- Email: [email protected]
Sutureless microvascular anastomosis with the aid of heparin loaded poloxamer 407
Accepted Manuscript Sutureless microvascular anastomosis with the aid of heparin loaded poloxamer 407 Fırat Özer, MD, Mustafa Nişancı, MD, Professor, Çetin Taş, PhD, Associate Professor, Jayakumar Rajadas, PhD, Doğan Alhan, MD, Yalçın Bozkurt, MD, Armağan Günal, MD, Associate Professor, Serdar Demirtaş, MD, Professor, Selçuk Işık, MD, Proffesor PII:
S1748-6815(16)30473-9
DOI:
10.1016/j.bjps.2016.10.012
Reference:
PRAS 5140
To appear in:
Journal of Plastic, Reconstructive & Aesthetic Surgery
Received Date: 17 November 2015 Revised Date:
22 September 2016
Accepted Date: 26 October 2016
Please cite this article as: Özer F, Nişancı M, Taş Ç, Rajadas J, Alhan D, Bozkurt Y, Günal A, Demirtaş S, Işık S, Sutureless microvascular anastomosis with the aid of heparin loaded poloxamer 407, British Journal of Plastic Surgery (2016), doi: 10.1016/j.bjps.2016.10.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Authors: Fırat Özer, MD1, Mustafa Nişancı, Professor MD1, Çetin Taş, Associate Professor PhD2, Jayakumar Rajadas, PhD3, Doğan Alhan, MD1, Yalçın Bozkurt, MD4, Armağan Günal,
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Associate Professor MD5, Serdar Demirtaş, Professor MD6, Selçuk Işık, Proffesor, MD1 1. Gulhane Military Medical Academy, Department of Plastic Surgery, Ankara, Turkey
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2. Gulhane Military Medical Academy, Deparment of Pharmacy, Ankara, Turkey 3. Stanford University, Department of Bioengineering, Stanford, California, USA
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4. Gulhane Military Medical Academy, Department of Radiology, Ankara, Turkey 5. Gulhane Military Medical Academy, Department of Pathology, Ankara, Turkey 6. Gulhane Military Medical Academy, Department of Biophysics, Ankara, Turkey
Fırat Özer, MD.
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Corresponding author:
Department of Plastic, Reconstructive and Aesthetic Surgery,
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Gülhane Military Medical Academy GSM: +90 5532601816
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Fax: 904423172264
E-mail: [email protected]
ACCEPTED MANUSCRIPT ABSTRACT Background Anastomosis with tissue adhesives is an alternative method for conventional anastomosis. However, this technique has several technical challenges. It requires using suture to
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prevent leakage into lumen and precise application on to all surfaces of the anastomosis site. In order to solve these problems poloxamer 407 (P 407) was used as a stent previously. In this study, we made heparinized P 407 (h-P 407) as a new formula. We aimed to use h-P 407 as a stent in sutureless anastomosis successfully in the rat abdominal
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aorta model.
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Methods
Sixty Sprague- Dawley rats were used. In the first group, end-to-end anastomoses were performed with suture, in the second and third groups; sutureless anastomoses were performed with 2-octyl cyanoacrylate. As an intraluminal stent, poloxamer 407 (P 407) was used in the second group heparinized poloxamer 407 (h-P 407) was used in the third group. Anastomosis time is measured. Lumen width, intimal hyperplasia and foreign body
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reaction were assessed histologically. Velocity flow rates and vessel diameters were measured radiological. Burst strength was measured. The results were evaluated statistically.
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Results
Sutureless anastomosis was more rapid than conventional anastomosis. Lumen width was
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narrower in the suture group. İnflammation and foreign body reaction was more severe in the suture group. There was no radiologic and biomechanical difference in all groups. We found intimal hyperplasia was less in h-P 407 than in P 407. Conclusions
Heparinized P407 can be used as an intraluminal stent for sutureless microvascular anastomosis with tissue adhesives successfully.
ACCEPTED MANUSCRIPT INTRODUCTION Vascular anastomosis is the most critical step in all microsurgery. The conventional hand- sewn method is still the most preferred technique. However, prolonged surgery, technical difficulties, intimal hyperplasia and foreign body reaction may all render this
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technique challenging with major disadvantages1, 2. In order to overcome technical difficulties and possible suture-related complications, many alternative techniques have been defined. However, no superior method has found when comparing with the conventional suture method3-6. Anastomosis with 2- octyl cyanoacrylate as a tissue
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adhesive is one of these alternative methods. This method still needs further refinement for its possible use in routine surgical practice because of adhesives leakage into the lumen1.
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Edward et al used Poloxamer 407 (P 407) gel as a stent in vessel ends and carried out sutureless anastomosis with 2- octyl cyanoacrylate7. Poloxamers are chemically a triblock of nanoparticles, which composed of two-polyethylene oxide (PEO) and a polypropylene oxide (PPO). Thermo reversibility is the worthy property of P 407. It means P 407 can be in gel phase at above the transition temperature and it can turn back to liquid phase at the body temperature. When it is in the gel form, P 407 has been shown to have
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adequate elastic modulus to maintain an open vessel lumen. Moreover, in pharmaceutical technology, poloxamer gels have been used as delivery vehicles for various agents such as chemotherapeutics, vaccines and antiviral drugs8-10.
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In this study, heparinized P 407 (h-P 407) solution is formulized, produced and
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used as stent for sutureless anastomosis with 2-octyl cyanoacrylate.
