transfer to the biceps in 32 patients experiencing upper nerve-root brachial plexus injury revealed a good or fair (grade-4 or grade-3) recovery in biceps strength of 30 patients in 5-months duration [26]. In another study (on 78 patients), 92% of the patients regained the intrinsic functions over an average duration of 3.7 months following an end-to-side anterior interosseous to ulnar motor nerve transfer [27]. Although nerve transfer allows the early restoration of injury induced lost functions, yet it requires the specifics of the physiological status of the donor nerve (from electromyographer) and precise nerve reconstruction microsurgery. Further, it is strictly monitored for electromyographic assessment of functional regains.

ends [13,14]. End-to-end suturing of the transected ulnar nerve demonstrated comparable thick fiber diameters 3–4 months following surgery to that of control rats [15]. It suggests the application of direct suturing to the transected nerve ends leads to substantial regeneration. However, suturing the transected nerve segments in proximal injuries reveals limited regain in motor functions and higher percentages of denervated neuromuscular junctions, as regenerating axons must reach the target tissues within a time window, beyond which recovery is compromised [16]. Thus, end-to-end suturing of the nerve is widely dependent on the site of injury and distance from the injury site to target tissues. 2.2. Nerve grafting The “gold standard” of the defected nerve repair is autologous nerve grafting, a technique involving the surgical excision of nerve such as cutaneous nerve (medial or lateral antebrachial, ulnar, femoral or radial nerve branches) and installation to the injured nerve gap such as damaged brachial plexus motor nerves in the same subject [17]. However, associated challenges include the donor-site morbidity, differences in the size and structure of the tissue and a second surgery. To overcome the graft-based limitations, the allografts (donor nerve) or cadaveric nerve tissues are considered as ideal alternatives, and individuals receiving such grafts undergo immunosuppression medication to avoid graft rejection [18]. The nerve grafting, being the gold standard for transected gap nerve injuries, is subjected to tremendous improvements. The development of vascularized nerve grafts provides advantages of supplying nutrients and oxygen to poorly vascularized regions of transected nerve over the non-vascularized nerve grafts (VNG), and hypoxia is avoided [19]. Patients experiencing failure of sensory recovery following conventional nerve grafts, when installed with the VNGs in upper extremity injury sites enjoyed good to excellent sensory recovery [19]. It provides a glimpse of better functional regains with the vasculature-augmentation of installed graft. With the development of a nerve graft lacking cells (acellular graft), the need of immunosuppression is eliminated. Decellularization of nerve segment is widely achieved through detergent treatment without altering the internal structure and basal lamina of the nerve [20]. Enhanced vascularization in the acellular nerve graft, installed in 10-mm sciatic nerve gaps in rat suggests blood microvasculature growth permeates and supplies nutrients to support the regeneration of NF-200 positive axons. However, the autologous graft installed group reveals higher levels of axonal regeneration comparing to acellular graft [21]. Importantly, decullularized nerve grafts provide opportunities for fabrication of regeneration-supporting cells into the lumen of the graft. The incorporation of dental pulp stem cells (DPSCs) into acellular nerve graft enhances the regenerative capacities comparing to the naïve acellular nerve graft in 10-mm sciatic nerve gap of rabbits. The persistent supplies of the growth factors in DPSCs rabbits group suggest higher regeneration supporting microenvironment comparing to naïve grafts [22].

3. Engineering approaches for nerve repair Emerging incidence of nerve injuries and limitations of nerve supplies for grafting and nerve transfer has led to the enormous advances in engineering approaches to use biocompatible, non-immunogenic and biodegradable (natural or synthetic) materials to facilitate nerve repair processes. 3.1. Fibrin glue Fibrin protein component of the glue self-assembles to form a polymeric network when applied directly to the transected nerve ends and alleviates the needs of the microsurgical procedures which require precise end-to-end nerve suturing or nerve grafting. Fibrin glue bonded nerves are reported to have faster recovery rates, as significantly higher axon density, length and branches of axons compared to end-to-end sutured nerves in rats [28]. Additionally, fibrin glue in combination with nerve growth factor, poly(lactic-co-glycolic acid) and polyglycolic acid has successfully used to repair peripheral nerves in rodents [29,30]. A recent study reports the fibrin glue coupled delivery of mesenchymal stem cells (MSCs) to the injured nerve area for augmenting nerve regeneration [31]. 3.2. Cell-based techniques Under chronic denervation failure of the regenerative environment in the distal axonal segments is reported. The probable reason is the progressive loss of trophic support (such as nerve growth factor) of distal nerve SC, leading to the failure of target re-innervation [32]. Cellbased techniques aim to augment the regeneration process of the injured nerve, in an attempt to regain functional recovery. The nerve growth factor, vascular endothelial growth factor, brain derived nerve growth factor and glial cell derived growth factors from such cells may enhance the nerve regeneration [33]. Cultured SCs administration to the distal stump of end-to-end sutured transected nerve enhances the axonal regeneration in rats [34], seemingly through augmenting trophic support. The limitation of primary SC harvest and culture for cell-therapy is markedly resolved through the production of self-renewing SC precursors from human pluripotent stem cells (hPSCs), enabling rapid expansion and timely availability for applications [35]. Comparatively, olfactory ensheathing cells secrete nerve growth factor and their application to an injured nerve enhances functional recovery following peripheral nerve damage in rats [36]. The bone marrow stromal cell-induced SC administration to the injured median nerve of non-human primate accelerated the functional recovery through

