DOI:10.1017/S0029665109990206

Reviews Molecular Cell Biology. 2013;**14**(8):467-473. DOI: 10.1038/nrm3620

10.1007/s40778-016-0030-z

**Provisional chapter**

### **Peripheral Nerve Reconstruction Using Enriched Chitosan Conduits Chitosan Conduits**

**Peripheral Nerve Reconstruction Using Enriched**

DOI: 10.5772/intechopen.69882

Shimon Rochkind, Mira M. Mandelbaum-Livnat, Stefania Raimondo, Michela Morano, Giulia Ronchi, Nicoletta Viano, Moshe Nissan, Akiva Koren, Tali Biron, Yifat Bitan, Evgeniy Reider, Mara Almog, Ofra Ziv-Polat, Abraham Shahar and Stefano Geuna Stefania Raimondo, Michela Morano, Giulia Ronchi, Nicoletta Viano, Moshe Nissan, Akiva Koren, Tali Biron, Yifat Bitan, Evgeniy Reider, Mara Almog, Ofra Ziv-Polat, Abraham Shahar and Stefano Geuna Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Shimon Rochkind, Mira M. Mandelbaum-Livnat,

http://dx.doi.org/10.5772/intechopen.69882

#### **Abstract**

The repair of peripheral nerve traumatic lesions still represents a major cause of permanent motor and sensory impairment. In case of substance loss, a nerve guide should be used to bridge the proximal with the distal stump of the severed nerve. The effectiveness of hollow nerve guides is limited by the delay of axonal growth due to the absence of a regeneration substrate inside the conduit. To fasten up nerve regeneration, nerve guides should thus be enriched by a luminal filler. In this study, we investigated, in a 12-mm rat sciatic nerve defect experimental model, the effectiveness of chitosan-based conduits of different acetylation filled either with a hyaluronic acid gel (NVR gel) or with a magnetic fibrin hydrogel, in comparison with traditional autografts. Results showed that all types of artificial nerve conduits led to functional recovery not significantly different from autografts. By contrast, morphological and morphometrical analyses showed that the best results among nerve guides were found in medium degree of acetylation (DAII: ∼5%) chitosan conduits enriched with the NVR gel.

**Keywords:** peripheral nerve injury, chitosan guidance conduit, conduit acetylation, hyaluronic acid gel, magnetic fibrin hydrogel

### **1. Introduction**

Chitosan is a biopolymer derived from chitin that can be extracted in large quantities from the shells of the crustaceans. Among the different applications, the use of chitosan

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

as a scaffold for neural repair is receiving growing interest from the scientific community and, in particular, many experimental studies have shown that chitosan-based conduits provide an effective scaffold for the reconstruction of peripheral nerve defects [1]. Recently, chitosan hollow nerve guides have been approved for clinical use (Reaxon® Nerve Guide) [2].

In spite of these positive results, the early growth of axons along hollow nerve guides is delayed since the conduit's lumen should first be colonized by connective tissue and Schwann cells migrating from the distal nerve stump [3, 4]. To prevent this occurrence, a luminal filler must be used for providing a substrate for early nerve fiber regeneration [5–7]. A number of different materials have been investigated as luminal fillers for nerve guides, including biological or artificial substrates [8–11].

The aim of the present study was to investigate rat sciatic nerve reconstruction with chitosan conduits filled up with two types of substrates which might potentially improve axon regeneration, namely (i) NVR gel and (ii) magnetic fibrin hydrogel.

NVR gel is a gel based on hyaluronic acid (HA), a non-immunogenic biopolymer that has been proposed for various tissue engineering applications [12, 13]. HA is a glycosaminoglycan composed of disaccharide D-glucuronic acid and N-acetyl-D-glucosamine produced by hyaluronan synthases, a membrane-bound enzyme. It is a component of ECM where it modulates cell adhesion, migration, and neuronal sprouting, and it controls tissue homeostasis and absorbs mechanical shock. HA action is related to its molecular weight; a long polymer (>500 kDA) is generally known as high molecular weight HA (HMW HA) and is found predominantly in the brain where it has a structural role and takes part in various biological process like silences inflammation, angiogenesis and neural differentiation [14]. In the peripheral nerves, HA is one of the prominent components of the Bunger band formed during Wallerian degeneration, and the ablation of HA receptors can reduce cell adhesion and migration [15]. Several studies showed that HA has a positive effects on peripheral nerve regeneration [16, 17].

Fibrin hydrogels are natural polymers made by mixing two blood coagulation components, fibrinogen and thrombin, which form a clot upon mixing.

In previous studies, it was demonstrated that appropriate conjugation of thrombin to iron oxide (γ-Fe2O3) magnetic nanoparticles preserved the thrombin-clotting activity, stabilized the thrombin against its major inhibitor, antithrombin III, and improved its storage stability [18, 19]. Moreover, these thrombin-conjugated γ-Fe2 O3 nanoparticles were used to fabricate novel magnetic fibrin hydrogel scaffolds [20].

### **2. Materials and methods**

### **2.1. Chitosan tube preparation**

Chitosan conduits were provided by Medovent GmbH (Mainz, Germany). They were produced under ISO 13485 conditions from chitin tubes made by extrusion process. Distinctive washing and hydrolysis steps were then carried out to adjust the required low, medium, and high degree of acetylation (DA): (1) DAI tubes (low DA, ∼2%); (2) DAII tubes (medium DA, ∼5%); and (3) DAIII tubes (high DA, ∼20%). Tubes were finally cut and sterilized by electron beam.

### **2.2. Preparation of NVR gel**

as a scaffold for neural repair is receiving growing interest from the scientific community and, in particular, many experimental studies have shown that chitosan-based conduits provide an effective scaffold for the reconstruction of peripheral nerve defects [1]. Recently, chitosan hollow nerve guides have been approved for clinical use (Reaxon®

In spite of these positive results, the early growth of axons along hollow nerve guides is delayed since the conduit's lumen should first be colonized by connective tissue and Schwann cells migrating from the distal nerve stump [3, 4]. To prevent this occurrence, a luminal filler must be used for providing a substrate for early nerve fiber regeneration [5–7]. A number of different materials have been investigated as luminal fillers for nerve guides, including bio-

The aim of the present study was to investigate rat sciatic nerve reconstruction with chitosan conduits filled up with two types of substrates which might potentially improve axon regen-

NVR gel is a gel based on hyaluronic acid (HA), a non-immunogenic biopolymer that has been proposed for various tissue engineering applications [12, 13]. HA is a glycosaminoglycan composed of disaccharide D-glucuronic acid and N-acetyl-D-glucosamine produced by hyaluronan synthases, a membrane-bound enzyme. It is a component of ECM where it modulates cell adhesion, migration, and neuronal sprouting, and it controls tissue homeostasis and absorbs mechanical shock. HA action is related to its molecular weight; a long polymer (>500 kDA) is generally known as high molecular weight HA (HMW HA) and is found predominantly in the brain where it has a structural role and takes part in various biological process like silences inflammation, angiogenesis and neural differentiation [14]. In the peripheral nerves, HA is one of the prominent components of the Bunger band formed during Wallerian degeneration, and the ablation of HA receptors can reduce cell adhesion and migration [15]. Several studies showed that HA has a positive effects on peripheral nerve

Fibrin hydrogels are natural polymers made by mixing two blood coagulation components,

In previous studies, it was demonstrated that appropriate conjugation of thrombin to iron oxide (γ-Fe2O3) magnetic nanoparticles preserved the thrombin-clotting activity, stabilized the thrombin against its major inhibitor, antithrombin III, and improved its storage stability

Chitosan conduits were provided by Medovent GmbH (Mainz, Germany). They were produced under ISO 13485 conditions from chitin tubes made by extrusion process. Distinctive washing

O3

nanoparticles were used to fabricate

Nerve Guide) [2].

regeneration [16, 17].

logical or artificial substrates [8–11].

eration, namely (i) NVR gel and (ii) magnetic fibrin hydrogel.

164 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

fibrinogen and thrombin, which form a clot upon mixing.

[18, 19]. Moreover, these thrombin-conjugated γ-Fe2

novel magnetic fibrin hydrogel scaffolds [20].

**2. Materials and methods**

**2.1. Chitosan tube preparation**

NVR gel was prepared by mixing high molecular weight hyaluronic acid (3×106 Da, BTG Polymers, Kiryat Malachi, Israel) and laminin (Sigma, Rehovot, Israel). The NVR gel was then diluted with nutrient medium (composed of 86% Dulbecco's modified Eagle medium-nutrient mixture F-12), 10% heat-denatured fetal calf serum (FCS), 6 g/L D-glucose, 2 nM glutamine, 25 μg/mL gentamycin, and 50 ng/mL IGF-I, all purchased from Biological Industries, Israel, to a final concentration of 0.5%. NVR gel was then injected into the chitosan tubes immediately before surgical implantation.

### **2.3. Preparation of the magnetic fibrin hydrogel**

Magnetic fibrin hydrogel was prepared by mixing 30 μL of bovine fibrinogen (from stock solution of 100 mg/mL, in PBS without Ca2+ and Mg2+), 460 μL of a culture medium, and 10 μL of a CaCl2 aqueous solution (from stock solution of 25 mM). To achieve coagulation, thrombin was conjugated to iron oxide nanoparticles [19–21] and added to the gel (50 μL from a stock solution of 100 μg/mL). The final liquid mixture was then injected into the chitosan conduits immediately before surgical implantation.

### **2.4. Experimental design and surgical technique**

All animal experiments were approved by the Institutional Animal Care and Usage Committee (IACUC) and adhered strictly to the Animal Care Guidelines of University Tel Aviv Sourasky Medical Center (number of approval for animal testing 9-3-12). Female Wistar rats were brought to the vivarium 2 weeks prior to the surgery and housed two per cage with a 12-h light/dark cycle, with free access to food and water. The study was conducted on 45 female Wistar rats (weight 200–250 g), using an experimental model for producing a complete peripheral nerve injury with massive nerve defect that has been recently described [22].

General anesthesia was induced with intraperitoneal injection of xylazine (15 mg/kg) and ketamine (50 mg/kg). Surgical procedures were performed using a high magnification microscope. The left sciatic nerve was exposed and separated from biceps femoris and semimembranosus muscles beginning from the area of branches to the glutei and hamstring muscles and distally to the trifurcation into peroneal, tibial, and sural nerves. The sciatic nerve was completely transected at the third femur level using microsurgical scissors and a 6-mm nerve segment was removed. A 14-mm chitosan hollow conduit of different degrees of acetylation (DAI ~2%; DAII ~5%; DAIII ~20%) filled with 0.5% NVR gel was placed between the proximal and the distal parts of the transected nerve for reconstruction, enabling the nerve to enter the conduit 1 mm on each side, while providing a 12-mm gap between the proximal and distal ends. Two 9–0 non-absorbable sutures were used to anchor the conduit to the epineurium at the proximal and distal nerve stumps (**Figure 1**).

**Figure 1.** In vivo image of sciatic nerve reconstruction using chitosan-based scaffold immediately after its implantation.

Only one experimental group was used for testing magnetic fibrin hydrogel injected inside DAI tubes.

Autologous nerve graft reconstruction was adopted as control. The left sciatic nerve was exposed as described above and then sharply incised with microscissors at the femur level below the superior gluteal nerve and above the division of the sciatic nerve to the tibial nerve and the peroneal nerve. A nerve segment of 12 mm was harvested, turned upside down, and immediately replaced to bridge the nerve gap using 2–3 non-absorbable 10-0 sutures for each stump. Coaptation of nerve fascicles was carried out to preserve all the fascicles within the epineural sac. The muscular, subcutaneous and skin layers were closed.

Experimental groups can be then summarized as follows: DAI tubes filled with NVR gel (n = 10); DAII tubes filled with NVR gel (n = 10); DAIII tubes filled with NVR gel (n = 10); DAI tubes filled with magnetic fibrin hydrogel (n = 10); and autologous nerve grafts (n = 5).

### **2.5. Functional analysis and electrophysiological evaluation**

Preoperative evaluation and postoperative follow-up were performed and consisted of motor assessment of the sciatic nerve utilizing sciatic functional index (SFI) (data not shown) and somatosensory evoked potentials (SSEP). SFI and SSEP were tested preoperatively and retested at 90 and 120 days postoperatively.

All assessments were carried out in a blinded manner without disclosure of rat's affiliation to the evaluating team.

SSEP were recorded in both the operated and intact hind limbs using a Dantec™ KEYPONT® PORTABLE. Conductivity of rat sciatic nerve and spinal cord was studied by stimulation of the sciatic nerve at the level of the tarsal joint with simultaneous recording from the skull over the somatosensory cortex in anesthetized rats. Two subcutaneous needle electrodes were inserted under the skin of the scalp with the active electrode over the somatosensory cortex along the midline and reference electrode between the eyes. The ground electrode was placed subcutaneously on the dorsal neck. The sciatic nerve was stimulated by a set of two polarized electrodes placed on the lateral aspect of the tarsal joint. Three hundred stimulation pulses of 0.2 ms in duration were generated at a rate of 3 Hz. The stimulus intensity was set on 2–5 mA, and a slight twitching of the limb was noted in all rats. The appearance of an evoked potential in at least two consecutive tests as a response to a stimulus was considered positive.

