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Peripheral nerve injury: Current challenges, conventional treatment approaches and new trends on biomaterials-based regenerative strategies Rita López Cebral, Joana Silva-Correia, Rui L. Reis, Tiago H. Silva, and Joaquim M. Oliveira ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00655 • Publication Date (Web): 27 Sep 2017 Downloaded from http://pubs.acs.org on September 29, 2017

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ACS Biomaterials Science & Engineering

Peripheral nerve injury: Current challenges, conventional treatment approaches and new trends on biomaterials-based regenerative strategies R. López-Cebral,1,2,* J. Silva-Correia,1,2 R. L. Reis,1,2 T. H. Silva,1,2 and J. M. Oliveira1,2 Affiliations 1

3Bs Research Group, Biomaterials, Biodegradables and Biomimetics, University of

Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark - Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal; 2

ICVS/3Bs, PT Government Associate Laboratory, Braga/Guimarães, Portugal.

*Corresponding author: Rita López-Cebral, PhD E-mail: [email protected] Postal address: 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, AvePark – Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal

1 Environment ACS Paragon Plus


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Index 1. Fundamentals 2. Biological challenges 2.1.

Processes after injury

2.2.

Immunomodulation

2.3.

Barriers for axon re-growth

3. Current treatment options 4. Biomaterials 4.1.

Natural-origin biomaterials

4.2.

Endogenous biomaterials

4.3.

Synthetic biomaterials

5. Concluding remarks and future perspectives


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Abstract Damage to peripheral nerves is a widely extended health problem, causing important socio-economic costs worldwide. Indeed, peripheral nerve injuries (PNI) have been concerning the medical community for many decades. Nevertheless, despite the increase in knowledge in the injury physiopathology and the great research efforts being undertaken, the current standard grafting strategies used to repair PNI are not as efficient as desired. Although alternative engineered nerve grafts are already commercialized, their clinical performance is suboptimal. In this review, a general description of the circumstances and repercussions surrounding the PNI pathological state are presented, together with the treatment limitations and current challenges when addressing both short- and long-gap defects. In addition, potential therapeutic molecules are considered, while innovative regenerative strategies have been identified. Finally, the most relevant advances on the use of a wide range of biomaterials for the development of novel medical devices are also overviewed in depth, either considering strategies making use of empty or filled nerve conduits for guided tissue regeneration.

Keywords: Biomaterials, peripheral nerve injury, regeneration, growth factors, cell therapy, gene therapy.



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1. Fundamentals: From the anatomical point of view the nervous system is divided into the central nervous system (CNS) and the peripheral nervous system (PNS) (Figure 1A). Contrary to the CNS, the PNS is neither shielded by bone tissue nor protected by the blood-brain barrier, being much more vulnerable to traumatisms and to harmful substances1. The basic functional unit in the nervous system is the neuron. Although the different types of neurons present some variations among them, four parts constitute the common denominator: the cell body, the dendrites, the axon and the axon terminals. Each one of these parts has a well-defined function. In addition, some neurons are myelinated, presenting gaps between the myelin sheaths called Nodes of Ranvier2 (Figure 1B). Schwann cells (SCs) are the glia of the PNS, and they are responsible for the generation of the axonal myelin (Figure 1C). SCs can be also found surrounding clusters of un-myelinated smaller nerves3-4. Figure 1 should be inserted here. In the peripheral nervous system each individual axon is protected by the endoneurium, a layer of collagen and elastic elements. A bunch of endoneurium protected axons group into nerve fascicles, which are sheathed by the perineurium (mainly connective tissue). Finally, several fascicles are gathered together by the epineurium (mainly connective tissue). A complex microcirculation system, the vasa nervorum (VN), runs the nerve longitudinally. The VN descends from the epineurium to the perineurium, ultimately establishing transperineural connections with the vessels in the endoneurium. Extensive anastomotic connections can be found at all levels. The endothelium of the endoneurial microvessels and the perineurium that protects the endoneurial space form the blood nerve barrier (BNB)5-6. The BNB helps in the maintenance of the particular endoneurial homeostasis and determines an immunologically and biochemically privileged space. Indeed, the breakdown of the BNB permits the infiltration of immunoglobulins, cytokines, chemokine, immune cells and pronociceptive molecules7-8. Either trauma or disease can cause a peripheral nerve (PN) injury (PNI)9-11. PN damage is also a frequent collateral effect of surgical interventions12, i.e. in pelvic surgery the damage to visceral nerves frequently causes bladder, bowel and/or sexual


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dysfunction13. The extent of harm in the described nerve layers gives rise to the classification used in clinics for PNIs cataloging. This classification was established in 1943 by Seddom and expanded in 1951 by Sunderland. It gives clinicians an orientation of what therapeutic strategies to adopt and the level of recovery to expect14 (Table 1). Table 1 should be inserted here. The PNS presents certain ability for regeneration, an uncommon property in mammalian tissues15. Indeed, not even the CNS equals this PNS characteristic. Nevertheless, the PNS spontaneous regeneration capacity is not as efficient as desirable, often leading to poor recovery and lifelong disabilities. Chronic neuropathic pain is also a frequent outcome16-18. The cases where the nerve lesion causes a nerve gap are the most critical. Indeed, spontaneous regeneration does not occur in cases of nerve gap. Of course, the wider the gap, the greater the challenge. This is due to several factors, such as the presence of growth-inhibiting molecules along the injury site, the slow rate of axon growth (1–5 mm per day, depending on the part of the body), the reduction in the regeneration ability with time or the higher probabilities of axon mismatch13, 19-20. The neural loss that occurs after PNI also contributes to an inefficient functional recovery. The anatomic position influences this neural loss, i.e. proximal transection in the sciatic nerve means a 3-fold higher loss, when compared with the distal injury13. Finally, axons regenerate slower and to a less extent with increased age21-22. These factors, together with the anatomic complexity of peripheral nerves, make of the implementation of strategies to stimulate peripheral nerve regeneration (PNR) a real challenge. PNIs are a worldwide clinical problem18. Recent publications have considered the incidence rate of PNIs as 1 in 1,000 individuals per year23. If it is taken into account that the European population in September 2017 was 739.271 million (Worldometers data), we will be taking about 739,271 cases per year only in Europe. Meanwhile, according to the same website, in U.S.A the registered number of habitants were 326.896 million people, in India 1.345 billion and in China 1.389 billion. In fact, PNI get translated into approximately a $150 billion dollars health-care expense per year just in the USA24. Indirect expenditures also add to these expenses, as this type of



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lesions affect adult people of working age, resulting in reduced employer output and elevating the socioeconomic costs17, 25. These numbers clearly show the relevance and repercussion of PNI in society.

2. Biological challenges 2.1. Physiopathology of the PNI After PNI a series of physiopathological events occur at different levels of the injury site. A brief description of these phenomena can be found in Table 23-4, 26-38. Table 2 should be inserted here. Succinctly, there is an up-regulation of regenerative-associated genes (RAGs) in the cell body, in the so-called cell body response. An elemental schema of this activation can be found in Figure 2A. Figure 2 should be inserted here. In the proximal injury end a growth cone (GC) emerges (Figure 2B), which is of fundamental importance for the advance of the regenerating axon. Figure 2B represents the different elements that constitute a GC. It can also be observed the example of the GC of retinal ganglion cells after fluorescent staining39. In the distal injury end a process called Wallerian degeneration (WD) occurs. This process involves a cascade of events leading to the clearance of undesired debris, paving of the way for regeneration. Figures 3A and 3B depicts two ex vivo examples of WD40-41. The role of SCs during WD is critical30, 32-33. Nevertheless, as appreciated in the schematic representation of Figure 3C, diverse immune cells also participate in the course of WD. The role of the immune system during WD will be described in detail in the next Section. Figure 3 should be inserted here. Ultimately, the alterations suffered by the targets as a result of the denervation will difficult the re-innervation. These alterations will be more pronounced over time. Interestingly, close axon-SC contact seems of pivotal importance for regeneration,


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due to mutual signaling phenomena4, 42. Indeed, SC phenotype differs between motor and sensory axons. Marquardt LM et al. and Wood MD et al. described that sensory neurons extend longer and more axons in the presence of sensory nerve-derived SCs. The same happened for motor neurons co-cultured with muscle-derived SCs42-43. Wood et al. pointed that after equal access to motor and sensory pathways, motor axons regenerate preferentially along the motor pathway. Chan KM et al. hypothesized that the different neurotrophic factors secreted by motor and sensory SCs determine these phenomena20, 42, being this hypothesis also corroborated by other authors4, 22.

2.2. Immunomodulation Inflammation The immune system plays a critical role during PNR. Regardless of the primary cause, PNI always involves an inflammatory process. Almost immediately after PNI SCs dissociate from axons, de-differentiate, and along with fibroblasts secrete cytokines and chemokines (e.g. TNFα, LIF, Il-1α and Il-1β, Il-6, and MCP-1). Matrix metalloproteinases contribute to the generation of chemotactic gradients along the injury site and throughout the BNB. As a consequence, different immune cell types are recruited, including granulocytes (neutrophils and mast cells) and agranulocytes (monocytes/macrophages and lymphocytes). Neutrophils are the first immune cells to infiltrate. They accumulate in the distal stump within 8 hours after injury, but their presence is short-lived. Subsequently, circulating monocytes are attracted to the injury site, where they differentiate into macrophages. These hematogenous macrophages become the dominant leukocyte population, playing a critical role in ensuring complete WD26, 30, 33 (see the scheme in Figure 3C). In addition, other inflammatory cells, including T-lymphocytes, also enter the injury site throughout the damaged BNB within hours after injury44-45. There are two activation states for macrophages, M1 (classically activated) and M2 (alternatively activated). M1 macrophages are pro-inflammatory and cytotoxic, whereas M2 macrophages participate in tissue repair and wound healing26,

46

.

