International Journal of Oral & Maxillofacial Surgery
Volume 39, Issue 7 , Pages 705-712, July 2010

Cyanoacrylate in nerve repair: transient cytotoxic effect

  • T. Landegren

      Affiliations

    • Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset, Section of Hand Surgery, Sweden
    • Corresponding Author InformationAddress: Thomas Landegren, Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset, Section of Hand Surgery, SE-118 83 Södersjukhuset, Stockholm, Sweden. Tel.: +46 86 16 20 41; fax: +46 884 65 99.
    • ,
  • M. Risling

      Affiliations

    • Department of Neuroscience, Experimental Traumatology Unit, Retzius Laboratory, Karolinska Institutet, Sweden
    • ,
  • J.K.E. Persson

      Affiliations

    • Karolinska Institutet, Department of Clinical Neuroscience, Karolinska Universitetssjukhuset Solna, Sweden
    • ,
  • A. Sondén

      Affiliations

    • Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset, Section of Surgery, Stockholm, Sweden

Accepted 16 March 2010. published online 03 May 2010.

Article Outline

Abstract 

Cyanoacrylate adhesive has been suggested as an alternative to suturing when repairing severed peripheral nerves. The authors examined the cytotoxic effect of ethyl-cyanoacrylate on the human neuroblastoma cell line SH-SY5Y and compared it with the effects of butyl-cyanoacrylate (Histoacryl®), an adhesive approved for skin closure. Ethyl-cyanoacrylate or butyl-cyanoacrylate was applied in confluent SH-SY5Y cultures. Immediately, at 24h and at 7, 14, 21 and 28 days, cultures were photographed and analysed digitally. At corresponding intervals, cell death was quantified using a 51Cr release assay. In cultures exposed to ethyl-cyanoacrylate or butyl-cyanoacrylate, cell death was observed predominantly in conjunction with the adhesive, causing a halo devoid of cells. Surviving cells showed neurodegenerative properties with loss of neuritis and reduction of body size up to 3 days post exposure. The inhibition halo diminished over time in both groups and at 28 days cells reached the margin of the adhesive in the ethyl-cyanoacrylate group. 51Cr assay indicated significant cell death in exposed cultures, which rapidly decreased during the first 14 days. No significant differences were found between the adhesives. This study demonstrates that ethyl-cyanoacrylate and butyl-cyanoacrylate have a transient cytotoxic effect, which may explain the promising results when using cyanoacrylate for nerve repair.

Keywords: nerve repair, cyanoacrylate, synthetic adhesive, cytotoxic, neuroblastoma cell, SH-SY5Y

 

Microsurgical suturing is the standard technique for repairing transected peripheral nerves. The technique requires surgical excellence and may be difficult to apply when surgical access is limited. Microsurgical suturing does not result in perfect coaptation of the internal fascicular structures, which hampers the growth of regenerating axons and, ultimately, complete nerve function recovery4. There is a need for complementary surgical techniques that provide rapid and reliable primary repair of transected nerves.

Different strategies have been tried to readapt the nerve endings using tissue adhesive. Fibrin sealants are the most successful surgical adhesives and sealants, but they have limitations in their mechanical and biological properties. In peripheral nerve repair, the primary disadvantage is the low cohesive strength, which makes the anastomosis insecure during the healing phase18. To avoid gaps at the repair site, a nerve anastomosis made with standard fibrin adhesive is often complemented, in practice, with microsutures. Other shortcomings preventing the acceptance of fibrin glue as a surgical tissue adhesive are: short-term persistence (<2 weeks) in vivo; risk of infectious blood-borne disease transmission; and low viscosity prior to polymerization with thrombin, which makes the adhesive difficult to apply14, 26, 32.

