Injectable and rapidly expandable thrombin-decorated cryogels achieve rapid hemostasis and high survival rates in a swine model of lethal junctional hemorrhage

Abstract

Effective therapies are urgently needed to stabilize patients with marginally compressible junctional hemorrhage long enough to get them to the hospital alive. Herein, we report injectable and rapidly expandable cryogels consisting of polyacrylamide and thrombin (AT cryogels) created by cryo-polymerization for the efficient management of lethal junctional hemorrhage in swine. The produced cryogels have small pore sizes and highly interconnected porous architecture with robust mechanical strength. The cryogels exhibit rapid shape memory properties and prove to be resilient against fatigue. These cryogels also show high water/blood absorption capacity, fast blood clotting effect, and enhanced adhesion of red blood cells and platelets in vitro. Further, in vivo, hemostatic efficacy tests in a lethal swine junctional hemorrhage model suggest that treatment with AT cryogels, especially AT-2 cryogels, achieves the least blood loss and the highest survival rate (100 %) compared to currently employed products such as XStat® and combat gauze. The high hemostatic performance of the cryogels may be attributed to highly interconnected porous architecture with small pore size and the use of thrombin as a pro-coagulant agent. Collectively, injectable and rapidly expandable thrombin-decorated polyacrylamide-based cryogels show significant promise as hemostatic material, offering effective management of marginally compressible junctional hemorrhages in prehospital settings.

Graphical abstract

Injectable and rapidly expandable cryogels consisting of polyacrylamide and thrombin (AT cryogels) are fabricated by cryo-polymerization for the efficient management of lethal junctional hemorrhage. Such cryogels show significant promise as hemostatic material, offering easy scale-up production and effective management of marginally compressible and irregularly shaped junctional hemorrhages in prehospital settings.

Highlights

• •Synthesis of cost-effective cryogels for the treatment of lethal swine junctional hemorrhage.
• •Cryogels exhibit highly interconnected porous network and robust mechanical stability.
• •AT cryogels are injectable and rapidly expandable to accelerate hemostasis.
• •AT cryogels stopped massive bleeding rapidly in the lethal swine hemorrhage model.

1. Introduction

Traumatic injuries leading to massive hemorrhage emerge as the foremost avoidable cause of fatalities within military combat, with the potential to prevent up to 90 % of these deaths [1]. A range of injuries contributes to this substantial hemorrhage, with the majority of the wound cases stemming from truncal (67.3 %) followed by junctional (19.2 %) and extremity (13.5 %) [[1], [2], [3]]. Within the civilian context, traumatic hemorrhage represents the second most prominent cause of mortality among trauma patients, as studies report rates ranging from 26 % to 40 %. In addition, the complete treatment of such injuries predominantly involves surgical procedures and requires transporting the patients to a point of care within a narrow time window of up to 3 h [1,4]. Hence, this underscores the critical significance of having highly effective hemostatic materials to promptly control bleeding, providing a vital bridge until patients can access hospital care. Unfortunately, there remains a significant gap in the availability of satisfactory hemostatic materials for addressing marginally/non-compressible hemorrhages.

A variety of hemostatic materials have been developed with distinct designs and functionality to stop massive bleeding and several were clinically approved, such as Celox™, Axiostat®, QuikClot®, HemCon®, etc. [[5], [6], [7]]. However, in the truncal or groin regions, high blood pressure and extensive bleeding pose a major challenge for their failure [7]. XStat®, an FDA-approved product consisting of compressed cellulose-based sponges, functions as a hemostatic adjunct with a distinct focus on controlling bleeding emanating from junctional wounds in the groin or axilla. Although XStat® shows some efficacy, several limitations, including lack of clotting ability, prolonged recovery time, risk of damaging surrounding tissues due to its high mechanical stress, high cost, and non-biodegradability, are still of grave concern [3,8,9]. Another FDA product for controlling bleeding in penetrating wounds is QuikClot® Combat Gauze (CG), but the hemostasis is ineffective with a long application time [8,10]. In addition, several types of materials with shape memory properties have been studied for controlling massive bleeding in deep irregular wounds, which were fabricated using multiple strategies such as 3D printing, freeze drying, gas foaming, and electrospinning in the form of hydrogels, cryogels, and nanofiber peanuts [5,[11], [12], [13], [14]]. However, these hemostats are associated with several potential drawbacks, such as unsuitable mechanical properties, lack of vital blood coagulation factors, limited testing primarily in small animal or non-critical hemorrhage models, and non-optimal delivery methods for the intended purpose [4,15].

