- Email: [email protected]
Ultrapure alginate anti-adhesion gel does not impair colon anastomotic strength
Accepted Manuscript Ultrapure alginate anti-adhesion gel does not impair colon anastomotic strength Ankit A. Chaturvedi , MS Roger M.L.M. Lomme , Thijs Hendriks , PhD Harry van Goor , M.D, PhD PII:
S0022-4804(14)00548-4
DOI:
10.1016/j.jss.2014.05.085
Reference:
YJSRE 12781
To appear in:
Journal of Surgical Research
Received Date: 16 September 2013 Revised Date:
8 April 2014
Accepted Date: 29 May 2014
Please cite this article as: Chaturvedi AA, Lomme RMLM, Hendriks T, van Goor H, Ultrapure alginate anti-adhesion gel does not impair colon anastomotic strength, Journal of Surgical Research (2014), doi: 10.1016/j.jss.2014.05.085. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised: 31/03/2014 Ultrapure alginate anti-adhesion gel does not impair colon anastomotic strength Ankit A. Chaturvedi a, b, MS, Roger M.L.M. Lomme a, Thijs Hendriks a, PhD and Harry van Goor a, M.D, PhD Department of Surgery, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
b
European Medical Contract Manufacturing B.V, Nijmegen, The Netherlands.
RI PT
a
Category: Original scientific article
SC
Subject Category: Gastrointestinal Correspondence to:
Department of surgery, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. Email: [email protected]
M AN U
Mr. Ankit Akshayesh Chaturvedi,
TE D
Telephone no: + 31-024-36-13956; + 31-0633633809
Author Contribution:
EP
Ankit Chaturvedi: Conception, Design, Data collection, Analysis, Interpretation, writing article and revision Roger Lomme: Data collection, Analysis, Interpretation
AC C
Dr. Thijs Hendriks: Conception, Design, Writing article and Critical revisions Prof. Harry Van Goor: Conception, Design, Interpretation, Writing article and Critical revision
Communications: The work has been presented at: 22nd European Tissue Repair Society Congress. October 4-5, 2012, Athens, Greece. Funding: The research leading to these results has received funding from the European Community's Seventh Framework Programme; MultiTERM, grant agreement nr 238551.
1
ACCEPTED MANUSCRIPT ABSTRACT Background: Ultrapure alginate gel is promising in terms of adhesion prevention. Since antiadhesive barriers have been shown to disturb healing of bowel anastomoses the effect of ultrapure alginate gel on the repair of colon anastomoses was studied.
RI PT
Materials and Methods: In 102 male Wistar rats a 0.5 cm segment was resected from the descending colon and continuity was restored by an inverted single-layer end-to-end
anastomosis. Animals were randomized into a control, an alginate gel and a sodium hyaluronate
SC
carboxymethyl cellulose (HA/CMC) film group, each n=34. Half of each group was sacrificed at day 3 and 7 postoperatively. Anastomotic strength was assessed by measuring both bursting
M AN U
pressure and breaking strength. Hydroxyproline content was measured and histological analysis was performed. The incidence of adhesion and abscess formation was scored at sacrifice. Results: No difference in either anastomotic bursting pressure or breaking strength was found between experimental groups and the controls at any time point. Both the incidence of adhesion
TE D
formation (35% vs 71%, P=0.007) and the adhesion score (0.38 vs 0.79, P=0.009) were significantly lower in the alginate gel group than in the controls. The abscess rate was higher
alginate gel group.
EP
(46% vs 18%, P=0.030) in the HA/CMC group than in the controls and unchanged in the
Conclusion: While reducing adhesion formation, ultrapure alginate gel does not interfere with
AC C
the development of colonic anastomotic strength during the crucial early healing period.
Keywords: Ultrapure alginate gel, HA/CMC, Colon anastomosis, abdominal adhesion
2
ACCEPTED MANUSCRIPT Introduction Abdominal adhesion formation occurs in about 65 to 100 percent of patients after intraabdominal surgery. It constitutes an important cause of long term morbidity including bowel obstruction, adhesiolysis induced organ damage at repeat surgery, female infertility, and chronic
5
RI PT
pain.1-4 These morbidities incur significant patient discomfort and come at great financial costs.4, Results from ‘good’ surgical techniques, including laparoscopy, have not yielded convincing
evidence of reduced adhesion morbidity.6
SC
The use of anti-adhesive barriers seems the best way to prevent adhesion formation and reduce morbidity and mortality. So far, only one product consisting of sodium hyaluronate
M AN U
carboxymethyl cellulose (HA/CMC) membrane has been shown to significantly reduce the incidence of postoperative adhesion formation in general surgery.7-10 However, the use of HA/CMC appears to be associated with an increased incidence of anastomotic leakage when the membrane is applied in direct contact with the anastomosis.11, 12 When wrapped around the
TE D
anastomosis, the anti-adhesive effect of the HA/CMC membrane is believed to convert subclinical into clinical leaks. This effect, which may be considered as prevention which is too rigorous, has not consistently been confirmed experimentally or in other clinical studies. Still, it
EP
emphasizes the need to demonstrate uncompromised anastomotic healing with the use of antiadhesive barriers in visceral surgery.
AC C
We recently showed excellent anti-adhesive effects of a new ultrapure, easy to apply, alginate gel.13 Adhesion formation was reduced by 80% in a cecal abrasion and peritoneal side wall excision rat model. There was also a tendency to reduce adhesion formation at locations distant from the site of actual gel application. While such a remote effect would be advantageous because it covers adhesiogenic areas not primarily injured by dissection, it could also become disadvantageous if it reaches an anastomotic area thereby disturbing normal healing.
3
ACCEPTED MANUSCRIPT The first postoperative week is crucial to anastomotic repair. During the first few days wound strength is low while after day 3 strength increases reaching pre-operative values around day 7.14 Therefore, it should be demonstrated that perioperative application of the gel does not lead to healing impairment in this period when the anastomosis is relatively vulnerable. The present
RI PT
study aims to investigate the effect of ultrapure alginate gel, applied around the suture line, on the healing of a rat colonic anastomoses. In a separate group the effect of a HA/CMC membrane
AC C
EP
TE D
M AN U
SC
is examined.
4
ACCEPTED MANUSCRIPT Methods Animals and experimental design One hundred and two male Wistar (Charles River, Sulzfeld, Germany) rats were used in the experiment. Rats weighed between 250 and 280 g and were housed in filter topped cages (two
RI PT
per cage) under clean, non-sterile, standardized conditions for at least five days prior to the experiment. Animals were checked daily and had free access to water and standard rodent chow (Ssniff R/M-H, Bio Services BV, Uden, The Netherlands) during the entire experimental period.
SC
All animals underwent resection and anastomosis of the distal colon. The rats were allocated randomly to any of three groups (n=34 each). Animals in the first group received no further
M AN U
treatment and served as controls. In the second group, animals received ultrapure alginate gel and in the third group HA/CMC film. Half of each group was killed on postoperative days 3 and 7, respectively, for analysis of wound strength (n=17) and wound hydroxyproline (n=11) and histology (n=6).
TE D
The study protocol was approved by the Institutional Animal Ethics Review Committee (Approval No: RU-DEC 2012-003) and performed in the GLP certified animal research laboratory of the Radboud University Nijmegen Medical Centre, The Netherlands. Prior to the
AC C
study period.
EP
experiment humane endpoints were defined to avoid unnecessary suffering of animals during the
Operative procedure
Each animal was prepared for surgery by shaving and cleaning the lower abdomen with 70 percent alcohol. For analgesia, rats were given buprenorphine subcutaneously (Temgesic; Schering-Plough, Houten, The Netherlands, 0.02 mg/kg) starting 30 min before surgery and repeating the same dose every 12 h for the next 48 h. The rats were anesthetized by inhalation of a mixture of isoflurane (3 %) and 1:1 oxygen-nitrous oxide. Surgical procedures were performed
5
ACCEPTED MANUSCRIPT using a Zeiss operation microscope (Carl Zeiss AG, Oberkochen, Germany). During the operation and shortly thereafter rats were placed on a warm plate to avoid hypothermia. All rats underwent a 4 cm midline laparotomy and a 0.5 cm segment of the descending colon was resected 3 cm proximal to the peritoneal reflection. Continuity was restored by an inverted
RI PT
single-layer end-to-end anastomosis with eight interrupted sutures of 8-0 monofilament material (Ethicon, Norderstedt, Germany). Controls received no further treatment. In the second group 1 ml of ultrapure alginate gel (Rebasol™, EMCM, Nijmegen, The Netherlands) was applied on the
SC
anastomotic suture line. In the third group a 10 x 30 mm sheet of HA/CMC bioresorbable film (Seprafilm®; Genzyme, A Sanofi Company, Cambridge (MA), USA) was wrapped around the
M AN U
anastomosis. The abdomen was closed in two layers using Vicryl 3-0 suture (Ethicon products, Amersfoort, the Netherlands) for the fascia/muscle layer and wound staples for the skin. Animals were resuscitated immediately after abdominal closure with 10 ml isotonic sodium chloride
TE D
(0.9%) solution administered subcutaneously.
