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Does a muscle flap accelerate wound healing of gastric wall defects compared with an omental flap?
International Journal of Surgery 18 (2015) 41e47
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International Journal of Surgery journal homepage: www.journal-surgery.net
Original research
Does a muscle flap accelerate wound healing of gastric wall defects compared with an omental flap? Masashi Hishida a, b, *, Kazuhiro Toriyama b, Shunjiro Yagi b, Katsumi Ebisawa b, Tsuyoshi Morishita b, Keisuke Takanari b, Yuzuru Kamei b a b
Department of Plastic and Reconstructive Surgery, Kasugai Municipal Hospital, 1-1-1 Takagi-cho, Kasugai 486-8510, Japan Department of Plastic and Reconstructive Surgery, Nagoya University Graduate School of Medicine, 65 Turumai-cho, Showa-ku, Nagoya 466-8550, Japan
h i g h l i g h t s Clinical reports have described successful use of muscle flaps to promote healing of visceral defects. We histologically and immunohistochemically compared muscle vs. omental flaps in rats with experimental gastric wall defects. Both flap types promoted healing, but omental flaps provoked less fibrous scarring in the long term.
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Article history: Received 15 August 2014 Received in revised form 14 March 2015 Accepted 25 March 2015 Available online 10 April 2015
Introduction: Most often used for reconstruction at superficial sites, a muscle flap recently was reported to promote clinical wound healing in a duodenal defect. We therefore examined whether a muscle flap could promote wound healing comparably to an omental flap in rats with gastric wall defects. Methods: After perforation of the centre of the anterior gastric wall, rats were divided into 2 groups. In the muscle group, a muscle flap was fixed to the defect; in the omentum group, an omental flap was placed over the defect. We histopathologically compared tissue responses during gastric wall healing. Results: While stratified villi had completely covered the defect by day 7 in both groups, scar maturation differed. Scar tissue persisted in the muscle group, but was gradually replaced by adipose tissue in the omentum group. Discussion: Both muscle and omental flaps accelerated gastric wall wound healing. Conclusion: A muscle flap is an excellent alternative for repair of gastric defects when no omental flap is available. © 2015 Published by Elsevier Ltd on behalf of IJS Publishing Group Limited.
Keywords: Muscle flap Omental flap Gastric wall defects Wound healing Rats
1. Introduction The omentum, a fatty peritoneal fold that extends caudally from the greater curvature of the stomach to overlie most abdominal organs, has the ability to migrate to an inflammatory site, cover it, and contain the process, as in reactions to acute appendicitis or digestive tract perforation [1]. Considering this remarkable property, general surgeons mobilize the omentum to treat gastric [2,3], duodenal [4], and ileal perforations [5], and even aortoesophageal fistulae [6]. Matoba et al. [7] histopathologically demonstrated that
* Corresponding author. Department of Plastic and Reconstructive Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail address: [email protected] (M. Hishida). http://dx.doi.org/10.1016/j.ijsu.2015.03.022 1743-9191/© 2015 Published by Elsevier Ltd on behalf of IJS Publishing Group Limited.
omentum drawn into the stomach through a perforation accelerated wound healing in a gastric ulcer model. Similarly, we recently reported that patching a gastric wall defect with omentum accelerated wound healing [8,9]. However, surgeons sometimes encounter cases where no omental flap is available because of repeated previous open laparotomies or severe peritonitis. Offering substantial volume and rich vascular flow, a muscle flap has a variety of clinical applications in reconstructive surgery [10,11]. For instance, a muscle flap is used commonly to fill large defects of the chest wall after debridement of sternal osteomyelitis [12,13]. Occasionally, a muscle flap is used clinically at deeper sites, such as in repair of a duodenal fistula [14], reconstruction following total pelvic exenteration [15], patching of a rectovaginal fistula [16], and covering of a bladder rupture [17]. However, few experimental studies have investigated the effect of muscle flaps within the
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peritoneal cavity. We therefore compared wound healing in rats after applying a muscle flap to an experimental gastric wall defect to healing after applying an omental flap. After making a central perforation in the anterior wall of the stomach, we elevated and placed a muscle flap or an omental flap over the defect. We compared these 2 groups concerning promotion of granulation, reepithelialisation, and scar maturation using haematoxylin and eosin, Masson trichrome, and immunohistochemical staining. 2. Materials and methods 2.1. Experimental animals and maintenance Animals received humane care in compliance with the Nagoya University Guidelines for Animal Care and Use, which are based on the National Research Council's criteria outlined in the Guide for the Care and Use of Laboratory Animals (USA, 1985). Eight-weekold male SpragueeDawley rats (Chubu Kagaku Shizai, Nagoya, Japan) weighing 250e300 g at surgery (n ¼ 66) were housed 3 to a cage in an air-conditioned room maintained at 23 C with 60% humidity on an alternating 12-h light/12-h dark cycle. Rats were given food and running water ad libitum. 2.2. Surgical procedure Animals initially received inhalation anaesthesia with diethyl ether, followed by additional anaesthesia with an intraperitoneal injection of pentobarbital sodium (25 mg/kg body weight; Nenbutal, Abott, Chicago, IL). Laparotomy was performed to expose the anterior portion of the stomach. We perforated the centre of the anterior wall of the stomach between the fundus and the body with a needle (2.0-mm diameter; KAI, Seki, Japan) (Fig. 1a). We preliminarily investigated the optimal defect size produced using a single needle puncture. When the stomach wall was perforated with a 1.0-mm needle, the defect was not visible after a few days, because it contracted immediately. Larger-diameter defects made it the easier to observe the healing process. When the diameter of defect exceeded 3 mm, however, the defect could not be reliably patched with an omental flap. Therefore, a 2.0-mm defect was chosen as the optimal defect size. In the muscle group (n ¼ 33), the right chest skin was detached from the abdominal wall. The muscle flap, measuring 50 7 mm, was planned on the right rectus abdominis muscle. The muscle flap included the superior epigastric artery and vein, which was identified beneath the muscle. The muscle flap was elevated and transposed to the gastric wall defect. The tip of flap was folded, and the fascial side was affixed to the defect, using 4 sutures with 60 nylon (Fig. 1b). In the omentum group (n ¼ 33), an omental flap
was separated from the pancreas. The branches from the gastroepiploic vessels were ligated or cauterized, and the omental flap was isolated to be supplied by the right gastroepiploic vessels. The omental flap was transposed to the gastric wall defect (Fig. 1c). The flap was affixed to the stomach in a perpendicular direction along the long axis from the fundus to the corpus, using 5 to 6 sutures placed with 10-0 nylon. The abdominal incision was closed primarily, using 4-0 nylon. Animals were fed beginning on the day of operation. 2.3. Gross examination Laparotomy was performed on 3 rats each at postoperative days 1 through 7, 10, 21, 28, and 60. Before this specimen-harvesting procedure, rats were killed by diethyl ether overdose. We incised the stomach along the greater curvature and removed specimens containing stomach contents together with each muscle or omental flap. After gross examination of specimens, photographs of each wound were taken with an EX-Z57 camera (Casio, Tokyo, Japan). 2.4. Histology After fastening with pins to a corkboard to expose tissues adequately to the fixative, specimens were immersed in 4% paraformaldehyde for 24 h at room temperature. After samples were embedded in paraffin blocks, serial sections 5- thick ㎛ were cut along the sagittal plane through the widest margin of the wound. After staining deparaffinised sections with haematoxylin and eosin, photomicrographs of each defect were taken with an Olympus BX50 stereoscopic microscope (Olympus, Tokyo, Japan). Sections also were stained using Masson trichrome to evaluate collagen organisation within the granulation tissue and the scar. 2.5. Quantitative analysis of vascular lunina On each postoperative day, patent small vessels were counted under 120 magnification after staining deparaffinised sections with a rabbit polyclonal antibody against rat factor VIII-related antigen (DakoCytomation, Glostrup, Denmark). Findings are expressed as the mean number of small-vessel cross-section profiles per microscopic field in the central portion of the defect (approximately 0.64 mm2). Mean values were calculated for each of the 3 rats representing each post operative time point. 2.6. Immnohistochemical observation In both groups, a rabbit anti-fibroblast growth factor (FGF) -2 polyclonal antibody (Calbiochem, Gibbstown, CA), a rabbit anti-
Fig. 1. Surgical techniques. a, The centre of the anterior wall of the stomach was perforated between the fundus and body (arrow). G: gastric wall, O: omentum, P: pancreas. b, The muscle flap was elevated and transposed to the gastric wall defect. The tip of flap was folded, and the fascial side was affixed to the defect. M: right rectus abdominis muscle, MF: muscle flap. c, The omental flap was transposed to the gastric wall defect. The omental flap was fixed to the stomach perpendicular to the long axis (asterisk).
