Magnetically Aligned Collagen Gel Filling a Collagen Nerve Guide Improves Peripheral Nerve Regeneration

Experimental Neurology 158, 290–300 (1999) Article ID exnr.1999.7111, available online at http://www.idealibrary.com on

Magnetically Aligned Collagen Gel Filling a Collagen Nerve Guide Improves Peripheral Nerve Regeneration Dolores Ceballos,*,† Xavier Navarro,† Naren Dubey,‡ Gwen Wendelschafer-Crabb,* William R. Kennedy,* and Robert T. Tranquillo‡ *Department of Neurology and ‡Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455; and †Department of Cell Biology and Physiology, Universitat Auto`noma de Barcelona, Barcelona, Spain Received October 6, 1998; accepted April 9, 1999

Bioresorbable collagen nerve guides filled with either magnetically aligned type I collagen gel or control collagen gel were implanted into 4- or 6-mm surgical gaps created in the sciatic nerve of mice and explanted 30 and 60 days postoperation (dpo) for histological and immunohistochemical evaluation. The hypothesis was that contact guidance of regenerating axons and/or invading nonneuronal cells to the longitudinally aligned collagen fibrils would improve nerve regeneration. The criterion for regeneration was observation of regenerating myelinated fibers distal to the nerve guide. Consistent with previous studies showing poor regeneration in 6-mm gaps at 60 dpo with entubulation repair, only one of six mice exhibited regeneration with control collagen gel. In contrast, four of four mice exhibited regeneration with magnetically aligned collagen gel, including the appearance of nerve fascicle formation. The numbers of myelinated fibers were less than the uninjured nerve in all groups, however, which may have been due to rapid resorption of the nerve guides. An attempt to increase the stability of the collagen gel, and thereby the directional information presented by the aligned collagen fibrils, by crosslinking the collagen with ribose before implantation proved detrimental for regeneration. r 1999 Academic Press

INTRODUCTION

The most widely used method for repair of severe injuries of peripheral nerves resulting in a gap between the nerve stumps is interfascicular suture of an autologous nerve graft. However, sometimes it is impossible to obtain a sufficient graft without significantly compromising important sensory or motor functions of the donor nerves (2). Moreover, coaptation between fascicles of the injured nerve and those of the nerve graft are frequently far from perfect, which can obstruct the progress of regenerating axons and lead to the formation of a neuroma-in-continuity. Synthetic tubular nerve guides (‘‘tubes’’) were de0014-4886/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

signed as an alternative repair method in order to replace the need for an autograft and to avoid the immunological reaction caused by heterologous grafts (8, 21). Regeneration in nerve guides is dependent on the formation of an initial fibrin matrix that bridges the gap between the nerve stumps. The fibrin matrix provides a surface for the ingrowth of fibroblasts, endothelial cells, and Schwann cells migrating from both proximal and distal nerve stumps (19, 34, 42). Fibrin is lysed in a few days by endogenous mechanisms and replaced by longitudinally oriented collagen fibrils. Regenerating axons then grow from the proximal nerve stump, presumably promoted by Schwann cells that already invaded the intratubular connective cable (12, 25, 36) and neuritotrophic factors that accumulate in the tubular fluid (20). Extracellular matrix components, mainly collagen, laminin, and fibronectin, localized in the endoneurium and basal membranes, are presumptive tropic factors that guide the growth cones (18). When used to repair sufficiently short gaps, tubulization has been shown to perform as well as nerve autografts in rodent and nonhuman primate experimental models (2, 3, 5, 27). However, a limit to regeneration exists depending upon the gap length (the gap limit does not apply to autografts). Previous studies have shown that regenerating axons are able to bridge tubes implanted in the rat sciatic nerve if the gap is 10 mm or less, but fail in most cases with 15-mm gaps (21, 22, 35, 43). In the mouse, regeneration occurs in most instances with a 4-mm gap, but does not occur with gaps 6 mm or longer (4, 10, 13, 24, 37). Failure of regeneration across long gaps seems to be due to an inadequate initial formation of an extracellular matrix scaffold (48); prefilling the tubes with dialyzed plasma (44), which forms a fibrin gel, or with collagen gel or a laminin-containing gel (17, 23, 24, 33, 47) supports axonal growth across longer gaps than when the chamber is filled with saline solution. (By gel we mean a highly hydrated network of entangled protein fibrils.) Despite such promising reports, regeneration using