Adhesion formation
Animals were euthanized by carbon dioxide asphyxiation. The abdomen was reopened and
EP
inspected for adhesion and abscess formation at the anastomotic site. Adhesions were scored by an investigator blinded to the group assignment (HvG), using Zuhlke’s grading system.15
AC C
Adhesions were classified macroscopically as 0 = no adhesions, 1 = filmy adhesions: easy to separate by blunt dissection; no vascularization, 2 = mild adhesions: blunt dissection possible but partly sharp dissection required, 3 = strong adhesions: lysis possible by sharp dissection only; clear vascularization, 4 = very strong adhesions: lysis possible by sharp dissection only (organ strongly attached with severe adhesion). The organs and structures involved in the adhesion formation with the anastomosis were also noted. An abscess was defined as any walled off collection containing pus.
6
ACCEPTED MANUSCRIPT Bursting pressure and breaking strength Colonic segments, approximately 4 cm in length containing the anastomosis were resected and adhesions were dissected carefully without affecting the anastomosis. After removal of intraluminal faecal material, the colonic segment was attached to an infusion pump and a manometric
RI PT
device recording the internal pressure for measuring the bursting pressure, which represents the capability of the suture line to withstand intraluminal forces. Pressure was increased by the constant infusion (2 ml/min) of water containing methylene blue into the segment, which was
SC
kept under water. The maximum pressure (mmHg) recorded immediately before the sudden loss of pressure was taken as the bursting pressure. The site of perforation (within or outside the
M AN U
anastomotic line) was indicated by the loss of methylene blue through the intestinal wall. Subsequently, the breaking strength (which represents the capability of the suture line to withstand longitudinal forces) was measured in the same segment.16 The segment was clamped in a tensiometer (AIKOH model (9500 series), Aikoh Engineering Co. Ltd, Higashi-Osaka,
TE D
Japan) that exerts a steadily increasing force in longitudinal direction. The peak force required for tearing the segment apart was recorded as the breaking strength (g). Thereafter, the anastomotic segment was cleaned carefully from any remaining adhering tissue
EP
and a 5 mm sample, containing the suture line, was frozen in liquid nitrogen and stored at -80° C
AC C
for further biochemical processing.
Biochemical analysis
Tissue samples were lyophilized, weighed and pulverized. The hydroxyproline content, as a measure of the collagen content, was measured by high-performance liquid chromatography (HPLC) after hydrolysis with 6-N hydrochloric acid and coupling to dabsyl chloride.
7
ACCEPTED MANUSCRIPT Histological analysis Intestinal samples of approximately 1 cm, containing the entire anastomosis in the middle, were collected, opened at the mesenteric side, washed gently with 0.9 percent sodium chloride and spread out in a cassette for paraffin embedding. From paraffin-embedded tissues, 4-µm sections
RI PT
were prepared and stained with hematoxylin and eosin (H & E). Sections were analyzed using a binocular light microscope.
Another set of sections was stained with picrosirius red to identify collagen fibers in the
SC
anastomotic area, which were quantified using digital image analysis.17. Images were recorded using a 3-chip CCD RGB camera (DXC-325P, Sony Corporation, Tokyo, Japan) mounted on a
M AN U
conventional light microscope (Axioskop, Carl Zeiss), using a 5x magnifying objective. Image acquisition and analysis were performed using a complimentary software program (Zeiss KS 400® Axio Vision 3.0). Microscopic images were digitized, and the positive area for picrosirius red staining was identified by segmentation using fixed threshold values. The anastomotic area
TE D
between the two inverted opposite muscular layers was interactively defined on the computer screen. The ratio between picrosirius red-positive and total number of pixels yielded the
Statistical analysis
EP
percentual area of anastomotic collagen.
AC C
Primary endpoints of the study were anastomotic strength and collagen density. Secondary endpoints were presence of adhesions and abscesses. Historical data from our group showed the breaking strength of a colonic anastomosis at days 3 and 7 typically to be 117 ± 27 (SD) g and 232 ± 52 (SD) g, respectively. Sample size for the current experiment was determined as being sufficient to detect a loss of wound strength of 30 g (25 %) at day 3. This decrease is considered, albeit arbitrarily, meaningful in terms of increased risk of leakage. Using an α of 0.025, a power of 0.8 and an one-tail test the group
8
ACCEPTED MANUSCRIPT size is calculated (G*Power 3.1.2) to be 11. For histological analysis an important parameter is the density of collagen fibrils (as percentage of the wound surface area), which in the colon at day 7 typically is 30 ± 5 %. To detect a reduction of 25% a group size of 6 animals in each group was calculated. Altogether 17 animals per group were thus included for each of the two
RI PT
time points.
Differences between the incidences of adhesion and abscess formation in the various groups were analyzed by a (two-tailed) Fisher’s exact test (two groups) or a Chi square test (three
SC
groups). Statistical analysis of continuous variables was performed using a one-tailed Mann-
AC C
EP
TE D
0.05 was considered significant.
M AN U
Whitney U test or a Kruskal-Wallis test with Dunn post test (three groups). A p-value less than
9
ACCEPTED MANUSCRIPT Results All animals survived the surgical procedure and none had to be taken out prematurely for reaching the humane endpoint. No abnormalities, signs of anastomotic leakage or infection were seen in the animals at autopsy. Up to day 3, animals lost approximately 8 % of their preoperative weight. From
RI PT
day 4 onwards, rats in all groups started to gain weight. (Figure 1) The body weight was not significantly different between groups at any time point.
SC
Bursting pressure and breaking strength
In all groups the bursting pressure (Figure 2) and breaking strength (Figure 3) of the anastomotic
M AN U
segment were significantly higher on day 7 than at day 3 (P < 0.05). At day 3 the bursting sites were almost invariably within the anastomosis (Figure 2). There was no significant difference in bursting pressure between groups treated with alginate gel or HA/CMC film and controls. Similar results were found at day 7, where the bursting site was mostly outside the suture line. When analyzing the
TE D
breaking strength, tearing of the tissue invariably occurred within the anastomotic area at both day 3 and day 7. Treatment with either alginate gel or HA/CMC film did not significantly affect the
EP
breaking strength at either time point.
Collagen content and histology
AC C
In all groups the anastomotic hydroxyproline content in colon was significantly higher at day 7 than at day 3 (P < 0.05). There was no significant difference in hydroxyproline content between groups treated with alginate gel or HA/CMC film and controls at day 3 or day 7 (Figure 4a). In all groups the average percentage of wound collagen in colon was significantly higher at day 7 than at day 3 (P <0.05), as quantified histologically using the picrosirius red staining. At day 3, there was no significant difference in wound collagen between the three groups. At day 7, the
10
ACCEPTED MANUSCRIPT average percentage of wound collagen in colon seemed somewhat lower in the alginate gel group, but the difference remained non-significant. (Figure 4b) Comparison of wound histology after 3 days and 7 days did not indicate any gross differences in microscopic wound architecture between groups. Typical examples of colonic anastomoses at
RI PT
day 3 and day 7 are presented in Figure 5.
Incidence of adhesions and abscess
SC
Seventy one percent (24/34) of control rats displayed adhesions, compared to 35 percent (12/34, P=0.007) in the alginate gel group and 47 percent (16/34) in the HA/CMC film group. (Table 1)
M AN U
Also, the adhesion score was significantly lower in the alginate gel group than in the control group (P = 0.009). There were no significant differences between the HA/CMC film group and controls or the alginate gel group. The type of structure adhered to the anastomotic site was also examined. (Table 2) A significantly reduced incidence of adhesion formation between the anastomosis and the
TE D
fad pad or the ileum was observed in the alginate gel group as compared to controls. The incidence of abscess formation was not significantly different in rats treated with alginate gel (9/34) and controls (6/34). In the HA/CMC film group the incidence of abscess formation
AC C
EP
(14/34) was higher (P=0.030) than in controls.
11
ACCEPTED MANUSCRIPT Discussion Anti-adhesive barriers act by separating injured peritoneal and serosal tissues preventing fibr(in)ous attachments and allowing uncomplicated peritoneal healing. It has been suggested that proper anastomotic healing depends on surrounding adhesions and that adhesion prevention
RI PT
leads to clinically overt leakage.11 Ultrapure alginate gel does not interfere with early
anastomotic repair while reducing the formation of postoperative intra-abdominal adhesions. The gel seems safer than HA/CMC membrane with respect to promotion of intra-abdominal
SC
infection.
Prevention of postsurgical adhesion formation is particularly relevant after colorectal procedures
M AN U
which carry a 10 to 25% lifetime risk of small bowel obstruction and a marked adhesiolysis morbidity at repeat surgery.4, 18 The contaminated environment and presence of an anastomosis demand an anti-adhesive barrier that does increase neither intra-abdominal infectious complications nor anastomotic leak rates. The prerequisite of uncompromised anastomotic
TE D
wound healing is even more important for a liquid or viscous barrier, such as the ultrapure alginate gel used in this study, that spreads from the application site,13 Based on the present findings showing normal anastomotic healing, adhesion reduction and absence of infection
EP
propagation, alginate gel is a promising anti-adhesive barrier for colorectal surgery. This is more so because the current data extend our previous findings obtained in a cecal abrasion model13, to
gel.
