Polybutylene succinate artificial scaffold for peripheral nerve regeneration

Abstract Regeneration and recovery of nerve tissues are a great challenge for medicine, and positively affect the quality of life of patients. The development of tissue engineering offers a new approach to the problem with the creation of multifunctional artificial scaffolds that act on various levels in the damaged tissue, providing physical and biochemical support for the growth of nerve cells. In this study, the effects of the use of a tubular scaffold made of polybutylene succinate (PBS), surgically positioned at the level of a sciatic nerve injured in rat, between the proximal stump and the distal one, was investigated. Scaffolds characterization was carried out by scanning electron microscopy and X‐ray microcomputed tomography and magnetic resonance imaging, in vivo. The demonstration of the nerve regeneration was based on the evaluation of electroneurography, measuring the weight of gastrocnemius and tibialis anterior muscles, histological examination of regenerated nerves and observing the recovery of the locomotor activity of animals. The PBS tubular scaffold minimized iatrogenic trauma on the nerve, acting as a directional guide for the regenerating fibers by conveying them toward the distal stump. In this context, neurotrophic and neurotropic factors may accumulate and perform their functions, while invasion by macrophages and scar tissue is hampered.


| INTRODUCTION
Damage to the central and peripheral nervous system causes irreversible effects and current treatment strategies do not offer reliable results. In particular, peripheral nerve injuries (PNI) include a wide range of disorders in neurologic and neurosurgical practice, and they are still today a serious medical and public health problem. 1 Diseases involving the peripheral nervous system, particularly in the younger population, often originate from motor vehicle accidents or high velocity trauma, leading to life-long disabling neurologic dysfunction and devastating impacts on patients' daily functions and routines. Up to 33% of all PNI shows incomplete nerve recovery and poor functional outcomes, resulting in motor and sensory disabilities, neuropathic chronic pain, end target muscle atrophy and profound weakness. 2 Despite noticeable advancements in instrumentation and microsurgical techniques, long-term prognosis in patients with severe nerve lesions and extended axonal degeneration remains discouraging. 3 Therefore, it is often difficult to achieve complete peripheral neural regeneration (rejoin the nerve gaps) and to restore function of target nerve-related muscles. 4 A nerve gap is defined as the distance between two ends of a severed nerve, resulting from nerve retraction or loss of tissue from injury. 5 Management of nerve gaps depends on the length of the nerve defect, the nerve diameter, the availability of the proximal stump and the proximal or distal site of the lesion. Different types of surgical therapeutic approaches are commonly used for sensory and motor functional recovery following PNIs. 6 With nerve gaps less than 1 cm, in the absence of tension between the ends of the severed nerve, the gold-standard method for treatment of nerve damage is the direct nerve repair with microsurgical techniques.
This approach hopes to provide continuity between the distal and the proximal part of the transected nerves. 7 In particular, epineural repair provides for achieving the tension-free natural connection of the nerve tissue and accurate alignment of the nerve fascicles. In this case, a rapid functional recovery is possible, especially if the denervation time is less than 6 months and the age of the patient is less than 50 years. 8,9 In the past, fibrin glue has been utilized for primary sutureless nerve cooptation by using an adhesive material known as fibrin sealants. 10 In clinical practice, it is considered as an efficient technique, quick and easy to use, as it ensures versatility for different nerve repair situations, a shorter recovery time and no-induction of inflammation or fibrosis. 11 Recent studies in small animal models focused on the engineering of nerve conduits by using natural (biological conduits) or artificial (synthetic conduits) materials. Nerve conduits serve as a bridge between the proximal and distal stump, providing a scaffold upon which cells can migrate between the two nerve stumps. The most significant advantage of a nerve conduit is its ability to create an ideal microenvironment for neuronal recovery and nerve growth, especially for complex defects. 12 Moreover, nerve conduits make the repair site less susceptible to perineural fibrosis and infiltration by inflammatory cells. For all these reasons, an ideal nerve conduit should have properties like porous, flexible, thin, biocompatibility, permeability, flexibility, biodegradability, neuroinductivity, and neuroconductivity with an appropriate surface. 13 To date, in order to avoid the possibility of external body reaction, scar formation and inflammation of neighboring tissues, the choice of material for nerve conduits and scaffolds has shifted toward the more biocompatible, biodegradable, and bioresorbable synthetic polymers such as polyglycolic acid (PGA), polylactidecaprolactone (PLCL), polycaprolactone (PCL), and recently also polyurethane, which induce only minimal foreign body reaction and excellent nerve regeneration. 14,15 In other case, myelination and collagen IV deposition were also observed. 16 Collagen based scaffold also shown to be effective in nerve repairing, in rat model and in humans as FDA-approved materials. 17,18 New biomaterial processing techniques, such as electrospinning or bioprinting, allow the development of special neural guides, designed to simulate the structure of the extracellular matrix, increase the contact surface for the regenerated axon and further stimulate its growth. Currently, electrospinning technique is available to produce degradable artificial nerve conduits with aligned or random nanotopographies. 19,20 In particular, as reported in several studies, aligned polymer fiber-based constructs present sub-micron scale structure. This characteristic improve peripheral nerve repair by promoting Schwann cell migration. 21 Poly(1,4-butylene succinate) (PBS) is another example of water insoluble biopolymer synthesized by the polycondensation of 1,4-butandiol with succinic acid. Given its chemical structure, PBS shows excellent melt processability, a proven biocompatibility and biodegradability, 22 and a good versatility when employed as material for various biomedical applications. Its versatility includes application in bone regeneration or myocardial tissue replacement and different manufacturing approaches, including salt leaching, electrospinning or extrusion techniques. [23][24][25][26][27][28] In this study, PBS was tested as biomaterials for the production of nanostructured conduits for severed nerve regeneration. To this aim, microfibrillar PBS-based 3D scaffolds, produced by electrospinning technique, 29     Rats were randomly divided into two experimental groups. In Group 1 (G1; Control; n = 10), sciatic nerves were transected and repaired with standard epineural microsurgical sutures (simple primary repair). In Group 2 (G2; Nanofiber wrap; n = 10), a PBS-based scaffold was implanted following neurotmesis at the severed nerve stumps without epineural repair. The outcomes were evaluated by electrophysiological assessment, magnetic resonance images (MRI), muscle atrophy evaluation and histological analysis after two post-surgery survival periods of 30 and 120 days, respectively.

