Bovine Pericardium Patch Wrapping Intestinal Anastomosis Improves Healing Process and Prevents Leakage in a Pig Model

Bovine Pericardium Patch Wrapping Intestinal Anastomosis Improves Healing Process and Prevents Leakage in a Pig Model
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  Bovine Pericardium Patch Wrapping IntestinalAnastomosis Improves Healing Process and PreventsLeakage in a Pig Model Mario Testini 1 * , Angela Gurrado 1 , Piero Portincasa 2 , Salvatore Scacco 3 , Andrea Marzullo 4 ,Giuseppe Piccinni 1 , Germana Lissidini 1 , Luigi Greco 5 , Maria Antonietta De Salvia 6 , Leonilde Bonfrate 2 ,Lucantonio Debellis 7 , Nicola Sardaro 3 , Francesco Staffieri 8 , Maria Rosaria Carratu ` 6 , Antonio Crovace 8 1 Department of Biomedical Sciences and Human Oncology, Unit of Endocrine, Digestive and Emergency Surgery, University Medical School ‘‘A. Moro’’, Bari, Italy, 2 Department of Biomedical Sciences and Human Oncology, Unit of Medicine ‘‘A. Murri’’, University Medical School ‘‘A. Moro’’, Bari, Italy,  3 Department Basic MedicalSciences, University Medical School ‘‘A. Moro’’, Bari, Italy,  4 Department of Emergency Surgery and Organ Transplantation, Unit of Pathology, University Medical School ‘‘A.Moro’’, Bari, Italy,  5 Department of Emergency Surgery and Organ Transplantation, Unit of General Surgery and Liver Transplantation, University Medical School ‘‘A. Moro’’,Bari, Italy,  6 Department of Biomedical Sciences and Human Oncology, Section of Pharmachology, University Medical School ‘‘A. Moro’’, Bari, Italy,  7 Department of Biosciences, Biotechnology and Pharmacological Sciences, University Medical School ‘‘A. Moro’’, Bari, Italy,  8 Department of Emergency Surgery and OrganTransplantation, Division of Veterinary Clinics and Animal Productions, University Medical School ‘‘A. Moro’’, Bari, Italy Abstract Failure of intestinal anastomosis is a major complication following abdominal surgery. Biological materials have beenintroduced as reinforcement of abdominal wall hernia in contaminated setting. An innovative application of biologicalpatch is its use as reinforcement of gastrointestinal anastomosis. The aim of study was to verify whether the bovinepericardium patch improves the healing of anastomosis, when  in vivo  wrapping the suture line of pig intestinal anastomosis,avoiding leakage in the event of deliberately incomplete suture. Forty-three pigs were randomly divided: Group 1 (control,n=14): hand-sewn ileo-ileal and colo-colic anastomosis; Group 2 (n=14): standard anastomosis wrapped by pericardiumbovine patch; Group 3 (n=1) and 4 (n=14): one suture was deliberately incomplete and also wrapped by patch in the lastone. Intraoperative evaluation, histological, biochemical, tensiometric and electrophysiological studies of intestinalspecimens were performed at 48 h, 7 and 90 days after. In groups 2 and 4, no leak, stenosis, abscess, peritonitis, meshdisplacement or shrinkage were found and adhesion rate decreased compared to control. Biochemical studies showedmitochondrial function improvement in colic wrapped anastomosis. Tensiometric evaluations suggested that the patchpreserves the colic contractility similar to the controls. Electrophysiological results demonstrated that the patch alsoimproves the mucosal function restoring almost normal transport properties. Use of pericardium bovine patch asreinforcement of intestinal anastomosis is safe and effective, significantly improving the healing process. Data of preventionof acute peritonitis and leakage in cases of iatrogenic perforation of anastomoses, covered with patch, is unpublished. Citation:  Testini M, Gurrado A, Portincasa P, Scacco S, Marzullo A, et al. (2014) Bovine Pericardium Patch Wrapping Intestinal Anastomosis Improves HealingProcess and Prevents Leakage in a Pig Model. PLoS ONE 9(1): e86627. doi:10.1371/journal.pone.0086627 Editor:  Giovanni Li Volti, University of Catania, Italy Received  November 4, 2013;  Accepted  December 16, 2013;  Published  January 29, 2014 Copyright:    2014 Testini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  The authors have no support or funding to report. Competing Interests:  The authors have declared that no competing interests exist.