ABSTRACT
The present study describes the repair of a 10 mm rat sciatic nerve defect by a tubular collagen conduit. This technique was used in a group of 19 animals and was compared with the standard nerve graft reconstruction in another group of 11 rats. At 100 days after surgery the regenerated nerve was evaluated by macroscopic examination, electromyography and histology. Macroscopic examination of the collagen tube reconstructed nerve shows good regeneration in 9 cases and no regenerated nerve in two cases; electromyography examination indicates comparable levels of motor muscle innervations between the animals with collagen tube and nerve graft repair, with higher conduction capacity through the fibers regenerated in the collagen tube. Histological examination shows nerve regeneration through the collagen conduits, with epi and perineural-like tissue proliferation. The present study concluded that collagen tube produced similar nerve regeneration compared to standard nerve graft when used for the repair of a 10 mm peripheral nerve defect in the rat.

INTRODUCTION

Data available in the clinical literature indicate that only a small percent of the patients with peripheral nerve lesions regain full motor and sensory function following nerve repair. For example, in patients with repair of the median nerve, less than 25% regain normal motor function and less than 3% recover normal sensory function.¹
There is no disagreement that nerve sections without tissue loss, such as clean transections of the nerve, should be repaired by direct suture, thus having the best chances to regain much of the function of the injured nerve.² There are cases though where nerve tissue loss is present and the gap between the nerve ends must be bridged. In such cases the common choice is the autologous nerve graft harvested from another peripheral nerve.^{3, 4}
Alternatives to this solution have developed during the last decades, several natural or synthetic biocompatible materials having been investigated for their ability to promote nerve regeneration. Among natural materials used to bridge the nerve defect, the following have been shown to be effective: vein,⁵ striated muscle (after thermal or chemical manipulation),^6,7 vein prefilled with muscle,^{8, 9} tendon,¹⁰ etc. Nerve conduits made from these structures showed some degree of nerve regeneration, but their main disadvantage is that they must be harvested from the patient, thus prolonging and complicating the reconstructive operation.
In order to eliminate this drawback some synthetic biocompatible materials are evaluated as nerve conduits:^11-14 nonresorbable materials such as polytetrafluorethylene, silicone, or resorbable materials such as polyglycolic acid, (poly)lactic acid, polycaprolactone, polyurethane, polyorganophosphazene, polyhidroxybutyrate, alginate gel.
Some natural substances can be shaped in structures that are able to promote nerve regeneration: purified extracellular matrix, collagen, fibronectin and glycosaminoglycans.^15-18 Collagen plays an important role in nerve regeneration^18-22 and can be shaped in structures that can support nerve fiber regeneration. It is also possible to have control over the porosity of these collagen structures, thus influencing to some degree the environment where regeneration takes place.²³ A new development in this field is represented by natural or artificial conduits containing extra cellular matrix molecules and seeded with cells that enhance nerve regeneration.^24,25

MATERIAL AND METHOD

The collagen tubes
We used multilayered collagen tubes with outer diameter of 2.3 mm and inner diameter of 2 mm.

Experimental design
The study involved 30 Wistar rats weighting between 178 and 230g (average = 227.8g) divided in two groups: Group 1 with 19 animals, in which the sciatic nerve was repaired using collagen tube, and Group 2 with 11 animals, in which the sciatic nerve was repaired with a nerve graft.
Results were evaluated at 100 days after the operation by macroscopic examination of the regenerated nerve, by electromyography and histological examination.

Operative technique
Animals were anesthetized by intramuscular injection of 0.13ml/100g of a Ketamine and Xylazine mixture (2/1 mixture of Ketamine 5% and Xylazine 2%).
Right sciatic nerve was exposed in both groups by dissection of the posterior aspect of the thigh from the sciatic notch to the popliteal region. After careful dissection from the surrounding tissues the nerve was transected at 5 mm below the sciatic notch; a second nerve section was made at 10 mm below the first one. A 10 mm nerve segment was excised. Nerve diameter was measured at the proximal and distal stumps and recorded.
The resulting defect was repaired with collagen tube in Group 1. For the Group 2 the excised nerve segment was used as nerve graft placed in anatomical position.
The repair was performed in both groups using microsurgical techniques with Ethylon 10-0 suture material.
Group 1 (repair with collagen tube): Collagen tubes of 14 millimeters in length were used so that the two nerve ends could be slided for 2 mm into the tube and anchored with two epi-perineural sutures. A 10 mm gap was thus maintained between the nerve stumps inside the tube. (Fig. 1-3)
Group 2 (repair with nerve graft): The nerve gap was repaired using the excised 10 mm nerve segment with epi-perineural sutures. (Fig. 4)

Figure 1. A 10 mm segment of the sciatic nerve is removed. Arrows point to nerve ends: (*) - proximal stump, (**) - distal stump.

