Comparative Effectiveness of Cyanoacrylate Bioadhesives and Monofilament Suture in Wound Healing : A Histopathological and Physicochemical Study in New Zealand

Comparative Effectiveness of Cyanoacrylate Bioadhesives and Monofilament Suture in Wound Healing: A Histopathological and Physicochemical Study in New Zealand White Rabbit Angulo A1*, Sebastián I2, Martínez FJ2, Torregrosa R3, Martín-Martínez JM3 and Madariaga AM2 1Optics, Pharmacology and Anatomy Department, University of Alicante, 03080 Alicante, Spain 2Animal House Facilities, Research Technical Services, University of Alicante, 03080 Alicante, Spain 3Adhesion and Adhesives Laboratory, University of Alicante, 03080 Alicante, Spain


Introduction
The rabbit is an important animal model in biomedical research and has a valuable economic role in animal production as much of the information on rabbit health and care were related primarily to those animals bred for meat, or kept in research laboratories. In the last decades rabbits became companion animals and their owners demand the highest standards of care for their rabbits, a demand of more and better information on the veterinary treatment of this species has been created [1].
Furthermore, the growing interest and respect for animal welfare, the application of the rule of the 3Rs, of Replacement, Reduce and Refinements the animals used in research [2], the refinement of techniques to prevent animal suffering, impart greater value in having methods of non-invasive wound closure which should be instantaneous, shortening the downtime and/or anaesthesia of the animal, producing better recovery and being less traumatic, especially considering that rabbit skin is very sensitive as its tear easily when stretched or cut its hair with razor or shaving. Since Elizabethan collars are not well tolerated by these animals, the use of subcuticular sutures for skin closure are recommended, or alternatively, the application of tissue adhesive or skin staples [3].
One of the advantages of using tissue adhesives for joining animal or human tissues as compared to the traditional suture is the creation of homogeneous and uniform distribution of stresses all along the joint. Furthermore, the tissue adhesives are easy to apply, they are less traumatic to patient, their use reduces the surgical time, avoids the mechanical damage produced in the tissues by suture, inhibit scar formation, and there is no need of sutures or bandages removal after surgical practice. Several tissue cyanoacrylate adhesives have been developed. These adhesives polymerize in the presence of water and because skin has a high concentration of water, these adhesives can be used for surgical practice and wound closure. Cyanoacrylate adhesives also produce strong and quick adhesion in wound closure.
Since 1950 cyanoacrylate adhesives have been used [4]. Several studies [5][6][7][8][9][10][11] have shown their effectiveness as an alternative to suture practice. Particularly, cyanoacrylate adhesives have shown excellent performance as skin adhesives, surgical glues and embolic materials [5,6], in skin closure in plastic surgery or in osteosynthesis (ethyl cyanoacrylate polymerized by ultrasounds) [7,8], in the therapeutic embolization of cerebral arteriovenous malformations, in gastric variceal bleeding or corneal perforations [9], in ocular strabismus surgery [10], in eyelid wound closure in rabbits [11] and as skin sealants for plastic surgery. In all these applications, similar performance in wound closure was obtained by using the suture and the adhesives.
Although medical use of cyanoacrylate adhesives goes back to the half of the last century, especially for army applications, only in the last two decades the US Food and Drug Administration (FDA) approved the use of cyanoacrylates in humans [12]. However, despite producing effective sealing, the use of cyanoacrylate adhesives showed exothermal cure and high stiff polymerized product of considerable strength but fragile at the same time. In addition, erosion, ulceration, and areas of necrosis in surrounding (or adjacent) tissues may occur. One easy route to reduce the exothermal reaction and stiffness of the cyanoacrylate adhesives is the increase of the length of the alkyl hydrocarbon chain.
To the best of our knowledge a comparison of the performance of suture versus ethyl, n-butyl and n-octyl cyanoacrylate adhesives for closing wounds in-vivo has not been carried out yet. Furthermore, the monitoring of haematological and biochemical tests in animals having skin wounds closed with cyanoacrylate adhesives of different alkyl chain 1000, Merial Laboratory, Barcelona, Spain + Xilagesic 2%, Calier Laboratory, Barcelona, Spain). The whole procedure was performed on a sterile, clear, non-slippery, non-wrinkle surface and animals were placed in prone position. The lumbar region of the rabbit was shaved and disinfected with chlorhexidine (Lifo-Scrub ® , BІBRAUN, Melsungen, Germany). For comparing the influence of traction on the process of wound healing, two superficial incisions were made with a sterile surgical blade (Aesculap ® , BІBRAUN, Tuttlingen, Germany), one on each side of the lumbar region, 2 cm long, one longitudinal (L) and other transversal (T) to main axis of the animal body [19].
The wounds (incisions) were closured using monofilament suture (Dafilon ® , BІBRAUN, Tuttlingen, Germany) or with adhesive ethyl cyanocrylate (ECN), butyl cyanocrylate (BCN) or octyl cyanocrylate (OCN). In the case of the adhesives, the wound edges were drawn and stretched longitudinally, applying the minimum amount of adhesive with a syringe. In the case of the suture, 5-6 points were performed ( Figures 1A and 1B).
The closed wounds were bandaged in order to prevent further contamination and minimize the manipulation of the animal during healing process ( Figures 1C and 1D). Animals received Meloxicam 0.1 mg/kg p.o. (Metacam ® , Boehringer Laboratory, Ingelheim, Germany) in water for 48 hours to relieve pain and reduce inflammation.
Healing process was rated according to several clinical manifestations such as dehiscence, bleeding, infection, inflammation, and itching. Photographs were taken throughout the healing process with a digital camera Canon-Power-Shot-S45 (Canon Inc., Japan) in order to assess the degree of inflammation and tissue reaction (double blind observation). One semiobjective assessment in wound healing process in rabbit skin was applied. Each wound received an assessment from 1 (appalling) to 6 (excellent) according the parameters in Table 1.

