The influence of adhesion prophylactic substances and taurolidine/heparin on local recurrence and intraperitoneal tumor growth after laparoscopic-assisted bowel resection of colon carcinoma in a rat model
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- Opitz, I., van der Veen, H., Braumann, C. et al. Surg Endosc (2003) 17: 1098. doi:10.1007/s00464-002-9161-7
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Backgroud: The goal of the study was to investigate the influence of adhesion prophylactic substances (Interceed/lntergel) as well as taurolidine/heparin on intraperitoneal tumor growth and the local recurrence rate after laparoscopic cecum resection in a rat tumor model. Methods: Sixty BDIX rats were randomized in three therapy groups and one control group. A laparoscopic-assisted cecum resection was performed via three-trocar method after intraperitoneal tumor cell application (10,000 cells) of a colon carcinoma cell line (DHD/K1/TRb) in all animals. According to the randomization, the cecum suture and a 1 × 1-cm peritoneal defect were either covered with Intergel/Interceed or 1 ml of 0.5% taurolidine 10 IU heparin. The control group underwent instillation of 1 ml 0.9% NaCl solution. After 4 weeks the animals were euthanized and intraperitoneal tumor growth, local recurrence rate, and the number of intraperitoneal adhesions were determined. Results: The local recurrence rate was not significantly affected by any of the substances. Nevertheless, taurolidine/heparin significantly reduced the total number and weight of intraperitoneal metastases. The formation of adhesions was not significantly influenced by adhesion prophylaxis substances or by taurolidine/heparin. Conclusions: Taurolidine/heparin led to a significant reduction of intraperitoneal tumor growth after intraperitoneal application, whereas local tumor recurrence was not significantly influenced. This might be due to the number of injected tumor cells in this cell suspension model. Interceed and Intergel did not reduce intraperitoneal tumor growth. Furthermore, adhesion formation was not reduced by any of the substances.
KeywordsLaparoscopyIntraperitoneal tumor growthPort-site metastasesTaurolidineAdhesion prophylaxis
Laparoscopic surgery for the resection of malignant tumors is controversial mainly because of the development of port-site metastases. In the literature, the incidence of port-site metastases after laparoscopic surgery of malignomas varies from 0 to 21% . Recent studies have documented much lower incidences of port-site metastase (approximately 1%) . The major difference is related to the experience of the surgical team and an increase in the learning curve of the surgeons performing laparoscopic cancer resection between 1994 and 1999 [8, 9, 25, 29]. Furthermore, port-site metastases have not only been reported for advanced cancer disease but also are described after laparoscopic colorectal resection of early stage carcinomas . Thus, many other factors beside surgical technique and experience have been found to influence tumor growth during laparoscopy: aerolization of tumor cells ; increased intraabdominal pressure ; CO2 [5, 19, 32], helium , or gasless pneumoperitoneum [4, 27, 46]; and local immunosuppression [11, 24, 39, 43]. Nevertheless, the incidence of abdominal wound recurrence after open surgery for colorectal cancer does not seem to be significantly different from that of the laparoscopic approach [13, 34]. If strict oncologic surgical principles (minimal instrumental movement, precise surgical technique, etc.) are followed, no difference between open and laparoscopic surgery could be demonstrated in the number of free viable tumor cells in the abdominal cavity .
In addition to wound metastases, local recurrence of colorectal cancer has been a common problem, with rates of 10–20% for open surgery [12, 14, 28, 33]. Long-term results including the rate of local recurrence after laparoscopic colorectal resection are not available, but rates of 1 or 2% have been published . Prospective randomized trials show only little difference between open and laparoscopic-assisted surgery, with tumor recurrence rates of 15 and 16.1% respectively . Franklin’s group showed no evidence of recurrence in the laparoscopic group (n = 84) compared with 3 patients of the groups of patients undergoing conventional procedures (n = 110) . However, mechanisms underlying the formation of local recurrence and intraperitoneal tumor growth after curative resection of colorectal cancer are poorly understood. Incomplete resection and inappropriate operative technique are considered the main causes. Malignant cells giving rise to local recurrence or intraperitoneal metastases may, in theory, derive from several sources. The hypothesis of tumor cell implantation by exfoliated cancer cells has been proven in different experimental studies. Their metastatic potential has been documented by the capacity of these cells to grow in vivo and in vitro [7, 38, 44].
