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Controlled release of growth factors from multi-layered fibrous scaffold for functional recoveries in crushed sciatic nerve Min-Ho Hong, Hye Jin Hong, Haejeong Pang, Hyo-Jung Lee, Seong Yi, and Won-Gun Koh ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00801 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 6, 2018

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ACS Biomaterials Science & Engineering

Controlled Release of Growth Factors from Multi-layered Fibrous Scaffold for Functional Recoveries in Crushed Sciatic Nerve

Min-Ho Honga.1, Hye Jin Hongb,1, Haejeong Pangb, Hyo-Jung Leea, Seong Yia,*, Won-Gun Kohb,**

a Department of Neurosurgery, Spine and Spinal Cord Institute, College of Medicine, Yonsei University, Seoul, 03722, Republic of Korea b Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, 03722, Republic of Korea

* Corresponding author, Prof. Seong Yi, Department of Neurosurgery, Spine and Spinal Cord Institute, College of Medicine, Yonsei University, Seoul, 03722, Republic of Korea, E-mail: [email protected] ** Corresponding author, Prof. Won-Gun Koh, Department of Chemical and Biomolecular Engineering,

Yonsei

University,

Seoul,

03722,

Republic

of

Korea,

E-mail:

[email protected]

1

These authors contributed equally to this work.

KEYWORDS: multi-layered fibrous scaffold, controlled release of growth factor, sciatic nerve regeneration, electrospinning, hydrogel micropattern

ACS Paragon Plus Environment

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ABSTRACT In this study, we designed and fabricated a multi-layered fibrous scaffold capable of the controlled release of multiple growth factors for sciatic nerve regeneration in rats. The scaffold consists of three layers prepared by sequential electrospinning, where the first layer is fabricated by polycaprolactone (PCL)-aligned electrospun nanofibers for the attachment and differentiation of cells toward the direction of the sciatic nerve. The second and third layers are fabricated by poly(lactic-co-glycolic acid) 6535 (PLGA 6535) and 8515 (PLGA 8515), respectively. The resultant three nanofiber layers were stacked together and fixed by incorporating hydrogel micropatterns at both ends of nanofiber scaffold, which also facilitated the surgical handling of the multi-layered scaffolds. The PLGA layers acted as reservoirs to release growth factors: neurotrophin (NT-3), brain-derived neurotrophic factor (BDNF), and platelet-derived growth factor (PDGF). The different biodegradation rate of each PLGA layer enabled the controlled release of multiple growth factors, such as NT-3, BDNF, and PDGF, with different patterns. In a rat model, the injured nerve was rolled up with the multi-layered scaffold loading growth factors, and behavior tests were performed 5 weeks after surgery. Sciatic functional index (SFI) and mechanical allodynia analysis revealed that the fast release of NT-3 and BDNF from PLGA 6535 and subsequent slow release of PDGF from PLGA 8515 proved to be the greatest aid to neural tissue regeneration. In addition to the biochemical cues from growth factors, the aligned PCL layer that directly contacts the injured nerve could provide topographical stimulation, offering practical assistance to new tissue and cells for directional growth parallel to the sciatic nerve. This study demonstrated that our multi-layered scaffold performs a function that can be used to promote locomotor activity and enhance nerve regeneration in combination with align-patterned topography and the controlled release of growth factors.


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■ INTRODUCTION The nervous system is broadly divided into the central nervous system (CNS, two major structures: brain and spinal cord) and the peripheral nervous system (PNS, nerves located throughout the rest of the body). In terms of axonal regeneration after injury, the PNS is more capable than the CNS. However, the spontaneous repair of peripheral nerves is still deficient in poor functional recovery.1-2

PNS injury causes the atrophy of effector muscles and the

impairment of behaviors (such as gait) because there is axon demyelination and degradation induced by distal stump.3 Generally, the most widely used technique for regenerating an injured nerve is using an autograft in the clinical field.4 However, the functional results of using an autograft are unstable and the lack of a donor nerve also retards the application of the autograft in a clinical setting.5 Moreover, there are many intrinsic drawbacks to autograft implantation, including pain and donor site morbidity, mismatch of donor nervous tissue size, fascicular inconsistency between the autograft and the proximal and the distal stumps of the injured site, the need for additional surgery, and a limited supply of suitable nerves.6 For these reasons, the development of artificial nerve grafts is thought to be a promising alternative to autologous nerve grafts. In the last few decades, a variety of polymer-based nerve grafts, mostly in the form of nerve guidance conduits, has been developed and tested in animal and/or clinical practice for neural tissue regeneration.7