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vascular endothelial growth factor (VEGF) Ab-1 polyclonal antibody (Thermo Fisher Scientific Anatomical Pathology, Fremont, CA), and a Proliferating cell nuclear antigen (PCNA) monoclonal antibody (Invitrogen PCNA Staining Kit, Invitrogen, Paisley, UK) were used for immunostaining by an indirect immunohistochemical method. Specificity of immunohistochemical procedures was confirmed by incubation of sections with non-immune serum substituted for the primary antibody. 3. Results 3.1. Gross observation In the muscle group on day 2, defects took on linear profiles because of contraction. By day 6 depth of the defect had decreased significantly, presenting a shallow lesion lined with mucosal tissue (Fig. 2a). No further changes were observed after day 7. In the omentum group on days 1 and 2, we confirmed a deep mucosal defect, which appeared like a small dimple, in the mucosa. After day 3, defects appeared oval or linear as a result of further contraction. By day 6, the defect had become shallow and was lined by mucosa (Fig. 2b). No further changes were apparent grossly after day 7. 3.2. Histologic observation and quantitative analysis On day 1 in both groups, although the mucosal edges were oedematous and everted vertically, fibrin matrix was observed to fill the defect. Inflammatory cells had infiltrated the superficial surface of both flaps at the defect. In the muscle group on day 3, granulation tissue appeared at the edge of the defect surrounding the fascia, with irregular, poorly developed villi apparent along the lateral margins (Fig. 3a). Vessels surrounding the defect were dilated, containing red blood cells and polymorphonuclear leucocytes. Beginning on day 4, the fascia of the muscle flap was thickened by increasingly dense arrays of collagen fibres. Many immature vessels had arisen in relation to the fascia (Fig. 4). By day 3, the omentum group showed extensive inflammatory cell infiltration dominated by polymorphonuclear leucocytes and macrophages; this reaction was most intense at the centre of the defect (Fig. 3b). Part of the omental flap itself was being replaced rapidly by granulation tissue on day 4, at which time the number of inflammatory cells within the defect decreased significantly while some vessels could be seen in the centre of the omental flap (Fig. 4). On day 5, the muscle group showed development of granulation tissue at the centre of the fascia (Fig. 3c). At that time, the number of vascular lumina reached a peak, decreasing gradually thereafter
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(Fig. 4). In the omentum group, granulation tissue had proliferated to fill the defect. Irregular, elongated villi with several glandular cysts were present at the mucosal edges. New vessels clearly originating from the adjacent area of the omental flap entered the centre of the defect (Fig. 3d). By day 6, granulation tissue had thickened significantly. Collagen fibres had widened and were increased in numbers, while cells had decreased significantly. On day 7 in the muscle group, the mucosal epithelial layer demonstrated stratified villi completely covering the defect (Fig. 3e). Granulation tissue was replaced gradually by scar tissue (Fig. 5a). In the omentum group, mucosal oedema decreased significantly, and stratified villi had completely covered the defect (Fig. 3f). Granulation tissue was gradually replaced by scar tissue composed of densely packed, disorganised collagen fibre bundles (Fig. 5b). The number of vascular lumina increased, peaking on day 5 and then decreasing slowly (Fig. 4). From day 10 on, the density of collagen in the scar tissue under the regenerative epithelium had decreased, as scar tissue was replaced by adipose tissue. In the muscle group on day 21, distorted, irregular villi were observed, and fundic glands were seen in the depths of the regenerated mucosal layer (Fig. 5c). The muscularis mucosae and the muscular layer had not regenerated by day 60 (Fig. 3g). Scar tissue still was present at day 60 (Fig. 5e). In the omentum group on day 21, connective tissue was replaced gradually by adipose tissue (Fig. 5d). On day 60 many elongated well-developed villi were observed and many more fundic glands were present in the depths of the regenerated mucosal layer (Fig. 3h). The muscularis mucosae and the muscular layer had partially regenerated in areas near the margin of the defect (Fig. 5f). 3.3. Immunohistochemical observation 3.3.1. PCNA staining In the mucosal epithelium of both groups, some PCNA-positive cells were observed from day 2 to day 7. In the submucosal layer, significant numbers of PCNA-positive cells were observed from day 4 to day 7 (Fig. 6a, b). In the muscle group, the submucosa contained more PCNA-positive cells than the omentum group. In the muscle group PCNA-positive cells converged around the fascia, while in the omentum group PCNA-positive cells were most abundant in granulation tissue. Once the defect was completely covered, numbers of PCNA-positive cells decreased gradually in both groups (Fig. 7a, b). 3.3.2. FGF-2 staining From day 3 in the muscle group, numbers of FGF-2-positive cells increased in and around the fascia (Fig. 6c). Once the defect was reepithelialised, numbers of FGF-2-positive cells decreased gradually
Fig. 2. Gross examination on day 6. a Muscle group, showing a shallow lesion filled with mucosal tissue. A deep ulcer defect appears as a small dimple (arrow). b Omentum group, showing a shallow lesion filled with mucosal tissue. Ulcer depth has decreased (arrow).