MATERIAL AND METHODS 1. Preparing formulation The poloxamer is prepared as described by Edward et al7. Briefly, in order to
prepare the formulation, 1.65 g P 407 (BSAF, Parsipanny, NJ, USA) and 0.025 g BSA (Millipore, Bedford, MA, USA) were mixed, and phosphate buffered saline (PBS) was added to yield a 10 ml of solution. Only for third group, 5000 unit (IU) heparin (St Louis, MO, USA) was added into this basic formulation. Before using this new solution, we
ACCEPTED MANUSCRIPT tested heparin whether it mixed liquid phase of the poloxamer in a diffuse manner. The formulation, that we finally achieved (16.5% P 407 and 0.25% BSA) has been shown to provide adequate elastic modulus at 39-40˚C to maintain vessel lumen open and it could revert to its liquid phase at the physiologic body temperature safely in vitro studies.
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2. Animals
Rat abdominal aorta model was preferred for our experimental study11. Sixty Sprague- Dawley rats, weighing 300-350 gr were used after being divided into hand- sewn suture group, pure P 407 group and heparin loaded P 407 group. Each group is consisted of
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20 rats. The study was carried out according to the procedures approved by the Animal Research Committee of Gülhane Military Medical Academy. All the animals were used in
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this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the ethic council.
3. Surgical procedure
All surgical procedures were performed under general anesthesia. General anesthesia was induced with intramuscular ketamine 10% (90 mg/kg) and xylasine 2% (10
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mg/kg). Abdominal aorta dissections and anastomoses were performed under X20 surgical magnification (Carl Zeiss, OPMI pentero, Germany). Under general anesthesia median laparotomy incision was done. Intestines were lateralized and covered with warm sterile gauze dressing. Abdominal aorta segment (so-
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called infra renal aorta which is approximately 13 mm length) between renal branches and iliaca bifurcation was dissected. As abdominal aorta gives two iliolumbal branches in the
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middle of this segment, these two branches were ligated with sutures instead of coagulation to prevent thermal damage to the anastomosis site. Consequently, ligated iliolumbal vessels, which located near the anastomosis site, were not related with anastomosis (Figure 1). After this step, infra renal aorta was surgically exposed and its adventitia was denuded off. End- to- end anastomoses were performed at this level of the aorta. The lumens were irrigated with serum saline before anastomosis in all groups. No agent for vasodilation was used in this step of procedure. Acland type approximator was used for anastomoses.
ACCEPTED MANUSCRIPT In the first group (suture group), hand-sewn anastomoses were carried out with 1012 interrupted 10/0 nylon sutures (Doğsan®, Turkey). In the second group, sutureless anastomoses were performed with using 2-octyl cyanoacrylate (Glubran®, Italy) and in this group P 407 was used as a stent. In the third group, sutureless anastomoses were performed with 2- octyl cyanoacrylate (Glubran®) and h-P 407 was used as a stent.
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In the second group, totally 0,3 ml P 407 gel was injected into the vessel lumens. In the third group, 0,3 ml h-P 407 (contained 150 IU heparin12) was injected into the vessel lumens. In these poloxamer groups, both P 407 formulations (pure and heparinized) were heated above the transition temperature (39-40˚C) before their infusion into the divided
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vessel ends to maintain an open lumen (Figure 1). In order to achieve maximum elastic modulus during the procedure, radiant halogen heat source was used to keep the
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temperature of the surgical field just above the phase-transition temperature. The temperature was monitorized and was held it below the 43 0C threshold of thermal damage13. After exact approximation of gel-filled lumens of the vessel ends, 2octylcyanoacyrlate was applied in a circumferential manner (Figure 2) to complete the anastomosis. Before releasing microvascular clamps, we waited approximately five minutes for polymerization of 2-octylcyanoacyrlate.
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At the end of the surgery, patencies were confirmed by milking test after implementation of anastomosis. No intraoperative or postoperative complication was encountered in either group. All rats survived without surgery related complications (Video 1).
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4. Evaluation
In each group, data pertaining to the measurements of anastomosis time
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(intraoperative), radiologic imaging (at the sixth week), histological investigations (at the first and sixth weeks), and biomechanical tests (at the sixth week) were obtained and recorded for comparison. Anastomosis time was described as the time elapsed from arteriotomy until removal
of vessel clamps following implementation of the anastomosis and measured intraoperatively (n=20). At the first week 10 rats were sacrificed in each groups for histological investigations. The vessel segments with 18 mm included anastomosis sites were sampled and fixed in % 10 formalin solution for 24 hours. The specimens were embedded in
ACCEPTED MANUSCRIPT paraffin block, after that 4 micron thickness sections were taken and stained with hematoxylin and eosin (H&E). Histologically, lumen width and vessel wall thickness were measured (DP Manager- Olympus BXSI, Olympus Co, Japan) and the numbers of inflammatory cells and multinuclear giant cells were counted at the anastomosis site in high power field (x400) (Figure 3).
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At sixth week, the abdominal aortas were assessed between diaphragmatic hiatus to iliac bifurcation with using Doppler-Ultrasound (General electric logiq ultrasound and 14 mHz lineer prob). This examination enabled us to obtain intra vital measurements of the vessel diameters and the blood flow rate (n=10). CT angiogram images were taken with
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TOSHIBA Aquilion spiral CT with one 320 detector. The images were obtained with 1 mm vessel slice thickness, 0.5 intersection gaps. These angiograms were utilized to assess
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the vessel diameters more precisely (n=10).
