Materials

Acetic acid (glacial), anhydrous dimethyl sulfoxide (DMSO), benzene, phosphate-buffered saline tablets, 3,3′,4,4′-biphenyltetracarboxylic dianhydride (BPDA), ethanol, methyl benzoate, heparin sodium, formalin solution (neutral buffered, 10%), triethylamine (Et3N), urethane, sodium chloride (NaCl), paraffin, deuterium oxide (D2O), acetic acid-d4, sodium hydroxide (NaOH), and chitosan (molecular weight 310–375 kDa) were purchased from Sigma-Aldrich and used as received. β-Cyclodextrin was obtained from TCI America and dried in vacuo at 70 °C for 12 h prior to use. Nanopure water (18.2 MΩ cm) was acquired from an EMD Millipore Milli-Q water filtration system.

Characterization

Solution-state 1H and 13C NMR spectra were collected on a Varian Inova 500 spectrometer at room temperature. Solid-state cross-polarization magic angle spinning (CP-MAS) 13C NMR spectra were obtained on a Bruker Avance 400 spectrometer with a 4 mm rotor at a spin rate of 10.0 kHz. The instruments were interfaced to a LINUX computer using VNMR-J software, and spectra were processed with MestReNova version 9.0.1–13254 by Mestrelab Research S.L. Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) was performed on a Shimadzu IR-Prestige-21 spectrometer at a 1.0 cm−1 resolution. Thermogravimetric analysis (TGA) was performed on a Mettler-Toledo TGA 2 with a heating rate of 10 °C min−1 under argon atmosphere. Scanning electron microscopy (SEM) images were collected on a JEOL JSM-7500F FE-SEM equipped with a high brightness conical FE gun and a low aberration conical objective lens after platinum/palladium sputtering of the sample. Powder X-ray diffraction (PXRD) spectra were obtained on a Bruker D8 ADVANCE instrument with a Bruker LYNXEYE detector, with a 1 kW Cu X-ray tube, maintained at an operating current of 40 kV and 25 mA, operated in Bragg-Brentano para-focusing mode. Turbidity measurements were performed on a Shimadzu UV-2550 spectrophotometer at 450 nm with polystyrene cuvettes. Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer Nano ZS with polystyrene cuvettes. Elemental analysis was performed at Midwest Microlab, LLC in Indianapolis, IN.

CDPE hydrogel template synthesis

To a flame-dried round-bottom flask equipped with a stir bar was added β-cyclodextrin (1.750 g, 1.542 mmol), triethylamine (0.468 g, 4.62 mmol), and anhydrous dimethyl sulfoxide (14.42 mL). After dissolution, BPDA (1.361 g, 4.626 mmol) was added in one portion. The mixture was stirred for 5 min until the solids were evenly distributed throughout the mixture. The suspension was then poured into a shallow glass dish and heated to 70 °C on a hot plate for 2.5 h. After complete gelation, the system was allowed to return to room temperature. The gel was removed and cut to the desired shape with a razor blade and soaked in an excess of dimethyl sulfoxide for 1 h. The solvent was removed and exchanged with fresh dimethyl sulfoxide three additional times for 1 h each before placing the gel in an excess of water. The gel was then soaked in a saturated aqueous sodium chloride solution three times for 1 h each, before soaking three additional times in water for 1 h each. Samples were stored in water, or dried by lyophilization for characterization. 13C CP-MAS NMR (101 MHz, spin rate 10.0 kHz, ppm) δ 237.65–225.48, 179.16–160.74, 145.71–120.24, 105.51–92.82, 87.50–52.05. FTIR (cm−1): 3678–3015, 2927, 1776, 1710, 1603, 1576, 1363, 1289, 1245, 1145, 1077, 1032, 1002, 944, 845, 792, 769, 705. TGA: 25–90 °C, 6% mass loss; 90–235 °C, 4% mass loss; 235–280 °C, 29% mass loss; 280–330 °C, 11% mass loss; 330–390 °C, 14% mass loss; 390–465 °C, 8% mass loss; 465–500 °C, 10% mass loss; 18% mass remaining above 500 °C. Anal. calcd. for (C6H10O5)1·(C14H12O8Na)6·(H2O)4·(NaOH)0.6: C, 47.33; H, 4.00; N, 0.00; Na, 6.64. Found: C, 47.64; H, 4.23; N, 0.00; Na, 6.80.

CDPE stability and degradation study

A phosphate-buffered saline tablet was dissolved in the corresponding amount of deuterated water to obtain a solution of 0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium chloride. A small, intact portion of lyophilized CDPE was cut to size by razor blade and placed in an NMR tube with the PBS D2O solution to obtain a concentration of 15.0 mg mL−1. The sample was then gently agitated at 37 °C (60 rpm), and analyzed by 1H NMR spectroscopy at 24 h intervals. After 7 days, the solution was transferred to a cuvette, and transmittance at 450 nm was measured to quantify turbidity, followed by dynamic light scattering measurements. A second sample was analogously prepared from lyophilized CDPE and 1% acetic acid in deuterated water, at a 15.0 mg mL−1 concentration, and analyzed by 1H NMR spectroscopy at 24 h intervals, with storage at room temperature without agitation.

Chitosan loading to afford composite CDPE-Cs hydrogels

A chitosan solution was prepared by the dissolution of 5.000 g chitosan in 500.0 g acetic acid solution (1 wt% in nanopure water) to obtain a 1 wt% solution. After stirring for 24 h at room temperature, aqueous sodium hydroxide (5 M) solution was added dropwise, with vigorous stirring, until a pH of 5.5 was obtained. Pre-cut CDPE gels were submerged in the chitosan solution in a glass dish, and allowed to stand at room temperature for 7 days. Loaded gels were rinsed with water and lyophilized for characterization by IR spectroscopy and TGA. FTIR (cm−1): 3678–3015, 2927, 2853, 1710, 1653, 1576, 1363, 1295, 1245, 1154, 1077, 1032, 944, 894, 845, 769, 705. TGA: 25–135 °C, 12% mass loss; 205–360 °C, 43% mass loss; 360–500 °C, 12% mass loss; 33% mass remaining above 500 °C.

CDPE template removal

Template removal was achieved by soaking lyophilized loaded gels in 100 mL aqueous NaOH solution (5 M) for 15 min, followed by rinsing with an excess of water. The resulting chitosan monolith was then dried in vacuo before characterization. The masses of the lyophilized CDPE-Cs composite gel and resulting dried templated chitosan mat were both measured to determine the mass loss during template removal, and to estimate the chitosan concentration in the composite material. For solution-state NMR spectroscopy of the templated chitosan, ca. 15 mg of the material was sonicated in deuterated trifluoroacetic acid until dissolved. 1H NMR (500 MHz, CF3COOD, ppm) δ 5.56–8.85 (m, 2H), 4.74–3.13 (m, 12H), 2.26–2.12 (m, 3H). 13C CP-MAS NMR (101 MHz, spin rate 10.0 kHz, ppm) δ 172.82, 110.78–95.86, 87.86–69.17, 66.02–55.74, 25.49–18.06. FTIR (cm−1): 3647–2997, 2920, 2853, 1647, 1571, 1471, 1417, 1378, 1319, 1153, 1065, 1030, 992, 947, 895, 805. TGA: 25–90 °C, 9% mass loss; 90–200 °C, 3% mass loss; 200–325 °C, 43% mass loss; 325–500 °C, 16% mass loss; 29% mass remaining above 500 °C.

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 previously24,36 (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.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Source