The indispensable role of interconnected pores for hemostatic application has received considerable attention and such hemostats are becoming the material of choice [16]. In this context, cryogels possess an inherent property of forming interconnected porous structures that impart cryogels with characteristics of high blood absorption, mechanical flexibility, shape memory and injectable properties. Additionally, cryogels can be fabricated from a wide range of highly adaptable polymeric precursors, making them an ideal platform for integrating desired hemostatic agents [11,17]. Zhao et al. developed injectable cryogels to cease fatal noncompressible and coagulopathic bleeding [16]. The degradable cryogels presented promising results when evaluated for hemostatic efficiency in rabbit liver and subclavian swine models, apart from promoting wound healing compared to the control groups. In another study, Yoo et al. prepared cryogels from quaternized chitosan and mesoporous bioactive glass to cope with massive bleeding [18]. The hemostatic potential of these cryogels was demonstrated in a mouse liver model. Similarly, considering other ideal characteristics of cryogels, previous studies have substantiated the effectiveness of cryogels in various animal models of bleeding, but excluding majorly those related to lethal junctional hemorrhage [5,9,13,19,20]. However, these cryogels typically exhibit low mechanical strength and various strategies, including the use of multiple polymers/components, composites, and cotton, have been explored to enhance mechanical strength. Nevertheless, these endeavors frequently result in heightened complexity, prolonged fabrication time, and increased costs [6,11].

Hence, it is highly anticipated to fabricate economically feasible polyacrylamide (pAAm) -based shape memory and injectable cryogel with desired mechanical strength for lethal hemorrhages, especially junctional hemorrhage. pAAm was chosen owing to its highly hydrophilic nature, biocompatibility, and ease in tunability to achieve desired mechanical robustness, apart from its proven role in many biomedical applications, including bio-separation, tissue engineering, and drug delivery [[21], [22], [23], [24]]. Recent studies have suggested that pAAm-based materials exhibit quick and effective hemostatic applications but are still largely unexplored [[24], [25], [26]]. Moreover, incorporating bioactive/clotting factors is quintessential to elevate the procoagulant activity of hemostats. Thrombin, one of the central components of the clotting pathway, is crucial for fibrin generation besides promoting aggregation of platelets, necessary for endorsing rapid clotting not only in normal but also in coagulopathies [[27], [28], [29]]. In clinical settings, thrombin is directly employed as a freshly prepared solution to the targeted site, which, however, is associated with specific challenges such as risk of contamination, short half-life, and use of high doses [10,27,30,31]. Therefore, thrombin incorporation into the relevant material can reduce pre-application time, improve its storage shelf-life, and help overcome other limitations [27,30].

In this study, we aimed to fabricate injectable and quickly responsive shape-memory pAAm-based cryogels with high blood absorption and excellent clotting abilities for effectively managing lethal junctional hemorrhage in swine. The pAAm cryogels were loaded with thrombin (AT cryogels) to enhance comprehensive hemostatic efficacy. The developed shape memory cryogels exhibited the desired small pore size and interconnected porous architecture apart from good mechanical strength and shorter recovery time in contrast to commercial products XStat®and CG. The AT cryogels were further evaluated for physiochemical properties in vitro and the pre-clinical testing was carried out in a lethal swine junctional hemorrhage model. Next, the pre-clinical hemostatic efficacy of the developed thrombin-loaded pAAm (AT-2) cryogels and other commercial hemostats was measured in terms of blood loss, rebleeding time, post-treatment survival time, and survival rate.

