Stem cells and the effects of platelet- rich fibrin in attenuated colonic anastomosis , experimental study

Colorectal procedures have an important place among the gastrointestinal system procedures with those carried out for colorectal tumors in the first place. New techniques and different treatment modalities are developed as a result of the studies carried out. While the surgical procedures have satisfactory results, the complications occurring result in bothersome problems. One of the most important complications is the leakage from the anastomosis line. The aim of many studie carried out is the ensure safe colon anastomosis to reduce complications. In colorectal cancers, wound healing after surgery becomes impaired as a result of neoadjuvant or adjuvant chemotherapy, and therefore, the ratio of leakage from anastomosis increases. The most important one among the targets for the healing anastomosis is protecting the integrity of tissue with correct techniques to increase the blood supply for the tissue and to improve the strength of the anastomosis. A rather important factor is the platelet concentration for the platelet-rich fibrin (PRF) to ensure biologic integrity in tissues, to make the endothelial cell migration easier and to regulate the infectious and inflammatory events through the growth factors and cytokines it contains by effecting the angiogenesis and wound healing. Stem cells originating from the adipose tissue are among the stem cells with the greatest potential with their method of harvesting and ability of differentiating to multiple tissues. It has been shown in many studies that adipose tissue-origin stem cells can differentiate to new cells and also increase the formation of new vessels. Strength of anastomosis is reduced and leakage becomes inevitable as a result of impairment of wound healing in in anastomosis areas after neoadjuvant or adjuvant therapies and surgery and prevention of the new vessel formation. Ninety Sprague-Dawley type male rats were used in the study. Four groups were created each containing 20 rats. Ten rats were used for stem cells and platelet-rich fibrin. After incising and making a resection on the descending colon of the rats in all the groups for the full thickness, anastomosis was created. In the control group, resection and only intraperitoneal 3-5cc saline following the anastomosis were applied in the surroundings of the anastomosis, intraperitoneal 5-Fu was applied to the 5-Fu group in the surroundings of the anastomosis, platelet-rich fibrin and intraperitoneal 5-Fu was applied in the surroundings of the anastomosis to the PRF+5-Fu group, and platelet-rich fibrin + stem cell and intraperitoneal 5-Fu group was applied to the PRF + stem cell + 5-Fu group in the surroundings of the anastomosis; and after this, administration of intraperitoneal 5-Fu was continued for 1 week for the 3 groups except for the control group. The dosage of 5-FU administered intraperitoneally was adjusted according to the weights of the rats. All the rats were sacrificed at day 7 following the procedure. Each of the four groups comprised two subgroups including 10 rats. It was planned to evaluate the anastomosis bursting pressure, histopathologic evaluation and vascularization with angiography. In the immünohistochemical study, fluorescent microscope was used to examine the follow-up of the adipose tissue-origin stem cells labeled with DiI in the study group. When weights of the rats in the study were evaluated before the experiment, there are no significant differences among the 4 groups (p=0.852). Significant differences were found between the groups in the baseline and final weights (p<0,001). The weight loss was found significantly higher in the 5-Fu and 5-Fu+PRF groups as compared to the control group (p<0.001 and p<0.001), while the weight loss was smaller in the 5-Fu+PRF and 5-Fu+PRF+ Stem cell groups as compared to the 5-Fu group (p=0.009 and p<0.001, respectively) and the observed weight loss in the 5-Fu+PRF+ Stem cell group was smaller as compared to the 5-Fu+PRF group (p<0.001). The weight loss between the control and 5- Fu+PRF + Stem cell groups was similar (p=0,753). As regards the intraabdominal adhesions, statistically significant differences were found among groups. Adhesions in the 5-Fu and 5-Fu+PRF groups were statistically higher as compared to the control group (p=0.007 and p<0.001, respectively), and the level of adhesion in the 5-Fu+PRF group was significantly higher as compared to the 5-Fu and 5-Fu+PRF + Stem cell groups (p=0.039 and p=0.005). No statistically significant differences were seen between the control group and the 5-Fu+PRF + Stem cell group (p=0.051), marked perforation, fistulization, intraabdominal abscess formation and breaking of the anastomosis were observed in the 5-Fu and 5-Fu+PRF groups as compared to the 5-Fu+PRF + stem cell and control groups. Upon comparison regarding the anastomosis bursting pressures, statistically significant differences were found between the control group and the PRF+5-Fu + stem cell groups as compared to the other two groups, while it was not possible to measure pressure in the 5-Fu and 5-Fu+PRF groups because of marked dissociation of the anastomosis. The mean bursting pressure was 147.9 ± 20.02 mmHg in the control group, and 150 ± 28.1 mmHg in the PRF+5 Fu + stem cell group, and no statistically significant differences were found between these two groups. In the angiographic evaluation, the number of vessels per square millimeters were calculated: 21.7 ± 3.2 vessels were observed in the control group, 15 ± 4,6 vessels were observed in the 5-Fu group, and 23.9 ± 4.1 vessels were observed in the PRF + stem cell+5-Fu group, and it was found that vascularization in control and stem cell groups were qualitatively increased as compared to the other two groups. In the histopathologic examination, no statistically differences were found in re-epithelization (p=0.002), fibrosis (p<0,001), ischemic necrosis (p<0.001) and inflammation, and neutrophils, lymphocytes and giant cells (p=0,177-p=0.562 respectively). Statistically significant (p=0.019) difference was found in the 5-Fu and 5-Fu+PRF+Stem cell group as compared to the control group. In the evaluation made using fluorescent microscopy, it was found that stem cells were present in the conversion to endothelial cells and within the villous structures. There are statistically significant differences between the groups as regards mortality rates (p<0.001), and mortality rate was significantly higher in the 5-Fu and 5-Fu+PRF groups as compared to the control group (p=0.018 and p=0.003), while the mortality was significantly higher in the 5-Fu and 5-Fu+PRF groups as compared to the PRF+5-Fu+stem cell group (p=0.018 and 0.003). No statistically significant differences were found between the control group and the PRF+5-Fu+stem cell group. It has been concluded in our study that increase of inflammation and decrease in angiogenesis by 5-Fu causes impairment of wound healing on anastomosis level. Integrity of the anastomosis was ensured through the growth factors secreted by PRF and through the differentiation of stem cells to endothelial cells and increasing the sitokin secretion. It has been found that effectiveness of growth factors was rather small when platelet-rich fibrin (PRF) was used only with 5-FU without stem cells. Stem cells have created significant difference in the wound healing after the effects of the chemotherapeutical agents following surgery. Keywords: Colon anastomosis, 5-Fu, Platelet-rich fibrin, Stem cell