Local effects of epidermal growth factor on the wound healing in esophageal anastomosis: An experimental study

https://doi.org/10.1016/j.ijporl.2017.05.013Get rights and content

Abstract

Objective

In this study with the experimental model of primary repair of esophageal atresia(EA), we investigated the effects of the epidermal growth factor(EGF) on wound healing in the anastomosis of EA.

Materials and methods

Forty rabbits that were performed a resection of a 1-cm segment of the cervical esophagus followed by the end-to-end anastomosis were divided into four groups. Group I (7-day group) and III (21-day group), the control groups, had no EGF applied to the anastomosis. In group II (7-day group) and group III (21-day group), all around the anastomoses were locally covered with 100 μg/kg EGF. Group 1, 2 and 3, 4 were sacrificed on the 7th and 21st day, respectively. We investigated and compared with bursting pressure, vascularization around the anastomotic line by histopathology, inflammation, and fibroblast granulation as well as hydroxyproline level by biochemical.

Results

No significant difference was found at vascularization and inflammation between all of the groups (p > 0.05). Considering the critical parameters of the groups, at bursting pressure difference was found between group 1 and 2 as well as 3 and (p < 0.05). At fibroblast granulation and hydroxyproline level, no difference was found between group 1 and 2 (p > 0.05) but the difference between group 3 and 4 (p < 0.05).

Conclusion

EGF might be useful in preventing anastomotic leakage of esophageal atresia in primary anastomosis of esophageal atresia by accelerating wound healing process and increasing the stability of the anastomotic line.

Introduction

Esophageal atresia (EA) occurring in one in 2500–3000 live births has successfully been treated for several decades in many countries [1]. However, anastomotic leakage remains currently to be one of the main problems in the surgery of esophageal atresia despite innovations and improvements in the surgical procedure such as the type of anastomosis employed and the extra-pleural approach [2].

Anastomotic leaks occur in 5–7% of cases in some series; however, there are comparable with the incidence figures ranging from 3% to 25% in previous studies. Major leaks occur within 48 h and may cause to life-threatening tension pneumothorax. Besides, these leaks are one of the important reason for anastomotic strictures in the long term [3], [4]. Disadvantages caused by physiological and anatomical features of the esophagus have as much importance as the efficacy of surgical procedures regarding the anastomotic leak. Because of the lack of serosal membrane and the natural thinness of the tunica muscularis as well as poor blood flow to the organ, the esophagus is at risk of anastomotic leak regardless of surgical procedure [5]. Furthermore, the degree of mobilization of the blind esophageal pouches, tension in the anastomosis zone, and the distance between the proximal and distal esophageal ends are of critical importance for wound healing after the primary anastomosis in EA [6], [7]. No matter what technique is used, accelerating wound healing in the esophageal anastomosis can reduce unwanted complications. When we look at the subject from this perspective; in the inflammation phase of acute wound healing, unless activated neutrophils disappear after the first 72 h, neutrophils continue to be present throughout the healing process due to tissue trauma by pressure, bacterial overgrowth, leukocyte trapping, or ischemic-reperfusion injury. If the events come to this stage, the transition to chronic wound healing and complications can be inevitable [8], [9].

In previous studies have been suggested that anastomotic leak and fistula resulting from the failure of surgical procedure or vulnerable structure of the digestive system can be reduced by using exogenous physiological substances locally such as Tisseel, a fibrin sealant [10], Cyanoacrylate [11] and Fibroblast Growth Factor [12]. Growth factors play a significant role in wound healing in the digestive system. EGF, being one of them, stimulates the proliferation and migration of all types of epithelial cells. It has been reported that EGF also has a significant role in the healing of the intestinal mucosa [13], [14], [15]. Of all specific articles related to EGF, the effect of EGF on acute and chronic wound healing showed that the promising effect for enhanced acute wound healing but had been accused of due to the limited impact on chronic wound healing and a connection between invasive neoplasm [16].

In anywhere of the body, the process of wound healing occurs in three stages: inflammation, proliferation, and remodeling, respectively [17]. EGF produced by a variety of sources, including platelets plays an important role in this process by stimulating fibronectin synthesis, angiogenesis, fibroplasia and collagenase activity. It also has a mitogenic effect on the epithelial cells, endothelial cells, and fibroblasts. After the formation of hemostatic plage in the inflammation phase, EGF regulates this process by promoting wound healing [8], [18].

For these reasons, we aimed to investigate the effects of local EGF applied to anastomosis line on the acute wound healing of an esophageal anastomosis in an animal model.