In developing a polymer-based nerve conduit, topographical and

biochemical cues have been incorporated, since they are known to play important roles in the regeneration of the nervous system. According to previous studies, the aligned topography that mimics the aligned architecture of native nerve anatomy is a critical parameter in the design of nerve conduits, showing the ability to promote the directional growth of nerve tissues.8-9


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Compared with topographical cues, biochemical cues (for example, growth factors loaded into the scaffold) could offer a more progressive microenvironment that allows cell migration and neuronal differentiation.10-12 Among diverse types of nerve conduits, electrospun nanofiber-based scaffolds have received great attention for nerve regeneration.13 Electrospinning produced porous scaffolds consisting of fibers with nano or micrometer scale, which recapitulate aspects of the native extracellular matrix (ECM).

Especially, the aligned electrospun fibers are well-suited for neural tissue

engineering because they can provide the spatial guidance for neurite outgrowth and axonal elongation.14 On the other hand, various strategies has been studied to incorporate biochemical cues, such as growth factors, into the electrospun fibers.15-16 Despite the successful results of developing fiber-based nerve conduits with either topographical or biochemical cues, little effort has been made to create scaffolds combining drug-releasing and aligned features, which would provide not only biochemical cues but also topographical guidance for peripheral nerve regeneration.17-18 In particular, to the best of our knowledge, there has been no report on the development of an aligned fibrous nerve conduit from which multiple drugs are released in a controlled manner. In the present study, we designed and fabricated multi-layered fibrous scaffolds for sciatic nerve regeneration in rats. The multi-layered scaffolds consisted of three layers that were combined together by a hydrogel patterning process. The first layer was prepared as aligned PCL fibers to provide a topographical cue, while the second and third layers were prepared from randomly oriented PLGA fibers with different lactide/glycolide ratios. The different biodegradation rates of each PLGA layer enabled the controlled release of multiple growth factors, such as NT-3, BDNF, and PDGF, with different patterns, providing spatiotemporal


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biochemical cues.19-20 After confirming the successful fabrication of the multi-layered scaffolds and their capability to control the release behavior of multiple growth factors, in vivo studies, such as behavior and biological tests, were performed to demonstrate the successful regeneration of neural tissue at the site of the injured sciatic nerve.

■ EXPERIMENTAL SECTION

Materials. Polycaprolactone (PCL; MW 80,000), polyethylene glycol diacrylate (PEG-DA; MW 575), poly(DL-lactide-co-glycolide) 6535 (PLGA 6535; MW 40,000-75,000), PLGA 8515 (MW 50,000-75,000), 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), Tween 20, Triton X-100, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF) and all the biotinylated detection antibodies for ELISA were purchased from PeproTech (Rocky Hill, NJ, USA). Horseradish peroxidase (HRP) for ELISA, and 3,3′,5,5′tetramethylbenzidine (TMB) were purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). The mouse neural stem cells (mNSC) were purchased from CRL-2925TM, American Type Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle Medium (DMEM/F12), fetal bovine serum (FBS), trypsin-EDTA, and penicillin were purchased from Gibco (Grand Island, NY, USA). Phosphate buffered saline (PBS) were purchased from HyCloneTM Laboratories Inc. (South Logan, UT, USA). Ketamine hydrochloride was purchased from Yuhan (Seoul, Korea), and xylazine hydrochloride was purchased from Bayer Korea (Ansan, Korea). A protein extraction solution for Bradford was purchased from PRO-


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PREPTM, Intron Biotechnology (Sungnam, Korea). Polyvinylidene difluoride membrane was purchased from Millipore (Schwalbach, Germany). Class III β-tubulin antibody (Tuj1; ab18207), anti-NT3 antibody (ab53685), anti-PDGF antibody (ab125268), β-actin antibody (ab8226), and microtubule associated protein-2 (MAP2; ab32454) were purchased from Abcam (Cambridge, UK). HRP-conjugated secondary antibodies, and anti-BDNF antibody (sc-65514) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All the secondary antibodies used in immunohistochemistry were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

Figure 1. Schematic representation for fabrication of growth factor-loaded multi-layered nanofiber scaffold.