2.3. Nerve transfer Nerve transfer, a technique used to lessen the impact of proximal nerve damage and fasten the reinnervation of denervated target is practiced in brachial plexus injuries and hand injuries (Fig. 1). The fascicle from the healthy nerve is redirected to the distal portion of the injured nerve, in proximity to the neuromuscular hilum [23]. In contrast to nerve grafting, it relaxes the time window required to perform surgery and allows for nerve reconstruction over a longer span (around 4 months) [24]. The distal segment of freshly transected rat common peroneal nerve was inserted into intact tibial nerve and following 3months, electromyography studies revealed muscular contractions after the stimulation. Interestingly, when chronically denervated (but 2

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Fig. 1. Surgical strategies for nerve repair. A. Direct end-to-end suture repair. Illustration of the epineurial sutures for neurorrhaphy B. Nerve grafting. Process of nerve grafting indicating the donor nerve installation at gap injury site C. Nerve transfer. Transfer of the intact nerve fascicle to the distal stump of the transected nerve D. Nerve guidance conduit installation. Schematic representation of the implantation of the NGC to nerve gap injury.

support as well. Recently, conduits are constructed from electroconducting polymers, such as polypyrrole, poly (3,4-ethylenedioxythiophene) and polyaniline to offer chemical properties for the propagation of electrical signals [43]. Additionally, tissue-derived conduits involving the porcine small intestine submucosa (SIS) is gaining increased attention to be used as biomaterial for NGC. The SIS composition reveals collagen rich ECM, > 90% collagen contents are of types I and III, with excellent biocompatibility and non-immunogenicity [44]. A study reveals the SIS as an exceptional biomaterial (for developing a nerve guidance conduit) to enhance the peripheral nerve regeneration following its installation in 5 mm rat sciatic nerve gap [45]. The rationale behind designing NGCs is to direct the cellular elements of the proximal nerve stump towards respective paths for successful target re-innervation. The major challenge of avoiding off-target re-innervation still persists in the case of hollow conduits despite limiting the nerve scar and inflammation [46]. Mounting evidence unveils that the axonal growth cone (distal tip expansions of regenerating axons) extends temporary processes known as lamellipodia and filopodia to interact with the extracellular matrix (e.g., collagen, laminin) for pathfinding and extension [47]. Thus, the conduit interior with ECM components or guidance scaffolds likely to have tremendous potential for supporting regeneration of higher axon density [48]. Additionally, the interior of conduit may be coated with topographical cues such as Arg-Gly-Asp (RGD), a tripeptide identified from ECM of the nerve believed to be required for axonal guidance [49]. A variety of the materials currently used to build NGC, could be considered as a measure to access the development and active research in the NGC-based neuroregenerative investigations (Table 1). A variety of synthetic and biologically-derived polymers have been fabricated to yield a diversity of NGCs. The challenge of limited availability of the donor nerve is successfully coping with the unlimited supplies of the synthetic polymers. Owing to the known properties and manufacturing protocols, the required properties of mechanical strength and controlled biodegradation are successfully optimized using synthetic