Figure 2. The collagen tube is 4 mm longer than the defect, so that the nerve ends can be slided inside the tube for 2 mm each. Arrows point to [...]

Figure 3. Epiperineural sutures are used to maintain the tube in the correct position. Arrows point to suture sites: (*) - proximal suture, (** [...]

Figure 4. The nerve graft is sutured in anatomical position. Arrows point to suture sites: (*) - proximal suture, (**) - distal suture.

Postoperative care and follow up
The animals were placed in cages after the operations with free access to food and water and checked daily for their general condition and the appearance of the operated limb.

Macroscopic examination
At 100 days after surgery the animal was anesthetized with 0.2ml/100g of 5% Ketamine and the repaired nerve was examined.

Electromiography
The sciatic nerve of anesthetized animals was exposed in both posterior limbs. The motor response of the gastrocnemius muscle after electric stimulation was observed on both normal (left) and operated (right) nerves. The stimulating electrode was placed on the sciatic nerve as it emerges from the sciatic notch (for the operated nerve this corresponds to the segment above the repaired segment) and the active (recording) electrode was placed on the gastrocnemius muscle belly; the distance between the stimulating and recording electrodes was 25 mm. The nerve was stimulated with 33-44 mA electrical stimuli at a frequency of 1 stimulus per second in order to obtain a maximal motor reaction; in each case the best response was recorded. The results obtained on the left normal sciatic nerve were used to estimate the normal mean values for the recorded parameters (nerve latency - NL and compound muscular action potential - CMAP). The values obtained on the normal sciatic nerve were compared with those recorded on the sciatic repaired with collagen tube and nerve graft, respectively.

Histopathology
After completion of the electromyographic examination the nerves were harvested and fixed in formalin 7% and embedded in paraffin. Tissue section were performed at 1, 5 and 9 millimeters from the proximal nerve suture, and stained with hematoxylin-eosin and Van Gieson. The slides were examined for the presence of the inflammatory response, conjunctive tissue, and the presence of nerve fibers. The presence of any foreign body or foreign body reaction was also noted.

Statistics
The electromyographic data are reported as mean ± standard error of the mean. Statistical analysis between two different groups was performed using the nonparametric U-test (Mann-Whitney). P<0.05 was considered significant.

Study approval
The experiments were approved by the local animal ethics committee and were carried out according to the principles and procedures outlined in the NIH Guide and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985).

RESULTS

Postoperative clinical follow-up
No postoperative infections were noted. Two animals from Group 1 developed significant clinical muscular atrophy in the calf and leg ulcers.

Macroscopic examination
The mean diameter for the sciatic nerve at the time of initial surgery was 1.6 mm at the proximal stump and 1.8 mm at the distal stump.
At 100 days after nerve repair no residual tube could be identified in Group 1. In 9 animals of this group a well –developed regenerated nerve cable bridged the distance between the nerve stumps. In 2 cases it was not possible to distinguish regenerated nerve fibers between the stumps. It was also noted the conjunctive reaction at the site of the repair which was more intense in Group 1(Fig. 5B) than in Group 2(Fig. 5A).

Figures 5. The nerve cable that regenerated through nerve graft (A) and collagen tube (B) at 100 days after surgery. The proliferation of the c [...]

Figures 5. The nerve cable that regenerated through nerve graft (A) and collagen tube (B) at 100 days after surgery. The proliferation of the c [...]

Electromiography
The mean values obtained for NL and CMAP are shown in Table 1.
Nerve latency: In Group 1 NL was found to be significantly different between the normal and repaired nerve (P=0.01). There is also a statistically significant difference between the normal nerve and the grafted nerve in Group 2 (P=0.001). The nerve repaired with collagen tube was found to have a significantly (P=0.0004) smaller NL when compared with the one repaired with nerve graft (Fig. 6).
CMAP: The results (Fig. 7) show a significant difference between the normal and collagen tube repaired nerve (P=0.00004) and between the normal and the grafted nerve (P=0.02). Although there is a difference between the grafted and the collagen tube repaired nerve, the difference is not statistically significant (P=0.07).

Table 1. Nerve latency (NL) and compound muscle action potential (CMAP) for both normal and repaired nerve in the two groups. Values are expres [...]

Figure 6. Electromyography performed at 100 days after surgery.

Figure 7. Nerve Latency for the regenerated nerves and Compound Muscle Action Potential (CMAP), compared with values obtained on normal sciatic [...]