2. Materials and methods

2.1. Synthesis of polyacrylamide and thrombin-loaded cryogels

The pAAm cryogels were prepared to free radial polymerization involving acrylamide (AA) as monomers units and N′, N′- methylene-bis-acrylamide (MBAAm) as crosslinker. A series of pAAm cryogels named pAAm-1, pAAm-2, pAAm-3 and pAAm-4 were prepared from different acrylamide concentrations, 1.5 %, 2.5 %, 3.5 % and 4.5 %, respectively. To prepare pAAm-1 cryogels, 150 mg of AA was added to 8 ml of degassed distilled water and dissolved completely using a magnetic stirrer. After complete dissolution, 50 mg of MBBAm in the ratio of 3:1 (AA: MBAAm) was added to this solution and stirred further. Next, 10 mg of ammonium persulfate (APS) and 100 μl of Tetramethylethylenediamine (TEMED) were separately dissolved in 1 ml of degassed distilled water. All the solutions were allowed to precool at 4 °C for 30 min. The precooled AA and MBBAm solution was first added with APS with quick mixing, followed by the addition of TEMED. The solution was immediately mixed, poured into the desired molds, and transferred to a cryostat, maintained at −14 °C for 16 h. Before pouring the solution into the molds, plastic syringes (2.5 ml), in this case, were completely sealed with parafilm at the bottom nozzle to avoid any leakage into or out of the mold. The cryogels were later freeze-dried for future use. Subsequently, all other pAAm cryogels, i.e., pAAm-2, pAAm-3, and pAAm-4, were fabricated by following the same procedure.

Similarly, for synthesizing the thrombin-loaded cryogels, 350 mg of AA (optimal concentration) was dissolved in degassed distilled water, followed by adding MBAAm in the same ratio. The solution was first precooled and added with varying concentrations of thrombin (T-1: 10 IU; T-2: 50 IU; T-3: 100 IU). The precursor solution was mixed with precooled APS and then TEMED solutions sequentially, and the rest of the steps were similar to those described above. The prepared AT cryogels (AT-1, AT-2, and AT-3) were washed three times to remove loosely bound polymer/thrombin and dried and stored at −80 °C for future use. The cryogel samples were evaluated to determine the presence of any residual acrylamide using liquid chromatography-mass spectrometry as detailed in the Supplementary Materials (Materials and Methods).

2.2. Characterization of morphology, microstructure and porosity

The developed pAAm and AT cryogels were assessed for morphology, pore size and porosity using SEM and Micro-CT analysis, as detailed in the Supplementary Materials (Materials and Methods).

2.3. Water/blood absorbability

The fluid absorption rate and swelling ratio of the developed cryogels were evaluated by immersing the samples in water and anticoagulated blood, as detailed in the Supplementary Materials (Materials and Methods).

2.4. Mechanical characterization

The mechanical strength of the cryogels was determined in both dry and wet states by cyclic compression testing. The details are in the Supplementary Materials (Materials and Methods).

2.5. Shape memory property and injectability

The shape-memory capability of the AAm, AT-1, AT-2, and AT-3 cryogels was examined, as reported previously [16]. The AAm and AT cryogels were prepared in cylindrical form and were initially cut to 1 cm in length. The samples were submerged in water and squeezed to drain water to attain a shape fixed state. Next, the samples in the fixed form were exposed to water/blood to measure recovery time and shape recovery ratio. Further, compression testing was performed to accomplish the shape-fixed state of AAm and AT cryogels. The testing was conducted by applying an 80 % compression strain to the cryogels for 5 min and then adding water while still under the same strain. Compression force was gradually released and the ability of the cryogels to revert to a normal state was observed and photographed. In addition, the change in surface morphology in original form, shape fixed state and recovery state was analyzed by SEM imaging.