improvements in electrophysiology, behavior and histology with no signs of abnormal proliferation [37]. A recent study suggests the incorporation of MSCs directly to the traumatic injury site using fibrin glue to stimulate myelination and axonal growth for enhanced functional regains [31]. Additionally, the intravenous and injury site administration of bone marrow-derived MSCs enhanced the density of regenerating axons and contributed in functional regains [38]. The presentation of mesenchymal characters from dedifferentiating SC isolated from wound and distal nerve stumps signifies and points to the relevance and scope of the use of MSCs based cellular strategies for effective nerve repair [39]. The neural crest cells (NCC) derived from human periodontal ligament has great potential to express the markers of neural and glia cells [40] to meet the supply needs and substitute the primary SC in nerve repair strategies. 4. Nerve guidance conduits Transection injuries without gaps could be dealt with surgical techniques, however, injuries with gaps (> 5 mm) involve installation of nerve guidance conduits (NGC) [41]. Transected nerve stumps are reported to join together through a heterogeneous structure largely composed of extracellular matrix known as “the bridge” and could be several millimeters in length. However, the bridge offers a non-directional environment for the axonal regrowth, and misdirection could result [42]. Thus, conduits precisely offer an advantage over natural healing/regeneration process of injured nerve by guiding the advancing axons to distal stump. The NGCs engineering reveals tremendous structural development as First generation conduits were hollow tubes developed from synthetic materials such as silicone or polytetrafluoroethylene and were removed later owing to their non-resorbable nature. Later, Second generation conduits were developed as hollow tubes from polyglycolic acid, poly(lactic-co-glycolic acid), polyvinyl alcohol, type I collagen and silk fibroin mostly with short degradation time (4 to 8 months). Third generation NGCs were engineered in such a way to incorporate the cellular elements such as SC to provide trophic 3

3–4 mm diameter processed nerve

5 cm long Saphenous vein filled with SCs Extracellular matrix, semi-permeable, AxoGuard (FDA approved)

Processed nerve

Vein graft Porcine small intestine submucosa

4

1.1 cm long PCL conduit (Neurolac™)

1.5 cm long PCLEEP tube conjugated with GDNF

PDLLA with polypyrrole conduit, 1 cm long

2-4 cm long PHB nerve conduits 1 cm long PU nerve conduits

Silk Fibroin

Synthetic PCL

PCLEEP

PDLLA

PHB PU

Rabbit, Peroneal nerve Rat, Sciatic nerve

Rat, Sciatic nerve

Human, arm nerve lesions Rat, Sciatic nerve

Rat, Sciatic nerve Macaque, median nerve Rat, Sciatic nerve

Human, Digital and Ulnar nerves Rat, Sciatic nerve

Goat, Peroneal nerve

Human, sural nerve allografts Human, large caliber nerve Rabbit, sciatic nerve Rat, sciatic nerve

Animal, nerve

Peroneal nerve 2-4 cm long Sciatic nerve, 10 mm gap

Sciatic nerve 10 mm gap

Fingers, palm, wrist, forearm, elbow nerves, 11.03 mm gap Sciatic nerve, 15 mm gap

Sciatic nerve, 10 mm gap

Sciatic nerve, 10 mm Gap Median nerve, 10 mm gap

Sciatic nerve, 10 mm Gap

Digital nerve, 30 mm gap

Peroneal nerve, 30 mm gap

Upper extremity nerve injury, 33 mm (mean) gap Sciatic nerve, 4 cm gap Sciatic nerve, 15 mm gap

Brachial plexus injury

Injury/repair

4 months 0.75–1.5 months

3–6 months

3 months

21.9 months

3 months

12 months 12 months

3 months

12 months

12 months

2 months 4 months

13 months

12 months

Experiment duration

44% Electrophysiological recovery in GDNF-PCLEEP installed rats Comparable functional recovery with autologous nerve graft Enhanced myelination and reduced loss of muscle mass Augmented functional recovery and cross-sectional area of nerve

64.62% grip strength regain in ipsilateral side

Higher density of myelinated fibers in collagen filled silk conduits

Successful nerve regeneration and muscle atrophy reduction Enhanced functional recovery and regeneration Larger nerve area in keratin hydrogel filled conduit

Comparable electrophysiology, histology and behavioral outcomes with autologous nerve grafts 80% patients regained sensory recovery in study duration

Myelinated nerve fibers in SC-filled vein grafted groups Higher axonal density comparing to PCLA conduits

Augmented functional recovery with limited immunosuppression 67% and 85% recovery of sensory and motor functions

Outcome

[72] [73]

[71]

[70]

[69]

[68]

[66] [67]

[65]

[64]

[63]

[61] [62]

[60]

[59]

Ref.