AC C
a model for anastomotic repair thereby emphasizing the general anti-adhesive properties of the
We deliberately selected both 3 and 7 days after surgery as time points to measure the primary outcome parameters. Experimental data have shown that anastomotic strength is lowest in the first three days after construction.14, 19 Thus, a subtle adverse effect by any adhesion barrier on anastomotic healing would most likely be measurable within this early time frame. The period from day 3 onwards is characterized by matrix synthesis and restoration of pre-operative
12
ACCEPTED MANUSCRIPT strength. It is also the period that clinical leaks start and anti-adhesive activity is needed to prevent permanent adhesion formation.14, 20 Yet, there were no signs of leakage and no reduction in wound strength and wound collagen content at either 3 or 7 days after surgery. In some experimental studies on adhesion barriers, anastomotic strength was measured at day 7 and
RI PT
beyond.21-25 It is questionable if later time points adequately represent the relation between adhesion prevention and anastomotic healing. A postoperative period of one week is long enough for development of fibr(in)ous adhesive bands that remain beyond this period. It is not
SC
expected that additional adhesions develop from one week onwards because mesothelial healing after injury is complete within one week. Therefore, investigating anastomotic strength and
M AN U
adhesion formation in the first postoperative week seems certainly warranted. In our model we opted to apply the anti-adhesive agent in close contact to the suture line. While this is probably not the place where such an agent would be deliberately placed in patients, this way conditions are created for any detrimental effect to be maximized. The proof that no
stem from such a model.
TE D
negative effects on repair are to be expected is believed to be most convincing if supporting data
Present findings suggest that anastomotic strength does not depend on the presence of adhesions.
EP
It has been shown that promoting adhesion formation by stimulating fibrin deposition does not increase wound strength of normal or compromised colonic anastomoses 26-28 Clinical data
AC C
suggest that adhesions keep anastomotic leaks contained, which is an appealing theory for many surgeons.9 The hypothesis that adhesions wall off leaks and that anti-adhesive barriers impair this defense mechanism could not be addressed in our model because no spontaneous leaks occurred. We did not show overt anastomotic leakage with HA/CMC but the higher number of peri-anastomotic abscesses indicates an increased infection load which was not observed with alginate gel. It should be noted that this can only be a suggestion since, due to the cohort size, the increased frequency in the HA/CMC group (14/34 vs 9/34) remained non-significant. The
13
ACCEPTED MANUSCRIPT assumed antibacterial effect of ultrapure gel may explain the lower infection propagation. 29 The higher number of abscesses after HA/CMC membrane treatment has been found in previous experimental and clinical studies, although two recent randomized controlled trials did not show an increase of abscess rate. 8, 11, 12, 30, 31
RI PT
At least eight rat studies, including our study, suggest that wrapping HA/CMC film around the anastomosis does not affect anastomotic healing. 19, 21-24, 32-34 Perhaps, the use of a large animal model would be more appropriate for this particular study purpose. A recent study in dogs
SC
demonstrated weakened anastomotic strength at day 3 after application of HA/CMC film.31 A rat model of ‘spontaneously’ weakened anastomoses may be a good alternative to a more expensive
M AN U
large animal model investigating adverse effects of anti-adhesives on anastomotic leakage.35 Despite discrepancies in results between various models, testing a new anti-adhesive barrier such as ultrapure alginate in an experimental anastomotic model remains a prerequisite for determining safety in general surgery.36 While a negative effect on healing would caution against
TE D
its clinical use, it should be kept in mind that the absence of such an effect is not yet a guarantee for its clinical safety.
In conclusion, alginate gel preserves early strength of colon anastomoses while reducing
EP
adhesion formation around the anastomosis and leaving abscess formation unchanged. The gel
AC C
holds promise as an easy to apply and safe anti-adhesive barrier in colorectal surgery.
14
ACCEPTED MANUSCRIPT References: 1.
Kossi J, Salminen P, Rantala A, Laato M. Population-based study of the surgical workload and
economic impact of bowel obstruction caused by postoperative adhesions. Br J Surg 2003;90(11): 1441-1444. Van Der Krabben AA, Dijkstra FR, Nieuwenhuijzen M, Reijnen MM, Schaapveld M, Van
RI PT
2.
Goor H. Morbidity and mortality of inadvertent enterotomy during adhesiotomy. Br J Surg 2000;87(4): 467-471. 3.
Vrijland WW, Jeekel J, van Geldorp HJ, Swank DJ, Bonjer HJ. Abdominal adhesions:
ten Broek RP, Strik C, Issa Y, Bleichrodt RP, van Goor H. Adhesiolysis-related morbidity in
M AN U
4.
SC
intestinal obstruction, pain, and infertility. Surgical endoscopy 2003;17(7): 1017-1022.
abdominal surgery. Annals of surgery 2013;258(1): 98-106. 5.
Ray NF, Larsen JW, Jr., Stillman RJ, Jacobs RJ. Economic impact of hospitalizations for
lower abdominal adhesiolysis in the United States in 1988. Surg Gynecol Obstet 1993;176(3): 271276.
Ten Broek RP, Kok-Krant N, Bakkum EA, Bleichrodt RP, van Goor H. Different surgical
TE D
6.
techniques to reduce post-operative adhesion formation: a systematic review and meta-analysis. Human reproduction update 2013;19(1): 12-25.
Ouaissi M, Gaujoux S, Veyrie N, Deneve E, Brigand C, Castel B, Duron JJ, Rault A, Slim K,
EP
7.
Nocca D. Post-operative adhesions after digestive surgery: their incidence and prevention: review of
8.
AC C
the literature. Journal of visceral surgery 2012;149(2): e104-114. Becker JM, Dayton MT, Fazio VW, Beck DE, Stryker SJ, Wexner SD, Wolff BG, Roberts PL,
Smith LE, Sweeney SA, Moore M. Prevention of postoperative abdominal adhesions by a sodium hyaluronate-based bioresorbable membrane: a prospective, randomized, double-blind multicenter study. J Am Coll Surg 1996;183(4): 297-306. 9.
Fazio VW, Cohen Z, Fleshman JW, van Goor H, Bauer JJ, Wolff BG, Corman M, Beart RW,
Jr., Wexner SD, Becker JM, Monson JR, Kaufman HS, Beck DE, Bailey HR, Ludwig KA, Stamos MJ, Darzi A, Bleday R, Dorazio R, Madoff RD, Smith LE, Gearhart S, Lillemoe K, Gohl J. Reduction
15
ACCEPTED MANUSCRIPT in adhesive small-bowel obstruction by Seprafilm adhesion barrier after intestinal resection. Dis Colon Rectum 2006;49(1): 1-11. 10.
Vrijland WW, Tseng LN, Eijkman HJ, Hop WC, Jakimowicz JJ, Leguit P, Stassen LP, Swank
DJ, Haverlag R, Bonjer HJ, Jeekel H. Fewer intraperitoneal adhesions with use of hyaluronic acid-
11.
RI PT
carboxymethylcellulose membrane: a randomized clinical trial. Ann Surg 2002;235(2): 193-199. Beck DE, Cohen Z, Fleshman JW, Kaufman HS, van Goor H, Wolff BG, Adhesion Study
Group Steering C. A prospective, randomized, multicenter, controlled study of the safety of Seprafilm adhesion barrier in abdominopelvic surgery of the intestine. Dis Colon Rectum 2003;46(10): 1310-
12.
SC
1319.
Bowers D, Raybon RB, Wheeless CR, Jr. Hyaluronic acid-carboxymethylcellulose film and
M AN U
perianastomotic adhesions in previously irradiated rats. Am J Obstet Gynecol 1999;181(6): 1335-1337; discussion 1137-1338. 13.
Chaturvedi AA, Lomme RM, Hendriks T, van Goor H. Prevention of postsurgical adhesions
using an ultrapure alginate-based gel. The British journal of surgery 2013;100(7): 904-910. Hendriks T, Mastboom WJ. Healing of experimental intestinal anastomoses. Parameters for
TE D
14.
repair. Diseases of the colon and rectum 1990;33(10): 891-901. 15.
Zuhlke HV, Lorenz EM, Straub EM, Savvas V. [Pathophysiology and classification of
EP
adhesions]. Langenbecks Archiv fur Chirurgie Supplement II, Verhandlungen der Deutschen Gesellschaft fur Chirurgie Deutsche Gesellschaft fur Chirurgie Kongress 1990: 1009-1016. de Waard JW, Wobbes T, de Man BM, van der Linden CJ, Hendriks T. Post-operative
AC C
16.
levamisole may compromise early healing of experimental intestinal anastomoses. Br J Cancer 1995;72(2): 456-460. 17.
Willems MC, van der Vliet JA, de Man BM, van der Laak JA, Lomme RM, Hendriks T.
Persistent effects of everolimus on strength of experimental wounds in intestine and fascia. Wound Repair Regen 2010;18(1): 98-104. 18.
Nieuwenhuijzen M, Reijnen MM, Kuijpers JH, van Goor H. Small bowel obstruction after
total or subtotal colectomy: a 10-year retrospective review. Br J Surg 1998;85(9): 1242-1245.
16
ACCEPTED MANUSCRIPT 19.
Reijnen MM, de Man BM, Hendriks T, Postma VA, Meis JF, van Goor H. Hyaluronic acid-
based agents do not affect anastomotic strength in the rat colon, in either the presence or absence of bacterial peritonitis. Br J Surg 2000;87(9): 1222-1228. 20.
Boland GM, Weigel RJ. Formation and prevention of postoperative abdominal adhesions. J
21.
RI PT
Surg Res 2006;132(1): 3-12. Sheldon HK, Gainsbury ML, Cassidy MR, Chu DI, Stucchi AF, Becker JM. A sprayable
hyaluronate/carboxymethylcellulose adhesion barrier exhibits regional adhesion reduction efficacy and does not impair intestinal healing. J Gastrointest Surg 2012;16(2): 325-333.
