Polyvinyl Alcohol Hydrogel Decreases Formation of Adhesions in a Rat Model of Peritonitis

Abstract

Background:

Adhesion formation after surgery for peritonitis-related conditions, with such associated complications as intestinal obstruction, pain, and infertility, remains an important problem. Applying a liquid barrier intra-peritoneally might reduce initial adhesion formation.

Methods:

A combination of the cecal ligation and puncture model of peritonitis with the side-wall defect (SWD) model of adhesion formation was performed. Forty rats were assigned randomly to receive no barrier or 1 mL or 2 mL of the cross-linked polyvinyl alcohol and carboxymethylcellulose (PVA/CMC) hydrogel A-Part^® Gel (B. Braun Aesculap AG, Tuttlingen, Germany). After 14 days, the animals were sacrificed, and adhesion formation and abscess formation were scored.

Results:

Thirty animals survived, distributed equally among the groups. There were significantly fewer adhesions to the SWD in the PVA/CMC groups (median 0) than in the control group (median 26%–50%) (p<0.05). The median tenacity of the adhesions was significantly higher in the control group (Zühlke score 2) than in the PVA/CMC groups (Zühlke score 0) (p<0.05). The amount and size of intra-abdominal abscesses were not significantly different in the three groups.

Conclusion:

In this experiment, PVA/CMC hydrogel reduced the amount of adhesions to the SWD and between viscera significantly with equal risk of abscess formation.

The formation of intra-abdominal adhesions remains a common serious problem in surgical practice. Formation of adhesions is observed after both open and laparoscopic surgery, with a reported incidence as high as 97% [1,2]. Complications of intra-abdominal adhesions include intestinal obstruction, chronic pain, infertility, and troublesome reinterventions. Of all patients undergoing open abdominal surgery, 35% will be readmitted for complications related to adhesions [3]. The cumulative risk of adhesive small-bowel obstruction after colectomy is 11%–25% after one year and 30% after 10 years [4,5]. During reintervention, some extent of adhesiolysis is unavoidable, leading to inadvertent enterotomy in 19% of patients owing to the severity of the adhesions or to iatrogenic bowel injury [6].

Adhesion formation is initiated by the inflammatory response following visceral damage as a result of intra-abdominal surgery and infection [7]. The underlying mechanism is local tissue ischemia and mesothelial injury. The mesothelial and submesothelial cells express proteases and protease inhibitors, disrupting the balance between coagulation and fibrinolysis and creating fibrous bands that form adhesions [7,8]. Furthermore, inflammation intensifies this reaction by attracting and activating fibroblasts and disrupting fibrinolysis [9,10]. Adhesion formation is controlled by macrophages and T lymphocytes and takes place mainly during the first two weeks of healing, before the defect has been covered with mesothelium [11 –13]. Adhesion formation can be prevented by a physical barrier that separates the injured tissue from adjacent organs and the peritoneum during the crucial healing period [2,14 –16].

Abdominal surgery for intra-abdominal infection with peritonitis is associated with high morbidity and mortality rates and complicated frequently by abscess formation. In peritonitis, severe inflammation of the peritoneum occurs, increasing adhesion formation. Prevention of adhesion formation after peritonitis seems the ultimate challenge for proving the effectiveness of an anti-adhesion barrier. It also is crucial that the barrier does not promote infection or abscess formation [17,18]. Cross-linked biocompatible polyvinyl alcohol (PVA) hydrogels reduce adhesion formation after non-infectious abdominal surgery [16,19,20,21]. The aim of our experiment was to investigate the effects of PVA hydrogel on adhesion and abscess formation in an experimental bacterial peritonitis and adhesion model in rodents.

Materials and Methods

To study the effects of PVA hydrogel on adhesion and abscess formation, a combination of the cecal ligation and puncture (CLP) peritonitis model and the cecal abrasion side-wall defect (SWD) adhesion model was used. We believe that our combination model resembles adhesion formation in a clinical situation after abdominal surgery in the presence of peritonitis. The protocol of the experiment was approved by the Animal Experiments Committee of the Erasmus University, Rotterdam.