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Fig. 3. Haematoxylin and eosin staining of the muscle group (a, c, e, g) and the omentum group (b, d, f, h) on day 3 (a, b), 5 (c, d), 7 (e, f) and 60 (g, h). a Granulation tissue has developed at the edge of defect and surrounding the fascia. b Extensive inflammatory cell infiltration is dominated by polymorphonuclear leucocytes and macrophages at the centre of the defect. c The granulation tissue at the mucosal edges has fused in the centre of the defect on the fascia. d Granulation tissue has proliferated to fill the defect. e The mucosal epithelial layer shows stratified villi completely covering the defect. f Mucosal oedema has decreased significantly, and stratified villi have completely covered the defect. g Irregular villi are observed. Scattered fundic glands are seen deep in the regenerate mucosa. h Many elongated well-developed villi were observed, while deep in the mucosa many fundic glands are seen.
in areas near the fascia (Fig. 7c). In the omentum group, numbers of FGF-2-positive cells increased in granulation tissue at the centre of the defect from day 4 to day 7 (Fig. 6d). By day 10, remaining positive cells were seen mostly around blood vessels (Fig. 7d). 3.3.3. VEGF staining From Day 3 in the Muscle group, the number of VEGF-positive cells increased near the fascia (Fig. 6e). Once the defect was reepithelialised, the numbers of VEGF-positive cells decreased significantly near the fascia. On day 10, some endothelial cells were still positive (Fig. 7e). From day 4 to day 7 in the omentum group, numerous strongpositive cells were seen in granulation tissue at the centre of the defect (Fig. 6f). Positive cells remained numerous on day 10 (Fig. 7f). 4. Discussion Both muscle and omental flaps supply excellent vascularised
tissue to fill defects. Some clinical reports have compared these flaps for indications such as pleural empyema [18], mediastinitis [13], non-healing perineal wounds [19], and complex cardiothoracic surgical problems [12]. Some of these pointed out situations where the omental flap might be more appropriate [12,13]. Since few reports have described different histopathologic findings associated with these flaps, we compared these flaps in terms of promotion of granulation, re-epithelialisation, and scar maturation. We found that while both flaps permitted considerable granulation tissue formation in our gastric wall defect model, the 2 types of flaps differed in pattern of formation of granulation tissue. In our muscle group, granulation tissue proliferated rapidly near the fascia. Spyrou et al. [20] reported that fibroblasts forming granulation tissue entered mainly from adjacent fascia. This observation agreed with own. Fascia associated with muscle, then, may be important as a source of granulation tissue. On the other hand, after inflammation had diminished in our omentum group, granulation tissue originated from part of the omental flap itself. Adams et al. [21] reported that granulation tissue arose from omentum used to
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Fig. 4. Numbers of vascular lumina in the centre of the defect. In the muscle group, the number of vascular lumina peaked on day 5 and decreased gradually thereafter. In the omentum group, the number of vascular lumina peaked on day 5 and then decreased slowly thereafter, becoming essentially constant by day 10.
wrap intestinal anastomoses. Wilkosz et al. [22] also has demonstrated that omental tissue showed intense inflammation and reduction of adipose tissue 3e7 days after surgery, with omentum giving rise granulation tissue with increased cellularity and collagen production. In short, those authors considered an omental flap to be an important source of granulation tissue. In both groups, FGF-positive cells and VEGF-positive cells were
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detected in granulation tissue. Our previous study suggested that omentum contains a potent angiogenic factor in its lipid fraction [23]. In vitro, Zhang et al. [24] identified VEGF as the major angiogenic factor in omentum. Matoba et al. [7] reported bFGFmediated angiogenesis after omental implantation in a rat gastric ulcer model. As for muscle flaps, Pallua et al. [25] detected bFGF in surgical wound fluids of patients with muscle flaps. Erdmann et al. [26] identified VEGF in porcine latissimus dorsi musculocutaneous flaps following ischaemia-reperfusion injury. These findings are consistent with our own. Ability of both flaps to give rise to granulation tissue may involve induction and formation of mature fibroblasts and endothelial cells by such growth factors produced by the flaps. We demonstrated that a muscle flap, like an omental flap, enhanced re-epithelialisation in our model. With either flap, stratified villi completely covered the defect by day 7. Our previous study demonstrated that the defect remained uncovered at day 10 when a silicon sheet lining was used as a control [8]. In the muscle flap, Wada et al. [27] described that epithelialisation progresses gradually along the surface of granulated fascia from surrounding tissues. As for the omental flap, Yamaki et al. [28]found such a flap to serve as an excellent supporting structure for regeneration of mucosal epithelium in the nasal cavity following skull base reconstruction. Both flaps may promote epithelialisation even though neither contains epidermal elements. As for scar maturation, we found a great difference between muscle and omentum groups. In our omentum group, the extent of collagen fibres was greatest on day 21, followed by a steady decrease. Substantial extracellular matrix was replaced by adipose tissue. Wilkosz et al. [22] demonstrated that affected omentum
Fig. 5. Masson trichrome staining of the muscle group (a, c, e) and the omentum group (b, d, f) on day 7 (a, b), 21 (c, d) and 60 (e, f). a Granulation tissue is being replaced gradually by scar tissue. b Granulation tissue is being replaced gradually by scar tissue. c Scar tissue persists in the submucosa. d Scar tissue has been replaced peripherally by adipose tissue. e Scar tissue persists. f Regenerated muscularis mucosae and muscular layer are observed at the edge.
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Fig. 6. Immunohistochemical staining on day 4 of the muscle group (a, c, e) and the omentum group (b, d, f) showing the expression of PCNA (a, b), FGF-2 (c, d) and VEGF (e, f). a PCNA-positive cells are seen mainly surrounding the fascia. b PCNA-positive cells are seen mainly in granulation tissue. c Numbers of FGF-2-positive cells are increased in the area of the fascia. d Numbers of FGF-2-positive cells are increased in granulation tissue at the centre of the defect. e Numbers of VEGF-positive cells are increased in the area of the fascia. f Many strongly VEGF-positive cells are densely present in the granulation tissue at the centre of the defect.
undergoes a remodelling process characterised by loss of adipose tissue, increased cellularity and collagen deposition, and return of adipose tissue with a decrease in cellularity and collagen deposition. In our muscle group after re-epithelialisation, scar tissue remained unchanged for at least 60 days. Brunelli et al. [29]
reported the degree of perineural fibrosis to be 5 times greater when muscle rather than omentum was placed in contact with nerve tissue. An omental flap may incite less fibrosis than a muscle flap as the scar matures. Since a muscle flap can accelerate wound healing as readily as
Fig. 7. Immunohistochemical staining on day 10 of the muscle group (a, c, e) and the omentum group (b, d, f) showing the expression of PCNA (a, b), FGF-2 (c, d) and VEGF (e, f). With the defect now completely covered, numbers of PCNA-positive cells are decreasing gradually in muscle (a) and omentum (b) groups. c With the defect now re-epithelialised, numbers of FGF-2-positive cells are decreasing gradually in the area of the fascia. d Numbers of FGF-2-positive cells are decreasing gradually in the granulation tissue at the centre of the defect. FGF-2-positive cells are seen mostly surrounding blood vessels. e With the defect now re-epithelialised, numbers of VEGF-positive cells have decreased considerably in the area of the fascia; some endothelial cells remain positive. f Numerous VEGF-positive cells are observed, though the number of positive cells has decreased in granulation tissue.