Baca B, Boler DE, Onur E, Akca O, Hamzaoglu I, Karahasanoglu T, Erdamar S, Atukeren P,
SC
22.
Dirican A. Icodextrin and Seprafilm do not interfere with colonic anastomosis in rats. Eur Surg Res
23.
M AN U
2007;39(5): 318-323.
Erturk S, Yuceyar S, Temiz M, Ekci B, Sakoglu N, Balci H, Dirican A, Cengiz A, Saner H.
Effects of hyaluronic acid-carboxymethylcellulose antiadhesion barrier on ischemic colonic anastomosis: an experimental study. Dis Colon Rectum 2003;46(4): 529-534. Medina M, Paddock HN, Connolly RJ, Schwaitzberg SD. Novel antiadhesion barrier does not
TE D
24.
prevent anastomotic healing in a rabbit model. J Invest Surg 1995;8(3): 179-186. 25.
Hadaegh A, Burns J, Burgess L, Rose R, Rowe E, LaMorte WW, Becker JM. Effects of
EP
hyaluronic acid/carboxymethylcellulose gel on bowel anastomoses in the New Zealand white rabbit. Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract
26.
AC C
1997;1(6): 569-575.
Shinohara K, Kobayashi E, Yoshida T, Toyama N, Kiyozaki H, Fujimura A, Miyata M. Effect
of fibrin glue on small and large bowel anastomoses in the rat. Eur Surg Res 1998;30(1): 8-12. 27.
Karahasanoglu T, Alcicek S, Altunkaya E, Sahinler I, Goksel S, Sirin F, Ozbal A. Effect of
fibrin glue on irradiated colonic anastomoses. Dis Colon Rectum 1997;40(10): 1240-1243. 28.
Houston KA, Rotstein OD. Fibrin sealant in high-risk colonic anastomoses. Archives of
surgery 1988;123(2): 230-234. 29.
Contreras A, Vazquez D, Carrasco L. Inhibition, by selected antibiotics, of protein synthesis in
cells growing in tissue cultures. J Antibiot (Tokyo) 1978;31(6): 598-602. 17
ACCEPTED MANUSCRIPT 30.
Zeng Q, Yu Z, You J, Zhang Q. Efficacy and safety of Seprafilm for preventing postoperative
abdominal adhesion: systematic review and meta-analysis. World journal of surgery 2007;31(11): 2125-2131; discussion 2132. 31.
Tsujimoto H, Tanzawa A, Matoba M, Hashimoto A, Suzuki S, Morita S, Ikada Y, Hagiwara
RI PT
A. The anti-adhesive effect of thermally cross-linked gelatin film and its influence on the intestinal anastomosis in canine models. Journal of biomedical materials research Part B, Applied biomaterials 2013;101(1): 99-109. 32.
Oncel M, Remzi FH, Senagore AJ, Connor JT, Fazio VW. Comparison of a novel liquid
SC
(Adcon-P) and a sodium hyaluronate and carboxymethylcellulose membrane (Seprafilm) in
postsurgical adhesion formation in a murine model. Dis Colon Rectum 2003;46(2): 187-191. Holzman S, Connolly RJ, Schwaitzberg SD. Effect of hyaluronic acid solution on healing of
M AN U
33.
bowel anastomoses. J Invest Surg 1994;7(5): 431-437. 34.
van Oosterom FJ, van Lanschot JJ, Oosting J, Obertop H. Hyaluronic
acid/carboxymethylcellulose membrane surrounding an intraperitoneal or subcutaneous
35.
TE D
jejunojejunostomy in rats. The European journal of surgery = Acta chirurgica 2000;166(8): 654-658. van der Vijver RJ, van Laarhoven CJ, de Man BM, Lomme RM, Hendriks T. Perioperative
pain relief by a COX-2 inhibitor affects ileal repair and provides a model for anastomotic leakage in
36.
EP
the intestine. Surgical innovation 2013;20(2): 113-118. Guidance for Resorbable adhesion barrier devices for use in abdominal and/or Pelvic surgery;
AC C
Guidance for industry. In: Administration DFaD, editor. U.S.: Center of Devices and Radiological health; 2008.
18
ACCEPTED MANUSCRIPT Legend to Tables:
Table 1: Adhesions, adhesion score and abscesses in the control and experimental groups.
AC C
EP
TE D
M AN U
SC
RI PT
Table 2: Number of the animals, with adhesions between the anastomosis and other organs
19
ACCEPTED MANUSCRIPT Legend to Figures:
Figure 1
Postoperative course of body weight. Data represent average relative body weight, in relation to the weight prior to operation for all the three groups.
Figure 2
Anastomotic bursting pressure. Individual values and medians (horizontal bars)
RI PT
are given in the controls (C), alginate gel group (ALG) and HA/CMC film group. The bursting site was either within (filled circle) or outside (open circle) the suture line. *p< 0.05 vs. day 3
SC
Figure 3 Anastomotic breaking strength. Data represent mean and SD in rats treated with
Figure 4
M AN U
alginate gel, HA/CMC film and controls. * p< 0.05 vs. day 3
Anastomotic collagen. A: Data represent mean and SD for the anastomotic hydroxyproline content in rats treated with alginate gel, HA/CMC film and controls. B: Data represent mean and SD for the wound collagen content, as determined by quantitative morphology, in the three groups of rats. * p< 0.05 vs.
Anastomotic histology. Each panel shows a tissue segment with the anastomosis in the middle at a magnification of approximately 40 x, representing typical examples obtained at day 3 (a. control; b. alginate gel; c. HA/CMC film) and day
EP
7 (d. control; e. alginate gel; f. HA/CMC film)
AC C
Figure 5
TE D
day 3
20
ACCEPTED MANUSCRIPT
Table 1: Adhesions, adhesion score and abscesses in the control and experimental groups
Adhesion score
Controls
24 (71%)
0.79 ± 0.59
Alginate gel
12 (35%)*
0.38 ± 0.55**
HA/CMC film
16 (47%)
0.55 ± 0.66 p= 0.0158
6 (18%)
9 (26%)
14 (46%)*** p= 0.0943
M AN U
p= 0.0124
with abscesses
RI PT
with adhesions
SC
Groups ( n = 34 each)
P-values under columns result from comparing the three groups with a chi square test. Two by two comparisons (Fisher’s exact test) yield significance versus controls as noted by * p = 0.0071,
AC C
EP
TE D
** p = 0.0092 and *** p = 0.0305.
21
ACCEPTED MANUSCRIPT
Table 2: Adhesions between the anastomosis and various other structures
Anastomosis – fat pad
15 (44%)
7 (20%)
Anastomosis - cecum
5 (15%)
2 (5%)
Anastomosis - ileum
9 (26%)
2 (5%)
Anastomosis - sidewall
0 (0%)
1 (3%)
P value
0.0342 NS 0.0223 NS
HA/CMC film (n=34)
RI PT
Alginate gel (n=34)
SC
Control (n=34)
P value
8 (23%)
NS
6 (18%)
NS
4 (12%)
NS
2 (5%)
NS
M AN U
Adhesions
Numbers denote the number of animals with adhesions present between the structures indicated. P-values concern the difference between either of the experimental groups and the control group
AC C
EP
TE D
(one-sided Fisher’s exact test). NS = non significant.
22
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
Figure. 1 Postoperative course of body weight.
23
ACCEPTED MANUSCRIPT
*
TE D
M AN U
SC
*
RI PT
*
AC C
EP
Figure. 2 Anastomotic bursting pressure.
24
EP
SC
TE D
M AN U
*
* *
RI PT
ACCEPTED MANUSCRIPT
Anastomotic breaking strength.
AC C
Figure.3
25
ACCEPTED MANUSCRIPT
4A.
RI PT
* *
TE D
M AN U
SC
*
*
AC C
EP
4B.
* *
26
ACCEPTED MANUSCRIPT Day 3
Figure 4. Anastomotic collagen
d
b
e
M AN U TE D
Alginate gel
f
AC C
EP
c HA/CMC film
SC
RI PT
Control
a
Day 7
Figure 5. Anastomotic histology
27
ACCEPTED MANUSCRIPT
Table 1:Adhesions, adhesion score and abscessesin the control and experimental groups
Adhesion score
with abscesses
6 (18%)
Controls
24 (71%)
0.79 ± 0.59
Alginate gel
12 (35%)*
0.38 ± 0.55**
HA/CMC film
16 (47%)
0.55 ± 0.66 p= 0.0158
9 (26%)
M AN U
p= 0.0124
RI PT
with adhesions
SC
Groups ( n = 34 each)
14 (46%)*** p= 0.0943
P-values under columns result from comparing the three groups with a chi square test. Two by two comparisons (Fisher’s exact test) yield significance versus controls as noted by * p = 0.0071, ** p =
AC C
EP
TE D
0.0092 and *** p = 0.0305.
