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Ann Thorac Surg 2005;79:1522-1528
© 2005 The Society of Thoracic Surgeons
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Research Center for Traumatology AUVA, Vienna, Austria
Accepted for publication November 2, 2004.
* Address reprint requests to Mr Fürst, Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstrasse 13, A-1200 Vienna, Austria (E-mail: office{at}lbitrauma.org).
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Abstract |
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METHODS: BioGlue was prepared according to the product insert, allowed to polymerize, and then overlaid with saline solution. The supernatant was analyzed for its content of glutaraldehyde. The cytotoxic effect of BioGlue was evaluated by adding the supernatants to either cultured human embryo fibroblasts (MRC5) or mouse myoblasts (C2C12). In vivo toxicity was assessed on three different tissues by applying BioGlue onto a partial lung resection, a liver abrasion, or an intact abdominal aorta in rabbits. Tissue samples were histologically evaluated 2 and 7 days after application.
RESULTS: Saline supernatants from polymerized BioGlue contained 100 to 200 µg/mL glutaraldehyde and were cytotoxic to both cell lines tested. Application of BioGlue to lung and liver tissue evoked serious adverse effects consisting of high-grade inflammation, edema, and toxic necrosis. Intact aortic tissue showed only low-grade or medium-grade inflammation.
CONCLUSIONS: Polymerized BioGlue releases amounts of glutaraldehyde that are capable of inducing cytotoxic effects both in vitro and in vivo. Use of BioGlue should be restricted to the aortic dissection procedure, as other tissues are sensitive to the amounts of glutaraldehyde released from the glue.
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Introduction |
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The relatively new adhesive, BioGlue [2], is composed of bovine serum albumin (45% solution) and glutaraldehyde (10% solution). This product is currently being used as an adjunct for securing hemostasis at cardiovascular anastomoses and for repair of acute proximal aortic dissection [3–5]. Instead of formaldehyde, as is used in gelatin-resorcinol-formaldehyde glue, BioGlue uses glutaraldehyde, which is thought to be less histotoxic. Furthermore, superior bonding and sealing capabilities to tissues as well as to synthetic graft materials allowing air-tight and blood-tight repair [4, 6] are claimed for BioGlue. This strong adherence is achieved mainly by glutaraldehyde, which is a highly reactive chemical that covalently cross-links the albumin molecules to each other and, on application, to the tissue proteins at the repair site.
BioGlue was approved by the US Food and Drug Administration in 1999 under a Humanitarian Device Exemption for use as an adjunct to sutures and staples in the repair of acute aortic dissections. The US Food and Drug Administration approval, however, includes warning statements that application of BioGlue to exposed tissues may cause serious nerve injury, coagulation necrosis that extends into the myocardium, and enhanced propensity for mineralization [7]. Furthermore, warnings have been raised against its use in pediatric patients, and inflammatory responses were reported [8, 9]. Application of BioGlue in a pig study caused nerve injury and paralysis of the diaphragm [10], and special procedures to protect the myocardium during application of BioGlue were suggested [11].
In addition to the adverse effects described above, unreacted, volatile glutaraldehyde is also a risk factor for staff involved in preparation and application of BioGlue. Glutaraldehyde is classified as a toxic substance (EU TN, UN 6), and repeated exposure to glutaraldehyde causes irritation of eye, nose, throat, or skin resulting in dermatitis and asthma [12].
Glutaraldehyde is widely used as a tissue fixative for histology. It has been suggested that the same properties that make it a good tissue fixative may account for the adverse effects noted in vivo when BioGlue is used clinically. The aim of the present study was to investigate both the potential release of (free) glutaraldehyde from the polymerized glue and its toxic potential in vitro and in vivo. In addition to studies on cell cultures, we assessed the effects of BioGlue applied to three different rabbit models: partial lung resection, liver abrasion, and aortic application models. In the lung and liver models a fibrin sealant was used as control.
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Material and Methods |
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In Vitro Application of BioGlue
Cellulose sheets were moistened (two layers, 3 x 2 cm, 200 µL of saline solution) and prewarmed to 37°C to simulate a tissue surface before application of BioGlue. According to the manufacturer's instructions, the mixing device was primed by expelling a narrow ribbon of approximately 3 cm length before application. This should ensure optimal mixing of the adhesive components. Similar amounts (half a step with the application device; approximately 1 mL) of both material used for priming the device and properly mixed BioGlue were applied consecutively onto the moist and prewarmed cellulose sheets in 4 separate Petri dishes. The glue was allowed to polymerize for 2 minutes, overlaid with 5 mL of saline solution, and incubated for 1 minute. Supernatants were then harvested into cryotubes in aliquots of 1 mL, snap frozen, and stored at –20°C.
