Danique Heuvelings

307 Intraperitoneal cytostatic-loaded supramolecular hydrogel and intestinal anastomotic healing After obduction of the prematurely sacrificed rats, 16/20 rats (n = 5 unloaded hydrogel, n = 11 MMC-loaded hydrogel; 69% male) had signs of intraluminal blood loss at the anastomotic site (Figure 3A). It seemed like the blood clots accumulated at the site of the anastomosis, which was not seen in animals without blood loss (Figure 3B), and sometimes blood clots in the colon or small intestine proximal of the anastomosis were observed. We did not observe hemoperitoneum in any of the rats, nor did we identify any intraluminal blood loss in the animals surviving the whole experiment. In 50% (n = 8) of the animals that had intraluminal blood loss, the blood loss was present on postoperative day (POD) 2, and in 33% on POD 3; two other rats were diagnosed with blood loss on POD4 and 5 (Figure 3C). In 50% (n = 8), microscopic signs of blood loss around the anastomotic site were seen, e.g., necrotic blood vessels (Figure 3D,E), hemorrhagic spots in different layers (serosa, muscularis mucosa and the mucosa) or congestion in some villi. The small intestine and stomach did not show any signs to which the blood loss could be related. Blood samples were taken from animals with (8 rats treated with MMC-loaded hydrogel) and without (8 rats treated with unloaded hydrogel and 3 with MMC-loaded hydrogel) intestinal blood loss (Supplementary S2, Figure S4 and Table S2). Thrombocyte numbers were not different between animals with and without blood loss. In addition, coagulation factors (prothrombin time, international normalized ratio, and activated partial thromboplastin time) were estimated in 5 of the previous animals (all MMC-loaded hydrogel) of which 4 were presenting with blood loss and one did not. No abnormalities related to coagulation outcomes were found. Weight Loss and Welfare Scores The results of the daily body weight monitoring in all animals who successfully underwent the operation are shown in Supplementary Figure S5. All animals had an initial weight gain on day 1 related to hydrogel and saline administration, followed by weight loss. From day 3, recovery to mean baseline weight was observed in saline treated animals, while animals who had hydrogel administered (unloaded hydrogel or MMC-loaded hydrogel) kept on losing weight. In both hydrogel groups, the course of the body weight was comparable, although we observed a little higher weight loss in the MMC-loaded hydrogel treated animals. A mixedeffects model showed a significant difference for both female and male rats (p < 0.0001 for both sexes) in favor of the saline-treated group. Animals in both the unloaded hydrogel and MMC-loaded hydrogel had higher post-operative welfare scores, implicating more discomfort compared to animals treated with saline. Seven rats opened their laparotomy wound (n = 5 unloaded hydrogel, n = 1 MMC-loaded hydrogel, n = 1 saline). 13

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