ACCEPTED MANUSCRIPT
Table 2: Adhesions between the anastomosis and various other structures
Alginate gel (n=34)
15 (44%)
7 (20%)
Anastomosis - cecum
5 (15%)
2 (5%)
Anastomosis - ileum
9 (26%)
2 (5%)
Anastomosis - sidewall
0 (0%)
1 (3%)
0.0342 NS 0.0223 NS
M AN U
Anastomosis – fat pad
P value
HA/CMC film (n=34)
RI PT
Control (n=34)
SC
Adhesions
P value
8 (23%)
NS
6 (18%)
NS
4 (12%)
NS
2 (5%)
NS
Numbers denote the number of animals with adhesions present between the structures indicated. Pvalues concern the difference between either of the experimental groups and the control group (one-
AC C
EP
TE D
sided Fisher’s exact test). NS = non significant.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S0022-4804(14)00548-4
DOI:
10.1016/j.jss.2014.05.085
Reference:
YJSRE 12781
To appear in:
Journal of Surgical Research
Received Date: 16 September 2013 Revised Date:
8 April 2014
Accepted Date: 29 May 2014
Please cite this article as: Chaturvedi AA, Lomme RMLM, Hendriks T, van Goor H, Ultrapure alginate anti-adhesion gel does not impair colon anastomotic strength, Journal of Surgical Research (2014), doi: 10.1016/j.jss.2014.05.085. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised: 31/03/2014 Ultrapure alginate anti-adhesion gel does not impair colon anastomotic strength Ankit A. Chaturvedi a, b, MS, Roger M.L.M. Lomme a, Thijs Hendriks a, PhD and Harry van Goor a, M.D, PhD Department of Surgery, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
b
European Medical Contract Manufacturing B.V, Nijmegen, The Netherlands.
RI PT
a
Category: Original scientific article
SC
Subject Category: Gastrointestinal Correspondence to:
Department of surgery, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. Email: [email protected]
M AN U
Mr. Ankit Akshayesh Chaturvedi,
TE D
Telephone no: + 31-024-36-13956; + 31-0633633809
Author Contribution:
EP
Ankit Chaturvedi: Conception, Design, Data collection, Analysis, Interpretation, writing article and revision Roger Lomme: Data collection, Analysis, Interpretation
AC C
Dr. Thijs Hendriks: Conception, Design, Writing article and Critical revisions Prof. Harry Van Goor: Conception, Design, Interpretation, Writing article and Critical revision
Communications: The work has been presented at: 22nd European Tissue Repair Society Congress. October 4-5, 2012, Athens, Greece. Funding: The research leading to these results has received funding from the European Community's Seventh Framework Programme; MultiTERM, grant agreement nr 238551.
1
ACCEPTED MANUSCRIPT ABSTRACT Background: Ultrapure alginate gel is promising in terms of adhesion prevention. Since antiadhesive barriers have been shown to disturb healing of bowel anastomoses the effect of ultrapure alginate gel on the repair of colon anastomoses was studied.
RI PT
Materials and Methods: In 102 male Wistar rats a 0.5 cm segment was resected from the descending colon and continuity was restored by an inverted single-layer end-to-end
anastomosis. Animals were randomized into a control, an alginate gel and a sodium hyaluronate
SC
carboxymethyl cellulose (HA/CMC) film group, each n=34. Half of each group was sacrificed at day 3 and 7 postoperatively. Anastomotic strength was assessed by measuring both bursting
M AN U
pressure and breaking strength. Hydroxyproline content was measured and histological analysis was performed. The incidence of adhesion and abscess formation was scored at sacrifice. Results: No difference in either anastomotic bursting pressure or breaking strength was found between experimental groups and the controls at any time point. Both the incidence of adhesion
TE D
formation (35% vs 71%, P=0.007) and the adhesion score (0.38 vs 0.79, P=0.009) were significantly lower in the alginate gel group than in the controls. The abscess rate was higher
alginate gel group.
EP
(46% vs 18%, P=0.030) in the HA/CMC group than in the controls and unchanged in the
Conclusion: While reducing adhesion formation, ultrapure alginate gel does not interfere with
AC C
the development of colonic anastomotic strength during the crucial early healing period.
Keywords: Ultrapure alginate gel, HA/CMC, Colon anastomosis, abdominal adhesion
2
ACCEPTED MANUSCRIPT Introduction Abdominal adhesion formation occurs in about 65 to 100 percent of patients after intraabdominal surgery. It constitutes an important cause of long term morbidity including bowel obstruction, adhesiolysis induced organ damage at repeat surgery, female infertility, and chronic
5
RI PT
pain.1-4 These morbidities incur significant patient discomfort and come at great financial costs.4, Results from ‘good’ surgical techniques, including laparoscopy, have not yielded convincing
evidence of reduced adhesion morbidity.6
SC
The use of anti-adhesive barriers seems the best way to prevent adhesion formation and reduce morbidity and mortality. So far, only one product consisting of sodium hyaluronate
M AN U
carboxymethyl cellulose (HA/CMC) membrane has been shown to significantly reduce the incidence of postoperative adhesion formation in general surgery.7-10 However, the use of HA/CMC appears to be associated with an increased incidence of anastomotic leakage when the membrane is applied in direct contact with the anastomosis.11, 12 When wrapped around the
TE D
anastomosis, the anti-adhesive effect of the HA/CMC membrane is believed to convert subclinical into clinical leaks. This effect, which may be considered as prevention which is too rigorous, has not consistently been confirmed experimentally or in other clinical studies. Still, it
EP
emphasizes the need to demonstrate uncompromised anastomotic healing with the use of antiadhesive barriers in visceral surgery.
AC C
We recently showed excellent anti-adhesive effects of a new ultrapure, easy to apply, alginate gel.13 Adhesion formation was reduced by 80% in a cecal abrasion and peritoneal side wall excision rat model. There was also a tendency to reduce adhesion formation at locations distant from the site of actual gel application. While such a remote effect would be advantageous because it covers adhesiogenic areas not primarily injured by dissection, it could also become disadvantageous if it reaches an anastomotic area thereby disturbing normal healing.
3
ACCEPTED MANUSCRIPT The first postoperative week is crucial to anastomotic repair. During the first few days wound strength is low while after day 3 strength increases reaching pre-operative values around day 7.14 Therefore, it should be demonstrated that perioperative application of the gel does not lead to healing impairment in this period when the anastomosis is relatively vulnerable. The present
RI PT
study aims to investigate the effect of ultrapure alginate gel, applied around the suture line, on the healing of a rat colonic anastomoses. In a separate group the effect of a HA/CMC membrane
AC C
EP
TE D
M AN U
SC
is examined.
4
ACCEPTED MANUSCRIPT Methods Animals and experimental design One hundred and two male Wistar (Charles River, Sulzfeld, Germany) rats were used in the experiment. Rats weighed between 250 and 280 g and were housed in filter topped cages (two
RI PT
per cage) under clean, non-sterile, standardized conditions for at least five days prior to the experiment. Animals were checked daily and had free access to water and standard rodent chow (Ssniff R/M-H, Bio Services BV, Uden, The Netherlands) during the entire experimental period.
SC
All animals underwent resection and anastomosis of the distal colon. The rats were allocated randomly to any of three groups (n=34 each). Animals in the first group received no further
M AN U
treatment and served as controls. In the second group, animals received ultrapure alginate gel and in the third group HA/CMC film. Half of each group was killed on postoperative days 3 and 7, respectively, for analysis of wound strength (n=17) and wound hydroxyproline (n=11) and histology (n=6).
TE D
The study protocol was approved by the Institutional Animal Ethics Review Committee (Approval No: RU-DEC 2012-003) and performed in the GLP certified animal research laboratory of the Radboud University Nijmegen Medical Centre, The Netherlands. Prior to the
AC C
study period.
EP
experiment humane endpoints were defined to avoid unnecessary suffering of animals during the
Operative procedure
Each animal was prepared for surgery by shaving and cleaning the lower abdomen with 70 percent alcohol. For analgesia, rats were given buprenorphine subcutaneously (Temgesic; Schering-Plough, Houten, The Netherlands, 0.02 mg/kg) starting 30 min before surgery and repeating the same dose every 12 h for the next 48 h. The rats were anesthetized by inhalation of a mixture of isoflurane (3 %) and 1:1 oxygen-nitrous oxide. Surgical procedures were performed
5
ACCEPTED MANUSCRIPT using a Zeiss operation microscope (Carl Zeiss AG, Oberkochen, Germany). During the operation and shortly thereafter rats were placed on a warm plate to avoid hypothermia. All rats underwent a 4 cm midline laparotomy and a 0.5 cm segment of the descending colon was resected 3 cm proximal to the peritoneal reflection. Continuity was restored by an inverted
RI PT
single-layer end-to-end anastomosis with eight interrupted sutures of 8-0 monofilament material (Ethicon, Norderstedt, Germany). Controls received no further treatment. In the second group 1 ml of ultrapure alginate gel (Rebasol™, EMCM, Nijmegen, The Netherlands) was applied on the
SC
anastomotic suture line. In the third group a 10 x 30 mm sheet of HA/CMC bioresorbable film (Seprafilm®; Genzyme, A Sanofi Company, Cambridge (MA), USA) was wrapped around the
M AN U
anastomosis. The abdomen was closed in two layers using Vicryl 3-0 suture (Ethicon products, Amersfoort, the Netherlands) for the fascia/muscle layer and wound staples for the skin. Animals were resuscitated immediately after abdominal closure with 10 ml isotonic sodium chloride
TE D
(0.9%) solution administered subcutaneously.
Adhesion formation
Animals were euthanized by carbon dioxide asphyxiation. The abdomen was reopened and
EP
inspected for adhesion and abscess formation at the anastomotic site. Adhesions were scored by an investigator blinded to the group assignment (HvG), using Zuhlke’s grading system.15
AC C
Adhesions were classified macroscopically as 0 = no adhesions, 1 = filmy adhesions: easy to separate by blunt dissection; no vascularization, 2 = mild adhesions: blunt dissection possible but partly sharp dissection required, 3 = strong adhesions: lysis possible by sharp dissection only; clear vascularization, 4 = very strong adhesions: lysis possible by sharp dissection only (organ strongly attached with severe adhesion). The organs and structures involved in the adhesion formation with the anastomosis were also noted. An abscess was defined as any walled off collection containing pus.