Quantification of Glutaraldehyde in Supernatants
Supernatants were thawed and cleared by centrifugation. Two hundred microliters of the sample or a glutaraldehyde standard solution were mixed with 1 mL of phenol reagent (40 µL of 5% phenol in water in 10 mL of 70% perchloric acid) and incubated at room temperature for 15 minutes. Absorbance was measured versus the phenol reagent at 479 nm with a UV/Vis Spectrophotometer (Beckman DU-70; Beckman, Fullerton, CA) [13].
Cytotoxicity of BioGlue and Glutaraldehyde
MRC5 human embryo lung fibroblasts and C2C12 mouse myoblasts (ECACC, Porton Down, UK) were seeded into 24-well plates and grown to confluency at 37°C and 5% CO2 in modified MEM alpha medium with 10% fetal calf serum (MRC5) or DMEM medium with 5% fetal calf serum (C2C12), respectively. The culture medium for the MRC5 cells was then replaced with fresh serum-free medium supplemented with dilutions of glutaraldehyde. The culture medium for the C2C12 cells was replaced with fresh serum-free medium supplemented with 10% (vol/vol) supernatant fluid from the BioGlue incubation. After incubation for 100 minutes, the MRC5 cells were stained with carbolfuchsin, and the C2C12 cells were assessed for viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test.
Carbolfuchsin Staining
Cells were washed with phosphate-buffered saline and stained with Ziehl-Neelson-carbolfuchsin solution (1:10 diluted with water). Excess staining reagent was removed by washing twice with 0.9% saline solution.
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide Test
Cells were overlaid with fresh culture medium supplemented with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to a final concentration of 650 mg/L and incubated for 2 hours. Medium was then aspirated and formazan extracted from the cells with 1 mL of dimethyl sulfoxide. Optical density was measured at 540 nm against dimethyl sulfoxide.
Animals and Anesthesia
Healthy New Zealand White rabbits, weighing 2.5 to 3.5 kg, were obtained from Harlan-Winkelmann (Borchen, Germany). Animals were caged according to sex at 18°C (± 2°C) and a relative humidity of approximately 55% (± 10%). Light was provided at 12-hour intervals, and animals had free access to food and water.
Anesthesia was induced by subcutaneous injection of 60 mg/kg ketamine hydrochloride (Pharmacia & Upjohn, Freiburg, Germany) and 16 mg/kg xylazine hydrochloride (Sanofi, Libourne, France). Anesthesia was maintained with Thiopental intravenously (thiopental sodium, Biochemie, Kundl, Austria).
Animal Models
RABBIT PARTIAL LUNG RESECTION MODEl
Each test animal was anesthetized, intubated, and placed on a respirator (20 respirations/min, 0.7 to 0.9 L/min, maximal inspiratory pressure of 13 to 15 cm of water). Access to lung was through the third intercostal space, cutting skin and muscles parallel to the ribs and enlarging the intercostal space with a retractor. Respiratory volume was reduced to half of the initial volume, the left median lobe was exposed and clamped at the base, and the apex was resected. The cut parenchymal surface was then sealed with a maximum of 2 mL of BioGlue or fibrin sealant. Five minutes after application of the test material, the clamp was removed, and the respiratory volume was increased slowly to baseline values. In control animals (sham), neither resection nor application of sealants was performed. The thoracic incision was closed with single sutures (Synthofil 2-0; Braun, Melsungen, Germany) in several layers.
LIVER ABRASION MODEL
The left lobe of the liver was exposed, and a superficial circular lesion (diameter 2 cm, depth 2 mm) was created by abrading the surface of the liver lobe using a drilling machine with a grinding disc attachment (bore grinder PROXXON FBS 230/E; Proxxon, Germany; grit size P40, rotational speed 5,000/min). The resulting hemorrhage (characterized as small vessel or capillary bleeding) was then treated with 1.5 mL of BioGlue or fibrin sealant. After hemostasis was achieved, the liver lobe was returned to its anatomic position in the abdominal cavity, and the abdomen was closed. The muscle and skin incision were sutured separately using Synthofil 2-0 in an interrupted pattern in a two-level manner.