Standardized therapeutic interventions to prevent tumor metastases in laparoscopic surgery have not been established in clinical practice. Nevertheless, different cytotoxic agents have been investigated and show a reduction of intraperitoneal metastases or local recurrence after tumor resection [18, 49]. Substances used for adhesion prophylaxis in abdominal and fertility surgery, such as hyaloronic acid [21, 22, 42] and oxygenized cellulose, have not been considered. Because the tumor cells undergo a complex cascade of single steps during the development of metastases beginning with adhesion of the tumor cells, theoretically there could be a therapeutic approach for the prevention of intraperitoneal tumor growth. Another substance that has been shown to be effective against the development of intraperitoneal metastases is taurolidine, a derivate of the amino acid taurine . Its combination with heparin resulted in a synergistic effect on tumor growth suppression .
This study investigated the influence of the adhesion prophylaxis substances Intergel (hyaloronic acid) and Interceed (oxygenized cellulose) as well as taurolidine/heparin on intraperitoneal tumor growth after laparoscopic-assisted cecal resection in a standardized rat tumor model.
Materials and methods
A total of 60 male rats, type BD IX, weighing 250–300 g (Charles River Institute, Sulzfeld, Germany) were acclimated to a climate- and light cycle-controlled environment for at least 7 days prior to operation. They were allowed standard laboratory food and water ad libitum. The study was performed under protocols approved by the local Committee for Animal Use and Care.
Colon adenocarcinoma cells (DHD/K12/TRb) (European Collection of Cell Cultures, Salisbury, UK) were cultured in Dulbecco’s MEM (Biochrom, Berlin) and Hams F10 medium (Biochrom) 1:1 supplement with 10% fetal bovine serum (Gibco BRL, Eggenstein, Germany), 2 mmol/L glutamine (Biochrom), and 1000 IU/ml penicillin–streptomycin (Gibco BRL). Cells were removed from the plates with 0.25% trypsin/0.02% EDTA (Biochrom), washed twice with PBS (Charité, Berlin), suspended in medium, centrifuged at 1000 rpm for 10 min, and resuspended in medium to the desired concentration.
Laparoscopic-assisted cecum resection
Sixty rats were randomized into one control group and three therapy groups (Table 1). The animals were operated during a deep barbiturate narcosis (40 mg/kg body weight) while remaining to spontaneous breath. At the beginning of the operation, an intraperitoneal tumor cell application with 1 × 104 colon carcinoma cells in 1.0 ml culture medium was performed in order to simulate intraoperative tumor cell seeding.
Randomization of rats in three therapy groups and one control group (n = 60)
Laparoscopic-assisted cecal resection (each group, n = 15)
Control group with instillation of 0.9% NaCl
1 × 1-cm Interceed application
1-ml Intergel application
1-ml 0.5% taurolidine/10 IU heparin instillation
The laparoscopic-assisted cecal resection was carried out with a three port-site method. After successful induction of anesthesia, the rats were placed in the Trendelenburg position and the peritoneal cavity was insufflated with carbon dioxide through a 3.5-mm trocar placed in the middle of the lower abdomen. A pneumoperitoneum with a pressure of 8 mmHg was established. After insertion of the camera, a second and third port (both approximately 3.5 mm) were then created in the right and left lower quadrant to allow introduction of laparoscopic forceps and bipolar instruments into the peritoneal cavity. The abdomen was laparoscopically explored for the terminal ileum and cecum, which are usually located in the left lower quadrant. The cecum, which extends 1 cm beyond the ileocecal valve, was grasped with forceps. There is no mesentery, nor are there ligamentous attachments tethering the cecum; however, an artery and vein course longitudinally along the cecum to its end. These vessels were coagulated with bipolar forceps and divided. After mobilization of the cecum and dissection of the vessels, the cecum was exteriorized through the operative port incision in the left lower quadrant, which was widened in order to carefully mobilize the cecum. Extracorporally, the cecum was resected. Before the cecal stump was returned to the peritoneal cavity, the bowel was sutured in two layers. Furthermore, a 1 × 1-cm parietal peritoneum defect zone was created in the upper left abdomen. According to the randomization, the bowel suture and the peritoneal defect were covered with 1 ml Intergel, Interceed (1 × 1 cm), or 1 ml of 0.5% taurolidine solution with 10 IU heparin applied directly. The control group underwent instillation of 1 ml 0.9% NaCl at the localizations mentioned previously.
