Comparison of nerve regenerative efficacy between decellularized nerve graft and nonwoven chitosan conduit

Abstract

Background:

Recently decellularized nerves with various methods are reported as highly functional nerve grafts for the treatment of nerve defects.

Objective:

To evaluate the efficacy of decellularized allogeneic nerve, compared with oriented chitosan mesh tube, and an autologous nerve.

Methods:

Sciatic nerves harvested from Sprague-Dawley (SD) rats were decellularized in combination with Sodium dodecyl sulfate and Triton X-100. A graft into the sciatic nerve in Wistar rats was performed with the decellularized SD rat sciatic nerves or oriented chitosan nonwoven nanofiber mesh tubes (15 mm in length, $N = 5$ in each group). A portion of sciatic nerve of Wistar rat was cut, reversed and re-sutured in-situ as a control. Nerve functional and histological evaluations were performed 25 weeks postoperatively.

Results:

It was revealed that functional, electrophysiological and histological recoveries in the decellularized nerve group match those in the autograft group. Recovery of sensory function and nerve maturation in the decellularized nerve group were superior to those in the chitosan mesh tube group.

Conclusions:

Nerve regeneration in the decellularized nerves could match that in the autografts and is somehow superior to artificial chitosan mesh tube. Detergents wash of SDS and Triton X-100 could obtain highly functional nerve grafts from allografts.

Keywords

Decellularized nerve peripheral nerve regeneration chitosan mesh tube

1. Introduction

Although an autologous nerve graft, autograft, is a gold standard to bridge a peripheral nerve gap, the donor nerve should be sacrificed. Therefore, development of an equally effective replacement for the autograft, artificial nerve is desired and many kinds of biomaterials with various shapes have been investigated [1]. We have constructed a chitosan nonwoven nanofiber mesh tube consisting of oriented fibers, oriented chitosan mesh tube, by the electrospinning method [2]. Functional recovery and electrophysiological recovery occurred in time and approximately matched those in the isograft. Furthermore, histological analysis revealed that the sprouting of myelinated axons occurred vigorously followed by axonal maturation in the isograft and oriented chitosan nanofiber mesh tube group in the order. Even this artificial nerve, however, cannot strictly match autogenous nerve graft. In addition to a good biocompatibility, suitable biodegradability, sufficient mechanical strength and elasticity, the ideal artificial nerve should have a three-dimensional configuration on which growth cone of regenerating axons and Schwann cells can easily attach and migrate. To date, however, no alternatives as effective as the autograft on nerve regeneration have been developed.

To remove the cellular components efficiently from donor tissue with extracellular matrix (ECM) components preserved, decellularization treatment has been noticed [3–7]. It is expected that the immunogenicity of donor organs is decreased substantially by eliminating cell remnants after decellularization, however, cell adhesion molecules (CAMs) such as laminin and fibronectin on the basement membrane, which have critical roles for cell attachment and migration, are preserved. Thus, decellularized nerve graft is expected to induce axon elongation and Schwann cells immigration directly into its empty basal lamina channel, resulting in accelerated nerve regeneration.

In this study, we decellularized allogeneic nerves in combination with SDS and Triton X-100 and confirmed that the three-dimensional structure of basal lamina column on which CAMs are integrated is preserved, and compared its efficacy as a scaffold to bridge a nerve gap with oriented chitosan mesh tubes and autografts.

2. Materials and methods

All procedures in this study were performed in accordance with the guidelines of the National Institutes of Health (USA) regarding the care and use of animals for experimental procedures (NIH Publications, 2011 revision), as well as those for the care and use of laboratory animals of the Tokyo Medical and Dental University.