### **2.6. Morphological and stereological analysis**

Nerve samples were collected 120 days after surgery. The complete conduits were harvested together with parts of proximal and distal nerves, and the whole specimens were fixed for 4–6 h at 4°C in 2.5% glutaraldehyde prepared in 0.1 M phosphate buffer (pH 7.4). A postfixation was then performed using 2% osmium tetroxide for 2 h, followed by dehydration in ethanol from 30 to 100%. After two washings with propylene oxide of 7 min each, samples were left for an hour in a mixture of propylene oxide and Glauerts' mixture of resins (50% Araldite HY964, 50% Araldite M, and 0.5% dibutylphthalate) mixed in equal parts. Then, samples were immersed in Glauerts' mixture of resins and left overnight. A second immersion with Glauerts' mixture of resin was then performed, leaving samples at 37°C for 1 h. Samples were then immersed twice for 30 min in resin with the addition of 2% of accelerator 964 and finally left in resin for three days at 60°C. Resin-embedded nerves were then processed for stereological analysis: transverse sciatic nerve sections were prepared starting from the distal part of the samples. For optical analysis, semi-thin sections (2.5 μm of thickness) were prepared using Ultracut UCT ultramicrotome (Leica Microsystems, Wetzlar, Germany) and stained with 1% toluidine blue.

The stereological analysis [23] was performed on semi-thin transverse nerve section stained with toluidine blue using DFC320 digital camera on DM4000B microscope. A random selected semi-thin section was analyzed for each sample with the help of specific image software (IM50 image manager system, Leica Microsystems). The total cross-sectional area of the nerve was measured. Then, 12–16 fields of the section were randomly selected following a systematic random protocol and proceeded as described previously [24] to measure the following parameters: total number of myelinated fibers, axon diameter and fiber diameter, myelin thickness, and g-ratio. Finally, the correlation between g-ratio and axon diameter of individual fibers was carried out as scatter plot graphs.

### **2.7. Statistical analysis**

Only one experimental group was used for testing magnetic fibrin hydrogel injected inside

**Figure 1.** In vivo image of sciatic nerve reconstruction using chitosan-based scaffold immediately after its implantation.

Autologous nerve graft reconstruction was adopted as control. The left sciatic nerve was exposed as described above and then sharply incised with microscissors at the femur level below the superior gluteal nerve and above the division of the sciatic nerve to the tibial nerve and the peroneal nerve. A nerve segment of 12 mm was harvested, turned upside down, and immediately replaced to bridge the nerve gap using 2–3 non-absorbable 10-0 sutures for each stump. Coaptation of nerve fascicles was carried out to preserve all the fascicles within the epineural sac. The muscular, subcutaneous and skin layers were

Experimental groups can be then summarized as follows: DAI tubes filled with NVR gel (n = 10); DAII tubes filled with NVR gel (n = 10); DAIII tubes filled with NVR gel (n = 10); DAI tubes filled with magnetic fibrin hydrogel (n = 10); and autologous nerve grafts (n = 5).

Preoperative evaluation and postoperative follow-up were performed and consisted of motor assessment of the sciatic nerve utilizing sciatic functional index (SFI) (data not shown) and somatosensory evoked potentials (SSEP). SFI and SSEP were tested preoperatively and

All assessments were carried out in a blinded manner without disclosure of rat's affiliation to

SSEP were recorded in both the operated and intact hind limbs using a Dantec™ KEYPONT® PORTABLE. Conductivity of rat sciatic nerve and spinal cord was studied by stimulation of the sciatic nerve at the level of the tarsal joint with simultaneous recording from the skull over the somatosensory cortex in anesthetized rats. Two subcutaneous needle electrodes were inserted under the skin of the scalp with the active electrode over the somatosensory cortex along the midline and reference electrode between the eyes. The ground electrode was placed subcutaneously on the dorsal neck. The sciatic nerve was stimulated by a set of two polarized electrodes placed on the lateral aspect of the tarsal joint. Three hundred

**2.5. Functional analysis and electrophysiological evaluation**

166 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

retested at 90 and 120 days postoperatively.

the evaluating team.

DAI tubes.

closed.

*Functional and electrophysiological* analyses were done using MATLAB software (v.2008b, The MathWorks, Inc.). Non-parametric statistics were used in this study. Hence, all figures are presented with Median ± MAD. Significance levels were calculated using a Mann-Whitney U test and a Wilcoxon signed-rank test. SSEP responses were analyzed as categorical parameters using χ2 test.

*For stereological analysis*, one-way ANOVA followed by Bonferroni post hoc test was performed using SPSS Statistic Program. The GraphPad software was used to obtain the regression line in scatter plot and to test whether slopes are significantly different. Results are reported as a mean ± standard deviation.

### **3. Results**

### **3.1. Functional analysis and electrophysiological evaluation**

Due to autotomy which often occurs in the operated hind limb [25], the number of rats available for meaningful SFI testing was not sufficient for reliable statistics (data not shown). The group of low acetylation conduit (DAI) filled with NVR gel had the lowest number of autotomies (4 out of 10), while the group of low acetylation (DAI) conduit filled with magnetic fibrin hydrogel had the highest number of autotomies (8 out of 10 rats).

For the electrophysiological evaluation, the SSEP peak-to-peak (P2P) amplitude was calculated. Two consecutive electrophysiological recordings were performed on 38 and 39 rats during 90 and 120 days, respectively. In order to evaluate P2P amplitude in the operated and intact limbs, we calculated the results from each limb separately. No significant differences were found between limbs (data not shown). Rats with P2P measurement in only one of the limbs were ignored in calculations from limb comparison. Regarding the operated limb, no significant differences were observed between all groups after both 90 and 120 days (**Figure 2**).

The P2P values of the intact limb were subtracted from the values of the operated limb in order to evaluate the effectiveness of the various conduits and fillers. A value closer to "0" indicates similar activity in both intact and operated limbs, while a major shift is an indicator of neurological dysfunction. As can be seen in **Figure 3**, all groups evolved with a value close to "0," and no significant difference among the various acetylation conduits and fillers was detected.

### **3.2. Morphological and stereological analysis**

During harvesting (120 days postoperation), a macroscopic observation was performed in order to evaluate the degradation conditions of the conduit and the presence of nerve filaments inside the conduit reconnecting the two nerve stumps. This qualitative analysis showed that 2 rats out of 9 of DAI-NVR gel group and 6 rats out of 10 of DAIII-NVR gel group showed conduits with sign of degradation. Moreover, 5 rats out of 7 of DAI-magnetic fibrin clot group, 7 rats out of 9 of DAI-NVR gel group, 8 rats out of 10 of DAII-NVR gel group, and 8 rats out of 10 of DAIII-NVR gel group showed macroscopically signs of regeneration. This macroscopic observation was then confirmed after histological analysis.

The morphological evaluation was carried out on regenerated nerves immediately downstream to the conduit, 120 days after nerve repair. Representative high-resolution light microscopy images of transverse semi-thin sections of regenerated sciatic nerves of all experimental groups are shown in **Figure 4**. A good regeneration was observed in most of

in scatter plot and to test whether slopes are significantly different. Results are reported as a

Due to autotomy which often occurs in the operated hind limb [25], the number of rats available for meaningful SFI testing was not sufficient for reliable statistics (data not shown). The group of low acetylation conduit (DAI) filled with NVR gel had the lowest number of autotomies (4 out of 10), while the group of low acetylation (DAI) conduit filled with magnetic fibrin

For the electrophysiological evaluation, the SSEP peak-to-peak (P2P) amplitude was calculated. Two consecutive electrophysiological recordings were performed on 38 and 39 rats during 90 and 120 days, respectively. In order to evaluate P2P amplitude in the operated and intact limbs, we calculated the results from each limb separately. No significant differences were found between limbs (data not shown). Rats with P2P measurement in only one of the limbs were ignored in calculations from limb comparison. Regarding the operated limb, no significant differences were observed between all groups after both 90 and 120 days

The P2P values of the intact limb were subtracted from the values of the operated limb in order to evaluate the effectiveness of the various conduits and fillers. A value closer to "0" indicates similar activity in both intact and operated limbs, while a major shift is an indicator of neurological dysfunction. As can be seen in **Figure 3**, all groups evolved with a value close to "0," and no significant difference among the various acetylation conduits and fillers was

During harvesting (120 days postoperation), a macroscopic observation was performed in order to evaluate the degradation conditions of the conduit and the presence of nerve filaments inside the conduit reconnecting the two nerve stumps. This qualitative analysis showed that 2 rats out of 9 of DAI-NVR gel group and 6 rats out of 10 of DAIII-NVR gel group showed conduits with sign of degradation. Moreover, 5 rats out of 7 of DAI-magnetic fibrin clot group, 7 rats out of 9 of DAI-NVR gel group, 8 rats out of 10 of DAII-NVR gel group, and 8 rats out of 10 of DAIII-NVR gel group showed macroscopically signs of regeneration. This macroscopic

The morphological evaluation was carried out on regenerated nerves immediately downstream to the conduit, 120 days after nerve repair. Representative high-resolution light microscopy images of transverse semi-thin sections of regenerated sciatic nerves of all experimental groups are shown in **Figure 4**. A good regeneration was observed in most of

**3.1. Functional analysis and electrophysiological evaluation**

168 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

hydrogel had the highest number of autotomies (8 out of 10 rats).

**3.2. Morphological and stereological analysis**

observation was then confirmed after histological analysis.

mean ± standard deviation.

**3. Results**

(**Figure 2**).

detected.

**Figure 2.** Somatosensory evoked potentials (SSEP) peak-to-peak (P2P) amplitude in operated limbs in various treatments during 90 and 120 days postoperatively. Values are presented with median ± MAD.

**Figure 3.** Evaluation of the effectiveness of various acetylation conduits and fillers. The P2P values of the intact limb were subtracted from the values of the operated limb.

the samples for each experimental group: autologous nerve graft (**Figure 4A**); DAI-magnetic fibrin clot (**Figure 4B**); DAI-NVR gel (**Figure 4C**); DAII-NVR gel (**Figure 4D**); DAIII-NVR gel (**Figure 4E**).

Semi-thin sections were also used to perform stereological analysis for the evaluation of the number of myelinated fibers, the axon and fiber size, and the myelin thickness.

**Figure 4.**  A good regeneration after nerve repair is detectable in all groups. Scale bar: 20 μm.

**Figure 5.** The graphs show the results of stereological analysis on semi-thin sections of the distal part of regenerated nerves. The number of myelinated fibers (A), axon diameter (B), the myelin thickness (C), the fiber diameter (D), and the g-ratio (E) were evaluated 120 days after surgery. All data are presented as means±SD; statistical analysis: one-way ANOVA with post hoc Bonferroni test.

As we can observe from the graph (**Figure 5A**), the total number of myelinated fibers in DAI magnetic fibrin clot group is about two-thirds smaller than the autograft group (*p* = 0.05). Moreover, the number of myelinated fibers in all NVR gel groups is similar to each other and about one-third reduced compared to the autograft group. Although the differences are evident, they can only be interpreted as a statistical trend of *p* < 0.1 due to the high standard deviation (that is probably due to the high variability in the regeneration success observed for all-gel-filled conduits).

Axon and fiber diameter, myelin thickness, and g-ratio are not statistically different among all groups (**Figure 5B–E**).

Scatter plot graphs in **Figure 6** represent the correlation between g-ratio and axon diameter of individual fibers. All groups are compared to the autograft, considered as control. Data show that in DAI-magnetic fibrin clot, DAI-NVR gel, and DAIII-NVR gel group, the slope of the regression line is significantly different from the autograft (**Figure 6A, B, D**). Contrary, there is no difference between autograft and DAII-NVR gel group (**Figure 6C**) since the slope of the two regression lines is not significant different.

**Figure 6.** Scatter plot graphs display g-ratio (y-axis) in relation to axon diameter (x-axis) of individual fiber. More than 250 myelinated axons were considered for each group. All the experimental groups are compared to autograft. The slope of linear regression for (A) DAI conduit filled with magnetic fibrin clot, (B) DAI conduit filled with NVR gel, and (D) DAIII conduit filled with NVR gel is significantly different from the autograft. On the contrary, there are no significant differences between autograft and the group DAII conduit filled with NVR gel (C). The statistical analysis was performed using Prism Program, comparing the slope of the linear regression.