M. Hishida et al. / International Journal of Surgery 18 (2015) 41e47
an omental flap, muscle flaps have been applied clinically in various settings such as closure of large chest wall defects after debridement of sternal osteomyelitis, reconstruction following total pelvic exenteration and treatment of empyema after pneumonectomy [30]. Advantages of a muscle flap include a relatively large available volume; provision of a mechanical barrier between tissues such as trachea and artery in tracheo-innominate artery fistula [31]; and direct application as a substitute for a muscle defect such as focally absent bladder wall in urinary bladder rupture [17]. Additionally, a muscle flap would be appropriate for patients who have undergone previous abdominal operations. On the other hand, a muscle flap has certain limitations, being ill-suited for obliteration of an irregularly contoured deep defect, such as those remaining after debridement of sternal osteomyelitis [32]. In such cases, the defect left behind is beyond the reach of systematic antibiotics or immune cells.32 Also, a muscle flap is less elastic than an omental flap, making it less effective for implantation into a fistula where wound healing is occurring, such as in a gastric ulcer defect [7]. 5. Conclusion In conclusion, this study demonstrated that like an omental flap, a muscle flap accelerated gastric wall wound healing. A muscle flap can be used for repair of gastric defects when an omental flap is not available. Ethical approval The animal care was in accordance with the institution guidelines (file number26250). Funding None. Author contribution M Hishida, K Toriyama: Surgical interventions editing the manuscript. S Yagi, K Ebisawa: Surgical intervention. T Morishita, K Takanari: Histological evaluation. Y Kamei: Editing the manuscript. Conflicts of interest None. Guarantor Masashi Hishida. References [1] S. Wilkosz, G. Ireland, N. Khwaja, et al., A comparative study of the structure of human and murine greater omentum, Anat. Embryo (Berl) 209 (2005) 251e261. [2] M.J.O.E. Bertleff, J.F. Lange, Perforated peptic ulcer disease: a review of history and treatment, Dig. Surg. 27 (2010) 161e169. [3] M. Matsuda, M. Nishiyama, T. Hanai, S. Saeki, T. Watanabe, Laparoscopic omental patch repair for perforated peptic ulcer, Ann. Surg. 221 (1995) 236e240. [4] A.E. Nicolau, V. Merlan, V. Veste, B. Micu, M. Beuran, Laparoscopic suture repair of perforated duodenal peptic ulcer for patients without risk factors, Chir. (Bucur) 103 (2008) 629e633.
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[5] M. Husain, R.N. Khan, B. Rehmani, H. Haris, Omental patch technique for the ileal perforation secondary to typhoid fever, Saudi J. Gastroenterol. 17 (2011) 208e211. [6] N. Wang, S.R. Sparks, L.L. Bailey, Staged repair using omentum for posttraumatic aortoesophageal fistula, Ann. Thorac. Surg. 58 (1994) 557e559. [7] Y. Matoba, H. Katayama, H. Ohami, Evaluation of omental implantation for perforated gastric ulcer therapy: findings in a rat model, J. Gastroenterol. 31 (1996) 777e784. [8] M. Hishida, Y. Kamei, K. Toriyama, et al., Healimg of gastric wall defects using omental flap, Sosyo 1 (2010) 88e94. [9] M. Hishida, K. Toriyama, Y. Yamashita, et al., Omental flap closure of refractory wounds: rat model, Nagoya J. Med. Sci. 72 (2010) 91e99. [10] H. Bajpai, D. Saikrishna, The versatility of temporalis myofascial flap in maxillo-facial reconstruction: a clinical study, J. Maxillofac. Oral Surg. 10 (2011) 25e31. [11] J.M. Serletti, Breast reconstruction with the TRAM flap: pedicled and free, J. Surg. Oncol. 94 (2006) 532e537. [12] J.B. Shrager, J.C. Wain, C.D. Wright, et al., Omentum is highly effective in the management of complex cardiothoracic surgical problems, J. Thorac. Cardiovasc. Surg. 125 (2003) 526e532. [13] B.H. Ghazi, G.W. Carlson, A. Losken, Use of the greater omentum for reconstruction of infected sternotomy wounds: a prognostic indicator, Ann. Plast. Surg. 60 (2008) 169e173. [14] J. Chander, P. Lal, V.K. Ramteke, Rectus abdominis muscle flap for high-output duodenal fistula: novel technique, World J. Surg. 28 (2004) 179e182. [15] S. Chan, M. Miller, R. Ng, D. Ross, P. Roblin, E. Carapeti, Use of myocutaneous flaps for perineal closure following abdominoperineal excision of the rectum for adenocarcinoma, Colorectal Dis. 12 (2010) 555e560. [16] O. Zmora, H. Tulchinsky, E. Gur, G. Goldman, J.M. Klausner, M. Rabau, Gracilis muscle transposition for fistulas between the rectum and urethra or vagina, Dis. Colon Rectum 49 (2006) 1316e1321. [17] C.-W. Wu, C.-H. Lin, C.-H. Lin, One-stage posttraumatic bladder reconstruction and soft-tissue coverage of the lower abdomen or perineum, Ann. Plast. Surg. 64 (2010) 65e68. [18] G. Stamatis, L. Freitag, M. Wencker, D. Greschuchna, Omentopexy and muscle transposition: two alternative methods in the treatment of pleural empyema and mediastinitis, Thorac. Cardiovasc. Surg. 42 (1994) 225e232. [19] A. Menon, M.A. Clark, T. Shatari, C. Keh, M.R.B. Keighley, Pedicled flaps in the treatment of nonhealing perineal wounds, Colorectal Dis. 7 (2005) 441e444. [20] G.E. Spyrou, D.A. Watt, I.L. Naylor, The origin and mode of fibroblast migration and proliferation in granulation tissue, Br. J. Plast. Surg. 51 (1998) 455e461. [21] W. Adams, G. Ctercteko, M. Bilous, Effect of an omental wrap on the healing and vascularity of compromised intestinal anastomoses, Dis. 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Moquin, et al., Immunohistochemical identification of vascular endothelial growth factor in pig latissimus dorsi musculocutaneous flaps following ischemia-reperfusion injury, Ann. Plast. Surg. 53 (2004) 398e403. [27] T. Wada, K. Okamoto, Y. Nakanishi, Y. Iwagami, N. Morita, Myofascial flap without skin for intra-oral reconstruction. 1: animal studies, Int. J. Clin. Oncol. 6 (2001) 40e44. [28] T. Yamaki, T. Uede, A. Tano-oka, K. Asakura, S. Tanabe, K. Hashi, Vascularized omentum graft for the reconstruction of the skull base after removal of a nasoethmoidal tumor with intracranial extension: case report, Neurosurgery 28 (1991) 877e880. [29] G.A. Brunelli, F. Brunelli, F. Di Rosa, Neurolized nerve padding in actinic lesions: omentum versus muscle use. An experimental study, Microsurgery 9 (1988) 177e180. [30] K. Takanari, Y. Kamei, K. Toriyama, et al., Management of postpneumonectomy empyema using free flap and pedicled flap, Ann. Thorac. Surg. 89 (2010) 321e323. [31] T. Komatsu, T. 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International Journal of Surgery journal homepage: www.journal-surgery.net
Original research
Does a muscle flap accelerate wound healing of gastric wall defects compared with an omental flap? Masashi Hishida a, b, *, Kazuhiro Toriyama b, Shunjiro Yagi b, Katsumi Ebisawa b, Tsuyoshi Morishita b, Keisuke Takanari b, Yuzuru Kamei b a b
Department of Plastic and Reconstructive Surgery, Kasugai Municipal Hospital, 1-1-1 Takagi-cho, Kasugai 486-8510, Japan Department of Plastic and Reconstructive Surgery, Nagoya University Graduate School of Medicine, 65 Turumai-cho, Showa-ku, Nagoya 466-8550, Japan
h i g h l i g h t s Clinical reports have described successful use of muscle flaps to promote healing of visceral defects. We histologically and immunohistochemically compared muscle vs. omental flaps in rats with experimental gastric wall defects. Both flap types promoted healing, but omental flaps provoked less fibrous scarring in the long term.