Cyanoacrylates (CAs) are synthetic adhesives that polymerize rapidly on contact with water or blood. The adhesives are inexpensive, relatively easy to apply, do not carry any risk of viral transmission and have sufficient strength to maintain a nerve anastomosis, even under tension8, 16. The cytotoxicity of CA adhesives in surgical applications is debated. When used as a tissue adhesive, CA has been shown to induce a stronger tissue reaction than non-resorbable sutures, resulting in a more pronounced foreign-body inflammatory reaction, while others have found it causes tissue necrosis in vivo15, 28. In some reports the inflammatory reaction around the repair site has been shown to be harmful32 and to cause a focal hindrance to the recreation of tissue25. In other studies, the inflammatory reaction has been shown to require active communication between the injured tissue and the recruited macrophages needed for degradation and tissue regrowth and, consequently, to be an integral part of the healing process20, 23.

The advantages of CA as a tissue adhesive are described by authors in a variety of medical fields. In microsurgery, successful use of CA for microneural and microvascular anastomoses has been reported3, 8. The authors have compared ethyl-cyanoacrylate (ECA) with microsurgical suturing for microneural anastomosis in two different studies. The two techniques gave rise to equivalent recovery of motor and sensory conduction velocities as well as motor nerve action potentials when measured 6 months after surgery16. 7 days after surgery, the adhesive stimulated an increased appearance of macrophages around the repair site, which could explain the accelerated Wallerian degeneration distal to the seam after nerve repair with ECA17. The results are promising and have prompted the authors to investigate the toxicity of ECA further. They have used SH-SY5Y, a widely employed neuronal cell line for studying neuronal toxicity and neurocytoprotection7. For comparison, the cytotoxicity of Histoacryl® (butyl-2-cyanoacrylate) was studied, this is considered to be one of the least cytotoxic cyanoacrylate derivatives and it is one of the most frequently used CAs in general surgery.

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Material and methods 

SH-SY5Y culture and incubation conditions 

SH-SY5Y cells, a human neuroblastoma cell line, obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% foetal bovine serum and 1% 200mM l-glutamine (National Veterinary Institute of Sweden, Uppsala, Sweden) and seeded in T 75cm2 cell culture flasks (Nunc, Roskilde, Denmark). The culture medium was changed the following day and thereafter twice a week. When confluent, cultures were trypsinized (Trypsin 0.5% EDTA, Invitrogen Life Technologies, Paisley, UK), seeded in 24-well cell culture dishes and maintained in a 5% CO2/95% humidified air atmosphere at 37°C. Plated cells were allowed to grow overnight prior to the treatment. Confluent cultures were used for experiments.

Cyanoacrylate exposure in culture 

Cell cultures were exposed to the adhesive by adding 0.1μl of ethyl- or butyl-cyanoacrylate to the centre of each well after the medium had been removed. The ECA used in this study is commercially available (Evobond®, Tong Shen Enterprise Co., Ltd., Taiwan), whereas the butyl-cyanoacrylate (BCA) is a tissue adhesive produced for sutureless skin closure (Histoacryl®, B. Braun Surgical GmbH, D-34209 Melsungen, Germany). The adhesive was set to polymerize on the surface of the confluent cell culture for 5s, whereupon 500μl of fresh medium was added to the well without letting the cell culture run dry. The culture medium was changed twice a week during the whole observation period. Eight wells in each 24-well dish were exposed to either ECA or BCA, whereas eight wells were unexposed to adhesive and used as controls.

Evaluation of the halo devoid of cells 

After application, 24h were allowed for full polymerization of each droplet of adhesive to occur. Cell cultures were followed up to 28 days (n=8 wells in each group for each time point). At 24h and 7, 14, 21 and 28 days after incubation, cultures were examined by phase contrast microscopy (Nikon Diaphot 300, Nikon Corp., Tokyo, Japan). A calibration grid was photographed at the corresponding magnification.