* E-mail: Introduction Failure of gastrointestinal anastomosis results in dehiscence,leaks and fistulas, and is considered a major complicationfollowing abdominal surgery. Despite improved surgical tech-nique, the reported incidence of gastrointestinal anastomosisleakage ranges from 2% to 10% [1–5], and is associated withboth increased morbidity (20–30%) and mortality (7–12%) [1–5].Hypoalbuminemia, chronic obstructive pulmonary disease,colon cancer and IBD have been identified as significant risk factors for anastomotic leakage [1,3,4,6]. However, wide resectionmargins, absence of tension at the level of the suture site andresection along anatomic blood supply may decrease the risk [1,6].By contrast, hypovolemia, blood transfusions, besides surgical skill,prolonged operative time and difficult operative procedures, havebeen associated with anastomotic leakage development [1,6].Biological materials have been introduced in general surgery asreinforcement of abdominal wall hernia in contaminated setting,when the use of alloplastic meshes is contraindicated [7]. Hand tohand with the success of bovine pericardium for valves and patchgrafts in cardiac surgery [8,9], this biomaterial has beenconsidered suitable in place of dura mater in anterior abdominalwall defects in pediatric surgery [10,11] and then for the treatmentof the patients affected by incisional hernia particularly in thecontaminated or urgent context. An innovative application in thisrespect is the use of the biomaterials as reinforcement of thegastrointestinal anastomotic suture line [12–21]. In particularly,the available experimental data showed that the mechanicalanastomoses buttressed with bovine pericardium [13,14] or smallintestinal submucosa had greater bursting strength as compared tonon-buttressed anastomoses [15,19] and a wound healing improvement [14,17] has been demonstrated. Moreover, a colic PLOS ONE | 1 January 2014 | Volume 9 | Issue 1 | e86627  perforation treated by positioning a resorbable bilayer collagenband of bovine origin in a pig model showed results veryencouraging [21].The aim of the study was to verify whether the bovinepericardium patch improves the healing of anastomosis, whenaffixed in vivo on the hand-sewn suture line of ileo-ileal and colo-colic anastomosis of the pigs. A further end-point was to seewhether the patch is able to avoid the anastomotic leakage in thecase of deliberately incomplete suture. For this, we used a pig model undergoing intraoperative and histological evaluation, andbiochemical, tensiometric and electrophysiological measurementsof intestinal specimens because these animals recapitulate severalkey features of human anatomy and physiology of the grastroin-testinal tract. Materials and Methods Animals and Ethics Statement  After approval by the Italian Ministry of Health (protocolnumber: 02/2010) and in strict accordance with the recommen-dations in the Guide for the Care and Use of Laboratory Animalsof the National Institutes of Health, between September 2010 and April 2012, forty-three domestic pigs (Landrace; female; mean age5.3 6 2.2 months; weight 38.7 6 9.2 Kg) were sourced from com-mercial piggery affiliated with the Division of Veterinary Clinicsand Animal Productions of University Medical School ‘‘A. Moro’’of Bari (Italy), and were included in this study and underwent twosurgical procedures. Vendor health reports indicated that theanimals were free of known viral, bacterial and parasiticpathogens. The pigs were weighed before surgery and at regularinterval during the experimental period. After a preoperativefasting time of 24 hours, all surgery was performed under anaseptic setting and general anesthesia, and all efforts were made tominimize suffering. At the end of the second surgical procedure,pigs were euthanized with a bolus of thiopental followed by a bolusof KCl. All pigs received ampicilline (25 mg/kg every 12 hours)and tramadol (5 mg/kg daily) for seven days after surgery. Pigswere fed, leaving water  ad libitum,  and were monitored daily inorder to detect any alteration of the clinical conditions (food intakeand weight loss; urine and feces production; rectal temperatureand behavior changes). Study groups and Surgical Procedure I The peritoneal cavity was entered  via   a midline incision and asegment of colon 30 cm from the anal verge and of ileum 30 cmfrom the cecum verge were resected. The animals were randomlyassigned to four groups: 1) Group 1 (control, n=14): the hand-sewn ileo-ileal and colo-colic anastomosis were performed using single layer of interrupted suture (PDS 3/0, Ethicon, Germany)according to  Gambee;   the distance between the single sutures were3–4 mm; 2) Group 2 (n=14): the standard anastomosis werewrapped by a 50 6 20 mm single layer pericardium bovine patch(Tutomesh H , Tutogen Medical GmbH, Germany); in order toallow some swelling during healing, 360 u  anastomotic sealing wasachieved by a 5 mm over-lapping of the two ends of the patch atthe mesenterial side of the anastomosis without fixing the ends toeach other (Fig. 1A); 3) Group 3 (n=1): one suture of theanastomosis was deliberately performed incomplete; 4) Group 4(n=14): one suture of the anastomosis was deliberately performedincomplete and wrapped by the pericardium bovine patch. Theabdominal cavity was closed in layers with absorbable sutures in allpigs. Procedures were carried out by the same surgeon (M.T.) andthe randomization was performed by using numbered and sealedenvelopes that were opened at the beginning of the operation. Surgical Procedure II and Macroscopic examination In Group 1, 2 and 4, a relaparotomy was performed 48 h (4 pigsfor each group), 7 and 90 days after (5 pigs for each group); theanimals of the Group 3 were reoperated 48 h thereafter only. Theabdominal cavity was inspected for any kind of free fluid collectionand anastomotic leakage, stenosis, abscess, peritonitis or intra-abdominal adhesions. Quantity and quality of adhesions wereexamined in a scoring system (0–3 points). In order to perform themicroscopic, biochemical, tensiometric and electrophysiologicalstudies, an intestinal tract was dissected four centimeters upstreamand downstream the surgical site. Preparation of histological specimens and Microscopicexamination  All the samples were fixed with neutral buffered formalin for24–48 hours and paraffin- embedded; consecutive sections of 3  m m thickness were longitudinally cut to include the anastomosisand the adjacent intestinal tracts, in order to observe the wholesection from the mucosa to the serosa, and were stained withhaematoxylin-eosin and periodic acid-Schiff stains.Evaluation was carried out by the same pathologist blinded forthe experimental protocol. For each section the following histological parameters were evaluated: grade of inflammation,parietal fibrosis, integrity of the mucosal layer, serosal status, andthe grade of granulocyte infiltration of the membrane in cases withthe biological mesh. Phlogosis, fibrosis and mesh infiltration wereemployed for scoring system (0–4 points). Biochemical studies Immediately after surgery, the tissue of representative segmentsof ileo-ileal and colo-colic anastomosis (Group 1, 2) was disruptedby a glass-Teflon homogenizer and mitochondria were isolatedaccording to  Bookelman et al  . [22]. The mitochondrial respirationwas measured polarographically with a Clark-type oxygenelectrode in a water-jacketed chamber, magnetically stirred at37 u C. Respiration rates were expressed as nanomoles of molecularoxygen consumption  per   minute  per   milligram of mitochondrialprotein [23], after addition of substrates and inhibitors of oxidativephosphorylation. To measure the rotenone sensitive reducednicotinamide adenine dinucleotide (NADH)-ubiquinone oxidore-ductase activity, mitochondria were exposed to ultrasound energyfor 15 s at 0 u C [23]. Cytochrome c oxidase activity wasdetermined on mitochondria [23], following the oxidation of ferrocytochrome c 10  m M. Citrate synthase activity [23] was usedas mitochondrial matrix enzymatic marker. For detection of reactive oxygen species (ROS) in ileo-ileal and colo-colicanastomosis whole tissue homogenate, fluorimetric analysis wasused [23] with a Jasco FP6200 spectrofluorimeter. All