Interestingly, Davis MJ et al. described that an interconversion of these two phenotypes occurs both in vitro and in vivo, in response to changes in the cytokine 7 Environment ACS Paragon Plus


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environment47. Effect also described by Chen P et al.48. The induction of M1 to M2 polarization after PNI may be of high therapeutic value. Fatty acids released after myelin breakdown can contribute to the inflammatory response, for instance by their transformation into prostaglandins and leukotrienes that stimulate immune cell infiltration33. Endogenous mechanisms exist that intend to control PNI inflammation, i.e. SCs produce the anti-inflammatory cytokine IL-1049 and the SC re-myelination of regenerated axons stimulates macrophage efflux33. Nevertheless, these mechanisms are not efficient enough, and prolonged inflammation becomes a challenge during PNR. The complex immune response after PNI also involves the CNS. During WD, the dorsal horn receives input from the affected axons and is infiltrated by macrophages and T-lymphocytes44. Dominguez CA et al. hypothesize that leukocyte presence may amplify the inflammatory activation of CNS resident glial cells, triggering exaggerated pain16. Meanwhile, Manjavachi MN et al. postulated that the migration of neutrophils to the spinal cord strengthens the neuropathic state50. Hyperalgesia The pro-inflammatory cytokines and chemokines released into the injury site cause hyperalgesia. Nevertheless, to this day there are no pharmacotherapies with a reasonable effect-consequence rate to treat persistent pain7, 50-51. It is interesting to mention that neuropathic pain is a rare phenomenon in infants, with only a few reports existing before 5 - 6 years of age. The same happens in rat and mice models. Thirteen years is the median age for the onset of pain after pediatric neuropathic syndromes. The exact reasons for this are unknown, as these children are able to feel other kind of pains. Nevertheless, McKelvey R et al. demonstrated that the pro-inflammatory immune response after infant PNI is counteracted by an antiinflammatory response triggered in the dorsal horn, which switches to proinflammatory in adolescence52. These data suggest that the modulation of the inflammatory response generated after PNI may be an efficient therapeutic solution against neuropathic pain.


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Specific immune response Results obtained by Vargas ME et al. indicate that preexisting IgG and IgM present in naïve WT mice bind to specific epitopes in myelin debris, targeting them for rapid clearance53. These results suggest that the humoral immune system can actively promote wound healing in the absence of an exogenous immunogenic stimulus, such as vaccination. Thus, these findings evidence two distinct physiological mechanisms for myelin debris cleaning: 1) one antibody-independent, mediated by Schwann cells, and 2) one antibody-dependent, mediated by hematogenous macrophages. In addition, there is evidence that SCs play an immune surveillance role, detecting pathogens and orchestrating an ensuing immune response49.

2.3. Barriers for axon re-growth The regenerating axons need to overcome a series of obstacles on their way across the injury site. Myelin and axonal debris Debris from degenerating neurons impairs axon regeneration. Kang H et al. described a tendency of regenerating axons to divide in two circumventing branches when they encounter debris, leading to an anatomically and functionally abnormal nerve21. These same authors also indicated that slower clearance of axonal and myelin debris is, at least in part, responsible for hindering regeneration in aged animals21. DeFrancescoLisowitz A et al.26 and Brosius LA et al.33 pointed that one of the major differences in the response to injury between CNS and PNS is the rate of myelin debris clearance. Scar tissue Fibrosis is a widespread response of the organism to many different pathological conditions. It intends to avoid injury spreading and to preserve the functionality of damaged structures. Nevertheless, prolonged fibrosis leads to excessive extracellular matrix (ECM) remodeling and to the formation of permanent scars54. Fibrotic tissue normally develops during the first weeks following PNI and continues to mature thereafter55-56. Tissue fibrosis is known to hamper PNR57-58, by acting both



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as a physical and chemical barrier for axonal regeneration. Indeed, recent reviews consider the avoidance of scar tissue formation as an important part in the design of artificial nerve conduits58. Oxidative stress Oxidative stress is caused by an overproduction of reactive oxygen species (ROS), a collective of highly reactive oxygen metabolism byproducts. These chemical species have essential roles in numerous biological processes. Nevertheless, an excessive production can cause cellular damage, even leading to cell apoptosis or tumor development59-60. The results obtained by Bros H et al. revealed that ROS are responsible for mitochondrial dysfunction in PNS myelinated axons, causing energy failure and triggering axon degeneration61. Meanwhile, Chang HM et al. concluded from previous studies that exposure to oxidative stress impairs SCs differentiation and can even cause SCs apoptosis32. Abnormal amounts of ROS are generated after different types of PNI62, impairing neuronal recovery62-63. The administration of anti-oxidative therapies may importantly help to overcome this obstacle. Unfortunately, these kinds of therapies have not been much explored60, 64-65. This Section gathers some of the most important physiopathological phenomena that impair PNR and should be overcome to achieve a successful nerve recovery. Currently there is a lack of effective treatment, able to efficiently solve them. One probable reason for this is that they have not been granted the critical importance that they really have. Nevertheless, it is also probable that a lack of meticulous knowledge, accurate therapeutic molecules and appropriate technological approaches are behind this situation. Indeed, some scientific conclusions are based on experimental data, like that assuming that the modulation of the inflammation may alleviate the neuropathic pain. The advances in basic knowledge should bring confirmation of these phenomena, as well as the detailed mechanisms governing these events. Consequently, making possible to develop accurate solutions. Accordingly, improvements in the PNR area will be seen.


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3. Current treatment options Despite the high incidence of PN lesions, their clinical relevance and the efforts undertaken, the therapeutic advances have not been much within the last 30 years. Only minor surgical refinements have been made since in 1964 Kurze and Smith independently applied the principles of microsurgical nerve repair described by Sunderland (1945). Meanwhile, the epineural nerve repair technique described by Huenter (1873) remains in use today24,

37, 66-67

. Table 34,

22-23, 25-26, 37, 44, 68-74

summarizes the main clinical strategies in use today for the treatment of peripheral nerve injuries. Table 3. Should be inserted here. As previously stated, nerve transection lesions do not regenerate spontaneously. In these cases surgical intervention is needed. When the gap is a long one, the actual PNI golden standard treatment is autologous nerve grafting. Nevertheless, among other disadvantages, it can only guarantee seldom-suboptimal functional outcomes. Sensibility abnormalities and painful conditions are also frequent fallouts, condemning the patient to a close relation with painkiller therapy. Other biologic structures explored as substitutes of nerve grafts (i.e., blood vessels, skeletal muscles or tendons) present the same drawbacks, but intensified67. A biomimetic strategy based on the implantation of Nerve Guidance Conduits (NGCs) has shown clinical potential as nerve grafting replacement75. This strategy seems the only viable alternative to nerve grafts. Nevertheless, commercially available NGCs fail completely to regenerate long gap injuries. Probably, because they are hollow tubes or nerve wraps, neither optimized for axonal guidance nor loaded with appropriate supportive cues18,

72

. Several authors described different

aspects to take into account in order to improve NGCs performance. Zheng J asserted that topographical cues play an essential role in neurite outgrowth76, while Pateman et al. emphasized the relevance of the microstructure25. Gu X et al. pointed that NGCs should have adequate physicochemical, mechanical and biological properties18. The absence of luminal fillers seems a negative aspect, being pointed as the main reason for the poor performance of commercial NGCs. Luminal fillers already explored for PNR are grooves, electrospun fibers, gels, films, sponges, filaments or multichannel and conductive structures17, 37.



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Chen P et al. highlighted the importance of a proper interplay between glial cells, growth factors, cell adhesion molecules and ECM proteins48. Meanwhile, vascularization seems a critical factor. Vascular irrigation supplies cells with oxygen and nutrients, at the same time that removes waste products18. It was more than 50 years ago that inadequate blood supply was described as a cause of nerve regeneration failure, due to cell death or malfunction. Indeed, necrosis and fibrosis phenomena are much more probable if there is no vascularization77-78. Advances in biotechnology permitted the large-scale production of bioactive endogenous molecules, making possible their incorporation within PNR NGCs. The advantages and limitations of the most interesting ones are described in Table 412, 18, 32, 40, 72, 79-119

. This table also includes conventional drugs with PNR potential.

Table 4. Should be inserted here. The advances described in the previous paragraph also enable cell isolation and expansion, as well as cellular genetic modification both ex vivo and in vivo. Table 518, 23, 30, 51, 57, 73, 120-164

summarizes a compendium of the most novel cell and gene therapy

possibilities regarding PNR. Table 5. Should be inserted here. This Section groups the main therapeutic PNR cues, their strengths and their limitations. It is reflected in this Section that new biotechnological approximations can bring new therapeutic possibilities, as it already happened with the growth factors and the genetic material. Nevertheless, the required technological tools for their proper therapeutic exploitation may not be available yet. This is for instance the case of the lack of an accurate drug delivery vehicle for the vehiculization of DNA plasmids. Also advances in the basic knowledge are still ongoing, as reflected by the recent identification of new miRNAs with clinical interest, indicated in Table 5. In addition, it can occur that some of the conventional drugs prove to have some unexpected secondary effect that is positive for PNR. This happened in the past with several drugs. It is for example the case of the acetylsalicylic acid used as an analgesic, which years later proved to have the important therapeutic effect of helping prevent heart attacks in patients with heart disease165. Even, new small chemical drugs can be synthetized in the lab that gather together the positive effects of several of the


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conventional drugs. It can also be the case that the combined administration of diverse above presented drugs provokes a synergistic effect. Meanwhile, the possible adverse effects of these drugs should be controlled, by mans of the incorporation in the proper drug delivery vehicle. Indeed, developments in biomaterials and their combinations for the preparation of specific, highly adapted, drug delivery vehicles will have an important part in the PNR therapeutic success. Accordingly, it is not difficult to foresee that the advances in relation to all the aforementioned aspects will have a deep impact in the PNR area.

4. Biomaterials As extracted from the previous Sections, the materials utilized in the preparation of the NGC should possess a compendium of as much convenient properties as possible, in order to cope with all the high PNR requirements. Table 675, 166-212 summarizes a selection of different materials with potential for application in PNR, their origin, as well as their main advantages and disadvantages are described. A more detailed explanation about the possible processing methods and roles of these materials in PNR will be further provided. Naturally occurring polymers usually present intrinsic bioactive properties that can positively contribute to the regenerative process. In addition, due to biocompatibility, biodegradability and biomimetic features, natural materials are preferred over synthetic or semi-synthetic ones. Table 6 should be inserted here. 4.1. Natural-origin biomaterials Table 6 introduces some of the most interesting natural-origin biomaterials for PNR applications, from proteins (silk or keratin) to polysaccharides (chitosan, alginate and gellan gum). During the following paragraphs more details about the application of these materials in the PNR field will be given, including the particular experiences of the review authors. In addition, their combinations with each other, as well as with other compounds of interest, will be covered.



