INTRODUCTION
The adhesion of organs and tissues after surgery is a natural phenomenon caused by the proliferation and regeneration of damaged tissue cells. However, excessive or unintended adhesion to other organs and tissues can lead to dysfunction and threaten life [1]. Various drug-based methods have been used to prevent adhesions [2]. Representative anti-adhesive agents include Guardix-sol (hyaluronic acid [HA] and sodium carboxymethyl cellulose [CMC]; Hanmi Pharmaceutical Co., Ltd.) available in a film form, and Interceed (oxidized regenerated cellulose, Johnson & Johnson Medical Ltd.) available in a fabric form [3,4].
Anti-adhesive agents form a physical barrier, known as an adhesion barrier, that blocks contact between the wound and surrounding tissues. This is achieved by wrapping or covering the wound area, which is expected to adhere after surgery [5]. These agents continue to act as physical barriers in the area where adhesion is expected for a certain period, preventing adhesions between adjacent tissues. After a certain period, they should be decomposed or absorbed into the body without leaving any foreign substances in the intra-abdominal cavity [6].
Despite the proven anti-adhesion effects of currently available anti-adhesion agents in previous studies, few products have instilled clinicians with a strong belief in their effectiveness [7–12]. Prior research has suggested that solution-type anti-adhesion agents typically exhibit a consistent anti-adhesion effect between the abdominal wall and organs within the abdominal cavity [13–15]. Unfortunately, there is still a lack of research comparing and evaluating the exact amount of anti-adhesive agents necessary to effectively prevent postoperative adhesions. Because the standardized dose of anti-adhesion agents is typically limited to 10 g, surgeons tend to selectively apply anti-adhesion agents to areas prone to adhesions, such as abdominal wall incisions or around the organs they primarily operate on.
Therefore, this animal study aimed to investigate the distribution of anti-adhesion agents when different doses of anti-adhesion agents are used, and to explore the additional doses required for comprehensive intra-abdominal cavity.
METHODS
Labeling the fluorescent dye Flamma Fluors 552 to the Guardix-sol
The BioActs Corp. was commissioned to perform the synthesis work of attaching Flamma Fluors 552 to Guardix-sol. Guardix-sol is composed of HA and CMC. Flamma was labeled on each, as shown in Supplementary Fig 1. At room temperature, 1 g of each HA and CMC was added to distilled water (DW) and stirred gently. EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) were added to each solution and stirred gently for 30 minutes at room temperature. Flamma 552 amine (50 mg) was added to the DW. The resulting Flamma 552 amine solution (50 mg) was added to HA (1 g)+EDC+NHS and CMC (1 g)+EDC+NHS solutions and stirred for 24 hours at room temperature. Each HA and CMC reaction solution was added to the dialysis membrane (size: 3.5 kDa). Before dialysis, thin-layer chromatography (TLC) was performed to confirm that the Flamma 552 amine compound was labeled with HA and CMC (Supplementary Fig. 2). Dialysis was performed for 7 days to remove the unresponsive dye, EDC, NHS, and other impurities. During dialysis, the composition of the dialysis solution was changed in the order of ethanol 3: DW1 to ethanol 1: DW1 to DW. After dialysis, TLC was performed, and the fluorescence signal of each compound was confirmed using a Fluorescence In Vivo Imaging System (FOBI) at 520 nm, as shown in Supplementary Fig. 3. The intensities of Flamma 552, HA-Flamma 522, and CMC-Flamma 552 were confirmed using ultraviolet (UV) spectrometry (Supplementary Fig. 4).
Animals and the study design
Two domestic male pigs (G1 and G2), each weighing approximately 10 kg, were used in this experiment to avoid selection bias. Each pig was administered a different amount of anti-adhesive solution: 15 mL of Guardix-sol labeled with a fluorescent dye (Guardix-Flamma) in G1 and 35 mL of the same solution in G2. Pigs were selected for this study because they have a similar metabolic process and organ structure to the human body and are simple to experiment on with laparoscopy and follow-up. In addition, pigs were chosen because numerous studies had previously used them, making it easy to interpret and evaluate experimental results using accumulated data. The number of pigs was determined in consideration of the minimum animal sacrifice and research funding limits. After obtaining the pigs, general symptoms were observed during a 7-day quarantine and adaptation period to check their health conditions; only healthy animals were used in the experiments. During the adaptation and experimental periods, each pig was housed in a stainless-steel breeding box (W 1,200×L 1,700×H 1,700 mm) in the 1st Animal Raising Zone of KNOTUS Co., Ltd. The animal facility was maintained at a temperature of 23±3 °C, relative humidity of 55%±15%, a ventilation rate of 10–20 times/hr, and 12 hours of lighting time (8:00 AM to 8:00 PM) with illumination ranging from 150 to 300 lx. Formulated feed mixture for pigs and water were provided all day through feeding machines and an automatic water supply system, allowing the pigs access and the ability to consume freely.