Cyanoacrylate adhesives
Three cyanoacrylate adhesives were used in this study ( Figure  2). Ethyl cyanoacrylate (ECN) was prepared in the Adhesion and Adhesives Laboratory of the University of Alicante (Alicante, Spain). n-butyl cyanoacrylate (BCN) monomer was Vetbond ® manufactured by 3M (StPaul, Minnesota, USA) and contains hydroquinone stabilizer and blue dye. n-octyl cyanoacrylate (OCN) was Dermabond ® manufactured by Ethicon (Somerville, New Jersey, USA) and contains thickening agent, stabilizer and violet dye.
The structure of the cyanoacrylate adhesives was characterized by thermal gravimetric analysis (TGA). TGA studies were carried out in TA TGA Q500 instrument (TA instruments, New Castle, Delaware, USA) under nitrogen atmosphere (flow rate: 100 ml/min). Samples of length has not been considered yet. Therefore, in this study, suture and three cyanoacrylate adhesives are used for skin wound closure in New Zealand White rabbits were carried out, paying particular attention to their physicochemical characterization and their performance in-vivo including histological, haematological and biochemical monitoring.

Animals
The study was performed under the terms of the previously existing Spanish Animal Protection Laws [13-15], European Directive [16] and approved by IACUC of the University of Alicante (Spain). Assays for medical device were conducted according to UNE-EN ISO 10993-1:2004 [17].
In the spirit of the 3Rs, a reduced number of 36 New Zealand White rabbit males were used, 6 groups of 6 animals each one. Animals were purchased from National Production Centre (Granja San Bernardo, Navarra, Spain) and allowed to acclimatize for one week after arrival, the transport quarantine period. Rabbits were 8 weeks age at the moment of arrival, and had an average body weight of 1982.4 g. The body weight was recorded weekly (the greater weight gain/week, average of 414.29 g, occurred during the transport quarantine period).
Rabbits were maintained individually on a constant 12 hr light/dark cycle, under controlled temperature (22°± 1.5°C) and relative humidity (55 ± 15%), and 15 to 20 air renewal/hr (according to the European legislation). Rabbits were fed with 2030 Teklad Global Rabbit Diet (Harlan Laboratories, elaborated by Mucedola s.r.l., Italy) ad libitum. Water was softened by 4030 Dual Demand Softener (Ecowater Systems, Inc. USA) and freely accessible.
In addition, blood samples (1 ml) were collected into tubes containing Li Heparin. These samples were centrifuged at 2500 rpm for 15 min in Centrifuge 5810R (Eppendorf, Hamburg, Germany) and the serum samples obtained were examined for the following parameters: albumin, alkaline phosphatase, alanine aminotransferase, amylase, total bilirubin, blood urea nitrogen, calcium, phosphorus, creatinine, glucose, sodium, potassium, total protein, globulin. Automatic analyzer VetScan ® (Union City, CA, USA) was used.