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Silk fibroin Silk fibroin is a water insoluble protein present in silk. Due to its unique mechanical properties, accompanied by good biocompatibility and low toxicity, it has caught the eye of the scientific community for regenerative purposes213. The fact that silk fibroin can be successfully processed by several different techniques adds to the interest awakened by this biomaterial. Several of the developed experimental works are related to the preparation of NGCs for PNR. For instance, Teuschl AH et al. observed good anatomical and functional nerve recovery in a rat sciatic nerve injury model with their silk fibroin NGCs214, while Tang X et al. obtained promising reconstruction results in a similar injury model with their silk fibroin-based scaffolds215. Figure 4A shows a model silk fibroin tube designed by our Group for PNR purposes. After the physicochemical and biological characterization of diverse prototypes, in vivo compatibility tests are being performed. Figure 4 should be inserted here. Keratin The protein keratin is the major component of wool, hair, horn, nail, hooves of mammals, as well as of bird feathers169. Indeed, millions of tons of keratinous wastes are generated annually around the world172. The use of Keratin as a compound with potential for PNR was proposed years ago. For instance, in 2008 Sierpinski P et al. observed that keratin induced a chemotactic action over SCs, also favoring their development216. The authors postulated then that keratins are neuroinductive and can improve PNR outcomes. In 2013 Pace LA et al. broadened this information by discovering that keratin biomaterial hydrogels fastened SC dedifferentiation in the distal stump, also fastening myelin debris clearance by these SCs217. As aforementioned, the efficient debris clearance plays an important role for the successful PNR. Despite these positive results, keratin has not been much exploited for PNR purposes, with only a bunch of spread works published along the last years. Advanced NGCs comprising the functionalization with keratin can lead to improved SCs attachment, proliferation and gene expression.

Chitosan


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Due to its biodegradability, biocompatibility, non-antigenicity and biofunctionality, this polysaccharide is one of the most often utilized components in the preparation of polymeric implants. Several groups have studied the use of chitosan for PNR. It has been described in the literature that low degrees of chitosan acetylation (DA) are more efficient promoting SCs development than intermediate ones. Based on this observation, our Group compared the ability of membranes prepared from three different low DA chitosans to sustain SCs development. The studied DAs were 1, 2 and 5%. The results showed the preference of these cells for the 5%DA membranes, where their proliferation was higher and their phenotypic expression more accurate218. In addition, these membranes avoided excessive fibroblast infiltration, a quality expected to hamper the formation of the fibrotic tissue described in Section 2.3. Lin CY et al. obtained 3D tubular chitosan scaffolds, very efficient in providing the spatial features for SCs attachment, migration and proliferation. These cells secreted higher amounts of laminin and collagen than those seeded on the plane un-rolled formulations34. This study demonstrates the importance of the scaffold shape. Figure 4B shows a chitosan tube prepared by our Group following a similar procedure than Lin CY et al. In brief, a 2% chitosan solution (in 2% acetic acid solution) was casted over a Petri dish. After solvent evaporation the obtained membranes were neutralized using 0.1 M NaOH. Finally, the dry neutralized membrane was rolled over itself to form a tube for PNR. The proper 2% chitosan solution acted as glue. Li G et al. also focused on the porosity and micropatterning of their chitosan-NGCs, prepared using a combination

of

micromodelling

and

lyophilization.

They

observed

that

micropatterning contributed to SCs alignment, without influencing the physiological function of these cells175. Wang W et al. utilized the potential of chitosan for electrospinning to obtain chitosan nanofiber mesh tubes by this procedure, getting promising results in a rat sciatic nerve injury model219.

Alginate Alginate is a natural polysaccharide produced by brown algae and the bacterial genera Pseudomonas and Azotobacter. It is frequently used in the food industry, for wound management or for the entrapment of cells220. It has been described that calcium ions induce specific associations between alginate chains, consequently forming



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hydrogels. Even a specific term was coined to describe the characteristic 3D structure formed, popularly acknowledged as the “egg-box model”221. Making the most of this property, different types of biomedical investigations have been carried on along the years, also in the area of PNR. For instance, Matsuura S et al. demonstrated that cavernous nerve could be regenerated, by simply filling the nerve gap with an alginate sheet222. Meanwhile, this polymer was also used to fill NGCs prepared using other biomaterials, with interesting results223-224.

Gellan gum This polysaccharide is produced by the bacteria Sphingomonas elodea225. Meanwhile gellan gum has been widely studied for different regenerative applications, to our knowledge, there are currently no research works exploiting its potential with PNR purposes. Similarly to alginate, gellan gum forms hydrogels by ionic cross-linking with divalent cations. Although gellan gum hydrogels are stable, able to act as drug delivery systems and to sustain cell growth, authors often resourced to its methacrylated derivatives to control different formulation parameters226. This is the case of our Group, where interesting results were obtained in an animal model of rat sciatic nerve defect with gellan gum and gellan gum-methacrylated hydrogels. The example of one of these gellan gum scaffolds is depicted in Figure 4C. More specifically, these hydrogels were envisioned as longitudinal channeled fillers for chitosan hollow tubes. Their inner porosity was controlled by means of freeze-drying. For the studied animal model, tibialis anterior functional muscle re-innervation was observed 12 weeks after nerve reconstruction.

Blending /functionalization of biomaterials The combination of different biomaterials has also been explored by diverse authors. Yao M et al. achieved similar results to those obtained using autologous nerve grafts, when bridging a rat sciatic nerve defect with chitosan/silk fibroin NGCs that incorporated bone marrow mononuclear cells227. Gu Y et al. followed a complex strategy. They seeded SCs into combined chitosan/silk fibroin NGCs that were decellularized after ECM deposition. The status of repair achieved with these NGCs


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was similar to that obtained with an acellular nerve graft228. Combinations of chitosan and alginate were also analyzed, as it is the case of the investigations carried our by Pfister LA et al. More in detail, in 2007 Pfister LA et al. developed chitosan-alginate NGCs that matched PNR requirements in terms of hydrophilicity, permeability, stiffness and surgical handling229. Meanwhile, in 2008 these same authors coated their chitosan/alginate NGCs with poly(lactide-co-gly-colide), getting to control the release kinetics of a embedded nerve growth factor230. Calcium has been identified as an important element for peripheral nerve regeneration. More specifically, calcium-mediated signaling pathways influence the development of nervous cells and the establishment of neural connections between them, as well as dendritic growth and arborization. Furthermore, calcium signaling determines the advance of growth cones and the rate of axonal outgrowth. Hydroxyapatite (HyA) is a source of this element, as it acts as a donor of calcium. Based on these considerations, Nawrotek K et al. designed a strategy for obtaining chitosan-based hydrogels enriched with hydroxyapatite. These NGCs presented adequate suitability as implants for PNR231. In a further developed approach, these same authors added carbon nanotubes (CNs) to the chitosan-HyA NGCs, improving the system mechanical properties. CNs have also been described to promote nerve growth232. Das S et al. developed a silk fibroin tube incorporating gold nanoparticles (GN), which improved the NGC structural properties. In addition, the NGC resistance to electrical conductivity decreased, facilitating the signal transmission across the nerve gap. Consequently, the overall neuro-muscular regeneration and functionality was ameliorated with respect to silk fibroin and untreated groups233-234. Following a similar path, Huang J et al. combined their chitosan scaffolds with polypyrrole, which make them conductive and created a local electrical environment235. After one hour of electrical stimulation (ES), applied for 2 weeks after surgery, there was a significant axonal regeneration improvement. The use of ES has been recently strengthened as a strategy to promote PNR, due to the positive effects of ES over axon outgrowth236.

4.2. Endogenous biomaterials As previously mentioned, technological advances permitted the exploitation of



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endogenous molecules. Table 6 includes a selection of the most significant endogenous biomaterials regarding PNR, from proteins (e.g., collagen, fibrin and laminin) to polysaccharides (e.g., hyaluronic acid and chondroitin sulfate). Herein, more details about the application of these materials in the PNR field will be given. In addition, a detailed description of decellularized endogenous structures as biomaterials for PNR will be also provided. It is important to clarify that although GFs are endogenous materials, they were considered as part of Section 3 (Current treatment options). This is due to their specific pro-regenerative capabilities, which confer them with specific therapeutic properties, being thus considered therapeutic molecules per se. The same rationale was applied for genetic material. Collagen Collagen is a structural protein dominant in the connective tissue of animals. Up to this date, 28 types of collagen proteins have been identified. Conventional sources are bovine and porcine slaughter by-products. Nevertheless, in recent years new sources are gaining ground, like marine-origin residues186 or duck’s feet237. Collagen matrices have been used during years for the preparation of NGCs, some of them showing really promising results. This is the case of NeuraGen® (Integra LifeSciences) NGC, a type I collagen commercialized product. As literally described in the specific web page, “NeuraGen® nerve guide provides a protective environment for peripheral nerve repair after injury, and is designed to be an interface between the nerve and surrounding tissue and to create a conduit for axonal growth across a nerve gap”. Other successful example is that of Fujimaki H et al.

238

, who utilized oriented

collagen tubes (OCTs) for the repair of long gap (15 mm) sciatic nerve defects in a rat model, even reaching motor function recovery. In addition, the functionalization of the OCTs with bFGF permitted to accelerate the process. Meanwhile, Van Neerven SGA et al. utilized Neuromaix (collagen hollow tube filled with a porous collagen scaffold) to bridge 20 mm sciatic nerve defects in rats. The authors observed successful axon regeneration, remyelination and reinnervation of skeletal muscle targets. This formulation is currently under clinical trials239. On the contrary, other authors got unsatisfactory results with their collagen NGCs. For instance, Lu C et al. functionalized

their

chemically

cross-linked

(1-ethyl-3-(3-dimethyl

aminopropyl)carbo-diimide and N-hydroxysuccin-imide) collagen NGCs with ciliary


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neurotrophic factor to treat 10 mm gaps in the buccal branch of mini-pigs’ facial nerve. Although the addition of CNTF showed improved results with respect to the control without CNTF, there were major differences between regenerated and the healthy nerve tissues240. The reason for these results is probably related to the chemical NGC preparation procedure, because, as previously indicated, endogenous molecules are very labile. Related to the Sub-Section Inflammation (Section Inmmunomodulation), and more specifically to the suggestion that the induction of M1 to M2 polarization after PNI may be of high therapeutic value, recent investigations have demonstrated the critical role of collagen for the presence of M2 macrophages. In more detail, in 2015 Chen P et al. discovered that collagen VI was critical for macrophage migration and M2 polarization via AKT and PKA pathways. Indeed, they verified that PNR was strikingly impaired in collagen VI null mice48. The authors also highlighted the interest in adding collagen VI to future PNR strategies. In 2017 Lv D et al. confirmed that local delivery of collagen VI increased the recruitment of macrophages, as well as their M2 polarization. Thus promoting nerve regeneration and functional recovery241.