The microscopic images were captured with a digital camera (Nikon CoolPix 990, Nikon, Tokyo, Japan). All micrographs were displayed on a computer monitor and analysed using morphometric software (ImageJ). The outer edge of the cell-free halo around the adhesive (see “Results”) was approximated to a circle and the distance from the adhesive border to the cell layer margin was measured. For each well, measurements were done in the four quadrants perpendicular to each other and mean values were calculated. In order to confirm the results obtained from cultures followed throughout the study period, complementary measurements were carried out in cultures intended for another analysis (i.e. adhesive-induced cell death measured using a 51Cr assay; see below). These measurements were carried out at 24h and 7 and 28 days.

Immunofluorescence staining and analysis of cell morphology 

A cytoskeletal assessment was made on samples exposed to ECA or BCA as well as time-matching controls after 24h and 3 and 7 days. The staining method for tubulin has been described in detail elsewhere31. Briefly, cells were fixed with methanol for 5min at −20°C and permeabilized with acetone at −20°C for 10s. Cells were incubated with primary antibodies to tubulin (BioGenex Laboratories, Mainz, Germany) (dilution 1:20) and subsequently with FITC-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA) (dilution 1:20). The specimens (n=8 wells in each group for each time point) were examined by fluorescence microscopy (Nikon Eclipse E600, Nikon, Tokyo, Japan) and photographed with a digital camera (Nikon Digital Sight DS-U1). Images were captured from each culture in four different quadrants perpendicular to each other (total of four images per cell culture) from the area immediately outside the cell-free halo (see “Results”). Five separately located cells (displayed on a computer monitor) were selected randomly from each micrograph and cell sizes were analysed using morphometric software (ImageJ® 1.33u, National Institutes of Health, USA) at 24h and 7 days post exposure. Cell bodies and neurites were analysed descriptively with respect to shape and texture at all time points.

Detection of cell death with 51Cr 

Cytotoxicity designating necrotic cell death was assessed by the release of 51Cr as described in detail elsewhere5, 9. Briefly, SH-SY5Y cells were grown to confluence in five 24-well dishes. Eight cell cultures in each 24-well dish were exposed to either ECA or BCA while eight wells were left unexposed to adhesive and used as controls. At 7, 14, 21 or 28 days the cultured cells (adhesive exposed), as well as time-matching controls, were labelled with 51Cr (Perkin Elmer, Boston, MA, USA) for 24h. Exceptions were cells analysed 24h after exposure to adhesive, which were labelled prior to exposure. After labelling, cultures were washed and left in the cell incubator for 24h. Supernatants were carefully aspirated and the remaining SH-SY5Y in each well was lysed with 500μl 1M NH4OH. The radioactivity of the supernatants and the remaining SH-SY5Y was measured in a gamma counter (Packard Cobra II from CIAB, Stockholm, Sweden). Cell death, expressed as percent of 51Cr release, was calculated as follows:

where A stands for mean counts per min in the supernatant and B stands for mean counts per min from the remaining SH-SY5Ys. Finally, the specific 51Cr release (i.e. 51Cr release due to the adhesive) was obtained by subtracting the mean 51Cr release in controls from the mean 51Cr release in any exposed well.

Statistical analysis 

Data are presented as the mean and standard deviation (SD). Differences within and between groups were analysed using a Mann–Whitney test, one-way ANOVA or repeated measures ANOVA when appropriate. The analyses were included in the statistical package GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, USA). Differences were considered to be significant at P<0.05.

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Results 

Adhesive-induced halo devoid of cells 

After incubation for 24h there was evidence of a cytotoxic milieu in conjunction with the adhesive border that produced a halo without cells. Although not significant, the halo in the ECA group was initially wider than that in the BCA group. The zone without cells around the adhesive ranged from 700 to 1300μm. For up to 7 days, these cell-free zones were significantly enlarged, whereupon they decreased significantly between 7 and 14 days with both adhesives (Fig. 1, Fig. 2). Between 14 and 28 days the halos continued to diminish continuously, and by 28 days some cells reached the margin of the adhesive in several wells and the zone was barely measurable in the ECA group (Fig. 1, Fig. 2). The cell-free zones in the BCA group were significantly wider than those of the ECA group at the end of the study period (Fig. 2). Complementary measurements carried out at 24h and 7 and 28 days in cultures used for the 51Cr assay corresponded with these estimates (data not shown).