**Figure 5.** The graphs show the results of stereological analysis on semi-thin sections of the distal part of regenerated nerves. The number of myelinated fibers (A), axon diameter (B), the myelin thickness (C), the fiber diameter (D), and the g-ratio (E) were evaluated 120 days after surgery. All data are presented as means±SD; statistical analysis: one-way

**Figure 4.**  A

good regeneration after nerve repair is detectable in all groups. Scale bar: 20 μm.

170 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

ANOVA with post hoc Bonferroni test.

### **4. Discussion**

Peripheral nerves are very often subjected to traumatic lesions both because of accidents (e.g., at work, on the road, and at home) and also because of iatrogenic damage (e.g., oncologic surgical excisions) [26, 27]. When the lesion causes substance loss, the two nerve stumps must be connected by a nerve guide in order to allow regenerating axons to bridge the gap [28, 29]. The nerve guide can be represented by an autologous nerve segment (traditional autograft), e.g., from the sural nerve or by a non-nervous conduit [5, 30]. Along the last years, an increasing number of papers describing innovative bio-artificial nerve guides has been published [7]. In general, nerve guides are composed of two main components: a tubular scaffold which can be sutured (or glued) to the nerve stumps and a luminal filler which provides the substrate for cell migration and axon regeneration inside the conduit.

In this study, we selected the chitosan as the biomaterial of choice for fashioning the nerve conduit based on the previous *in vitro* and *in vivo* experimental evidences. *In vitro*, it has been shown that chitosan membranes are a suitable substrate for survival and orientation of Schwann cell growth as well as survival and differentiation of neuronal cells [31, 32]. *In vivo*, studies showed that chitosan tubes can efficiently bridge peripheral nerve defects [33–36].

On the other hand, two types of luminal filler have been investigated in this study: (i) NVR gel and (ii) magnetic fibrin hydrogel.

The selection of NVR gel as a potentially bioactive luminal filler has been based on a number of studies that have proposed his employment for various tissue engineering applications [12, 13]. In peripheral nerves, HA has positive effects on peripheral nerve regeneration modulating glial cell adhesion and migration and neuronal sprouting [15–17]. Yet, topical application of HA is able to reduce the scar formation and create a more favorable environment for nerve regeneration [37, 38].

The results of this study, which compared HA gel enrichment of three different types of chitosan conduits with traditional autografts, showed no significant inter-group differences in the electrophysiological functional outcome and with respect to all histomorphometrical predictors of axon regeneration. By contrast, g-ratio and axon diameter correlation plots (a strong predictor of nerve fiber maturation) showed significant differences between autograft and all groups of conduits except for NVR gel-enriched medium degree of acetylation (DAII). The better performance of DAII (w5%) conduits is in line with a previous report which compared chitosan hollow conduits with different degrees of acetylation in the rat sciatic nerve model [35] and might be explained with the need of a medium-degree degradation time (several months). In fact, the degree of acetylation is directly related to the degradation velocity of the biomaterial, resulting in too slow degradation (more than 1 year) under low acetylation conditions and too fast degradation (several weeks) under high acetylation conditions [35].

As regards the choice of magnetic fibrin hydrogel, the second potentially bioactive luminal filler investigated in this study, this has been based on its previous successful use for tissue engineering applications in various tissues and organs [39–43]. Fibrin hydrogels combine some important advantages such as inherent flexibility, soft, high seeding efficiency, and they cast easily into three-dimensional shapes, can be injected directly into the site of an injury, and contain cell-binding sites which enhance cell adhesion [44, 45].

In this study, thrombin-conjugated γ-Fe2 O3 nanoparticles were used to fabricate the magnetic fibrin hydrogel in order to enrich low acetylation (DAI) chitosan conduits. The selection of DAI conduits instead of DAII conduits, which showed a better performance in other experiments (35), was due to the fact that, when these experiments started, full data on the comparison among different acetylation chitosan scaffolds were not available yet.

Results of the electrophysiological assessment showed that no significant difference in functional recovery can be detected between magnetic fibrin hydrogel-enriched DAI conduits, NVR gel-enriched DAI conduits and traditional autografts. By contrast, as regards morphometrical analysis, magnetic fibrin hydrogel-enriched DAI conduits showed significantly less fibers than autograft.

### **5. Conclusion**

**4. Discussion**

gel and (ii) magnetic fibrin hydrogel.

conditions [35].

Peripheral nerves are very often subjected to traumatic lesions both because of accidents (e.g., at work, on the road, and at home) and also because of iatrogenic damage (e.g., oncologic surgical excisions) [26, 27]. When the lesion causes substance loss, the two nerve stumps must be connected by a nerve guide in order to allow regenerating axons to bridge the gap [28, 29]. The nerve guide can be represented by an autologous nerve segment (traditional autograft), e.g., from the sural nerve or by a non-nervous conduit [5, 30]. Along the last years, an increasing number of papers describing innovative bio-artificial nerve guides has been published [7]. In general, nerve guides are composed of two main components: a tubular scaffold which can be sutured (or glued) to the nerve stumps and a luminal filler which pro-

In this study, we selected the chitosan as the biomaterial of choice for fashioning the nerve conduit based on the previous *in vitro* and *in vivo* experimental evidences. *In vitro*, it has been shown that chitosan membranes are a suitable substrate for survival and orientation of Schwann cell growth as well as survival and differentiation of neuronal cells [31, 32]. *In vivo*, studies showed that chitosan tubes can efficiently bridge peripheral nerve defects [33–36].

On the other hand, two types of luminal filler have been investigated in this study: (i) NVR

The selection of NVR gel as a potentially bioactive luminal filler has been based on a number of studies that have proposed his employment for various tissue engineering applications [12, 13]. In peripheral nerves, HA has positive effects on peripheral nerve regeneration modulating glial cell adhesion and migration and neuronal sprouting [15–17]. Yet, topical application of HA is able to reduce the scar formation and create a more favorable environment for nerve regeneration [37, 38]. The results of this study, which compared HA gel enrichment of three different types of chitosan conduits with traditional autografts, showed no significant inter-group differences in the electrophysiological functional outcome and with respect to all histomorphometrical predictors of axon regeneration. By contrast, g-ratio and axon diameter correlation plots (a strong predictor of nerve fiber maturation) showed significant differences between autograft and all groups of conduits except for NVR gel-enriched medium degree of acetylation (DAII). The better performance of DAII (w5%) conduits is in line with a previous report which compared chitosan hollow conduits with different degrees of acetylation in the rat sciatic nerve model [35] and might be explained with the need of a medium-degree degradation time (several months). In fact, the degree of acetylation is directly related to the degradation velocity of the biomaterial, resulting in too slow degradation (more than 1 year) under low acetylation conditions and too fast degradation (several weeks) under high acetylation

As regards the choice of magnetic fibrin hydrogel, the second potentially bioactive luminal filler investigated in this study, this has been based on its previous successful use for tissue engineering applications in various tissues and organs [39–43]. Fibrin hydrogels combine some important advantages such as inherent flexibility, soft, high seeding efficiency, and they

vides the substrate for cell migration and axon regeneration inside the conduit.

172 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

Altogether, the results of our study showed that the enrichment of chitosan tubes with both NVR gel and magnetic fibrin hydrogel for the repair of 12 mm long rat sciatic nerve gaps leads to a degree of functional recovery (measured by electrophysiology) not significantly different from traditional autograft. The functional results were not completely matched by the histomorphometric investigation of nerve fibers that showed that best results were found in medium degree (w5%) of acetylation chitosan conduits enriched with the NVR gel. This occurrence, which is not surprising since several studies previously showed that morphological and functional predictors of nerve regeneration are often unrelated [46], must be taken into consideration when translating experimental results to the clinics.

### **Acknowledgements**

This study was supported by the European Community's Seventh Framework Programme (FP7-HEALTH-2011) under grant agreement n° 278612 (BIOHYBRID) and Compagnia di San Paolo (InTheCure Project).

Medical grade chitosan for manufacturing the chitosan films and nerve guides was supplied by Altakitin SA (Lisbon, Portugal). The chitosan materials were supplied by Medovent GmbH (Mainz, Germany). We thank Alex Litvak DVM for technical assistance in animal treatment and care.

### **Conflict of interest**

O. Ziv-Polat and A. Shahar were employees of NVR Research Ltd., while the experiments were conducted, that owns the patent of the NVR gel used in this study.

### **Author details**

Shimon Rochkind1 , Mira M. Mandelbaum-Livnat1 , Stefania Raimondo2 \*, Michela Morano2 , Giulia Ronchi2 , Nicoletta Viano2 , Moshe Nissan1 , Akiva Koren1 , Tali Biron1 , Yifat Bitan1 , Evgeniy Reider1 , Mara Almog1 , Ofra Ziv-Polat3 , Abraham Shahar3 and Stefano Geuna2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Division of Peripheral Nerve Reconstruction, Department of Neurosurgery, Research Center for Nerve Reconstruction, Tel Aviv Sourasky Medical Center, Tel Aviv University, Israel

2 Department of Clinical and Biological Sciences, Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Italy

3 NVR Research Ltd., Ness Ziona, Israel

### **References**

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**Author details**

Shimon Rochkind1

Giulia Ronchi2

**References**

Israel

Evgeniy Reider1

, Mira M. Mandelbaum-Livnat1

\*Address all correspondence to<EMAIL_ADDRESS>
174 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

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**Provisional chapter**

**Scaffolds for Peripheral Nerve Regeneration, the Importance of** *In Vitro* **and** *In Vivo* **Studies for the Development of Cell-Based Therapies and Biomaterials: State of the Art Importance of** *In Vitro* **and** *In Vivo* **Studies for the Development of Cell-Based Therapies and Biomaterials: State of the Art**

**Scaffolds for Peripheral Nerve Regeneration, the**

DOI: 10.5772/intechopen.69540

Sílvia Santos Pedrosa, Ana Rita Caseiro, José Domingos Santos and Ana Colette Maurício Ana Colette Maurício

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Sílvia Santos Pedrosa, Ana Rita Caseiro,

http://dx.doi.org/10.5772/intechopen.69540

José Domingos Santos and

#### **Abstract**

Human adult peripheral nerve injuries are a high incidence clinical problem that greatly affects patients' quality of life. Although peripheral nervous system has intrinsic regenerative capacity, this occurs in an incomplete or poorly functional manner. When a nerve fiber loses its continuity with consequent damage of the basal lamina tubes, axon spontaneous regeneration is disorganized and mismatched. These phenomena translate in an inadequate nerve functional recovery and consequent musculoskeletal incapacity. Nerve grafts still remain the gold standard in peripheral injuries treatment. However, this approach contains its disadvantages such as the necessity of primary surgery to harvest the autografts, loss of a functional nerve, donor site morbidity and longer surgery procedures. Therefore, biomaterials and tissue engineering can provide efficient resources and alternatives to nerve injury repair not only by the development of biocompatible structures but also, introducing neurotrophic factors and cellular systems to stimulate optimum clinical outcome. In this chapter, a comprehensive state-of-the art picture of tissue-engineered nerve grafts scaffolds, their application in nerve regeneration along with latest advances in peripheral nerve repair and future perspectives will be discussed, including our own large experience in this field of knowledge.

**Keywords:** nerve regeneration, peripheral nerve, biomaterials, hydrogels, tube-guides, neurotrophic factors, cell-based therapies, functional assessment, mesenchymal stem cells, Schwann cells, tissue engineering, scaffolds

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

Tissue engineering was originally defined by Skalak and Fox, in 1988 [1] as 'the application of the principles and methods of engineering and life sciences towards the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve functions'. Later, Langer and Vacanti [2], in a widespread review paper, defined three main pillars of tissue-engineering principles, the (a) isolated cells and substitutes—cellular systems; (b) tissue-inducing substances—bioactive molecules; and (c) scaffolds, biomaterials and/or matrices.

The first strategy concerns cell-based therapies in which cells in a small volume or in cell sheets are transplanted into the body. Cellular system includes a wide range of cells and most significantly stem cells [3–7]. Stem cells are responsive undifferentiated cells with varying degrees of self-proliferation and differentiation plasticity [8]. Although the number of stem cells is higher before birth, in adults there are still several 'niches' with significant number of stem cells [9]. The second pillar focuses on the [5, 7, 10] bioactive molecules that can be signalling molecules, proteins and oligonucleotides that can enhance cell migration, cell growth and/or differentiation. These bioactive molecules are roughly divided into mitogens, growth factors and morphogens. Finally, the third pillar is the three-dimensional structure that provides shelter and structure for the cellular system [5–7, 11]. Usually, the biomaterials or scaffold mimics the environment and natural extracellular matrix (ECM) of the place of implantation and should be biocompatible such as their metabolites. Also, scaffolds can be used as drug-delivery system in the controlled release of bioactive molecules [9, 12, 13].