At the sixth week, after radiologic imaging, histological evaluation was made at the sixth week (n=5). Same technique was used and same parameters were evaluated as at the first week histologic investigation.
Burst strengths of the anastomoses were measured using a custom-designed system at sixth week (n=5). 18 mm aorta segments were used. One end of the vessel was fixed and
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other end was pulled with 0.05 cm/second to opposite direction with custom made device. The values were measured and recorded at the time when the anastomosis sites were broken down.
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5. Statistical analysis
Statistical analysis was performed with SPSS version 15.0 statistical package
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program. Descriptive statistics were presented as mean and standard deviation. One-way ANOVA and posthoc Tukey test were used to compare groups. A p value of <0.05 was accepted as statistically significant.
RESULTS 1. Anastomosis-time
ACCEPTED MANUSCRIPT The recorded times elapsed for anastomoses revealed that traditional hand-sewn anastomosis was much more time consuming with a mean anastomosis-time of 35.5±1.8 min, in comparison to the sutureless technique that had a mean anastomosis time of 8±0.8
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min (p<0.001).
2. Imaging techniques
Ultrasound-Doppler examinations displayed no observable differences in vessel
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diameters (1.1 mm in the all groups, p=1.00), (Figure 4), and only verified the patency of anastomoses site with similar volumetric flow rates (14.3±0.5 cm/min in suture group, 14.6 ±0.5 cm/min in P 407 group, and 14.5±0.6 cm/min in h-P 407 group), (p=0.568). In
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addition to that, the patencies were also confirmed with CT-angiogram studies without any statistically important difference between the groups (1.1 mm in all groups), (p=1.00), (Figure 5).
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2. Histologic Evaluations
In both early and late histological investigations, lumen widths were found to be narrower in suture-group compared to the two sutureless groups at first week (p<0.001), and at sixth weeks (p=0.001) (Table 1), while there was no statistically significant
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difference between P 407 and heparin-loaded P 407 groups (p>0.05).
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Histological measurements of the vessel-wall thickness revealed that vessel walls were thicker in the suture-group both at first week (p<0.001) and at sixth week (p<0.001), (Table 1). In addition to that, the measurements obtained at first week disclosed that vessel walls were thinner in the heparin loaded P 407 group than the ones in the P 407 group with a statistically significant difference (p<0.001) (Figure 6). At the sixth week there is no statistical difference between P 407 and h-P 407 groups (p=1.00).
More inflammatory cells were counted in the suture group than in the suturelessgroups at first week (p<0.001). More inflammatory cells were in P 407 group than in h-P 407group (p<0.001). No statistically significant difference was found between the groups
ACCEPTED MANUSCRIPT in terms of inflammatory-cell counts obtained at sixth week (p>0.05) (Figure 7). On the contrary, the quantity of the giant cells were observed to have been higher in suture-group than the sutureless-groups with a statistically significant difference at sixth week (p<0.001), whereas there was no statistically significant difference between the giant-cell
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counts of all groups at first week (p=1.00).
4. Biomechanical tests
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The mean burst-strength forces were measured 40 gram- forces in all groups (p=1.00). With respect to burst-strength force, the only statistically significant difference was obtained when a native vessel (200 gram- forces) was compared with the study groups
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(p=0.001).
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DISCUSSION
When bio adhesives came out to be magic products that were able to facilitate many surgical procedures, researchers were intrigued by the idea of joining the vessels with an available tissue adhesive in a sutureless manner14, 15. Indeed, use of tissue adhesives instead
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of sutures might be a fascinating technique of performing anastomosis that could circumvent the major disadvantages associated with the classical suture-technique
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including time-consuming and challenging surgical labor, vessel wall trauma and foreign body reaction against suture material. On the other hand, requirement for precise alignment of the vessel ends while open
lumens are maintained and almost inevitable leakage of the adhesive into the vessels appeared to be the major drawbacks of any technique in which tissue adhesives may be used instead of sutures16. Even though it could be used, employment of anchoring sutures in order to ensure a precise end-to-end alignment with open lumens will certainly wipe out the advantages attributed to using tissue adhesives instead of sutures. Hence, ongoing
ACCEPTED MANUSCRIPT researches have recently focused on improving a better technique that can rule out the limitations of using tissue adhesives for anastomosis. In an effort to achieve a patent anastomosis by joining vessel ends with tissue adhesives, using a transient intravascular stent would be a wise approach not only in terms
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of maintaining an open lumen during the procedure but also in terms of preventing adhesive leakage into the vessel. Accordingly, searching for a proper intraluminal stent has become a new subject of interest for the researchers. Recently, Edward et al7 studied on Poloxamer 407 gel in detail for its potential use in sutureless anastomosis, and then carried
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out first completely sutureless microvascular anastomosis with success in rat abdominalaorta model by using this gel as an intraluminal stent. In addition, Ying-Zheng et al17 designed novel P 407- low molecular weight heparin solution and used this new hydrogel
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at the sutureless anastomosis successfully. They had report no any undesirable changes with the thermo reversible feature of P 407.