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a b s t r a c t
Article history: Received 15 August 2014 Received in revised form 14 March 2015 Accepted 25 March 2015 Available online 10 April 2015
Introduction: Most often used for reconstruction at superficial sites, a muscle flap recently was reported to promote clinical wound healing in a duodenal defect. We therefore examined whether a muscle flap could promote wound healing comparably to an omental flap in rats with gastric wall defects. Methods: After perforation of the centre of the anterior gastric wall, rats were divided into 2 groups. In the muscle group, a muscle flap was fixed to the defect; in the omentum group, an omental flap was placed over the defect. We histopathologically compared tissue responses during gastric wall healing. Results: While stratified villi had completely covered the defect by day 7 in both groups, scar maturation differed. Scar tissue persisted in the muscle group, but was gradually replaced by adipose tissue in the omentum group. Discussion: Both muscle and omental flaps accelerated gastric wall wound healing. Conclusion: A muscle flap is an excellent alternative for repair of gastric defects when no omental flap is available. © 2015 Published by Elsevier Ltd on behalf of IJS Publishing Group Limited.
Keywords: Muscle flap Omental flap Gastric wall defects Wound healing Rats
1. Introduction The omentum, a fatty peritoneal fold that extends caudally from the greater curvature of the stomach to overlie most abdominal organs, has the ability to migrate to an inflammatory site, cover it, and contain the process, as in reactions to acute appendicitis or digestive tract perforation [1]. Considering this remarkable property, general surgeons mobilize the omentum to treat gastric [2,3], duodenal [4], and ileal perforations [5], and even aortoesophageal fistulae [6]. Matoba et al. [7] histopathologically demonstrated that
* Corresponding author. Department of Plastic and Reconstructive Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail address: [email protected] (M. Hishida). http://dx.doi.org/10.1016/j.ijsu.2015.03.022 1743-9191/© 2015 Published by Elsevier Ltd on behalf of IJS Publishing Group Limited.
omentum drawn into the stomach through a perforation accelerated wound healing in a gastric ulcer model. Similarly, we recently reported that patching a gastric wall defect with omentum accelerated wound healing [8,9]. However, surgeons sometimes encounter cases where no omental flap is available because of repeated previous open laparotomies or severe peritonitis. Offering substantial volume and rich vascular flow, a muscle flap has a variety of clinical applications in reconstructive surgery [10,11]. For instance, a muscle flap is used commonly to fill large defects of the chest wall after debridement of sternal osteomyelitis [12,13]. Occasionally, a muscle flap is used clinically at deeper sites, such as in repair of a duodenal fistula [14], reconstruction following total pelvic exenteration [15], patching of a rectovaginal fistula [16], and covering of a bladder rupture [17]. However, few experimental studies have investigated the effect of muscle flaps within the
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peritoneal cavity. We therefore compared wound healing in rats after applying a muscle flap to an experimental gastric wall defect to healing after applying an omental flap. After making a central perforation in the anterior wall of the stomach, we elevated and placed a muscle flap or an omental flap over the defect. We compared these 2 groups concerning promotion of granulation, reepithelialisation, and scar maturation using haematoxylin and eosin, Masson trichrome, and immunohistochemical staining. 2. Materials and methods 2.1. Experimental animals and maintenance Animals received humane care in compliance with the Nagoya University Guidelines for Animal Care and Use, which are based on the National Research Council's criteria outlined in the Guide for the Care and Use of Laboratory Animals (USA, 1985). Eight-weekold male SpragueeDawley rats (Chubu Kagaku Shizai, Nagoya, Japan) weighing 250e300 g at surgery (n ¼ 66) were housed 3 to a cage in an air-conditioned room maintained at 23 C with 60% humidity on an alternating 12-h light/12-h dark cycle. Rats were given food and running water ad libitum. 2.2. Surgical procedure Animals initially received inhalation anaesthesia with diethyl ether, followed by additional anaesthesia with an intraperitoneal injection of pentobarbital sodium (25 mg/kg body weight; Nenbutal, Abott, Chicago, IL). Laparotomy was performed to expose the anterior portion of the stomach. We perforated the centre of the anterior wall of the stomach between the fundus and the body with a needle (2.0-mm diameter; KAI, Seki, Japan) (Fig. 1a). We preliminarily investigated the optimal defect size produced using a single needle puncture. When the stomach wall was perforated with a 1.0-mm needle, the defect was not visible after a few days, because it contracted immediately. Larger-diameter defects made it the easier to observe the healing process. When the diameter of defect exceeded 3 mm, however, the defect could not be reliably patched with an omental flap. Therefore, a 2.0-mm defect was chosen as the optimal defect size. In the muscle group (n ¼ 33), the right chest skin was detached from the abdominal wall. The muscle flap, measuring 50 7 mm, was planned on the right rectus abdominis muscle. The muscle flap included the superior epigastric artery and vein, which was identified beneath the muscle. The muscle flap was elevated and transposed to the gastric wall defect. The tip of flap was folded, and the fascial side was affixed to the defect, using 4 sutures with 60 nylon (Fig. 1b). In the omentum group (n ¼ 33), an omental flap
was separated from the pancreas. The branches from the gastroepiploic vessels were ligated or cauterized, and the omental flap was isolated to be supplied by the right gastroepiploic vessels. The omental flap was transposed to the gastric wall defect (Fig. 1c). The flap was affixed to the stomach in a perpendicular direction along the long axis from the fundus to the corpus, using 5 to 6 sutures placed with 10-0 nylon. The abdominal incision was closed primarily, using 4-0 nylon. Animals were fed beginning on the day of operation. 2.3. Gross examination Laparotomy was performed on 3 rats each at postoperative days 1 through 7, 10, 21, 28, and 60. Before this specimen-harvesting procedure, rats were killed by diethyl ether overdose. We incised the stomach along the greater curvature and removed specimens containing stomach contents together with each muscle or omental flap. After gross examination of specimens, photographs of each wound were taken with an EX-Z57 camera (Casio, Tokyo, Japan). 2.4. Histology After fastening with pins to a corkboard to expose tissues adequately to the fixative, specimens were immersed in 4% paraformaldehyde for 24 h at room temperature. After samples were embedded in paraffin blocks, serial sections 5- thick ㎛ were cut along the sagittal plane through the widest margin of the wound. After staining deparaffinised sections with haematoxylin and eosin, photomicrographs of each defect were taken with an Olympus BX50 stereoscopic microscope (Olympus, Tokyo, Japan). Sections also were stained using Masson trichrome to evaluate collagen organisation within the granulation tissue and the scar. 2.5. Quantitative analysis of vascular lunina On each postoperative day, patent small vessels were counted under 120 magnification after staining deparaffinised sections with a rabbit polyclonal antibody against rat factor VIII-related antigen (DakoCytomation, Glostrup, Denmark). Findings are expressed as the mean number of small-vessel cross-section profiles per microscopic field in the central portion of the defect (approximately 0.64 mm2). Mean values were calculated for each of the 3 rats representing each post operative time point. 2.6. Immnohistochemical observation In both groups, a rabbit anti-fibroblast growth factor (FGF) -2 polyclonal antibody (Calbiochem, Gibbstown, CA), a rabbit anti-
Fig. 1. Surgical techniques. a, The centre of the anterior wall of the stomach was perforated between the fundus and body (arrow). G: gastric wall, O: omentum, P: pancreas. b, The muscle flap was elevated and transposed to the gastric wall defect. The tip of flap was folded, and the fascial side was affixed to the defect. M: right rectus abdominis muscle, MF: muscle flap. c, The omental flap was transposed to the gastric wall defect. The omental flap was fixed to the stomach perpendicular to the long axis (asterisk).