Fabrication of multi-layered scaffold loaded with multiple growth factors. The growth factor-loaded, multi-layered fibrous scaffolds were fabricated by combining sequential


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electrospinning and photolithographic hydrogel patterning as shown in Figure 1. Each nanofiber matrix was electrospun separately and combined into one whole scaffold by patterning with a PEG-DA hydrogel. The incorporation of hydrogel micropatterns into nanofibers made handling convenient and firmly fixed the different layers of nanofibers together. The resultant nanofiber scaffolds were vertically divided into three different regions (“triple-layered nanofiber scaffolds”), which consist of aligned PCL fibers, PLGA 6535 fibers, and PLGA 8515 fibers as the first, second, and third layer, respectively. For the first layer, electrospinning of 20 wt% of PCL in TFE was conducted around a rotating drum at a speed of 850 rpm for 2 hr. The flow rate of PCL solution injection was 0.5 mL/h with a voltage of 8.0 kV, and the distance between the 23G needle and the collecting plate was approximately 80 mm. For the second PLGA 6535 nanofiber layer, 40 wt% PLGA 6535 in TFE was electrospun with the following condition: 0.5 mL/h, 11 kV, 10 cm distance, and 23G needle for one hour. The third layer was fabricated through electrospinning of 50 wt% PLGA 8515 in HFIP under the same condition as the one for PLGA 6535. When the growth factor-loaded nanofibers were prepared, 1 µg each of NT-3, BDNF, and PDGF was added separately or together to different PGLA solutions. After all the nanofiber sheets were obtained, they were stacked in a proper order and ready for the PEG hydrogel patterning. The multi-layered scaffold sheet would go through O2 plasma treatment (FemtoScience, Seoul, Korea) to facilitate the infiltration of hydrogel precursor solution. After precursor solution permeated through the nanofiber scaffold, the photomask was placed onto the resultant nanofibers and exposed to 365 nm, 300 mW/cm2 UV light (EFOS Ultracure 100ss Plus, UV spot lamp, Mississauga, Ontario, Canada) for 0.5 sec. The resulting scaffold was washed with distilled water to remove the unreacted precursor solution. The resultant triple-layered


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nanofiber scaffolds were divided into four groups depending on the composition of growth factors within the different PLGA nanofiber layers (Table 1). The formation of multiple layer structure was visualized with a confocal laser scanning microscope (LSM700, Carl Zeiss, Oberkochen, Germany), where each layer was incorporated with rhodamine B (red), toluidine blue (blue), and FITC (green) fluorophores, respectively. Scanning electron microscopy (SEM; JSM-7001F, JEOL, Tokyo, Japan) was performed to examine the thickness and structure of layers (aligned-PCL, PLGA 6535, PLGA 8515, and PEGDA hydrogel). Porosity of PCL layer and average pore size was obtained using Porosimeter (Quantachrome Inst. PM33GT, Boynton Beach, FL, USA). Table 1. The representative groups in this study. Control group

6535 group

8515 group

Experimental group

Aligned PCL layer

-

-

-

-

PLGA 6535 layer

-

NT-3, BDNF, and PDGF

-

NT-3 and BDNF

PLGA 8515 layer

-

-

NT-3, BDNF, and PDGF

PDGF

Growth factors release test. The release behavior of growth factors from multi-layered scaffold with (6535, 8515, and experimental groups) or without growth factors (control group) were monitored in vitro for 10 weeks. The scaffold was immersed in a well plate containing 2 mL of PBS and stored at 37°C in an incubator (n = 15). The amount of the released growth factors was determined through ELISA at predetermined time points for 10 weeks. A 96-wellplate was coated with capture antibodies of either NT-3, BDNF, or PDGF. The sample solution,