AORTIC APPLICATION MODEL
A median laparotomy was performed, 2 cm of the abdominal aorta was exposed distal to the renal artery branch, and 0.5 mL of either BioGlue or fibrin sealant was applied.
Two animals were studied in each of the treatment groups of the lung, liver, and aorta models. All study animals received 0.05 mg buprenorphine subcutaneously (Schering-Plough, NJ) after surgery. All animal experiments were approved by the Animal Protocol Review Board of Vienna. The requirements defined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (publication NIH 86-23, revised 1985) were strictly fulfilled.
Histologic Specimens and Analyses
After either 2 or 7 days, animals were sacrificed with an overdose of pentobarbitone sodium intravenously (Intervet, Vienna, Austria), and a necropsy was performed. All specimens were fixed in 4.5% buffered formaldehyde solution (>24 hours), dehydrated, and embedded in paraffin according to standard techniques. Transverse sections were mounted onto glass slides. After removal of the paraffin by xylene, sections were stained with hematoxylin and eosin. Evaluation was performed using a light microscope and was semiquantitative. The evaluator was blinded as to treatment.
At necropsy of the lung resection group, the left median lung lobe, including resection site and any test material present, was harvested for histologic evaluation. The right median lung lobe was taken as control.
At necropsy of the liver abrasion group, the section of the liver that included the lesion and any test material present was harvested for histologic evaluation.
At necropsy of the animals from the aortic application group, the 2-cm length of the abdominal aorta treated with either of the two sealants was harvested for histologic evaluation.
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Results |
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In Vitro Cytotoxicity of Glutaraldehyde Released From Polymerized BioGlue
Using carbolfuchsin staining, it was determined that the supernatants obtained from overlaying polymerized BioGlue with saline solution were cytotoxic to MRC5 human embryo lung fibroblasts. Glutaraldehyde concentrations as low as 12 µg/mL led to significant changes in morphology and density of cells (Figs 2a, 2b). This concentration of glutaraldehyde corresponds to that achieved in the viability tests by supplementation of medium with 10% supernatant. Concentrations of 111 µg/mL and above, as detected in the supernatants, resulted in fixation of the cells (Figs 2c, 2d). Even after incubation with trypsin, these cells could not be detached from their culture plates.
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In the control (sham) group, a thoracotomy was performed and the lung manipulated without resection. Dystelectasis, atelectasis, and pleural thickening were seen at the 2-day time (Fig 4c). At the 7-day time, superficial inflammation was detected.
No pathologic findings were noted in the right, untreated control lungs of all groups, except 1 animal, which had dystelectasis and emphysema.
LIVER ABRASION MODEL
In animals treated with BioGlue, necrosis was seen at the 2-day time (Fig 5). At the 7-day time, toxic necrosis, hemorrhage, medium-grade inflammation, granulation tissue, and giant cells were detected.
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AORTIC APPLICATION MODEL
No difference was noted between animals treated with BioGlue or fibrin sealant. The reaction to the test materials was characterized as low-grade or medium-grade inflammation (Fig 6).
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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We determined the amounts of glutaraldehyde released from BioGlue, which had been allowed to polymerize, and found concentrations of 100 to 200 µg/mL in the supernatants. This is the same range as that found in the supernatants from the first trigger performed to prime the cartridge. According to the manufacturer's instructions, this first trigger material has to be discarded because of the likelihood of increased glutaraldehyde release rates.
The cytocompatibility or cytotoxicity of supernatants from polymerized BioGlue was tested on cultured cells, allowing for rapid screening of supernatants from different phases during application of BioGlue without ethical concerns. Direct contact of cultured cells with undiluted supernatants led to immediate fixation of the cells as a result of the high concentrations of glutaraldehyde. Even diluted supernatants nearly abolished cell viability, and already 10% of the concentration of glutaraldehyde found in supernatants of polymerized BioGlue affected morphology of cultured cells.
Although several observations of adverse effects have been reported [5, 8, 9, 14, 15], broadening of clinical applications of BioGlue has been evaluated in several investigations [6, 10, 11, 16]. Therefore, the second aim of our study was to characterize the sensitivity of different tissue types to the potential adverse effects of BioGlue.