Fibrin Fibrin is a protein involved in the formation of the blood clot. It is formed by protein fibrinogen polymerization, caused by the protease thrombin. Fibrin has been applied as a sealant in neurosurgery for decades, without inducing neural adverse effects. Indeed, some authors described it to have a protective effect over the nerve242. The actual source of commercial fibrin sealants is the human blood. Nevertheless, the search for alternative sources has already born fruit. A new heterologous fibrin sealant from snake venom has been proposed as substitute. Also bubaline-derived blood serum cryoprecipitate has been used as fibrinogen source243. Fribrin Glue (FG) was used by Bhatnagar D et al. to coat porous NGCs. The authors observed that the FG attracted non-neuronal cells, facilitating the presence of scar tissue into the lumen. In Section 2.3. the presence of scar tissue was signaled as one of the barriers for axonal re-growth. As a result, axonal regeneration and functional recovery were poor. The



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authors contrasted these data with the positive data obtained by other authors that used FG in conjunction with non-porous NGCs. Hypothesizing that the system porosity was the cause of the discouraging results244. Other interesting works include that of Reichenberger MA et al., who embedded stem cells into a fibrin matrix for sciatic nerve repair. The cells were preferentially situated in the nerve parenchyma and in close proximity to the vasa nervorum. This fact was translated into an improved nerve repair due to the promoted angiogenesis. These results are further sustained by studies that describe a role of MSCs in the secretion of pro-angiogenic factors245. This study by Reichenberger MA et al. stresses the importance of angiogenesis for efficient nerve regeneration. Man AJ et al. examined the effect of physical and mechanical properties of fibrin gels on dorsal root ganglia neurite extension. To this end, fibrinogen concentration or NaCl content within the matrices was modulated, implicating variations in the previous parameters. The obtained results provide new insights to improve the PNR efficacy of these scaffolds246. The importance of the physicochemical and mechanical properties has been highlighted in relation to the properties that should be taken into account in the design of a NGC. Laminin Laminin is a cell-recognition ECM protein that plays crucial roles in cell migration, differentiation and axonal growth. It also acts as a neuronal cue, promoting the advance of the growth cone. For these reasons, laminin was incorporated as a motif to improve the PNR ability of different engineered systems247-250. Collagen NGCs are a specific example, as different collagen PNR systems have been functionalized with laminin. For instance, Swindle-Reilly KE et al. supplemented collagen gels with different laminin concentrations. The results showed that, at low concentrations, the laminin influence on neurite growth depended on the mechanical stiffness of the 3D scaffold. Meanwhile, at higher laminin concentrations, the growth became independent of the gel stiffness251. Gonzalez-Perez F et al. also functionalized their collagen NGCs with laminin, getting to improve the nerve repair process both qualitatively and quantitatively. Nevertheless, it should be mentioned that these authors repeated the process using fibronectin instead of laminin and got even better results252.


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Hyaluronic Acid Hyaluronic acid (HA) is an important building block of the ECM, and is responsible for maintaining tissue homeostasis. HA mediates many important cell-signaling events by interacting with cell surface receptors. HA also plays a significant role in scar-free wound healing, tissue remodeling, morphogenesis and angiogenesis when degraded into smaller fragments253. In this sense, Zor F et al. covert with HA film sheaths coaptated sciatic nerves, proving to reduce the scar formation in rats254. HA hydrogels have been widely used for PNR purposes, as drug delivery systems, regeneration supportive matrices or fuctionalization cues. Among the different works present in the literature, some of them represent out of the box approaches. For instance, Vilariño-Feltrer G et al. developed what they call a truly engineered tissue. It consisted in hyaluronic acid NGCs with a three-layered porous structure that impeded the leakage of the cells seeded in its interior, while making it impervious to cell invasion from the exterior. At the same time, it allowed free transport of nutrients and other molecules needed for cell survival. The conduits were tested with Schwann cells providing interesting results3. HA was also used in combination with the previously described endogenous materials. For instance, Suri S and Schmidt CE engineered 3D hydrogels formed by interpenetrating collagen and hyaluronic acid. SCs effectively spread and proliferate within this system, actively secreting nerve growth factor and brain-derived neurotrophic factor. Furthermore, the incorporation of laminin increased the overall production of both growth factors. In addition, when dissociated neurons were cocultured with Schwann cells, they were able to extend neurites. Some of these neurites were observed to follow Schwann cells. The authors concluded that these biomimetic scaffolds, exclusively composed by native ECM components, may help to understand Schwann cell interactions with neurons and

various extracellular matrix

components255. Zhan H et al. developed neural stem cell-embedded NT-3 supplemented HA-collagen composite scaffolds in order to bridge nerve gaps in the facial nerves using a rabbit model. They obtained promising re-innervation results256.

Chondroitin sulfate



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Despite all the positive results previously described, care should be taken, as not all the intrinsic properties of endogenous molecules are positive for PNR. It is the case of the

sulfated

polysaccharide

chondroitin

sulfate.

Despite

being

an

ECM

257

glycosaminoglycan implicated in relevant biological functions , this polysaccharide is a well-known inhibitor of axonal growth258. It is possible to perform chemical modifications over the natural-origin biomaterials and endogenous biomaterials described along the previous sub-Sections, originating materials with novel characteristics, known as semi-synthetic biomaterials. That strategy has been highly exploited by different research groups. For instance, for the obtaining of keratin allyl thioether169, methacrylated collagen259 or methacrylated hyaluronic acid260, among others. Nevertheless, despite the potential of these materials for specific particulars (i.e., modification of the degradation rate or cell attachment promotion), possible alterations of the original structures and concerns regarding their safe biodegradability can difficult their approval by the regulatory agencies.

Acellular matrices When it comes to endogenous materials, decellularized 3D structures could not go without being mentioned. As previously discussed, the golden standard treatment in nerve gap PNIs is autologous nerve grafting, a solution with significant drawbacks. Sacrifice of healthy nerves or limited nerve availability are among them. The storage and utilization of cadaveric nerve allografts with different lengths and sizes can provide a solution. This strategy will also avoid the extraction surgery in the patient, as well as the subsequent donor-site morbidity. Nevertheless, the cellular components in allografts induce rejection by the host immune system. The elimination of this cellular component with their antigenic domains should generate a biomaterial free of immunogenic associated rejections. Of course, decellularization techniques should be infallible in their purpose, at the same time that they preserve the structure and chemical composition of the neural structures, including the cell adhesion molecules261.


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The first reference to an acellular nerve for peripheral nerve regeneration in the Medline database dates back from 1986. It corresponds to a work from Hall SM, where he compared regeneration after cellular and acellular autograft transplantation in mice. The author observed that regeneration was less efficient in the second case, and he asserted that this was due to the impairing effects of the remaining decellularization

debris

(advance

difficulties,

macrophages

activation

and

attraction)262. The research on this topic continued during the following years. In 1988 Gulati AK evaluated the utilization of cellular and acellular nerve allografts for long gap PN injuries by utilizing isogeneic rats to avoid immune rejection. The author observed that cellular grafts stood for a more efficient regeneration. It was postulated that this was due to the presence of Schwann cells all along the cellular grafts. Contrary to what happened in the case of acellular grafts, where host Schwann cells could not efficiently populate the entire length of the graft263. The important role of these cells in PNR was described in previous Sections of this manuscript, and these type of results further confirm it. In 1994 Gulati AK, in a work in collaboration with Cole GP, compared the PNR performance of cellular isografts, cellular allografts and acellular allografts in rats and rabbits. The cellular allografts were in all cases rejected. Acellular nerve allografts showed capable of supporting axonal regeneration. Nevertheless, once more cellular isografts promoted a faster regeneration and reinnervation, and once more the presence of Schwann cells was hypothesized as the cause of the better results264. These authors also postulated that further specialization of decellularization methods could enhance the performance of acellular allografts264. Indeed, these initial promising results pushed the research in the area of decellularized neural structures. Repeated freezing and thawing was the most common method utilized during years for decellularization. In 2004, Hudson TW et al. clearly highlighted the limitations of the conventional thermal decellularization processes in eliminating cellular remains, which, as in the previous examples, promote the invasion of SCs and macrophages to clean the debris. Chemical treatments had been implemented to further reduce the immunogenicity of decellularized nerve grafts. However these procedures were more aggressive, greatly damaging the ECM265. Consequently, the search for the perfect decellularization technique continued. For instance, in 2014 Vasudevan S et al. explored the use of detergent-free decellularized nerve grafts, with more favorable results than that obtained by using detergentprocessed grafts across long-gap defects in a rat model266. 23 Environment ACS Paragon Plus


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The 4th of May of 2015, AxoGen, Inc. announced that they were granted clearance by the U.S. Food and Drug Administration (FDA) to proceed with a multicenter, prospective and randomized study to evaluate PNR recovery with the Avance® Nerve Graft product. Avance® is a decellularized nerve graft proceeding from human cadavers. Meaningful functional recovery was achieved with this material in 86% of motor, 89% of sensory and 77% of mixed nerve injuries261. Indeed, this product is now commercialized. However, Avance® is not without its limitations. For instance, as pointed by Biriani F et al., it is imported from US, thus having high costs. Moreover, American donor selection criteria do not always match the European regulations and standards267. Boriani F et al. also pointed that the decellularization methods employed for Avance® obtaining are time-consuming, while postmanufacturing gamma-ray sterilization is utilized. Gamma-ray sterilization is polemic, as it has been pointed that it can compromise tissue functionality. Indeed, various European countries (i.e., Germany) require a special marketing authorization for irradiated tissue derivatives267. Accordingly, these authors explored a novel strategy, complying clean room needs for acellular nerve obtaining. They prepared a completely decellularized and aseptic nerve graft, and they did it within a shorter time comparing with the pre-existing methods. The obtained graft lead to regenerated fascicles and bundles, comparable to that obtained with autografts in a rabbit model267. Other authors also continue the search towards optimal nerve decellularization and host cells colonization268-269. As previously mentioned, other endogenous structures were also tested as grafts for PNR, i.e. skeletal muscles, tendons, veins or arteries. Although in all cases the performance was lower than that obtained with nerve grafts. Following this rationale, some authors explored the utilization of decellularized vascular structures with PNR purposes. For instance, Crouzier T et al. decellularized human umbilical arteries, turned them inside out to prevent collapse and studied their in vitro interaction with PC12 cells. The results were encouraging, with these cells growing and migrating within the acellular scaffold. Nevertheless, the authors recognize that further optimization is required to obtain a more uniform cellular distribution within the scaffold, and that the ideal wall thickness that prevents collapse still needs to be determined. However, the authors highlight the interest of this system for investigating neuronal growth factors, electrical stimulation and other environmental factors in PNR270. Sun F et al. combined decellularized artery 24 Environment ACS Paragon Plus

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allografts with adipose-derived stem cells

in a rat model. This strategy showed an

improved performance with respect to that of an artery conduit alone. Nevertheless, the outcomes were inferior when compared to that obtained with a nerve autograft271. Hassan NH et al. decellularized human muscle stuffed veins and seeded them with differentiated mesenchymal stem cells. These structures were implanted in athymic mice. It was observed that 8 weeks after implantation, the muscular tissue was fully degraded and replaced with new matrix produced by the seeded cells. In addition, the vein was intact and no inflammation was detected. However, the authors did not compare their system with a native nerve272.

