Reduction of Anastomotic Time Through the Use of Cyanoacrylate in Microvascular Procedures

Abstract

Background: Ever since the description of the first microvascular anastomosis, numerous alternative methods have been described to the classical approach. Tissue adhesive has shown promising result in previous studies and can be a fast and efficient alternative which still requires more studies to allow its clinical implementation. Methods: A randomized comparative experimental study was conducted on rats’ femoral arteries and an end-to-end anastomosis was performed in order to compare 2 anastomosis techniques. In one group, a simple interrupted suture was utilized, whereas in the second group a combination between fewer sutures and tissue adhesive was used. The anastomotic time, total operative time, blood flow velocity before, immediately after and 48 hours after the procedure, as well as an independent grading of the anastomosis immediately after the procedure were performed. Magnetic resonance imaging (MRI) was performed in order to assess the degree of stenosis. After euthanasia, histology and scanning electron microscopy (SEM) were performed on the vessels in order to assess possible complications. Results: A total of 24 anastomoses were performed, of which 12 with a classic technique and 12 with an adhesive technique. All the anastomoses were patent with a significant reduction of anastomotic and total operative time. The grading of the anastomoses showed better results in the classic suture group. The blood flow velocities were not statistically significant between the 2 groups. On MRI there was one stenotic anastomosis, whereas histology and SEM showed more complications on the adhesive group. Conclusion: Anastomotic times were significantly lower with a non-significant trend toward more thrombotic complications in the adhesive group. Further improvement of the glue properties and refinement of the technique will likely make it a viable alternative to interrupted suturing in the future.

Introduction

Ever since microsurgery started to be contoured as a technique, there has been a tendency for various methods of vascular anastomosis to be described. Despite all available methods, interrupted suturing remains the gold standard, and to date no technique has been scientifically proven superior to it. ^{1 -3} The major drawbacks that have been associated when performing a vascular anastomosis using the so-called classic approach are represented by the vascular wall trauma, foreign body reaction, risk of bleeding/leakage, and prolonged procedural time. ^{3 -6}

In order to overcome the disadvantages previously described, and in an attempt to simplify the technique of microvascular anastomosis, numerous techniques using vessel clips, stents, couplers, and laser-assisted bonding have been described. Despite presenting a certain degree of success in terms of overall procedural time, and in some cases a lower stenosis rate when compared with the suture only technique, they all have been associated with difficulties or complications which would hinder their wide distribution in clinical practice. ^{1,2,7 -13}

Medical adhesives are widely used experimentally as a sutureless alternative when performing an anastomosis. Initially being used for superficial wound repair, as well as for haemostasis and embolization, medical adhesives have made their way into the experimental field and have proven their ease of use at the cost of a potentially higher prevalence of lumen obstruction and wall hypertrophy. ^{13 -17}

The medical adhesives that can be used in vascular anastomosis can be divided, according to their material properties, into biologic tissue adhesives and chemical synthetic tissue adhesives. Fibrin adhesive has been shown to have good histocompatibility, haemostasis, and adhesive properties; furthermore, several studies have reported that its use could reduce the foreign body reactions by reducing the total number of sutures, shorten the operation time, and improve the vascular patency rate. ^12,13,18 However, fibrin adhesives are relatively expensive and more difficult to prepare, do not provide adequate strength and can potentially transmit blood-borne disease. ^10,13,19

Chemical synthetic adhesives such as cyanoacrylates have been widely used in various fields and have gained popularity over the past years. Methyl-, ethyl-, or butyl-cyanoacrylates have been thoroughly studied but due to their associated toxicity and patency issues as a result of vessel distortion, their use has been limited. ^13,20 The newly elaborated adhesives such as n-butyl-cyanoacrylate and 2-octylcyanoacrylate (Dermabond®; Ethicon Inc) seem to have answered to the previously mentioned drawbacks. 2-octylcyanoacrylate is a long-chain tissue adhesive containing plasticizers, and has stronger flexible bond with a greater breaking strength that makes it easier to handle when compared to short-chain cyanoacrylates. ^13,21,22 To date, experimental studies using 2-octycyanoacrylate adhesive have achieved satisfying results which include overall shorter anastomotic time, less vessel damage, and foreign body reaction together with better patency rates. ^13,21,23,24

The aim of the present study was to compare the conventional interrupted suture on vessels under 1 mm diameter with a technique using chemical synthetic adhesives—2-octylcyanoacrylates—in terms of anastomosis time, anastomosis quality, and prevalence of complications. We hypothesized a reduction in anastomotic time and a reduction in the complication rate using the adhesive. In order to establish the feasibility of the technique, a thorough monitorization of the nature of post-operatively complications and the complication rates themselves were considered as to further promote it, if suitable, for clinical practice.

