Intra-abdominal adhesions are a common complication following abdominal surgery, characterized by the structural adhesion of organs due to fibrin formation at the surgical site (4,24,27). Studies have shown that 90-95% of patients develop intra-abdominal adhesions after such procedures (17). While fibrin deposition is part of the normal healing process, an imbalance favoring fibrin formation over fibrinolysis can lead to an inflammatory response and the development of adhesions (24,30). These postoperative adhesions can result in further complications, including chronic abdominal pain, intestinal obstruction, infertility, and intestinal perforation, sometimes necessitating additional surgery (26).

Various strategies are being explored to prevent adhesions following abdominal surgery. Adhesions can be minimized by reducing tissue damage through microsurgical techniques, inhibiting inflammation during wound healing, or creating a physical barrier between peritoneal surfaces. Among these approaches, the intraperitoneal application of solution-based materials has been widely employed to prevent postoperative adhesions (8,23). Numerous materials have been studied for this purpose, including carboxymethyl cellulose, carboxymethyl chitosan, hyaluronic acid, and dextran (7,12,14,25,29). Additionally, products like Seprafilm^® and Interceed^®, which are absorbable membranes made from these components, are commonly used in clinical practice (9,15,16).

Hyaluronic acid is well-known for its effectiveness in preventing abdominal adhesions, and it is available in various forms depending on the manufacturing process (5,21,28). The viscosity and absorption rate of hyaluronic acid in the body can vary depending on the type of bonding, which affects its duration of action. To extend the short duration of standard hyaluronic acid, it can be modified into a high-molecular material by cross-linking the hyaluronic acid molecules, thereby enhancing its intraperitoneal retention. This modification delays absorption and maintains the coating effect at the adhesion site for a longer period, increasing the anti-adhesion effect (11). Additionally, it is known that even when cross-linked, the monophasic form has a higher volume augmentation due to its swelling capacity compared to the biphasic form (18,20).

This study, therefore, aimed to compare the effects of intraperitoneal hyaluronic acid and monophasic cross-linked hyaluronic acid on adhesions caused by scraped ileal serosa and parietal peritoneal wounds.

Experimental design

Eighteen 7-week-old Sprague-Dawley rats (weighing 240-290 g) were used in the experiment. The rats were housed in three polycarbonate cages with daily access to water and solid food, while environmental factors such as temperature, humidity, and light were controlled. After a 7-day acclimation period, the rats were fasted overnight before the surgery. Adhesions between the ileum and the abdominal wall were induced in all rats under anesthesia. The rats were divided into three groups of six: a non-treated control group, a HA group that received 1% intraperitoneal hyaluronic acid (HA) (Araganplus injection^TM, Dongkwang Pharm, Korea), and a MCHA group that received 1% intraperitoneal monophasic cross-linked hyaluronic acid (MCHA) (BIDAN Volume^TM, Aribio, Korea). The difference between HA and MCHA is that HA is made by simply combining hyaluronic acid with distilled water, whereas MCHA is synthesized through a crosslinking process of hyaluronic acid to enhance biocompatibility, improve viscoelasticity, and slow down the absorption rate in the body. This study received ethical approval from the Kyungpook National University Ethical Committee (approval number: 2021-0221).

Surgical procedure

For anesthesia, ketamine hydrochloride (40 mg/kg, YUHAN Ketamine 50 Injection^TM, YUHAN, Korea) and xylazine (10 mg/kg, Rompun^TM, Bayer HealthCare, Germany) were administered intraperitoneally. The surgical site was disinfected with 70% alcohol followed by 10% povidone iodine. A midline abdominal incision approximately 2 cm long was made from the xiphoid process to the umbilicus, and the abdominal wall was opened to perform a laparotomy. The ileum and ileocecal junction were then exteriorized. The serosa of the ileum near the ileocecal junction was gently scraped over an area of about 1 × 0.5 cm with a sterile No. 15 scalpel blade until petechial bleeding was observed. A similar-sized area of the parietal peritoneum was excised using Metzenbaum scissors. The edges of the excised parietal peritoneum and the scraped ileal serosal surface were sutured together using 6-0 prolene (PROLENE^TM, Ethicon, USA). The abdominal wall was closed with a simple continuous suture using 4-0 vicryl (Vicryl^TM, Ethicon, USA), and immediately before the final suture, 2 mL of 0.9% saline for control group, 1% hyaluronic acid for HA group, or 1% monophasic cross-linked hyaluronic acid for MCHA group was injected intraperitoneally. The skin was closed with 4-0 nylon (Blue Nylon^TM, Ailee Co, Korea), and the surgical site was sterilized with 10% povidone iodine. Postoperative care included daily disinfection with 10% povidone iodine for 7 days.