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which had been incubated with experimental fiber sheets, was loaded in a volume of 100 µL/well, and then, 100 µL/well of biotinylated detection antibodies were conjugated after 2 hr of incubation. After additional incubation for 2 hr, the further conjugation of HRP and reaction with TMB in the dark would finally result in a colorimetric change of the solutions. The final result was measured with spectrophotometric plate reader at 450 nm (VersaMaxTM ELISA microplate reader, Molecular Devices, Sunnyvale, CA). In vitro cytotoxicity assay. The mNSC was used for the in vitro assay. The cells were cultured in DMEM/F12 supplemented with 10% FBS and 5% penicillin under 5% CO2 at 37°C. Cells were sub-cultured every 2 days before reaching confluence using PBS and trypsin/EDTA, and used for the experiment between passage 5 and 10. To culture the cells on the scaffolds, the scaffolds were placed in 6-well plates, and sterilized with 70% ethanol. The in vitro biocompatibility of the scaffolds was tested with an MTT assay, which is a colorimetric assay that measures the activity of enzymes that reduce MTT to formazan dyes, giving purple colors. The MTT assay was performed according to the manufacturer’s protocol. In detail, 7 × 104 cells/scaffold was seeded on the scaffold and cultured in 6-well culture plate under 5% CO2 at 37°C. On the 1st, 3rd, 5th and 7th day of culture, the culture medium was removed, and MTT reagent was added to the wells and then incubated for 1 hr. After removing the MTT reagent, DMSO was added to each well, and the plates were shaken for 15 min. The optical density (OD) at 570 nm was measured using the spectrophotometric plate reader. Surgical procedures and animal care. This research was carried out in strict accordance with the recommendations contained in the Guideline of Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) and approved by the Institutional Animal Research Ethics Committee of the Yonsei Medical Center (IACUC


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Approval No. 2015-0375). In this study, male adult Sprague-Dawley rats (300 ± 30 g, Orient, Korea) were used in accordance with the approved guidelines. The animal room was artificially illuminated from 8:00 am to 8:00 pm and conditioned at 24 ± 3°C with 40–60% humidity. The rats were anesthetized with ketamine hydrochloride (1.15 µL/g) and xylazine hydrochloride (0.5 µL/g). After exposing the left sciatic nerves of rats, they were clamped by a 100g force micro clamp (jaw width = 2 mm) for 10 sec. The injured sciatic nerves were rolled up by the multilayered scaffold (10 × 15 mm2). We wrapped the crushed nerve three times with the scaffold. Following the injury and scaffold implantation, the muscle and skin were closed with a running suture of polyglactin 3/0 (Vicryl®, Johnson and Johnson, Markham, ON). The above-mentioned surgical procedure was shown in Figure 2.

Figure 2. Photos showing the surgical procedure for animal studies.


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Behavior studies. All rats were subjected to a series of behavior analysis after injury to their sciatic nerves. Recovery of the left hindlimb locomotor activity was considered as a proof of adequate muscle reinnervation and functional recovery of the sciatic nerve lesion, and was monitored by analyzing the free-walking pattern, which was the method referenced from a previous study.21 For this test, rats were placed on a track (length of 500 mm and width of 100 mm, covered with a white sheet of paper at the bottom and ending in a dark box) and were allowed to walk after painting a dark dye on the plantar surface of the hind paws (Supplementary video 1). The rats’ footprints were used to calculate the sciatic functional index (SFI). Three values were measured from the footprints: a distance from the heel to the third toe, which is indicated as the print length (PL), a distance from the first to the fifth toe (TS), and a distance from the second to the fourth toe, which is named as the intermediary toe spread (ITS) (Figure 3). All 3 measurements were obtained from the experimental (E) and the normal (N) sides. The measured values were inserted in the equations, and the SFI values were calculated (n = 12).21 As an additional behavior study, a basal pain sensitivity was measured before and after the implantation of the scaffolds.22 The rats were placed on an elevated wire grid. For mechanical allodynia, the plantar surface on the hind paw was stimulated with a series of von Frey hairs. In this study, mechanical paw withdrawal threshold (PWT) measurement was assessed by a dynamic plantar esthesiometer (Ugo Basile, Varese, Italy). The threshold was taken as the lowest force that evokes a brisk withdrawal response (1–50g in 50 sec). When the rat withdrew its hind paw, the mechanical stimulus was automatically withdrawn (n = 12) (Supplementary video 2).