Section snippets

Materials and methods

Our experimental study was performed according to the Guide for the Care and Use of Laboratory Animals of the Necmettin Erbakan University. The Ethical Committee approved the study (Protocol; 2009/37). Forty 6-month-old female New Zealand rabbits weighing between 1650 and 3000 g (mean; 2240 ± 345.52 g) were used. Animals were housed in individual cages at a steady temperature of 21 °C and humidity of 55% in a 12-h light/dark (7:00 a.m.- 7:00 p.m.) cycle and were fed standard laboratory chow and

Surgical procedure

Once the rabbits fasted for 12 h, they were anesthetized with intramuscular 2 mg/kg xylazine (Alfazyne, Alfasan, Turkey) and 30 mg/kg ketamine hydrochloride (Ketalar, Pfizer, Turkey). Thirty minutes before the surgery, rabbits were given ceftriaxone (50 mg/kg; Iesef, Turkey) intramuscularly as a single dose. Because thoracic surgery would be more invasive than the cervical approach for rabbit, we preferred the cervical approach [9]. Under sterile conditions, the cervical esophagus was isolated

Determination of the mechanical strength of the anastomosis

As soon as a 3-cm segment of the esophagus with anastomosis was taken out, it was prepared for measuring the bursting pressure of the anastomosis as follows: the lumen of the removed segment was washed with saline through a fine catheter without any tissue injury. BP was measured pneumatically using a sphygmomanometer [19]. After two silicone catheters were fixed within the cleaned lumen and tied with a 2-0 silk suture at both ends, one of them was connected to an air infusion pump; the other

Histochemical analysis of the esophageal anastomosis

Immediately following the measurement of the bursting pressure, a 2-cm specimen with the anastomosis area of the resected esophageal segment was equally and vertically divided into two samples. One of them was placed in a neutral buffered formaldehyde solution for histopathologic evaluation. Another part was stored at −80 °C for determination of OHP levels.

Following the fixation of the specimens, paraffin sections were stained with Masson trichrome (HT15; Sigma, Milan, Italy) and

Statistical analysis

In all statistical analysis, SPSS (version 16.0; SPSS Inc, Chicago, USA) was used. All parameters were expressed as mean ± S.D. Pearson's chi-square test (χ2 test) was used for evaluating of categorical data with the histopathological results. One-way ANOVA followed by Tukey HSD test was performed to compare groups with the quantitative value (biochemical values) conforming to the normal distribution. Comparisons between all four groups with numeric values (bursting pressure) not fitting into

Results

When compared with control groups (I and III) and EGF administered groups (II and IV) in terms of BP, there were differences between I and II as well as III and IV (p = 0.000 and p = 0.000), respectively. Namely, the bursting pressures of study groups were higher than those of control groups. The considerable differences between the groups were not found as regard to the vascularization (p = 0.595) and inflammation (p = 0.341) scores at the anastomosis area.

In the comparison of tissue HP

Discussion

Various studies have demonstrated that EGF accelerated wound healing of full thickness skin lesions, corneal epithelial cell regeneration, and in the collagen deposition as well as the tensile strength of full thickness wounds [23]. After successful studies regarding application of exogenous EGF in the healing of acute surgical wounds had been published, it was reported in the literature that it showed limited efficacy in the chronic wound healing [24], [25], [26]. Neutrophil numbers in acute

Ethics approval and consent

Our experimental study was performed according to the Guide for the Care and Use of Laboratory Animals of the Necmettin Erbakan University. The Ethical Committee approved the study (Protocol; 2009/37).

Funding

This work was supported by the Necmettin Erbakan University Institution of Scientific Research Projects [grant numbers, 09202074].

Conflicts of interest

Nil.

Acknowledgments

Nil.

References (36)

  • J.F. Woessner

    The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid

    Arch. Biochem. Biophys.

    (1961)
  • F.J. Thornton et al.

    Healing in the gastrointestinal tract

    Surg. Clin. North Am.

    (1997)
  • I. Bergman et al.

    The determinations of hydroxyproline in urine hydrolysates

    Clin. Chim. Acta

    (1970)
  • U. Koltuksuz et al.

    Effects of caffeic acid phenethyl ester and epidermal growth factor on the development of caustic esophageal stricture in rats

    J. Pediatr. Surg.

    (2001)
  • J.Y. Tsai et al.

    Esophageal atresia and tracheoesophageal fistula: surgical experience over two decades

    Ann. Thorac. Surg.

    (1997)
  • L. Spitz

    Oesophageal atresia

    Orphanet J. Rare Dis.

    (2007)
  • T. Kovács et al.

    Healing of esophageal anastomoses performed with the biofragmentable anastomoses ring versus the end-to-end anastomoses stapler: comparative experimental study in dogs

    World Surg.

    (2003)
  • R.F. Diegelmann

    Excessive neutrophils characterize chronic pressure ulcers

    Wound Repair Regen.

    (2003)
  • Cited by (11)

    • Association of clinical factors with postoperative complications of esophageal atresia

      2021, Pediatrics and Neonatology
      Citation Excerpt :

      Only a restricted number of studies about EA therapy examined different strategies and described EA therapy in detail. Reduced wound healing and AF are important complications after EA surgery.6–8 This study aims at identifying clinical factors associated with postoperative complications, based on assessment of clinical features and outcome of patients treated in our children's hospital between January 2010 and September 2018.

    • The impact of implant abutment surface treatment with TiO<inf>2</inf> on peri-implant levels of angiogenesis and bone-related markers: a randomized clinical trial

      2019, International Journal of Oral and Maxillofacial Surgery
      Citation Excerpt :

      EGF is related to osteoblast differentiation and mineralization, as well as DNA synthesis, and is directly associated with rapid tissue regeneration after injuries30. It is a marker found in osteoblasts, osteoclasts, and endothelial cells31. An in vitro study found covalent immobilization of EGF in TiO2 nanotubes, demonstrating that the number and activity of mesenchymal cells increased significantly in the presence of this molecule32.

    View all citing articles on Scopus
    View full text