Preparations of samples

After general anesthesia and euthanasia, tissue samples were collected from the abdominal wall and ileum at the sites where adhesions had formed, measuring approximately 3 cm in length and 2 cm in width. The collected tissue was fixed in 10% neutral buffered formalin for histological analysis. The samples were then processed and embedded in paraffin using an automated tissue processor (Shandon Citadel 2000^TM, Thermo Scientific, Waltham, MA, USA) and embedding center (Shandon Histostar^TM, Thermo Scientific, Waltham, MA, USA). Serial sections, 3-4 μm thick, were prepared from each paraffin block using an automated microtome (RM2255^TM, Leica Biosystems, Nussloch, Germany). The sections were stained with Hematoxylin and Eosin (H&E) for general histological evaluation and with Masson’s Trichrome (MT) for collagen fiber analysis.

Histological evaluations

The histological profiles of individual cross sectioned sample were observed under a light microscope (Model Eclipse 80i, Nikon, Tokyo, Japan), equipped with a camera system (ProgRes^TM C5, Jenoptik Optical Systems GmbH, Jena, Germany) and a computer-assisted automated image analyzer (iSolution FL ver 9.1, IMT i-solution Inc., Bernaby, BC, Canada), with the analysis conducted in a blinded manner to the group distribution.

The thickness of the adhesion layers (μm), the number of inflammatory cells in the adhesion layers (cells/mm²), and the percentage of collagen fiber-occupied regions in the adhesion layers (%/mm²) were measured using a computer-assisted image analysis program. Additionally, the slides were graded for histological fibrosis (collagen deposition) and inflammation (extent of inflammatory cell infiltration) using a scoring system previously described by other researchers (1,3). This system includes four severity scores for fibrosis and inflammation, ranging from 0 to 3: no fibrosis or inflammation (grade 0); minimal and loose fibrosis, with giant cells, occasional scattered lymphocytes, and plasma cells (grade 1); moderate fibrosis, with giant cells and an increased number of admixed lymphocytes, plasma cells, eosinophils, and neutrophils (grade 2); and florid and dense fibrosis, with numerous admixed inflammatory cells and micro-abscesses (grade 3) (1,3).

Statistical analysis

All histomorphometrical values were expressed as mean ± standard deviation (SD). If the Levene test indicated no significant deviations from variance homogeneity, the data were analyzed by one way ANOVA test followed by Tukey’s Honest Significant Difference (THSD) test to determine which pairs of group comparison were significantly different. When a significant difference is observed in the Kruskal-Wallis H test, the Mann-Whitney U (MW) test was conducted to determine the specific pairs of group comparison, which are significantly different. Statistical analyses were conducted using SPSS for Windows (IBM SPSS Inc., Armonk, NY, USA). Differences were considered significant at p < 0.01.

To evaluate the degree of inflammation and fibrosis in the adhesion area, histological assessment was performed using H&E staining and MT staining. Histological examination revealed that the thickness between the abdominal wall and intestinal mucosa at adhesion sites was 1,591.25 ± 263.10 μm in the control group, which was the greatest compared to the other two groups: 989.12 ± 163.46 μm in the HA group and 716.83 ± 94.01 μm in the MCHA group. In HA and MCHA groups, the thickness at adhesion sites was significantly reduced compared to the control group. (p < 0.01, Fig. 1).

Figure 1. The thickness of adhesion formed between the ileal serosa and the parietal peritoneum on day 10 after operation (A) and histological images by Hematoxylin-eosin staining (B). The thickness of HA group and MCHA group significantly decreased compared to that of control group. *p < 0.01 compared with the control. HA, hyaluronic acid; MCHA, monophasic cross-linked hyaluronic acid; AW, abdominal wall; SI, small intestine; LU, lumen. Two-sided arrow: the thickness of adhesion. Scale bar: 160 μm.

The infiltration of inflammatory cells was 341.67 ± 115.69 cells/mm² in the control group, compared to 175.00 ± 29.14 cells/mm² in the HA group and 99.67 ± 17.22 cells/mm² in the MCHA group. Both treatment groups showed a significant decrease in inflammatory cell infiltration compared to the control group. (p < 0.01, Fig. 2).

Figure 2. The inflammatory cell infiltration of adhesion on day 10 after operation (A) and histological images by Hematoxylin-eosin staining (B). The inflammatory cell infiltration of HA group and MCHA group were significantly lower than that of control group. *p < 0.01 compared with the control. **p < 0.01 compared with the HA. HA, hyaluronic acid; MCHA, monophasic cross-linked hyaluronic acid. Scale bar: 160 μm.

The degree of collagen fiber formation at the adhesion site was 71.77 ± 10.26%/mm² in the control group, compared to 50.12 ± 5.41%/mm² in the HA group and 42.83 ± 7.30%/mm² in the MCHA group. Both treatment groups showed a significant decrease compared to the control group (p < 0.01, Fig. 3).

Figure 3. The collagen fiber of adhesion on day 10 after operation (A) and histological images by Masson’s Trichrome staining (B). The collagen fiber of HA group and MCHA group were significantly lower than that of control group. *p < 0.01 compared with the control. HA, hyaluronic acid; MCHA, monophasic cross-linked hyaluronic acid. Scale bar: 160 μm.