Under the light of above-mentioned knowledges18- 21, we added a therapeutic dose of heparin into the P 407-formulation to obtain an antithrombotic-loaded intravascular gelstent that is to be used in sutureless microvascular anastomosis, hoping that it would
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release heparin locally while it dissolves in the body temperature. In the heparin-loaded P407 group, we injected totally 0.3 ml of ploxamer gel containing 150 U of heparin into vessel ends and experienced no heparin-related hemorrhagic complication12, 22.
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Total anastomosis time was four times more rapid in sutureless technique than in hand- sewn technique. No significant difference between P 407 and heparin added P 407 groups. We did not find any difference between the all groups with imaging evaluations
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and burst strength. Unlike Edward et al.7, we found burst strength 5- fold weaker when comparing with native untouched vessel. In histologic evaluations, we found statistically no significant differences in terms
of lumen width, inflammation, and foreign body reaction at first and sixth weeks. The most striking result, we achieved, was the statistically significant difference between the P 407 group and heparin loaded P 407 in terms of vessel wall thickness at first week. We inferred that this was due to thinner pseudo-intima formation in heparin loaded P 407 group. But it would better when we labeled heparin and showed its releasing from the gel. Therefore, a disadvantage of this study is we could not estimate effect of heparin is topical or systemic.
ACCEPTED MANUSCRIPT We believe that residue heparin is likely to be at the anastomosis site, but this idea requires to be verified with further investigation. However, before application this method to human, some questions about fate of poloxamer in the distal circulation and end organs should be clarified. Consequently, further studies should be necessary. Furthermore, we performed anastomosis with same
This method also can be applicable to both arteries and veins.
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size vessels. This anastomosis technique is more suitable for the vessel with same size.
In conclusion, P 407 could be used as a reliable intraluminal stent to facilitate sutureless microvascular anastomosis with tissue adhesives, and when it is added with
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heparin, early probable thrombotic complications due to thick pseudo intima formation
Acknowledgement
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may be reduced.
We would like to thanks to Dr. Fatih Zor, Dr. Selim Kılıç and Dr. Guven Uysal for their
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Funding: None
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helps with performing this study.
Conflicts of interest: None declared
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Ethical approval: Not required
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Lefeng Q, Zaiping J, Yuqi W. Sutureless anastomosis of small and medium size vessels by medical adhesive. European J Vasc and Endovasc Surg. 2004;28(5):526533. Berggren A, Ostrup LT, Ragnarsson R. Clinical experience with the Unilink/ 3M
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Roques, C., Salmon, A., Fiszman, M.Y., Fattal, E. & Fromes, Y. Intrapericardial administration of novel DNA formulations based on thermosensitive poloxamer 407 gel. Int. J. Pharm. 2007;331: 220–223.
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Habal SM, Fitzpatrick HF, Gree GE. Training in microvascular surgery. Surgery. 1977;81:596-8.
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Braam MJ, Cooley BC, Gould JS. Topical heparin enhanced patency in a rat model of arterial thrombosis. Ann Plast Surg.1995;34(2):148-151.
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Hume, S.P., Marigold, J.C. & Michalowski, A. The effect of local hyperthermia on
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Bot GM, Bot KG, Ogunranti JO, Onah JA, Sule AZ, Hassan I, Dung ED. The use of
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Ong, Y.S. et al. 2-Octylcynanoacrylate-assisted microvascular anastomosis in a rat
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model: long-term biomechanical properties and histological changes. Microsurgery. 2004; 24, 304–308. 17.
Ying-Zheng et al. Evaluation of a novel thermosensitive heparin- poloxamer
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hydrogel for improving vascular anastomosis quality and safety in a rabbit model. Plos One. 2013;8(8):e73178. Debra H, Martin E, Joann M, Omer I, Jawed F. Effected of purified poloxamer 188
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and various dextrans on erythrocyte sedimentation rate in healthy subjects and
patients with sickle cell disease. Blood.2013;122(21):4764.
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Gu JH, Ge JB, Li M, Xu HD, Wu F, Qin ZH. Poloxamer 188 protects neurons against ischemia/ reperfusion injury through preserving integrity of cell membranes and blood brain barrier. PloS one.2013;8(4):1641.
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Butt Am, Mohd Amin MC, Katas H. Synergistic effect of pH- responsive folatefunctionalized poloxamer 407- TPGS- mixed micelles on targeted delivery of anticancer drugs. Int J Nanomedicine.2015;13(10):1321-1334. Grindel J.M, Jaworski T, Piraner O, Emanuele R.M, Balasubramanian M.
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Distribution, metabolism, and excretion of a novel surface-active agent, purified poloxamer 188, in rats, dogs and humans. J Pharm Sci.2002;91:1936-1947.
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Rumbolo, P. M., Cooley, B. C., Hanel, D. P., and Gould, J. S. Comparison of the influence of intralumenal irrigation solutions on free flap survival. Microsurgery
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.1992;13:45.
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22.
ACCEPTED MANUSCRIPT Figure legends
Figure 1: Poloxamer gel which is injected at 40 °C (above transition temperature) maintain open vessel ends during the 2- octylcyanoacrylate application. Iliolumbal
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vessels that were ligated with sutures located near the anastomosis site. Consequently, sutures, which are seen in the figure, are not related with anastomosis.
Figure 2: 2-octylcyanoacrylate was applied in a circumferential manner.
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Figure 3: H&E staining of hand-sewn anastomosis tissue section at 6 weeks after
operation. Arrow shows anastomosis site. Lumen width, vessel wall thickness, the
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numbers of inflammatory cells and multinuclear giant cells were evaluated at the anastomosis site in high power field (x400).