activitieswere expressed as nanomoles of substrates  per   minute  per   milligramof mitochondrial protein. Tensiometric studies Tensiometric studies of freshly excised colic and ileal smoothmuscle specimens (Group 1, 2) were conducted at 48 hours, 7 and90 days after surgery. Specimens were mounted in an organ bathfilled with modified Krebs’ solution (20 ml at 37 u C gassed withO2/CO2 (95%/5%), pH 7.4). Isometric tension was measuredwith a strain gauge transducer (cat. 7003 Basile, Milan, Italy)connected to data acquisition system (PowerLab and Chart 4.1.2 ADInstruments, Castle Hill, Australia). After 45 minutes equili-bration, an initial load of 1.0 g tension was applied to the tissue.Contractile responses were measured to acetylcholine (ACh,10 2 7  –10 2 4 M) and KCl (80 mM). Bovine Pericardium Wrapping AnastomosesPLOS ONE | 2 January 2014 | Volume 9 | Issue 1 | e86627  Electrophysiological measurements Transepithelial potentials (V T  ), resistance (R T  ) and short circuitcurrent (I SC  ) were measured as markers of colic and ileal mucosatransport efficiency at baseline, early stage (days 2 and 7, tissuespool), and late stage (day 90) after surgery in Groups 1, 2. Freshlyexcised mucosa was placed in a cooled modified Krebs’bicarbonate/phosphate buffer solution (at 37 u C gassed with O2/CO2 (95%/5%), pH 7.4) and mounted in a Ussing chambers(Mussler Scientific Inst., Aachen, Germany) with an exposed areaof 1 cm 2 . Two pairs of Ag/AgCl electrodes were used to monitorV T  (mV) and R T  (  V .cm2). I SC  (  m  A/cm 2  ) was measured with theV T  clamped to 0 at 5 min intervals. At fixed intervals of 1 min atransepithelial bipolar current pulse (I) of 1  m  A amplitude and200 msec duration was applied to tissue and R T  calculated fromthe change in open-circuit voltage (  D V T  ) according to Ohm’s law(R T = D V T /I). Experiments were conducted simultaneously onmultiple specimens, each from an individual pig. Electricalparameters were measured by the software Clamp (v. 2.14, Aachen, Germany) and recorded for 60 min after an initialequilibration time of about 30 min. Statistical analysis Data are given as means  6  SEMs. Infra-group comparisonswere made using median test with the two sided p-value computedusing Fisher’s exact test. For biochemical studies, one-way or two-way analysis of variance, as appropriate, followed by pairedStudent  t  -test or Newman-Keuls multiple comparison tests wereused. For electrophysiological studies, data of the measurementsduring 60 min were compared between control and treated tissueswere assessed using the Student  t  -test for unpaired data betweenthe data average of early and late follow-up days  vs   day 0 and foranastomosis with patch  vs   anastomosis without patch. Data wereanalyzed by Stata 12 software (StataCorp LP, College Station,Tex, USA).  P   values of   , 0.05 were considered statisticallysignificant. Results Macroscopic examination Tables 1 and 2 summarize the intraoperative findings atrelaparotomy. In the control group, one leakage was revealed in acolo-colic anastomosis 48 h after surgery. No stenosis, abscess orperitonitis was found in this group and adhesions were progres-sively observed in the subgroups. In Group 2, the intraoperativeevaluation of the abdominal cavity lacked anastomotic leaks,intraabdominal abscess, stenosis or peritonitis. Moreover, adhesionrates (quantity and quality; Table 2) were significantly less than thecontrol group (p=0.006 and p=0.018, respectively). As expected,in Group 3, peritonitis was evident and adhesions were extremelysolid. By contrast, in Group 4, no leaks, stenosis, abscess andperitonitis were seen in any of the pigs and adhesion ratesprogressively increased, but less than Group 1 again (p=0.016and =0.006, respectively). No shrinkage and displacement of prosthesis was seen either in Group 2 or 4. Histological examination Table 3 shows the microscopic findings of the anastomosis at therelaparotomy. In control group, a heavy inflammatory infiltratewas observed at early stage, decreasing progressively and being  virtually absent on day 90 postoperatively. Conversely, thefibrogenic process was initially evident from the 7 th postoperativeday and completed on the 90 th . The mucosal surface and theserosal layer rapidly recovered. In Groups 2 (Fig. 1B) and 4(Fig. 1C), there was no statistical difference in comparison with thecontrol group in terms of the phlogosis. Moreover, the fibroticreaction was statistically less evident in Groups 2 and 4 (bothp=0.033) compared to the control. Indeed, a significant Figure1.