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gel-filled tubes across long gaps remains inferior to that typically obtained with nerve autografts (32). While nerve guides provide global direction to the regenerating axons and invading nonneuronal cells, in that escape into the surrounding tissue is prevented (34, 37), they do not provide local direction for axons and cells within the tube. A recent study by Dubey et al. (7) reported that magnetically aligned type I collagen gel directed the invasion of neurites and Schwann cells from dorsal root ganglia cultured on the gel surface and consequently increased the depth of invasion relative to control collagen gel. This enhancement was attributed to a contact guidance response of the growth cones and Schwann cells to the aligned collagen fibrils. Therefore, by prefilling a tube with magnetically aligned collagen gel, the desired local direction would be provided by fibrils aligned along the tube axis. In principle, the rate and degree of regeneration should improve due to contact guidance, with maximal structural arrangement of regenerated axons and minimal neuroma formation. Such a scenario has only been reported for the fibrin clot formed in empty tubes (40) and for tubes prefilled with dialyzed plasma (44), where longitudinal alignment of fibrin fibrils occurs in an uncontrolled manner. When the fibrin gel is not longitudinally aligned, regeneration can actually be impaired (17, 44). Similarly, tubes filled with collagen gel that were not prealigned yielded fewer myelinated axons (38) and poorer functional recovery (17) compared to salinefilled tubes in entubulation repair of a 4-mm gap in the mouse sciatic nerve. Therefore, using magnetically aligned collagen gel, achieved by exposing the forming collagen gel to a high-strength magnetic field, was an attractive approach to improve the regeneration that has been obtained using collagen gel thus far. Consideration had to be given to the fact that the collagen gel would be proteolytically degraded and the local direction conferred by the aligned fibrils possibly lost prematurely during nerve regeneration. In the short-term in vitro study of Dubey et al. (7), this was not an issue. While the optimal degradation rate for improving entubulation repair was unknown, the rate could be decreased by crosslinking the collagen. Numerous methods have been reported for crosslinking collagen in its various forms as a biomaterial, all of which were unacceptable for two reasons: residual cytotoxicity and the difficulty of crosslinking the gel within the tube (where it is formed in situ). A potentially acceptable method was glycation, the nonenzymatic crosslinking of proteins with free amine groups, such as the lysine and hydroxylysine residues of type I collagen, by reducing sugars such as glucose and ribose, which normally occurs during aging, particularly in diabetics (15). This simply required incubating the collagen gel-filled tube in a sugar solution prior to use, with the elevated sugar content and glycation products posing little threat of

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toxicity. We have shown that incubation of collagen gel in medium with elevated glucose or ribose increases the time until solubilization in collagenase (9). Thus, the purpose of this study was to assess by histological analysis whether filling a nerve guide with magnetically aligned collagen gel improved the initial phase of nerve regeneration compared to control collagen gel. It was also our aim to assess whether increasing the in vivo stability of the collagen gel via glycation mediated crosslinking would be necessary for or beneficial to any improvement of nerve regeneration conferred by the aligned collagen fibrils. MATERIALS AND METHODS