Combinations of natural-origin biomaterials and endogenous biomaterials Making the most of all the possibilities, some authors combined these materials in the preparation NGCs. For instance, Zhu N et al. blended CHT and laminin to obtain substrates for dorsal root ganglion neurites outgrowth. The cells showed preference for the formulations with laminin273. By their part, Huang YC et al. modified their CHT membranes with laminin, obtaining a significant improvement in SCs affinity and attachment274. Li G et al. tailored their chitosan scaffolds with heparin, thus promoting the attachment, proliferation and biological functioning of SCs275. Xu Y et al. co-cultured Schwann cells and adipose-derived stem cells in a combined SF/collagen nerve scaffold276. These authors utilized this scaffold to bridge a 10 mm gap defect in the rat sciatic nerve transection model. Nerve regeneration was successfully achieved. Nevertheless, function recovery was deficient. Dinis TM et al. bi-functionalized their silk electrospun conduits with two growth factors (nerve growth factor and ciliary neurotrophic factor), in order to enhance PNR by the sustained release of these molecules277. Instead of silk fribroin, Barreiros VC et al. evaluated the association of natural latex protein and hyaluronic acid hydrogels, revealing the regenerative capacity of these systems in a rat sciatic nerve injury model278.

4.3. Synthetic biomaterials As depicted in table 6, the four synthetic biomaterials that were at some point 25 Environment ACS Paragon Plus


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considered among the most promising materials for PNR are: silicone, poly-glycolic acid (PGA), poly-lactic-co-glycolic (PLGA) and poly-caprolactone (PCL). All of them are FDA approved materials. These materials were widely investigated for PNR purposes, even in combination with natural-origin biomaterials279 or endogenous biomaterials280. Indeed, they were used in the preparation of several commercialized NGCs (i.e. Neurotube (PGA), Neurolac (PLCL))75. This is due to several reasons. For example, they frequently give rise to more reproducible results after their processing into NGCs, than natural-origin biomaterials or endogenous biomaterials do. In addition, their specific characteristics suit better certain processing techniques. Despite all the positive characteristics of synthetic polymers, some drawbacks are also associated. The main negative aspects are related to sub-optimal biodegradation or toxic biodegradation by-products. These inconveniences hamper their extended use in clinics. A specific example comprises the first generation of clinical implants used as substitutes of functional nerves, which were made of silicone. Important complications were observed, such as inflammation or the so-called compression syndrome. To the point that a second surgery was often necessary for the extraction of the implant. This was due to the non-permeability and non-resorbability of silicone74. Although some of the PNR limitations presented along this manuscript have been referred in the Biomaterials Section, this does not mean that a particular material could solve a given limitation, let alone the PNR therapeutic. Nevertheless, the utilization of diverse concomitant strategies, able to target as much of these limitations as possible, can importantly contribute to the ultimate PNR solution. Indeed, due to the high number of factors interplaying, a combined combat approach seems the only strategy able to reverse the global pathological state. As indicated in this Section, there are different biomaterials with great potential in the area of peripheral nerve regeneration, which have a lot to offer as biomedical tools. Nevertheless, there is still much to do in order to find the ideal alternative to substitute nerve grafts. The further exploitation of biomaterials like keratin or gellan gum, or their combinations, as well as the development of novel techniques for the preparation of therapeutic 3D structures, should be embraced. However, when doing this, it will be very important to carefully control the experimental conditions, with


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the purpose to avoid variability and guarantee reproducibility in the results. Of course, when reproducing experiments from other authors their experimental conditions should be carefully mimicked. As experimentally observed by ours and other groups, biological variations between individuals will make for variations in the response to the applied therapeutic treatment. Due to the standardized conditions utilized during our studies, only the genetic and epigenetic individual endowments are considered to blame for this. Indeed, genetic and epigenetic inter-individual differences are considered in the pharmaceutical industry as responsible for the time consuming process to put a new medicament in the market (media of 15 years). These differences are also the ones responsible for the large prospects indicating “possible secondary adverse effects”. Because one specific interpersonal-variability can lead to an effect in one individual that does not manifest itself in another. The complete sequencing of the human genome in 2003 opened the door to a new medicine, the personalized medicine. This way of practicing medicine is based in the adaptation of the therapeutic strategy to one or more genetic and epigenetic characteristics of the patient in particular281. In the last years several were the works that followed this direction, i.e. the prescription of the anticoagulant drug warfarin282. For now, a limitation of this type of medicine is the high costs involved. Nevertheless, the lowering in the prize of genetic tests and personalized care could make it compete with the high costs of producing a medicament with adequate risk-benefit for all the patients.

5. Concluding remarks and future perspectives The efficient treatment of peripheral nerve injuries, leading to proper anatomical and functional recovery, has worried the medical and scientific communities during years. Regenerative medicine has presented itself as the most efficient solution to cope with this problem. Nevertheless, this is a challenging strategy, where many anatomical, physiological and technological obstacles must be overcome. The increase in the knowledge about the deterioration of the neural structures or the processes triggered after a peripheral nerve injury occurs, have shed some light on the biological targets.



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Also the biotechnological advances have cleared the way towards an efficient regenerative clinical solution, alternative to the conventional nerve grafts. This solution will be the design of complex bioengineered artificial Nerve Guidance Conduits. However, despite the fact that the intense research efforts made have led to very interesting achievements, there is still much work to do. Commercialized Nerve Guidance Conduits have showed a fair amount of success for short gap nerve transection injuries, but this outcome has not repeated itself in the case of long gap injuries, with clear failure. To improve their biofunctionality, NGCs should be loaded with diverse therapeutic cues, intended to interplay in vivo. Many of these cues are endogenous labile molecules or cells, which should be included into an adequate vehicle previous to their administration, for accurate environmental protection and timeframe delivery. For the success of this strategy an important deal of attention should be paid to the starting materials used in the preparation of the NGCs. In particular, naturally occurring polymers seem to present several advantages over synthetic ones. Consequently, the method of elaboration should be adapted to the special characteristics of this type of biomaterials. Green processing strategies that avoid the use of harsh solvents and energy sources should be pursued. Moreover, the use of novel automated techniques, such as rapid prototyping, or adjuvants, such as electric stimulation, may greatly contribute to the therapeutic success of these approaches. It will not be however after the ongoing research in basic knowledge, new possibilities regarding drug therapies and technological improvements bear fruit, that the definite PNR solution will finally be within reach.

Acknowledgements This work has received funding from the European Community’s Seventh Framework Programme (FP7-HEALTH-2011) under grant agreement no 278612 (BIOHYBRID), from ERDF/POCTEP 2007-2013 under project 0687_NOVOMAR_1_P, from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number REGPOT-CT2012-316331-POLARIS, and from the ComplexiTE – An integrated multidisciplinary tissue engineering approach combining novel highthroughput screening and advanced methodologies to create complex biomaterials-


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stem cells constructs (ERC-2012-ADG_20120216-321266). Portuguese Foundation for Science and Technology (FCT) is also acknowledged for the funds provided under the program Investigador FCT to JSC (IF/00115/2015) and JMO (IF/00423/2012 and IF/01285/2015).