6
ACCEPTED MANUSCRIPT Bursting pressure and breaking strength Colonic segments, approximately 4 cm in length containing the anastomosis were resected and adhesions were dissected carefully without affecting the anastomosis. After removal of intraluminal faecal material, the colonic segment was attached to an infusion pump and a manometric
RI PT
device recording the internal pressure for measuring the bursting pressure, which represents the capability of the suture line to withstand intraluminal forces. Pressure was increased by the constant infusion (2 ml/min) of water containing methylene blue into the segment, which was
SC
kept under water. The maximum pressure (mmHg) recorded immediately before the sudden loss of pressure was taken as the bursting pressure. The site of perforation (within or outside the
M AN U
anastomotic line) was indicated by the loss of methylene blue through the intestinal wall. Subsequently, the breaking strength (which represents the capability of the suture line to withstand longitudinal forces) was measured in the same segment.16 The segment was clamped in a tensiometer (AIKOH model (9500 series), Aikoh Engineering Co. Ltd, Higashi-Osaka,
TE D
Japan) that exerts a steadily increasing force in longitudinal direction. The peak force required for tearing the segment apart was recorded as the breaking strength (g). Thereafter, the anastomotic segment was cleaned carefully from any remaining adhering tissue
EP
and a 5 mm sample, containing the suture line, was frozen in liquid nitrogen and stored at -80° C
AC C
for further biochemical processing.
Biochemical analysis
Tissue samples were lyophilized, weighed and pulverized. The hydroxyproline content, as a measure of the collagen content, was measured by high-performance liquid chromatography (HPLC) after hydrolysis with 6-N hydrochloric acid and coupling to dabsyl chloride.
7
ACCEPTED MANUSCRIPT Histological analysis Intestinal samples of approximately 1 cm, containing the entire anastomosis in the middle, were collected, opened at the mesenteric side, washed gently with 0.9 percent sodium chloride and spread out in a cassette for paraffin embedding. From paraffin-embedded tissues, 4-µm sections
RI PT
were prepared and stained with hematoxylin and eosin (H & E). Sections were analyzed using a binocular light microscope.
Another set of sections was stained with picrosirius red to identify collagen fibers in the
SC
anastomotic area, which were quantified using digital image analysis.17. Images were recorded using a 3-chip CCD RGB camera (DXC-325P, Sony Corporation, Tokyo, Japan) mounted on a
M AN U
conventional light microscope (Axioskop, Carl Zeiss), using a 5x magnifying objective. Image acquisition and analysis were performed using a complimentary software program (Zeiss KS 400® Axio Vision 3.0). Microscopic images were digitized, and the positive area for picrosirius red staining was identified by segmentation using fixed threshold values. The anastomotic area
TE D
between the two inverted opposite muscular layers was interactively defined on the computer screen. The ratio between picrosirius red-positive and total number of pixels yielded the
Statistical analysis
EP
percentual area of anastomotic collagen.
AC C
Primary endpoints of the study were anastomotic strength and collagen density. Secondary endpoints were presence of adhesions and abscesses. Historical data from our group showed the breaking strength of a colonic anastomosis at days 3 and 7 typically to be 117 ± 27 (SD) g and 232 ± 52 (SD) g, respectively. Sample size for the current experiment was determined as being sufficient to detect a loss of wound strength of 30 g (25 %) at day 3. This decrease is considered, albeit arbitrarily, meaningful in terms of increased risk of leakage. Using an α of 0.025, a power of 0.8 and an one-tail test the group
8
ACCEPTED MANUSCRIPT size is calculated (G*Power 3.1.2) to be 11. For histological analysis an important parameter is the density of collagen fibrils (as percentage of the wound surface area), which in the colon at day 7 typically is 30 ± 5 %. To detect a reduction of 25% a group size of 6 animals in each group was calculated. Altogether 17 animals per group were thus included for each of the two
RI PT
time points.
Differences between the incidences of adhesion and abscess formation in the various groups were analyzed by a (two-tailed) Fisher’s exact test (two groups) or a Chi square test (three
SC
groups). Statistical analysis of continuous variables was performed using a one-tailed Mann-
AC C
EP
TE D
0.05 was considered significant.
M AN U
Whitney U test or a Kruskal-Wallis test with Dunn post test (three groups). A p-value less than
9
ACCEPTED MANUSCRIPT Results All animals survived the surgical procedure and none had to be taken out prematurely for reaching the humane endpoint. No abnormalities, signs of anastomotic leakage or infection were seen in the animals at autopsy. Up to day 3, animals lost approximately 8 % of their preoperative weight. From
RI PT
day 4 onwards, rats in all groups started to gain weight. (Figure 1) The body weight was not significantly different between groups at any time point.
SC
Bursting pressure and breaking strength
In all groups the bursting pressure (Figure 2) and breaking strength (Figure 3) of the anastomotic
M AN U
segment were significantly higher on day 7 than at day 3 (P < 0.05). At day 3 the bursting sites were almost invariably within the anastomosis (Figure 2). There was no significant difference in bursting pressure between groups treated with alginate gel or HA/CMC film and controls. Similar results were found at day 7, where the bursting site was mostly outside the suture line. When analyzing the
TE D
breaking strength, tearing of the tissue invariably occurred within the anastomotic area at both day 3 and day 7. Treatment with either alginate gel or HA/CMC film did not significantly affect the
EP
breaking strength at either time point.
Collagen content and histology
AC C
In all groups the anastomotic hydroxyproline content in colon was significantly higher at day 7 than at day 3 (P < 0.05). There was no significant difference in hydroxyproline content between groups treated with alginate gel or HA/CMC film and controls at day 3 or day 7 (Figure 4a). In all groups the average percentage of wound collagen in colon was significantly higher at day 7 than at day 3 (P <0.05), as quantified histologically using the picrosirius red staining. At day 3, there was no significant difference in wound collagen between the three groups. At day 7, the
10
ACCEPTED MANUSCRIPT average percentage of wound collagen in colon seemed somewhat lower in the alginate gel group, but the difference remained non-significant. (Figure 4b) Comparison of wound histology after 3 days and 7 days did not indicate any gross differences in microscopic wound architecture between groups. Typical examples of colonic anastomoses at
RI PT
day 3 and day 7 are presented in Figure 5.
Incidence of adhesions and abscess
SC
Seventy one percent (24/34) of control rats displayed adhesions, compared to 35 percent (12/34, P=0.007) in the alginate gel group and 47 percent (16/34) in the HA/CMC film group. (Table 1)
M AN U
Also, the adhesion score was significantly lower in the alginate gel group than in the control group (P = 0.009). There were no significant differences between the HA/CMC film group and controls or the alginate gel group. The type of structure adhered to the anastomotic site was also examined. (Table 2) A significantly reduced incidence of adhesion formation between the anastomosis and the
TE D
fad pad or the ileum was observed in the alginate gel group as compared to controls. The incidence of abscess formation was not significantly different in rats treated with alginate gel (9/34) and controls (6/34). In the HA/CMC film group the incidence of abscess formation
AC C
EP
(14/34) was higher (P=0.030) than in controls.
11
ACCEPTED MANUSCRIPT Discussion Anti-adhesive barriers act by separating injured peritoneal and serosal tissues preventing fibr(in)ous attachments and allowing uncomplicated peritoneal healing. It has been suggested that proper anastomotic healing depends on surrounding adhesions and that adhesion prevention
RI PT
leads to clinically overt leakage.11 Ultrapure alginate gel does not interfere with early
anastomotic repair while reducing the formation of postoperative intra-abdominal adhesions. The gel seems safer than HA/CMC membrane with respect to promotion of intra-abdominal
SC
infection.
Prevention of postsurgical adhesion formation is particularly relevant after colorectal procedures
M AN U
which carry a 10 to 25% lifetime risk of small bowel obstruction and a marked adhesiolysis morbidity at repeat surgery.4, 18 The contaminated environment and presence of an anastomosis demand an anti-adhesive barrier that does increase neither intra-abdominal infectious complications nor anastomotic leak rates. The prerequisite of uncompromised anastomotic
TE D
wound healing is even more important for a liquid or viscous barrier, such as the ultrapure alginate gel used in this study, that spreads from the application site,13 Based on the present findings showing normal anastomotic healing, adhesion reduction and absence of infection
EP
propagation, alginate gel is a promising anti-adhesive barrier for colorectal surgery. This is more so because the current data extend our previous findings obtained in a cecal abrasion model13, to
gel.