Preparation of the Nerve Guides The tubes were composed of processed type I collagen, with 1 mm i.d. and 0.1-mm wall thickness (Integra Life Sciences). The tubes were prepared so as to avoid acidification upon hydration, which precluded collagen fibrillogenesis necessary for gel formation in the lumen. Briefly, type I collagen fibers were precipitated from a 0.5% lactic acid solution containing 0.75% (w/v) collagen solids. Precipitation was achieved by bringing the solution to the isoelectric point with the addition of 0.35% ammonium hydroxide. The resulting fibers were formed into 1-mm diameter tubes and lyophilized. The tubes were crosslinked for 30 min in a buffered 0.25% formaldehyde solution, and residual formaldehyde was removed by repeated washings in deionized water. The tubes were finally lyophilized, packaged, and sterilized by ethylene oxide treatment. For experimental use, 10-cm-length sections of these tubes were hydrated in phosphate-buffered saline (PBS) for 1 h and then filled with a type I collagen solution at 2 mg/ml final concentration (Vitrogen 100, Collagen Corp.) and warmed to 37°C to induce fibrillogenesis, a process largely complete within 2 h. To induce alignment of the fibrils, the tubes were placed perpendicular to a 9.4-T magnetic field during fibrillogenesis (9). For glycation mediated crosslinking, tubes containing collagen gel were incubated at 37°C in a 100 mM ribose solution for 5 days and then multiply rinsed in PBS for a 48-h period just prior to implantation. The tubes were cut using a sharp scalpel to the required lengths (6 mm for 4-mm nerve gaps and 8 mm for 6-mm nerve gaps, to allow for insertion of 1 mm of both nerve stumps into the tube). Assessment of Collagen Gel Alignment Birefringence is an objective, noninvasive measure of fibril alignment. However, the opaque nature of the tubes precluded measurement of birefringence of the gel in the lumen using polarized light microscopy, and the gel could not be removed from the tube to circumvent this problem as was done by Dubey et al. (7) who

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used Teflon tubes to which the gel did not adhere. Thus, 1-mm i.d. glass tubes were used to form the gel under identical conditions and then the gel was removed from the tubes to enable birefringence measurement using the method detailed by Guido and Tranquillo (11). Four fields were chosen along the axis of each sample and the mean birefringence of these four fields represented the mean value for a sample. The standard error was calculated from the mean value of three different samples (n ⫽ 3). Surgical Procedure Operations were performed under Nembutal anesthesia (50 mg/kg ip) on female CF1 mice, aged 2.5 months (Charles River). Under a dissecting microscope, the right sciatic nerve was exposed at the midthigh and transected at a constant point, 45 mm from the tip of the third digit, and a segment of the distal stump was resected. The gap was repaired by fixing the nerve stumps 1 mm inside the ends of a collagen tube, by means of one 10-0 nylon monofilament suture (Ethicon) stitch at each end, leaving an interstump gap of 4 or 6 mm. The wound was closed by muscle and skin sutures and topically disinfected with povidone–iodine solution. In order to avoid autotomy after denervation, animals were pretreated with amitriptyline (28). Mice were divided among nine groups according to the treatment made on the collagen gel filling the collagen tube and the gap length, with an additional group of unoperated mice as a normal control, as defined in Table 1. All the mice used in the study were procured, housed, and handled in compliance with the University Regents’ Policy on Animal Care and Use, NIH policy, Federal Animal Welfare Act, and other applicable standards. Histological Evaluation At two time points, 30 and 60 days postoperation (dpo), half of the mice of each group were reanestheTABLE 1 Definition of Study Groups Groups

n

Gap (mm)

Tube content

Unoperated S4 C4 C6 M4 M6 R4 R6 MR4

9 3 11 11 6 8 6 8 7

— 4 4 6 4 6 4 6 4

8

6

— Saline solution Control collagen gel Control collagen gel Magnetically aligned collagen gel Magnetically aligned collagen gel Ribose cross-linked collagen gel Ribose cross-linked collagen gel Magnetically aligned ribose cross-linked collagen gel Magnetically aligned ribose cross-linked collagen gel