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259. Gaudet, I. D.; Shreiber, D. I., Characterization of Methacrylated Type-I Collagen as a Dynamic, Photoactive Hydrogel. Biointerphases 2012, 7 (1), 25. DOI: 10.1007/s13758-012-0025-y. 260. Ondeck, M. G.; Engler, A. J., Mechanical Characterization of a Dynamic and Tunable Methacrylated Hyaluronic Acid Hydrogel. J Biomech Eng 2016, 138 (2), 0210031-0210036. DOI: 10.1115/1.4032429. 261. Wang, W.; Itoh, S.; Takakuda, K., Comparative study of the efficacy of decellularization treatment of allogenic and xenogeneic nerves as nerve conduits. J Biomed Mater Res A 2016, 104 (2), 445-454. DOI: 10.1002/jbm.a.35589. 262. Hall, S. M., Regeneration in cellular and acellular autografts in the peripheral nervous system. Neuropathol Appl Neurobiol 1986, 12 (1), 27-46. DOI: 10.1111/j.1365-2990.1986.tb00679.x. 263. Gulati, A., Evaluation of acellular and cellular nerve grafts in repair of rat peripheral nerve. J Neurosurg. 1988, 68 (1), 117-123. 264. Gulati, A.; Cole, G., Immunogenicity and Regenerative Potential of Acellular Nerve Allografts to Repair Peripheral Nerve in Rats and Rabbits. Acta Neurochir (Wien) 1994, 126 ((2-4)), 158-164. 265. Hudson, T.; Zawko, S.; Deister, C.; Lundy, S.; Hu, C.; Lee, K.; Schmidt, C., Optimized acellular nerve graft is immunologically tolerated and supports regeneration. Tissue Eng. 2004, 10 (11-12), 1641-51. DOI: 10.1089/ten.2004.10.1641. 266. Vasudevan, S.; Huang, J.; Botterman, B.; Matloub, H. S.; Keefer, E.; Cheng, J., Detergent-free Decellularized Nerve Grafts for Long-gap Peripheral Nerve Reconstruction. Plast Reconstr Surg Glob Open 2014, 2 (8), e201. DOI: 10.1097/GOX.0000000000000118. 267. Boriani, F.; Fazio, N.; Fotia, C.; Savarino, L.; Nicoli Aldini, N.; Martini, L.; Zini, N.; Bernardini, M.; Baldini, N., A novel technique for decellularization of allogenic nerves and in vivo study of their use for peripheral nerve reconstruction. J Biomed Mater Res A 2017, 105 (8), 2228-2240. DOI: 10.1002/jbm.a.36090. 268. Sridharan, R.; Reilly, R. B.; Buckley, C. T., Decellularized grafts with axially aligned channels for peripheral nerve regeneration. J Mech Behav Biomed Mater 2015, 41, 124-135. DOI: 10.1016/j.jmbbm.2014.10.002. 269. Cai, M.; Huang, T.; Hou, B.; Guo, Y., Role of Demyelination Efficiency within Acellular Nerve Scaffolds during Nerve Regeneration across Peripheral Defects. Biomed Res Int 2017, 2017, 4606387. DOI: 10.1155/2017/4606387. 270. Crouzier, T.; McClendon, T.; Tosun, Z.; McFetridge, P. S., Inverted human umbilical arteries with tunable wall thicknesses for nerve regeneration. J Biomed Mater Res A 2009, 89A (3), 818-828. DOI: 10.1002/jbm.a.32103. 271. Sun, F.; Zhou, K.; Mi, W.-j.; Qiu, J.-h., Repair of facial nerve defects with decellularized artery allografts containing autologous adipose-derived stem cells in a rat model. Neurosci Lett 2011, 499 (2), 104-108. DOI: 10.1016/j.neulet.2011.05.043. 272. Hassan, N. H.; Sulong, A. F.; Ng, M.-H.; Htwe, O.; Idrus, R. B. H.; Roohi, S.; Naicker, A. S.; Abdullah, S., Neural-differentiated mesenchymal stem cells incorporated into muscle stuffed vein scaffold forms a stable living nerve conduit. J Orthop Res 2012, 30 (10), 1674-1681. DOI: 10.1002/jor.22102.



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273. Zhu, N.; Li, M.; Guan, Y.; Schreyer, D.; Chen, X., Effects of laminin blended with chitosan on axon guidance on patterned substrates. Biofabrication 2010, 2 (4), 045002. DOI: 10.1088/1758-5082/2/4/045002. 274. Huang, Y.-C.; Huang, C.-C.; Huang, Y.-Y.; Chen, K.-S., Surface modification and characterization of chitosan or PLGA membrane with laminin by chemical and oxygen plasma treatment for neural regeneration. J Biomed Mater Res A 2007, 82A (4), 842-851. DOI: 10.1002/jbm.a.31036. 275. Li, G.; Zhang, L.; Yang, Y., Tailoring of chitosan scaffolds with heparin and γ-aminopropyltriethoxysilane for promoting peripheral nerve regeneration. Colloids Surf B Biointerfaces 2015, 134, 413-422. DOI: 10.1016/j.colsurfb.2015.07.012. 276. Xu, Y.; Zhang, Z.; Chen, X.; Li, R.; Li, D.; Feng, S., A Silk Fibroin/Collagen Nerve Scaffold Seeded with a Co-Culture of Schwann Cells and Adipose-Derived Stem Cells for Sciatic Nerve Regeneration. PLoS One 2016, 11 (1), e0147184. DOI: 10.1371/journal.pone.0147184. 277. Dinis, T. M.; Elia, R.; Vidal, G.; Dermigny, Q.; Denoeud, C.; Kaplan, D. L.; Egles, C.; Marin, F., 3D multi-channel bi-functionalized silk electrospun conduits for peripheral nerve regeneration. J Mech Behav Biomed Mater 2015, 41, 43-55. DOI: 10.1016/j.jmbbm.2014.09.029. 278. Barreiros, V. C. P.; Dias, F. J.; Iyomasa, M. M.; Coutinho-Netto, J.; de Sousa, L. G.; Fazan, V. P. S.; Antunes, R. d. S.; Watanabe, I.-s.; Issa, J. P. M., Morphological and morphometric analyses of crushed sciatic nerves after application of a purified protein from natural latex and hyaluronic acid hydrogel. Growth Factors 2014, 32 (5), 164-170. DOI: 10.3109/08977194.2014.952727. 279. Wang, Y.-l.; Gu, X.-m.; Kong, Y.; Feng, Q.-l.; Yang, Y.-m., Electrospun and woven silk fibroin/poly(lactic-co-glycolic acid) nerve guidance conduits for repairing peripheral nerve injury. Neural Regen Res 2015, 10 (10), 1635-1642. DOI: 10.4103/1673-5374.167763. 280. Lin, Y.; Ramadan, M.; Van Dyke, M.; Kokai, L.; Philips, B.; Rubin, J.; Marra, K., Keratin gel filler for peripheral nerve repair in a rodent sciatic nerve injury model. Plast Reconstr Surg 2012, 129 (1), 67-78. DOI: 10.1097/PRS.0b013e3182268ae0. 281. Mathur, S.; Sutton, J., Personalized medicine could transform healthcare. Biomed Rep 2017, 7 (1), 3-5. DOI: 10.3892/br.2017.922. 282. Johnson, J. A.; Cavallari, L. H., Pharmacogenetics and Cardiovascular Disease—Implications for Personalized Medicine. Pharmacol Rev 2013, 65 (3), 987-1009. DOI: 10.1124/pr.112.007252.


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Figures index Figure 1 Neural anatomy. A) Delimitation of the human CNS (yellow) and PNS (blue) structures. B) Image showing different types of neurons: multipolar interneurons (1 and 2), a motor neuron (3) and a sensory neuron (4). All these neurons present 4 general parts: dendrites (α), cell body (β), axon (γ) and axon terminals (δ). The axons of neurons (3) and (4) present myelin sheaths (ε). The spaces between myelin sheath are callled Nodes of Ranvier (ζ). C) 3D (left) and 2D (right) crosssections of a myelinated axon. In the imagen the Schwann cell responsible for the myelin sheath formation can be clearly seen sourrounding the axon (center of the spiral). Figure 2 Events triggered after peripheral nerve damage. A) Via cell surface signaling and retrograde axonal transport, occurs the nucleic induction of specific transcription factors. This up-regulation implies the expression of regenerationassociated genes (RAGs). In the proximal axonal injury end a growth cone emerges: B) Schematic representation of the main cellular elements implicated in the growth cone forward movement (left) and image corresponding to the growth cones of retinal ganglion cell axons (right; observation after fluorescent staining: ALCAM protein can be seen in green and adhesion molecule CAML1 in red) (with permission of Copyright Clearance Center). Figure 3 Different ongoing actions during Wallerian degeneration. A) Image showing the Wallerian degeneration process of retinal ganglion cells axons at the indicated times post-transection (projection images of TdTomato-labeled). The authors reported a rapid depletion in the protein calpastatin in the transected axons (with permission of Elsevier and Copyright Clearance Center). B1) 3 days ex vivo culture of non-treated axotomized sciatic nerves from adult mice B2) 3 days ex vivo culture of axotomized sciatic nerves from adult mice treated with ATP. The authors observed that an increase in extracellular ATP delayed myelin fragmentation and degradation (differential interference contrast, DIC, and myelin protein zero immunofluorescence, P0) (with permission of Springer and Copyright Clearance Center). C) In this schematic representation a harmful stimulus provokes the breakup of the neural axon. Within the first hours after injury dissociated SCs and fibroblasts secrete diverse cytokines and chemokines that activate resident macrophages, while circulating phagocytic neutrophils infiltrate the injury site. Several days after injury phagocytic activated macrophages are the predominant leukocyte population present in the distal nerve stump, and play a critical role in ensuring complete WD. Figure 4 Examples of nerve guidance conduits obtained by our Group from natural origin materials. A) Example of a silk fibroin tube obtained by our research for its utilization in PNR. Also in this case silk fibroin is the only component used in the elaboration of this tube (patent pending). B) Images of a chitosan tube developed by our Research Group with PNR purposes. Chitosan is the only component used in the elaboration of this tube. C) Gellan gum scaffold designed by our Research Group for PNR. The right image shows the natural hydrogel, while in the left image the freeze-dried formulation can be appreciated. These scaffolds were prepared using just low acyl gellan gum and mixtures of gellan gum and methacrylated gellan gum, which permitted to modify the degradation rate. Although SCs responded positively



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to all three systems, the ones made by silk presented a higher potential for Schwann cell development.


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Tables index Table 1 Categorization of peripheral nerve injuries. Table presenting the characterization of peripheral nerve lesions according to Seddon (1943) and Sunderland (1951). This classification is the one used nowadays in clinics. Table 2 Description of the physiopathological events triggered after peripheral nerve injury. This table resumes the most important processes occurring after damage to a peripheral nerve was inflicted. These series of events proved to be of fundamental importance for the regeneration to take place. Peripheral nerve strategies should focus on strengthening the positive ones and palliate those that are negative. Table 3 Main clinical strategies for peripheral nerve injuries management. The three types of possibilities available at the moment for the treatment of peripheral nerve lesions are presented in this Table (stumps suture, autografting and nerve guidance conduits). For long gap lesions autografting remains the gold standard. Nevertheless, the limited degree of success of this strategy has led to the emergence of bioengineered nerve guidance conduits as the most promising alternative. Table 4 Therapeutic molecules with peripheral nerve regeneration potential. This table is a compendium of different drugs with potential to promote PNR. Both biotechnologically obtained and conventional drugs are included in the Table. Some representative examples of the positive effects of these molecules over PNR are also described. These examples are extracted from high quality research works developed by different authors in the area of peripheral nerve treatment strategies. Table 5 Novel biotechnological approaches in peripheral nerve regeneration. This table summarizes the possibilities brought to the PNR area by the advances in the fundamental knowledge and the biotechnological developments. Approaches related to cell therapy and gene therapy are included. Table 6 Biomaterials for nerve engineering. This table includes the preeminent natural-origin biomaterials, endogenous biomaterials and synthetic biomaterials studied for PNR. In each particular case, a brief description of the material is given, together with an overview of the main advantages and disadvantages supporting or impairing its clinical use.