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Figure 3. The formula to calculate the sciatic functional index (SFI) which is derived by multiple linear regression (PL; print length, ITS; intermediary toe spread, TS; toe spread).

Immunohistochemistry. The immunohistochemistry (IHC) staining was performed to detect specific expression patterns of Tuj1 and MAP2 exhibited by proteins. In detail, animals were terminally sacrificed in a CO2 chamber and perfused through the ascending aorta with saline solution followed by 4% paraformaldehyde (pH 7.4) after defined survival times (2 and 5 weeks). After the perfusion, the sciatic nerve rolled with the scaffold was removed and postfixed in the same fixative for 6 hr, then dehydrated with 30% sucrose overnight. The cryostat samples were cut at 10∼20 µm both perpendicularly and longitudinally to the main nerve axis. The cryostat sections were rinsed with 0.3% Tween 20. Non-specific primary antibody binding was blocked using 10% normal donkey serum diluted in 0.3% Triton X-100 (Tuj1; 1:2000 and


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MAP2; 1:2000). The samples were incubated with primary antibodies for 2 hr at room temperature. After rinsing with 0.3% Tween 20, the primary antibodies were bound by secondary antibodies for an hour. After rinsing with 0.3% Tween 20, the sections were examined under a confocal laser scanning microscope. Statistical analysis. The data of the behavioral experiments and a quantitative analysis of immunohistochemical evaluation were statistically analyzed by one-way ANOVA using SPSS 23 statistical software (IBM, Armonk, NY), and a post hoc analysis was performed according to Tukey’s method. The significant difference between each group was determined at p < 0.05, whereas p < 0.001 was considered a highly significant difference.

■ RESULTS

Characterization of multi-layered scaffold. The structure and configuration of the scaffolds were observed with SEM and confocal microscopy (Figure 4).

As shown in Figure 4A,

hydrogels were successfully incorporated at both ends of a triple-layered nanofiber scaffold. Because all the nanofiber layers were prepared with hydrophobic polymers, the multi-layered scaffold without hydrogel formed an aggregated clump after contact with water as shown in Figure 4B. However, the incorporation of a PEG hydrogel micropattern facilitated the handling of nanofibers as shown in Figure 4B. The upper PCL layer that directly contacted the injured nerve was electrospun in an aligned form to support the elongated guidance of neuronal cells when inserted within the in vivo environment as demonstrated in previous studies,23-25 while the other two layers releasing growth factors had randomly oriented nanofibers. The aligned and


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random orientations of each fibrous sheet were clearly distinguished and observed through the SEM results as shown in Figure 4C. The porosity was approximately 52.48% and average pore size was 34.06 µm, respectively. These results indicate that porosity of PCL layer was large enough to allow the diffusion of growth factors through the fibrous scaffold.

The formation of

triple-layered nanofibers was verified by incorporating different fluorescent dyes into each layer (first PCL layer with rhodamine B, second PLGA 6535 layer with toluidine blue, and third PLGA 8515 layer with FITC).

A cross-sectional fluorescence image of the scaffold

demonstrated that the nanofiber scaffold was clearly divided into three different vertical regimes (Figure 4D). The SEM image shown in Figure 4D reveals that the overall thickness of the scaffold was approximately 140 µm, where the aligned PCL layer was approximately 30 µm thick, and the PLGA 6535 and PLGA 8515 layers represented similar thicknesses (55∼60 µm). Hydrogel micropatterning plays the role of a stapler, firmly fixing different layers of nanofibers together. The in vitro biocompatibility of the multi-layered scaffolds was evaluated using an MTT assay as shown in Figure 4E, where absorbance values are proportional to the number of viable cells seeded to the scaffolds. It is apparent that the mNSCs remained viable and could proliferate within the all scaffold groups.