MR6

tized, except for group S4 that was only evaluated at 30 dpo. The operated nerve was carefully dissected from surrounding tissues and a long segment including several millimeters proximal and distal to the tube was harvested. The specimens were divided into two halves by transverse cutting at midtube. The distal half of each specimen was fixed for 4 h by immersion in glutaraldehyde–paraformaldehyde (3– 3%) cacodylate-buffered solution (0.1 M, pH 7.4, 4°C), postfixed with 2% osmium tetroxide, dehydrated through a graded series of ethanol solutions, stained with uranyl acetate (2.5%), and embedded in Epon. Semithin sections (0.5 µm) cut at midtube and at the distal nerve segment on an ultramicrotome and stained with toluidine blue were used for study under light microscopy. Quantitation of the number of myelinated fibers regenerated at each section was performed on randomly selected fields representing at least 30% of the nerve cross-sectional area, projected on a video screen connected to the light microscope (Nikon Microphot-SA) at 1000⫻ magnification. Morphometric evaluation, including axon diameter and myelin thickness, was made from prints of randomly selected fields at 2000⫻ magnification, with the help of a computerlinked digitizing tablet and software designed for the Macintosh. The proximal half of each specimen was processed for immunofluorescent localization of tissue antigens. Specimens were fixed with Zamboni’s solution, cryoprotected, frozen, and sectioned at 40 µm. Sections were processed for triple staining of the following antigens: nerve was localized with rabbit polyclonal antibody to protein gene product 9.5 (rPGP 9.5) (1/1000) (Ultraclone, Wellow, UK), myelin basic protein was localized with mouse monoclonal antibody (mMBP) (1/800) (Ultraclone), and type I collagen or Schwann cells were localized (in alternate sections) with goat polyclonal antibodies to type I collagen (gCol I) (1/40) (Southern Biotechnology Associates, Inc., Birmingham, AL) or S-100 (gS-100) (1/300) (Biogenesis, Sandown, NH). Secondary donkey anti-IgG antibodies directed against each species source of primary antibody (rabbit, mouse, or goat) were labeled with cyanine fluorophores Cy 2, Cy3, and Cy 5 (Jackson ImmunoResearch, West Grove, PA). Nonspecific antibody adhesion was blocked by incubation in normal donkey serum. Background staining with the mouse antibody was reduced by preincubating sections (prior to applying mMBP) in unlabeled donkey anti-mouse IgG to block naturally existing IgG within the sections of mouse tissue. Sections were dehydrated and coverslipped with DPX (Fluka, Ronkonkoma, NY). The fluorescently stained antigens were imaged simultaneously with a laser scanning confocal microscope (Bio-Rad MRC-100, Boston, MA).

IMPROVED PERIPHERAL NERVE REGENERATION

Data Analysis All data are expressed as means ⫾ SEM. As a working definition in this study, regeneration was considered to have occurred when a newly formed nerve cable containing regenerating myelinated axons was present distal to the tube. Comparisons between groups exhibiting regeneration were made by the ␹2 test. The quantitative parameters of nerve cable crosssectional area, number of regenerated myelinated axons, and associated morphometric values were compared by nonparametric Kruskal–Wallis and Mann–Whitney U tests, and the differences were considered significant when P ⬍ 0.05. RESULTS

Fate of Collagen Tube and Collagen Gel During postoperative dissection, the tube was found progressively resorbed. By 30 dpo it was partly resorbed and by 60 dpo it was completely resorbed in most cases. Prior to implantation, the magnetically aligned collagen gel formed in the simulating glass tube was highly aligned based on a measured birefringence of 1.21 ⫾ 0.11 ⫻ 10⫺4 compared to control collagen gel which had none measureable. This magnitude of birefringence has been correlated with strong contact guidance of growing axons in vitro (7). In all groups at 30 dpo, the remaining collagen gel had a dense appearance, being either disperse throughout the lumen or surrounding the regenerated nerve fascicles. In these cases, a large proportion of the nerve cable area was occupied by collagen gel (Fig. 1A). When axonal regeneration was poor or absent, such as in C6 and R groups, the remaining collagen gel appeared densely segregated and barely penetrated by regenerated axons (Fig. 1B). At 60 dpo, the resorption of the collagen gel was more evident, but in samples where axonal regeneration was poor, the collagen remnants had a similar appearance as in 30 dpo samples. In C and R groups, most of the remnants were located outside the regenerated nerve cable. On the other hand, in groups with magnetically aligned collagen gel (M and MR) the collagen gel showed a different appearance at both time points (Figs. 1C and 1D); the remnants, if any, appeared disposed between the regenerated nerve fibers and covering the reformed nerve fascicles rather than being densely segregated. General Morphology At 30 dpo, regeneration was found only in group C4 (Table 2). In the three mice of five exhibiting regeneration, there was a central reformed nerve cable, containing a dense core of connective tissue surrounded by adipocites with small blood vessels and a few nerve fascicles (Fig. 1A). These small nerve fascicles con-