| Surgical procedure and scaffold implantation
Surgical procedures were performed under aseptic conditions using a power focus surgical microscope (Carl Zeiss, Germany). Animals were induced to anesthetic depth with inhaled isoflurane at 2% and then anesthetized with intramuscular (i.m.) injection of Zoletil(r) (tiletamine/zolazepam; 10 mg/kg) and Domitor(r) (medetomidine hydrochloride; 0.5 mg/kg). 30 All rats were operated by the same surgeon and only on a limb, so that mobility, self-sufficiency in eating and drinking were allowed. Before surgery, the hair was clipped over the thigh and surgical area was scrubbed with a 70% alcohol solution. A small skin incision of 40 mm was created in the right limb of each rat over the gluteal muscle along the femoral axis. With a muscle-splitting approach, that is the biceps femoris and superficial gluteal muscles were detached with blunt dissection, the sciatic nerve located 4 mm below the skin was exposed and then sharply transected at the midthigh level, proximal to the tibial and peroneal bifurcation, using microscissors. After transection, a 7 mm long nerve gap was created only in the nanofiber wrap group (G2) injured nerves, resulting from a "facilitated" nerve retraction. In the control group (G1), the proximal and distal nerve stumps of the injured nerve were sutured using three 6/0 monofilament nylon epineural sutures (Ethicon). In the experimental group G2, the proximal and distal nerve ends (included the interstump gap) were wrapped with the PBS nanofiber scaffold (12 Â 12 mm) to surround the whole repair site, with no primary repair. The 12 mm long polymeric wrap enabled a 7 mm nerve gap when used as guidance tube, due to the 2.5 mm overlap needed on each end of the severed nerve. The wrap was 0.5-1 mm larger than the nerve diameter. The sciatic nerve was kept moist with sterile saline solution throughout the surgical procedure. In all groups, muscle wound beds were sutured with 2/0 Vicryl. The incised skin was closed with surgical staples with 6-7 sutures and disinfected with povidoneiodine (Betadine) solution. I.m. atipamezole (Antisedan) (300 μg/kg) was used in order to awaken all rats. Carprofen analgesia (5 mg/kg) and Enrofloxacin (5 mg/kg) were daily administered for 1 week to each rat immediately after surgery to prevent infection. Animals were then transferred and housed one per cage and given an identification number. They were monitored on a daily basis for infection, self-mutilation, and signs of distress. Subsequent postoperative observations and procedures were performed at 30 and 120 days respectively.