Figure 4: Anastomoses sites are shown with Ultrasound- Doppler. Figure 5: CT- angiogram images of infra renal aorta. Anastomoses sites were marked
poloxamer groups.
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with arrows. (A) Anastomosis site was at suture group. (B) Anastomosis site was at
Figure 6: Vessel wall was thicker in suture group than in sutureless P 407 and h-P
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407 groups. In addition, vessel walls were thinner in the heparin loaded P 407 group
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than the ones in the P 407 group. Figure 7: Inflammatory cells are shown at the anastomoses sites. (A) Inflammatory cells located at the anastomosis site in suture group (B). Inflammatory cells accumulated around the 2- octyl cyanoacrylate application site than at the anastomosis site in the poloxamer groups.
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Vessel wall thickness
(mean±SD)
(micrometer)
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Lumen width (micrometer)
(mean±SD)
6.week
1. week
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1. week
6. week
465.0±4.0
570.0±1.414
140.0±2.748
120.0±2.000
P 407
474.0±6.1a
575.0±1.581a
122.0±1.247a
90.0±1.581a
h-P 407
472.2±1.7a
574.0±2.645a
95.0±1.563a,b
90.0±1.581a
P
<0.001
0.001
<0.001
<0.001
b p<0.05 versus P 407 group
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a p<0.05 versus suture group
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Suture
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Table 1: Vessel lumen is narrower in the suture group than in the poloxamer groups at the first and sixth weeks. Vessel wall is thicker in the poloxamer group than in the
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poloxamer groups at the first and sixth weeks. In addition, vessel wall is thinner in the h-P 407 group than in the P 407 group.
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S1748-6815(16)30473-9
DOI:
10.1016/j.bjps.2016.10.012
Reference:
PRAS 5140
To appear in:
Journal of Plastic, Reconstructive & Aesthetic Surgery
Received Date: 17 November 2015 Revised Date:
22 September 2016
Accepted Date: 26 October 2016
Please cite this article as: Özer F, Nişancı M, Taş Ç, Rajadas J, Alhan D, Bozkurt Y, Günal A, Demirtaş S, Işık S, Sutureless microvascular anastomosis with the aid of heparin loaded poloxamer 407, British Journal of Plastic Surgery (2016), doi: 10.1016/j.bjps.2016.10.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Authors: Fırat Özer, MD1, Mustafa Nişancı, Professor MD1, Çetin Taş, Associate Professor PhD2, Jayakumar Rajadas, PhD3, Doğan Alhan, MD1, Yalçın Bozkurt, MD4, Armağan Günal,
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Associate Professor MD5, Serdar Demirtaş, Professor MD6, Selçuk Işık, Proffesor, MD1 1. Gulhane Military Medical Academy, Department of Plastic Surgery, Ankara, Turkey
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2. Gulhane Military Medical Academy, Deparment of Pharmacy, Ankara, Turkey 3. Stanford University, Department of Bioengineering, Stanford, California, USA
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4. Gulhane Military Medical Academy, Department of Radiology, Ankara, Turkey 5. Gulhane Military Medical Academy, Department of Pathology, Ankara, Turkey 6. Gulhane Military Medical Academy, Department of Biophysics, Ankara, Turkey
Fırat Özer, MD.
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Corresponding author:
Department of Plastic, Reconstructive and Aesthetic Surgery,
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Gülhane Military Medical Academy GSM: +90 5532601816
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Fax: 904423172264
E-mail: [email protected]
ACCEPTED MANUSCRIPT ABSTRACT Background Anastomosis with tissue adhesives is an alternative method for conventional anastomosis. However, this technique has several technical challenges. It requires using suture to
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prevent leakage into lumen and precise application on to all surfaces of the anastomosis site. In order to solve these problems poloxamer 407 (P 407) was used as a stent previously. In this study, we made heparinized P 407 (h-P 407) as a new formula. We aimed to use h-P 407 as a stent in sutureless anastomosis successfully in the rat abdominal
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aorta model.
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Methods
Sixty Sprague- Dawley rats were used. In the first group, end-to-end anastomoses were performed with suture, in the second and third groups; sutureless anastomoses were performed with 2-octyl cyanoacrylate. As an intraluminal stent, poloxamer 407 (P 407) was used in the second group heparinized poloxamer 407 (h-P 407) was used in the third group. Anastomosis time is measured. Lumen width, intimal hyperplasia and foreign body
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reaction were assessed histologically. Velocity flow rates and vessel diameters were measured radiological. Burst strength was measured. The results were evaluated statistically.
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Results
Sutureless anastomosis was more rapid than conventional anastomosis. Lumen width was
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narrower in the suture group. İnflammation and foreign body reaction was more severe in the suture group. There was no radiologic and biomechanical difference in all groups. We found intimal hyperplasia was less in h-P 407 than in P 407. Conclusions
Heparinized P407 can be used as an intraluminal stent for sutureless microvascular anastomosis with tissue adhesives successfully.
ACCEPTED MANUSCRIPT INTRODUCTION Vascular anastomosis is the most critical step in all microsurgery. The conventional hand- sewn method is still the most preferred technique. However, prolonged surgery, technical difficulties, intimal hyperplasia and foreign body reaction may all render this
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technique challenging with major disadvantages1, 2. In order to overcome technical difficulties and possible suture-related complications, many alternative techniques have been defined. However, no superior method has found when comparing with the conventional suture method3-6. Anastomosis with 2- octyl cyanoacrylate as a tissue
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adhesive is one of these alternative methods. This method still needs further refinement for its possible use in routine surgical practice because of adhesives leakage into the lumen1.