Study design

In 40 male Wistar rats, the CLP model was created by ligation and puncture of the cecum. The following day, the necrotic cecum was resected and the SWD model created. The hydrogel studied in this experiment was A-Part^® Gel (B. Braun Aesculap AG, Tuttlingen, Germany), which is a cross-linked PVA and carboxymethylcellulose (CMC) formulation. After completion of the SWD model, the rats were assigned randomly to one of three experimental groups: A (control), B (1 mL of A-Part^® Gel), or C (2 mL of A-Part^® Gel). The viscous PVA/CMC gel was administered with a special nozzle to the sites of peritoneal damage; that is, the SWD and around the cecal stump. The abdominal wall and skin were closed separately with continuous, absorbable suture. After 14 days, the animals were sacrificed humanely and scored on adhesion and abscess formation.

Animals

Inbred male rats of the Wistar strain weighing 294–355 g were obtained from a licensed breeder (Harlan, The Netherlands) and accustomed to laboratory conditions two weeks before the start of the experiment. They were bred under specified pathogen-free conditions, kept under standard laboratory conditions in individually ventilated cages (temperature 20–24°C; relative humidity 50%–60%; 12 h light/dark cycles), and fed standard rat chow and water ad libitum during the entire experimental period.

Procedure

Day 0: CLP model

The surgery was performed under aseptic conditions. At the start of the experiment, the animals were anesthetized using isoflurane/O₂ by inhalation, and buprenorphine analgesia (0.05 mg/kg) was administered subcutaneously. The abdomen was shaved and cleaned with isopropyl alcohol 70%, after which a 3-cm midline incision was made. The cecum was manipulated carefully to the outside of the abdominal cavity and ligated just distal to the ileocecal valve with a monofilament non-absorbable suture (4-0 Ethilon,^® Ethicon, Inc., Somerville, NJ), maintaining the continuity of the bowel. Distally, the cecum was punctured once with an 18-gauge needle. Some fecal material was expressed through the puncture hole and cultured. The cecum was replaced intra-peritoneally, and the abdominal wall and skin were closed separately with running polyglycolic acid sutures (5-0 Safil^®, B. Braun Melsungen AG, Melsungen, Germany). For resuscitation, 5 mL isotonic sodium chloride solution was administered, and animals were placed under a heating lamp during the immediate post-operative period.

Day 1: CLP and SWD model

The animals were anesthetized using isoflurane/O₂ inhalation, and buprenorphine analgesia (0.05 mg/kg) was administered subcutaneously. The abdomen was re-opened through the midline incision, and a culture swab of the abdominal cavity was obtained to confirm fecal peritonitis. The necrotic cecum was resected and the abdominal cavity rinsed with at least 20 mL of phosphate-buffered saline warmed to 37°C. The remaining cecum was abraded lightly with dry sterile gauze until punctate bleeding was observed. From the left anterior abdominal wall, a tissue strip of 0.5×2.0 cm, including peritoneum and muscle, was excised. Before closure of the abdomen, animals were assigned randomly to one of the three experimental groups. All animals received gentamicin (6 mg/kg intramuscular), buprenorphine (0.05 mg/kg subcutaneously), and isotonic sodium chloride solution (5 mL subcutaneously).

Day 1: Application of PVA/CMC hydrogel

Animals in Group A served as the control group and received no additional treatment. Group B received 1 mL of PVA/CMC hydrogel, and Group C received 2 mL of hydrogel. The hydrogel was applied to the SWD and the abraded cecum. Finally, the abdominal wall and skin were closed separately with running polyglycolic acid sutures (5-0 Safil^®).

Day 14: Sacrifice

The animals were anesthetized using isoflurane/O₂ by inhalation and shaved. After disinfection, the ventral abdominal wall was opened through a U-shaped incision, and a swab of the abdominal cavity was taken. The amount and tenacity of the adhesions was scored, and pictures were taken (5.0-megapixel digital camera; Sony Cybershot^®, Tokyo, Japan). The abdominal cavity was inspected for abscesses, which were scored and cultured when found. The animal was then euthanized by cardiac cut.

Measurements

Wellness and survival

During the experiment, animals were weighed daily and scored for their wellness using an objective 12-point scoring system [24]. Animals with a weight loss of 20% or more or a wellness score of <5 were euthanized. A necropsy was performed on all euthanized and deceased animals.

Adhesions

Two independent observers assessed adhesion coverage of the surface of the abdominal wall defect in a blinded manner using a scoring system, dividing the defect into four squares of 0.5×0.5 cm (0%, 1%–25%, 26%–50%, 51%–75%, or 76%–100%) and categorized the tenacity of the adhesions using the objective Zühlke score [24], which is based on histologic and morphologic criteria (Table 1). In case of inter-observer disagreement, the mean Zühlke score was calculated. The amount of adhesions (between viscera or between viscera and the abdominal wall, including the midline scar) was scored and the severity of the adhesions was graded by the Zühlke score.