Biomaterials according to the American National Institute of Health describes 'any substance or combination of substances, other than drugs, synthetic or natural in origin, which can be used for any period of time, which augments or replaces partially or totally any tissue, organ or function of the body, in order to maintain or improve the quality of life of the individual'. Earlier, the Williams Dictionary of Biomaterials [14] defined biomaterial as 'any substance intended to interact with the biological system in order to replace living matter which has lost its function. It can serve as a vehicle or not, matrix, support, or for stimulating new tissue growth'. The selection of the most adequate material for a given application should fulfil several requirements of physical, mechanical, chemical and biological properties. The most important features of biomaterials must be [7] (i) biocompatibility, that is, the biomaterial itself must not cause any harm in the living system; (ii) biofunctionality, since the biomaterial must feature mechanical and physico-chemical properties adequate to the function and intended application; and (iii) sterilizability, while materials must be able to undergo sterilization procedures, especially the polymeric materials. Biocompatibility is considered the main feature of a biomaterial and represents the response of the living system to the introduction of a foreigner material. It can be defined as the 'ability of a biomaterial to perform its desired function with respect to a medical therapy, without eliciting any undesirable local or systemic effects in the recipient or beneficiary of that therapy, but generating the most appropriate beneficial cellular or tissue response to that specific situation, and optimizing the clinically, relevant performance of that therapy' [14]. However, biocompatible biomaterials are not useful if they are not biofunctional. Similarly, bioactive devices cannot be used if they are not biocompatible. The term biofunctionality can be simply explained as the suitability to the function [7]. Scaffold porosity is an important desirable feature in the majority of scaffolds, as it promotes cell seeding and cell-matrix interaction and leads to increased neovascularization. However, the exact pore size depends on application, the average pore diameter of 20–125 µm is adequate of skin tissue and >300 µm, in bone tissue [11, 15]. The macro- and micro-topography and other physico-chemical properties of the scaffolds influence cell attachment, migration, proliferation and differentiation and promote protein and other factors adsorption and therefore the success of the system [11, 16]. The mechanical properties and specially the degradation kinetics also is a key feature, especially for musculoskeletal and neuromuscular repair due to the slower repair rates.

In this chapter, a comprehensive state of the art of tissue engineering focused on peripheral nerve repair and the advances on materials and nerve grafts will be discussed, as well as our own large experience in this field of knowledge and the future perspectives.

### **2. Peripheral nerve regeneration**

**1. Introduction**

matrices.

[9, 12, 13].

Tissue engineering was originally defined by Skalak and Fox, in 1988 [1] as 'the application of the principles and methods of engineering and life sciences towards the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve functions'. Later, Langer and Vacanti [2], in a widespread review paper, defined three main pillars of tissue-engineering principles, the (a) isolated cells and substitutes—cellular systems; (b) tissue-inducing substances—bioactive molecules; and (c) scaffolds, biomaterials and/or

180 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

The first strategy concerns cell-based therapies in which cells in a small volume or in cell sheets are transplanted into the body. Cellular system includes a wide range of cells and most significantly stem cells [3–7]. Stem cells are responsive undifferentiated cells with varying degrees of self-proliferation and differentiation plasticity [8]. Although the number of stem cells is higher before birth, in adults there are still several 'niches' with significant number of stem cells [9]. The second pillar focuses on the [5, 7, 10] bioactive molecules that can be signalling molecules, proteins and oligonucleotides that can enhance cell migration, cell growth and/or differentiation. These bioactive molecules are roughly divided into mitogens, growth factors and morphogens. Finally, the third pillar is the three-dimensional structure that provides shelter and structure for the cellular system [5–7, 11]. Usually, the biomaterials or scaffold mimics the environment and natural extracellular matrix (ECM) of the place of implantation and should be biocompatible such as their metabolites. Also, scaffolds can be used as drug-delivery system in the controlled release of bioactive molecules

Biomaterials according to the American National Institute of Health describes 'any substance or combination of substances, other than drugs, synthetic or natural in origin, which can be used for any period of time, which augments or replaces partially or totally any tissue, organ or function of the body, in order to maintain or improve the quality of life of the individual'. Earlier, the Williams Dictionary of Biomaterials [14] defined biomaterial as 'any substance intended to interact with the biological system in order to replace living matter which has lost its function. It can serve as a vehicle or not, matrix, support, or for stimulating new tissue growth'. The selection of the most adequate material for a given application should fulfil several requirements of physical, mechanical, chemical and biological properties. The most important features of biomaterials must be [7] (i) biocompatibility, that is, the biomaterial itself must not cause any harm in the living system; (ii) biofunctionality, since the biomaterial must feature mechanical and physico-chemical properties adequate to the function and intended application; and (iii) sterilizability, while materials must be able to undergo sterilization procedures, especially the polymeric materials. Biocompatibility is considered the main feature of a biomaterial and represents the response of the living system to the introduction of a foreigner material. It can be defined as the 'ability of a biomaterial to perform its desired function with respect to a medical therapy, without eliciting any undesirable local or systemic effects in Peripheral nerve injury remains a major public health problem with an estimated incidence of 13–23 cases per 100,000 persons [17, 18]. These injuries may have a traumatic or an iatrogenic cause and usually are associated with pain, decrease of function and sensory sensibility with devastating effects on patients and family lives [3, 17, 19, 20].

Aegineta et al. [21] for the first time performed nerve repair and wound closure in wounded soldiers, as a military surgeon. Then, in 1873, Hunter [22] first described the epineural nerve repair procedure, still in use today. Sunderland portrayed the principles of nerve repair resourcing to microsurgical techniques, and Kurze and Smith were able to apply those principles in 1964, thanks to the advance in microscopy [23–25].

Peripheral nerve injuries treatment understands the most challenging surgical procedures, and despite the major breakthroughs in this area, complete nerve recovery and nerve function in all clinical cases have not yet been achieved [17, 20, 26]. Despite the exquisite surgical techniques, poor recovery outcome results from nervous system intrinsic and extrinsic factors, such as the integrity of the surrounding tissues post lesion, type and level of the injury itself, the effect on the spinal cord and neurons, the compromising of end organs and with key importance the timing of the surgery [17, 27–29]. Also, although peripheral nervous system has spontaneous regeneration ability, there is a very limited prospective of spontaneous recovery, mostly concerning the complete functional neuromuscular recovery [20].

#### **2.1. Peripheral nerve anatomy**

The peripheral nerve system is composed by neurons, Schwann cells (SCs), fibroblasts, macrophages and interconnected blood system [20, 30]. Motor and sensory neurons are polarized cells whose bodies reside in the spinal cord and with long cytoplasm called axon. Their terminations, called dendrites, target a site of innervation. The signal conduction originates in the axon hillock, in the cell body and projects itself in synapses with target end organs. Axons plasma membrane is partially enclosed by the SCs that produce myelin that encapsulates the axon and helps with signal transmission. Myelin is therefore an insulator that enhances the signal transmission efficiency down the axon. Then, there is a connective tissue net that surrounds the individual axons called the endoneurium. An arrangement of axons, designed fascicles, is surrounded by the perineurium, and groups of fascicles are separated by the epineurium. External to this layer is the blood supply derived from major arteries and the latter involved by the mesoneurium (**Figure 1**). The conservation of fascicles patterns and connective tissue is vital for optimal nerve repair and regeneration. Therefore, more commonly, the epineurium is sutured in end-to-end suture (called epineural end-toend suture), and all nerve surgical interventions are strictly directed at these connective tissue layers. The most important feature is the fact that these sutures must be tension free; otherwise, they will compromise the nerve blood supply and the process of regeneration itself [20, 30–32].

**Figure 1.** Peripheral nerve anatomy. The figure was adapted from Ref. [30].

#### **2.2. Nerve response to injury**

**2.1. Peripheral nerve anatomy**

182 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

itself [20, 30–32].

**Figure 1.** Peripheral nerve anatomy. The figure was adapted from Ref. [30].

The peripheral nerve system is composed by neurons, Schwann cells (SCs), fibroblasts, macrophages and interconnected blood system [20, 30]. Motor and sensory neurons are polarized cells whose bodies reside in the spinal cord and with long cytoplasm called axon. Their terminations, called dendrites, target a site of innervation. The signal conduction originates in the axon hillock, in the cell body and projects itself in synapses with target end organs. Axons plasma membrane is partially enclosed by the SCs that produce myelin that encapsulates the axon and helps with signal transmission. Myelin is therefore an insulator that enhances the signal transmission efficiency down the axon. Then, there is a connective tissue net that surrounds the individual axons called the endoneurium. An arrangement of axons, designed fascicles, is surrounded by the perineurium, and groups of fascicles are separated by the epineurium. External to this layer is the blood supply derived from major arteries and the latter involved by the mesoneurium (**Figure 1**). The conservation of fascicles patterns and connective tissue is vital for optimal nerve repair and regeneration. Therefore, more commonly, the epineurium is sutured in end-to-end suture (called epineural end-toend suture), and all nerve surgical interventions are strictly directed at these connective tissue layers. The most important feature is the fact that these sutures must be tension free; otherwise, they will compromise the nerve blood supply and the process of regeneration Nerve injuries can be from a chronic or acute nature, and SCs are the major cause of chronic injuries [20, 30]. Acute injuries are mediated by axonal degeneration and occur in a sequence of events proximally and distally from the zone of trauma. The initial stages of degeneration occur proximally to the lesion as programmed cell death, called chromatolysis [33, 34], and distally to the lesion, through Wallerian degeneration of the distal axonal segment [35, 36]. Within 24–48 h of the injury, SCs degrade the myelin and phagocytes debris from distal axons [37, 38]. The proximal portion of the axon also degenerates up to the node of Ranvier, where the axonal regrowth occurs. Then macrophages recruitment occurs, originating growth factor release and fibroblast and SCs proliferation. The SCs form organized longitudinal structures inside the endoneurium—called bands of Büngner [30, 34]—that are critical to the axonal regeneration. At the distal site of lesion, the node of Ranvier, around 50–100 finger-like sprouts, starts to form a growth cone directed to the distal nerve stump [39]. Proteases are also released from the growth cone by the influence of several factors, clearing the way towards the target tissue. Nerve growth factor (NGF), brain-derived growth factor (BDNF) and other neurotrophic factors are upregulated by SCs, and their expression is increased which promotes the migration and proliferation of the SCs [20]. Axon existing actin allows axon elongation and which occurs in a 1–3 mm/day rate [36] until a receptor is reached. If no receptor or endoneurial tube is reached, axon continues to grow but in a disorganized manner, causing neuroma and clinically painful lesions [40]. In severe nerve injury, axon regeneration is further disorganized with additional scaring and pourer regeneration.

### **2.3. Nerve injury grading**

Nerve injuries were firstly classified into neuropraxia, axonotmesis and neurotmesis by Seddon [41], after his World War II experience in treating nerve-injured soldiers. Neuropraxia is characterized by the segmentation of the myelin without disturbance of the axon, usually a consequence of a compression. Typically, it resolves itself, once the myelin is restored, within 12 weeks. Axonotmesis concerns axonal injury and occurs from a crush mechanism. In this case, connective tissue and nerve continuity are not affected but are followed by Wallerian degeneration. Axonal regeneration occurs at 1–3 mm/day rate [36], and depending on the distance of the lesion, an incomplete recovery may happen. Neurotmesis comprehends the anatomical and physiological section of the axons and connective tissues. Therefore, no spontaneous regeneration may occur and needs surgical reconstruction [30, 42]. Sunderland later expanded this classification by including five types of injury, based on histological knowledge that allowed further distinction between axonotmesis injuries [25, 43]. Sunderland grade Type I classification is equivalent to neurapraxia. The Type II, III and IV classifications differentiate axonotmesis injuries based on the commitment of the connective tissues. In Type II class injury, there is axonal damage without commitment of the endoneurium, and therefore, it is possible to achieve a full recovery. In Type III lesions, we find axon impairment affecting the endoneurium, and in Type IV besides endoneurium, there is perineurium damage. Sunderland grades III and IV may heal spontaneously, but there is attendant scaring and increasing axon and connective tissue damage that causes incomplete recovery. Type IV lesion usually implies surgical intervention and results in extensive scaring. Scars are associated with pain and nerve conduction impairment and may require reconstructive surgery. Type V Sunderland classification corresponds to neurotmesis [30, 42]. Finally, Mackinnon and Dellon [44] described a mixed type of lesion degree, a Type VI addition to the Sunderland classification. This classification represents probably the most common type of lesions, with several layers of injury and not necessarily traditional model as described by Sunderland. The recovery potential and also the treatment approach vary, according to the type of lesion, considering the three classifications mentioned earlier.

In **Table 1**, the major findings in nerve injury grading according to Seddon [41], Sunderland [25, 43] and Mackinnon and Dellon [44] addition are described.