Histopathology
At 100 days after nerve repair the full length of the collagen tube was crossed by nerve fibers from the proximal to the distal stump (Fig. 8).
The tube appeared to be replaced by an atmosphere of connective tissue which had a lot of collagen fibers and few cells at the surface of the regenerated nerve but was richly vascularized inside the nerve cable. There was one case in which the tube was replaced with areas of thick, dense connective tissue, with few cells and pierced by small caliber blood vessels.
The slides taken at 1 mm from the proximal stump showed that the regenerated nerve maintains the trifascicular structure, with the nerve fascicles separated by thin bands of loose, well-vascularized connective tissue. The slides prepared from sections taken in the middle portions of the tube (5 mm from the proximal suture) showed changes in nerve architecture. The nerve fibers were arranged in small diameter fascicles over the entire section area and were surrounded by the same well vascularized loose connective tissue.
The nerve fibers were also visible on the distal sections (9 mm from the proximal stump) except for 2 cases in which nerve fibers were missing. The regenerated nerve appeared as minifascicles of different sizes, but significantly smaller than those found on 1 mm and 5 mm sections, separated by well vascularized connective tissue, which was found in greater proportion than on the other sections.
Proximal and distal suture material is surrounded by an infiltration of macrophage cells.

DISCUSSION

The attempt to replace the lack of certain damaged or entirely destroyed anatomic structures is a challenge and a problem of continuous present interest in many medical fields. In this respect, the research concerning nerve regeneration can not overlook the vast field of biocompatible materials, as some of them may be suitable to be used as nerve conduits. The aim is to produce a structure that promotes nerve regeneration to a level at least comparable to that found in nerve grafts, and to eliminate the need of sacrificing healthy nerves in order to repair a damaged one.
From that perspective the collagen tube is a candidate that might replace the nerve graft, at least for the repair of small nerve defects.
The main quality of the collagen tube is that it is biodegradable, but it maintains at the same time its structure for long enough time to ensure the environment in which neurotrophic factors required by the growing nerve fibers can concentrate. Its structure also offers biochemical support for nerve regeneration, as it has been proven that collagen is one of the factors required in the process of nerve regeneration.^27-31 The tube is very easy to be handled; it can be easily placed and sutured on the nerve, thus eliminating the need of a very accurate nerve suture technique. The collagen prosthesis is flexible and easy to trim (e.g. with scissors or scalpel) to the required dimensions, and at the same time is strong enough to prevent the collapse of its walls after the operation.
Collagen tubes can be produced with different diameters, according to the size of the receptor nerves.
At least in theory collagen tubes have another major advantage: because the nerve stumps are slided inside the tube during the suturing process, the regenerating axons can not escape into the surrounding tissues, which is a common situation in the case of nerve grafts.27
In this respect, neuromas had not been observed in collagen tube repaired nerves in which regeneration was successful (9 cases). In other 2 cases regeneration did not occur and the nerve stumps were bridged by connective tissue; this situation might have been caused by postoperative suture rupture. In one of these two cases neuromas occurred.
At 100 days after surgery in Group 1 an epineural-like tissue ensheathed the regenerated nerve fascicle, although this neo-epineurium was thicker and more vascularized than the one of the normal nerve or the epineurium covering the nerve graft.
The fact that nerve fibers are found on histological sections proves the ability of the nerve fibers to regenerate well inside the collagen tube even without direct contact between nerve ends. Nevertheless, this is proven only for small nerve defects. When compared with the nerve graft, the collagen tube appeared to induce an important proliferation of the collagen tissue and blood vessels. This was probably due to capillary invasion into the collagen material of the tube.
It was also noted a less intense foreign body reaction due to suture material at the proximal and distal nerve stumps in Group 1 than in Group 2. This was correlated with the fact that the collagen tube requires significantly less suture material than the nerve graft for fixation.
The regenerated nerve appears to have a smaller nerve latency value (and, presumably, a higher conduction velocity) in the case of collagen tube than the nerve graft. The compound muscle action potential (CMAP) had similar values in both animal groups (Group 2 had slightly better results than Group 1 – trend, but not statistically significant), meaning similar reinnervation levels. Our study investigated only the motor response of the nerve; the regeneration of sensory fibers was not evaluated. Other studies reported that sensory fibers regeneration occurs at a higher level than the motor fibers regeneration.³¹

CONCLUSIONS

The repair of peripheral nerve using collagen tube can be a therapeutic option, especially in the case of small nerve defects, when the sacrifice of another healthy nerve for nerve graft harvesting may not be justified.
Our study shows similar levels of muscular reinnervation for collagen tubes and nerve grafts for a 10 mm rat sciatic nerve defect.

Acknowledgments

We thank Mr. Didier Guinard for kindly providing the collagen tubes used in this study, Mrs. Magda Petrescu for the help with the preparation of histological sections and Mrs. Irina Caruntu for morphopatological analysis. We also thank Mr. Dragos Pieptu for his support and advice and Mr. Septimiu Toader for the care for the animals and assistance during the entire study.

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