300 Chapter 13 METHODS Ethics and Safety Protocol This animal study was performed at the animal center of Maastricht University (Maastricht, The Netherlands). The experimental protocol followed the Dutch Animal Experimental Act and was approved by the Animal Experimental Committee of Maastricht University Medical Center (project license AVD1070020198765). The ARRIVE guidelines 18 for reporting animal research were followed and additional information can be found in the Supplementary Materials. During the experiment, we maintained a local cytostatic protocol developed by the animal center of Maastricht University to ensure appropriate safety measures while working with chemotherapy. Animals and Housing A total of 42 healthy adult Wistar rats (21 males/21 females) aged 10–12 weeks with a body weight of 400 g–500 g (males) and 230 g–330 g (females) were used (all characteristics can be found in Supplementary Table S1). All rats were bred by Charles River Laboratories (Sulzfeld, Germany). An acclimatization period of at least one week was maintained. All animals were socially housed in individually ventilated cages in a temperature- and humidity-controlled room with 12 h light/dark cycles. All animals had ad libitum access to food (10 mm Sniff rat/ mouse sterilized food compressed into pallets) and acidified drinking water. Postoperatively, animals were weighed daily, and welfare was scored systematically based on predefined standardized welfare scoring sheets (Supplementary Materials). Human endpoints (HEPs) were defined prior to the experiment. Study Design, Randomization and Blinding The aim of this study was to investigate if intraperitoneal administration of MMC-loaded and unloaded hydrogel affected the anastomotic healing compared to animals receiving a peritoneal injection with saline. To study the effect on anastomotic healing, all animals received a sufficient colon–colon anastomosis. Subsequently, the animals were randomly assigned to one of the three following intervention groups receiving a single injection with either saline (n = 6), unloaded hydrogel (n = 18) or MMC-loaded hydrogel (n = 18). The random allocation of the animals was performed by a computer-based random order generator. After a follow-up period of seven days (most ALs show up within the first week after surgery), the anastomotic healing was assessed. During the allocation, the conduct of the experiment and the outcome assessment, the research team, the veterinarian and the people working in the animal facility were blinded for the group allocation. Supramolecular Hydrogel For this experiment, we used a supramolecular hydrogel based on polyethylene glycol (PEG) chains end-modified with fourfold hydrogen bonding the ureido-pyrimidinone (UPy) units (UPy–PEG Hydrogel), previously described by Wintjens et al. 12, 13, 19, 20. Identical to the previous
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