The rats were euthanized 4 weeks after the operation. After opening of the abdomen via laparotomy from the xyphoid to the mons pubis, the adhesion score of Moreno  was determined with number, site, thickness, tenacity, and vascularization of the intraabdominal adhesions. Afterwards, all metastases were resected and their number and total weight were determined. The obduction was performed by two assessors and documentation by a third person; all investigators were blinded regarding the treatment of the animals that were examined. Furthermore, the resection line, port-site scars, and the former peritoneal defect were resected and histologically examined.
Data are given as mean ± SD for normal distribution and as median and 95% confidence interval for abnormal distributed values. For abnormal distributed values, significance was tested using Kruskal–Wallis and Mann–Whitney U-test for continuous data and the chi-square test for categorical data. p values less than 0.05 were considered to be significant.
None of the 60 rats died during the observation period. The mean operation time was 21 ± 6 min.
Local recurrence and intraperitoneal and distant metastases
The incidence of local recurrence at the bowel suture line did not differ in all groups. Although Intergel (n = 14) and taurolidine/heparin (n = 11) had almost the same percentage of local recurrence as the control group (n = 13), only 7 rats in the Interceed group developed local recurrence. Nevertheless, this was not significant (Table 2). Intraperitoneal tumor growth was significantly reduced by the application of taurolidine/heparin. The total number of metastases was significantly (p < 0.001) lower in this group (4.3 ± 3.6) when compared to the control group (10 ± 5.8) (Fig. 1). Taurolidine/heparin also significantly (p = 0.09) decreased the total tumor weight (171.1 ± 291.4 mg) in comparison to that of the control group (283.2 ± 181.2 mg) (Fig. 2). Intraperitoneal tumor growth in the Interceed and Intergel groups did not significantly differ from that of the control group.
Incidence of local recurrence
No local recurrences
Description of adhesions according to the therapy groups and the control group
Control, n = 15
Interceed, n = 15 (%)
Intergel, n= 15 (%)
Taurolidine/heparin, n = 15 (%)
Pelvic fat–abd walla
Pelvic fat or om–intd
The analysis of the localization of the metastases was performed by combining metastases at the peritoneal defect and the port sites into one group called abdominal wall metastases. The group, intraperitoneal metastases, included tumor nodes at the minor pelvis, small bowel, diaphragm as well as paraaortic, pancreatic, liver, and renal metastases. The group omentum + mesenteric metastases included tumor nodes situated at the smaller and greater omentum. Comparison of the mean number of metastases at the three different localizations showed that the application of tauroldine/heparin led to a significant tumor reduction at the omentum and bowel mesenterium (p < 0.002) as well as the abdominal wall (p < 0.005) in comparison to the control group (Fig. 3). Analyzing in detail the abdominal wall recurrences, the number of metastases at the peritoneal defect was decreased in the taurolidine/heparin group (0.3 ± 0.62) and in the Intergel group (0.67 ± 1.23) compared to the control group (1.1 ± 1.5). The reason for this reduction could not be determined. Similar results were observed for the number of metastases at the port-sites: taurolidine/heparin decreased the number of metastases at the camera and the instrumental port sites when compared to the control group, but differences were not significant.
Intraperitoneal adhesion formation
The total number, site, thickness, tenacity, and vascularization of the adhesions, according to the Moreno score , are given in numbers and percentages (Table 4). The final mean scores and the analysis of the significance between the different therapy groups showed the following results.
Mean adhesion score and standard deviation of the therapy groups and the control group for each variable
Number of adhesions
There was a remarkable predominance of adhesion formation at the site pelvic fat body/omentum–intestine (pelvic fat or om–int). Less adhesions were observed at the pelvic fat body–abdominal wall site (pelvic fat–abd wall), and the lowest number of adhesions was found at the localizations omentum–abdominal wall (om–abd wall) and intestine–abdominal wall (intest–abd wall) (Table 3). The therapy groups showed no significant differences in the number of adhesions at the different sites (Table 4).