(A – C)Intraoperativeimage andhistologicalfindingsofintestinalanastomosiswrappedbypericardiumbovinepatch. Ileo-ileal anastomosis wrapped by a 50 6 20 mm one layer pericardium bovine patch (A); at day 7 (B) the patch is enveloped by an heavy lympho-isthyocitic infiltrate without essudation on the serosal surface (Group 2) and after 48 h (C) the necrotic tissue is well contained into the intestinal wallby the patch (Group 4), with minimal leukocyte infiltration of the serosal surface (Haematoxylin-Eosin 200X srcinal magnification).doi:10.1371/journal.pone.0086627.g001Bovine Pericardium Wrapping AnastomosesPLOS ONE | 3 January 2014 | Volume 9 | Issue 1 | e86627  granulation tissue was initially present at the border of the bovinepericardium patch and subsequently in the deeper areas. On the90 th day there were only small, hardly recognizable fragments of the patch surrounded by macrophages and lymphocytes. Whilethe mucosal surface rapidly recovered, the serosa became smoothonly 90 days after the operation. In the Group 3, the inflammatorycell infiltrate and the fibrin deposition were massive and diffuse. Biochemical studies We measured the mitochondrial respiratory activity by endog-enous substrates (NADH-dependent respiration through complexI, III and IV; Fig. 2A) and from added substrates, like succinate(respiration through complex II, III and IV; Fig. 2B) and ascorbate(final respiratory step through complex IV; Fig. 2C), in ileo-ilealand colo-colic anastomosis samples (Group 1, 2). In Fig. 2D,respiratory control ratios indicates the coupling of mitochondria,oxidative phosphorylation efficiency and adenosine triphosphate(ATP) production. In the same samples, at each time point, theenzyme marker for mitochondrial matrix (citrate synthase;Fig. 3A), and the enzymatic activities of complex I (NADH-ubiquinone oxidoreductase; Fig. 3B) and IV (cytochrome coxidase; Fig. 3C) were measured. Within 48 hours after anasto-mosis, mitochondrial respiration (Fig. 2A–C) and enzymaticactivities (Fig. 3A–C) showed a marked decline of all functionalparameters in ileo-ileal and colo-colic anastomosis, which lastedfor one week. After this phase, the recovery showed a differencebetween the two intestinal parts. Specifically, the biochemicalparameters of ileo-ileal anastomosis showed a gradual recoveryand in three months were almost fully restored to the initial values,without a significant influence by the patch (Group 2). On thecontrary, the parameters of colo-colic anastomosis showed norecovery even after three months, except when the patch wasapplied, with a partial early recovery after two days, which was fullafter three months. Uncoupling of mitochondria from two days toone week after anastomosis was observed in both colo-colic andileo-ileal anastomosis, with the latter being more affected (Fig. 2D).The Tutomesh H , once again, was shown to improve the recoveryof mitochondrial coupling in colo-colic anastomosis from one -week to three months. Measurement of hydrogen peroxidegeneration in whole tissue homogenates after anastomosis showedsignificant differences between ileo-ileal and colo-colic anastomosis(Fig 3D). In particular, in ileo-ileal anastomosis a significantincrease of H 2 O 2  production was detected from two days toone week after anastomosis, before returning to basal levels afterthree months and this did not seem to be significantly influencedby the patch. In classic colo-colic anastomosis, however, theincrease of H 2 O 2  generation was significantly higher two daysafter anastomosis and decreased progressively in the subsequentdays reaching normal basal values after three months. Unlike thisapplication of Tutomesh H , in colo-colic anastomosis it appeared tolimit the early increase of H 2 O 2  production, two days afteranastomosis, which was delayed to a threefold increase atone week and gradually decreased to normal basal values inthree months. Tensiometric studies  At baseline, ACh induced dose-response contractions in ileumand colon specimens (controls), which were slightly morepronounced in ileum. The response to KCl was comparable inboth bowel tracts. Forty-eight hours after surgery (Fig. 4A), the Table 1.  