Materials and Methods

Our comparative study took place at the Centre for Experimental Medicine within the “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania. The animals used in the study were provided by the University’s animal research facility. The Wistar rats were being kept under strict regulations regarding animals used in biomedical research (Romanian Law Nr. 43/2014). The housing of the animals was adequate and in conformity with all required standards while also being provided with free access to food and water.

All of the procedures were made by the main author (AVO), highly experienced in microvascular anastomosis. Before commencing the study, the adhesive technique was practiced in the microsurgery laboratory on both living and non-living training models. Because of its simplicity compared to the classic approach to a microvascular anastomosis, the operator had no trouble adapting to the new technique, with an insignificant learning curve. All of the post-operative evaluations were performed blindly by the senior author (VV).

Twenty-four male rats weighting between 250 and 300 g were included in the study (n = 24) with ages ranging from 12 to 16 weeks, divided into 2 groups. The anaesthesia was done by intramuscular injection of Ketamine (Vetased 10%) 80 mg/kg and Xilazine (Xylazin BIO 2%, Bioveta) 8 mg/kg. After the induction of anaesthesia, the right inguinal region was shaved with clippers, the animal was positioned supine, and a small 2 cm incision was placed in the inguinal crease. After the dissection of the superficial fascia and inguinal fat-pat, the femoral artery was exposed and dissected from the vascular sheath, ligating a muscular branch always present at this level (Murphy’s branch). A piece of blue latex was placed underneath the vessel for contrast purpose. After the placement of the microapproximator clamp (S&T® ABB-2 V), the artery was cut in the midpoint between the clamps, flushed with heparinized saline solution, the adventitia was trimmed and the 2 vascular ends dilated with vascular dilator forceps (S&T® D-5a.3).

The animal subjects were randomized into 2 groups of 12 animals each, as following:

In the first group (S), the control group, a simple end-to-end arterial anastomosis was performed on the femoral artery, using the 180° technique, with 8 interrupted nylon 10.0 sutures (BBraun Aesculap® 70 μ needle; Figure 1A, B). In the second group (A), the study group, an end-to-end anastomosis was performed using only 3 simple interrupted sutures 120° apart, with tissue adhesive being applied between the sutures to complete the anastomosis, a technique that has previously been described in the literature. ¹⁰ The adhesive was applied with a 8.0 nylon by tensioning 2 of the 3 the sutures (Figure 1C, D).

Figure 1.

Anastomosis technique. A and B, 180° classic technique. C and D, Triangulation technique with adhesive.

For each subject, the total operative time—time elapsed between incision and skin closure—and anastomotic time were recorded. After the completion of the anastomosis and removal of the clamps, an independent grading of the anastomosis was done by evaluating the following: patency—milking test described by Acland; the degree of stenosis and suture spacing. For each of the 3 criteria, a score ranging from 1 to 5 was assigned and the mean score was calculated in order to compare the results. In order to assess the velocity of the blood flow in the femoral artery (cm/s), Doppler ultrasound (Ultrasonix SonixTablet—linear 40-8 MHz probe, 16 mm depth of field) was performed before-, immediately after-, and at 48 hours post-procedural and the results were compared.

The animals were euthanized at 6, 7, 10, and 14 days, respectively, after the procedure was done in order to examine the blood vessel’s wall structure. We expected uniformity in terms of performed anastomoses, which is why we chose at random samples for histological, scanning electron microscopy, and magnetic resonance imaging (MRI) analysis, as each anastomosis was representative of the whole group. Euthanasia was performed under general anaesthesia by cervical dislocation.

Histology was performed in order to evaluate the level of thrombosis at the anastomotic site. Transverse sections of the vessel near the anastomosis were made and haematoxylin-eosin staining was used. From each group the arteries were harvested as following: 3 vessels at 7 days post-operatively and 3 vessels at 14 days post-operatively.