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tained numerous Schwann cells inside a thin perineurial membrane, some of them surrounding axons, whereas others were denervated. However, there was no relation between the number of Schwann cells and the number of myelinated fibers in the regenerated cable. The number of macrophages and mast cells was low. In samples without regenerated nerve fibers, the connective cable contained Schwann cells and fibroblasts, suggesting that they were the first cellular constituents that migrated into the tube. In crosssections distal to the tube, there were abundant degenerated myelinated fibers, as well as an increased number of macrophages and denervated Schwann cells (Fig. 2C). By 60 dpo, the regeneration process midtube had generally advanced. A regenerated nerve cable containing myelinated nerve fibers (Fig. 2B) surrounded by Schwann cells (Figs. 1D and 3D) was found bridging the gap in 100% of the mice examined of groups M4 and M6 (four of four) in contrast to only 50% of the control groups C4 and C6 (three of six) (Table 2). Regeneration occurred in all mice of groups M4 and M6, but in lower proportion in the other groups. Crosslinking the collagen gel with 100 mM ribose worsened regeneration as seen by comparing all R groups to the corresponding non-R groups. In cases without nerve regeneration, the lumen was occupied by a dense connective cable populated by blood vessels, mononuclear cells, Schwann cells, and fibroblasts. At the nerve distal to the tube, some degenerated myelinated fibers were still present and denervated Schwann cells, macrophages, and fibroblasts were abundant. Morphometric Results At 30 dpo, the regenerated cables at midtube, when present, were considerably smaller than the normal sciatic nerve based on cross-sectional nerve area (Table 3). Mean values of the diameter of myelinated axons and the myelin thickness were lower in M4 than in C4, although the differences were not significant (Table 4). Distal to the tube, the cross-sectional area was near normal values for all groups, corresponding to a nerve at an early phase of degeneration. There were regenerated myelinated fibers in three mice of C4, but in none of the other groups (Table 2). At 60 dpo there was regeneration in at least one mouse of each of the different groups, except in R6 (Table 2). At midtube, the cross-sectional nerve cable area was of variable thickness, ranging in individual cases from 6 to 84% with respect to the normal sciatic nerve area. The mean value for all mice in group M6 was larger than in the other groups, being statistically significant with respect to group C6 (Table 3). The mean number of regenerated myelinated fibers was higher in groups with mice exhibiting regeneration. While there were no significant differences between

FIG. 1. Laser scanning confocal micrographs of transverse sections of the nerve cables regenerated across 4-mm gaps using collagen tubes prefilled with collagen gel at 60 dpo. (A) Sample from one mouse of group C4 (control collagen gel). (B) Sample from one mouse of group R4 (ribose crosslinked collagen gel). (C) Sample from one mouse of group C4. (D) Sample from one mouse of group M4 (magnetically aligned collagen gel). Green, rPGP 9.5; red, mMBP; and blue, gCol I for A and B and s100 for C and D. ⫻400.

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FIG. 3. Laser scanning confocal micrographs of transverse sections of the nerve cables regenerated across a 6-mm gap using collagen tubes prefilled with collagen gel at 60 dpo. (A) Sample from one mouse of group C6 (control collagen gel). (B) Sample from one mouse of group R6 (ribose crosslinked collagen gel). (C and D) Samples from one mouse of group M6 (magnetically aligned collagen gel). Green, rPGP 9.5; red, mMBP; and blue, gCol I in A, B, and C and s100 in D. ⫻400.