The cryogel samples were prepared in cylindrical shapes (8 mm diameter) to validate their injectable property. Once produced, the water from the samples was impelled out to establish a shape-fixed form. Subsequently, the shape-fixed croygels were packed into an applicator with a 20 mm diameter and injected into an enclosed water system. The ability of the croygels to absorb the fluid and recover to their original form was recorded and photographed.

2.6. Release of thrombin from AT cryogels

The thrombin release from the AT cryogels was evaluated using an ELISA kit assay, as reported previously [14]. The details are provided in the Supplementary Materials (Materials and Methods).

2.7. Hemocompatibility study

The compatibility of the developed cryogels was examined by a hemolysis assay using anticoagulated blood. The details are provided in the Supplementary Materials (Materials and Methods).

2.8. Cell viability study

The toxicity of the AT cryogels was carried out in-vitro using human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVEC) and are detailed in the Supplementary Materials (Materials and Methods).

2.9. Hemostatic efficacy in vitro

The whole blood clotting index (BCI) was employed to validate the clotting efficiency of the developed cryogels [9]. The cryogels and XStat® were precisely cut and transferred in the glass vials and prewarmed at 37 °C. Each scaffold containing vial was added with 100 μl of anticoagulated human blood and incubated at 37 °C. At pre-set time points ranging from 30 s to 10 min, 15 ml of deionized water was added to the vials without disturbing the clot. Adding water ensures that the loosely bound erythrocytes are released, which undergo hemolysis. Consequently, to quantify the released hemoglobin from lysed erythrocytes, absorbance at 542 nm was measured using a microplate reader. For reference, 100 μl of blood in 10 ml of deionized water was also measured to be an absorbance reference. The BCI was calculated using the formula as follows:

$$BCI(\%)=\frac{Is-Io}{Ir-Io}\times 100$$

Where Is, Io and Ir are the absorbance of a scaffold containing group, PBS, and reference, respectively.

2.10. Material-cell interaction: in vitro adhesion of platelets and red blood cells

The effect of material interface on blood cells was assessed by platelet and red blood cells (RBCs) adhesion test [13,16]. The various groups examined in this study include AAm, AT-1, AT-2, AT-3 cryogel groups and commercial product XStat® as positive control. All samples were fabricated in cylindrical shape (8 mm diameter) and cut into discs of 3 mm thickness. The anticoagulated human blood was used to produce platelet-rich plasma (PRP), following centrifugation at 2500 rpm for 15 min. The platelet adhesion on the AT cryogel samples and other groups was tested by adding 100 μl of PRP on their surfaces. The PRP-added samples were incubated for 1 h at 37 °C. The platelet-adhered scaffolds were gently washed with PBS (three times) to wipe out the material surface from non-adherent platelets. Afterward, the samples underwent fixation with 2.5 % glutaraldehyde for a period of 4 h. Following fixation, the samples were subjected to dehydration using varying ethanol gradients (50 %, 70 %, 90 % and 100 %), with each step lasting 10 min. Once the samples dried, the adhered platelets and their morphology were observed under SEM. RBC adhesion was determined using a similar method; however, separation of erythrocytes was done by centrifuging human blood at 1000 rpm for 15 min and then gently PBS washed. Next, 100 μl of RBCs were dropped on the surface of cryogels and XStat®. The samples were PBS washed post 3 min incubation at 37 °C and the subsequent steps closely followed those mentioned earlier.

2.11. In vivo hemostatic efficacy test in a lethal swine junctional hemorrhage model

The efficacy of hemostatic cryogels in addressing marginally compressible junctional hemorrhages was assessed through a swine model involving the complete transection of the femur artery and vein, following the established reference procedures [16,32]. The in vivo studies were conducted in strict adherence to the protocol approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC) (Protocol No.: 22-051-08-EP). The studies involved a lethal swine femur artery-venous complete transection hemorrhage model as a preclinical model. Swine were randomly distributed into 5 groups to have average weight; each group had 5 animals.