The Institutional Animal Care and Use Committee of KNOTUS (KNOTUS IACUC 21-KE-657) approved the study in terms of its potential scientific contributions and ethics in animal protection. All experiments were performed in accordance with relevant guidelines and regulations. The study is reported in accordance with Animal Research: Reporting of In Vivo Experiments guidelines.
Procedures
Experimental day 1: application of an anti-adhesive agent into the intra-abdominal cavity of pigs through a laparoscopic procedure
The pigs fasted for 24 hours before surgery but had free access to water. Alfaxalone (2 mg/kg) was injected intravenously for induction, and endotracheal intubation was performed. Anesthesia was maintained by the inhalation of 100% oxygen and isoflurane (2%–3% effect) until the end of the surgical procedure. Hartmann’s solution (5 mL/kg/hr) was infused intravenously during the operation after a bolus injection of a prophylactic antibiotic (cefazoline, 22 mg/kg) and analgesic (tramadol, 2 mg/kg).
After general anesthesia, the abdomen of the pigs was scrubbed with betadine and isolated with drapes for aseptic procedures. Three trocars were inserted: a 12 mm camera trocar at the umbilicus and two 5 mm assistant trocars in the right upper and right lower quadrant areas. The intra-abdominal cavity was divided into six areas, and photos and videos were taken several times in laparoscopic general RGB (red, green, blue) color mode. Fifteen milliliters of Guardix-Flamma were evenly applied in the intraperitoneal space of G1, while 35 mL of the same solution were similarly applied in G2 (Fig. 1). Six areas of the intra-abdominal cavity were photographed and recorded in near-infrared (NIR) mode in the same order. The trocar sites were closed using simple sutures. After the pigs fully recovered from anesthesia, they were exercised for more than 12 hours to ensure that the anti-adhesion agents were evenly distributed in the intra-abdominal cavity.
Experimental day 2: verification of the distribution of the anti-adhesive agent in the abdominal cavity through fluorescence and euthanasia to estimate the area of the intra-abdominal cavity
After 24 hours from the first experiment, the same procedure and order were followed, including general anesthesia, scrubbing and draping of the abdomen, and the insertion of three trocars. The photos and videos of the six areas in the intra-abdominal cavity were taken in the NIR mode, following the normal laparoscopic RGB color mode, in the same order as the previous day. The pigs were euthanized by bloodletting through an incision in the internal jugular vein while under general anesthesia as they needed to be dissected to measure the area of the abdominal cavity and organs (Fig. 2).
To calculate the surface area of the parietal peritoneum, the longest longitudinal and transverse lengths of the abdominal wall were measured. For the visceral peritoneum, the longest longitudinal and transverse lengths of the liver (anterior and basal), stomach, spleen, and bladder were measured. The duodenum, small intestine, large intestine, and rectum were dissected, arranged in a rectangular shape on the floor, and their lengths were measured. Finally, the area of each organ was measured by applying the formula for calculating the area of an ellipse, triangle, or rectangle to similarly shaped organs (Fig. 3). Parts of the omentum and the small and large intestine specimens were stored in an icebox and sent to the laboratory for immunofluorescence analysis.
Immunofluorescence analysis
Frozen sections were prepared from parts of the omentum, small intestine, and large intestine for immunofluorescence analysis. Three slides were prepared for each tissue. After staining with 4′, 6-diamidino-2-phenylindole (DAPI) for Guardix-Flamma, each slide was analyzed using confocal microscopy (LSM 900; Carl Zeiss) (Fig. 4). The thickness of the Guardix-Flamma was measured at three different sites on each slide, and the average was calculated and compared between two pigs.
Statistics analysis
The results were recorded at each time point during the experiment. The normality of the data for this experiment was determined, and the significance between the test groups was assessed using the Student t-test. Prism 7.04 (GraphPad Software Inc.) was used for the statistical analysis. The results were considered statistically significant if the P-value was less than 0.05.