AC C
a model for anastomotic repair thereby emphasizing the general anti-adhesive properties of the
We deliberately selected both 3 and 7 days after surgery as time points to measure the primary outcome parameters. Experimental data have shown that anastomotic strength is lowest in the first three days after construction.14, 19 Thus, a subtle adverse effect by any adhesion barrier on anastomotic healing would most likely be measurable within this early time frame. The period from day 3 onwards is characterized by matrix synthesis and restoration of pre-operative
12
ACCEPTED MANUSCRIPT strength. It is also the period that clinical leaks start and anti-adhesive activity is needed to prevent permanent adhesion formation.14, 20 Yet, there were no signs of leakage and no reduction in wound strength and wound collagen content at either 3 or 7 days after surgery. In some experimental studies on adhesion barriers, anastomotic strength was measured at day 7 and
RI PT
beyond.21-25 It is questionable if later time points adequately represent the relation between adhesion prevention and anastomotic healing. A postoperative period of one week is long enough for development of fibr(in)ous adhesive bands that remain beyond this period. It is not
SC
expected that additional adhesions develop from one week onwards because mesothelial healing after injury is complete within one week. Therefore, investigating anastomotic strength and
M AN U
adhesion formation in the first postoperative week seems certainly warranted. In our model we opted to apply the anti-adhesive agent in close contact to the suture line. While this is probably not the place where such an agent would be deliberately placed in patients, this way conditions are created for any detrimental effect to be maximized. The proof that no
stem from such a model.
TE D
negative effects on repair are to be expected is believed to be most convincing if supporting data
Present findings suggest that anastomotic strength does not depend on the presence of adhesions.
EP
It has been shown that promoting adhesion formation by stimulating fibrin deposition does not increase wound strength of normal or compromised colonic anastomoses 26-28 Clinical data
AC C
suggest that adhesions keep anastomotic leaks contained, which is an appealing theory for many surgeons.9 The hypothesis that adhesions wall off leaks and that anti-adhesive barriers impair this defense mechanism could not be addressed in our model because no spontaneous leaks occurred. We did not show overt anastomotic leakage with HA/CMC but the higher number of peri-anastomotic abscesses indicates an increased infection load which was not observed with alginate gel. It should be noted that this can only be a suggestion since, due to the cohort size, the increased frequency in the HA/CMC group (14/34 vs 9/34) remained non-significant. The
13
ACCEPTED MANUSCRIPT assumed antibacterial effect of ultrapure gel may explain the lower infection propagation. 29 The higher number of abscesses after HA/CMC membrane treatment has been found in previous experimental and clinical studies, although two recent randomized controlled trials did not show an increase of abscess rate. 8, 11, 12, 30, 31
RI PT
At least eight rat studies, including our study, suggest that wrapping HA/CMC film around the anastomosis does not affect anastomotic healing. 19, 21-24, 32-34 Perhaps, the use of a large animal model would be more appropriate for this particular study purpose. A recent study in dogs
SC
demonstrated weakened anastomotic strength at day 3 after application of HA/CMC film.31 A rat model of ‘spontaneously’ weakened anastomoses may be a good alternative to a more expensive
M AN U
large animal model investigating adverse effects of anti-adhesives on anastomotic leakage.35 Despite discrepancies in results between various models, testing a new anti-adhesive barrier such as ultrapure alginate in an experimental anastomotic model remains a prerequisite for determining safety in general surgery.36 While a negative effect on healing would caution against
TE D
its clinical use, it should be kept in mind that the absence of such an effect is not yet a guarantee for its clinical safety.
In conclusion, alginate gel preserves early strength of colon anastomoses while reducing
EP
adhesion formation around the anastomosis and leaving abscess formation unchanged. The gel
AC C
holds promise as an easy to apply and safe anti-adhesive barrier in colorectal surgery.
14
ACCEPTED MANUSCRIPT References: 1.
Kossi J, Salminen P, Rantala A, Laato M. Population-based study of the surgical workload and
economic impact of bowel obstruction caused by postoperative adhesions. Br J Surg 2003;90(11): 1441-1444. Van Der Krabben AA, Dijkstra FR, Nieuwenhuijzen M, Reijnen MM, Schaapveld M, Van
RI PT
2.
Goor H. Morbidity and mortality of inadvertent enterotomy during adhesiotomy. Br J Surg 2000;87(4): 467-471. 3.
Vrijland WW, Jeekel J, van Geldorp HJ, Swank DJ, Bonjer HJ. Abdominal adhesions:
ten Broek RP, Strik C, Issa Y, Bleichrodt RP, van Goor H. Adhesiolysis-related morbidity in
M AN U
4.
SC
intestinal obstruction, pain, and infertility. Surgical endoscopy 2003;17(7): 1017-1022.
abdominal surgery. Annals of surgery 2013;258(1): 98-106. 5.
Ray NF, Larsen JW, Jr., Stillman RJ, Jacobs RJ. Economic impact of hospitalizations for
lower abdominal adhesiolysis in the United States in 1988. Surg Gynecol Obstet 1993;176(3): 271276.
Ten Broek RP, Kok-Krant N, Bakkum EA, Bleichrodt RP, van Goor H. Different surgical
TE D
6.
techniques to reduce post-operative adhesion formation: a systematic review and meta-analysis. Human reproduction update 2013;19(1): 12-25.
Ouaissi M, Gaujoux S, Veyrie N, Deneve E, Brigand C, Castel B, Duron JJ, Rault A, Slim K,
EP
7.
Nocca D. Post-operative adhesions after digestive surgery: their incidence and prevention: review of
8.
AC C
the literature. Journal of visceral surgery 2012;149(2): e104-114. Becker JM, Dayton MT, Fazio VW, Beck DE, Stryker SJ, Wexner SD, Wolff BG, Roberts PL,
Smith LE, Sweeney SA, Moore M. Prevention of postoperative abdominal adhesions by a sodium hyaluronate-based bioresorbable membrane: a prospective, randomized, double-blind multicenter study. J Am Coll Surg 1996;183(4): 297-306. 9.
Fazio VW, Cohen Z, Fleshman JW, van Goor H, Bauer JJ, Wolff BG, Corman M, Beart RW,
Jr., Wexner SD, Becker JM, Monson JR, Kaufman HS, Beck DE, Bailey HR, Ludwig KA, Stamos MJ, Darzi A, Bleday R, Dorazio R, Madoff RD, Smith LE, Gearhart S, Lillemoe K, Gohl J. Reduction
15
ACCEPTED MANUSCRIPT in adhesive small-bowel obstruction by Seprafilm adhesion barrier after intestinal resection. Dis Colon Rectum 2006;49(1): 1-11. 10.
Vrijland WW, Tseng LN, Eijkman HJ, Hop WC, Jakimowicz JJ, Leguit P, Stassen LP, Swank
DJ, Haverlag R, Bonjer HJ, Jeekel H. Fewer intraperitoneal adhesions with use of hyaluronic acid-
11.
RI PT
carboxymethylcellulose membrane: a randomized clinical trial. Ann Surg 2002;235(2): 193-199. Beck DE, Cohen Z, Fleshman JW, Kaufman HS, van Goor H, Wolff BG, Adhesion Study
Group Steering C. A prospective, randomized, multicenter, controlled study of the safety of Seprafilm adhesion barrier in abdominopelvic surgery of the intestine. Dis Colon Rectum 2003;46(10): 1310-
12.
SC
1319.
Bowers D, Raybon RB, Wheeless CR, Jr. Hyaluronic acid-carboxymethylcellulose film and
M AN U
perianastomotic adhesions in previously irradiated rats. Am J Obstet Gynecol 1999;181(6): 1335-1337; discussion 1137-1338. 13.
Chaturvedi AA, Lomme RM, Hendriks T, van Goor H. Prevention of postsurgical adhesions
using an ultrapure alginate-based gel. The British journal of surgery 2013;100(7): 904-910. Hendriks T, Mastboom WJ. Healing of experimental intestinal anastomoses. Parameters for
TE D
14.
repair. Diseases of the colon and rectum 1990;33(10): 891-901. 15.
Zuhlke HV, Lorenz EM, Straub EM, Savvas V. [Pathophysiology and classification of
EP
adhesions]. Langenbecks Archiv fur Chirurgie Supplement II, Verhandlungen der Deutschen Gesellschaft fur Chirurgie Deutsche Gesellschaft fur Chirurgie Kongress 1990: 1009-1016. de Waard JW, Wobbes T, de Man BM, van der Linden CJ, Hendriks T. Post-operative
AC C
16.
levamisole may compromise early healing of experimental intestinal anastomoses. Br J Cancer 1995;72(2): 456-460. 17.
Willems MC, van der Vliet JA, de Man BM, van der Laak JA, Lomme RM, Hendriks T.
Persistent effects of everolimus on strength of experimental wounds in intestine and fascia. Wound Repair Regen 2010;18(1): 98-104. 18.
Nieuwenhuijzen M, Reijnen MM, Kuijpers JH, van Goor H. Small bowel obstruction after
total or subtotal colectomy: a 10-year retrospective review. Br J Surg 1998;85(9): 1242-1245.
16
ACCEPTED MANUSCRIPT 19.
Reijnen MM, de Man BM, Hendriks T, Postma VA, Meis JF, van Goor H. Hyaluronic acid-
based agents do not affect anastomotic strength in the rat colon, in either the presence or absence of bacterial peritonitis. Br J Surg 2000;87(9): 1222-1228. 20.
Boland GM, Weigel RJ. Formation and prevention of postoperative abdominal adhesions. J
21.
RI PT
Surg Res 2006;132(1): 3-12. Sheldon HK, Gainsbury ML, Cassidy MR, Chu DI, Stucchi AF, Becker JM. A sprayable
hyaluronate/carboxymethylcellulose adhesion barrier exhibits regional adhesion reduction efficacy and does not impair intestinal healing. J Gastrointest Surg 2012;16(2): 325-333.
Baca B, Boler DE, Onur E, Akca O, Hamzaoglu I, Karahasanoglu T, Erdamar S, Atukeren P,
SC
22.
Dirican A. Icodextrin and Seprafilm do not interfere with colonic anastomosis in rats. Eur Surg Res
23.