  • View full-size image.
  • Fig. 1. 

    Photomicrographs showing SH-SY5Y cell cultures 7 days (A and B), 14 days (C and D) and 28 days (E and F) after exposure to 0.1μl ECA. The width of the halo devoid of cells (1) between the adhesive (2) and the margin of the confluent cell layer (3) was measured at 24h and 7, 14, 21 and 28 days after exposure to the adhesive. Over time the halo became narrower and after 28 days (i.e. at the end of the measuring period) cells were growing at the margin of the adhesive (4). Arrows indicate unaffected cells in the intermediate area between confluent culture and the zone devoid of cells (B and D). Scale bars: A and C, 1000μm; B, D and E, 100μm; F, 50μm.

  • View full-size image.
  • Fig. 2. 

    Exposure to ECA or BCA gave rise to a halo devoid of SH-SY5Y cells around the adhesive which grew significantly larger up to 7 days whereupon it decreased significantly in size to the end of the study period (P<0.001). Mean values with standard deviation of the measured halo width are given (n=8 cultures in each adhesive group for each time point). *P<0.05.

Adhesive-induced changes in cell morphology 

After 24h of incubation with either of the two adhesives, loss of neurites, cell shape changes and texture degeneration were seen in all cultures (Fig. 3). Groups of cells formed clusters, which were less evident at increased distances from the adhesive. These changes were not seen in controls. Cells located in the area close to the halo devoid of cells (see above) exhibited a significantly smaller size compared with cells in control cultures (Fig. 4). No difference in cell size could be detected between the two adhesive groups. After 3 days, adhesive-exposed cells regained shape and texture. Neurite outgrowth was frequently observed, but the neurites of adhesive-exposed cells were rougher and of shorter extension than those in controls (not shown). At 7 days, adhesive-exposed cells could not be distinguished from controls with respect to neurites, cell shape, texture and size (Fig. 3, Fig. 4). No differences in cell morphology, descriptive or quantitative, were observed between the two adhesive groups at any time point.

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  • Fig. 3. 

    SH-SY5Y cells labelled with tubulin antibodies following exposure to ECA (A and B) or BCA (C and D) and controls (E and F). Immunofluorescence photomicrographs were taken immediately outside the cell-free halo (A and C) 24h or (B and D) 7 days after exposure to the adhesive and at random spots at corresponding time delays in controls. Note the loss of neurites, the change in cell shapes and cell sizes and texture degeneration 24h after incubation (A and C). At 7 days, exposed cells had regained their normal appearance (B and D), including neurites (arrows), and could not be distinguished from controls (F). Scale bar=20μm.

  • View full-size image.
  • Fig. 4. 

    The sizes of SH-SY5Y cell bodies after exposure to ECA or BCA, as well as of time-matching controls (n=8), were measured at 24h and at 7 days after incubation. In the area close to the halo devoid of cells, 20 random SH-SY5Y cells in each culture were measured (total of 160 cells in each group for each time point). Values are means and standard deviation. **P<0.001.

Adhesive-induced cell death measured with 51Cr 

The two adhesives induced rapid cell death, which decreased during the first 2 weeks after exposure (Fig. 5). The relative cell death induced by the adhesive declined from 11.3±6.0% at 24h to 1.2±1.1% at 14 days in the ECA group (P<0.01). The corresponding values for the BCA group were 15.6±9.2% and 0.9±1.4% (P<0.01). From the second to the fourth week, cell death decreased continuously, but at a slower pace. At 28 days no significant differences in toxicity were seen between exposed wells and controls. No significant difference in cell toxicity between the two adhesives was seen at any time point.

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  • Fig. 5. 