The adhesions found in all groups were not easily distinguished with regard to tenacity. Most of the intraabdominal adhesions were type II or III (loosening with blunt or sharp dissection). Only in the Intergel group were most of the adhesions type I or II (Table 3). No statistical differences existed with respect to the use of adhesion prophylactica ortaurolidine/heparin (Table 4).
Overall adhesion scores
The overall mean adhesion scores were as follows: 13.8 (1.9) for the control group. 13.7 (1.6) for Interceed, 13.6 (2.5) for Intergel, and 13.4 (1.8) for taurolidine/heparin. There were no statistically significant differences between the control and therapy groups (Table 4).
Although the etiology and exact pathomechanisms of tumor recurrence after laparoscopic procedures in oncologic surgery have been widely investigated, standardized methods for the prevention of port-site metastases and local recurrence are still lacking. In this standardized animal model the influence of different substances on intraperitoneal tumor growth after laprascopic-assisted cecal resection was investigated.
The intraperitoneal instillation of the adhesion prophylactica Intergel and Interceed had no significant influence on the development of metastases. Analysis of the local recurrence rate also showed no significant reduction by the application of adhesion prophylactica compared to the control group. Moreover, the total number and total weight of intraperitoneal metastases were not significantly decreased by application of Interceed or Intergel. Until now, the effect of adhesion prophylactica on intraperitoneal tumor growth had not been evaluated in the context of laparoscopic resection of colon carcinoma. Intergel is a hyaluronic acid (HA) gel, a glycosaminoglycan, which is a natural component of the extracellular matrix and plays a major role in postoperative abdominal adhesion prophylaxis. As a natural ligand of CD44, which plays a central role in adhesion formation, its effectiveness against adhesions has been documented in experimental and clinical studies [21, 22, 30, 42]. When applied to the injured serosal surface, Intergel acts as a coating with protecting properties against deposition of fibrin, cells, and other tissues during the critical phase of peritoneal healing. The iron modification of Intergel leads to a longer half-life of HA, which results in a longer coating time and better effectiveness against adhesion formation. Oxygenized cellulose (Interceed) is a barrier agent, available in recent years for the purpose of adhesion prevention. It is a specifically designed fabric that converts into a gel and adheres to the peritoneal surface without the need for sutures. Thus, it acts as a barrier preventing direct tissue apposition to the injured peritoneal surface. In this animal study neither Intergel nor Interceed showed significant protective qualities against the adhesion of tumor cells to the injured surfaces, such as the bowel suture, the peritoneal defect, or the port-site incisions. This might be due to the difficulties of correctly placing the substances with laparoscopic instruments in a small animal model. Nevertheless, recent results from another experimental model with colorectal carcinoma cells showed that sodium hyaluronate significantly increased tumor cell proliferation and motility in vitro. In vivo studies by the same group showed significantly higher total tumor nodule counts in the peritoneal cavity compared to those of a control group . These results indicate that HA may provide a stable matrix for tumor cell proliferation. Ropponen et al.  described an increased potential for invasive growth and metastases when colorectal tumor cells synthesized their own hyaloronic acid. Furthermore, it is well established that HA is a major ligand to CD44. Some CD44 splices have been found on the surface of colorectal tumor cells and correlate with increased metastatic potential of the tumor . Thus, there seems to be a CD44-mediated increase in tumor cell motility by HA and an increase in tumor cell proliferation when a stable matrix is provided in which colorectal tumor cells can proliferate . Nevertheless, we cannot confirm these observations because in our model the application of sodium hyaloronate carboxymethylcellulose membranes did not enhance tumor growth. Underwood et al.  reinforced our results: Seprafilm neither reduced nor enhanced the tumor cell implantation of human colon cancer cells at abdominal wounds in a hamster model. The other substance investigated was taurolidine, a derivate of the amino acid taurine. It is commonly used as an adjuvant therapy for various infections. Furthermore, a significant suppression of tumor cell growth in vitro and in vivo has been demonstrated . Our results support these observations. In our animal model, the combination of taurolidine and heparin led to a significant reduction of the total number and total weight of intraperitoneal metastases after instillation in the peritoneal cavity. Nevertheless, we acknowledge that the small sample size in each therapy group (n = 15) makes interpretation of the results less reliable. Furthermore, although locally applied, the solution of taurolidine/heparin distributes all over the peritoneal cavity, in contrast to Interceed, the oxygenized cellulose barrier agent. This might be another