The Effects of Barrier Agents in Postoperative Pelvic Adhesion Formation: A Comparative Study of a Temperature-Sensitive Poloxamer-Based Solution/Gel and a Hyaluronic Acid-Based Solution in a Rat Uterine Horn Model

Abstract

Objective:

The aim is to evaluate the efficacy of poloxamer/alginate/CaCl₂ mixture (PACM) solution/gel and hyaluronic acid/carboxymethylcellulose (HA-CMC) solution for reducing pelvic adhesion in a rat uterine horn model.

Study Design:

A total of 48 females, Sprague Dawley rats, were divided into three groups according to the applied materials. A uterine horn adhesion model was created. The control group (group CO; n = 16) received no special materials except saline infusion. The experimental groups were treated with 1.0 mL HA-CMC solution (group HA-CMC; n = 16) or 1.0 mL PACM solution/gel (group PACM; n = 16). Adhesion scores were evaluated with macroscopic, microscopic, and immunohistochemistry grading 14 days postoperatively.

Results:

Macroscopic adhesion scores, including extent, severity, and total scores in group HA-CMC and group PACM, were significantly lower than those in group CO (P < .001). Among these three categories of scoring, group PACM had a significantly lower score than did group HA-CMC in adhesion severity (P = .025). In the microscopic adhesion score analysis, the fibrosis scores in group HA-CMC and group PACM were also significantly lower than that of group CO. In group PACM, the fibrosis score was significantly lower than that of group HA-CMC. There were no statistical differences across all three groups in the microscopic inflammation and immunohistochemistry staining.

Conclusion:

Both HA-CMC solution and PACM solution/gel effectively reduced adhesion formation. PACM solution/gel was superior to HA-CMC solution in preventing pelvic adhesion, especially in adhesion severity and microscopic fibrosis.

Introduction

Adhesions form in the peritoneal space when the inflammatory coagulation cascade is activated in response to tissue trauma, ischemia, infection, hemorrhage, or foreign bodies. Postoperative adhesion development is a complication of obstetric and gynecologic surgery. Approximately 60%–90% of women who undergo major gynecologic surgeries develop pelvic adhesions.¹

These adhesions provoke various medical problems, including chronic pelvic pain, dyspareunia, and secondary infertility; adhesions account for 20%–40% of all causes of female infertility.² A severe and potentially fatal complication of intra-abdominal adhesion is small bowel obstruction. Moreover, postoperative adhesions complicate future surgeries by extending operative time and increasing the risk of pelvic organ injury, particularly involving the intestines and ureters. In addition to their medical consequences, adhesions put a significant burden on healthcare expenditure.

Therefore, many attempts have been made to prevent postoperative adhesion formation. Among them, placing biocompatible materials between the viscera and peritoneum is to physically separate two areas of injury. These agents acting as “adhesion barrier” are typically two different types, a film type and a liquid type. However, there are many obstacles to using film type barriers, such as difficulty handling these materials, correct application, and the need for fixation suturing in laparoscopic surgery.^3,4 Furthermore, most of these film type agents used in the abdominal cavity are not as advantageous as expected.^5,6 Therefore, a liquid type antiadhesive agent is thought to be an alternative form suitable for various surgical applications.

Recent comparative studies demonstrated that liquid type barriers appear to be superior to film type agents.^5,7 Both hyaluronic acid/carboxymethylcellulose (HA-CMC) solution (Guardix-SOL^®; Hanmi, Seoul, South Korea) and poloxamer/alginate/CaCl₂ mixture (PACM) solution/gel (Guardix-SG; Hanmi) are viscous liquid form agents. PACM solution/gel has a distinctive feature that the viscosity varies with temperature. In several previous studies, HA-CMC solution has demonstrated safety and efficacy in reducing postsurgical adhesions.^5,8 To the best of our knowledge, no study has been performed for assessing efficacy of PACM solution/gel in the gynecologic field. In this regard, the aim of this study was to demonstrate the efficacy of the PACM solution/gel compared to the HA-CMC solution to reduce pelvic adhesions in a uterine horn model.

Materials and Methods

This study was approved by the Animal Review Committee of Chung-Ang University Hospital and performed in the Animal Research Laboratory (Reference number; 2015-00024, Date of approval; May 6, 2015). All of the procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Animal preparation and surgical technique

The research animals included 48, 2- to 3-month-old female, nonpregnant Sprague Dawley rats, weighing ∼180–220 g (SAMTAKO, Seoul, Korea). The rats were housed in cages (with 4 animals per one cage) in a temperature-controlled room (22°C) and a 12-hour light/dark cycle. They were fed a standard laboratory diet and tap water.

All of the procedures were performed in semisterile settings by the same surgical team. Anesthesia was provided with a 50 mg/kg IM injection of 10% ketamine hydrochloride (Ketasol; Richter Pharma AG, Wels, Austria) and 5 mg/kg of 2% xylazine (Rompun; Bayer Health Care LCC, Kansas, KS) to achieve appropriate sedation. The surgical teams wore surgical gloves with no powder and used aseptic instruments. The abdominal skin was shaved and scrubbed with povidone/iodine. Laparotomy was performed through a 4- to 5-cm-long midline incision. After gently exposing the uterine horn, a scalpel was used to make a 1 cm linear incision, 0.5 cm from the ovary, on the antimesenteric surface of the left uterine horn. Sandpaper was used for meticulous abrasion of the incision site. The surrounding tissues were handled as little as possible.

Two liquid type agents, HA-CMC and PACM, were evaluated as antiadhesive barriers. Each rat was randomly assigned to one of three different groups. Each respective agent was applied over the incision site before closing the abdominal wall. The group CO was composed of 16 rats that were free for any barrier agent except saline administration. The group HA-CMC consisted of 16 rats that were treated with 1.0 mL HA-CMC solution. The group PACM consisted of 16 rats that were treated with 1.0 mL PACM solution/gel.

Several preliminary experiments have been conducted to find the appropriate volume of the barrier agents. During the experiments, 0.5, 1, and 1.5 mL of barrier agents were tried on adhesion formation models. The solutions used in the experimental groups had high viscosity, but still the amount lost in the abdominal cavity had to be taken into account. The results revealed that 1.0 mL material volume was most appropriate to create sufficient coverage for the adhesion forming area, which was a 1-cm-long incision with surrounding abrasion. After all of the procedures were complete, the abdominal wall was closed layer by layer using no. 4-0 polypropylene monofilament, nonabsorbable sutures. Antibiotics were not administered.

Tissue sample collection and macroscopic examination

Two weeks after surgery, the rats underwent repeat laparotomy using the same anesthesia method. A U-shaped abdominal incision was made to examine all adhesions in detail. Two surgeons, both blinded to the study purpose, evaluated and scored the adhesion status (Fig. 1). Each score was then used for consensus data. A previously validated macroscopic score system was applied, as shown in Table 1.⁹ The extent of adhesion spread and the severity of attachment between the uterine horn and other abdominal organs were evaluated. After gross examination, the adhesion-carrying tissues were excised and preserved in 10% buffered formalin for microscopic examination and immunohistochemistry. Postoperatively, all of the experimental animals were sacrificed with a lethal dose of ether.

FIG. 1.

Gross adhesion scoring. (a) No uterine horn adhesion, (b) 26%–50% involvement, filmy avascular adhesion, (c) 1%–25% involvement, vascular and opaque adhesion.

Table 1.

Adhesion Scoring System for Macroscopic Evaluation

Score	Extent	Severity
0	No uterine adhesion	No adhesions
1	1%–25% involvement	Filmy avascular adhesions
2	26%–50% involvement	Vascular or opaque adhesions
3	51%–75% involvement	Cohesive adhesions
4	76%–100% involvement

Microscopic examination and immunohistochemistry

The formalin-fixed tissue samples were embedded in paraffin after routine tissue processing. Paraffin blocks were then cut into 5-μm sections and stained with hematoxylin and eosin. One histologist, who was blind to the groups, evaluated all of the tissue slides (Fig. 2). Tissues were graded according to the levels of inflammation and fibrosis (Table 2).

FIG. 2.

Histopathologic adhesion scoring (hematoxylin and eosin, × 100). (a) Inflammation grade 2; numerous giant cells and neutrophils, (b) moderate fibrosis.

Table 2.

Microscopic Adhesion Scoring System

Score	Fibrosis	Inflammation
0	None	None
1	Minimal, loose	Giant cells, lymphocytes, plasma cells
2	Moderate	Giant cells, eosinophils, neutrophils
3	Florid, dense	Many inflammatory cells, microabscess

In preparation for immunohistochemistry, 5-μm paraffin-embedded sections were dewaxed in xylene, rehydrated with graded alcohol, and placed in an endogenous peroxide block for 15 minutes. The sections were then subjected to antigen retrieval by autoclaving for 15 minutes in a citrate buffer (10% citrate buffer stock in distilled water, pH 6.0). Nonreactive blocking staining was blocked with 1% horse serum in Tris-buffered saline (pH 6.0), which was applied for 5 minutes. The sections were incubated with primary antibodies, transforming growth factor beta-1 (TGF beta-1, Clone-#9016, rabbit polyclonal, R&D Systems, 1:60), and vascular endothelial growth factor (VEGF, SC-152, rabbit polyclonal; 1:100; Santa Cruz), for 1 hour at 37°C in a humidified chamber.

Antibody binding was detected using a standard labeled Ultra Vision LP detection system (TL-125-HD; Thermo Fisher Scientific). The primary antibodies were omitted as a negative control. The semiquantitative scoring system for VEGF and TGF beta-1 is also shown in Table 3. Cytoplasmic immunostaining intensity and prevalence were semiquantitatively scored on a scale of 0–3.

Table 3.

Immunohistochemistry Scoring System

Score	VEGF, TGF beta-1
0	No staining
1	Weak, <33% positive staining
2	Moderate, 33%–66% staining
3	Strong, >66% staining

TGF beta-1, transforming growth factor beta-1; VEGF, vascular endothelial growth factor.

Statistical analyses

In a previous study comparing the efficacy of HA-CMC and PACM in pericardial adhesion formation,¹⁰ the gross adhesion score in the control group was 2.79 ± 0.79. This study was conducted based on the assumption that the macroscopic adhesion score in our control group is the same as that of the previously mentioned study. Additional premises were set such that the standard deviations and reduction rate of the macroscopic adhesion score in the two groups were the same as those in the reference study. Considering these conditions, sample sizes of 14 for each group were obtained through a one-way analysis of variance study. Ultimately, 16 rats were included in each group, allowing 10% loss.

The data are reported as mean values with standard deviations. For continuous variables, the Shapiro–Wilk test was used for normality. As all the variables were not normally distributed, we analyzed the data using the Kruskal–Wallis test followed by the Mann–Whitney U test with Bonferroni correction. P-values <.05 were considered statistically significant. Statistical analyses were performed using SPSS version 24.0 (SPSS, Inc., Chicago, IL).

Results

Two rats died 5 days after the first operation and they were excluded from the study groups. One mortality in group HA-CMC was due to infection and 1 rat in group PACM died of possible anesthetic problem. Finally, the group CO, group HA-CMC, group PACM were composed of 16, 15, and 15 rats, respectively.

There were no complications, including wound dehiscence or intra-abdominal abscess, on gross examination at the time of secondary laparotomy. Each group was evaluated for the extent, severity, and total scores of macroscopic adhesions (Table 4). There were statistically significant differences between the three groups with regard to the extent (P < .001), severity (P < .001), and total scores (P < .001). As shown in Table 5, group CO had a significantly higher adhesion score than did group HA-CMC and group PACM in all gross scores. Among these three categories of macroscopic examination, group PACM had significantly lower score than did group HA-CMC in the adhesion severity (P = .025).

Table 4.

Comparison of Macroscopic, Microscopic, and Immunohistochemistry Scores by Group

	Group CO	Group HA-CMC	Group PACM	P
Number	16	15	15
Macroscopic score
Extent	1.88 ± 1.31	0.47 ± 0.74	0.40 ± 0.51	<.001
Severity	2.31 ± 1.14	1.20 ± 0.94	0.42 ± 0.50	<.001
Total	4.19 ± 2.32	1.67 ± 1.63	0.82 ± 0.94	<.001
Microscopic score
Fibrosis	2.44 ± 1.15	1.47 ± 1.06	0.53 ± 0.64	<.001
Inflammation	1.75 ± 0.45	1.60 ± 0.51	1.53 ± 0.52	.456
Immunohistochemistry
VEGF	1.63 ± 0.72	1.40 ± 0.63	1.47 ± 0.64	.618
TGF beta-1	1.88 ± 0.72	1.47 ± 0.52	1.40 ± 0.51	.109

HA-CMC, hyaluronic acid/carboxymethylcellulose; PACM, poloxamer/alginate/CaCl₂ mixture; TGF beta-1, transforming growth factor beta-1; VEGF, vascular endothelial growth factor.

Table 5.

Comparison of P-Values for Macroscopic Adhesion Scores and Microscopic Fibrosis Scores by Group

	Group HA-CMC/Group CO	Group PACM/Group CO	Group PACM/Group HA-CMC
Extent	0.003	0.001	0.989
Severity	0.018	<0.001	0.025
Total	0.005	<0.001	0.243
Fibrosis	0.026	<0.001	0.038

HA-CMC, hyaluronic acid/carboxymethylcellulose; PACM, poloxamer/alginate/CaCl₂ mixture.

There was a statistically significant difference among the three groups in the microscopic fibrosis score (Table 4, P < .001). The fibrosis score in group CO was significantly higher than those of group HA-CMC and group PACM, as shown in Table 5. In group PACM, the fibrosis score was significantly lower than that of group HA-CMC (P = .038).

In comparison of inflammation scores, there were no statistical differences between all groups. On immunohistochemical examination, no significant differences were found in the results of VEGF and TGF beta-1.

Discussion

In this study, both HA-CMC and PACM effectively reduced adhesion formation after gynecologic surgery. In particular, PACM has proven to be superior to HA-CMC in terms of reducing adhesion severity and microscopic fibrosis formation. There were no significant differences across groups regarding microscopic inflammation, VEGF, and TGF beta-1 scores.

Peritoneal adhesion formation after pelvic surgery is an unavoidable and natural outcome of the tissue healing process. However, pelvic adhesions are problematic and may lead to many troublesome complications.^3,11,12 Therefore, many drugs, substances, and surgical interventions have been investigated to modify these responses in an effort to avoid adhesion. Some studied agents include fibrinolytics, anticoagulants, nonsteroidal anti-inflammatory drugs, corticosteroid, antioxidants, and bioabsorbable physical barriers.^13,14 There has been variable success using these agents. There is still no consensus for overcoming this challenge.

The basic principle of barrier agents is to separate the injured sites from the adjacent tissues or peritoneum during a critical, 2-week postoperative period.¹⁵ Since dense adhesion is already formed 2 weeks postoperatively, repeat laparotomy time was scheduled as the 15th day after initial surgery.¹⁵ With regard to the time intervals longer than 14 days, it was assumed that there would be no significant difference in the result compared to that of 14 days.

There are different types of barrier agents, including films, liquids, and the recently developed solution/gel transition type. It is most technically difficult to handle film type barriers and to apply them at the correct site. These film agents may even migrate from the intended site after application.^16,17 Furthermore, it is technically difficult to place these agents on the surface of ovary or fallopian tube, both of which are essentially freely floating in the pelvis.

In contrast to film type barriers, liquid barriers can be readily applied on the injured site. However, liquid barriers flow to the dependent portion of the site with gravity, such as the posterior cul de sac; therefore, they may be drained from the site, along with postoperative exudates by a drainage catheter.¹⁷ Consequentially, the barrier's efficacy is reduced if it is removed prematurely. Therefore, the viscosity of liquid agents is their most important aspect with respect to prolonging their effectiveness.

Both liquid type solutions used in this study have high viscosity scores for early-stage products. The remaining time of HA-CMC to the injured tissue surface is longer than that of initial form of hyaluronic acid alone barrier product.⁷ At room air temperature (22°C), the viscosity of HA-CMC is similar to that of honey or maple syrup, ∼3000 cP.

PACM solution/gel is based on poloxamer, which has been previously shown to prevent postoperative adhesion development.^18–20 Accordingly, it has proven to be safe and biocompatible in the human body.^21,22 In its poloxamer molecular structure, polyethylene glycerol has hydrophilic properties that prevent cell adhesion.²³ However, poloxamer is easily degraded and absorbed before it can exert its antiadhesive effects. The poloxamer/alginate mixture, when crosslinked with CaCl₂, becomes a highly stable gel. Fortunately, it still retains the antiadhesive effect of the poloxamer.¹⁰

At room air temperature (22°C), PACM also has about 3000 cP viscosity level similar to that of HA-CMC. However, when PACM is applied on the target site at human body temperature (37°C), its viscosity increases by 30–40 times. Therefore, this modified agent has the unique characteristic of phase shifting, solution to gel by thermal change. There are two advantages for each form. In the solution form, it is easily injectable at the correct site. After injection, it immediately transforms into a gel form, which adheres and lasts longer than a liquid would. It was proven in our study that PACM showed a better protective effect than that of HA-CMC in the adhesion severity and the microscopic fibrosis score.

In one experimental study, PACM was observed in gel form on postoperative day 7. This gel begins to undergo hydrolysis after 2 weeks and then metabolizes slowly. All degraded materials were excreted through the kidney.²³

VEGF is the strongest known angiogenic factor. TGF beta-1 has a well-described role in cell differentiation, in triggering angiogenesis, and in fibroblast activation.^9,24 These two key playing markers are increased in the development of adhesion band, highly vascularized dense collagen fibers, within 2 weeks.^15,25 However, the outcomes of immunohistochemistry staining in this study showed no statistical differences between three groups and these results are consistent with those of previous studies.^5,10 They support our hypothesis that the inflammatory reactions will occur equally in the three groups under similar experimental conditions. They also suggest that the effect of PACM and HA-CMC is mainly related to a barrier role, rather than anti-inflammatory or antifibroblastic effects.

Our study has several limitations. The first is to recognize the anatomical differences between human gynecologic organs and the rat uterine horn model. Ideally, we would have used an animal model that has a more similar pelvic organ structure to that of humans to provide a more realistic model for adhesion analysis. Another limitation is that the surgeons involved in this study may have some biases in surgical techniques, slide preparation, and gross assessment scoring. All of the specimens were assessed by the same, blinded pathologist. However, pathologic assessment is highly dependent on the site of the slide section and the subjective judgment of the observer. Therefore, there may have been bias associated with the pathological examinations. Regardless, our study protocol was designed as objectively as possible to avoid such biases.

In conclusion, both the HA-CMC solution and the PACM solution/gel effectively reduced adhesion formation after gynecologic surgery. PACM has demonstrated superiority to HA-CMC in preventing pelvic adhesion, especially in adhesion severity and microscopic fibrosis. The unique feature of phase shifting by thermal change may make PACM differences to HA-CMC. Further research in a more optimal animal model is required to determine which of these agents, if either, is more effective at preventing pelvic adhesion formation.

Footnotes

Acknowledgment

We are grateful for the support of Sun-Mi Kim at Chung-Ang University for statistical analyses of the data.

Disclosure Statement

No competing financial interests exist.

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