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Edward et al used Poloxamer 407 (P 407) gel as a stent in vessel ends and carried out sutureless anastomosis with 2- octyl cyanoacrylate7. Poloxamers are chemically a triblock of nanoparticles, which composed of two-polyethylene oxide (PEO) and a polypropylene oxide (PPO). Thermo reversibility is the worthy property of P 407. It means P 407 can be in gel phase at above the transition temperature and it can turn back to liquid phase at the body temperature. When it is in the gel form, P 407 has been shown to have
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adequate elastic modulus to maintain an open vessel lumen. Moreover, in pharmaceutical technology, poloxamer gels have been used as delivery vehicles for various agents such as chemotherapeutics, vaccines and antiviral drugs8-10.
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In this study, heparinized P 407 (h-P 407) solution is formulized, produced and
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used as stent for sutureless anastomosis with 2-octyl cyanoacrylate.
MATERIAL AND METHODS 1. Preparing formulation The poloxamer is prepared as described by Edward et al7. Briefly, in order to
prepare the formulation, 1.65 g P 407 (BSAF, Parsipanny, NJ, USA) and 0.025 g BSA (Millipore, Bedford, MA, USA) were mixed, and phosphate buffered saline (PBS) was added to yield a 10 ml of solution. Only for third group, 5000 unit (IU) heparin (St Louis, MO, USA) was added into this basic formulation. Before using this new solution, we
ACCEPTED MANUSCRIPT tested heparin whether it mixed liquid phase of the poloxamer in a diffuse manner. The formulation, that we finally achieved (16.5% P 407 and 0.25% BSA) has been shown to provide adequate elastic modulus at 39-40˚C to maintain vessel lumen open and it could revert to its liquid phase at the physiologic body temperature safely in vitro studies.
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2. Animals
Rat abdominal aorta model was preferred for our experimental study11. Sixty Sprague- Dawley rats, weighing 300-350 gr were used after being divided into hand- sewn suture group, pure P 407 group and heparin loaded P 407 group. Each group is consisted of
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20 rats. The study was carried out according to the procedures approved by the Animal Research Committee of Gülhane Military Medical Academy. All the animals were used in
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this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the ethic council.
3. Surgical procedure
All surgical procedures were performed under general anesthesia. General anesthesia was induced with intramuscular ketamine 10% (90 mg/kg) and xylasine 2% (10
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mg/kg). Abdominal aorta dissections and anastomoses were performed under X20 surgical magnification (Carl Zeiss, OPMI pentero, Germany). Under general anesthesia median laparotomy incision was done. Intestines were lateralized and covered with warm sterile gauze dressing. Abdominal aorta segment (so-
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called infra renal aorta which is approximately 13 mm length) between renal branches and iliaca bifurcation was dissected. As abdominal aorta gives two iliolumbal branches in the
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middle of this segment, these two branches were ligated with sutures instead of coagulation to prevent thermal damage to the anastomosis site. Consequently, ligated iliolumbal vessels, which located near the anastomosis site, were not related with anastomosis (Figure 1). After this step, infra renal aorta was surgically exposed and its adventitia was denuded off. End- to- end anastomoses were performed at this level of the aorta. The lumens were irrigated with serum saline before anastomosis in all groups. No agent for vasodilation was used in this step of procedure. Acland type approximator was used for anastomoses.
ACCEPTED MANUSCRIPT In the first group (suture group), hand-sewn anastomoses were carried out with 1012 interrupted 10/0 nylon sutures (Doğsan®, Turkey). In the second group, sutureless anastomoses were performed with using 2-octyl cyanoacrylate (Glubran®, Italy) and in this group P 407 was used as a stent. In the third group, sutureless anastomoses were performed with 2- octyl cyanoacrylate (Glubran®) and h-P 407 was used as a stent.
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In the second group, totally 0,3 ml P 407 gel was injected into the vessel lumens. In the third group, 0,3 ml h-P 407 (contained 150 IU heparin12) was injected into the vessel lumens. In these poloxamer groups, both P 407 formulations (pure and heparinized) were heated above the transition temperature (39-40˚C) before their infusion into the divided
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vessel ends to maintain an open lumen (Figure 1). In order to achieve maximum elastic modulus during the procedure, radiant halogen heat source was used to keep the
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temperature of the surgical field just above the phase-transition temperature. The temperature was monitorized and was held it below the 43 0C threshold of thermal damage13. After exact approximation of gel-filled lumens of the vessel ends, 2octylcyanoacyrlate was applied in a circumferential manner (Figure 2) to complete the anastomosis. Before releasing microvascular clamps, we waited approximately five minutes for polymerization of 2-octylcyanoacyrlate.
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At the end of the surgery, patencies were confirmed by milking test after implementation of anastomosis. No intraoperative or postoperative complication was encountered in either group. All rats survived without surgery related complications (Video 1).