Intraoperative results. Group Relaparotomy Leak Stenosis Abscess Peritonitis Pericardium bovine patchShrinkage Displacement 48h (4) 1* 0 0 0 - -1 (n) 7 pod (5) 0 0 0 0 - -90 pod (5) 0 0 0 0 - -48h (4) 0 0 0 0 0 02 (n) 7pod (5) 0 0 0 0 0 090 pod (5) 0 0 0 0 0 03 (n) 48h (1) 1** 0 0 1 - -48h (4) 0 0 0 0 0 04 (n) 7pod (5) 0 0 0 0 0 090 pod (5) 0 0 0 0 0 0h: hours; pod: post-operative day; *colo-colic anastomosis; **both anastomosis.doi:10.1371/journal.pone.0086627.t001 Table 2.  Mean Adhesion Score. Adhesion rate I quantity  * Group  P  1 2 3 4 2  vs   1 4  vs   1 48h 1 6 0 0 6 0 3 0.3 6 0.3 0.029 NA7 pod 2.4 6 0.2 0.2 6 0.2 - 1.0 6 0.0 0.008 0.00890 pod 2.8 6 0.2 1.4 6 0.2 - 2.0 6 0.0 0.048 0.048 Total   2.1 6 0.2 0.6 6 0.2 - 1.1 6 0.2 0.006 0.016  Adhesion rate II quality  **48h 1 6 0 0 6 0 3 0.5 6 0.3 0.029 NA7 pod 2.2 6 0.4 0.2 6 0.2 - 1.6 6 0.2 0.048 0.44490 pod 3 6 0 1.4 6 0.2 - 2 6 0 0.008 0.008 Total   2.1 6 0.3 0.6 6 0.2 - 1.4 6 0.2 0.018 0.006h: hours; pod: post-operative day. Mean score 6 SEM. *Adhesion rate I: 0= noadhesion; 1= adhesions with one structure; 2= adhesions with two structures;3= adhesions with three or more structures. **Adhesion rate II: 0= noadhesions; 1= light adhesions; 2= fixed adhesions; 3= solid adhesions, onlyremovable with damage.doi:10.1371/journal.pone.0086627.t002 Bovine Pericardium Wrapping AnastomosesPLOS ONE | 4 January 2014 | Volume 9 | Issue 1 | e86627  contractile response of the colon to ACh was lower (p , 0.05) inGroup 1  vs   control, while Tutomesh H  prevented this effect. Nodifference was found in ileal specimens (Fig. 4B). Seven days aftersurgery the contractile response of the colon to ACh increased inGroup 1 (p , 0.05)  vs   control, but was unchanged in Group 2(Fig. 4C). In the ileum, Tutomesh H  determined a reducedcontractile response to ACh (p , 0.05), while Group 1 responsewas similar to control (Fig. 4D). Ninety days after surgery, theresponse to ACh or KCl was comparable in different surgicaltreatments both in the colon and ileum (Fig. 4E,F). The responseto KCl was similar in colic and ileal specimens (Fig. 4A–F). Electrophysiological measurements  At baseline, the ileum showed I SC  2 12.1 6 1.3  m  A/cm 2 , R T 131.2 6 8.1  V . cm 2 , and V T  1.1 6 0.2 mV, lumen negative(N=15). The colon showed I SC  2 14.7 6 1.2  m  A/cm 2 , R T 126.6 6 6.9  V . cm 2 , and V T  0.59 6 0.1 mV, lumen negative(N=15). In the ileo-ileal anastomosis the early stage, wasassociated with significantly decreased I SC  (  2 74.1%, p , 0.0001 vs   control) and increased R T  (  + 25.7%, p , 0.05  vs   control)(Fig. 5A,B). A similar trend (i.e.  2 35%) existed for V T  (data notshown). In the late stage, I SC  remained below the control value(  2 40.9%, p , 0.05  vs   control; Fig. 5A), R T  increased significantlyup to 53% (p , 0.02  vs   control; Fig. 5B), and V T  decreased by57.5% (p , 0.02  vs   control). The presence of colo-colic anasto-mosis in the early stage was associated with significantlydecreased I SC  (  2 36,4%, p , 0.05  vs   control; Fig. 5C), a tendencyof increased R T  (Fig. 5D) and reduced V T . In the late stage, I SC remained stable, while R T  and V T  tended to increase comparedto the control. Affixing a pericardium bovine patch on ilealanastomosis prevented the electrophysiological changes seen withanastomosis alone (I SC  virtually unchanged in the early and latestages, Fig. 5A) while R T  (Fig. 5B) and V T  did not increase. Inthe colon, likewise, the prosthesis prevented the changes of I SC ,R T  and V T  (Fig. 5C–D). Discussion Our experimental study is the first in literature to investigatewhether a pericardium bovine patch, wrapping ileo-ileal and colo-colic hand-sewn anastomosis in pigs, seals the suture line