2.1. Decellularization of the peripheral nerve

Both the left and right sciatic nerves were harvested from SD rats under aseptic conditions after sacrifice by intraperitoneal injection of a lethal dose of pentobarbital (Somnopentyl, Kyoritsu Seiyaku Co. Ltd, Tokyo, Japan). Specimens were rinsed by phosphate buffered saline (PBS) and stirred in 1% SDS (Wako Pure Chemical Industries, Ltd, Tokyo, Japan) in deionized water at 25°C for 24 h, followed by 30 min of deionized water irrigation and 1 h stirring of 1% Triton X-100 (Wako Pure Chemical Industries, Ltd) in deionized water. After rinsing in deionized water for 30 min, these nerves were rinsed in 1x antibiotic–antimycotic containing PBS flow (100 U/ml penicillin-G, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B, Gibco, Carlsbad, CA) for 7 days.

The decellularized nerve specimens were rinsed in deionized water, dehydrated through a graded ethanol series and dried in a critical point drying apparatus (HCP-2; Hitachi, Tokyo, Japan) with liquid CO₂ and Platinum coated with a ion sputter (E-1045; Hitachi, Tokyo, Japan), followed by examining under a scanning electron microscopy (SEM) (Model S-4500; Hitachi, Tokyo, Japan).

The decellularized nerves were fixed in 4% paraformaldehyde in PBS, rinsed and dehydrated with a series of graded ethanol and xylene, followed by soaking and embedding in paraffin, and then sliced transversely to 4 µm thick sections for immunofluorescence staining. The sections were deparaffinized in xylene and hydrated in a reverse series of graded ethanol, rinsed in phosphate buffered saline with 0.05% Tween 20 (PBST), followed by incubation in 5% goat serum containing PBST (blocking; 25°C, 1 h). Then the sections were incubated with various primary antibodies respectively at appropriate dilution in blocking solution at 4°C overnight. Antibodies used in this procedure were as follows: Rabbit anti Collagen-1 (1:200, LB-1102, LSL), Rabbit anti Collagen-4 (1:200, LB-1407, LSL), Rabbit anti Laminin Ab-1 (1:100, RB-082-A0, LVC) and Mouse anti Fibronectin (1:200, SC-8422, SCB). After rinsed in PBST, incubation of secondary antibodies at appropriate dilution in blocking solution was performed at 25°C for 1 h. The secondary antibodies used were as follows: Alexa Fluor 555 goat anti rabbit IgG (1:1000, A21428, Invitrogen), Alexa Fluor 488 goat anti mouse IgG (1:1000, A11001, Invirogen). After washing in PBST, the sections were mounted with 4′,6-diamidino-2-phenylindole (DAPI) containing hardening mounting medium (1:1000, Vectashield, Vector Laboratories), covered with cover glass and sealed with manicure. Section were viewed and taken images by a fluorescent microscope (BX53-FL, Olympus, Japan).

2.2. Preparation of the oriented chitosan fiber mesh tubes

The nonwoven chitosan mesh tube at the size of 15 mm in length, 1.2 mm in inner diameter and 0.3–0.5 mm in wall thickness was prepared by electro spinning method [2,8]. Chitosan solution was prepared by adding 0.8 g of chitosan powder (HOKKAIDO SODA Co., Japan) with the deacetylation rate at 90% to 10 ml of trifluoroacetic acid (TFA) (0.5 mol/l, TCI, Japan) and incubating at 50°C overnight. 2.5 ml of methylene chloride (MC) (DOJINDO, Japan) was then added to this mixture and glass filtered (6.4 w/v% chitosan). For electro spinning, the chitosan-TFA-MC solution was filled in a plastic syringe fitted with a 17-mm-long needle with an inner diameter of 0.5 mm. A high voltage power supply provided a potential of 20–30 kV at a distance of 10–20 cm between the needle tip (anode) and the collector (cathode), a 100 mm thick rotating stainless drum with the linear rate at 0.52 m/s (100 rpm). A positive-charged jet ejected from the syringe at a rate of 2–8 ml/h was sprayed onto the negative-charged collector. Nonwoven Chitosan mesh constituted by numerous randomly crossed nano/micro fibers was formed on the drum surface, the peak (>50%) of the distribution of fiber diameters was between 200 to 600 nm (10). Obtained mesh with a thickness of 0.02 mm was carefully unrolled from the drum, reeled on a stainless bar with the diameter of 1.2 mm and immersed in 28% aqueous ammonia at room temperature overnight to neutralize the chitosan-TFA-MC. After being washed with distilled water at room temperature for 2 h, it was dipped into 100 v/v% ethanol for 3 min. As a result of slight shrinkage of the chitosan fiber, slim but sufficient clearance occurred between the bar and the tube; thus, the chitosan mesh tube was easily removed from the bar. The completed chitosan mesh tube had an inner diameter of 1.2 mm; an outer diameter of 2.0 mm and a length of 12 mm. Twenty-seven samples in total were made for further evaluations.

2.3. Bridging of the nerve gap

Male Wistar rats weighing 180–200 g were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight, Kyoritsu Seiyaku Co. Ltd). The right sciatic nerve was exposed and a section of 10 mm in length was excised at the center of the thigh. A graft of the decellularized SD rat sciatic nerves or oriented chitosan mesh tubes with a length of 15 mm was performed by end-to-end suturing with 8-0 mono-filament nylon to connect nerve ends (each $N = 5$ ; decellularized nerve or chitosan mesh tube group, respectively) for long term evaluation of function, electrophysiological and histomorphological recovery respectively. As a positive control, portions of sciatic nerve of Wistar rat were cut with 15 mm in length, reversed and re-sutured ( $N = 5$ ; autograft group).

2.4. Assessment of function recovery

To assess the recovery of motor and sensory function associated with the sciatic nerve, von Frey hair test and static toe spread factor (STSF) was evaluated respectively 25 weeks post-implantation [8,9].

2.4.1. Static toe spread factor

The tested animals were placed on a transparent plastic plate, and 3 frames of both hind feet were taken for each rat to quantify the distance between the spread 1st and 5th toe. The results of measurements were assessed by the following formula: $\begin{matrix} \begin{matrix} STSF = & (length between the 1st and 5th toe in the normal side \\ - length between the 1st and 5th toe in the experimental side) \\ / length between the 1st and 5th toe in the normal side . \end{matrix} \end{matrix}$

2.4.2. von Frey hair test

The tested rats were placed on a wire mesh plate and the sole of the hind feet was stimulated by nylon monofilaments (Touch-Test Sensory Evaluator, North Coast Medical, CA). The monofilament’s size was recorded when a reaction of paw withdrawal or lick was observed repeatedly. Both sides were tested and results were calculated by the following formula: $\begin{matrix} (size in the experimental side - size in the normal side) / size in the normal side . \end{matrix}$

2.5. Electrophysiological evaluation

Electrophysiological evaluations were carried out under anesthesia by an intraperitoneal injection of ketamine hydrochloride (40–50 mg/kg body weight). A bipolar stimulating electrode was placed at the proximal site of anastomosis, and evoked muscle action potentials (CMAPs) were recorded on the triceps surae muscle with super-threshold intensity (Neuropack 8, Nihon Kohden Co., Tokyo, Japan). The results were assessed by the following formulas [8,9]. $\begin{matrix} \begin{matrix} The amplitude: (amplitude in the normal side - amplitude in the experimental side) \\ / amplitude in the normal side . \\ The latency: (latency in the experimental side - latency in the normal side) \\ / latency in the normal side . \end{matrix} \end{matrix}$

2.6. Histomorphological evaluation

After electrophysiological evaluations, specimens of the graft were harvested and fixed in 2.5 vol.% glutaraldehyde in 0.1 M phosphate buffer, followed by post-fixation of 1% OsO₄ in 0.1 M phosphate buffer. Then they were rinsed and dehydrated in a graded ethanol series, and embedded in Epon 812 resin. The mid-portions of the specimens were cut into 1 µm thick cross-sections with an ultra-microtome (EM UC6, Leica Microsystems, Wetzlar, Germany), stained with 0.5% toluidine blue, and observed with a light microscopy (BH-2; Olympus). Images of whole sections of the implanted nerves were captured to measure the mean axon diameter and area including myelin sheath, as well as axon and vessel density. An Image Pro Plus 6.0 software (Media Cybernetics, Carlsbad, CA) for Windows was employed for image analysis [8,9].