reason for the results. Additionally, the number of abdominal wall metastases (peritoneal defect + port site) was significantly reduced by taurolidine/heparin in comparison to the control group. The incidence of port-site metastases in this therapy group was decreased, but differences were not significant, mainly due to the small number of animals in each group. The suppression of intraperitoneal tumor growth by instillation of taurolidine can be explained by several factors: it has been demonstrated that taurolidine inhibits the production of interleukin-1β (IL-1β), a strong growth-promoting cytokine. The intraperitoneal application of taurolidine might cause a decrease in tumor growth by inhibiting the IL-1β-mediated proliferation of tumor cells on injured surfaces and in the peritoneal cavity [3, 26, 50]. Furthermore, the results of in vitro studies indicate that the decrease in tumor growth is a direct toxic effect of taurolidine on tumor cells by inhibition of tumor cell proliferation . Moreover, taurolidine has significant antiadherent activity on pathogen microorganisms and peritoneal adhesion formations . Thus, these antiadherent qualities might also be responsible for the reduction of intraperioneal tumor growth by the prevention of tumor cell attachment. These results are supported by those of other in vivo experiments. In combination with heparin, taurolidine showed even greater suppression of tumor cell growth . The synergistic effect of heparin and taurolidine should be further investigated. It has been demonstrated that the adherence of tumor cells, bacteria, and other substances on injured urothelium was reduced after instillation of heparin into the bladder . It has been suggested that heparin binds to fibronectin and restores the antiadherence integrity of the bladder surface . Therefore, it is possible that taurolidine acts more on tumor cells and peritoneal macrophages, whereas heparin primarily acts on the peritoneal surface.
Nevertheless, tauroldine/heparin showed no significant effect on the incidence of local recurrence as well as the application of Intergel or Interceed. However, Interceed showed a slight suppression of local recurrence, which might become significant if larger numbers of animals are used. The high rate of local recurrence might also be explained by the tumor cell suspension model used, which had high numbers of colon carcinoma cells. Although these models are frequently used in experimental investigations of laparoscopic surgery in malignancies, it is unclear if they are representative for tumor cell spillage occurring during instrumental manipulation in cancer patients. In our experimental setting with a tumor cell suspension model, the incidences of metastases were unrealistically high in the control group. Thus, it does not seem to be representative of the clinical situation. Furthermore, it has been proven that the number of injected cells influences the tumor weight and development of metastases . Recently, our laboratory developed a solid tumor model of an orthotopic colonic carcinoma and further studies are under way to prove our results .
The development of intraabdominal adhesions after cecal resection was not significantly influenced by taurolidine/heparin or the adhesion prophylactica. For the overall adhesion score, the taurolidine/heparin group had the lowest values followed by the Intergel and lnterceed groups, but differences were not statistically significant in comparison to results from the control group. Only slight tendancies concerning the vascularization status and tenacity of the adhesions could be observed in favor of the adhesion–prophylactica but without significant influence. One reason for these results might be that the substances investigated were locally applied. Although Intergel and taurolidine/heparin are fluids that distribute into the peritoneal cavity, they showed only a slight but not significant effect on adhesion formation. Nevertheless, the highest levels of adhesion score were observed in the animal group treated with Interceed. Because this substance can only be applied locally, it is not surprising that it did not influence abdominal tumor growth in general. These observations are in contrast to results of previous clinical investigations. In a prospective randomized multicenter study of the Interceed (TC7) Adhesion Barrier Study Group, Interceed effectively reduced the incidence of postoperative adhesions, even in patients with severe endometriosis . Possibly, the quantities we used, 1 ml Intergel and 1 × 1 cm Interceed, were too small to prevent intraabdominal adhesion formation. Furthermore, Intergel and Interceed were only applied at the peritoneal injury site and the bowel suture. Most postoperative adhesions were between the pelvic fat body and the abdominal wall or between the pelvic fat body, the omentum, and intestinum, respectively. These are locations where the adhesion prophylactica were not directly applied, whereas adhesions near the application sites (between the intestinum and the abdominal wall or between the bowel loops) were rare. In conclusion, significant prevention of postoperative adhesions by taurolidine/heparin or adhesion prophylactica was not demonstrated in this experimental setting.
This study was supported by a grant from the General Association of Research (DFG).