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4. Evaluation
In each group, data pertaining to the measurements of anastomosis time
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(intraoperative), radiologic imaging (at the sixth week), histological investigations (at the first and sixth weeks), and biomechanical tests (at the sixth week) were obtained and recorded for comparison. Anastomosis time was described as the time elapsed from arteriotomy until removal
of vessel clamps following implementation of the anastomosis and measured intraoperatively (n=20). At the first week 10 rats were sacrificed in each groups for histological investigations. The vessel segments with 18 mm included anastomosis sites were sampled and fixed in % 10 formalin solution for 24 hours. The specimens were embedded in
ACCEPTED MANUSCRIPT paraffin block, after that 4 micron thickness sections were taken and stained with hematoxylin and eosin (H&E). Histologically, lumen width and vessel wall thickness were measured (DP Manager- Olympus BXSI, Olympus Co, Japan) and the numbers of inflammatory cells and multinuclear giant cells were counted at the anastomosis site in high power field (x400) (Figure 3).
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At sixth week, the abdominal aortas were assessed between diaphragmatic hiatus to iliac bifurcation with using Doppler-Ultrasound (General electric logiq ultrasound and 14 mHz lineer prob). This examination enabled us to obtain intra vital measurements of the vessel diameters and the blood flow rate (n=10). CT angiogram images were taken with
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TOSHIBA Aquilion spiral CT with one 320 detector. The images were obtained with 1 mm vessel slice thickness, 0.5 intersection gaps. These angiograms were utilized to assess
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the vessel diameters more precisely (n=10).
At the sixth week, after radiologic imaging, histological evaluation was made at the sixth week (n=5). Same technique was used and same parameters were evaluated as at the first week histologic investigation.
Burst strengths of the anastomoses were measured using a custom-designed system at sixth week (n=5). 18 mm aorta segments were used. One end of the vessel was fixed and
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other end was pulled with 0.05 cm/second to opposite direction with custom made device. The values were measured and recorded at the time when the anastomosis sites were broken down.
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5. Statistical analysis
Statistical analysis was performed with SPSS version 15.0 statistical package
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program. Descriptive statistics were presented as mean and standard deviation. One-way ANOVA and posthoc Tukey test were used to compare groups. A p value of <0.05 was accepted as statistically significant.
RESULTS 1. Anastomosis-time
ACCEPTED MANUSCRIPT The recorded times elapsed for anastomoses revealed that traditional hand-sewn anastomosis was much more time consuming with a mean anastomosis-time of 35.5±1.8 min, in comparison to the sutureless technique that had a mean anastomosis time of 8±0.8
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min (p<0.001).
2. Imaging techniques
Ultrasound-Doppler examinations displayed no observable differences in vessel
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diameters (1.1 mm in the all groups, p=1.00), (Figure 4), and only verified the patency of anastomoses site with similar volumetric flow rates (14.3±0.5 cm/min in suture group, 14.6 ±0.5 cm/min in P 407 group, and 14.5±0.6 cm/min in h-P 407 group), (p=0.568). In
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addition to that, the patencies were also confirmed with CT-angiogram studies without any statistically important difference between the groups (1.1 mm in all groups), (p=1.00), (Figure 5).
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2. Histologic Evaluations
In both early and late histological investigations, lumen widths were found to be narrower in suture-group compared to the two sutureless groups at first week (p<0.001), and at sixth weeks (p=0.001) (Table 1), while there was no statistically significant
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difference between P 407 and heparin-loaded P 407 groups (p>0.05).
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Histological measurements of the vessel-wall thickness revealed that vessel walls were thicker in the suture-group both at first week (p<0.001) and at sixth week (p<0.001), (Table 1). In addition to that, the measurements obtained at first week disclosed that vessel walls were thinner in the heparin loaded P 407 group than the ones in the P 407 group with a statistically significant difference (p<0.001) (Figure 6). At the sixth week there is no statistical difference between P 407 and h-P 407 groups (p=1.00).
More inflammatory cells were counted in the suture group than in the suturelessgroups at first week (p<0.001). More inflammatory cells were in P 407 group than in h-P 407group (p<0.001). No statistically significant difference was found between the groups
ACCEPTED MANUSCRIPT in terms of inflammatory-cell counts obtained at sixth week (p>0.05) (Figure 7). On the contrary, the quantity of the giant cells were observed to have been higher in suture-group than the sutureless-groups with a statistically significant difference at sixth week (p<0.001), whereas there was no statistically significant difference between the giant-cell
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counts of all groups at first week (p=1.00).
4. Biomechanical tests
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The mean burst-strength forces were measured 40 gram- forces in all groups (p=1.00). With respect to burst-strength force, the only statistically significant difference was obtained when a native vessel (200 gram- forces) was compared with the study groups
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(p=0.001).
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DISCUSSION
When bio adhesives came out to be magic products that were able to facilitate many surgical procedures, researchers were intrigued by the idea of joining the vessels with an available tissue adhesive in a sutureless manner14, 15. Indeed, use of tissue adhesives instead
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of sutures might be a fascinating technique of performing anastomosis that could circumvent the major disadvantages associated with the classical suture-technique
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including time-consuming and challenging surgical labor, vessel wall trauma and foreign body reaction against suture material. On the other hand, requirement for precise alignment of the vessel ends while open
lumens are maintained and almost inevitable leakage of the adhesive into the vessels appeared to be the major drawbacks of any technique in which tissue adhesives may be used instead of sutures16. Even though it could be used, employment of anchoring sutures in order to ensure a precise end-to-end alignment with open lumens will certainly wipe out the advantages attributed to using tissue adhesives instead of sutures. Hence, ongoing
ACCEPTED MANUSCRIPT researches have recently focused on improving a better technique that can rule out the limitations of using tissue adhesives for anastomosis. In an effort to achieve a patent anastomosis by joining vessel ends with tissue adhesives, using a transient intravascular stent would be a wise approach not only in terms
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of maintaining an open lumen during the procedure but also in terms of preventing adhesive leakage into the vessel. Accordingly, searching for a proper intraluminal stent has become a new subject of interest for the researchers. Recently, Edward et al7 studied on Poloxamer 407 gel in detail for its potential use in sutureless anastomosis, and then carried
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out first completely sutureless microvascular anastomosis with success in rat abdominalaorta model by using this gel as an intraluminal stent. In addition, Ying-Zheng et al17 designed novel P 407- low molecular weight heparin solution and used this new hydrogel
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at the sutureless anastomosis successfully. They had report no any undesirable changes with the thermo reversible feature of P 407.