### **2.4. Diagnosis**

Nerve injury may involve variable lengths of nerve impairment, and the degree of the lesion is affected by the type of lesion [45]. Also, the prognosis is dependent on the age of the patient, location of the lesion—distal fare better than proximal lesions—and also demographics [42]. In terms of nerve electrical signal and electrophysiology, the absence of electrical conduction may not indicate severe nerve damage, since conduction may be recovered after just 1 week. Clinical examination (including the functional evaluation) and surgical inspection still are the most accurate means to obtain diagnosis. However, non-invasive procedures such as nerve conduction studies (NCSs) and electromyograms (EMGs) have a diagnostic role in the delayed setting, when muscle fibrillation occurs [30, 42]. NCS assesses both motor and sensory functions through a voltage stimulator applied to the skin at different points of

**Table 1.** Nerve injury classification according to Sunderland and Seddon.

the nerve. A sensor detects response at the muscle (motor function) or nerve (sensory function). This is the initial screening test for the presence or absence of conduction signal. The EMG assesses only the motor function, and in this test, a needle is inserted in the muscle to assess the resting electrical activity and voluntary motor unit analysis [30]. In **Figure 2**, the representation of an algorithm of best treating approach selection according to the type of lesion, length and also complementary diagnostic results is shown. Sciatic Function Index (SFI) is one of the most widely used forms of functional assessment. It compares parameters from footprints and mathematically infers about sensory-motor gait function mediated by the sciatic nerve, without requiring terminal assessment [46, 47]. Sciatic Static Index (SSI) was first introduced by Bervar [48, 49] and is another way of assessing recovery of function after sciatic injury in animal models. It also uses the footprints in a static position and minimizes bias related to gait's velocity. Also, the SSI improves the acquisition of footprints and is more repeatable and accurate than the SFI. Other motor performance index is measuring

**Figure 2.** Peripheral nerve repair treatment diagram.

lesion usually implies surgical intervention and results in extensive scaring. Scars are associated with pain and nerve conduction impairment and may require reconstructive surgery. Type V Sunderland classification corresponds to neurotmesis [30, 42]. Finally, Mackinnon and Dellon [44] described a mixed type of lesion degree, a Type VI addition to the Sunderland classification. This classification represents probably the most common type of lesions, with several layers of injury and not necessarily traditional model as described by Sunderland. The recovery potential and also the treatment approach vary, according to the type of lesion,

In **Table 1**, the major findings in nerve injury grading according to Seddon [41], Sunderland

Nerve injury may involve variable lengths of nerve impairment, and the degree of the lesion is affected by the type of lesion [45]. Also, the prognosis is dependent on the age of the patient, location of the lesion—distal fare better than proximal lesions—and also demographics [42]. In terms of nerve electrical signal and electrophysiology, the absence of electrical conduction may not indicate severe nerve damage, since conduction may be recovered after just 1 week. Clinical examination (including the functional evaluation) and surgical inspection still are the most accurate means to obtain diagnosis. However, non-invasive procedures such as nerve conduction studies (NCSs) and electromyograms (EMGs) have a diagnostic role in the delayed setting, when muscle fibrillation occurs [30, 42]. NCS assesses both motor and sensory functions through a voltage stimulator applied to the skin at different points of

**Sunderland Seddon Characteristics Spontaneous recovery**

only

only

axon and disrupt the endoneurium

axon and disrupt the endoneurium and perineurium

III and IV

nerve; with the epineurium

**potential**

Poor to none

None

none

Usually slow or incomplete

Variable, can be poor to

Full

Full

considering the three classifications mentioned earlier.

184 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

**2.4. Diagnosis**

\*

[25, 43] and Mackinnon and Dellon [44] addition are described.

Type I Neuropraxia Injury to myelin sheath

Type II Axonotmesis Injuries involve the axon

Type III Axonotmesis Injuries involve the

Type IV Axonotmesis Injuries involve the

Type V Neurotmesis Complete disruption of the

Type VI\* Mixed Combination of Types II,

Mackinnon and Dellon [44] addition to the Sunderland [25, 43] classification.

**Table 1.** Nerve injury classification according to Sunderland and Seddon.

the extensor postural thrust (EPT) and nociceptive function using the withdrawal reflex latency (WRL). EPT is induced by lowering the affected hindlimb towards the platform of a digital balance supporting the animal by the thorax. During the test, the rat extends the hindlimb and the distal metatarsus and digits connect with digital platform balance [50–54]. Nociceptive function using the withdrawal reflex latency was described by Masters and colleagues [55] and is based on the fact that rats without sciatic nerve injury withdraw their paws from the hotplate within 4.3 s or less, when this period of time is increased, it is a symptom of impaired nerve conduction [51–53, 56–61]. Nuclear magnetic resonance (RMN) imaging could become a valuable tool in nerve injury diagnosis since it allows fine, detailed evaluation nerve anatomy and pathology due to excellent image resolution [42]. Also, functional diffusion tensor imaging (DTI), a new diagnosis tool, has been showing peripheral nerve sheath tumours; however, its application on differentiating various grades of injuries remains to be tested [42].

### **2.5. Timing of medical intervention**

After peripheral nerve injury, repair events begin to take place. Primary repair events occur within the first couple of days. However, the rate of axon regeneration is very slow, as previously referred—it is 1–3 mm/day [36]—and no therapeutic methods have yet improved this regeneration rate [30]. However, it is consensual that early nerve repair results in improved functional outcomes, as described by Mackinnon and Dellon [44]. Furthermore, there is a set period of 12–18 months in which muscle re-enervation can occur before irreversible motor end-plate degeneration occurs, and the neurogenic atrophy takes place [30]. Slow axonal regeneration associated with muscle structural changes and increasingly degraded stromal environment contribute for an incomplete functional recovery. Muscle fibrosis and atrophy phenomena begin immediately after denervation and are called neurogenic atrophy. After 4 months, a plateau is reached, when 60–80% of muscle mass is lost, and although motor end plates increase, beyond a 12-month period, a functional muscle re-enervation is highly unlikely [30, 39, 62]. The time frame for sensory re-enervation is longer but not endless, and early repair also grants better results [30, 39]. In **Figure 2**, we present the peripheral nerve injuries repair algorithm which helps to understand the variables taken into account in the selection of best nerve repair strategy.

#### **2.6. Nerve repair strategies**

#### *2.6.1. Direct nerve repair*

Direct nerve repair with epineural end-to-end sutures using microsurgery techniques is still the gold-standard surgical treatment for severe neurotmesis injuries, but only in cases where well-vascularized tension-free coaptation can be achieved [30, 37, 63–65]. The procedure involves rough fascicular matching between proximal and distal nerve ends and the alignment of nerve fascicles and epineural blood vessels [30, 66]. Other types of direct repair consist in fascicular repair or grouped fascicular repair. This requires intranerval dissection and direct matching and suturing of fascicular groups. Despite better fascicle alignment, this procedure is no better than epineural repair in functional outcomes and in fact is associated with more traumas and scaring [30, 63, 66]. Complementary assistance techniques of histologic staining using acetylcholine esterase and carbonic anhydrase, electrical stimulation during the procedure in awaken patients and visual observation of surface vessels, are visual orientations to the surgeon that grant the success of the procedure [63, 67]. Another possible approach is the use of tissue adhesives such as fibrin glue to supplement or replace sutures, creating a gel-like clot at the nerve ends. The advantages of this procedure are the efficiency and practicality, the reduced trauma and scaring due to a barrier effect. The major disadvantage of this technique is the inferior holding strength more subject to stress [63, 68].

#### *2.6.2. Nerve grafts*

the extensor postural thrust (EPT) and nociceptive function using the withdrawal reflex latency (WRL). EPT is induced by lowering the affected hindlimb towards the platform of a digital balance supporting the animal by the thorax. During the test, the rat extends the hindlimb and the distal metatarsus and digits connect with digital platform balance [50–54]. Nociceptive function using the withdrawal reflex latency was described by Masters and colleagues [55] and is based on the fact that rats without sciatic nerve injury withdraw their paws from the hotplate within 4.3 s or less, when this period of time is increased, it is a symptom of impaired nerve conduction [51–53, 56–61]. Nuclear magnetic resonance (RMN) imaging could become a valuable tool in nerve injury diagnosis since it allows fine, detailed evaluation nerve anatomy and pathology due to excellent image resolution [42]. Also, functional diffusion tensor imaging (DTI), a new diagnosis tool, has been showing peripheral nerve sheath tumours; however, its application on differentiating various grades of injuries

186 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

After peripheral nerve injury, repair events begin to take place. Primary repair events occur within the first couple of days. However, the rate of axon regeneration is very slow, as previously referred—it is 1–3 mm/day [36]—and no therapeutic methods have yet improved this regeneration rate [30]. However, it is consensual that early nerve repair results in improved functional outcomes, as described by Mackinnon and Dellon [44]. Furthermore, there is a set period of 12–18 months in which muscle re-enervation can occur before irreversible motor end-plate degeneration occurs, and the neurogenic atrophy takes place [30]. Slow axonal regeneration associated with muscle structural changes and increasingly degraded stromal environment contribute for an incomplete functional recovery. Muscle fibrosis and atrophy phenomena begin immediately after denervation and are called neurogenic atrophy. After 4 months, a plateau is reached, when 60–80% of muscle mass is lost, and although motor end plates increase, beyond a 12-month period, a functional muscle re-enervation is highly unlikely [30, 39, 62]. The time frame for sensory re-enervation is longer but not endless, and early repair also grants better results [30, 39]. In **Figure 2**, we present the peripheral nerve injuries repair algorithm which helps to understand the variables taken into account in the

Direct nerve repair with epineural end-to-end sutures using microsurgery techniques is still the gold-standard surgical treatment for severe neurotmesis injuries, but only in cases where well-vascularized tension-free coaptation can be achieved [30, 37, 63–65]. The procedure involves rough fascicular matching between proximal and distal nerve ends and the alignment of nerve fascicles and epineural blood vessels [30, 66]. Other types of direct repair consist in fascicular repair or grouped fascicular repair. This requires intranerval dissection and direct matching and suturing of fascicular groups. Despite better fascicle alignment, this procedure is no better than epineural repair in functional outcomes and in fact is associated with more traumas and scaring [30, 63, 66]. Complementary assistance techniques of histologic staining

remains to be tested [42].

**2.5. Timing of medical intervention**

selection of best nerve repair strategy.

**2.6. Nerve repair strategies**

*2.6.1. Direct nerve repair*

When peripheral nerve injury originates a significant gap (>3 cm) between the nerve ends with excessive tension for direct epineural repair and reversed interposition, nerve grafts are required [30, 63]. Such gaps may occur in severe neurotmesis lesions or in axonotmesis stretch injuries in which long regions of the nerve may be damaged in the setting of a lesion-in-continuity [63, 69]. Nerve grafts are single, cable, trunk, interfascicular or vascularized portions of the nerve with similar diameter to the affected [30, 70, 71]. Nerve grafting may be from autologous or allograph origin. Xenografts have been described as viable alternatives but require extensive immunosuppression and prionic diseases transmission if they are from ruminants [72, 73]. Nerve autografts are considered the gold standard since they provide appropriate neurotrophic factors and viable SCs, both essential for axonal regeneration without immune compromise [63, 74]. For the choice of the autologous grafts, many factors must be taken into account, such as the size of the nerve gap, the location of proposed nerve repair and associated donor-site morbidity [63, 74]. Grafts are either sutured to the epineurium of single nerves or more commonly to the perineurium of individual fascicles, depending on nerve calibre, type and location [30, 63, 74]. The interfascicular nerve graft was described by Millesi et al. [75]. Vascularized nerve graft was designed by Taylor and Ham, whereby the donor nerve is transposed with its arterial and venous supply into the graft site [76]. Terzis and Kostopoulos [71] clinically demonstrated that medium-sized trunk grafts, which would normally undergo central necrosis, could be transferred as vascularized nerve grafts and survive. However, autografts sacrifice a functioning nerve, usually a sensory nerve, to substitute a more important injured motor nerve. Therefore, sensory loss and scarring at the donor site, where neuroma and pain phenomena, are expected [30, 63, 77]. Autologous nerve graft undergoes Wallerian degeneration and therefore just provides support and guidance for the ingrowing axon. Also, fascicle mismatch, scarring and fibrosis of the repair site is unavoidable and is caused by the injury, tissue handling and suture itself. [30, 63, 77]. An alternative to autologous nerve grafting is the use of nerve allografts. The advantages of allografts are no donor supply limitations or donor-site morbidity, accessibility and unlimited supply of neuronal tissue. However, there are significant costs and complexity with their use, such as immunosuppression [30, 63, 72, 73, 77]. Several techniques have been used to reduce allograft antigenicity, such as cold preservation, irradiation and lyophilization and certainly patients' immunosuppressive therapy. However, it is proven that immune response is caused by SCs, and once their migration has occurred, approximately 24 months after nerve repair, systemic immunosuppression can be withdrawn [74, 78]. To avoid immunosuppression, nerve allografts are decellularized by a process of chemical detergent, enzyme degradation or irradiation, resulting in an acellular nerve scaffold [30, 63, 79]. Similarly, in tendon transfer, a distal function is treated at the expenses of a secondary function [30, 63, 79].