Sample preparation: A total of 25 pigs were used for this lethal junctional hemorrhagic model, with each group having 5 animals. The various groups included were no treatment group as control (Ctr), treatment groups: AAm group (pAAm cryogels without thrombin), AT-2 group (pAAm cryogels containing thrombin) and commercial products, XStat® and QuikClot Combat Gauze (CG). In treatment groups, AAm and AT-2 shape memory groups were compressed and packed in the syringes, while XStat® and CG were applied directly as received. The treatment administered to each swine of the respective groups involves 2 syringes of AAm cryogels (∼36 g), 1 syringe of AT-2 cryogels (∼15 g), 2 syringes of XStat®, and 1 pack of CG.

Swine preparation and surgical procedure: Before the surgical procedure, the animals underwent a 12 h fasting period, with unrestricted access to water and were pre-medicated on the morning of surgery. They were pre-anesthetized using an intramuscular injection of Telazol®-Ketamine-Xylazine and taken to the operating room, placed in the supine position with legs secured to an operating table, and intubated with 6–7 French endotracheal tube. The animals were anesthetized and maintained under isoflurane anesthesia with supplemental oxygen at a rate of 1–2 L/min throughout the entire procedure until the point of either death or euthanasia. A peripheral ear intravenous (IV) line (20-22G) was placed as needed for additional medications. A right neck cut down was carried down through subcutaneous tissue and platysma down to the sternocleidomastoid muscle (SCM) at which point dissection was carried laterally to expose the external jugular artery. The vein was canalized with 16G angiocath for fluid and medication administration, and the IV catheter was secured surrounding tissue. Dissection was then carried medial and deep to the SCM to expose the carotid artery, after which the anterior wall of the artery was canalized with a 20G angio cath, secured to surrounding tissue, and connected to an arterial line monitoring device.

To make the porcine hemorrhage model more closely resemble human physiology, a midline abdominal laparotomy followed by splenectomy was performed. This step aimed to reduce auto-transfusion by the contractile porcine spleen, following the recommendation from the US Army Institute of Surgical Research in San Antonio, TX. Following splenectomy, the free spleen was weighed, and warm lactated Ringers (LR) fluid was given 3 × the spleen weight to replace the reserved volume of blood in the spleen. Subsequently, a bladder cystostomy was done to prevent inadvertent compression of pelvic vascular structures from an over-distended bladder. The abdominal wall was then re-approximated with multiple penetrating clamps.

Finally, an 8 cm incision in the right groin was made to expose the underlying femoral vessels (artery and vein). A 2-0 silk vessel loop was used to encircle the femoral artery and vein proximally and a complete transaction was created to simulate junctional hemorrhage. Blood was freely allowed to flow for 30 s and suctioned to measure post-injury blood loss. After 30 s of free bleeding, in the case of treated groups, samples of each group fixed in the syringes were injected into the groin wound, and a slight manual pressure was applied for 3 min. Later, the pressure was removed, and blood was collected from the arterial line at 0, 15, 30, 60, 120, 180 min or pre-terminal point if the subject expired before 3 h. In addition, vitals were recorded every minute for the first 10 min, then at 15, 30, 60, 120, 180 or pre till the terminal point. The hemostatic potential of the AT-2 cryogels and other groups was measured in terms of blood loss (post-injury + post-treatment), bleeding time and survivability. After 3 h of observation, the abdomen was re-entered, the diaphragm was incised to expose the IVC; weight-based fetal plus (1ml/10 kg) of fatal plus was injected into the IVC and after cessation of cardiac activity, the IVC was transected.

2.12. Statistical analysis

All the experiments were carried out at least in triplicates and the results are presented as the means ± SD. GraphPad Prism 10 software was used to analyze the data. The statistical difference was determined by applying the student's t-test, one-way and two-way ANOVA test. For statistical significance, the value of p ≤ 0.05 was considered. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns = non-significant. To achieve a statistical power of 0.9 and maintain a significance level of 0.5, five rats were required for each treatment group.

