The thermosensitive poly (organophosphazene) hydrogel have a polymer backbone containing hydrophobic isoleucine ethyl esters (IleOEt), hydrophilic α-amino-ω-methoxy-polyethylene glycol (AMPEG) and hydrolysis-sensitive glycyl lactate ethyl ester (GlyLacOEt) as substitutes. These substitutes are related to controllable hydrogel characteristics such as gelation temperature, gel strength, and biodegradability. In this study, [NP(IleOEt)1.46(GlyGlyCOOH)0.06(AMPEG750)1.08]n was synthesized according to published methods with permission.(14) An aqueous polymer solution was prepared, dissolving 2 g of the polymer in Phosphate buffered saline (PBS, 0.01 M, pH 7.4) by continuous stirring at 4℃ for 3 days. The polymer solution (8 wt.%) was filtered using Millipore filters (Millipore, Madison, WI, USA, diameter 0.45µm) and sterilized with ultraviolet radiation over 6 hours. The solution was stored in sterile 1 ml syringes at 4℃. Guardix-sol®, which is a mixed solution of 0.25% (w/v) hyaluronate and 0.49% (w/v) carboxymethyl cellulose, was purchased from Hanmi Inc. (Seoul, Korea). Stock 10 ml solutions in a 10 ml Luer lock syringe were divided and stored in sterile 1 ml syringes. Twelve 2×5 cm sized pieces of Interceed® were prepared as the positive control from two purchased sterile 7.6×10.2 cm-sized sheets from Johnson & Johnson (Langhorne, PA, USA). PBS (0.01 M, pH 7.4) was used as the negative control.

Twenty-four male Sprague Dawley rats (310~410 g; Orient Bio Inc.) were used to induce adhesions. They were individually housed at a constant room temperature and humidity, with 12:12 h light and dark cycles, and were provided food and water ad libitum throughout. All animal experiments were performed at the Department of the Laboratory for Experimental Animal Research in the Clinical Research Institute at Seoul National University Hospital (AAALAC International accredited facility). The approval of the Institutional Animal Care and Use Committee (IACUC) was received, and National Research Council (NRC) guidelines for the care and use of laboratory animals were observed (revised 1996).

The ischemic button model using rats was chosen for comparison of anti-adhesive effect. In the ischemic button model using rats, eight buttons were created in one rat and adhesions on the button were analyzed, which makes this model require smaller number of rats. Moreover it is a standardized qualitative and quantitative model and has been shown to provide reproducible results and constant adhesion formation.(16,17) Reed et al.(17) reported that in control group with normal saline, the 72.2 (±4.7) to 82.0% ischemic buttons of Wister rats formed adhesions attached to various structures in the peritoneum including liver and small bowel after 1 week.

Peritoneal adhesions were created in rats as described. (16,17) After isoflurane inhalation anesthesia, each rat's weight was checked, and the abdomen was shaved and swabbed with 70% alcohol. A laparotomy was performed through a 5 cm midline incision. Four ischemic buttons, spaced 1 cm apart along the paracolic gutter, were made on both sides of the parietal peritoneum by grasping 5 mm of peritoneum with a hemostat and ligating the base of the segment with a 4-0 silk suture (Fig. 1A). Twenty-four rats were randomized to four experimental groups managed with 1 ml of the aqueous polymer solution (the Gel group, n=6), 1 ml of Guardix-sol® (the Gd group, n=6), two 5×2 cm pieces of Interceed® (the IC group, n=6), or 1 ml of PBS (the control group, n=6). Then, the agents in a syringe were applied on all ischemic buttons or the membrane was applied to cover all ischemic buttons on both sides (Fig. 1B). The laparotomy incision was closed in one layer with 3-0 coated Vicryl^*Plus® (Ethicon, Sommerville, NJ, USA) suture.

After two or four weeks, three rats in each group underwent a secondary laparotomy. The ischemic buttons were inspected for the presence or absence of adhesions to evaluate antiadhesive efficacy using the following measurements (Fig. 2)(18):

(1) The general overall health of the tested animals indicated by the percentage of their gained weight relative to basal weight.

(2) The extent of adhesions recorded as the number of the adhered buttons in a rat.

(3) The incidence of adhesion recorded as the existence of attached adhesion to each button in a group.

Two rats in the IC group were excluded because one rat had intraoperative mesenteric bleeding and required meticulous hemostasis and evacuation of hematoma, and the other died six days after initial surgery, and autopsy was not possible. Therefore, the number of adhered ischemic buttons was analyzed in the remaining 22 rats.

To analyze the histology of adhesion, peritoneal tissue from representative areas was obtained from one rat in each group under sterile conditions. It was fixed in buffered formalin (4%), embedded in paraffin, cut into 4 to 5µm sections and stained with hematoxylin and eosin under standardized conditions. Masson trichrome staining was used to demonstrate tissue fibrosis. All slides were analyzed using light microscopy (Olympus BX45, Olympus, Germany). Histological features including adipose tissue, collagen, fibrosis, and vascularization were analyzed by a pathologist.

All variables are expressed as the means with standard deviation. Differences among the four groups were evaluated by Kruskal-Wallis variance analysis, followed by a post hoc Mann-Whitney nonparametric U-test. Fisher's exact test was used to assess any differences in incidence. P<0.05 was considered statistically significant. All data were processed using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA).

The mean basal weight of 22 rats was 349±24 g (range 312~410) and there was no difference between 4 groups (P=0.402). The mean percentages of gained weight in each group at two and four weeks were showed in Table 1 (Gel vs. IC, P=0.083; Gel vs. Gd, P=0.050, respectively).

The mean number of buttons with adhesion in a rat in each group was showed in Table 2, and there was no significant difference between four groups at any time (P=0.084 after 2 weeks and P=0.197 after 4 weeks). When comparing between the mean number of adhered button in a rat after 2 weeks and that after 4 weeks, there was no difference in each groups. After the rats at two period were put together, the extent of adhesion in the IC group was significantly smaller than that in other groups (P=0.004). In addition, the Gel group also showed fewer adhesions than the Gel groups and the control group, although it was not statistically significant (P=0.074, and P=0.310, respectively).

The overall numbers of adhered ischemic buttons were significantly different between groups at two weeks (P=0.001) and at four weeks (P=0.003). After the rats at two period were put together, overall number of adhered ischemic buttons were 23 in the control group (n=48), 22 in the Gd group (n=48), 14 in the Gel group (n=48) and 0 in the IC group (n=32) and those of each group were significantly different (P<0.001). That of Gel group was significantly bigger than of the IC group (P=0.001) and it was smaller than the control group and the Gd group, although not statistically significant (P=0.093 and P=0.140, respectively). And that of Gd group was bigger than that of IC group (P<0.001) and not significantly different from that of the control group (P=1.000).

Characteristic acute and chronic inflammatory reactions caused by ischemia were observed around the sutures. There were dense infiltrations with mixed granulocytic and lymphoplasmocytic inflammatory cells with numerous multinucleated giant cells. However in the adhered tissue, there was no sign of acute inflammation, only slight lymphocytic infiltration without granulocytes or macrophages. Most of the adhesions examined consisted of varying amount of adipose tissue and tortuous vascularized bands of collagen. Loosely arranged collagen fibers of heterogeneous size were often found adjacent to regions of dense fibrotic tissue within the same adhesion (Fig. 3). There was no significant difference in each group at two and four weeks.