2.7. Statistical analysis

The variance among the experimental and control groups was determined and evaluated for statistical significance using the Bartlett test. Differences were assessed using one-way analysis of variance (one-way ANOVA). Thereafter, statistical significance was evaluated using the Bonferroni/Dunn’s multiple comparison test. The differences were considered as statistically significant when $p < 0.05$ .

3. Result

3.1. Evaluation of decellularized nerve

The SEM images of the decellularized sciatic nerves revealed that the communicating honey comb structure of nerve extracellular matrix was preserved well while axon and Schwann cell contents were eliminated substantially (Fig. 1(A)). Immunohistology of these sections stained for type 1 and 4 collagen (Fig. 2) as well as double stained for laminin and fibronectin (Fig. 3) showed that basal laminae onto which CAMs were integrated were preserved through the decellularization protocols; the ring-like structures in the nerve tissue were open columns of basal laminae. Nuclear stain of DAPI was completely negative indicating that cell contents were removed very efficiently.

Fig. 1.

The SEM micrograph of decellularized sciatic nerve and chitosan mesh tube. The left stand (A) is a decellularized nerve, suggesting that the communicating honey comb structure is preserved and cellular contents are eliminated substantially. The upper right stand (B) is a chitosan mesh tube and the lower left stand (C) is a magnification of the marked inner surface of the tube, which exhibits three-dimensional pores formed among oriented nanofibers, interconnected and distributed throughout the structure.

Fig. 2.

Type 1 and 4 collagen staining of the decellularized nerve. Immunohistochemistry of the same region as Fig. 1(A). The ring-like structures in the nerve tissue are open columns of basal laminae (scale bar is 50 µm). (A) Type 1 collagen, (B) type 4 collagen.

Fig. 3.

Laminin and fibronectin double staining of the decellularized nerve. These CAMs integrated into basal laminae are preserved. (A) Laminin, (B) fibronectin, (C) merge.

Fig. 4.

Results of von Frey hair test and Static Toe Spread Factor (STSF). (A) von Frey hair test. The von Frey hair test values in the decellularized nerve group is significantly smaller than that in the chitosan mesh tube group. (B) Static Toe Spread Factor. There is no significant difference among the groups. Mean and SD.

3.2. SEM images of oriented chitosan mesh tube

The SEM images of the inner wall of chitosan mesh tube with orientation revealed that structure comprised appreciably oriented nano fibers along axis (Fig. 1(B), (C)). Three-dimensional pores formed between fibers interconnected and distributed throughout the structure.

3.3. Recovery of sensory and motor sciatic nerve function after bridge grafting

The von Frey hair test values in the decellularized nerve group was significantly smaller than that in the chitosan mesh tube group ( $p < 0.05$ ) (Fig. 4(A)). There was no significant difference in the STSF values among the groups (Fig. 4(B)). These results indicate that the sensory nerve function had recovered more rapidly compared with motor function especially in the decellularized nerve group than in the chitosan mesh tube group.

3.4. Electrophysiological evaluation

The CMAPs were recorded in all 5 rats in each group at 25 weeks after operation. No statistical significance was detected in the amplitude and latency values among the groups (Fig. 5).

Fig. 5.

Results of amplitude and latency. No statistical significance is detected. (A) Amplitude, (B) latency. Mean and SD.

Fig. 6.

Histology of the grafted nerves, toluidine blue staining. Nerve transections of each group are filled with myelinated axons of various sizes in high density. Many myelinated axons have matured to mass on a large monofascicle (scale bar is 50 µm). (A) Decellularized nerve, (B) chitosan mesh tube, (C) autograft.