#### *2.6.3. Nerve transfers and free-functioning muscle transfer*

The definition of nerve transfer is the surgical coaptation of a healthy nerve donor to a denervated nerve [30, 63, 80]. The procedure was first described by Harris in 1921 [81], in the treatment of low median nerve injury suffered during World War I. The major disadvantage is finding an expendable donor nerve near the target muscle with a large enough motor fibre population [30, 63]. Free-functioning muscle transfer (FFMT) is another treatment approach, in severe injuries and especially in secondary reconstructions. The procedure entails the transfer of a healthy muscle and its neurovascular pedicle to a new location to assume a new function [71, 82]. Since it is a complex procedure, it is only considered as a secondary reconstructive surgery.

#### *2.6.4. Nerve conduits*

Nerve gap repair and nerve grafts have its complications. In the procedures described earlier, the nerve repair requires a second incision site for autograft harvest, donor-site morbidity, loss of a functional, usually sensory nerve, and long-surgery procedure [63, 65]. The described disadvantages triggered the development of nerve conduit or nerve guides to bring a new approach for nerve gap repair [40, 83]. Also, developments in tissue engineering and regenerative medicine, and research in artificial and natural biomaterials have enabled the development of the first nerve conduits [73]. Nerve tubulation approach to the repair of peripheral nerve gaps can be traced back to the nineteenth century. Gluck (Gluck, 1881) performed the first experiment of nerve tabulation, with a tube of decalcified bone to aid the approximation of transected nerve ends, in animals [78, 84, 85]. Later, Dahlin and Lundborg [86] developed the first synthetic tube, made of silicone. Their work was also pioneer in the characterization of the mechanism of regeneration within the lumen of the engineered tube [78, 86]. A nerve conduit is a tubular structure made of biological or synthetic materials designed to bridge the gap of a sectioned nerve. It is used when primary end-to-end direct repair is not possible, to protect the nerve from scar formation, to prevent fluid from leaking from the nerve stump and to guide the axon nerve cone into the distal nerve stump [63, 65, 78]. The fluid formed from the transected nerve ends is essentially made of fibrin, which forms a matrix or a hydrogel matrix between the nerve ends that is able to support cell migration. Cell migration within the fibrin matrix creates some linear bands—bands of Büngner—that steer the growth of the nerve cone [78, 86]. The mechanism through which neurite growth cone forms within the lumen of the conduit depends on the volumetric ratio [87, 88]. If the gap is too long or the diameter of the inner lumen is too large, the growth cables are too thin, and due to the fibrin matrix, the growth cone takes on an hourglass figure that affects axonal regeneration. Nectow et al. [89] also studied the effect of the defect size in the regenerative process through nerve conduits. Nerve conduits provide control environment to outgrowing axons, migration of SCs and neurotrophic stimulation by the distal stump crucial for optimal regeneration of nerve function [40, 90]. This approach is usually reserved for gap defects between 1.5 and 3 cm [91, 92]. As early as 1994, Brunelli et al. [91] defined four factors for an ideal nerve conduit material: (i) biocompatibility, (ii) easy preparation and tailoring, (iii) incorporation of neurotrophins and stimulating substances, and (iv) protection against scaring. Recently, Arslantunali et al. [93] defined the desirable features for a nerve conduit, as flexibility, biocompatibility, biodegradability, high porosity, neuroinductivity, neuroconductivity, easy handling and sufficient endurance. Nowadays, the second- and third-generation nerve conduits are becoming Food and Drug Administration (FDA) approved and reaching the marked, so, many more pre-clinical and clinical trials are demonstrating major breakthrough in this area. In **Figure 3**, we represent the major features of nerve conduits, and the modification researchers have made to introduce neurotrophic elements that enhance nerve regeneration and peripheral nerve repair.

#### *2.6.5. Biological conduits*

*2.6.3. Nerve transfers and free-functioning muscle transfer*

188 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

structive surgery.

*2.6.4. Nerve conduits*

The definition of nerve transfer is the surgical coaptation of a healthy nerve donor to a denervated nerve [30, 63, 80]. The procedure was first described by Harris in 1921 [81], in the treatment of low median nerve injury suffered during World War I. The major disadvantage is finding an expendable donor nerve near the target muscle with a large enough motor fibre population [30, 63]. Free-functioning muscle transfer (FFMT) is another treatment approach, in severe injuries and especially in secondary reconstructions. The procedure entails the transfer of a healthy muscle and its neurovascular pedicle to a new location to assume a new function [71, 82]. Since it is a complex procedure, it is only considered as a secondary recon-

Nerve gap repair and nerve grafts have its complications. In the procedures described earlier, the nerve repair requires a second incision site for autograft harvest, donor-site morbidity, loss of a functional, usually sensory nerve, and long-surgery procedure [63, 65]. The described disadvantages triggered the development of nerve conduit or nerve guides to bring a new approach for nerve gap repair [40, 83]. Also, developments in tissue engineering and regenerative medicine, and research in artificial and natural biomaterials have enabled the development of the first nerve conduits [73]. Nerve tubulation approach to the repair of peripheral nerve gaps can be traced back to the nineteenth century. Gluck (Gluck, 1881) performed the first experiment of nerve tabulation, with a tube of decalcified bone to aid the approximation of transected nerve ends, in animals [78, 84, 85]. Later, Dahlin and Lundborg [86] developed the first synthetic tube, made of silicone. Their work was also pioneer in the characterization of the mechanism of regeneration within the lumen of the engineered tube [78, 86]. A nerve conduit is a tubular structure made of biological or synthetic materials designed to bridge the gap of a sectioned nerve. It is used when primary end-to-end direct repair is not possible, to protect the nerve from scar formation, to prevent fluid from leaking from the nerve stump and to guide the axon nerve cone into the distal nerve stump [63, 65, 78]. The fluid formed from the transected nerve ends is essentially made of fibrin, which forms a matrix or a hydrogel matrix between the nerve ends that is able to support cell migration. Cell migration within the fibrin matrix creates some linear bands—bands of Büngner—that steer the growth of the nerve cone [78, 86]. The mechanism through which neurite growth cone forms within the lumen of the conduit depends on the volumetric ratio [87, 88]. If the gap is too long or the diameter of the inner lumen is too large, the growth cables are too thin, and due to the fibrin matrix, the growth cone takes on an hourglass figure that affects axonal regeneration. Nectow et al. [89] also studied the effect of the defect size in the regenerative process through nerve conduits. Nerve conduits provide control environment to outgrowing axons, migration of SCs and neurotrophic stimulation by the distal stump crucial for optimal regeneration of nerve function [40, 90]. This approach is usually reserved for gap defects between 1.5 and 3 cm [91, 92]. As early as 1994, Brunelli et al. [91] defined four factors for an ideal nerve conduit material: (i) biocompatibility, (ii) easy preparation and tailoring, (iii) incorporation of neurotrophins and stimulating substances, and (iv) protection against scaring. Recently, Arslantunali et al. [93] defined the desirable features for a nerve conduit, as The use of non-neuronal tissue as conduits was first reported by Büngner (Büngner, 1891), when he successfully used a segment of human brachial artery to regenerate a gap in sciatic nerve [84]. The use of arterial grafts has been demonstrated [94, 95] but is associated with high morbidity. Also, lack of donor vessels makes this a less popular approach in nerve repair. However, it has been described that neurovascular injuries in the hand have benefited from the use of homolateral arteries in the repair [40, 84]. Frerichs et al. and Kim at al. [96, 97] have used acellular allogenic nerve grafts effectively in the regeneration nerve gaps in a rat sciatic model. A similar approach was approved by such Food and Drug Administration and is commercially available as Avance®, by AxoGen, Inc. (Alachua, FL, USA) (**Table 2**). Veins have also been a viable option for nerve repair. The risk of vein collapse led to their filling with

**Figure 3.** Different types of nerve conduits and their main features. Nerve conduits modification that functions as nerve regeneration enhancers.

nerve or muscle tissue. This supplementation has the added advantage of supplying neurotrophic factors and ECM (muscle fibres) and has been described to facilitate nerve regeneration across longer gaps by promoting SCs migration, cell proliferation and guidance of the axonal growth cone [84]. Another type of biological conduit is tendon autograft. Although with only theoretically and historic interest, ECM macrostructure and the presence of hyaluronic acid have been described to enhance regenerating in the nerve cone [84]. AxoGuard™ is the only FDA-approved device composed by small intestine submucosa (SIS) extracellular matrix (**Table 2**). Preliminary studies in rat sciatic model showed distally directed growth of the proximal nerve [98]. Later further in vivo studies revealed better EMG response for distal motor latency and amplitude [99].

#### *2.6.6. Manufactured conduits*

Manufactured nerve conduits can be divided as first-, second- and third-generation conduits. The first-generation conduits are non-resorbable, synthetic tubes made of silicone or polytetrafluoroethylene (ePTFE, Gore-Tex®) [65]. These conduits require a second surgery in order to remove the non-resorbable material. The original idea was to provide support, structure to guide axonal regrowth and form a stable barrier against connective tissue infiltration [73, 100, 101]. Synthetic nerve conduits made of ePTFE were successfully applied in a 4-cm nerve gap, in human [102]. Second-generation nerve conduits are resorbable, biocompatible tubes and are FDA approved and commercially available through different materials. The main advantage of these conduits is their permeability and resorbability that spares patients of a second surgery procedure. Third-generation nerve conduits contrary to second-generation may incorporate controlled release/delivery of neurotrophic factors, electroconductive material, stem cells or SCs, extracellular matrix proteins, surface micropatterning or luminal fillers [73, 103]. Already, two third-generation products have been approved by the FDA, namely NeuraGen® 3D from Integra LifeSciences Corporation and Nerbridge from Toyobo Co., Ltd. NeuraGen® 3D (K130557, approved in 2014) is a bovine Type I collagen conduit with a porous inner hydrogel matrix of collagen and glycosaminoglycan (chondroitin-6-sulfate). Nerbridge™ (K152967, approved in 2016) is a flexible, resorbable and semipermeable tubular membrane matrix filled with porous collagen that provides a non-constricting encasement for injured peripheral nerves for protection of the neural environment.

### **2.7. Materials**

#### *2.7.1. Collagen*

Denaturated collagen conduits are available from a wide number of manufacturers and are in fact the most exploited material in nerve conduits [73, 78]. Collagen is a structural protein ubiquitous in the human body, particularly in the peripheral nerve system. Also, collagen supports cell proliferation and tissue regeneration [65, 73, 104]. As nerve conduits collagen allows the establishment of topographical cues that guide axons to regrow [105, 106] and has shown excellent cell adhesive properties that encourage cell attachment and proliferation [106, 107]. The degradation time of the collagen conduits is relatively prolonged and takes up to 48 months which can cause nerve compression and fibrosis [84]. The first commercially available collagen

nerve or muscle tissue. This supplementation has the added advantage of supplying neurotrophic factors and ECM (muscle fibres) and has been described to facilitate nerve regeneration across longer gaps by promoting SCs migration, cell proliferation and guidance of the axonal growth cone [84]. Another type of biological conduit is tendon autograft. Although with only theoretically and historic interest, ECM macrostructure and the presence of hyaluronic acid have been described to enhance regenerating in the nerve cone [84]. AxoGuard™ is the only FDA-approved device composed by small intestine submucosa (SIS) extracellular matrix (**Table 2**). Preliminary studies in rat sciatic model showed distally directed growth of the proximal nerve [98]. Later further in vivo studies revealed better EMG response for distal

190 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

Manufactured nerve conduits can be divided as first-, second- and third-generation conduits. The first-generation conduits are non-resorbable, synthetic tubes made of silicone or polytetrafluoroethylene (ePTFE, Gore-Tex®) [65]. These conduits require a second surgery in order to remove the non-resorbable material. The original idea was to provide support, structure to guide axonal regrowth and form a stable barrier against connective tissue infiltration [73, 100, 101]. Synthetic nerve conduits made of ePTFE were successfully applied in a 4-cm nerve gap, in human [102]. Second-generation nerve conduits are resorbable, biocompatible tubes and are FDA approved and commercially available through different materials. The main advantage of these conduits is their permeability and resorbability that spares patients of a second surgery procedure. Third-generation nerve conduits contrary to second-generation may incorporate controlled release/delivery of neurotrophic factors, electroconductive material, stem cells or SCs, extracellular matrix proteins, surface micropatterning or luminal fillers [73, 103]. Already, two third-generation products have been approved by the FDA, namely NeuraGen® 3D from Integra LifeSciences Corporation and Nerbridge from Toyobo Co., Ltd. NeuraGen® 3D (K130557, approved in 2014) is a bovine Type I collagen conduit with a porous inner hydrogel matrix of collagen and glycosaminoglycan (chondroitin-6-sulfate). Nerbridge™ (K152967, approved in 2016) is a flexible, resorbable and semipermeable tubular membrane matrix filled with porous collagen that provides a non-constricting encasement for

Denaturated collagen conduits are available from a wide number of manufacturers and are in fact the most exploited material in nerve conduits [73, 78]. Collagen is a structural protein ubiquitous in the human body, particularly in the peripheral nerve system. Also, collagen supports cell proliferation and tissue regeneration [65, 73, 104]. As nerve conduits collagen allows the establishment of topographical cues that guide axons to regrow [105, 106] and has shown excellent cell adhesive properties that encourage cell attachment and proliferation [106, 107]. The degradation time of the collagen conduits is relatively prolonged and takes up to 48 months which can cause nerve compression and fibrosis [84]. The first commercially available collagen

injured peripheral nerves for protection of the neural environment.

motor latency and amplitude [99].

*2.6.6. Manufactured conduits*

**2.7. Materials**

*2.7.1. Collagen*

**Table 2.** Commercially available and FDA-approved nerve conduits. nerve conduit was NeuraGen®, from Integra Lifesciences, Princeton, NJ, FDA approved in 2001 (**Table 2**). Several studies regarding the efficacy of collagen conduits in peripheral nerve injuries have been stated. Bushnell and colleagues [108] performed in 2008 a retrospective study of the utilization of collagen conduits in digital sensory nerve gaps of up to 20 mm and demonstrated a significant recovery rate of 89%. In a retrospective review, Wangensteen and Kalliaine [109] reported on a large number of sensory nerve gaps of 2.5–20 mm repaired with collagen conduits in multiple body regions and concluded that clinically successful outcomes were only observed in 43% of the cases. A prospective cohort study was performed by Lohmeyer and colleagues in 2009 [110] in digital and palmar nerve gaps of 6–18 mm, and results showed meaningful recovery in 75% of patients. Later, in 2011, Taras et al. [111] reported a 73% meaningful recovery in 5- to 15-mm isolated digital nerve lacerations repaired with collagen conduits. In a niche approach, collagen conduits have been used in children suffering from plexus brachialis injury during birth [112]. Also, a significant number of in vivo studies on collagen conduits showed good functional outcomes in nerve reconstructions in rat, cat, dog and primate models [113–115]. Researchers and surgeons have, however, raised their concern about these conduits due to its high cost, conduit stiffness, lack of flexibility and poor enhancement of nerve regeneration [116, 117]. Also, collagen conduit application on major peripheral nerve injuries is limited to median and ulnar nerve repairs at the wrist and only observed as an alternative to the classic epineural suture repair [78]. In a recent study, Monaco and colleagues [118] investigated the effect of three different sterilization methods, dry heat, ethylene oxide and electron beam radiation, on the properties of cylindrical collagen scaffolds with longitudinally oriented pore channels, specifically designed for peripheral nerve regeneration. Ethylene oxide exposure demonstrated to be the most suitable method for the sterilization of the proposed scaffolds, since β-sterilization significantly augmented scaffold enzymatic degradation. Currently, there are 10 commercially available and/or FDA-approved collagen nerve conduits (**Table 2**).

#### *2.7.2. Fibrin*

Fibrin is a fibrous, non-globular protein involved in the clotting of blood. It is a commonly used biomaterial as fibrin glue in nerve repair. Nonetheless, it can also be engineered into a hydrogel with aligned matrix or shaped into tubular conduits [84]. As fibrin glue, it is widely used in sutures, thanks to its semisolid structure that enhances haemostasis and integrity of the repair and also due to its angiogenesis, chemotaxis, leucocytosis and macrophage proliferation stimulation [119, 120]. When used in a conduit, fibrin has shown to promote axon regeneration and functional recovery in small gaps [116, 121, 122]. Rafijah and colleagues [123] have reported the use of collagen conduits filled with fibrin glue in 10-mm rat sciatic nerve defect. After 12 weeks, no inhibitory effect was observed on function, axonal regeneration and compound motor action potential compared to hollow collagen conduit.

#### *2.7.3. N-fibroin*

**Name** Nerve cuff Neuragen 3D

Reaxon Plus

Nerbridge

**Table 2.**

Commercially available and FDA-approved nerve conduits.

polyglycolic acid and

Type I and III collagen

Chitosan

Type I collagen and

Flexible, pliable

6.35

9–12

Integra LifeSciences

2014

Corporation

tube with collagenglycosaminoglycan inner

glycosaminoglycan

(chondroitin-6-sulphate)

matrix

Flexible, pliable tube

flexible, semipermeable

3–5

3–4

tubular membrane filled

with porous collagen

3

3

MEDOVENT GmbH

TOYOBO CO., LTD.

 2016

192 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

2015

Type I, III, IV and VI

Extracellular collagen

1–5

matrix

collagen

**Composition**

**Structure**

**Length (cm)**

**Degradation Time**

**Manufacturer**

**FDA approval**

**(months)**

Reabsorbable

Cook Biotech

2014

N-fibroin is a soluble protein, derived from silk with great potential due to their superior biocompatibility and low immunogenicity and mechanical stability upon degradation [73, 124]. Several studies have demonstrated the potential of N-fibroin in the regeneration of peripheral nerve injuries [124–128]. Silk fibroin-based nerve guidance conduit with oriented filaments was produced by Yang and colleagues [129] and originated successful results in rat sciatic model. Researchers have also shown that silk-based conduits [130] and silk nanofibres [131] enhance cell-material interactions like cell adhesion, proliferation and differentiation. A new approach entails the development of conduits with multi-walled silk fibroin/silk sericin internal lumen, beneficial to nerve regeneration and outer sheath (the hollow poly(lactic-coglycolic acid) conduits that provide strong mechanical protection. Engineered bionic conduit showed promising in vivo results [132]. Research has also reported the development of silk fibroin conduits loaded with nerve growth factors [133–138]. Despite the promising results, no silk conduit has yet been FDA approved.

### *2.7.4. Chitosan*

Chitosan is a natural polymer currently under investigation as nerve conduits due to its favourable biocompatibility, biodegradability and bioactivity [139]. Our group research tested the nerve-regenerative potential of chitosan membrane with N1E-115 cellular system in rat sciatic nerve crush injury. Results showed that freeze-dried chitosan Type III without N1E-115 cell addition was the only type of membrane that significantly improved post-traumatic axonal regrowth and functional recovery [52]. Recent study showed meaningful motor and sensory recovery in 30-mm defect in the nerve of the distal right forearm [140].

### *2.7.5. Polyglycolic acid*

Polyglycolic acid (PGA) was the first FDA approved and commercially available nerve conduit—NeuroTube®, from Synovis Micro Companies Alliance, Birmingham, Ala. PGA is a common suture material and is more flexible and porous than silicone. PGA is degraded into lactic acid in 6–12 months [73, 90]. Therefore, critics claim that PGA may degrade faster than the regeneration process and resulting lactic acid may a have toxic effects [84, 141–143]. Dellon and Mackinnon [144] were the first to report the use of PGA conduits as secondary reconstitution of digital nerve defects 3 cm or smaller in 15 clinical cases in monkeys. After 1 year, 86% meaningful recovery was reported. Later, in an attempt to compare PGA conduit results to conventional autograft repair, Weber et al. [145] conducted a randomized prospective multicentre trial in the reconstruction of long sensory nerve gaps up to 3 cm. After 1 year, 74% meaningful recovery was noted in the PGA group compared with 86% in the standard techniques group (*P* > 0.05). Further analysis showed that PGA conduits were equivalent or superior to traditional autografts in less than 4- and 9–30-mm gaps [145]. Other researchers also compared PGA conduits and vein grafts to repair digital nerve gaps up to 4 cm and equivalent or superior recovery was obtained [146, 147]. Further experiments have demonstrated the success of bioabsorbable PGA nerve conduits in the regeneration of nerve defects [148–151]. The most recent FDA-approved PGA conduit is Nerbridge®, from Toyobo Co., Ltd., which is a flexible, semipermeable tubular membrane filled with porous Type I and III collagens (**Table 2**).

#### *2.7.6. Poly (d, l lactide-co-ε-caprolactone)*

Poly-d, l lactide-co-epsilon-caprolactone (PCL) consists in lactic acid and caprolactone monomers. Their nerve conduits are resolvable polyester with the advantage of being transparent and with less acidic degradation product that therefore causes less toxic reaction [60, 84, 116, 152]. Also, PCL conduit is easy to produce, has a low-cost processing and has a long degradation time up to 16 months. However, resulting conduits have higher rigidity and more difficult to handle in clinical settings [84]. Also, Duda et al. [153] reported a strong foreign body response in PCL conduits. Currently, Neurolac® [154–156] is the only FDA-approved caprolactone conduit (**Table 2**). Several reports have been made in the application of PCL conduits in sciatic rat nerve repair [157–162]. Bertleff et al. [154] performed a randomized prospective multicentre study where PCL conduits were comparable to either primary end-to-end repair or nerve autograft. However, latter research showed no meaningful recovery in digital nerves repair [155, 156]. Secer and colleagues [163] studied the use of PCL in the recovery of 455 patients with ulnar nerve injuries. PCL conduits filled with muscle tissue showed superior results in comparison to single PCL conduits in 10- and 15-mm gap in sciatic nerve rat model [36, 156, 164–168].

### *2.7.7. Polyhydroxybutyrate*

filaments was produced by Yang and colleagues [129] and originated successful results in rat sciatic model. Researchers have also shown that silk-based conduits [130] and silk nanofibres [131] enhance cell-material interactions like cell adhesion, proliferation and differentiation. A new approach entails the development of conduits with multi-walled silk fibroin/silk sericin internal lumen, beneficial to nerve regeneration and outer sheath (the hollow poly(lactic-coglycolic acid) conduits that provide strong mechanical protection. Engineered bionic conduit showed promising in vivo results [132]. Research has also reported the development of silk fibroin conduits loaded with nerve growth factors [133–138]. Despite the promising results,

Chitosan is a natural polymer currently under investigation as nerve conduits due to its favourable biocompatibility, biodegradability and bioactivity [139]. Our group research tested the nerve-regenerative potential of chitosan membrane with N1E-115 cellular system in rat sciatic nerve crush injury. Results showed that freeze-dried chitosan Type III without N1E-115 cell addition was the only type of membrane that significantly improved post-traumatic axonal regrowth and functional recovery [52]. Recent study showed meaningful motor and

Polyglycolic acid (PGA) was the first FDA approved and commercially available nerve conduit—NeuroTube®, from Synovis Micro Companies Alliance, Birmingham, Ala. PGA is a common suture material and is more flexible and porous than silicone. PGA is degraded into lactic acid in 6–12 months [73, 90]. Therefore, critics claim that PGA may degrade faster than the regeneration process and resulting lactic acid may a have toxic effects [84, 141–143]. Dellon and Mackinnon [144] were the first to report the use of PGA conduits as secondary reconstitution of digital nerve defects 3 cm or smaller in 15 clinical cases in monkeys. After 1 year, 86% meaningful recovery was reported. Later, in an attempt to compare PGA conduit results to conventional autograft repair, Weber et al. [145] conducted a randomized prospective multicentre trial in the reconstruction of long sensory nerve gaps up to 3 cm. After 1 year, 74% meaningful recovery was noted in the PGA group compared with 86% in the standard techniques group (*P* > 0.05). Further analysis showed that PGA conduits were equivalent or superior to traditional autografts in less than 4- and 9–30-mm gaps [145]. Other researchers also compared PGA conduits and vein grafts to repair digital nerve gaps up to 4 cm and equivalent or superior recovery was obtained [146, 147]. Further experiments have demonstrated the success of bioabsorbable PGA nerve conduits in the regeneration of nerve defects [148–151]. The most recent FDA-approved PGA conduit is Nerbridge®, from Toyobo Co., Ltd., which is a flexible, semipermeable tubular membrane filled with porous Type I and III collagens (**Table 2**).

Poly-d, l lactide-co-epsilon-caprolactone (PCL) consists in lactic acid and caprolactone monomers. Their nerve conduits are resolvable polyester with the advantage of being transparent and with less acidic degradation product that therefore causes less toxic reaction [60, 84, 116, 152].

sensory recovery in 30-mm defect in the nerve of the distal right forearm [140].

no silk conduit has yet been FDA approved.

194 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

*2.7.4. Chitosan*

*2.7.5. Polyglycolic acid*

*2.7.6. Poly (d, l lactide-co-ε-caprolactone)*

Polyhydroxybutyrate (PHB) is a polyester polymer, also used in sutures and wound dressings [116, 169]. PHB has a long degradation time, up to 24–30 months [116, 169]. It has been reported that PHB has a neuroprotective effect and can help axon regeneration [170, 171]. Recently, Axongen Pharmaceuticals has a pending approval order of a PHB conduit. A study in which PHB wrap implants were used in human patients showed promising results compared to epineural suture [172].

#### *2.7.8. Polyvinyl alcohol*

Polyvinyl alcohol (PVA) [36, 89, 93] is the only nondegradable synthetic nerve conduit approved by the FDA—SaluBridge and SaluTunnel, from SaluMedica LLC, Atlanta, GA, USA (**Table 2**).

#### *2.7.9. Polyhedral oligomeric silsesquioxane*

Polyhedral oligomeric silsesquioxane (POSS) can be described as the smallest particles of silica [116]. The combination of POSS and PCL has been employed in the fabrication of peripheral nerve conduits and in vivo clinical studies to show the potential translation into clinics [117].

### **2.8. Optimizing nerve regeneration**

Although nerve conduits provide sufficient guidance for regeneration of nerve defects, the development of new generation of scaffolds is under way. Third-generation conduits include artificial conduits that may incorporate controlled release/delivery of neurotrophic factors, electroconductive material, stem cells, SCs, extracellular matrix proteins, surface micropatterning, luminal fillers and guidance structures [73, 103].

#### *2.8.1. Conduits structure modulation*

Surface micropatterning and the inclusion of extracellular matrix proteins are new approaches that can provide suitable nanostructure topography for adequate neural growth and simulate topographical dimensions that mimic native nerve extracellular matrix [103, 173]. Coating of

peripheral nerve conduits can enhance nerve regeneration process and solve longer nerve gaps repair. Resource to extracellular matrix materials, such as fibronectin, laminin and collagen, give naturally hydrophobic scaffolds a hydrophilic surface that promotes cell adhesion [73, 116, 174]. Collagen Type I conduits coated with laminin and fibronectin have shown improved neural regeneration [175, 176]. With that intend, new peptides have been engineered to mimic the active binding domains of various extracellular matrix molecules [177, 178]. Coating with arginylglycylaspartic acid (RGD) sequences (the tripeptide Arg-Gly-Asp) also obtained very good results [117, 179–182]. Structure pore design is a great strategy to promote nerve regeneration. Controlling the architecture of the conduit wall is possible to develop a microporous inner layer and macroporous outer layer and obtain a bidirectional permeability [86, 103]. The diameter of the pores plays a critical role in the efficiency of the scaffolds since it influences cell attachment, axon regeneration and diffusion of nutrients [183, 184]. Electrospinning is a frequently used technique in bioengineering to produce imprinted micropatterns and can be used as a luminal guidance strategy. The advantages associate with electrospun conduits are (i) highly flexible and porous materials; (ii) high surface area-to-volume ration, thereby great availability for protein absorption, stem cells migration and regeneration of axons; (iii) fibers can be preferentially aligned, resulting in increased SC alignment, proliferation and growth, and the promotion of guided axonal growth [116, 185, 186]. Uneven fibre distribution and nerve growth inhibition caused by fibre overlapping are some of the disadvantages associated with this type of conduits. However, association with wall guides helps to avoid this problem [105, 187, 188].

### *2.8.2. Luminal fillers*

Biomaterials and other strategies as conduits luminal fillers aim to change its microenvironment in a favourable way. The goal of this strategy is to promote axon regeneration inside the conduit and the restoration of motor and sensory nerve function.

#### *<IP_ADDRESS>. Cellular systems*

Stem cell therapies have received increased attention in regenerative medicine [189–201]. The use of cellular systems inside nerve conduits is intended to promote axon regeneration [202–206]. Several cells have been used with this intention, SCs, bone marrow stem cells (BMSCs) including more specifically the mesenchymal stem cells (MSCs). SCs are the natural glia of the peripheral nervous system and have been used successfully with beneficial effects in nerve reconstruction [204, 207–210]. However, there is limited availability, and it requires previous surgery. Stem cells have the opportunity to show their potential since they are able to secrete neurotrophic factors and provide a favourable microenvironment for neurogenesis and the proliferation of SCs in peripheral nerve repair [211] and also are able to differentiate into Schwann cell-like phenotype [200, 212, 213]. MSCs therefore seem the most attractive approach as cellular system in peripheral nerve regeneration. MSCs all share mesenchymal markers that differentiate them from other cells that are positive staining for CD10, CD13, CD29, CD44, CD90 and CD105, and negative expression of haematopoietic markers [214]. Their main feature is their ability to proliferate and self-renew in a sustained manner, high plasticity and low immunogenicity, and differentiate into multiple mesodermal cells, including neuron-like cells [215, 216]. Also, they are major histocompatibility complex (MHC) class II negative and also their MHC class I expression levels can be manipulated; therefore they do not require the use of immunosuppressive drugs [217, 218]. MSCs can be isolated from several origins, including skin, hair follicle, periosteum, amniotic fluid, umbilical cord blood adipose tissue and dental pulp [53, 56, 200, 215, 219–221]. Bone marrow represents the most commonly used tissue source of adult MSCs. BMSCs have limited availability especially in adult life and there is donor-site morbidity associated with its harvesting. Therefore, alternative sources have been reached. MSCs exist in the connective tissue (Wharton's jelly (WJ)) of human umbilical cord and can be harvested easily [214, 218, 222, 223]. Several researchers have demonstrated that umbilical cord–derived stem cells can be differentiated into neuronal phenotype [214, 218, 222, 223] and have demonstrated potential utility in neurodegenerative diseases [224, 225]. Our group research has vast experience in the application of mesenchymal stem cells in peripheral nerve regeneration [50, 52, 53, 56–61]. In our most recent work [56], we report the therapeutic value of MSCs isolated from the Wharton jelly in nerve repair associated to different tube guides made of biodegradable and biocompatible biomaterials. Biomaterials like PVA, PVA loaded with functionalized carbon nanotubes (PVA-CNTs), PVA loaded with polypyrrole (PVA-PPy) and PLC associated to MSCs were tested in terms of cytocompatibility and *in vivo* in the rat sciatic nerve neurotmesis injury model. The functional recovery was assessed serially for gait biomechanical analysis, by EPT, SFI and SSI, and by WRL. Results showed that MSCs enhanced the recovery of sensory and motor function in neurotmesis injuries showing a thicker myelin sheath. Other authors have reported the application of bone marrow stem cells and Schwann-like cells in the regeneration of facial nerves in rats [219].

Other sources of stem cells have been used in peripheral nerve injuries, such as adipose-derived stem cells [203, 206, 212, 226–231] and dental pulp stem cells (DPSCs) [232–236]. Dental pulp stem cells have also demonstrated differentiation capacity towards multiple other mesodermal and endodermal lineages, under appropriate conditions: adipogenic, osteo/dentinogenic, chondrogenic, neurogenic, endothelial, myofibroblastic [237] and hepatocytes [238]. DPSCs can be isolated from both the perivascular and the sub-odontoblastic compartments (the inner surface of the tooth and the outer part of the pulp tissue), by separate digestion of the tooth and extracted pulp tissue. Both populations presented identical cell size, doubling times and karyotype stability, differing only in morphology with rounder cells in the sub-odontoblastic compartment versus spindle-shaped cells with long processes in the perivascular one [239]. Pre-clinical experiments have demonstrated that stem cells show promising results in differentiation into neuronal-like cells [226, 228, 232, 233, 240] and their secretion of growth factors [229, 241, 242]. Shi and colleagues [243] stated the potential of glia-derived neurotrophic factor expressing neural stem cells in the regeneration of facial nerve gap in rats.

#### *<IP_ADDRESS>. Neurotrophic factors*

peripheral nerve conduits can enhance nerve regeneration process and solve longer nerve gaps repair. Resource to extracellular matrix materials, such as fibronectin, laminin and collagen, give naturally hydrophobic scaffolds a hydrophilic surface that promotes cell adhesion [73, 116, 174]. Collagen Type I conduits coated with laminin and fibronectin have shown improved neural regeneration [175, 176]. With that intend, new peptides have been engineered to mimic the active binding domains of various extracellular matrix molecules [177, 178]. Coating with arginylglycylaspartic acid (RGD) sequences (the tripeptide Arg-Gly-Asp) also obtained very good results [117, 179–182]. Structure pore design is a great strategy to promote nerve regeneration. Controlling the architecture of the conduit wall is possible to develop a microporous inner layer and macroporous outer layer and obtain a bidirectional permeability [86, 103]. The diameter of the pores plays a critical role in the efficiency of the scaffolds since it influences cell attachment, axon regeneration and diffusion of nutrients [183, 184]. Electrospinning is a frequently used technique in bioengineering to produce imprinted micropatterns and can be used as a luminal guidance strategy. The advantages associate with electrospun conduits are (i) highly flexible and porous materials; (ii) high surface area-to-volume ration, thereby great availability for protein absorption, stem cells migration and regeneration of axons; (iii) fibers can be preferentially aligned, resulting in increased SC alignment, proliferation and growth, and the promotion of guided axonal growth [116, 185, 186]. Uneven fibre distribution and nerve growth inhibition caused by fibre overlapping are some of the disadvantages associated with this type of conduits. However, association with wall guides

196 Scaffolds in Tissue Engineering - Materials, Technologies and Clinical Applications

Biomaterials and other strategies as conduits luminal fillers aim to change its microenvironment in a favourable way. The goal of this strategy is to promote axon regeneration inside the

Stem cell therapies have received increased attention in regenerative medicine [189–201]. The use of cellular systems inside nerve conduits is intended to promote axon regeneration [202–206]. Several cells have been used with this intention, SCs, bone marrow stem cells (BMSCs) including more specifically the mesenchymal stem cells (MSCs). SCs are the natural glia of the peripheral nervous system and have been used successfully with beneficial effects in nerve reconstruction [204, 207–210]. However, there is limited availability, and it requires previous surgery. Stem cells have the opportunity to show their potential since they are able to secrete neurotrophic factors and provide a favourable microenvironment for neurogenesis and the proliferation of SCs in peripheral nerve repair [211] and also are able to differentiate into Schwann cell-like phenotype [200, 212, 213]. MSCs therefore seem the most attractive approach as cellular system in peripheral nerve regeneration. MSCs all share mesenchymal markers that differentiate them from other cells that are positive staining for CD10, CD13, CD29, CD44, CD90 and CD105, and negative expression of haematopoietic markers [214]. Their main feature is their ability to proliferate and self-renew in a sustained manner, high plasticity and low immunogenicity, and differentiate into multiple

helps to avoid this problem [105, 187, 188].

conduit and the restoration of motor and sensory nerve function.

*2.8.2. Luminal fillers*

*<IP_ADDRESS>. Cellular systems*

Growth factors and cytokines have a complex enhancing effect in tissue regeneration which can be exploited with great potential in nerve regeneration [73]. To date, several neurotrophic factors have been identified: transforming growth factor beta superfamily (TGF-β), nerve growth factor, neurotrophins 3, 4 and 5 (NT-3/4/5), ciliary neurotrophic factor (CNTF), neuregulin-1 (NRG1), brain-derived neurotrophic factor, glial cell line–derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF). The controlled release of the neurotrophic factors in nerve conduits may take several approaches, namely topical application, subcutaneous injection, microosmotic pump and diffusion or affinity-based polymer microspheres [244–251]. In another approach, Cui and colleagues [252] loaded the neurotrophic factors into the wall of the nerve conduit with controlled diffusion into the lumen. NGF has been reported to promote neurite outgrowth in vitro in co-culture of neurons and astrocytes [253] and in vivo–enhanced axonal regeneration in sciatic nerve in rat model [248, 254]. Several researchers have demonstrated BDNF efficacy in the regeneration of rat motor nerves [253, 255, 256]. The dosage of BDNS has also been studied [257]. A high dosage, set as 12–20 mg/day, has been stated to interact with p75 receptors and therefore inhibits axonal regeneration. In fact, single exogenous dosage of BDNF has shown better results than continuous long-term applications [258]. It is reported that muscle regeneration causes the overexpression of GDNF which increases the number of motor axons in the neuromuscular junctions in vivo [259–266]. Also, GDNF is a potent protective factor against axotomy-induced motor and sensory neuron death [267–270]. CNTF has the ability to improve and regenerate muscle function after nerve injury in vivo [271, 272]. It has also demonstrated to enable peripheral nerve regeneration by promoting axon elongation and sprouting from axon distal stump [273]. Six main isoforms of NRG-1 are described. Due to their importance in nerve regeneration isoforms I, II and III are the most studied in the last few years [274]. Nerveregenerative effect of NRG-1 is highly dependent on the isoform and its dosage [275, 276]. Several studies indicate that NRG-1 isoforms are capable of stimulating SCs proliferation a remyelinization [275–278]. Gambarrota and colleagues [279] recommended that initial high dosages of NRGA-1 stimulate SCs differentiation.

#### *<IP_ADDRESS>. Pharmacological agents*