RESULTS
The experiment was successfully performed on two pigs without intraoperative or immediate postoperative mortality.
Distribution of anti-adhesive agents in the abdominal cavity through fluorescence
Fig. 1 shows that no abnormal findings were observed in either pig during the laparoscopic inspection on the first day of the experiment. The Guardix-Flamma was evenly applied throughout the abdominal cavity to ensure no areas were missed. In RGB mode, the Guardix-Flamma compound appeared pink. In the NIR mode, the fluorescence signal of the Guardix-Flamma compound was green. Guardix-Flamma, which appeared pink in the RGB mode, was not identified in either pig on the second day of the experiment (Fig. 2). However, a green fluorescence signal was detected in NIR mode. Fluorescence was observed in the greater omentum and ascites in the pelvis and right lower quadrant in G1, whereas fluorescence was detected throughout the area in G2.
Estimate the area of the intra-abdominal cavity
The total estimated area of the abdominal cavity in G2 was 1.30 times larger than that in G1 (Fig. 3). In G1, the total calculated abdominal cavity area was 2,399 cm2. Specifically, the surface area of the anterior abdominal wall was 439.8 cm2. The posterior abdominal wall was determined by adding up the calculated areas of the anterior surfaces of each organ in the abdominal cavity: stomach 125.7 cm2, liver anterior 117.8 cm2, liver base 126 cm2, spleen 42.4 cm2, bladder 27.5 cm2, and small and large intestines 1,520 cm2. When the same calculation was applied to G2, the total area of the abdominal cavity was estimated to be 3,110 cm2. Specifically, the surface area of the anterior abdominal wall was 523.1 cm2, the anterior surface of the liver was 76.5 cm2, and the basement of the liver was 66.5 cm2. The anterior surfaces of the stomach, spleen, bladder, and small and large intestines were 129.6 cm2, 59.7 cm2, 18.9 cm2, and 2,236 cm2, respectively.
Immunofluorescence analysis
Fig. 4 shows that the Guardix-Flamma compound stained with DAPI appeared red under confocal light at 500 nm. The average thickness of the greater omentum and small and large intestines in G1 and G2 were measured as 10.82±2.41, 2.99±0.24, 24.24±5.78, and 14.13±3.00, 4.34±1.02, 36.00±13.06, respectively (Table 1, Fig. 4). The thickness of the Guardix-Flamma applied to the omentum and small and large intestines of G2 were 1.31-, 1.45-, 1.49-times thicker than G1, respectively, and all were statistically significant (P<0.05) (Fig. 5). The total thickness of G1 and G2 were measured at 12.68±9.80 and 18.16±15.57, respectively (Table 1).
DISCUSSION
Anti-adhesive products are available in different commercialized packaging units, ranging from 1 to 10 g for each product. Guardix-sol, the product used in this study, is available in three different packaging units (1.5 g, 5 g, and 10 g per syringe). Using an anti-adhesive agent at the end of surgery usually depends on the surgeon’s preference or experience and the specific surgical field. Generally, 1.5 g or 5 g of the anti-adhesive agent is considered sufficient for surgeries with a narrow field or local applications such as thyroidectomy [16], vascular surgery, appendectomy, or cholecystectomy. However, in major intra-abdominal surgeries, such as gastrectomy [17], liver lobectomy, pancreatectomy, and colectomy [15], surgeons typically choose the maximum amount of anti-adhesion agent on the package, 10 g, and expect it to be applied as evenly as possible throughout the abdominal cavity. Nevertheless, research on the optimal amount of anti-adhesive agent required to prevent adhesion in the entire abdominal cavity is lacking. This animal study aimed to estimate the amount of anti-adhesive agent needed to cover the entire intra-abdominal cavity in pigs.
Anti-adhesive agents are available in the market in the form of solution, hydrogel, powder, spray, and film [18–23]. The Guardix used in this study was a viscous liquid [13–17]. Rather than comparing which type distributes more effectively, this study focused on a specific anti-adhesive agent, Guardix, which is widely used in Korea, and considered how it might be better distributed within the abdominal cavity. It is believed that in actual clinical practice, the anti-adhesive agent applied during surgery can be distributed evenly in the abdominal cavity owing to the relatively high intra-abdominal temperature and early ambulation after surgery. Therefore, experimental pigs were exercised after surgery to induce an even distribution of Guardix in the intra-abdominal cavity. However, most anti-adhesive agents are transparent, making it challenging to evaluate their distribution. Therefore, we labeled the Guardix fluorescence as shown in Supplementary Figs. 1–4.