Table 1.

Zühlke Scoring System for Adhesions

Score	Definition
0	No adhesions
1	Minimal, filmy adhesions requiring little blunt dissection
2	Moderate adhesions requiring blunt and partly sharp dissection; beginning of vascularization
3	Strong adhesions; lysis possible by sharp dissection only; clear vascularization
4	Very strong adhesions; lysis possible by sharp dissection only; organs attached (damage to organs difficult to prevent)

Translated and reprinted from Zühlke HV, Lorenz EM, Straub EM, Savvas V. [Pathophysiology and classification of adhesions](Ger). Langenbecks Arch Chir Suppl II Verh Dtsch Ges Chir 1990:1009–1016.

Abscesses

Two independent observers assessed the amount and size of abscesses at four sites in the peritoneum: liver, abdominal wall, bowel, and omentum using an objective size scoring system (Table 2)[26].

Table 2.

Abscess Scoring System

Score	Definition
0	No abscess present at the site
0.5	One small abscess present at the site
1	Several small abscesses present at the site
2	Medium abscess present at the site
3	Large or several medium abscesses present at the site
4	One very large or several large abscesses present at the site

Reprinted from Rodgers KE, Schwartz HE, Roda N, et al. Effect of Oxiplex® films (PEO/CMC) on adhesion formation and reformation in rabbit models and on peritoneal infection in a rat model. Fertil Steril 2000;73:831–838.

Cultures

The fecal sample, intra-abdominal swab, and abscess cultures were evaluated for the strains and amount of bacteria present.

Statistical analysis

Adhesion formation and tenacity, abscess formation, survival, animal weight, and wellness score were compared using non-parametric tests (Kruskal–Wallis, Mann–WhitneyU) because the data did not show a normal distribution. Therefore, all results are presented using the median and the interquartile range. All reported p values are two-sided and considered significant if p<0.05. Statistical analysis was performed using the PSAW statistical software package (IBM SPSS Statistics, Chicago, IL).

Results

No animals needed to be euthanized because of a weight loss of 20% or more or a wellness score <5. A total of 30 animals survived; 10/14 (72%) in both experimental groups (B and C) and 10/12 (83%) in the control group (A)(p=0.81). No differences in weight change or wellness score were observed among the three groups. Ten animals died within 24 h after the second operation. All were found to have died of sepsis secondary to fecal peritonitis.

Adhesions

Adhesions to the surface of the SWD were significantly different among the groups (p=0.009) (Table 3). There were significantly more adhesions (median 26%–50%) to the SWD in the control group than in the PVA/CMC groups (median 0)(p=0.02), but no difference in adhesions between the two PVA/CMC groups (p=1.00). The tenacity of the adhesions also was significantly different among the groups (p=0.004) (Table 4). There was a significant difference in adhesion tenacity between the PVA/CMC groups (both having a median Zühlke score of 0) and the control group (median Zühlke score 2)(p=0.01 and p=0.02), but no difference between the PVA/CMC groups (p=0.74). Visceral adhesions between intestinal loops or between intestinal loops and the abdominal wall (including the midline scar) appeared in 70% of the control group, 50% of the PVA/CMC 1-mL group, and 40% of the PVA/CMC 2-mL group (p=0.31). The tenacity was the greatest in the control group (median Zühlke score 3.5) compared with the PVA/CMC 1 mL (median Zühlke score 0.5) and PVA/CMC 2 mL (median Zühlke score 0), but this difference was not statistically significant. In all surviving animals, adhesions to the cecal stump occurred with comparable tenacity in all groups (median Zühlke score 3–3.5)(p=0.32).

Table 3.

Adhesion Coverage of Side Wall Defect by Treatment Group

Adhesion coverage (%)	Control	A-Part ^® 1 mL	A-Part 2 mL
0	3	9	8
1–25	2	0	1
26–50	1	0	1
51–75	0	0	0
76–100	4	1	0

A-Part=cross-linked polyvinyl alcohol–carboxymethylcellulose gel.

Table 4.

Zühlke Score of Adhesions on Side Wall Defect by Treatment Group

Score	Control	A-Part ^® 1 mL	A-Part 2 mL
0	3	9	8
1	2	0	1
2	0	1	0
3	1	0	1
4	4	0	0

A-Part=cross-linked polyvinyl alcohol–carboxymethylcellulose gel.