Surgical method
Animals were weighted and anaesthetized by intramuscular administration of Ketamine + Xylazine (50 + 10 mg/kg) (Imalgène Raspeig, Alicante, Spain). The pig skin test samples were immersed for 5 minutes in 60 ml physiological serum solution (Fleboplast, Grifols, Barcelona, Spain) to impart homogeneous and controlled humidity. 10 seconds after removal of the test pieces from the solution, they were surface dried with filter paper for 10 seconds and the test samples were maintained under ambient conditions for 30 seconds. 0.05 ml of cyanoacrylate adhesive was applied by means of a syringe on one pig skin test piece and the adhesive was spread over a bonding area of 20×30 mm by means of the syringe needle. The second test piece was immediately placed in contact and press by using two fingers for 30 seconds ( Figure 3A). 60 seconds later, the single lap-shear test was carried out in Instron 4411 universal testing machine (Instron Ldt. Buckinghamshire, UK) by using a pulling rate of 100 mm/min ( Figure  3B). Five replicates were measured and averaged.

Histopathological method
Animals were sacrificed with an overdose of pentobarbital i.v. (Dolethal ® , Vétoquinol, Cedex, France). Two skin samples of dimensions 2×1×0.3 cm approximately were collected from each rabbit and they were placed in a container with formalin 10% (Formaldehyde 37-38%w/w, Panreac, Barcelona, Spain) for 6 hours at least; the volume of formalin was threefold higher than the size of the skin samples.
Skin samples were removed from formalin and processed by conventional histological embedded paraffin (Histosec ® pastilles, Merck, Darmstadt, Germany) method. The paraffin blocks with the samples were cut perpendicularly to the skin surface in 10-15 µm histological sections using automatic microtome Leica-RM-2065 (Wetzlar, Germany). Each sample was spread on glycerinated albumin slide with distilled water at 35-40°C, followed by dewaxing, rehydration, cyanoacrylate (10-15 mg) were heated from room temperature up to 400ºC by using a heating rate of 10ºC/min. Almost normallooking skin in the joint and adjacent area Table 1: Healing evaluation procedure criteria used for assessing the degree of inflammation and tissue reaction under macroscopic and microscopic.  the existence of different structures in the polymer. As a consequence, the structural heterogeneity of the cyanoacrylate monomers becomes more marked by increasing their alkyl hydrocarbon chain length likely due to slower kinetics of reaction. This issue may affect the time for polymerization of the cyanoacrylate adhesive on the wound in the rabbit skin. Therefore, the immediate adhesion of the cyanoacrylate adhesives was tested.

Immediate adhesion of the cyanoacrylate adhesives
The immediate adhesion (i.e., 60 seconds after joint formation) of the cyanoacrylate adhesives was measured by single lap-shear tests of pig skin/cyanoacrylate adhesive/pig skin joints as an estimation of their performance in wound closure. Figure 5 shows the adhesive strength values of the pig skin/cyanoacrylate adhesive joints and, in general, it decreases by increasing the length of the alkyl hydrocarbon chain of the cyanoacrylate adhesive. The adhesive strength values obtained in the joints made with ethyl and n-butyl cyanoacrylate are high and sufficient to maintain the tissues joined in wound closure.