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TABLE 2 Proportions of Mice with Regenerated Nerves, as Judged by the Presence of Myelinated Nerve Fibers Regenerated at Midtube and Distal to the Tube after 30 and 60 dpo No. reg/total at 30 dpo

No. reg/total at 60 dpo

Group

Mid

Distal

Mid

Distal

S4 C4 C6 M4 M6 R4 R6 MR4 MR6

1/3 3/5 0/5a,c 3/4 0/4a 0/3a 0/4a 0/3a 0/4a

0/3 3/5 0/5c 0/4 0/4 0/3 0/4 0/3 0/4

— 3/6 3/6 4/4 4/4 1/3 0/4a,b 1/4a,b 2/4

— 2/6b 1/6a,b 3/3 d 4/4 1/3 0/4a,b 1/4a,b 1/4a,b

Note. P ⬍ 0.05 vs aM4, bM6, and cC4 (␹2 test). d One sample was unavailable for analysis.

groups with a 4-mm gap, the number of myelinated fibers was significantly higher in group M6 than in groups C6 and MR6 (Table 3). These remarks also apply distal to the tube, where the values for crosssectional area ranged between 72 and 126% of the normal sciatic nerve area; the number of myelinated fibers was quite variable, with some mice showing lower numbers than at midtube and a few higher numbers, indicative of distal sprouting in these cases. The regenerated fibers were in all cases of smaller diameter and had thinner myelin sheaths than in control nerves. The distribution of axonal diameters was unimodal, with peak frequencies midtube between 1 and 2 µm at 30 dpo and between 1 and 3 µm at 60 dpo. Distal to the tube, the distribution was more skewed to smaller values in all groups, indicating a maturation gradient of the regenerated axons. DISCUSSION

Our results demonstrate that filling the collagen tube with magnetically aligned type I collagen gel

FIG. 2. Light microscopy of transverse sections of the regenerated nerves at 60 dpo. (A) Section at the middle of the collagen tube in one mouse of group M6 (magnetically aligned collagen gel, 6 mm gap); ⫻260. (B) Detail of the regenerated fascicles in the same nerve. (C) Section at the distal nerve in one mouse of group C4 (control collagen gel, 4-mm gap). (D) Section at the distal nerve in one mouse of group M6. ⫻1380.

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TABLE 3 Cross-sectional Area (NA, in 10 mm2 ) and Number of Myelinated Fibers (MF) in the Regenerated Nerve Cable at Midtube and Distal to the Tube at 30 and 60 dpo 30 dpo Group

Parameter

Unoperated

NA MF NA MF NA MF NA MF NA MF NA MF NA MF NA MF NA MF NA MF

S4 C4 C6 M4 M6 R4 R6 MR4 MR6

Midtube

0.21 ⫾ 0.21 218 ⫾ 218 0.65 ⫾ 0.31 1041 ⫾ 533 0 0 0.25 ⫾ 0.09 233 ⫾ 100 0 0 0 0 0 0 0 0 0 0

60 dpo Distal

Midtube

Distal

0.36 ⫾ 0.21 651 ⫾ 383 0.07 ⫾ 0.05a 112 ⫾ 78a 0.26 ⫾ 0.10 520 ⫾ 188 0.55 ⫾ 0.13 1266 ⫾ 789 0.08 ⫾ 0.08 148 ⫾ 148 0 0 0.35 ⫾ 0.35 326 ⫾ 326 0.22 ⫾ 0.22 178 ⫾ 160a

0.54 ⫾ 0.34 675 ⫾ 606 0.20 ⫾ 0.21a 41 ⫾ 41a 0.96 ⫾ 0.40 377 ⫾ 167 1.56 ⫾ 0.18 1171 ⫾ 649 0.37 ⫾ 0.37 298 ⫾ 298 0 0 0.42 ⫾ 0.42 489 ⫾ 489 0.52 ⫾ 0.52 30 ⫾ 30a

1.64 ⫾ 0.11 4158 ⫾ 170 0.92 ⫾ 0.30 486 ⫾ 234 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Note. Values are expressed as means ⫾ SEM. a P ⬍ 0.05 vs group M6. NA was considered 0 if the sample did not contain regenerated nerve fibers.