3. Results

3.1. Fabrication of acrylamide-based cryogels without and with the incorporation of thrombin

In this study, we fabricated a series of cryogels based on hydrophilic pAAm and thrombin using cryogelation technology, followed by their characterization and application in a lethal swine hemorrhagic model (Fig. 1). The cryogels should fulfill the following design criteria: (i) having a uniformly interconnected macroporous structure; (ii) having high porosity and suitable mechanical strength; and (iii) having quickly responsive shape-recovery and fast blood clotting properties. Based on these criteria, we initially synthesized pAAm-based cryogels using four concentrations of AA (e.g., 1.5 %, 2.5 %, 3.5 %, and 4.5 %) and crosslinker MBAAm (AA: MBAAm = 3:1) via free radical polymerization. APS/TEMED were incorporated into the polymer solution as free radical initiators/accelerators before subjecting it to subzero temperature (−14 °C) for cryo-polymerization. During the cryogelation process at sub-zero temperatures, the homogenized polymeric solution undergoes phase separation, forming aqueous frozen and unfrozen polymeric phases. The ice crystals, later on thawing/drying create interconnected porous structures in the cryogels with high porosity (Fig. 1a). Using LCMS, the cryogel samples showed a very minimal amount of residual acrylamide after the first wash (1.2 μg/g) and was clear after 3rd wash. The synthesized cryogels exhibit shape memory properties triggered by water/blood, rapidly expanding to fill the injured bleeding site for effective hemostasis (Fig. 1b and c).

Fig. 1.

Schematic illustration of synthesizing pAAm-based cryogels, their shape recovery property and application in lethal junctional hemorrhage. (a) The synthetic procedure of pAAm-based cryogels. (b) The shape recovery property of cryogels. (c) The application of injectable and expandable cryogels for managing marginally compressible junctional hemorrhage in swine.

The developed pAAm cryogels were validated to identify the optimal concentration for the desired morphological architecture, mechanical stability, and clotting ability. The cryogels (dry state) showed uneven and irregular shapes at lower concentrations (pAAm-1 and pAAm-2), while uniform cylindrical shape was maintained in the cryogels prepared at higher concentrations (pAAm-3 and pAAm-4) (Fig. S1). SEM images revealed larger and non-uniform porous structures in pAAm-1 and pAAm-2, while uniform porous structure was observed in pAAm-3 and pAAm-4 (Fig. S1). These variations were further confirmed by micro-CT analysis, showing more irregular morphology and larger pores (marked by arrows) for the cryogels, pAAm-1 and pAAm-2, when compared to the cryogels, pAAm-3 and pAAm-4 (Fig. S2a). The variations in morphology and porous architecture among the developed cryogels can be ascribed to differences in concentration, as reported previously [23]. In addition, all the pAAm cryogels displayed high porosity, while varying pore sizes were observed in the pAAm cryogels, specifically pAAm-1 and pAAm-2, exhibiting large pore sizes (Figs. S2b and c).

Next, the developed cryogels were examined for water/blood absorption rate and swelling capacity to validate their potential application in hemostasis. The results showed that pAAm-3 and pAAm-4 cryogels absorbed water/blood more rapidly and exhibited higher absorption capacity than pAAm-1 and pAAm-2 (Figs. S3a–d). Moreover, physical observation of the developed cryogels (wet state) revealed significant frailty of pAAm-1 followed by pAAm-2 in contrast to the pAAm-3 and pAAm-4 cryogels (Fig. S4a). Due to the weak mechanical properties owing to large pore sizes, slow fluid uptake rate, and low fluid absorption capacity of pAAm-1 and pAAm-2, no detailed mechanical study was performed and these cryogels were unsuitable for the desired application. The cyclic compressive testing of pAAm-3 exhibited good mechanical strength and shape recovery properties. In the case of pAAm-4, the strength was higher, but this resulted in a loss of recovery (Figs. S4b and c). Hence, considering these results, pAAm-3 (now named AAm) was selected for further study.

