Effects of chitosan on healing and strength of colonic anastomosis in rats

PURPOSE: To investigate whether chitosan application over colonic anastomosis line, provide reinforcement, and subsequently improve anastomotic healing. METHODS: Forty eight Wistar albino female rats were used and were randomly divided into four groups, 12 rats in each: The control groups (1 and 3) received no further treatment. The experimental groups (2 and 4) received chitosan application over the colonic anastomosis. After sacrifying rats at the end of the experiment (either on day three or on day seven, depending on the group), colonic bursting pressure, a hihydroxyproline level and histopathologic characteristics of the perianastomotic tissue were examined. RESULTS: At three days, chitosan and control groups had similar values for histopathologically. On day seven, chitosan group had significantly higher mean score of collagenization (p=0.007) and a significantly higher bursting pressure (p=0.038). CONCLUSION: Our study emphasizes the positive effect of chitosan in the process of collagenation in colonic anastomosis healing.


Introduction
Gastrointestinal anastomoses are among the most frequently performed procedures in general surgery units throughout the world.From a clinical viewpoint, anastomoses are an essential part of the surgical management for many benign or malignant conditions of the gastrointestinal system and considerable effort has been devoted by colorectal surgeons to prevent anastomosis-associated complications.
Anastomotic leak is the single most important determinant for mortality, morbidity, and the length of hospital stay 1,2 after anastomoses.Within the gastrointestinal system, the risk of anastomotic leak is highest in the large intestine.However, the risk is not uniformly distributed in this anatomical location: the more distal the anastomosis in the large intestine the greater the likelihood of anastomotic leak 1,3 .Relative scarcity of collateral circulation together with a very rich bacterial flora may help to explain the higher probability of leaks in this area 4 .Another factor associated with increased risk is the frequent occurrence of coexistent pathologies in most of the patients undergoing colon surgery 5 .
Chitosan is a linear copolymer of ß 1,4 linked 2-acetamido-2-deoxy-ß-D-glucopyranose and 2-amino-2-deoxyß-D-glycopyranose.It is easily obtained by deacetylation of chitin, an abundant polysaccharide found in nature as a component of exoskeletons of crustaceans and insects.Chitosan has been reported to be a biocompatible, biodegradable and non-toxic substance with antifungal, hemostatic, antimicrobial, analgesic, and wound healing accelerating effects 6,7 .Several studies have shown the activity of chitosan in the healing process through enhanced infiltration of inflammatory cells in the area of injury.Furthermore, chitosan-mediated stimulation of macrophage and fibroblast activity during wound healing has resulted in a more pronounced formation of granulation tissue 8,9 .Other studies have provided evidence for accelerated wound healing with chitosan gel formulations 10,11 .
Fibroblasts produce the main constituents of the connective tissue, i.e. collagen, proteoglycans, and elastins, and play a role in wound healing.Collagen, the most abundant protein in human body, is responsible for the integrity and durability of tissues.Collagen content of the tissues, which is a surrogate marker for anastomotic healing, is estimated on the basis hydroxyproline assay 6,12 .
This experimental study aimed to investigate any potential favorable effects of chitosan application over colonic anastomoses on anastomosis strength and reactions taking place during healing process.All rats were sacrificed at the end of the experiment to assess bursting pressure, hydroxyproline level and histopathologic characteristics of the perianastomotic tissue.

Surgical procedures
Surgical procedures were performed under strictly sterile conditions and all rats were fasted for 12 hours prior to the operation.After general anesthesia was administered with 10 mg/ kg of subcutaneous ketamine (Ketalar, Eczacıbaşı, Ist.), anterior abdominal wall was shaved and cleansed with povidon iodine and covered with sterile surgical drapes.A 3 cm midline incison was made to access the abdominal cavity.Left colon was exposed and a full-layer cut was made in a right angle to its longitudinal axis.
Then, end-to-end colo-colic anastomosis was performed using 5/0 absorbable monofilamentous polydiaxanon (PDS; Johnson & Johnson Professional Export Company Ethicon Ltd, Edinburgh, England) with inverting sutures.All anastomoses were performed by the same surgeon using seven to eight sutures for each anastomosis.The fascia and skin were closed with continuous sutures using 3/0 silk suture material.Postoperatively the animals were fed with standard rat food and drinking water.

Preparation of chitosan
Medium-molecular-weight chitosan in powder form purchased from Sigma-Aldrich (St. Louis, MO, USA) was directly applied to the line of anastomosis.No other processes were applied to the commercial chitosan preparation prior to application.

Assessments
Rats were sacrificed on their respective postoperative days for assessments.A re-laparotomy was done in the previously used line of incision in sacrificed rats.

Evaluation of adhesions
At post-mortem examination, adhesions were graded in a scale between 0 and 3 according to the system developed by van Der Ham et al. 14 : 0, no adhesions; 1, minimal adhesions, primarily between the omentum and the anastomosis; 2, moderate adhesions, i.e., between the anastomotic site and omentum or between the anastomosis and a loop of the small bowel or abdominal wall; and 3, severe and extensive adhesions, i.e., between the anastomotic site and several loops of the small bowel and abdominal wall.