(patent pending) significantly improved nerve regeneration over tubes filled with control collagen gel. The beneficial effect was more obvious with 6-mm gaps, where regeneration usually fails, than with 4-mm gaps where regeneration usually occurs with tubulization in the mouse model (4, 10, 13). Although the number of regenerated myelinated axons was quite variable among the mice, as expected for a relatively long gap of 6 mm, all the mice examined in the group using magnetically aligned collagen gel (Table 2; M6, four of four) possessed regenerating myelinated fibers distal to the TABLE 4 Morphometric Results of the Regenerated Myelinated Fibers at Midtube (M) and Distal to the Tube (D) at 30 and 60 dpo

Groups Level C4 C6 M4 M6

M D M D M D M D

30 dpo axon diameter

Myelin thick

60 dpo axon diameter

1.56 ⫾ 0.02 0.52 ⫾ 0.01 2.04 ⫾ 0.02 1.49 ⫾ 0.03 0.63 ⫾ 0.01 1.83 ⫾ 0.03 1.81 ⫾ 0.03 1.41 ⫾ 0.09 1.34 ⫾ 0.03 0.42 ⫾ 0.01 1.68 ⫾ 0.03 1.24 ⫾ 0.03 1.93 ⫾ 0.02 1.74 ⫾ 0.02

Note. Values (in µm) are expressed as means ⫾ SEM.

Myelin thick 0.68 ⫾ 0.01 0.71 ⫾ 0.01 0.66 ⫾ 0.01 0.60 ⫾ 0.02 0.66 ⫾ 0.01 0.61 ⫾ 0.01 0.69 ⫾ 0.01 0.67 ⫾ 0.01

tube, our definition of regeneration in this study. This was significant (P ⬍ 0.05) compared to the control group (Table 2; C6, one of six). Interestingly, there was little improvement in the proportion of mice exhibiting regeneration in C4 or C6 going from 30 to 60 dpo, but a complete increase in M4 and M6 (Table 2). In addition, regenerating nerves in the magnetically aligned collagen gel exhibited better fascicular organization (Figs. 2 and 3); the collagen gel remnants appeared intermingled among the regenerated axons and surrounding the fascicles, instead of appearing as dense acellular regions as frequently seen in nonregenerates. These results were presumably due to contact guidance of regenerating axons and nonneuronal cells, with Schwann cells being considered key in this respect (12, 25, 36), as was observed in the in vitro correlate of entubulation repair by Dubey et al. (7). This study could not distinguish whether directed invasion of nonneuronal cells or directed axonal growth was the driving force for dramatically improving regeneration across the 6-mm gap. By crosslinking the collagen gel, we attempted to increase its structural stability by reducing its rate of resorption. However, the ribosylation conditions used for crosslinking in this study decreased the likelihood of nerve regeneration (Table 2) and the number of regenerated fibers when it occurred (Table 3). It is known that intratubular exogenous gels, even if containing neuro-