Fluorescence labeling of biomolecules with fluorescent dyes enables the visualization of small amounts of labeled molecules in a complex mixture of biomolecules in the spectral range from UV to NIR wavelengths [24–28]. Flamma Fluor dyes, used in this study, maintain good fluorescence activity and stability after conjugation to biomolecules and allow the detection of low-abundance biological structures with great sensitivity. In this study, Flamma Fluors 552 was successfully labeled with HA and CMC, as shown in Supplementary Fig. 1, and the distribution of Guardix-Flamma was confirmed in the NIR mode. As shown in Fig. 2, in G1, using 15 mL of Guardix-Flamma, Guardix-Flamma was weakly visible in some parts. However, in G2, using 35 mL of Guardix-Flamma, fluorescence expression was confirmed in almost all areas of the abdominal cavity. This result may indicate that 15 mL of Guardix is insufficient to evenly cover the entire abdominal cavity of a 10 kg pig with an approximately 2,755 cm2 area, whereas 35 mL of Guardix can cover the entire area. The study results suggest that at least 100 mL of Guardix are needed to cover the abdominal cavity (17,103±0.15 cm2, unpublished data measured by 3-dimentional computed tomography) of a 64-kg human, considering that 15 mL of Guardix barely covered the abdominal cavity (2,755 cm2) of a 10-kg pig. However, further safety studies are needed to determine the amount of Guardix in the intra-abdominal cavity.
Anti-adhesive agents must be maintained in the abdominal cavity for a certain period to create a physical barrier that prevents adhesion between adjacent tissues and organs. Simultaneously, they should decompose or be absorbed into the body without causing harm because they are foreign substances [29,30]. Fig. 3 shows the thickness of the Guardix-Flamma measured using immunohistochemical staining. In both pigs, the thickness of the Guardix-Flamma increased in the following order: the small intestine, greater omentum, and large intestine. Interesting is that the thickness was over 10 times greater in the large intestine than the small intestine (Table 1). This is probably due to the fact that the small intestine has thicker bowel walls, a smoother surface, and more active peristalsis movement compared to the large intestine, so there is a high possibility that the anti-adhesive agent may not stay where it was applied. On the other hand, the large intestine has a larger diameter and surface area, and slower peristalsis compared to the small intestine, and structures such as tenia coli and hastra form folds on the surface, which can cause anti-adhesive agent to stagnate in these areas. Comparing the two pigs, the average thickness of the Guardix-Flamma applied to G2 was approximately 1.5 times thicker than that of G1, and the difference was statistically significant. This indicates that even if some of the applied Guardix is absorbed in the body, a thicker application is achieved in larger amounts. In this study, the minimum thickness of Guardix needed to create a physical barrier for a certain period without causing harm to the human body unfortunately not determined due to lack of research funding, thus, this should be evaluated in future studies.
This study has few limitations. The experiment was performed on only two pigs because of funding limitations. Although this small sample size is a fundamental limitation of this study, our study clearly shows that a standardized 10 mL of Guardix is insufficient to cover an adult human’s entire abdominal cavity. Another limitation is the rough measurement of the surface area of the intra-abdominal cavity, as there is no formula for accurately calculating the area of the abdominal cavity. Additionally, there may have been a loss of the Guardix because of manipulation by the experimenters while dissecting the pig to obtain the area of the visual peritoneum. Therefore, there is a possibility that the thickness of the Guardix was underestimated in this study. Nevertheless, the study’s strength is that it is the first to investigate whether 10 mL of Guardix, routinely used in actual clinical practice, is sufficient to apply to the entire abdominal cavity. Additionally, it is the first study to use fluorescence to overcome the experimental difficulty of Guardix being invisible to the naked eye.
In conclusion, 15 mL of Guardix was insufficient to evenly cover the entire abdominal cavity of a 10 kg pig with approximately 2,755 cm2 of surface area. Although 35 mL of Guardix is sufficient to cover the same area, with an average thickness of 18 μm, the minimum thickness required for the effective anti-adhesive function must be further evaluated in future studies.
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