The histopathological grades of adhesion sites were 2.90 ± 0.32 in the control group, 2.30 ± 0.48 in the HA group, and 1.00 ± 0.47 in the MCHA group. The HA and MCHA groups showed significant decreases compared to the control group, and the MCHA group showed a significant decrease compared to the HA group (p < 0.01, Fig. 4).

Figure 4. The histopathological score of adhesion on day 10 after operation. The assessment of the adhesion site was conducted by analyzing the amount of adhesion tissue, the infiltration of inflammatory cells, and the presence of collagen fibers. The grade of HA and MCHA groups were significantly lower than that of control group. The grade of MCHA group was significantly lower than that of HA group. *p < 0.01 compared with the control. **p < 0.01 compared with the HA. HA, hyaluronic acid; MCHA, monophasic cross-linked hyaluronic acid.

Abdominal adhesions can arise from an inflammatory response within the abdominal cavity triggered by surgical manipulation, infection, or the presence of a foreign body (4,24). This process involves a complex interplay of biochemical, cellular, and immune factors (30). Although various strategies have been explored to prevent intra-abdominal adhesions following surgery, they remain a significant complication. Adhesion formation can be minimized or prevented by mitigating the inflammatory response, diluting fibrous exudate, or physically separating potential adhesion sites through coating. The anti-adhesion effect has been extensively studied using post-surgical intra-abdominal adhesion models, and this experiment was conducted following established research protocols (2,6,22).

A method to reduce intra-abdominal adhesions involves creating a barrier between adhesion sites. By applying a film or solution, the damaged tissue surface can be physically separated from other structures, preventing adhesion formation. Hyaluronic acid is a commonly used anti-adhesion agent for this purpose (13). However, one limitation of solution-based barriers is that they can be easily washed away due to decomposition and absorption at the injury site, leading to reduced durability. To address these challenges, the development of more effective materials is ongoing, with hydrogels-polymers formed by cross-linking natural hyaluronic acid-emerging as a promising option (10). Hydrogels with a 3-dimensional polymeric structure are notable for their high water absorption capacity, offering advantages such as increased viscosity and prolonged retention within the abdominal cavity (19). In this study, a monophasic cross-linked hyaluronic acid gel was employed as the polymer form of hyaluronic acid. To assess the anti-adhesion effect, hyaluronic acid-recognized for its efficacy-was used as a positive control (HA group), while the experimental group utilized monophasic cross-linked hyaluronic acid (MCHA group). The anti-adhesion effectiveness was evaluated by measuring the thickness of the adhesion site, counting the infiltrating inflammatory cells, and assessing the proportion of collagen fibers following the induction of adhesions in the small intestine. The histopathological grade was determined using the method described by Åkerberg et al. (1,3).

In this study, the adhesion area between the intestinal serosa and the abdominal wall was significantly thicker in the control group compared to the other two groups. Both treatment groups demonstrated an anti-adhesion effect by serving as a physical barrier at the adhesion site. Notably, the adhesion thickness in the MCHA group was lower than that in the HA group, suggesting that MCHA was more effective, likely due to its prolonged presence in the abdominal cavity.

This study found that both inflammatory cell infiltration and collagen fiber formation were significantly reduced in the HA and MCHA groups compared to the control group. The significant decrease in inflammatory cells is likely due to the slow absorption and prolonged retention of cross-linked hyaluronic acid in the abdominal cavity during the experiment. The notable reduction in collagen formation in both groups is believed to result from the prevention of adhesion formation through the dilution of fibrous exudate. In the MCHA group, further prevention of collagen formation is thought to have been achieved by the extended retention of fluid in the abdominal cavity, facilitated by its high absorbency.

Histopathological examination has revealed focal fibrosis and inflammatory cell infiltration around intra-abdominal adhesions, which are graded using specific scoring systems (1,3). In this histological grading system, higher scores indicate greater inflammatory cell infiltration and fibrin deposition. When this system was applied in the present study, the HA group showed significantly lower scores compared to the control group, and the MCHA group exhibited significantly lower scores compared to the HA group. These scoring results suggest that the MCHA group is more effective in preventing abdominal adhesions.

These findings suggest that monophasic cross-linked hyaluronic acid effectively prevents adhesion formation by reducing adhesion thickness, inflammatory cell infiltration, and collagen fiber formation in a rat peritoneal adhesion model. Further research is needed on the intraperitoneal application of an appropriate amount of monophasic cross-linked hyaluronic acid for preventing adhesions.

This study aimed to evaluate the anti-adhesion effect of monophasic cross-linked hyaluronic acid (MCHA) in the abdominal cavity of rats. Histopathological examination consistently demonstrated that the MCHA-treated group exhibited a superior anti-adhesion effect compared to both the negative and positive control groups. The appropriate intraperitoneal administration of MCHA provided effective lubrication, anti-inflammatory, and anti-fibrotic benefits, proving to be more effective at preventing abdominal adhesions than hyaluronic acid.

The authors have no conflicting interests.