Measurement of the colonic bursting pressure
After the integrity of the anastomosis was ascertained upon exposure of the line of anastomosis and removal of adhesions in the surrounding tissues, two cuts, one 3 cm proximal and one 3 cm distal to the anastomosis were made to obtain a 6-cm long colonic segment.The distal end of this segment was ligated using 3/0 silk sutures after an infusion set was inserted into the proximal end.The water pressure was increased gradually and appearance of air bubbles was recorded as the bursting pressure in mm Hg.
Following this measurement, the intestinal segment was cut longitudinally, divided into two segments while preserving the distal sutures, and one-half was placed in 10% formaline solution.
The other half of the sample segment was stored at -70°C for tissue hydroxyproline assay.

Histopathological examination
Anastomotic segments were stained with hematoxylin and eosin (H&E) stain and examined with the light microscopy at 20x and 200x magnification power by the same pathology specialist blinded to the groups.Inflammatory cell infiltration (leukocyte count), fibroblast activity, neoangiogenesis, and collagen content were determined using Ehrlich and Hunt scale as modified by Philips et al. 17 .

Tissue hydroxyproline assay
The tissue hydroxyproline concentration, which represents the perianastomotic collagen concentrations, was measured using the spectrophotometric method of Bergman and Loxley 15,16 .The results were expressed in micrograms of hydroxyproline per milligram of tissue (μg/mg).

Statistical analyses
Results were expressed as mean ± SEM.Betweengroup differences were evaluated by Kruskal-Wallis test.Multiple comparisons between groups were performed with post-hoc Tukey's HSD test.All analyses were conducted using SPSS 10 software (SPSS, Inc., Chicago, IL, USA).A p value less than 0.05 was considered indication of statistical significance.

Overall difference between groups
Tables 1 and 2 show mean values for tested variables in the four experimental groups at three and seven days after the anatomoses.Based on non-parametric variance analysis results (Kruskal-Wallis test), the scores for acute inflammation and neovascularization did not significantly differ between the four groups.On the other hand, other variables revealed significant overall p values necessitating post hoc analysis.which is a highly complex biological process that involves cell division, chemotaxis, neovascularisation, extracellular matrix protein synthesis, and scar formation 18 .
Healing in intestinal anastomoses conform to the general principles of wound healing, such that edema and inflammation prevail in the initial four days.Inflammatory stage is very crucial for the scavenger activity of inflammatory cells in the wound site 18 and the presence of inflammatory cells is a sign of tissue repair 19 .As in all anastomoses, tissue circulation and oxygenation play a major role in the healing of colonic anastomoses 19,20 .
Chitosan has been found to have a myriad of biological properties such as antimicrobial effects, acceleration of wound healing, activation of macrophages, increasing fibroblast migration and proliferation, and stimulation of angiogenesis, collagen production, and interleukin-8 secretion by the fibroblasts [21][22][23][24][25] .For instance, in support of these abovementioned effects, Ishihara et al. 26 found a significant increase in angiogenesis at the wound site with a mixture of chitosan and FGF-2 compared to controls.
It is no surprise that neovascularization represents an important factor to combat infections, in the light of the fact that oxygen, leucocytes, and immunoglobulins are carried to the wound site by the very same vascular structures 27 .In our study however, a significant difference between chitosan and control group could not be detected with regard to neovascularization, probably due to the small duration of follow-up.Moreover, a number of previous studies have shown that different molecular weights of chitosan have a range of antiinflammatory actions, including inhibition of the production of TNF-α, IL-6, prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), VCAM-1 and ICAM-1 in vitro 28,29 .
Qiao Y. et al. 8 reported decreases in oxidative tissue injury, septic injury, organ injury, and cytokine levels 8 .These somehow conflicting findings may be explained on the basis of differential effects of chitosan with varying doses.Likewise, rats sacrificed Comparison of chitosan and control groups three and seven days after anastomoses At three days, chitosan and control groups had similar values for all indices.At seven days, ( although the synthesis and source of collagenase enzyme are yet to be defined more precisely.Elucidation of this latter point may provide deeper insights into the wound healing process [31][32][33] .An in-vitro study by Nastasescu et al. 34 showed decreased collagen breakdown after addition of a collagen-chitosan mixture into a body fluid containing collagenase.In the present study, we believe that the emergence of a significant difference between the two groups at day 7 and day 3. Colonic bursting pressure is a sign of the mechanical endurance of the anastomosis 35 .In a study by Xiao H. et al. 36 the impact of a carboxymethylchitosan-carboxymethylcellulose (CMCH-CMC) film applied to the surface of anastomosis on wound healing was explored in rats.They found an increased rate of adhesions nearby the anastomosis without an observable effect on anastomotic healing.Nursal et al. 37 suggested that agents that cause excessive increase or decrease in inflammation may cause undesired effects on wound healing 38,39 .Our study shows that the bursting pressure of the chitosan application group on day 3 and day 7 were significantly higher when compared to the control groups.However no differences with regard to intra-abdominal adhesions between chitosan and control groups.However, significantly higher rate of adhesions were observed in chitosan 2 group compared to chitosan 1, which was probably due to slow absorption of chitosan in the abdominal cavity leading to fibrous capsule formation and enhanced foreign body reaction.
Although our results have not lend support to clinical use of chitosan to improve the safety of anastomoses, further studies may help to better define the role of chitosan in the healing of colonic anastomoses since the significant increase in collagenization among chitosan groups may deserve investigation.

Conclusion
Our study emphasizes the positive effect of chitosan in the process of collagenation in colonic anastomosis healing.

TABLE 1 -
The comparison between chitosan and control group via histopathological analysis in day 3.

TABLE 2 -
The comparison between chitosan and control group via histopathological analysis in day 7.

TABLE 3 -
Result of bursting pressure, hidroxyproline and adhesion score.