Under the light of above-mentioned knowledges18- 21, we added a therapeutic dose of heparin into the P 407-formulation to obtain an antithrombotic-loaded intravascular gelstent that is to be used in sutureless microvascular anastomosis, hoping that it would
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release heparin locally while it dissolves in the body temperature. In the heparin-loaded P407 group, we injected totally 0.3 ml of ploxamer gel containing 150 U of heparin into vessel ends and experienced no heparin-related hemorrhagic complication12, 22.
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Total anastomosis time was four times more rapid in sutureless technique than in hand- sewn technique. No significant difference between P 407 and heparin added P 407 groups. We did not find any difference between the all groups with imaging evaluations
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and burst strength. Unlike Edward et al.7, we found burst strength 5- fold weaker when comparing with native untouched vessel. In histologic evaluations, we found statistically no significant differences in terms
of lumen width, inflammation, and foreign body reaction at first and sixth weeks. The most striking result, we achieved, was the statistically significant difference between the P 407 group and heparin loaded P 407 in terms of vessel wall thickness at first week. We inferred that this was due to thinner pseudo-intima formation in heparin loaded P 407 group. But it would better when we labeled heparin and showed its releasing from the gel. Therefore, a disadvantage of this study is we could not estimate effect of heparin is topical or systemic.
ACCEPTED MANUSCRIPT We believe that residue heparin is likely to be at the anastomosis site, but this idea requires to be verified with further investigation. However, before application this method to human, some questions about fate of poloxamer in the distal circulation and end organs should be clarified. Consequently, further studies should be necessary. Furthermore, we performed anastomosis with same
This method also can be applicable to both arteries and veins.
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size vessels. This anastomosis technique is more suitable for the vessel with same size.
In conclusion, P 407 could be used as a reliable intraluminal stent to facilitate sutureless microvascular anastomosis with tissue adhesives, and when it is added with
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heparin, early probable thrombotic complications due to thick pseudo intima formation
Acknowledgement
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may be reduced.
We would like to thanks to Dr. Fatih Zor, Dr. Selim Kılıç and Dr. Guven Uysal for their
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Funding: None
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helps with performing this study.
Conflicts of interest: None declared
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Ethical approval: Not required
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ACCEPTED MANUSCRIPT Figure legends
Figure 1: Poloxamer gel which is injected at 40 °C (above transition temperature) maintain open vessel ends during the 2- octylcyanoacrylate application. Iliolumbal
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vessels that were ligated with sutures located near the anastomosis site. Consequently, sutures, which are seen in the figure, are not related with anastomosis.
Figure 2: 2-octylcyanoacrylate was applied in a circumferential manner.
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Figure 3: H&E staining of hand-sewn anastomosis tissue section at 6 weeks after
operation. Arrow shows anastomosis site. Lumen width, vessel wall thickness, the
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numbers of inflammatory cells and multinuclear giant cells were evaluated at the anastomosis site in high power field (x400).
Figure 4: Anastomoses sites are shown with Ultrasound- Doppler. Figure 5: CT- angiogram images of infra renal aorta. Anastomoses sites were marked
poloxamer groups.
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with arrows. (A) Anastomosis site was at suture group. (B) Anastomosis site was at
Figure 6: Vessel wall was thicker in suture group than in sutureless P 407 and h-P
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407 groups. In addition, vessel walls were thinner in the heparin loaded P 407 group
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than the ones in the P 407 group. Figure 7: Inflammatory cells are shown at the anastomoses sites. (A) Inflammatory cells located at the anastomosis site in suture group (B). Inflammatory cells accumulated around the 2- octyl cyanoacrylate application site than at the anastomosis site in the poloxamer groups.
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Vessel wall thickness
(mean±SD)
(micrometer)
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Lumen width (micrometer)
(mean±SD)
6.week
1. week
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1. week
6. week
465.0±4.0
570.0±1.414
140.0±2.748
120.0±2.000
P 407
474.0±6.1a
575.0±1.581a
122.0±1.247a
90.0±1.581a
h-P 407
472.2±1.7a
574.0±2.645a
95.0±1.563a,b
90.0±1.581a
P
<0.001
0.001
<0.001
<0.001
b p<0.05 versus P 407 group
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a p<0.05 versus suture group
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Suture
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Table 1: Vessel lumen is narrower in the suture group than in the poloxamer groups at the first and sixth weeks. Vessel wall is thicker in the poloxamer group than in the
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poloxamer groups at the first and sixth weeks. In addition, vessel wall is thinner in the h-P 407 group than in the P 407 group.
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