303 Intraperitoneal cytostatic-loaded supramolecular hydrogel and intestinal anastomotic healing Statistical Analysis General characteristics of the animals can be found in Supplementary Table S1. Statistical analysis was performed using SPSS (IBM SPSS Statistics for Apple, Version 27, Armonk, New York, NY, USA) and GraphPad Prism (GraphPad software for Apple, version 8.0.0, San Diego, CA, USA). Numerical variables were presented as median with interquartile range (IQR, Q1–Q3). To evaluate the statistical significance of numerical variables differences observed between groups, non-parametric tests (overall Kruskal–Wallis and post hoc Mann–Whitey U-tests for pairwise comparison) were applied. In case of significant overall tests (α = 0.05), a Bonferroni correction was used for the pairwise comparisons (α = 0.0167). The percentage of body weight change was calculated by subtracting the daily measured weight from the baseline weight of each animal. Group comparison of mean body weight was performed with mixed-effect models. RESULTS A total of 42 healthy rats underwent the surgical procedure (saline n = 6, unloaded hydrogel n = 18 and MMC-loaded hydrogel n = 18) of which 22 completed the follow-up period of 7 days (Supplementary Figure S3, saline = 6, unloaded hydrogel = 10, MMC-loaded hydrogel = 6) and were included in the final analysis. Anastomotic Adhesion and Leakage Scores The macroscopic anastomotic adhesion and leakage scores are displayed per intervention group in Figure 1A,B. Representative images of the anastomoses with corresponding scores are shown in Figure 1C–E. The median (IQR) adhesion scores were 1.5 (1–2), 1.5 (1–2) and 1 (1–2) for saline, unloaded hydrogel and MMC-loaded hydrogel groups, respectively. There were no significant differences between the groups (Table 1). Severe and extensive adhesions were only present in one animal that had unloaded hydrogel administered. The median (IQR) AL scores were 1 (1–1), 2 (1–2) and 1 (1–1.25) for saline, unloaded hydrogel and MMCloaded hydrogel groups, respectively. A difference was observed for the AL score (p = 0.034), for which pairwise comparison showed a difference for AL score comparing the saline and unloaded hydrogel subgroup (p = 0.020, Figure 1B). This difference was not significant after Bonferroni correction (α = 0.0167). Table 1. Macroscopic adhesion and AL scores. Saline (n = 6) Unloaded Hydrogel (n = 10) MMC-Loaded Hydrogel (n = 6) p Value Adhesion score – median (Q1–Q3) 1.5 (1–2) 1.5 (1–2) 1 (1–2) 0.741 a AL score – median (Q1–Q3) 1 (1–1) 2 (1–2) 1 (1–1.25) 0.034 a,* a Kruskal–Wallis test; * p = 0.020 after pairwise comparison of saline and unloaded hydrogel group with Mann–Whitney U test, which was not significant after Bonferroni correction. 13
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