During cryogelation, pAAm forms the essential backbone of the cryogel network for hemostatic applications by providing an interconnected porous network with suitable flexibility, mechanical and rapid shape recovery properties. In addition to possessing these properties ideal for hemostatic applications, the ability to swiftly staunch excessive bleeding is a prerequisite for practical use in managing lethal junctional hemorrhage. Therefore, thrombin, a potent coagulation factor, was incorporated into the AAm cryogels in three different concentrations (e.g., AT-1, AT-2, and AT-3) to enhance their hemostatic efficacy, which was comprehensively validated both in vitro and in vivo. Fig. 2a shows the digital images of the AAm cryogels with and without the incorporation of thrombin and XStat®, indicating their cylindrical shape. The strategy involving acrylamide as the material choice and the use of the cryogelation process provides a straightforward approach, facilitating the integration of hemostatic agents and enabling scale-up production for commercialization.

Fig. 2.

Morphology and structure of pAAm-based cryogels and XStat®. (a) Digital images, SEM images, and micro-CT images of pAAm-based cryogels and XStat®. (b) and (c) The porosity and pore size of pAAm-based cryogels and XStat®.

3.2. Characterizations of morphology, microstructure and porosity

The surface morphology and porous architecture of AAm and AT cryogels were examined by scanning electron microscopy (SEM) and micro-CT (Fig. 2). All the cryogels (AAm, AT-1, AT-2 and AT-3) exhibit uniform pore distributions and well-interconnected porous architectures, and thin, and smooth pore walls, while the commercially available hemostat XStat® display rough and thick pore walls, along with uneven pore size distributions (Fig. 2a). The results were further confirmed by the micro-CT analysis, which revealed a more uniform porous structure of cryogels as compared to XStat® (Fig. 2a). This is illustrated through 3D and cross-sectional images, respectively. A nonhierarchical porous surface with a substantial difference in pore size distribution was evident from the micro-CT images (longitudinal and cross-sectional) (Fig. 2a). Further micro-CT analysis demonstrated that all the cryogels exhibit high and similar porosity of approximately 85 ± 7 %, significantly higher than the porosity of XStat® (∼75 ± 4 %) (Fig. 2b). In addition, pore size estimation revealed comparable results among the cryogel groups, with pore sizes ranging from 45 to 60 μm. In contrast, XStat® exhibited larger pore size in the range of 130–190 μm (Fig. 2c). These results also revealed that thrombin incorporation does not affect the morphology of AAm cryogels. The cryogels’ uniform and interconnected porous architecture, coupled with the desired small pore size, may contribute to enhancing the hemostatic effect. This enhancement is achieved by boosting the fluid absorption rate and facilitating the entrapment of clotting factors, thereby rapidly activating the coagulation cascade.

3.3. Water/blood absorbability

For an ideal hemostatic material, a fast liquid absorptive rate and high absorption capacity are the primary factors conducive for promoting hemostasis. The well-interconnected porous structure of cryogels essentially facilitates fluid absorption capacity and provides a high ability to amass blood cells/clotting factors, promoting rapid coagulation [11,13]. Fig. 3 shows the fluid absorption rate/capacity of the AAm, AT cryogels (AT-1, AT-2 and AT-3), and XStat®. All the cryogels, both with and without incorporation of thrombin, exhibited approximately 96 ± 7 % water absorption within just 5 s. In comparison, the absorption rate of XStat® was slower, demonstrating only about 75 ± 5 % water absorption in 5 s, reaching approximately 97 ± 4 % in 10 s (Fig. 3a). As expected, the cryogels initially displayed a higher water absorption capacity. However, once swelling equilibrium was reached, there was no difference in water absorption capacity between the cryogels and XStat®, but estimated at approximately 1.6 ± 0.1 g/cm³ (Fig. 3b). A similar trend was observed for blood absorption, with cryogels showing a faster blood absorption rate initially compared to XStat®. The blood absorption rate in cryogels reached approximately 90 ± 5 % within 5 s (Movie S1), while for XStat®, only around 60 ± 6 % blood uptake was observed after 5 s (Fig. 3c). The blood absorption of XStat® gradually increased over time, reaching approximately 98 ± 8 % absorption in 15 s. In all cryogel groups and XStat®, the blood absorption capacity became comparable after reaching the swelling equilibrium point, demonstrating a capacity of approximately 1.52 g/cm³ (Fig. 3d).