andpromotes processes of anastomotic healing. Dehiscence of anastomosis is a disturbing, common and severe complicationafter bowel resection, with an incidence ranging from 0.5 to 30%after large bowel resection and to about 6% after small bowelresection [1–5].Several studies have demonstrated that the early integrity of theanastomosis depends not only on the correct surgical performance,but also on the suture-holding property of the sub-mucosal layerand on formation of a fibrin seal on the serosa [24]. Furthermore,the process of anastomotic healing is related to the structure andarrangement of the collagen matrix [24].Despite the fact that many risk factors for postoperative leakagehave been analyzed in literature [1,3,4,6], few clinical andexperimental studies have focused on the best preventiontechniques of anastomotic dehiscence [25,26]. However, operativeand post-operative treatments, allowing suitable oxygenation andblood perfusion of the anastomotic side and stimulating theangiogenesis, with infusion of grow factors or inhibitors of metalloproteinase, have been investigated [25,26]. Some studiesinvestigated whether the reinforcement of anastomosis withbiological or synthetic materials was able to prevent theanastomotic leakage. Reports regarding the use of syntheticmaterials are, however, uncommon and with discordant outcomes[27,28]. Hand in hand with the use of the biologic mesh inabdominal wall repair, many experimental studies on theirapplication as reinforcement of anastomosis have spread, andthe bovine pericardium [13–16], small intestinal submucosa [17– 19] and porcine dermis [20] are the biomaterials used. Thebiological materials are all basically composed of an extracellularmatrix deprived of its cellular components. The extracellularmatrix serves as scaffold for the remodeling process by hostthrough the connective tissue ingrowth and cellular colonizationand proliferation. Reports regarding the use of the bovinepericardium are experimental [13–15] or clinical studies [16]and the anastomoses have been performed using a circular staplerwith the introduction of bovine pericardium as a buttressing material to reinforce staple lines.In this study, the anastomosis was performed using a single layerof interrupted suture according to  Gambee.  In accordance withknowledge of the healing phases of anastomosis, the surgicalprocedure II was carried out in the early and late phase (at 48 h, 7,and 90 days, respectively after the first operation). However, theprevious experimental studies analyzed the early effect (after a fewhours) of the pericardium bovine buttress on the anastomosis[13,15] and only  Hagerman et al   [14] performed the evaluation inlate phase of suture healing. In this study, we provide a completeoverview of key events involved in anastomosis healing with andwithout bovine patch. By using highly integrated and translationalmethodologies, we describe a detailed intraoperative evaluation, Table 3.  Microscopic Findings and Mean Histological Score. Phlogosis*   Group  P  1 2 3 4 2  vs   1 4  vs   1 48h 4 6 0 3.8 6 0.3 4 4 6 0 NA NA7 pod 3.4 6 0.2 3.4 6 0.2 - 3.4 6 0.2 1.000 1.00090 pod 0.2 6 0.2 0.4 6 0.2 - 0.4 6 0.2 1.000 1.000 Total   2.4 6 0.5 2.4 6 0.2 - 2.5 6 0.5 1.000 1.000 Fibrosis*  48h 0.5 6 0.3 0.3 6 0.3 4 0.3 6 0.3 1.000 1.0007 pod 2.2 6 0.4 1.8 6 0.2 - 1.2 6 0.2 0.444 0.20690 pod 3.8 6 0.2 2.2 6 0.2 - 2.2 6 0.2 0.048 0.048 Total   2.3 6 0.4 1.5 6 0.2 - 1.3 6 0.2 0.033 0.033 Mucosa 48h ulcerated ulcerated ulcerated ulcerated - -7pod normal eroded - eroded - -90 pod normal normal - normal - - Serosa 48h thickened normal thickened normal - -7pod normal thickened - thickened - -90 pod normal normal - normal - - Meshinfiltration*  48h - 0.3 6 0.3 - 0.3 6 0.3 - -7pod - 1.4 6 0.2 - 1.4 6 0.2 - -90 pod - 0.2 6 0.2 - 0.2 6 0.2 - - Total   - 0.6 6 0.2 - 0.6 6 0.2 - -h: hours; pod: post-operative day; mean score  6  SEM. *Score 0-4: 0= absent;1= minimal; 2= moderate; 3= distinctive; 4= severe.doi:10.1371/journal.pone.0086627.t003 Bovine Pericardium Wrapping AnastomosesPLOS ONE | 5 January 2014 | Volume 9 | Issue 1 | e86627
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