| In-vivo magnetic resonance imaging (MRI) measurements
Rats were anesthetized with isoflurane and imaged by a Bruker 7-T MRI instrument (Germany) at 30 and 120 days after implantation (n = 5 per group). The parameters for T2 weighted sequence were: gradient echo with TR/TE/flip angle: 250 ms/33 ms/15 ms and matrix pixel 256 Â 256. Images could be taken from the sagittal and axial directions to observe in connection with the regenerated nerve.

| Electrophysiological assessment
To test the restoration of functionality of the regenerated nerve through the implant, electrophysiological recordings were performed at 120 days post-surgery (n = 4 per group), before the animals were sacrificed for histological analysis and muscles dissection, by means of motor unit number estimation (MUNE). MUNE is a non-invasive electrophysiologic technique, originally described by McComas and coworkers over three decades ago, that has been used to monitor the functional status of a motor unit pool in vivo and to estimate the number of functioning motor neurons innervating the muscles being tested. 31 This method is based on compound muscle action potential (CMAP) response that represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE was performed on all animals according to an adapted version of Gordon and co-workers. 32 Briefly, rats were anesthetized as described in detail previously and placed in the supine position. Surface temperature at 37 C was maintained with a thermostatic warming plate to avoid hypothermia. Animals were fixed with tape on a smooth table to prevent movement artifacts due to the electrical stimulation, the lower limbs gently stretched and spread forming an approximately 45 angle to the spine, the sciatic nerve was then stimulated by using a device with two mono polar needle electrode that were inserted subcutaneously at the root of the hind limb.
The muscular response to the electrical nerve stimulation was recorded with a pair of monopolar recording needle electrodes placed onto the belly and onto the tendon of the tibialis anterior (TA) and the gastrocnemious (GA) medialis muscles, respectively. Once the optimal position was found, as assessed by evoked CMAP on both muscles,   the sampling field by its area. 34 All values of morphometric parameters were expressed as mean ± SD.

| Statistical analysis
All experiments were performed in triplicate collecting for each experiment a number of samples n = 6, and values were expressed as mean ± standard deviation. ANOVA followed by a Tukey post hoc test was performed using the QI Macros SPC Software for Excel to determine the significance of results. p-value < .05 was defined as the level of statistical significance.

| Fabrication and characterization of PBS scaffolds
The electrospinning procedure used for the production of PBS scaffolds tested in this study was already explored in a previously published work. 29 For this study, the polymeric solution extrusion rate was increased from 0.6 to 0.8 ml/min, in order to improve the density of the final electrospun scaffold and the mechanical resistance to the surgical procedure. Scaffold were produced as flexible thin sheet (9 Â 12 cm), with an average thickness of 0.5 mm, as shown in the photograph of Figure 1 (Panel a).
After production, PBS scaffold were analyzed by SEM in order to highlight the microscopic features, porosity, and micro-fibrillar structure. As shown in SEM images, (Figure 1, Panel b), the inner structure of the scaffold is highly porous. Interestingly, the microstructure of the scaffold shows microfibers with a diameter between 1-5 μm, alternating with the presence of collapsed-balloons like structures along the microfibers. This finding may be related to the increased polymeric solution extrusion rate from 0.6 to 0.8 ml/min, during electrospinning process. Moreover, at higher magnification, a superficial microporosity is observable in the scaffold forming fibers. Interestingly, the 3D reconstruction obtained by μCT analysis evidenced the microfibrillar structure also in the internal part of the scaffold and an adequate porosity of the materials (Figure 2a). This analysis was also used to measure the exact thickness of the scaffold that resulted about 300 μm (Figure 2b).

| MRI of nerve repaired with PBS wraps
The 7 Tesla preclinical MRI tomography allowed to qualitative predict the absence of strong inflammatory reaction and any anomalies at a morpho-structural level, consistent with regenerative process of the sciatic nerve. Actually, the analysis of MRI images was focused in evaluating changes in signal intensity, in particular on T2-weighted images, in order to identify potential anomalies in the cross-sectional area and nerve course, as well as disorganization or absence of the typical fascicular pattern. Interestingly, the MRI scan of the region of the hind limbs, left and right, of the animal showed an improvement of the regenerative process from 30 to 120 days in the G2 group.
In particular, at 30 days post implant, it is possible to highlight a hyperintense signal in T2 (Figure 3a,b,e) in the right limb, expected when an inflammatory process occurs; it is also possible to view the presence of the tubular scaffold. The portion of the scaffold, with the severed nerve inside, is located between the GA muscle, the soleus muscle, and the cranial tibialis muscle.
Differently, the MRI scanning analysis after 120 days post implant, showed the reduction of the hyperintense signal in T2, which is coherent with absence of inflammation process, and almost the total reabsorption of the scaffold (Figure 3c,d,f).