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(A)

(B)

(C)

(D)

(E) Figure 4. Triple-layered nanofibrous scaffolds incorporated into hydrogel micropattern. (A) The photographic image and magnified SEM image of the scaffold (scale bar; 200 ㎛). (B) The photographic image of the scaffold with PEG hydrogel pattern (left), and without PEG hydrogel pattern (right) after wetting with water. (C) SEM images of aligned PCL fibrous layer and randomly oriented PLGA fibrous layers (scale bar; 10 ㎛). (D) The cross-sectional images taken by confocal microscopy and SEM (scale bar; 100 ㎛). (E) The MTT assay result of all groups (n=3). There is no significant difference.

In vitro release profile of growth factors. Three different growth factors (NT-3, BDNF, and PDGF) were loaded into the second and third nanofiber layers in the multi-layered scaffold,


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which were made with PLGA with a lactide/glycolide ratio 65:35 (PLGA 6535) and 85:15 (PLGA 8515), respectively, as grouped in Table 1. It is expected that growth factors in PLGA 6535 released faster than those in PLGA 8515 because the PLGA with the lower lactide/glycolide ratio degraded faster than the PLGA with the higher lactide/glycolide ratio. Therefore, our multi-layered scaffold can realize the independent release of multiple growth factors in a controlled manner. Figure 5 shows the controlled release of different growth factors, which has three different scenarios. First, all the growth factors were released quickly by loading all of them in PLGA 6535 layer as shown in Figure 5A (6535 group). Second, all the growth factors were loaded into PLGA 8515 (8515 group) and released more slowly than in the first case (Figure 5B). Finally, Figure 5C shows the release behaviors from the experimental group, where NT-3 and BDNF were loaded into the PLGA 6535 layer and PDGF was loaded into the PLGA 8515 layer. In this experimental group, NT-3 and BDNF were released faster than PDGF, which showed more sustained release behavior. For all three graphs, the profiles showed sustained release. Moreover, the release profile from PLGA 6535 reached a plateau after 6 weeks, while the one from PLGA 8515 began to exhibit a plateau after 8 weeks.

(A)

(B)

(C)

Figure 5. The release profiles of growth factors loaded into multi-layered scaffold from (A) 6535 group, (B) 8515 group, and (C) Experimental group for 10 weeks (n=15).


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Sciatic functional index and mechanical allodynia. To determine whether the local delivery of growth factors from the multi-layered scaffold at the injury site was effective in peripheral nerve regeneration, we first assessed motor function in rat with a biceps femoris injury of the sciatic nerve. Motor recovery after sciatic nerve injury was evaluated using the SFI, which is a reliable method to estimate the recovery of innervation of foot muscles. Figure 6 shows the SFI values of all four groups at 0, 1, 2, 3, 4, and 5 weeks after the scaffold implantation, which improved with time for all groups. The experimental group and 8515 group showed superior progressive improvements compared to the 6535 and the control groups after 2 weeks. The improvements in the outcome of the experiment group were found to be statistically significant compared to the control and 6535 groups at all times after implantation. A highly significant effect of the groups was observed for the SFI values (p < 0.001) with the experimental group being different from the control and the 6535 groups at 4 and 5 weeks. For the result at 5 weeks, there was a significant difference between the experimental and the 8515 groups (p < 0.01). Next, the sensory function was measured by the pain reaction elicited after a touch stimulus using von Frey hairs. As shown in Figure 7, the ipsilateral mechanical allodynia following the compression injury increased after the implantation of multi-layered scaffolds in all groups, and similar trends were observed with SFI results. The experimental group had significantly higher threshold values than the 6535 and control groups after 2 weeks. Although the experimental group also had higher values than the 8515 group, there was no significant difference between these two groups for all testing times. According to these two behavior tests, the fast release of three different growth factors simultaneously did not cause any improvement compared with the control group that did not have any growth factors. At the same time, the slow, more sustained, and simultaneous release of three growth factors brought much better results than the control


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group did. However, the best results were obtained from the sequential release of different growth factors, that is, the fast release of NT-3 and BDNF, and subsequent slow release of PDGF as we hypothesized.

Figure 6. Sciatic functional index (SFI) for the control, 6535, 8515, and experimental groups performed weekly through 5 weeks. Experimental group had the highest SFI after 2 weeks (indicating improved reinnervation compared to other groups by 5 weeks) (n=12; statistically significant difference with respect to experimental group. p

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