Scanning electron microscopy (SEM) was performed on the inner part of the anastomosis to assess the degree of endothelialization and the anastomosis line. A segment of the artery containing the anastomosis was cut longitudinally and fixed in glutaraldehyde in order to observe the sutures from the inner part of the vessel. Scanning electron microscopy was performed on 4 subjects from each group as following: 2 subjects at 6 days post-operatively and 2 subjects at 10 days post-operatively.

Magnetic resonance imaging was done in vivo, 10 days after the procedure on 2 subjects from each group in order to evaluate the overall stenosis of the artery at the anastomotic site. After the procedure, the animals were euthanized. MRI protocol: Flash 3D inflow angiography; field of view 5 cm, 4.9 cm, 5 cm; axial images; slice thickness 50 mm; matrix 384×384×256; 0.0130 cm/pixel, 0.0128 cm/pixel, and 0.0155 cm/pixel resolution; TR 15 ms; TE 2.6 ms; total acquisition time 18 min, 26 sec (BrukerBioSpec 70/16 USRscanner 7 T, “Babeş-Bolyai” University, Faculty of Physics, Cluj-Napoca, Romania).

Statistical Analysis

The data were collected in an Excel (Microsoft Corp.) database and the statistical analysis was done with SPSS software 23rd ed (SPSS Inc). Kruskal-Wallis, analysis of variance one-way test were applied. A value of P ≤ 0.05 was considered statistically significant.

Results

Both total operative time and anastomotic time were significantly lower in group A when compared to group S (87.92 ± 5.66 vs 65.42 ± 4.54; P = .05 and 22.58 ± 1.77 vs 15.83 ± 0.88; P = .003). Regarding the grading of the anastomosis, better results were obtained in group S with a higher total score (4.56 vs 3.92; P = .003; Figure 2). Post-operative blood flow velocity measurements (Doppler ultrasound) were not statistically significant between the 2 groups, neither immediately post-operatively (13.69 ± 0.87 vs 12.22 ± 1.01; P = .28) nor at 48 hours post-operatively (16.22 ± 1.14 vs 14.35 ± 1.22; P = .28). Histological evaluation indicated a higher rate of thrombotic complications in group A (5 subjects) compared to group S (2 subjects) but neither of these were statistically significant (P = .079). The occlusion of the artery with a thrombus larger than 50% of the cross-section was seen in 4 subjects in group A and in 1 subject in group S (Figure 3). Scanning electron microscopy revealed good endothelialization in both groups (Figure 4). On MRI, a slight stenosis was seen in one subject from group A (Figure 5).

Figure 2.

Anastomosis grading.

Figure 3.

Histological evaluation. A, Vessel without thrombosis. B, Small thrombus (t) with collagen strand (c). C, Small thrombus (t) adherent to the vessel wall. D, Large thrombus (t), >50% occlusion. a indicates artery; v, vein; s, suture.

Figure 4.

Scanning electron microscopy (SEM) on the inner part of the anastomosis. A, Sutures at the anastomosis (arrow). B, Endothelialization on the suture. C, Good alignment of the anastomosis (arrow). D, Uneven alignment of the anastomosis (arrow).

Figure 5.

Angio-MRI. A, Femoral artery without stenosis at the anastomosis (arrow). B, Femoral artery with stenosis at the anastomosis (arrow). CIA indicates common iliac artery; IIA, internal iliac artery; FA, femoral artery; MRI, magnetic resonance imaging.

Discussion

Since the introduction of the first microvascular anastomosis, numerous other techniques have been described to help improve certain aspects. Among them, tissue adhesive plays an important role and has been previously documented. ^{1 -3}

The results of our study together with those presented by other similar studies suggest 2 main points which could be taken into consideration when approaching this topic. Firstly, the shorter anastomotic time is highlighted in our study as well as within the available literature results whenever comparing the technique using adhesives with the classic approach. This aspect is crucial in microvascular surgery as to avoid undesirable complications. ^{11,13,15,23,25 -27} Moreover, this could be of great addition for trainees with little experience since it does not require substantial training as the gold standard technique does; however, this does not imply that adequate training should not be acquired throughout one’s surgical curriculum. ¹