| Electrophysiological findings
Sciatic nerve functional recovery was estimated by performing electrophysiological analysis only at 120 days postoperatively, before the animals were sacrificed, as reinnervation phenomena need several weeks to months to be seen. 35 The number of estimated motor units (MUNE) was calculated both for operated (right) and for healthy con-  Actually, a general increase in weight of all tested subjects was expected during evaluation period, due to normal growth, not being animals on a restricted diet regime. Results are reported in Table 1.

| Histological findings: Counting of regenerated fibers
The analysis of the normal sciatic nerve with HE staining allowed us to observe the typical undulated and parallel organization of the nerve fibers ( Figure S3). After 30 and 120 days, the total fiber number  Table S1.  As expected following nerve transections, neurophysiological findings showed a significant reduction of MUNEs in operated limb when compared to contralateral uninjured nerve. 36 However, this difference was limited to GA muscle in G2 and to the TA muscle in G1. It was observed that even proximal sciatic neuropathy preferentially affect peroneal fibers, leading to worse deficit in TA muscle. This finding in G1 is coherent with the literature. 35 Conversely, we found better recovery in TA muscle in the G2 (PBS scaffold group); while we strongly believe that the limited number of subjects enrolled severely affects the results, and only qualitative considerations can be made, this could suggest a specific effect of PBS scaffold in guiding peroneal fibers. If confirmed in a larger series this data can represent a basis for using scaffolds when peroneal fibers are more severely injured.
Actually, muscles mass indexes of gastrocnemius muscle (GMWR) and tibialis anterior (TAMWR) supported the tropism of muscles inner- The statistical analysis of the GMWR with the T-test showed no statistically significant difference between G1 and G2 either at 30 days or 120 days after surgery (30 days: p = .4008; 120 days: p = .2938).
In addition, the corresponding analysis of the TAMWR showed similar results as well (30 days: p = .3449; 120 days: p = .2975).
The above findings are certainly related to a normal gait regained by the animal as early as 30 days after the PBS implant.
Histological analysis of the nerves provided two further parameters supporting the positive advantages of PBS scaffolds in nerve regeneration: the number of fibers and the density of the fibers. Given that the ability to synthesize proteins is largely owned by the neurosome, the distal segment quickly loses its ability to transmit action potentials and undergoes a series of degenerative changes called Wallerian degeneration. Otherwise, the proximal stump generates axonal gems that, organized in growth cones, will try to reach the target organs for reinnervation. This process can take place only if the continuity between the proximal and distal stump is maintained. This explains why surgical repair is always necessary. Finally, the observation of locomotor activity, despite an altered semi-erect position, revealed a progressive normalization of the posterior locomotor system movements (legs movement did not appear impaired) just starting from 30 days post implant.

| CONCLUSIONS
The present study demonstrates that the use of the planar PBS scaffold is a more effective method of fixing the injured two portions (proximal and distal) of the sciatic nerve, to preserve nerve continuity and promote its regeneration. The interpretation of the difference obtained by electroneurography, the weight of GA and TA muscles, histological examination of regenerated nerves and locomotor activity of animals leaves no doubt that there is a real improvement in the regeneration process of the sciatic nerve, used here as a nerve model, if the animals treated with the scaffold are compared with those in which the severed nerve has been sutured with a traditional technique.
The results demonstrated that there is an important nerve regeneration action due to both mechanical and vehicle support of the scaffold and in the same way an adequate biodegradability, as observed from high resolution MRI investigation, that highlight the potentiality of PBS as biomaterial for nerve regeneration.
The results obtained encourage new research perspectives aimed at testing the use of a three-dimensional structure such as the PBS planar scaffold, on a larger nerve sample model, to subsequently promote its use in clinical practice, considering an advancement of the standard surgical technique and the advantage of a reduction in clinical healing times and therefore also in costs.
ATeN Center of University of Palermo-Laboratory of Preparazione e Analisi di Biomateriali, for SEM and microCT analysis of scaffolds.