Haematological and histopathological features of wound healing in rabbit skin by using suture and cyanoacrylate adhesives
Most biochemical parameters analyzed and haemogram (Tables 3  and 4) are within the normal range given by the different references for rabbits [1], with a marked increase until 12 weeks of age, associated with higher growth and body weight gain, except calcium and phosphorus that decreases with decreasing growth and bone metabolism.
Total protein are in the normal range and increase with age, from 5.23 to 5.92 g/dL although the albumin gives low values, from 1.64 to 2.14 g/dL, an increase of 0.48 g/dL, however, globulin has high levels of 3.57 to 3.78 g/dL, a smaller increase, 0.21 g/dL. Alkaline phosphatase increased significantly up to 12 weeks old, 115 to 162.78 IU/L, and then decreases gradually. The amylase gave low levels throughout the study, although gradually increase along this, of from 122.75 to 160.2 IU/L, reference values in the literature are from 200 to 500 IU/L. The literature associates the low level of some parameters with the confinement in cages and the lack or poor caecotrophy, a physiological mechanism of the rabbit.
The white blood cells (WBC), the highest value is observed at 12 weeks of age, 8,67 × 109/L, and the ratio lymphocyte / granulocyte in this age, is of 30/60 as the literature points reference. staining and dehydration. Haematoxylin and eosin-stained sections were examined with transmitted light microscope (Leica DMLS) and photomicrographs were taken with digital camera. Samples were reevaluated according the parameters of the Table 1.

Results and Discussion
In this section, the structure of the cyanoacrylate monomers with different alkyl hydrocarbon length was analyzed by TGA and their inherent immediate in-vitro adhesion was measured by single lap-shear strength tests of pig skin/cyanoacrylate adhesive/pig skin joints. In the third part of this section, the haematological and histopathological features of wounds made in rabbits which were healed with suture and different cyanoacrylate adhesives was carried out.

Structural characterization of the cyanoacrylate monomers
In this study TGA was found useful and sensitive to analyze the structure of the cyanoacrylate adhesives. Figure 4A shows the variation of the weight loss as a function of the temperature (TGA thermograms) of the three cyanoacrylate adhesives and the corresponding derivative curves are given in Figure 4B; the derivative curves are useful for showing more precisely the thermal decompositions in the cyanoacrylates which correspond to different structures.
The TGA thermograms of the three cyanoacrylate monomers show the first decomposition at about 120ºC (Table 2), likely due to formaldehyde evolution. This first decomposition becomes less marked by increasing the length of the alkyl hydrocarbon chain of the cyanoacrylate, i.e. the weight loss at this temperature decreases from ethyl to n-octyl cyanoacrylate. On the other hand, the lowest thermal stability corresponds to the ethyl cyanoacrylate and the highest one to the n-octyl cyanoacrylate.
The presence of more than one decomposition step in the cyanoacrylate monomers is an indication of the presence of oligomers in their structure. Whereas the ethyl cyanoacrylate is mainly composed of monomer (98.7 wt% is lost at 117°C), the n-butyl cyanoacrylate contains monomer and some dimmers (17.8 wt% is lost at 157°C), and the n-octyl cyanoacrylate has only a minor content of monomer (only 25.3 wt% is lost at 126°C) and shows four additional decompositions ( Figure 4 and Table 2). These results are in agreement with literature [20] that also described the existence of different decompositions in the TGA thermograms of n-butyl cyanoacrylate polymer ascribed to          The incisions made in the surgical procedure were healed with conventional suture and by using the three cyanoacrylate adhesives of different alkyl chain length. The general appearance and clinical course of all animals after surgery was good, with no apparent disease register data. Biochemical and haematological values were within the normal range for rabbits [18,[21][22][23], which suggests good biocompatibility of the adhesives. Histological features of the tissues of the layers of healthy normal skin rabbit are given in Figure 6.
At the end of the surgical procedure when suture or adhesives were used, most of the incisions in the rabbit skin were closed in full by primary closure [24]. The normal skin appearance, as showed in Figure  6, was recovered when ECN ( Figures 7A, 7D, 8A and 8E) or OCN ( Figures 8C, 8G, 9B and 9D-right) adhesives were used. However, using the evaluation criteria described in Table 1, the CT and HF were not well identified in wound closed with BCN ( Figures 9A and 9D-left) and suture ( Figures 8D, 8H, 9C, 9E,10C, 10D and 10F). The results were worse when the rabbit removed itself off the dressing and gnawing the stitches from the incision (Figures 1D and 9E) or the adhesive was very aggressive by reacting with the tissue (Figures 7B, 7E, 8B and 8F).
An excess of bioadhesive quantity or a deficient confronting edges of the wound caused delay in closure (Table 1 and Figures 7A,  7B, 7D, 7E, 8C and 8G). In the most of those cases, a secondary closure [24] of the wound were produced until healing develops a granulation tissue (with several inflammatory reaction degrees and varied   Table 1) (A and C pictures). In B picture one edge of open wound with remains of adhesive is shown (Degree 1 in Table 1).  Table 1) as compared to the use of suture (Degree 4 in Table 1). When BCN adhesive is used (B picture), the wound edges are totally separated (Degree 2 in Table 1).  Table 1) as compared with A (Degree 4 in Table 1) and C (Degree 5 in Table 1) pictures.  (Table 1). The inflammatory reaction, the absence of HF and disorganization of collagen fibres of the dermis was higher in the sutured junctions than in the cuts closed with cyanoacrylate adhesives (Figures 8D, 9C and 10D) show that the skin have recovered an almost normal aspect, but fibrosis is more evident in the wound closed with suture (Degree 5 in Table 1) than with bioadhesive (Degree 6 in Table 1).
Considering the evolution with time of the wound after surgery, in general, the performance obtained by using bioadhesives is better than by using suture. After only 3 days, ECN and OCN adhesives are rare efficient in spite of the most superficial layer is separated (Figure 7). Until 7 days the suture is absolutely inefficient for closing the wound (Figure 8). After 14 and 28 days, the use of both suture and adhesives for closing the wounds are very similar, but the skin tissues are better recovered when the adhesives are used (Figures 9 and 10).
Compared to conventional monofilament suture, the three cyanoacrylate adhesives (ECN, BCN, OCN) show some advantages in handling time, haemostasis achieved immediately upon application, excellent confronting edges and no incisions plaque retention. No significant differences were found between the binding efficiency comparing longitudinal and transverse incisions using whatever combination of suture and cyanoacrylate adhesives on the same or different animals (Figures 7-10). Furthermore, the inflammatory reaction and disorganization of collagen fibres of the dermis was higher in the wound sutured with respect to those closed with adhesive [11,19].
The structure and immediate adhesion of the cyanoacrylate adhesives with different alkyl hydrocarbon chain length can be related to their performance in wound healing of rabbit skin. The ethyl cyanoacrylate is mainly composed of monomers and it shows high adhesive strength causing an effective closure of the incisions on rabbit skin. Because of its quick polymerization, there is not the chance of having remaining unreacted monomers on the wound surface leading to a fast healing and effective tissue regeneration. On the other hand, the existence of dimers in n-butyl cyanoacrylate does not favour quick healing and they can remain on the wound surface delaying the healing and causing reactions with the neighbouring tissues. Finally, the wounds closed with n-octyl cyanoacrylate show slower tissue regeneration because the important existence of oligomers of different length which will retard the healing. As a consequence, the structure of the cyanoacrylate monomer can be related to its performance as adhesive in wound healing of rabbit skin, the optimal performance was obtained by using the ethyl cyanoacrylate adhesive.

Conclusions
The use of cyanoacrylate adhesive was as favourable as surgical suture on rabbit skin being faster, easy to handle and apply, painless, having less risk of infection, excellent aesthetic results, and significant cost savings before and after surgery to reduce the need for anaesthesia and postoperative cures.
The structure of the cyanoacrylate monomer can be related to its performance as adhesive in wound healing of rabbit skin, the optimal performance was obtained by using the ethyl cyanoacrylate adhesive. Whereas the ethyl cyanoacrylate was mainly composed of monomer, the n-butyl cyanoacrylate contained monomer and some dimmers, and the n-octyl cyanoacrylate had only a minor content of monomer.
The immediate adhesion of the cyanoacrylate adhesives decreases by increasing the length of the alkyl hydrocarbon chain. The adhesive strength of the joints produced with cyanoacrylates were high and sufficient to maintain the tissues jointed during wound closure and the cyanoacrylate adhesives with lower alkyl hydrocarbon chain length had greater efficiency in tissue repair.
Finally, the cyanoacrylate adhesives have a biological tolerance similar to the surgical monofilament suture, so they are an alternative to conventional suture in animal surgery.