3.5. Histology of the regenerated nerve

Many myelinated axons had matured to mass on a large monofascicle (Fig. 6). Mean axon diameter in the decellularized nerve group was significantly larger than that in the chitosan mesh tube group ( $p < 0.05$ ) (Fig. 7(A)), and mean axon area in the decellularized nerve and autograft group were significantly larger than that in the chitosan mesh tube group (each $p < 0.05$ ) (Fig. 7(B)). No statistical significance was detected among the groups in the mean axon density and mean vessel density (Fig. 7(C), (D)). These results suggest that maturation of the regenerating axons in the decellularized nerves matches with that in autografts, and it has progressed as compared to that in the chitosan mesh tubes.

4. Discussion

The three-dimensional ultrastructure, surface topology, and composition of the ECM influences cell activities; cell mitogenesis, chemotaxis and cell differentiation induces constructive host tissue remodeling responses [10]. To enhance the tissue remodeling process, decellularization treatment, that removes cytoplasm and nucleus of cells from the tissue but preserves its three-dimensional ECM scaffolds, may be promising approach for tissue engineering strategy [11]. T.W. Hudson et al. reported a novel decellularization procedure that removes immunological material while leaving the majority of the ECM structure intact. That may elicit an immune response equal to that elicited by the isograft in the rat model [12]. Furthermore, they evaluated a long-term functional regeneration using the sciatic functional index up to 52 weeks after graft implantation followed by 1 cm sciatic nerve resection. It was concluded that equivalent functional recovery to the isograft was obtained with their procedure [13]. In this study, we proposed simpler decellularization method in combination with Sodium dodecyl sulfate and Triton X-100 to treat sciatic nerves harvested from SD rats, and it was examined by SEM and immunofluorescence staining. SEM observation and immunofluorescence staining for type 1 and 4 collagen combined with nuclear stain of DAPI, as well as laminin and fibronectin. That showed the communicating honey comb structures of basal laminae, on to which CAMs are integrated, are preserved well, while cell contents are removed substantially after decellularized process of allogeneic nerve.

These results suggest that three-dimensional structure of peripheral nerve is effectively preserved after combined SDS and Triton X-100 treatment for decellularization. In the long-term evaluation of nerve regeneration at 25 weeks post implantation, it was revealed that functional, electrophysiological and histological recovery in the decellularized nerve group match with those in the autograft group. Furthermore, the sensory function recovery evaluated by von Frey hair test values and the regenerating nerve maturation evaluated by mean axon diameter and area in the decellularized nerve group were superior with those in the chitosan mesh tube group. It suggests that nerve regeneration in the decellularized nerves should compare favorably with the autografts at least in these fields. Although viable Schwann cells and neurotrophic factors are preserved in the autologous nerve, degenerated axons and myelin sheaths should be removed for regenerating axons to penetrate into the implanted Schwann cell columns, resulting in primary delay in the nerve regeneration process. In contrast, scaffold for nerve regeneration is set up from the first stage of implantation in both the decellularized nerves and chitosan mesh tubes. Furthermore, since basal laminae onto which CAMs are integrated are available in the decellularized nerve, not only growth cones of regenerating axons but also Schwann cells, which excrete neurotrophic factors as well as CAMs, can migrate into the Schwann cell columns without time lag. Thus, nerve regeneration may be enhanced in the decellularized nerve implantation up to outstrip the autograft.

5. Conclusion

Through a simple procedure of detergent wash in combination with SDS and Triton X-100, these results encouragingly indicate, a highly functional nerve conduit that is equally effective with autograft may be obtained from allograft. Furthermore, it is confirmed that when a well preserved communicating honey comb structure of ECM without immunological cytoplasm and nucleus of cells on integrated CAMs that is provided, autologous Schwann cells can easily migrate and growth cones penetrate into the anastomosed site to bridge the length of nerve gap experimented in the present study.

Fig. 7.

Results of histological analysis. (A) Mean axon diameter. Mean axon diameter in the decellularized nerve group is significantly larger than that in the chitosan mesh tube group. (B) Mean axon area. Mean axon area in the decellularized nerve and autograft group are significantly larger than that in the chitosan mesh tube group. (C) Mean axon density, (D) mean vessel density. No statistical significance is detected among the groups in the mean axon density and mean vessel density.

Conflict of interest

No benefit of any kind will be received either directly or indirectly by the authors.

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