Secondly, the use of chemical synthetic adhesives is thought to be highly thrombotic once they would leak inside the vessel whenever compared to the classic technique, compromising the anastomosis. ^{10,11,28 -30} However, there are studies that provide information with regard to avoiding the leakage of adhesive by overlapping the adventitia as well as applying the adhesive evenly in order to ensure that only the minimum amount of adhesive is applied. Moreover, the overuse of adhesive has been shown to simply harden around without increasing the adhesive power. ³⁰

Better results regarding the grading of the anastomosis in group S may be due to the fact that even a so-called “simple” technique has a learning curve which needs to be mastered. This aspect is to be considered for future studies, as it helps objectively assess one’s work. Grading the anastomosis in both groups could help diminish bias regarding the operator’s experience.

Despite achieving satisfying results regarding the patency of the anastomosis (milking and Doppler ultrasound) in group A, the lack of sutures placed circumferencially may allow the adhesive to enter the lumen through small imperfections—visible at SEM (Figure 4D)—and therefore induce thrombosis which can be seen on histological examinations on day 7 and 14. Moreover, the lack of sutures hinders an adequate eversion of the vessel walls as in the suture only technique, where eversion can be controlled more easily. Therefore, the collagen in the blood vessel wall is exposed and can lead to thrombi formation. Collagen strands with small thrombi can be seen on histological evaluation (Figure 3B). The addition of an intravascular stent may overcome this highly important issue. Numerous methods have been previously described, such as soluble stents or thermoreversible poloxamer gels that dissolve in the bloodstream after the completion of the anastomosis. ^{1,2,31 -33} Other methods include specially designed albumin tubes which are fixed on the vascular ends and then adhesive is used to complete the anastomosis without any sutures. ³⁴ All these experimental methods represent potential alternatives for a better and more reliable type of sutureless anastomosis, with little to no thrombotic complications.

Studies have shown the technique to be successful both on artery as well as on vein walls, suggesting it would be a suitable and reliable approach in clinical practice depending on the vessel diameter. ²¹ However, more experimental research would be advised to be conducted in order to clarify the terms and have suitable guidelines as to when the technique can be safely indicated. ^15,27 Given the fact that recent studies have shown less perivascular inflammatory tissue together with neointimal hyperplasia when comparing chemical synthetic adhesives to sutures only technique, raising the possibility that the reduced exposure to chronic inflammatory mediators may result in better long-term anastomotic patency rates by reduction in fibrogenesis and scarring, make it even more likely that with further research the aforementioned technique would be suitable for clinical practice. ¹⁵ The clinical use of chemical synthetic adhesives in vascular anastomosis, either used as adjuvants or as a stand-alone technique, could bring better overall results. ^{12,35 -38} A study that aimed to evaluate the long-term biomechanical properties as well as histological changes when using 2-octocyanoacrylate adhesive has shown better tensile strength as well as similar wall stiffness when comparing the study group with the control groups using the classic suture technique. In terms of histological changes, at a 6-month evaluation, there were no signs of toxicity caused to the vessel walls as well as less perivascular foreign body reaction in the adhesive group. ¹⁰ In addition to this, it was shown that cyanoacrylate acts as a barrier against microbial penetration and creates an environment that could enhance wound healing by serving as a reliable wound dressing. ³⁹

For a better understanding of the indications and applicability of the adhesive technique, further studies with a higher number of subjects are necessary to support the use of adhesive in a safe manner. Although anastomotic time is an important variable to take in consideration in microsurgery, it should not surpass the potential thrombotic complications which can lead to the failure of the anastomosis and catastrophic outcomes (eg, flap loss). The potential time saving may not be justified if there is any increased risk of thrombosis, but it should be taken into consideration.

Moreover, a longer follow-up period of the subjects can determine the possible late complications and patency rate of the anastomosis overtime.

Conclusion

Within a randomized study of 24 anastomoses performed on rat femoral arteries with either the simple interrupted suture or tissue glue, anastomotic times were significantly lower with a non-significant trend toward more thrombotic complications in the tissue glue group. Further improvement of the glue properties and refinement of the technique will likely make it a viable alternative to interrupted suturing in the future. Thrombotic events are considered critical in microsurgery; therefore, the reduction of the anastomotic time solely may not be justified if there is any increased risk of thrombosis, further studies are necessary in order to support the safety and efficacy of tissue glue in performing microvascular anastomosis.