M AN U
2007;39(5): 318-323.
Erturk S, Yuceyar S, Temiz M, Ekci B, Sakoglu N, Balci H, Dirican A, Cengiz A, Saner H.
Effects of hyaluronic acid-carboxymethylcellulose antiadhesion barrier on ischemic colonic anastomosis: an experimental study. Dis Colon Rectum 2003;46(4): 529-534. Medina M, Paddock HN, Connolly RJ, Schwaitzberg SD. Novel antiadhesion barrier does not
TE D
24.
prevent anastomotic healing in a rabbit model. J Invest Surg 1995;8(3): 179-186. 25.
Hadaegh A, Burns J, Burgess L, Rose R, Rowe E, LaMorte WW, Becker JM. Effects of
EP
hyaluronic acid/carboxymethylcellulose gel on bowel anastomoses in the New Zealand white rabbit. Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract
26.
AC C
1997;1(6): 569-575.
Shinohara K, Kobayashi E, Yoshida T, Toyama N, Kiyozaki H, Fujimura A, Miyata M. Effect
of fibrin glue on small and large bowel anastomoses in the rat. Eur Surg Res 1998;30(1): 8-12. 27.
Karahasanoglu T, Alcicek S, Altunkaya E, Sahinler I, Goksel S, Sirin F, Ozbal A. Effect of
fibrin glue on irradiated colonic anastomoses. Dis Colon Rectum 1997;40(10): 1240-1243. 28.
Houston KA, Rotstein OD. Fibrin sealant in high-risk colonic anastomoses. Archives of
surgery 1988;123(2): 230-234. 29.
Contreras A, Vazquez D, Carrasco L. Inhibition, by selected antibiotics, of protein synthesis in
cells growing in tissue cultures. J Antibiot (Tokyo) 1978;31(6): 598-602. 17
ACCEPTED MANUSCRIPT 30.
Zeng Q, Yu Z, You J, Zhang Q. Efficacy and safety of Seprafilm for preventing postoperative
abdominal adhesion: systematic review and meta-analysis. World journal of surgery 2007;31(11): 2125-2131; discussion 2132. 31.
Tsujimoto H, Tanzawa A, Matoba M, Hashimoto A, Suzuki S, Morita S, Ikada Y, Hagiwara
RI PT
A. The anti-adhesive effect of thermally cross-linked gelatin film and its influence on the intestinal anastomosis in canine models. Journal of biomedical materials research Part B, Applied biomaterials 2013;101(1): 99-109. 32.
Oncel M, Remzi FH, Senagore AJ, Connor JT, Fazio VW. Comparison of a novel liquid
SC
(Adcon-P) and a sodium hyaluronate and carboxymethylcellulose membrane (Seprafilm) in
postsurgical adhesion formation in a murine model. Dis Colon Rectum 2003;46(2): 187-191. Holzman S, Connolly RJ, Schwaitzberg SD. Effect of hyaluronic acid solution on healing of
M AN U
33.
bowel anastomoses. J Invest Surg 1994;7(5): 431-437. 34.
van Oosterom FJ, van Lanschot JJ, Oosting J, Obertop H. Hyaluronic
acid/carboxymethylcellulose membrane surrounding an intraperitoneal or subcutaneous
35.
TE D
jejunojejunostomy in rats. The European journal of surgery = Acta chirurgica 2000;166(8): 654-658. van der Vijver RJ, van Laarhoven CJ, de Man BM, Lomme RM, Hendriks T. Perioperative
pain relief by a COX-2 inhibitor affects ileal repair and provides a model for anastomotic leakage in
36.
EP
the intestine. Surgical innovation 2013;20(2): 113-118. Guidance for Resorbable adhesion barrier devices for use in abdominal and/or Pelvic surgery;
AC C
Guidance for industry. In: Administration DFaD, editor. U.S.: Center of Devices and Radiological health; 2008.
18
ACCEPTED MANUSCRIPT Legend to Tables:
Table 1: Adhesions, adhesion score and abscesses in the control and experimental groups.
AC C
EP
TE D
M AN U
SC
RI PT
Table 2: Number of the animals, with adhesions between the anastomosis and other organs
19
ACCEPTED MANUSCRIPT Legend to Figures:
Figure 1
Postoperative course of body weight. Data represent average relative body weight, in relation to the weight prior to operation for all the three groups.
Figure 2
Anastomotic bursting pressure. Individual values and medians (horizontal bars)
RI PT
are given in the controls (C), alginate gel group (ALG) and HA/CMC film group. The bursting site was either within (filled circle) or outside (open circle) the suture line. *p< 0.05 vs. day 3
SC
Figure 3 Anastomotic breaking strength. Data represent mean and SD in rats treated with
Figure 4
M AN U
alginate gel, HA/CMC film and controls. * p< 0.05 vs. day 3
Anastomotic collagen. A: Data represent mean and SD for the anastomotic hydroxyproline content in rats treated with alginate gel, HA/CMC film and controls. B: Data represent mean and SD for the wound collagen content, as determined by quantitative morphology, in the three groups of rats. * p< 0.05 vs.
Anastomotic histology. Each panel shows a tissue segment with the anastomosis in the middle at a magnification of approximately 40 x, representing typical examples obtained at day 3 (a. control; b. alginate gel; c. HA/CMC film) and day
EP
7 (d. control; e. alginate gel; f. HA/CMC film)
AC C
Figure 5
TE D
day 3
20
ACCEPTED MANUSCRIPT
Table 1: Adhesions, adhesion score and abscesses in the control and experimental groups
Adhesion score
Controls
24 (71%)
0.79 ± 0.59
Alginate gel
12 (35%)*
0.38 ± 0.55**
HA/CMC film
16 (47%)
0.55 ± 0.66 p= 0.0158
6 (18%)
9 (26%)
14 (46%)*** p= 0.0943
M AN U
p= 0.0124
with abscesses
RI PT
with adhesions
SC
Groups ( n = 34 each)
P-values under columns result from comparing the three groups with a chi square test. Two by two comparisons (Fisher’s exact test) yield significance versus controls as noted by * p = 0.0071,
AC C
EP
TE D
** p = 0.0092 and *** p = 0.0305.
21
ACCEPTED MANUSCRIPT
Table 2: Adhesions between the anastomosis and various other structures
Anastomosis – fat pad
15 (44%)
7 (20%)
Anastomosis - cecum
5 (15%)
2 (5%)
Anastomosis - ileum
9 (26%)
2 (5%)
Anastomosis - sidewall
0 (0%)
1 (3%)
P value
0.0342 NS 0.0223 NS
HA/CMC film (n=34)
RI PT
Alginate gel (n=34)
SC
Control (n=34)
P value
8 (23%)
NS
6 (18%)
NS
4 (12%)
NS
2 (5%)
NS
M AN U
Adhesions
Numbers denote the number of animals with adhesions present between the structures indicated. P-values concern the difference between either of the experimental groups and the control group
AC C
EP
TE D
(one-sided Fisher’s exact test). NS = non significant.
22
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
Figure. 1 Postoperative course of body weight.
23
ACCEPTED MANUSCRIPT
*
TE D
M AN U
SC
*
RI PT
*
AC C
EP
Figure. 2 Anastomotic bursting pressure.
24
EP
SC
TE D
M AN U
*
* *
RI PT
ACCEPTED MANUSCRIPT
Anastomotic breaking strength.
AC C
Figure.3
25
ACCEPTED MANUSCRIPT
4A.
RI PT
* *
TE D
M AN U
SC
*
*
AC C
EP
4B.
* *
26
ACCEPTED MANUSCRIPT Day 3
Figure 4. Anastomotic collagen
d
b
e
M AN U TE D
Alginate gel
f
AC C
EP
c HA/CMC film
SC
RI PT
Control
a
Day 7
Figure 5. Anastomotic histology
27
ACCEPTED MANUSCRIPT
Table 1:Adhesions, adhesion score and abscessesin the control and experimental groups
Adhesion score
with abscesses
6 (18%)
Controls
24 (71%)
0.79 ± 0.59
Alginate gel
12 (35%)*
0.38 ± 0.55**
HA/CMC film
16 (47%)
0.55 ± 0.66 p= 0.0158
9 (26%)
M AN U
p= 0.0124
RI PT
with adhesions
SC
Groups ( n = 34 each)
14 (46%)*** p= 0.0943
P-values under columns result from comparing the three groups with a chi square test. Two by two comparisons (Fisher’s exact test) yield significance versus controls as noted by * p = 0.0071, ** p =
AC C
EP
TE D
0.0092 and *** p = 0.0305.
ACCEPTED MANUSCRIPT
Table 2: Adhesions between the anastomosis and various other structures
Alginate gel (n=34)
15 (44%)
7 (20%)
Anastomosis - cecum
5 (15%)
2 (5%)
Anastomosis - ileum
9 (26%)
2 (5%)
Anastomosis - sidewall
0 (0%)
1 (3%)
0.0342 NS 0.0223 NS
M AN U
Anastomosis – fat pad
P value
HA/CMC film (n=34)
RI PT
Control (n=34)
SC
Adhesions
P value
8 (23%)
NS
6 (18%)
NS
4 (12%)
NS
2 (5%)
NS
Numbers denote the number of animals with adhesions present between the structures indicated. Pvalues concern the difference between either of the experimental groups and the control group (one-
AC C
EP
TE D
sided Fisher’s exact test). NS = non significant.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT