Cross-linked hyaluronate hydrogel prevents adhesion formation and reformation in mouse uterine horn model

Tomio Sawada1, Kazuhiko Tsukada1, Kiyoshi Hasegawa1, Yoshimasa Ohashi1, Yasuhiro Udagawa1 and Victor Gomel2,,3

1 Department of Obstetrics and Gynecology, Fujita Health University, School of Medicine, Toyoake, Aichi, Japan and 2 Department of Obstetrics and Gynecology, Faculty of Medicine University of British Colombia, Vancouver Hospital and Health Sciences Centre, Vancouver BC, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to evaluate the efficacy of cross-linked hyaluronate hydrogel (HA gel) as an adjuvant for postoperative adhesion prevention, in a mouse uterine horn model. In experiment 1 uterine horns were abrased with iodine. HA gel was applied to the injured surface before closure in the treatment group. In experiment 2, after injuring the uterine horns, three stitches were placed at equal distances around the uterine horns to appose the injured medial surfaces of the two horns during healing. HA gel was inserted between the uterine horns in the treatment group. In experiment 3 prevention of adhesion reformation was assessed. After lysis of adhesions that were induced as in experiment 2, HA gel was introduced between the serosal surfaces of apposing uterine horns. Untreated animals served as controls in each experiment. Statistical analysis was carried out using Student's t-test. The adhesion score was significantly lower in the HA gel group on the 14th day compared with controls in all the experiments: in experiment 1, 0.3 ± 0.4 versus 1.7 ± 1.2; in experiment 2, 0.9 ± 1.0 versus 2.6 ± 0.5; and in experiment 3, 1.5 ± 0.9 versus 2.2 ± 0.6 respectively. Cross-linked HA gel significantly reduced de-novo adhesions (P < 0.03) and adhesion reformation (P < 0.03).

Key words: adhesion prevention/adhesion reformation/hyaluronate hydrogel/mouse uterine horn model/reproductive surgery


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pelvic adhesions may cause many complications including infertility, chronic pelvic pain and bowel obstruction. Even when surgery is performed with strict adherence to microsurgical principles, postoperative adhesions occur in >50% of cases (Gomel, 1977Go).

Adhesion formation ascribable to surgery frequently results from the two injured surfaces that remain in contact during the healing period. A variety of medical agents have been used to reduce postoperative adhesions, such as systemic and intraperitoneal non-steroidal anti-inflammatory agents, intraperitoneal high molecular weight dextran, fibrinolytic agents, and extracellular matrix (Ellis, 1971Go; Holtz, 1984Go). Unfortunately, none have confirmed sufficient efficacy except hyaluronic acid solution. Various barriers designed to isolate damaged tissues have also been investigated. Absorbable barriers of oxidized regenerated cellulose (Interceed®) (Azziz, 1993Go), expanded polytetrafluoroethylene (Preclude®) (Haney and Doty, 1993Go), hyaluronic acid carboxymethylcelullose (Seprafilm®) (Diamond and The Seprafilm Adhesion Study Group, 1996Go) have been shown to be effective in reducing postoperative adhesion formation.

This report evaluates the efficacy of cross-linked hyaluronate hydrogel (HA gel; Seikagaku Kogyo Co., Tokyo, Japan), a hyaluronate gel that has a long retention time in the peritoneal cavity with a half-life of ~60 h after i.p. administration (Matsuda et al., 1993Go), in reducing postoperative adhesion formation using the mouse uterine horn model.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Eighty-nine female outbred ICR mice were used in these experiments. They were 7 weeks old and weighed 22–24 g. The surgical procedures used were approved by the Fujita Health University Institutional Animal Care and Use Committee. After they were housed in a cage for at least 2 days before undergoing surgery, their menstrual periods were synchronized with the i.p. administration of 5 IU of pregnant mare serum gonadotrophin (PMSG; Funakoshi Co, Tokyo, Japan). The mice were kept on a 12 h:12 h light:dark cycle, with food and water available until the operation. Because of the difficulties of general anaesthesia, this was induced by i.p. injection of Nembutal sodium solution (Abbott Laboratories, North Chicago, IL, USA) in both experimental and control groups and after the skin was cleaned with disinfectant, the surgical procedure was performed under sterile conditions. The mice had their abdominal wall opened through midline ventral incisions and the bowel was gently retracted upward. The surface of the uterine horn was abraded using the iodine method as previously described (Sawada et al., 1999Go). Briefly, a 1.5 cm section of the horn was abraded with a cotton ball permeated with iodine ethanol, moving the cotton ball forwards and backwards four times. A prescribed amount of the HA gel (Osada et al., 1999Go) was injected from a syringe to cover the whole surface of the injury site with a thickness of 2 mm, for which 0.15 ml of HA gel was used in the treatment group. The control group received no treatment.

This study was approved by the Institutional Animal Care Committee of Fujita Health University, and the Declaration of Helsinki and the Guiding Principle in the Care and Use of Animals was followed.

Preparation of HA gel
HA gel was prepared by introducing photo-reactive residues into sodium hyaluronate molecule at a concentration of 2% and was heat treated at 105°C for 10 min before irradiation with ultraviolet rays and at 121°C for 8 min after irradiation with ultraviolet rays (Matsuda et al., 1993Go). The product was provided by Seikagaku Kogyo Co. Because of the photo-cross-linking the gel remains on the application site for a long period according to the report by Osada using HA-specific binding protein (Osada et al., 1999Go) and thus the tissue separation effect is maintained for an equally long time.

Experiment 1. (De-novo adhesion formation on the uterine horns: HA gel versus no treatment)
Thirty-nine mice had their abdomens opened through midline incisions and the abdominal contents were carefully retracted upwards. Both uterine horns were carefully picked up and uterine surfaces were abraded by the iodine cotton method described above. After this procedure HA gel was pasted in 2 mm thickness on both injured surfaces in 18 animals selected from the 39 animals (HA gel group) before closing the abdominal wall by continuous running suture. The 21 untreated animals served as controls. Adhesion formation was evaluated at 7 days in 26 mice and 14 days in 13 mice.

Experiment 2. (De-novo adhesion formation on the uterine horns apposed by suturing: HA gel versus no treatment)
Twenty-five mice had their abdomens opened as in experiment 1. A single suture was placed through the wall of both uterine horns immediately below the junction with the oviducts with the view of approximating the two uterine horns. The medial surfaces of both uterine horns brought into direct apposition were subjected to iodine abrasion injuries. Two loose external sutures were then placed around both horns at their middle and lower ends to ensure that the two apposing injury sites remained in constant contact during the healing period. HA gel was carefully inserted between the uterine horns in 2 mm thickness in 14 mice selected arbitrarily from the 25 mice (HA gel group) and 11 mice served as controls.

Experiment 3. (Adhesion reformation in the approximated uterine horn model: HA gel versus no treatment)
Adhesions were created in 25 mice using the iodine abrasion method, as in experiment 2, without application of HA gel. Seven days later, all 25 mice were reopened and the adhesions were lysed electrosurgically using a microsurgical technique employing a very sharp monopolar electrocautery and forceps for microsurgery with magnification glasses. Simultaneously adhesion scores were evaluated in order to give baseline values from which to determine the reformation scores at laparotomy three. HA gel was inserted between the uterine horns in 13 mice arbitrarily selected from the group of 25 re-operated mice (HA gel group), and the horns were re-sutured. Adhesion reformation was assessed 7 days after lysis. All procedures were performed by the same researcher under the same conditons, however there was minimum intra-researcher variability.

Evaluation of adhesions
After dislocating and immediately entering the peritoneal cavity, the injury site was carefully inspected with magnification to assess blindly the extent and nature of adhesions according to the following scoring system reported by Rodgers et al. (Rodgers et al., 1998Go): 0 = no adhesion; 1 = mild and easily detachable adhesion; 2 = moderate and detachable adhesion; 3 = dense and undetachable adhesion.

In experiments 2 and 3, there were frequently filmy adhesions between the fat tissue, normally located in the bladder area which was not covered with HA gel, and the external surface of the uterine horns at the site of the loose circumferential sutures. These sutures were gently peeled away and only the adhesions directly between the uterine horns were considered in the assessment.

Statistics
Student's t-test was used to evaluate the significance of gel application on the injured surfaces. Data are expressed as mean ± SD. A P value of < 0.05 was considered to be statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experiment 1
On day 7, the HA gel group (n = 12) had an adhesion score of 0.5 ± 0.5. This score was significantly lower (P < 0.03) than the control group (n = 14) that had a score of 1.3 ± 1.1. On day 14, the HA gel (n = 7) and control groups (n = 6) demonstrated significantly different scores of 0.3 ± 0.4 and 1.7 ± 1.3 respectively (P < 0.03) (Table IGo). The scores showed no significant difference between day 7 and day 14 in each group.


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Table I. Adhesion scores 7 and 14 days after surgery
 
Experiment 2
In the approximated uterine horn model on day 7, the scoring was done after gently lysing away loose adhesion at the site on the outside of uterine horn to the fat tissue in front of the bladder area. The HA gel group (n = 14) had a score of 0.9 ± 1.0 that was significantly lower than the control group (n = 11) which had a score 2.6 ± 0.5 (P < 0.0003) (Table IIGo).


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Table II. Effect of HA gel administration 7 days after surgery in the approximated uterine horn model
 
Experiment 3
In this group of 25 mice the mean adhesion score was 1.7 ± 0.9 at re-operation, 7 days after the abrasion. When the third laparotomy was performed 7 days later, the adhesion scores were 1.5 ± 0.9 and 2.2 ± 0.6 for HA gel (n = 13) and control groups (n = 12) respectively. There was a significant difference (P < 0.03) between those scores, but not in the alternation of scores from 7 days to 14 days in the HA gel group or control group (Table IIIGo).


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Table III. Adhesion reformation scores 7 days after adhesiolysis
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many techniques have been used to induce adhesions in animal models and different animals have been used in adhesion experiments (Diamond, 1997Go). In this study outbred mice were used. For induction of adhesions the iodine application technique was employed as published previously (Sawada et al., 1999Go). However, this technique induces a high incidence of adhesions; in order to examine the efficacy in other adhesion models, in experiments 2 and 3 the uterine horns were approximated with sutures as reported by Haney and Doty (Haney and Doty, 1993Go). The apposition of injured surfaces is likely to increase the incidence and intensity of adhesion from the point of adhesion score. Moreover, in animal models like these small mice, it is quite important to use magnification glasses as were used in the current study, or use the microscope for evaluation of the nature of adhesion as reported by Yesildaglar et al. (Yesildaglar et al., 1999Go).

Subsequent to an inflammatory reaction, invasion by polymorphonuclear neutrophils (PMN), proliferation of fibroblasts and peritoneal tissue repair cells and the release of cytokines such as transforming growth factor ß (TGFß) are involved in the development of adhesions (Simpson and Ross, 1972Go; Vipond et al., 1990Go). These factors change drastically in the early stages after injury. Adhesions are completely formed within 3 to 5 days after the peritoneal injury. It has also been reported that it is crucial for the injured surfaces to be separated with a barrier during this period. The experiments in the current study demonstrated no difference in adhesion scores between day 7 and day 14, which suggests that adhesions were largely completed before day 7. These findings underline the importance of isolating the injured areas during the period of adhesiogenesis and the importance of the duration (peritoneal residence) of the adjuvant.

In a recent clinical study where second look laparoscopy was used to assess outcomes, the use of the combined hyaluronic acid, carboxymethylcellulose (CMC) membrane (Seprafilm®) has been reported to be effective in preventing de-novo adhesions after myomectomy (Diamond and The Seprafilm Adhesion Study Group, 1996Go). However, various absorbable adjuvants including oxidized regenerated cellulose and carboxymethylcellulose yield unsatisfactory results in clinical studies, due probably to insufficient concentration of peritoneal residence in CMC (Wurster et al., 1995Go) or not staying at a suitable position on injured surfaces in Interceed®.