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trophic factors, may impair the regeneration process by physically impeding the diffusion of soluble factors, the migration of nonneuronal cells, or the growth of axons. Previous reports have shown the dependence of nerve regeneration upon the density, concentration, and viscosity of the gels (16, 38, 41). More recently, diluted collagen gels were found to improve regeneration and functional reinnervation over more concentrated gels filling silicone tubes (17). In the groups of mice treated with ribose crosslinked collagen gel we observed that the gel remained for a longer time and appeared densely segregated, as reported when using higher concentrations of collagen gel (38). Collagenolysis has been shown to be important for neurite extension within collagen gel (30). The inhibiting effect could be compensated, at least in part, by magnetic alignment (compare MR with R groups in Table 2). These results must be considered in view of the fact that we achieved relatively poor success of regeneration in 4-mm gaps, with smaller nerve cables and fewer numbers of regenerated myelinated fibers, than expected in comparison to previous studies (1, 10, 21, 37, 39, 42), although in some of these studies the time allowed to evaluate regeneration was longer (up to 5 months). With medium length gaps, such as 4 mm in the mouse and 4–8 mm in the rat, tubulization is usually successful with permanent or resorbable tube materials, provided that the physical characteristics of the tube are adequate and do not occlude the lumen (4, 26). However, bioresorbable tubes that resorb too quickly may not provide an appropriate milieu for nerve regeneration and maturation during the time when the nerve fibers are more susceptible to compression and extraneural factors. If the tube resorbes too quickly, the tube wall may swell, the lumen may be distorted, and fibrous tissue may form inside the tube and impair further regeneration (6, 13). Because collagen fibrillogenesis is prevented in acidic environments, it was necessary to manufacture the collagen tubes having a pH above the isoelectric point for collagen fibrillogenesis, for this study, in order to form collagen gels within the tube lumen. This was achieved by using a buffered liquidphase crosslinking process instead of the standard vapor phase procedure. This change in the manufacturing procedure is the most likely reason for the relatively rapid resorption rate of the tubes in this study. In previous studies, the resorption rate of the collagen tubes manufactured with standard methods was lower, with tube remnants found around the regenerated nerve as long as 5 months after implantation (29). Use of collagen tubes with a slower resorption rate have yielded levels of regeneration comparable to those achieved by direct suture or an autograft in very short gaps in the monkey (2, 3). On the contrary, entubulation repair is considered to be useless when attempting to repair long gaps. Previ-

ous studies have indicated that the limit for nerve regeneration through synthetic tubes is about a 6-mm gap for the mouse sciatic nerve (4, 10, 13). However, bioresorbable collagen tubes allowed regeneration across a 6-mm gap in more mice and with slightly more axons compared to silicone tubes, although regeneration by our criterion only occurred in 50% of the cases (10, 29). Such results indicate that both tube permeability and tube durability influence the likelihood of regeneration across long gaps (1, 6, 14, 24). By filling the tube with magnetically aligned collagen gel we have achieved a 100% (four of four) success of regeneration across a 6-mm gap, and the number of regenerated axons was similar both midtube and distal to the tube, indicating an optimal axonal outgrowth through the gel. In theory, magnetic alignment of collagen gel is independent of the tube length, so that regeneration might occur for larger gaps than 6 mm in this model (in fact, tubes used in the M6 group were cut from the same magnetically aligned 10-cm section). It is also effective for tubes as large as 4 mm i.d. (7), which would be relevant for entubulation repair in primates. Stronger gel alignment should be realized in higher strength magnetic fields leading to further improvement in regeneration. Further investigations in this model will be designed to use different collagen concentrations and collagen tubes with a slower rate of resorption and to exploit the potential of cell and particle entrapment within collagen gel for controlled delivery of neurotrophic factors so that axonal growth will be stimulated as well as guided by the aligned collagen gel. Entrapment merely requires adding the cells (e.g., Schwann cells) or particles (e.g., controlled release polymer beads) to the neutralized collagen solution just prior to warming. This fundamentally distinguishes our use of magnetically aligned collagen gel from alternative approaches which have sought to exploit contact guidance to improve peripheral nerve regeneration (e.g., 31, 45). ACKNOWLEDGMENTS This study was supported by an NIH Public Health Service STTR Phase I Grant 95-4 to Integra LifeSciences Corp. and a NATO grant CRG971509 to X.N. We thank Kathy Wabner and Eric Hazen for technical assistance and the Center for Magnetic Resonance Research, University of Minnesota, for magnet use.

REFERENCES 1. Aebischer, P., V. Guenard, S. R. Winn, R. F. Valentini, and P. M., Galletti. 1988. Blind-ended semipermeable guidance channels support peripheral nerve regeneration in the absence of distal nerve stump. Brain Res. 454: 179–187. 2. Archibald, S. J., C. Krarup, J. Shefner, S-T. Li, and R. D. Madison. 1991. A collagen-based nerve guide conduit for peripheral nerve repair: An electrophysiological study of nerve regeneration in rodents and nonhuman primates. J. Comp. Neurol. 306: 685–696.

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