In vivo evaluation of CDPE-Cs hydrogels

Animals were purchased and kept at room temperature, and allowed food and water ad libitum. The protocol was approved by the Assiut University Animal Ethical Committee (Ethical approval number 1730030), and was in compliance with all relevant ethical regulations for animal testing and research. Animals were maintained under general anesthesia during the duration of experiments and euthanatized while under anesthesia at the end of the experiments.

Rats: The acute liver punch biopsy model was developed as described previously^24,36 (with slight modifications, to evaluate the biocompatibility/biodegradation of the developed hemostatic hydrogel and to examine the hemostatic efficiency via measurements of the time to hemostasis and blood loss). Adult male Sprague-Dawley rats (ca. 300 g) were anaesthetized with an intraperitoneal injection of sodium thiopental (50 mg kg⁻¹) and their abdominal hair was shaved. The rats were randomly selected from the laboratory population and divided into five groups (n = 6 animals per group). After opening the abdomen, the liver was exposed and an incision of ca. 1 cm in diameter, at an angle perpendicular to the tissue to a depth stop of ca. 0.3 cm, was produced on the liver. The tissue in the center of the injury site was removed using forceps and surgical scissors. Sterile gauze was used to absorb initial bleeding, and then, conventional gauze, Curaspon®, Surgicel®, CDPE and CDPE-Cs hydrogels were implanted into the injury site. A blade inserted onto a syringe was utilized to induce the injury, and to cut the gauze, sponge, and hemostatic hydrogels to match the dimensions of the wound (i.e., ca. 1 cm in diameter × 0.3 cm in depth). Several pieces were applied from the conventional gauze, Curaspon®, and Surgicel®, and bleeding was evaluated every 30 s for 10 min. CDPE and CDPE-Cs hydrogels were applied only once. The time to hemostasis and blood loss were measured. The hemostatic time was recorded as the last time of application where bleeding was not observed from the injury site. Weights of the dressings before and after applications were recorded to calculate the amount of blood loss. For hydrogels, blood loss was calculated based on the bleeding from the injury that was absorbed into the surrounding gauze.

For biocompatibility/biodegradation studies, the conventional gauze dressings, Curaspon®, Surgicel®, and CDPE and CDPE-Cs hydrogels were implanted and left inside the liver after the first application. The initial bleeding was absorbed with gauze. The abdomen was then closed with sutures, and the area was sterilized with povidone iodine. After one week, the sutures were removed and the liver was visually examined for wound healing. The animals were euthanized at the end of the experiments, and specimens from the liver tissues surrounding the injury sites were harvested for histological examination. Tissues were fixed immediately in 10% neutral formalin for 24 h, washed in running tap water for at least 2 h, and then immersed in 70% ethanol. Dehydration in ascending concentrations of ethanol (i.e., 70, 90, 95, and 100%) was carried out, followed by clearing the specimens in double embedding (1 g celloidin dissolved in 100 mL methyl benzoate, three changes for 3 days). The specimens were then washed in benzene (two changes, each for 15 min). Impregnation in paraffin was performed, followed by embedding the specimens in hard paraffin. Sections of 5 µm thickness were prepared using a rotary microtome (CUT 4050, microTec Laborgeräte GmbH, Walldorf, Germany). Specimens were stained with Hematoxylin and Eosin (H&E) stain for histological examination and imaged by Olympus BX53 light microscope (Olympus Co., Tokyo, Japan).

Rabbits: Adult male New Zealand white rabbits (ca. 2 kg) were kept in large cages and were anesthetized with intraperitoneal injection of urethane (1 g kg⁻¹) prior to the surgical procedures. Rabbits were randomly selected from the laboratory population and divided into five groups (n = 6 animals per group) and treated exactly as mentioned in the in vivo rat section (vide supra). In a separate study, the MAP was monitored during the surgical procedures (n = 3 animals per group). A 20-gauge catheter was surgically cannulated into the right carotid artery and the MAP was recorded continuously under general anesthesia before the liver injury and for 1 h after the injury. Blood pressure was monitored on a polygraph using the Universal Oscillograph (Harvard Apparatus, South Natick, MA). A heparinized rabbit model was utilized at a certain dose of heparin (i.e., 250 IU kg⁻¹) to achieve bleeding that would not stop spontaneously after the liver injury.

Pigs: Female Sus scrofa domesticus pigs (ca. 50 kg) were anesthetized with intraperitoneal injection of sodium thiopental (50 mg kg⁻¹). Identical procedures were performed to evaluate the time to hemostasis and blood loss, as mentioned previously. Six pigs were utilized, and five incisions were performed in each one, as previously described, and conventional gauze, Curaspon®, Surgicel®, and CDPE and CDPE-Cs hydrogels were applied.

Values are presented as the mean ± SD of at least six independent experiments. Significant differences between groups were evaluated by one-way ANOVA followed by Tukey’s multiple comparison tests. A sample size of six animals per group, for time to hemostasis and blood loss experiments, was expected to provide a power of ca. 0.9, with a Type I error probability for rejection of the null hypothesis of 0.05. Differences between different groups were considered significant for p values less than 0.05.