HA is an important component of the extracellular matrix of the peritoneal epithelium, and is produced by peritoneal epithelial cells. A number of investigators previously reported the effect of HA solution in adhesion prevention. One of the first of such studies by Urman and Gomel demonstrated that pre-coating of serosal surfaces with HA solution was effective in preventing de-novo adhesions in rats (Urman and Gomel, 1991Go). However, administration of HA at the end of surgery was not effective.

Modified forms of HA, that have a longer intraperitoneal half life, have also been studied. Shushan et al. observed an effect similar to the previous study by Urman and Gomel with the use of HA membrane in the rat model (Shushan et al., 1994Go). Previously a reduction of de-novo adhesions with the use of high molecular HA solution in the mouse uterine model was reported (Sawada et al., 1999Go). Ferric hyaluronate is another gel type of HA. Johns et al. and Thornton et al. reported this gel to be efficacious in preventing adhesions in animal models (Johns et al., 1997Go; Thornton et al., 1998Go). The greater efficacy of these gels compared to HA alone in preventing adhesions is very likely to be due to the longer intra-peritoneal residence time. Ferric hyaluronate gel is believed to have twice the half-life (51 h) of HA (Thornton et al., 1998Go). The photocured cross-linked HA gel used in the current experiments is considered to have a half-life of ~60 h due to low serum concentrations observed 6 days after administration (Matsuda et al., 1993Go).

In one of the rare clinical studies on adhesion reformation Sekiba et al. (Sekiba and Obstetrics and Gynecology Adhesion Prevention Committee, 1992Go) reported a reduction of reformation of adhesions, with the use of oxidized regenerated cellulose (Interceed®), after adhesiolysis for endometriosis. In animal studies, Haney and Doty reported the use of polytetrafluoroethylene (PTFE; Gore-Tex®) a non-absorbable barrier, to be efficacious on mouse uterine adhesion reformation models (Haney and Doty, 1993Go). Rodgers et al. reported that the use of absorbable films consisting of polyethylene glycol/polylactic acid, reduced de-novo adhesion formation and adhesion reformation on rabbit side wall models (Rodgers et al., 1998Go). Haney and Doty reported that use of chemically cross-linked hyaluronic acid (Incerf®), was effective in reducing de-novo adhesions on mouse approximated uterine horn models (Haney and Doty, 1998Go). However, these adjuvants have been much less effective in preventing reformation of adhesions as opposed to the prevention of de-novo adhesions. In contrast to these experiments, the efficacy of high content of sodium carboxymethylcelullose was evaluated for preventing adhesion reformation in rat model (Wurster et al., 1995Go), hyaluronate hydrogel had preventive effects of adhesion reformation in the rat model. The studies presented here in mice have also showed significantly (P < 0.03) lower adhesion reformation scores after adhesiolysis, associated with the use of photocured cross-linked hyaluronate hydrogel compared with no treatment group. But it was not possible to show the efficacy of prevention of adhesion reformation completely because of insufficient reduction of score in HA gel group after application.

In conclusion, although this study has the following limitations: (i) the mice were not inbred; (ii) not strictly randomized; (iii) lysed adhesions were not of the same type, tenacity, vascularity and extent in the reformation model; (iv) intra-reseacher variability, cross-linked hyaluronate hydrogel significantly reduced de-novo adhesions and adhesion reformation (P < 0.03 for both). Although encouraging, these results must be corroborated with further animal and clinical studies.


    Notes
 
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Fujita Health University, School of Medicine, 1–98 Dengakugakubo, Kutsukakecho, Toyoake, Aichi, Japan 470-1192. Back


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 Abstract
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 Results
 Discussion
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Submitted on June 23, 2000; accepted on October 10, 2000.





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