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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Table 1 Seddon (1943) classification

Sunderland (1951) classification

Anatomical consequences

Neurapraxia

Grade I

Focal demyelinisation

Axonotmesis

Grade II

Interruption of axons continuity Destruction of endoneurium architecture Destruction of perineurium architecture Interruption of all nerve structures continuity

Grade III Grade IV Seurotmesis

Grade V

58 ACS Paragon Plus Environment

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Table 2 Anatomic part affected Cell body

Proximal injury end

Distal injury end

Target


Processes occurring Signals from damaged axons induce the upregulation of regenerationassociated genes. The proximal axon stump degenerates to the node of Ranvier and an actin reorganization occurs. The distal axon stump undergoes a degradation process called Wallerian degeneration, which triggers a cascade of events32.

The ultimate chemotactic forces guiding the GC advance are produced by the final targets35. Nevertheless, the potential re-innervation depends on their nature.

Outcome - There is a switch from a transmission phenotype to a regenerative one. - This phenomenon is commonly denominated cell body response26-28.

- An axonal sprout emerges, called growth cone. - It detects permissive and inhibitory signals along the injury site, promoting axonal elongation by extension, retraction and turning of F-actin filopodia29-31. - SCs in the distal axon end (and in a short proximal segment), proliferate and suffer radical modifications in their gene expression, including: --- Down-regulation of structural proteins. --- Up-regulation of cell adhesion molecules and growth factors (like nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), neurotrophins 3 and 4 (NT-3 and NT-4) or glial-derived neurotrophic factor (GDNF))3-4, 33. - Myelin breakdown is triggered by SC phospholipases. These cells and macrophages will clear the myelin and axon debris. After most debris removal SCs align along the injury site, forming structures called Bands of Büngner. A guide for the growing axons4. - Production of ECM components by SCs is critical for regeneration34. - SCs palliate the restructuring of muscular receptors. This SCs capability diminishes with time, leading to muscular atrophy. At this stage, muscle reconstruction by surgical transfers may be the only option36. - Skin sensory receptors can be effectively re-innervated years after injury, with total sensation recovery37. - Histological alterations in the internal organs also impair the restoration of the synaptic inputs38.



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Table 3 THERAPY Conventional strategies

Artificial Nerve Guidance Conduits (NGCs)

SPECIFIC PROBLEM High requirements converge for nerve reunification (neural plasticity, axonal guidance, trophic support…), which increment with the gap length44.

SPECIFIC STRATEGY - Small nerve gap injuries (~2 mm): microsurgical suture. - Bigger gaps: autologous nerve grafting (gold standard)23, 25. - Commonly the sacrifice of pure sensory nerves (sural) is preferred over motor ones22.

High requirements.

- Connection of the two injured stumps by an artificial NGC, which will provide regenerative support72-73.

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ADVANTAGES

LIMITATIONS

OUTCOME

- Micro-suture links the two extremes of the injury. -Nerve autografts provide structural and guiding support for axon growth, also preventing fibrous tissue invasion37.

- Suture impairs axon advance, with risk of misdirection4. - Sacrifice of functional nerves, surgical intervention for extraction, size mismatch, presence of inhibitory molecules26. - SCs misaligning along the fragmented ECM lead to axonal misdirection4. - Immunological rejection of donor grafts. - Long recovery times68 promote compensatory disfuctionalities69. - Starting materials should be chosen carefully (i.e. silicone failure74). - High technological challenge.

- Risk of deficient recovery4.

- NGCs are considered a promising alternative to nerve grafts72-73. - The regulatory approval of certain NGCs for medical use reflects their potential25.


- Only a small number of axotomized axons grow entirely across grafts up to 4 cm70. - Inefficient recovery and pain are common. Indeed, less than 25% of patients regain proper motor function, with less than 3% regaining sensation71.

Commercialized NGCs match nerve grafts efficacy only for short gaps (< 30 mm in humans), having limited efficacy for gaps beyond 20 mm25, 71. In any case, they do not surpass nerve grafts.

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Table 4 DRUG THERAPY Endogenous molecules administration


DRUG

DESCRIPTION

Growth factors GFs for PNR are divided in two (GFs) families of proteins: Neurotrophins, including: NGF, BDNF, NT-3 and NT4. Neuropoietic cytokines, including: GDNF, FGFs and CNTF (ciliary neurotrophic factor)18, 79 .

POSITIVE EFFECT OVER PNR - Promotion of cell survival80-82. - Prevention of neural atrophy81. - Promotion of proximal axonal sprouting and 72, 79, 83-84 growth . - Promotion of proper SCs phenotypic expression85. - Induction of functional improvements86.

Although vascular belonging to a endothelial growth family of angiogenic factors (VEGFs). factors, VEGFs are also potent PNR mediators9. -

Protein

LIMITATIONS

OUTCOME (Representative examples)

- Critical importance of - Intensive research for the timing in the injury site82, development of accurate drug 86. delivery vehicles93-95. - High diffusion rate from One step ahead are the the injury site80, 87 89. techniques that pretend to mimic - Physiological instability, the in vivo GFs interplay, by leading to a low timeframe means of multiple concomitant of action87, 89-90. administration72, 87. Dosage-dependent - A novel tendency pursues the negative side effects, i.e. creation of GF gradients inside NGF injection into NGCs, as growth cones can peripheral tissues induces sense these gradients, modifying sensitivity to mechanical its shape and spatial orientation stimuli in animal models accordingly96-97. and humans, resulting in increased pain91-92. Same as for neurotrophins Recent discoveries indicate that: and neuropoietic - VEGF-B absence impairs nerve cytokines. regeneration in mice. - VEGF-B contributes to restore the innervation of target tissues. - VEGF-B effects are specific for injured tissues. - VEGF-B regeneration effects differ from those of VEGF-A9.



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Erythropoietin (EPO)

Melatonin

Triiodothyroni ne (T3)

Growth hormone

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Glycoprotein hormone Positive effects Same as for neurotrophins - Tan H et al. observed its ability over PNI and neuropoietic to promote axonal regeneration regeneration. cytokines. of retinal ganglion cells after optic nerve crush injury in rats98. - Rotter R et al. proved that EPO promoted muscle restoration and nerve recovery after combined muscle-nerve injury99. N-acetyl-5-methoxy Positive effects - Accurate therapeutic Chang HM et al. observed a tryptamine hormone. over PNI levels at the injury site. positive effect over SCs 32 regeneration. - Risk of undesired effects. proliferation . Thyroid hormone Positive effects - Accurate therapeutic - Mercier G et al. observed the over PNI levels at the injury site. T3 regulation of SCs regeneration. - Risk of undesired effects. functioning100. - Bessede T et al. described neuroregeneration and functional recovery improvements101. Peptide hormone Positive effects - Accurate therapeutic -Devesa P et al. saw promotion over PNI levels at the injury site. of sciatic nerve regeneration regeneration. - Risk of undesired effects. after GH treatment102. - Tuffaha SH et al. showed that GH therapy accelerates axonal regeneration, reduces muscle atrophy and promotes muscle reinnervation103. - Saceda J et al. observed improved nerve regeneration and better conduction velocities with GH104.


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Conventional drugs administration


Acetyl-Lcarnitine

Acetylated quaternary Positive effects - Accurate therapeutic amine (3-hydroxy-4- over PNI levels at the injury site. trimethylaminobutirat regeneration. - Risk of undesired effects. e). Transport molecule (fatty acids).

Zonisamide

- Anti-epileptic - Anti-parkinsonnian

Riluzole

Amyotrophic lateral Positive effects - Accurate therapeutic sclerosis treatment over PNI levels at the injury site. regeneration. - Risk of undesired effects.

Nacetylcysteine

- Mucus loosen Treatment of paracetamol overdose Anti-inflammatory

Dexamethasone*

Positive effects - Accurate therapeutic over PNI levels at the injury site. regeneration. - Risk of undesired effects.

Positive effects over PNI regeneration. Positive effects over PNI regeneration.

- Accurate therapeutic levels at the injury site. - Risk of undesired effects. - Accurate therapeutic levels at the injury site. - Risk of undesired effects.


- Its antihyperalgesic and neuroregenerative effects were proved by Vivoli E et al.105. - Karsidag S et al. verified that it enhanced the quality of neural recovery, also leading to a decline in nerve death106. - Yagi H et al. saw that it enhances neurite regeneration, also protecting against oxidative stress107-108. - Tanabe N et al. proved the zinosamide analgesic effect109. - Nógrádi A et al. observed an enhancement of motoneurons survival and axon regeneration, even when injury treatment was delayed for 10 days110. - Costa HJ et al. saw riluzole axon density preservation111. Hart AM et al.112 and West CA et al.113 described its neuroprotective effect. Mohammadi R et al. described the acceleration of nerve repair and target organ re114 innervation . Li H et al. asserted that it protects the blood-nerve


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*

Ibuprofen

Non-steroidal inflammatory

anti- Positive effects - Accurate therapeutic over PNI levels at the injury site. regeneration. - Risk of undesired effects.

Tacrolimus*

Immunosuppressant

Positive effects - Accurate therapeutic over PNI levels at the injury site. regeneration. - Risk of undesired effects.

Rapamycin*

Immunosuppressant

Enhancement of - Obtaining of accurate PNI recovery. therapeutic concentration in the site of action. - Risk of undesired effects.

*

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barrier115. - Pires LR et al. demonstrated its enhancement of axonal 116 regeneration . - Madura T et al. proved its effect over PNR functional improvement117. - Tajdaran K et al.12 and Gold BG et al.118 observed its enhancement of axonal regeneration. Ding T et al. proved that promotes nerve regeneration119.

The examples in this table reflect once more the important role of the immune system in the pathogenesis of PNI.


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Table 5 THERAPY Cell therapy

SPECIFIC STRATEGY SCs administration

Stem administration

cells

(i.e. Bone marrow122, olfactory ensheathing18, epidermal neural crest123-124, amniotic membrane124, adipose derived (ADSCs)18, 125 or dental pulp (DPSCs)126.