Abscesses

The amount and size of intra-abdominal abscesses were not significantly different among the three groups (p=0.48 and p=0.10). In the control group, four abscesses were found in three animals, with an abscess score ranging from 1 to 4. In the PVA/CMC 1-mL group, seven abscesses were found in six animals, with a score between 0.5 and 2. In the PVA/CMC 2-mL group, five abscesses were found in five animals, with a score of 0.5 and 1.

Cultures

The feces of the rats contained no bacteria other than what was expected in view of their specified pathogen-free status. The culture taken on Day 1 proved fecal peritonitis in all 40 rats. During sacrifice, no bacteria were found intra-abdominally. All abscesses contained one or more fecal bacteria.

Discussion

The formation of adhesions begins with injury to two opposing layers of the peritoneum, with exudate contributing to the deposit of fibrin. The PVA/CMC hydrogel separates the damaged peritoneal surfaces during the crucial remesothelialisation phase in the first two postoperative weeks and reduces initial adhesion formation [11 –13]. This hydrogel is completely biodegradable, and no residues are found after two weeks [27].

A serious concern about barriers is their influence on infection. It was especially interesting that in our experiment, the risk of abscess formation was equal in all groups, with or without PVA/CMC gel. We believe that our experimental model combining peritonitis and adhesions resembles adhesion formation after clinical abdominal surgery in the presence of peritonitis. The CLP model produces generalized peritonitis with an immunologic response secondary to the spill of the animal's own stool into the peritoneal cavity through the devascularized and punctured cecum. This model closely mimics the clinical situation of peritonitis induced by bowel perforation, and subsequent surgical source control followed by post-operative adhesion and abscess formation [28]. In peritonitis models, abscess formation increases until two weeks postoperatively and then decreases in the following weeks [22,23]. The CLP model combines an acceptable mortality rate of 15%–30% with a high incidence of abscess formation [29 –31]. The SWD model promotes adhesion formation and mimics peritoneal trauma caused by laparotomy. It is a dependable, predictable, and readily quantifiable model that allows objective measurement of the extent of adhesion coverage [32,33].

Adhesions are formed predominantly during the first two weeks of healing, before the defect is covered by mesothelium [11 –13]. The amount of adhesions does not increase after 14 days, and the situation at that time therefore is representative of the long-term effects. On the other hand, the tenacity of the adhesions is influenced by the chronic inflammatory response and therefore probably will change over time. The combination of the two models promotes a severe inflammatory response with subsequent adhesion formation. An inflammatory reaction occurs, causing an influx of inflammatory cells and activating mesothelial cells, leading to fibrinous exudate. The exudate becomes more dense as a result of persisting fibroblasts, and adhesions are formed [7].

In this experiment, the SWD and other visceral adhesions (except to the cecum) were significantly fewer in the groups in which the PVA/CMC hydrogel was used as a barrier. The PVA/CMC probably remained in place on the SWD because of the flat surface of the abdominal wall and the prostrate position of the rodents. There was no reduction of adhesion formation to the cecal stump. This might be the result of the PVA/CMC hydrogel not remaining at the site because of the roundness and gravity making the gel shift from the cecum, losing its barrier function. Also, the non-absorbable suture and possible microscopic fecal leakage from the cecal stump might have given rise to adhesion formation.

In this experiment, the PVA/CMC hydrogel was safe and efficient in reducing adhesion formation in a peritonitis model. Because both concentrations (1 mL and 2 mL) of the hydrogel provided the same reduction of adhesion formation and no difference in abscess formation, we conclude that 1 mL of A-Part^® is an adequate dosage to reduce adhesion formation in a Wistar rat weighing 300–350 mg. The promising efficacy demonstrated by the A-Part^® hydrogel in this experiment stimulated us to investigate this product further, focusing on its safety when applied around an intestinal anastomosis and in the presence of synthetic material such as intra-abdominally placed meshes [33]. The promising experimental results also led to initiation of a clinical trial of the safety and efficacy of the hydrogel [34].

Footnotes

Author Disclosure Statement

The authors declare no conflicts of interest. This experiment was supported financially by B. Braun Aesculap AG, Tuttlingen, Germany, manufacturer of the PVA/CMC hydrogel, which was not involved in the study design, analysis of the results, or preparation of the manuscript.

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