Short-term experiments of in vivo application of BioGlue to a partial lung resection or a liver abrasion in rabbits led to high-grade inflammation, edema formation, and necrosis at the site of application. Although the highly concentrated albumin in BioGlue is of bovine origin, it is not likely to be the reason for the massive inflammatory response because of the short observation period used in this study. Nevertheless, bovine serum albumin is known to be the major beef allergen, and several epitopes for immunoglobulin E binding as well as T-cell stimulation are reported [17]. Application of a fibrin sealant to the partial lung resection or liver abrasion exhibited significantly better biocompatibility and better resorption. Only mild signs of inflammation were detected in lungs or livers treated with fibrin sealant. Therefore, we conclude that the glutaraldehyde released from BioGlue is responsible for the lesions we observed on lung and liver tissue.
Reviewing other in vivo studies investigating BioGlue, a report by Herget and colleagues [6] confirmed our observations on lung tissue. They performed bronchial anastomoses with BioGlue in sheep and detected "an inflammatory tissue response consisting of polymorphonuclear neutrophils and macrophages infiltrating the glued bronchial anastomosis and parenchyma." Also tissues other than lung and liver seem to exhibit pronounced sensitivity to BioGlue. LeMaire and associates [10, 11] reported that contact between BioGlue and the phrenic nerve consistently caused acute nerve injury and paralysis of the diaphragm [10] and described similar toxicities involving the cardiac conduction tissue [11].
Interestingly, the local reaction of aortic tissue to BioGlue was less pronounced than that of lung and liver tissue. Only low-grade or medium-grade inflammation was observed. An obvious reason for higher sensitivity of lung and liver compared with aorta may be their damaged integrity after incision or resection. However, it was the aim of the study to mimic the use of BioGlue in clinical practice. There it is used as a sealant on lung and liver tissue and comes into contact with raw tissue surfaces. The clinical application on aortic dissections is on the outer, adventitial layer where, in contrast to liver or lung incisions, the tissue tends to be less damaged. Another reason for higher resistance of aortic tissue may be the different structure of the tissue with large amounts of extracellular matrix, mainly collagen and elastin, and fewer cellular components. In fact, aorta is the application site for which BioGlue is approved.
Similar to this evaluation, Hewitt and coworkers [18] and Gundry and associates [19] found only a weak inflammatory response in the aortas of sheep and goats treated with BioGlue. In contrast, LeMaire and colleagues [8] tested the effect of BioGlue on growing vessels in piglets and detected significantly reduced vessel diameters 7 weeks after treatment. They claimed tissue injury and severe fibrosis surrounding the aorta and the adventitia as major factors responsible for the impaired growth. Furthermore, increased rates of pseudoaneurysm formation in patients in whom BioGlue was used to repair dissections of the aorta have been reported [5, 14, 15]. The authors of these reports assume that this phenomenon might be related to local tissue damage evoked by toxic products in the glue. Also Erasmi and colleagues [9], who had to reoperate on a patient and examined a specimen from a dissecting aortic aneurysm 3 months after BioGlue application, observed "severe active inflammation surrounding the glue remnant with multiple granulocytes and histiocytes and a massive foreign-body reaction with numerous multinucleated giant cells." Together with our results, these data suggest that although aortic tissue seems to be less sensitive to the toxic effects of BioGlue, minor lesions caused by the glue may still involve a certain risk for subsequent complications.
Another critical point may be the rigidity of BioGlue. Polymerized BioGlue formes a stiff layer that is thoroughly bound to the tissue. Especially when larger areas have to be covered with the glue, this may result in substantial mechanical problems.
After evaluation of the data obtained in this study and those from the literature, we conclude that the use of BioGlue should be restricted to the aortic dissection procedure because tissues other than the aorta appear to be very sensitive to the high amounts of glutaraldehyde released from the glue. Even when BioGlue appears to represent a clear benefit, the significant toxic potential of the glue should be considered, and additional long-term studies should be conducted to evaluate these effects. A further potential risk, which was not tested in this study, but suggested as a potential danger by one of the reviewers, is the introduction of free glutaraldehyde into patient-sensitive circulatory systems such as bypass systems or cell-saving devices, although operating room personnel have been trained to avoid suctioning of biomaterials into those systems. In general, a comprehensive deliberation of benefits and risks should precede any application of BioGlue.
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W. Furst and H. Redl Conflict of Interest Disclosure Relating to "Release of Glutaraldehyde From an Albumin-Glutaraldehyde Tissue Adhesive Causes Significant In Vitro and In Vivo Toxicity. Ann Thorac Surg 2005;79:1522-8." Ann. Thorac. Surg., September 1, 2005; 80(3): 1159 - 1159. [Full Text] [PDF]
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