    Cell death caused by ECA or BCA assessed as the release of 51Cr. Cytotoxicity in both groups decreased rapidly between 24h and 14 days (*P<0.01, n=8 in each adhesive group for each time point). Values in the figure (means and standard deviation) were calculated by subtracting cell death values in controls from values in exposed cultures. No significant differences between groups were seen.

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Discussion 

Anastomosis with epineural microsutures is the gold standard for the repair of an injured peripheral nerve19, but this technique further traumatizes the nerve tissue24, takes a considerably long time21 and may be difficult to perform when surgical access is limited. It results in the formation of suture granulomas, which obstruct myelin regeneration and sprouting of axons25, factors that are thought to impair nerve regeneration13. There is a need for complementary surgical techniques that provide rapid and reliable primary repair of transected nerves.

In a recently published study, the authors compared local tissue reactions around the repair site after conventional microsuturing or repair using CA. In an effort to maximize the tensile strength and minimize the histotoxicity, Evobond®, a commercially available ECA with a minimum of additives, was chosen. The authors found a significant increase in macrophages at the repair site 7 days after repair with ECA compared with conventional microsuturing. Distal to the repair site, a significant concurrent reduction of neurofilaments was observed in the ECA group. Proximal to the seam, no evident differences between the two repair methods with respect to the number or distribution of neurofilaments could be seen. These findings, in combination with observations of nerve regeneration, starting in the proximal stump, with path-finding axons beginning to penetrate the ECA barrier, indicated that no acute axonal degeneration caused by the adhesive had taken place proximal to the seam17. In a parallel study, the authors demonstrated an improved motor, and a comparable sensory, recovery after experimental repair of a transected peripheral nerve with synthetic adhesive in comparison with microsutures16.

These results were contradictory to previous published reports32 claiming the explicit disadvantage of using CA as a tissue adhesive in peripheral nerve repair on account of its toxicity. To increase their knowledge of ECA cytotoxicity, the authors set up the current study in which the behaviour of human nerve cells after direct exposure to ECA or BCA was studied. The comparison with BCA was chosen because BCA is a widely used adhesive for various medical applications and is approved by the FDA for use in certain forms of neurosurgery1. Using a 51Cr release assay, the authors demonstrated a significant, but transient, cytotoxic effect of ECA and BCA immediately after exposure to the nerve cells. This is in accordance with a study by Thumwanit & Kedjarune27, which investigated the effect of a mixture of ethyl- and methyl-cyanoacrylate on cultured oral fibroblasts and showed a considerable reduction in cytotoxicity during the first 24h, followed by a continuous decrease in the inhibitory effect during the following 2 weeks. In the present model, maximum cytotoxicity was observed at 24h and significant cytotoxicity was seen up to 7 days after exposure; it was almost negligible 2 weeks after exposure.

In the present study, cytotoxicity was also evaluated with a morphometric assay measuring the halo devoid of cells around the adhesive. During the first week after exposure the halo increased, but gradually diminished during the following 3 weeks, which, in contrast to the 51Cr release method, indicated maximum cytotoxicity after 1 week. This delay may be explained by the fact that the 51Cr assay measures cytotoxicity at a specific time point, while the halo is also a result of prior cytotoxicity. At the end of the observation period, cells in the ECA group were found to reach the margin of the adhesive, evidently growing independently of the presence of the adhesive. In the BCA group, a small but visible distance between the adhesive and cells remained throughout the study period, indicating a persistent cytotoxic effect. This could not be confirmed by the 51Cr method. Although the 51Cr release method is known to be highly sensitive6, it is difficult not to ascribe this phenomenon to a sensitivity problem. Since Thumwanit & Kedjarune27 did not follow the cell line for more than 2 weeks, no conclusion about the cytotoxicity beyond this time point can be drawn from their study.