Abbreviations: PCL, poly(caprolactone); PLGA, poly(lactic-co-glycolic acid); PDLLA, poly-DL-lactic acid; PHB, poly(3-hydroxybutyrate); PLLA, poly-L-lactic acid; PU, polyurethane; PCLEEP, poly (e-caprolactone-co-ethyl ethylene phosphate.

15 mm long gelatin conduit containing fibers 1 cm long collagen conduit filled with keratin hydrogel 1 cm long collagen filled Silk fibroin based conduit

Gelatin Keratin

Fibrin

Collagen

3.4 cm long chitosan conduits filled with bone marrow cells 3 cm long collagen fiber filled collagen type I & III conduits (Collagen type I FDA approved) 1.4 cm long fibrin conduit filled with MSCs

2–3 cm long sural nerve grafts

Autogenic/allogeneic Nerve

Biopolymer Chitosan

Composition/design

Material

Table 1 Material, design and applications of currently available NGCs.

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Fig. 2. Strategies for augmenting regeneration of peripheral nerve through NGC. Illustration of different approaches for enhancing nerve regeneration through engineered NGC.

glycolic acid), polycaprolactone, silicone and gelatin hydrogels materials to yield NGC is successfully achieved for pre-clinical studies. The installation of such NGC in rat sciatic nerve gap (1 cm) revealed augmented functional recovery regains [55]. The surface area of the conduit interior is augmented through the application of fillers, thus enhancing the chances of attachment for a considerable number of cellular elements comparing to the hollow conduits. Conduit fillers may include SC, growth factors, gels, laminins, co-polymers (poly (lactic-co-glycolic acid) and polyurethane in the form of gel rod, gel layer, electro-spun fiber layer, microsphere-embedded layer, sponge, fiber mat roll or aligned nano-filaments [30,56]. In a study, a silicone based conduit filled with collagen containing allogeneic SC was installed in 12 mm sciatic nerve gap of rat. Functional evaluations revealed significant improvements in rats installed with filled conduits comparing to hollow silicone conduits [57]. Another study reports the enhancement of sensory functional recovery in aligned poly-L-lactide-co-caprolactone nanofiber scaffold in rats [58]. The application of NGC in clinics has grown since last three decades. The silicone conduits installed in 3 mm to 5 mm median nerve gaps of patients and muscle contraction force was evaluated following 3-years and revealed the muscles regained significant power [74]. Similarly, silicone tube installation in median and ulnar nerve injuries with up to 30 mm gaps demonstrated better functional results [75]. Expanded

polymers. A wide range of such polymers has been developed and employed in animal models with nerve transection and gap injuries. The non-degradable polymers (such as polyethylene, polypyrrole and poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) and biodegradable polymers (such as poly (ε-caprolatone), poly (lactic-co-glycolic acid), poly (ethylene glycol) and poly (ethylene oxide)) are extensively studied and fabricated for in vivo applications [50]. Three types of natural polymers namely polysaccharides (alginate, chitin, chitosan and hyaluronic acid), proteins (silk fibroin, collagen, fibrinogen, elastic and keratin) and polyesters (poly (3-hydroxybutyric acid-cp-3-hydroxyvaleric acid) are explored for NGC preparations [50]. Natural polymers offer excellent biocompatibility and cell attachment support, however, often tend to degrade at a faster rate, however require extensive purification [51,52]. With the development of three-dimensional (3D) bioprinting technology, it may be possible to engineer semi-permeable nerve guidance conduits to facilitate the vascularization, mass transport of materials and establishing regenerative conducive microenvironment [53]. In principle, 3D bioprinting involves the construction of layer-by-layer biological ingredients (such as biomaterials, growth factors and living cells) to engineer 3D structures. The biomaterial along-with the growth factors and cellular elements are integrated in the form of a liquid, known as bio-ink [54]. 3D bioprinting of alginate, poly(lactic-co5

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Fig. 3. Proposed strategy for NGC engineering. Fabrication of angiogenic cues, SC facilitation processes and augmenting axonal regeneration in the hydrogel containing aligned nanofibers or nanotubes could optimally enhance the regeneration of nerve transection gap injuries and enhance timely re-innervation of regenerating axons to establish functional synapses with target tissues.

neurotrophin-3 in rabbit facial nerves facilitated the re-innervation of axons demonstrating promoting effects of engineered NGCs [80]. Similarly, electroconductive conduits supported sciatic nerve regeneration in rats as assessed 8-weeks post-installation [81]. Topographical cues such as RGD-laden conduits installation enhanced the cell survival and axon extension from human neural stem cells (hNSC) [49]. Although NGCs application results in substantial improvements in the regeneration of transected peripheral nerves with gap injuries, however effective strategies are yet to be devised in longer nerve gap injuries (> 40 mm) [82]. The tissue engineering approaches are hoping to develop NGCs alternative to the nerve autograft with immunologically inert, intact ECM components, embedded SCs and fortified with growth factors for optimized functional regains [83]. The axonal guidance in NGCs lumen may be compromised, as higher numbers of disorganized axons, in the interior of 5 mm conduit installed in the sciatic nerve gap, comparing to nerve graft are seen 3weeks following installation [84]. A recent study reports the dynamic interaction of macrophages and SCs in a bridge of transected sciatic nerve through macrophage borne Slit3 interaction with the Robo-1 receptor on SCs for proper axon guidance. Consistent with the notion, knockout mice for Slit3 or Robo-1 demonstrate axonal misdirection in the transected nerve bridge [85]. Additionally, SCs in the injured nerve undergo reprogramming to establish SC-cords for axonal growth and the process is governed by the transforming growth factor beta (TGFβ) [39] and the involvement of EphB3 [86]. The expression of the Robo-1, EphB3 and TGFβ-receptor in peripheral nerve endothelial cells [86–88] reveals the crucial axonal guidance roles of nerve vasculature in regeneration. The role of neurotrophic factors in mediating peripheral nerve regeneration is well established and recent reports suggest the involvement of the angiogenesis process in the facilitation of axonal regeneration [89]. Specifically, vascular endothelial growth factors (VEGF-A and VEGF-B) demonstrate their influence on intraneural angiogenesis, and hence modulate the nerve regeneration process

polytetrafluoroethylene conduits in the repair of median and ulnar upper limb extremity gap injuries from 1.5 cm to 6 cm revealed significant functional sensory and motor recoveries, especially in distal nerve injuries [76]. The use of silicone tubes and polytetrafluoroethylene conduits in nerve repairs is abandoned owing to the development of resorbable conduits. The biodegradable conduits are preferred over non-degradable conduits in clinical trials. The collagen based biodegradable conduits were installed in 12 mm (average) gaps in 19 patients and follow-up till 20 months reported satisfactory restoration of nerve functions as assessed by an independent observer [77]. Polyglycolic acid composed nerve conduit installation in 42 patients with at least 4 mm gaps revealed similar sensory recovery comparing to autogenous vein graft and lesser post-operative concerns over 12-months [78]. Poly(DL-lactide-εcaprolactone) composed NGC installation in patients with forearm, elbow, wrist, palm and finger nerve injury sites revealed some advantages in the regain of sensory function, however, it also posed some serious disadvantages such as formation of neuroma in one out of 23 patients [69]. The clinical use of engineered biodegradable conduits seems promising in substantial recovery of PNI induced lost functions, and it subsides the nerve grafting techniques of nerve repair.

5. Opportunities offered by NGC and future research directions The profound advantages achieved through the NGCs engineering are not only limited to provide controlled guidance path for advancing axons but also include the strategies incorporating supportive cells, growth factors, conduit fillers, genetic manipulations, electroconduction and topographical cues (Fig. 2). A survey of in vivo literature reveals the promoting effects of SC seeded acellular nerve grafts in rat sciatic and femoral nerves in significant restoration of muscle strength [79]. The installation of neural stem cells incorporated hyaluronic acid-collagen conduits with 6

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following injury and functional recovery in rat [90–92]. A recent study reports the silk fibroin blended conduit enhanced nerve regeneration through vascularization [93]. Thus, the incorporation of such guidance cues in NGC may be warranted to evaluate their possible roles on nerve regeneration. The strategy may involve the gradient establishment from proximal to the distal nerve stump direction of the NGC and vice versa. A growing body of knowledge suggests the macrophage involvement in Wallerian degeneration, the proliferation of endothelial cells establishing a capillary network to guide the proliferated population of dedifferentiated SC and subsequent formation of guidance tubes (bands of bungner) for advancing axons to mediate regeneration of transected nerve [42]. Thus, a strategy inspired by nature's own design, could facilitate the intrinsic events and may provide a potential therapeutic approach to treat the unfortunate long gap transected nerve injuries. Hence, the development of strategy for a novel NGCs aiming to enhance the formation of functional blood vasculature, SC proliferation and migration, facilitate the SC alignment and promoting axonal regeneration is proposed (Fig. 3).

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