Cell engineering

ADVANTAGES

LIMITATIONS

OUTCOME

- Essential for PNR (Section 2.1.). - ~80% of the total cell count at day 1 after sciatic nerve injury30. - Ability to transform into Schwann-like cells. - Low immunogenicity. - Efficient in vitro expansion rates57, 120. - Preference for sites of inflammation and tissue damage, where they have immunosuppressive and antifibrotic effects73. - ADSCs are present in large amounts in liposuctionderived adipose tissue18, 125. - DPSCs can be easily isolated from discarded wisdom teeth, in a low invasive manner121, 126. - Render cells as vehicles of different therapeutics. - Confer cells with specific

- Sacrifice of healthy Fundamental for PNR nerves for SC isolation. - Hetero-immunogenic - Low rate of expansion in vitro120-121. - Their activity should be Improvement of PNR carefully controlled in order to avoid undesired adverse effects127. -The utilization of proper vehicles is recommended for their adequate administration120.

- Cells may become Physical, chemical and material immunogenic after being methods have lead to two main engineered. outcomes:



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Gene therapy

functionalities94.

- Obtaining in a large scale for widespread therapeutics is still a challenge.

DNA approaches

Delivery of genes of interest into the cell nucleus and its effective expression128.

RNA approaches

- RNA interference (RNAi) is an endogenous mechanism for gene activity regulation. Specific mRNA degradation leads to post-transcriptional

- The nuclear location of action implies the use of specialized delivery vectors: -- Viral vectors, with very high transfection efficiency. They present many adverse effects, such as: lack of control over host genome insertion, mutagenesis, immunogenicity…129. -- Although safer, nonviral vectors transfect in less extent128, 130. But more efficient ones are being created131. - Accurate expression shut down is critical132. Specific vehicles are needed for their administration, mainly due to: their poor biological stability (rapid degradation


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- Encapsulation of drugs inside cells. - Immobilization of molecules of interest onto cell membranes94. Therapeutic potential for PNR: - For instance, Karagyaur M et al. observed that the injection of plasmid encoding BDNF led to better restoration of neural structure and function in mice133. - Other illustrative examples are134-136.

- RNA is for PNR, observed silencing outgrowth

potentially beneficial i.e. Yao C et al. that uc.217 siRNA promoted neurite in cultured dorsal

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root ganglion neurons142. - New possibilities will appear in parallel with the increase in basic knowledge, i.e. Li S et al. investigated the lethal-7 miRNAs expression changes on SCs and axons following PNI, discovering positive effects of their down-regulation over regeneration143; miRNAs expression is being studied in diverse pain animal models51. - Synergistic effect of gene - That of cell and gene Therapeutic potential for PNR, and cell therapy. therapy. i. e.: - Opens the possibility of - Higher engineering - May F et al. transfected SCs using cells as drug delivery challenge. with retroviral vectors vehicles and biological expressing GDNF, reducing the factories of therapeutic time for cavernous nerve proteins, like growth functional recovery132. 144-145 factors - Hu Y et al. used lentiviral . vectors to transfect SCs with CNTF-encoding plasmids, leading to better retinal ganglion cells regeneration than with autograft therapy146. - Different stem cells were transfected with plasmids encoding diverse GFs, with interesting PNR results147-149.

silencing, which blocks the production of the related protein137-139. - In this case the cell nucleus membrane does not need to be overcame, as RNAs act in the cell cytoplasm.

Cellular gene therapy

Combination of cell and gene therapy

by nucleases), poor cellular specificity and inefficient cellular uptake140-141.



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Vascularization

Nitric oxide (NO) administration

GFs administration

Potent angiogenic inductor150. - Low half-life. - Site-concentration is critical. High NO concentrations are inhibitory151. VEGFs and FGF-2 are potent - The ones described in Table 3 for GFs angiogenic promoters154. administration. - Days to weeks are needed for GF-induced capillary formation, giving rise to cell dysfunctions and survival deficits during the first stages of regeneration23.


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Different strategies for NO administration showed promising results152-153.

Diverse studies have been dedicated to the administration of GFs with angiogenic purposes155. For instance, Zieris A et al. have developed a strategy for the dual combined delivery of VEGF and FGF2154.

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Cell therapy

Gene therapy

Cellular gene therapy Oxygen suppliers

Expound in the Cell therapy Expound in the Cell part of this same table. therapy part of this same table. Expound in the Gene therapy Expound in the Gene part of this same table. therapy part of this same table (i.e. side effects originated by dis-regulated VEGF expression158).

There is great potential in this area156-157.

- High potential of plasmid promoted angiogenesis159-160. - High interest of RNA strategies, i.e. Yue J et al. used miRNAs for managing vascular cell movement161 and Bonauer A et al. employed an antogomir (anti-miRs) to inhibit an endogenous miRNA known to degrade mRNA corresponding to pro-angiogenic factors162. Synergistic effect of gene and High engineering Growing number of works137, 163 cell therapy. challenge. . Alternative to solve the initial Maintenance of proper Zhu S et al. have added the avascularization problem. concentrations at the site perfluorocarbon PFTBA, a of action. compound with high oxygen solubility properties, to their NGC. This approach had successful angiogenic results23. A similar strategy was followed by Wang Y et al.164.



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Table 6 NATURALENDOGENOUS ORIGIN BIOMATERIALS BIOMATERIALS Silk fibroin

Keratin

Chitosan

SYNTHETIC BIOMATERIALS

COMPOSITION

Fibrous proteins fabricated by arthropods. They consist in short side-chain amino acids (including glycine, alanine or serine) and hydrophilic blocks with more complex sequences (including larger sidechain amino acids)166. Family of fibrous proteins. They can present several conformations: helical (α-keratin), rectal (βkeratin), and undefined169-170.

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ADVANTAGES

- Biocompatible - Biodegradable Tailorable mechanical properties - Degradation profile dependent on the conformation - Can be easily shaped in several architectures167. -Biocompatible - Biodegradable - Interaction with cell surface receptors169. - High mechanical properties due to the presence of numerous disulfide bonds171. Linear polysaccharide - Biocompatibility consisting of D- Non-toxicity glucosamine residues - Biodegradability with a variable number - Non-antigenicity of randomly located N- Mucoadhession acetyl-D-glucosamine - Antimicrobial groups. Obtained through - Biomechanics


DISADVANTAGES

Insolubility in water (due to the high concentration of hydrophobic amino acids)168.

- Insoluble in water, solutions of weak alkali and acids, and most organic solvents172.

Insoluble at neutral or basic pHs (weak acids are needed instead). Nevertheless, chemical reactions like carboxymethylation can transform chitosan

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alkaline N-deacetylation of chitin, a structural component in the exoskeleton of crustaceans173-174. Brown algae-derived polysaccharide composed of polyguluronate and polymannuronate repeating blocks179

Alginate

Gellan gum

Collagen

Linear exopolysaccharide produced by the bacteria Sphingomonas (formerly Pseudomonas) elodea, with a repeating unit consisting of α-Lrhamnose, β-D-glucose, and β-D-glucuronate183. Group of extracellular matrix structural proteins. They are formed by polypeptide chains constituted by repeating triplets of Glycine and two other amino acids, commonly proline and


- Vascularization into a semi-synthetic supporter water soluble - Anti-inflammatory material178. - Wound healing175, 177. - Biocompatible - Non-toxic -Non-immunogenic - Mucoadhesive180-181.

Mammals lack the enzyme (i.e., alginase) to cleave the polymer chains, as consequence the renal clearance is impaired182.

- Biocompatibility - Degradation into non-toxic byproducts Adjustable mechanical properties184.

Very high degradation rates185. As the authors experimentally observed, methacrylated semisynthetic gellan gum can help to reduce the degradation rates.

- Biocompatible - Biodegradable - Emulation of the ECM environment187. Indeed, collagen folding, self-assembly, and fibril formation can be easily mimicked in vitro188.

Insoluble in water187. Relatively fast in vivo degradation and loss of its mechanical strength189.


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hydroxyproline186. Fibrin is formed from the soluble plasma protein fibrinogen190.

-Neurogenic191. - Together with the enzyme thrombin present adhesive properties192. Trimeric protein - Biocompatible containing α-, β- and γ- - Biodegradable subunits194. - Important structural - Growth promoting functions194, 196. molecules for axons195. - Biocompatibility Co-polymer of Dglucuronic acid and N- - Biodegradability - Non-immunogenicity acetyl-D-glucosamine 198 present in all tissues . - Pivotal mediator of cell motility - Bacteriostatic - Fungistatic - Anti-inflammatory - Anti-edematous - Pro-angiogenetic198199 .

Fibrin

Laminin

Hyaluronic acid

Chondroitin sulfate

Composed by alternated Nacetylgalactosamine and glucuronic acid units. Silicone

- Antiinflammatory properties201.

Pro-inflammatory193.

Proteolysis. Although some authors described the angiogenic potential of laminin proteolysis197. Ability to resist degradation by hyaluronidases200.

- PNR inhibitor195.

Synthetic compound FDA approved Serious clinical made from repeating biomaterial. complications due to


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Poly-glycolic acid

Poly-lactic-coglycolic

Poly-caprolactone

siloxane units, frequently High combined with carbon biocompatibility. and/or hydrogen. - Oxygen permeability. - Optical transparency. - Tunable stiffness202. Is the simplest linear - Biocompatible and aliphatic polyester. It can biodegradable203. be prepared starting from Scaffolds with glycolic acid. controllable porosity, topographies, mechanical properties and degradation rates204. Co-polymer of glycolic - Biocompatible and acid and lactic acid. biodegradable206. - It permits the fabrication of scaffolds with controllable porosity, topographies, mechanical properties and degradation properties204. Polyester. It can be Biocompatible, prepared starting from ε- bioresorbable and lowcaprolactone or 2- cost209. - Excellent tensile methylene-1-3strength210. dioxepane.


its non-resorbable nature75.

Degradation subproducts are known to cause inflammatory responses205.

Serious local inflammatory responses induced by acid degradation byproducts207-208.

Slow rate211-212.

resorption


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For Table of Contents Use Only

Peripheral nerve injury: Current challenges, conventional treatment approaches and new trends on biomaterials-based regenerative strategies R. López-Cebral,1,2,* J. Silva-Correia,1,2 R. L. Reis,1,2 T. H. Silva,1,2 and J. M. Oliveira1,2 Affiliations 1

3Bs Research Group, Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark - Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal;

2

ICVS/3Bs, PT Government Associate Laboratory, Braga/Guimarães, Portugal.

*Corresponding author: Rita López-Cebral, PhD E-mail: [email protected] Postal address: 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, AvePark – Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal


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