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vascular endothelial growth factor (VEGF) Ab-1 polyclonal antibody (Thermo Fisher Scientific Anatomical Pathology, Fremont, CA), and a Proliferating cell nuclear antigen (PCNA) monoclonal antibody (Invitrogen PCNA Staining Kit, Invitrogen, Paisley, UK) were used for immunostaining by an indirect immunohistochemical method. Specificity of immunohistochemical procedures was confirmed by incubation of sections with non-immune serum substituted for the primary antibody. 3. Results 3.1. Gross observation In the muscle group on day 2, defects took on linear profiles because of contraction. By day 6 depth of the defect had decreased significantly, presenting a shallow lesion lined with mucosal tissue (Fig. 2a). No further changes were observed after day 7. In the omentum group on days 1 and 2, we confirmed a deep mucosal defect, which appeared like a small dimple, in the mucosa. After day 3, defects appeared oval or linear as a result of further contraction. By day 6, the defect had become shallow and was lined by mucosa (Fig. 2b). No further changes were apparent grossly after day 7. 3.2. Histologic observation and quantitative analysis On day 1 in both groups, although the mucosal edges were oedematous and everted vertically, fibrin matrix was observed to fill the defect. Inflammatory cells had infiltrated the superficial surface of both flaps at the defect. In the muscle group on day 3, granulation tissue appeared at the edge of the defect surrounding the fascia, with irregular, poorly developed villi apparent along the lateral margins (Fig. 3a). Vessels surrounding the defect were dilated, containing red blood cells and polymorphonuclear leucocytes. Beginning on day 4, the fascia of the muscle flap was thickened by increasingly dense arrays of collagen fibres. Many immature vessels had arisen in relation to the fascia (Fig. 4). By day 3, the omentum group showed extensive inflammatory cell infiltration dominated by polymorphonuclear leucocytes and macrophages; this reaction was most intense at the centre of the defect (Fig. 3b). Part of the omental flap itself was being replaced rapidly by granulation tissue on day 4, at which time the number of inflammatory cells within the defect decreased significantly while some vessels could be seen in the centre of the omental flap (Fig. 4). On day 5, the muscle group showed development of granulation tissue at the centre of the fascia (Fig. 3c). At that time, the number of vascular lumina reached a peak, decreasing gradually thereafter
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(Fig. 4). In the omentum group, granulation tissue had proliferated to fill the defect. Irregular, elongated villi with several glandular cysts were present at the mucosal edges. New vessels clearly originating from the adjacent area of the omental flap entered the centre of the defect (Fig. 3d). By day 6, granulation tissue had thickened significantly. Collagen fibres had widened and were increased in numbers, while cells had decreased significantly. On day 7 in the muscle group, the mucosal epithelial layer demonstrated stratified villi completely covering the defect (Fig. 3e). Granulation tissue was replaced gradually by scar tissue (Fig. 5a). In the omentum group, mucosal oedema decreased significantly, and stratified villi had completely covered the defect (Fig. 3f). Granulation tissue was gradually replaced by scar tissue composed of densely packed, disorganised collagen fibre bundles (Fig. 5b). The number of vascular lumina increased, peaking on day 5 and then decreasing slowly (Fig. 4). From day 10 on, the density of collagen in the scar tissue under the regenerative epithelium had decreased, as scar tissue was replaced by adipose tissue. In the muscle group on day 21, distorted, irregular villi were observed, and fundic glands were seen in the depths of the regenerated mucosal layer (Fig. 5c). The muscularis mucosae and the muscular layer had not regenerated by day 60 (Fig. 3g). Scar tissue still was present at day 60 (Fig. 5e). In the omentum group on day 21, connective tissue was replaced gradually by adipose tissue (Fig. 5d). On day 60 many elongated well-developed villi were observed and many more fundic glands were present in the depths of the regenerated mucosal layer (Fig. 3h). The muscularis mucosae and the muscular layer had partially regenerated in areas near the margin of the defect (Fig. 5f). 3.3. Immunohistochemical observation 3.3.1. PCNA staining In the mucosal epithelium of both groups, some PCNA-positive cells were observed from day 2 to day 7. In the submucosal layer, significant numbers of PCNA-positive cells were observed from day 4 to day 7 (Fig. 6a, b). In the muscle group, the submucosa contained more PCNA-positive cells than the omentum group. In the muscle group PCNA-positive cells converged around the fascia, while in the omentum group PCNA-positive cells were most abundant in granulation tissue. Once the defect was completely covered, numbers of PCNA-positive cells decreased gradually in both groups (Fig. 7a, b). 3.3.2. FGF-2 staining From day 3 in the muscle group, numbers of FGF-2-positive cells increased in and around the fascia (Fig. 6c). Once the defect was reepithelialised, numbers of FGF-2-positive cells decreased gradually
Fig. 2. Gross examination on day 6. a Muscle group, showing a shallow lesion filled with mucosal tissue. A deep ulcer defect appears as a small dimple (arrow). b Omentum group, showing a shallow lesion filled with mucosal tissue. Ulcer depth has decreased (arrow).