Basal cytotoxicity has also been evaluated in vitro by studying neurodegenerative properties in SH-SY5Y neuroblastoma cells during exposure to toxic substances12, 22. In the authors’ study a complete degeneration of neuritis and decreased cell size was observed 24h after exposure to adhesive. Signs of recuperation were seen after 3 days and, at 7 days after continuous exposure to the adhesive, a total recovery of the neuroblastoma cells, with a morphology equal to that of controls, could be demonstrated. No differences between the ECA and BCA groups were seen. These findings are in line with the results of the 51Cr and morphometric assays and further support the conclusion that the cytotoxic effect of CA is of a temporary nature10.

A comparative cytotoxicity study of BCA and ECA has also been conducted by de Azevedo et al.11, who applied polymerized adhesive to cultured fibroblasts (NIH 3T3 cells). In their study, the assays were divided into two groups: an immediate (short-term) response group and a group exhibiting long-term survival measured with respect to the retention of the self-renewal capacity of the cells. In the short-term experiment (up to 24h), lower cell viability was observed in the ECA group, but in the long-term experiment (up to 7 days) the group presented continuous and progressive cell growth, reaching the viability levels of the control cultures at the end of the experimental period. Cultures treated with BCA exhibited a drop in viability levels after day 3 in culture, and viability levels were significantly lower compared with ECA and control cultures. Their results are in accord with those of the authors showing that the cytotoxicity of ECA, although in close contact with cultured nerve cells, decreases during the first 7 days and thereafter does not constrain cell growth. The greater biocompatibility of ECA, compared with BCA, in the long-term experiment also supports the authors’ finding that BCA has a small but persistent cytotoxic effect, at least up to 28 days.

In this study, the authors used a cytotoxicity model in which cells were in direct contact with the adhesive during polymerization. Although there are similar models with cells in contact with the adhesive during polymerization or when polymerization is completed10, 11, earlier in vitro models generally focused on the cytotoxicity of the degradation products of the adhesives30. In addition to the cytotoxicity, the polymerization of CA is exothermic, and the release of heat has the potential to cause cellular damage and tissue necrosis2. Hence, the cell death measured in the authors’ experimental model may be a consequence of cytotoxicity as well as heat production, which, in the authors’ opinion, better mimics the in vivo situation. The heat of polymerization depends, among other things, on factors such as the polymerization rate, and is strongly influenced by the amount of adhesive instilled2. In practice, when repairing transected nerves the cross-sections of the injured nerve endings are seldom or never oriented horizontally. CA homologues with longer ester chains (e.g. BCA.) have low viscosity and tend to contaminate the surrounding tissues when applied to non-horizontal surfaces. CA homologues with shorter chains such as ECA have high viscosity and do not spread as readily but form a droplet of liquid29. These spreading characteristics allow precise application of the adhesive to a surface, whether vertically or obliquely oriented, without spreading in the wound. A logical consequence of this is that a minimal amount of the adhesive, just enough to form a solidified thin layer of liquid, can be applied between the two nerve endings. Cytotoxicity is proportional to the dose used2 and the cellular damage due to the exothermic reaction can be minimized.

The purpose of this study was to investigate the toxicity of ECA and compare the results with BCA, a widely employed adhesive for various medical uses. Both adhesives show evidence of transient cytotoxicity. In the light of previous experimental studies and according to this in vitro cell culture study, the authors conclude that there is no toxicological evidence indicating why ECA could not be used instead of BCA in peripheral nerve repair when a synthetic tissue adhesive is preferable.

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Competing interests 

None declared.

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Funding 

The present study was supported by the Swedish Defence Research Agency Innovation Foundation and Karolinska Institutet, Stockholm, Sweden.

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Ethical approval 

Not required.

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Acknowledgements 

The excellent technical assistance given by Ms. Elisabeth Malm is gratefully acknowledged.

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PII: S0901-5027(10)00101-3

doi:10.1016/j.ijom.2010.03.008

International Journal of Oral & Maxillofacial Surgery
Volume 39, Issue 7 , Pages 705-712, July 2010

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