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Fig. 3. Haematoxylin and eosin staining of the muscle group (a, c, e, g) and the omentum group (b, d, f, h) on day 3 (a, b), 5 (c, d), 7 (e, f) and 60 (g, h). a Granulation tissue has developed at the edge of defect and surrounding the fascia. b Extensive inflammatory cell infiltration is dominated by polymorphonuclear leucocytes and macrophages at the centre of the defect. c The granulation tissue at the mucosal edges has fused in the centre of the defect on the fascia. d Granulation tissue has proliferated to fill the defect. e The mucosal epithelial layer shows stratified villi completely covering the defect. f Mucosal oedema has decreased significantly, and stratified villi have completely covered the defect. g Irregular villi are observed. Scattered fundic glands are seen deep in the regenerate mucosa. h Many elongated well-developed villi were observed, while deep in the mucosa many fundic glands are seen.
in areas near the fascia (Fig. 7c). In the omentum group, numbers of FGF-2-positive cells increased in granulation tissue at the centre of the defect from day 4 to day 7 (Fig. 6d). By day 10, remaining positive cells were seen mostly around blood vessels (Fig. 7d). 3.3.3. VEGF staining From Day 3 in the Muscle group, the number of VEGF-positive cells increased near the fascia (Fig. 6e). Once the defect was reepithelialised, the numbers of VEGF-positive cells decreased significantly near the fascia. On day 10, some endothelial cells were still positive (Fig. 7e). From day 4 to day 7 in the omentum group, numerous strongpositive cells were seen in granulation tissue at the centre of the defect (Fig. 6f). Positive cells remained numerous on day 10 (Fig. 7f). 4. Discussion Both muscle and omental flaps supply excellent vascularised
tissue to fill defects. Some clinical reports have compared these flaps for indications such as pleural empyema [18], mediastinitis [13], non-healing perineal wounds [19], and complex cardiothoracic surgical problems [12]. Some of these pointed out situations where the omental flap might be more appropriate [12,13]. Since few reports have described different histopathologic findings associated with these flaps, we compared these flaps in terms of promotion of granulation, re-epithelialisation, and scar maturation. We found that while both flaps permitted considerable granulation tissue formation in our gastric wall defect model, the 2 types of flaps differed in pattern of formation of granulation tissue. In our muscle group, granulation tissue proliferated rapidly near the fascia. Spyrou et al. [20] reported that fibroblasts forming granulation tissue entered mainly from adjacent fascia. This observation agreed with own. Fascia associated with muscle, then, may be important as a source of granulation tissue. On the other hand, after inflammation had diminished in our omentum group, granulation tissue originated from part of the omental flap itself. Adams et al. [21] reported that granulation tissue arose from omentum used to
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Fig. 4. Numbers of vascular lumina in the centre of the defect. In the muscle group, the number of vascular lumina peaked on day 5 and decreased gradually thereafter. In the omentum group, the number of vascular lumina peaked on day 5 and then decreased slowly thereafter, becoming essentially constant by day 10.
wrap intestinal anastomoses. Wilkosz et al. [22] also has demonstrated that omental tissue showed intense inflammation and reduction of adipose tissue 3e7 days after surgery, with omentum giving rise granulation tissue with increased cellularity and collagen production. In short, those authors considered an omental flap to be an important source of granulation tissue. In both groups, FGF-positive cells and VEGF-positive cells were
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detected in granulation tissue. Our previous study suggested that omentum contains a potent angiogenic factor in its lipid fraction [23]. In vitro, Zhang et al. [24] identified VEGF as the major angiogenic factor in omentum. Matoba et al. [7] reported bFGFmediated angiogenesis after omental implantation in a rat gastric ulcer model. As for muscle flaps, Pallua et al. [25] detected bFGF in surgical wound fluids of patients with muscle flaps. Erdmann et al. [26] identified VEGF in porcine latissimus dorsi musculocutaneous flaps following ischaemia-reperfusion injury. These findings are consistent with our own. Ability of both flaps to give rise to granulation tissue may involve induction and formation of mature fibroblasts and endothelial cells by such growth factors produced by the flaps. We demonstrated that a muscle flap, like an omental flap, enhanced re-epithelialisation in our model. With either flap, stratified villi completely covered the defect by day 7. Our previous study demonstrated that the defect remained uncovered at day 10 when a silicon sheet lining was used as a control [8]. In the muscle flap, Wada et al. [27] described that epithelialisation progresses gradually along the surface of granulated fascia from surrounding tissues. As for the omental flap, Yamaki et al. [28]found such a flap to serve as an excellent supporting structure for regeneration of mucosal epithelium in the nasal cavity following skull base reconstruction. Both flaps may promote epithelialisation even though neither contains epidermal elements. As for scar maturation, we found a great difference between muscle and omentum groups. In our omentum group, the extent of collagen fibres was greatest on day 21, followed by a steady decrease. Substantial extracellular matrix was replaced by adipose tissue. Wilkosz et al. [22] demonstrated that affected omentum
Fig. 5. Masson trichrome staining of the muscle group (a, c, e) and the omentum group (b, d, f) on day 7 (a, b), 21 (c, d) and 60 (e, f). a Granulation tissue is being replaced gradually by scar tissue. b Granulation tissue is being replaced gradually by scar tissue. c Scar tissue persists in the submucosa. d Scar tissue has been replaced peripherally by adipose tissue. e Scar tissue persists. f Regenerated muscularis mucosae and muscular layer are observed at the edge.
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Fig. 6. Immunohistochemical staining on day 4 of the muscle group (a, c, e) and the omentum group (b, d, f) showing the expression of PCNA (a, b), FGF-2 (c, d) and VEGF (e, f). a PCNA-positive cells are seen mainly surrounding the fascia. b PCNA-positive cells are seen mainly in granulation tissue. c Numbers of FGF-2-positive cells are increased in the area of the fascia. d Numbers of FGF-2-positive cells are increased in granulation tissue at the centre of the defect. e Numbers of VEGF-positive cells are increased in the area of the fascia. f Many strongly VEGF-positive cells are densely present in the granulation tissue at the centre of the defect.
undergoes a remodelling process characterised by loss of adipose tissue, increased cellularity and collagen deposition, and return of adipose tissue with a decrease in cellularity and collagen deposition. In our muscle group after re-epithelialisation, scar tissue remained unchanged for at least 60 days. Brunelli et al. [29]
reported the degree of perineural fibrosis to be 5 times greater when muscle rather than omentum was placed in contact with nerve tissue. An omental flap may incite less fibrosis than a muscle flap as the scar matures. Since a muscle flap can accelerate wound healing as readily as
Fig. 7. Immunohistochemical staining on day 10 of the muscle group (a, c, e) and the omentum group (b, d, f) showing the expression of PCNA (a, b), FGF-2 (c, d) and VEGF (e, f). With the defect now completely covered, numbers of PCNA-positive cells are decreasing gradually in muscle (a) and omentum (b) groups. c With the defect now re-epithelialised, numbers of FGF-2-positive cells are decreasing gradually in the area of the fascia. d Numbers of FGF-2-positive cells are decreasing gradually in the granulation tissue at the centre of the defect. FGF-2-positive cells are seen mostly surrounding blood vessels. e With the defect now re-epithelialised, numbers of VEGF-positive cells have decreased considerably in the area of the fascia; some endothelial cells remain positive. f Numerous VEGF-positive cells are observed, though the number of positive cells has decreased in granulation tissue.
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an omental flap, muscle flaps have been applied clinically in various settings such as closure of large chest wall defects after debridement of sternal osteomyelitis, reconstruction following total pelvic exenteration and treatment of empyema after pneumonectomy [30]. Advantages of a muscle flap include a relatively large available volume; provision of a mechanical barrier between tissues such as trachea and artery in tracheo-innominate artery fistula [31]; and direct application as a substitute for a muscle defect such as focally absent bladder wall in urinary bladder rupture [17]. Additionally, a muscle flap would be appropriate for patients who have undergone previous abdominal operations. On the other hand, a muscle flap has certain limitations, being ill-suited for obliteration of an irregularly contoured deep defect, such as those remaining after debridement of sternal osteomyelitis [32]. In such cases, the defect left behind is beyond the reach of systematic antibiotics or immune cells.32 Also, a muscle flap is less elastic than an omental flap, making it less effective for implantation into a fistula where wound healing is occurring, such as in a gastric ulcer defect [7]. 5. Conclusion In conclusion, this study demonstrated that like an omental flap, a muscle flap accelerated gastric wall wound healing. A muscle flap can be used for repair of gastric defects when an omental flap is not available. Ethical approval The animal care was in accordance with the institution guidelines (file number26250). Funding None. Author contribution M Hishida, K Toriyama: Surgical interventions editing the manuscript. S Yagi, K Ebisawa: Surgical intervention. T Morishita, K Takanari: Histological evaluation. Y Kamei: Editing the manuscript. Conflicts of interest None. Guarantor Masashi Hishida. References [1] S. Wilkosz, G. Ireland, N. Khwaja, et al., A comparative study of the structure of human and murine greater omentum, Anat. Embryo (Berl) 209 (2005) 251e261. [2] M.J.O.E. Bertleff, J.F. Lange, Perforated peptic ulcer disease: a review of history and treatment, Dig. Surg. 27 (2010) 161e169. [3] M. Matsuda, M. Nishiyama, T. Hanai, S. Saeki, T. Watanabe, Laparoscopic omental patch repair for perforated peptic ulcer, Ann. Surg. 221 (1995) 236e240. [4] A.E. Nicolau, V. Merlan, V. Veste, B. Micu, M. Beuran, Laparoscopic suture repair of perforated duodenal peptic ulcer for patients without risk factors, Chir. (Bucur) 103 (2008) 629e633.
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[5] M. Husain, R.N. Khan, B. Rehmani, H. Haris, Omental patch technique for the ileal perforation secondary to typhoid fever, Saudi J. Gastroenterol. 17 (2011) 208e211. [6] N. Wang, S.R. Sparks, L.L. Bailey, Staged repair using omentum for posttraumatic aortoesophageal fistula, Ann. Thorac. Surg. 58 (1994) 557e559. [7] Y. Matoba, H. Katayama, H. Ohami, Evaluation of omental implantation for perforated gastric ulcer therapy: findings in a rat model, J. Gastroenterol. 31 (1996) 777e784. [8] M. Hishida, Y. Kamei, K. Toriyama, et al., Healimg of gastric wall defects using omental flap, Sosyo 1 (2010) 88e94. [9] M. Hishida, K. Toriyama, Y. Yamashita, et al., Omental flap closure of refractory wounds: rat model, Nagoya J. Med. Sci. 72 (2010) 91e99. [10] H. Bajpai, D. Saikrishna, The versatility of temporalis myofascial flap in maxillo-facial reconstruction: a clinical study, J. Maxillofac. Oral Surg. 10 (2011) 25e31. [11] J.M. Serletti, Breast reconstruction with the TRAM flap: pedicled and free, J. Surg. Oncol. 94 (2006) 532e537. [12] J.B. Shrager, J.C. Wain, C.D. Wright, et al., Omentum is highly effective in the management of complex cardiothoracic surgical problems, J. Thorac. Cardiovasc. Surg. 125 (2003) 526e532. [13] B.H. Ghazi, G.W. Carlson, A. Losken, Use of the greater omentum for reconstruction of infected sternotomy wounds: a prognostic indicator, Ann. Plast. Surg. 60 (2008) 169e173. [14] J. Chander, P. Lal, V.K. Ramteke, Rectus abdominis muscle flap for high-output duodenal fistula: novel technique, World J. Surg. 28 (2004) 179e182. [15] S. Chan, M. Miller, R. Ng, D. Ross, P. Roblin, E. Carapeti, Use of myocutaneous flaps for perineal closure following abdominoperineal excision of the rectum for adenocarcinoma, Colorectal Dis. 12 (2010) 555e560. [16] O. Zmora, H. Tulchinsky, E. Gur, G. Goldman, J.M. Klausner, M. Rabau, Gracilis muscle transposition for fistulas between the rectum and urethra or vagina, Dis. Colon Rectum 49 (2006) 1316e1321. [17] C.-W. Wu, C.-H. Lin, C.-H. Lin, One-stage posttraumatic bladder reconstruction and soft-tissue coverage of the lower abdomen or perineum, Ann. Plast. Surg. 64 (2010) 65e68. [18] G. Stamatis, L. Freitag, M. Wencker, D. Greschuchna, Omentopexy and muscle transposition: two alternative methods in the treatment of pleural empyema and mediastinitis, Thorac. Cardiovasc. Surg. 42 (1994) 225e232. [19] A. Menon, M.A. Clark, T. Shatari, C. Keh, M.R.B. Keighley, Pedicled flaps in the treatment of nonhealing perineal wounds, Colorectal Dis. 7 (2005) 441e444. [20] G.E. Spyrou, D.A. Watt, I.L. Naylor, The origin and mode of fibroblast migration and proliferation in granulation tissue, Br. J. Plast. Surg. 51 (1998) 455e461. [21] W. Adams, G. Ctercteko, M. Bilous, Effect of an omental wrap on the healing and vascularity of compromised intestinal anastomoses, Dis. 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Moquin, et al., Immunohistochemical identification of vascular endothelial growth factor in pig latissimus dorsi musculocutaneous flaps following ischemia-reperfusion injury, Ann. Plast. Surg. 53 (2004) 398e403. [27] T. Wada, K. Okamoto, Y. Nakanishi, Y. Iwagami, N. Morita, Myofascial flap without skin for intra-oral reconstruction. 1: animal studies, Int. J. Clin. Oncol. 6 (2001) 40e44. [28] T. Yamaki, T. Uede, A. Tano-oka, K. Asakura, S. Tanabe, K. Hashi, Vascularized omentum graft for the reconstruction of the skull base after removal of a nasoethmoidal tumor with intracranial extension: case report, Neurosurgery 28 (1991) 877e880. [29] G.A. Brunelli, F. Brunelli, F. Di Rosa, Neurolized nerve padding in actinic lesions: omentum versus muscle use. An experimental study, Microsurgery 9 (1988) 177e180. [30] K. Takanari, Y. Kamei, K. Toriyama, et al., Management of postpneumonectomy empyema using free flap and pedicled flap, Ann. Thorac. Surg. 89 (2010) 321e323. [31] T. Komatsu, T. Sowa, T. Fujinaga, et al., Tracho-innominate artery fistula: two case reports and a clinical review, Ann. Thorac. Cardiovasc. Surg. 19 (2013) 60e62. [32] V. Chittithavorn, C. Rergkliang, A. Chetpaophan, et al., Single- stage omental flap transposition: modality of an effective treatment for deep sternal wound infection, Interact. Cardiovasc. Thorac. Surg. 12 (2011) 982e986.