Fig. 3.

Absorption and swelling properties of the AT cryogels. (a)–(d) The absorption rate and capacity of the AAm, AT cryogels and XStat® in water/blood. The cryogels showed a significantly higher water/blood absorption rate compared to XStat®. (e) and (f) The swelling ratio of the AT cryogels in water and blood, respectively.

Fig. 3e and f shows the swelling ratio (%) of the cryogels and XStat®, indicating a high swelling ratio (%) of cryogel groups (AAm, AT-1, AT-2, and AT-3) and XStat® when submerged in water and blood. The cryogel groups showed a swelling ratio (%) of approximately 970 ± 130 in water and 1090 ± 110 in blood. The value of swelling ratio (%) of XStat® was slightly higher (1170 ± 165 in water and 1110 ± 175 in blood) compared to the cryogels groups but there was no statistically significant difference. Overall the results revealed that the cryogel groups and XStat® exhibited a similar fluid absorption rate and swelling behavior. The results also indicated thrombin loading did not influence the absorption and swelling behavior of the AT cryogels. The difference in absorption rate between cryogels and XStat® could be attributed to the disparity in porosity, pore size and interconnected porous structure. The water/blood absorbability and swelling properties may make AT cryogels an excellent candidate for hemostatic applications, especially in the context of junctional hemorrhage.

3.4. Mechanical characterization

Mechanical strength and flexibility are primary requirements of hemostats during practical scenarios, and the AT cryogels, as a hemostatic material, should possess suitable mechanical properties. The mechanical property of the AAm and AT cryogels was tested in both dry and wet states (Fig. S5). All the samples were compressed by 80 % with respect to the original length in a compressive strain test (Figs. S5a and b). The results indicated that the AT-1, AT-2 and AT-3 groups had compressive stress of 125 ± 24 kPa, 140 ± 30 kPa, and 105 ± 40 kPa, respectively, slightly lower than the AAm group (145 ± 35 kPa). Although there was a slight difference in the compressive stress between AAm and other AT groups, it was statistically non-significant. In addition, the subtle difference among AT cryogel groups might be accredited to thrombin loading, showing a marginal decrease in the mechanical strength with increasing thrombin amount. The compressive stress-strain curve of the samples revealed a non-linear behavior, primarily observed in flexible and shape memory sponges. From the curve, the cryogels during compression, sequentially undergo elastic and plastic deformations. This usually occurs by expelling air from pores, followed by subsequent flexing, collapse, or fracture in sample walls. Finally, a quick increase in the slope leading to maximum stress point is attained due to cryogel compactness, observed at a pre-set strain of 80 %. The compression test showed that all AT cryogels exhibit non-linear behavior and excellent mechanical strength in a dry state, essential for maintaining structural integrity during handling and storage.

Further, the AAm and AT cryogels were evaluated for the effect of thrombin concentration on mechanical stability in a wet state using cyclic compression at an 80 % compressive strain. The results showed that the cryogels maintained their original shape and were flexible enough to withstand compression (Figs. S5c–f). As expected, the cryogels in the wet state exhibited lower compressive stress, in contrast to the dry state, having more stiffness (Fig. S5c). The AAm group showed a compressive stress of ∼1.7 kPa, similar to thrombin-loaded groups. The compressive stress in AT cryogel groups was determined in the range of 1.5 kPa–1.8 kPa (Figs. S5d–f). The results demonstrated the sample's suitable mechanical strength, essential for withstanding the strain during injection at the bleeding site and retaining structural integrity to restore the original shape. The excellent mechanical properties of the AAm and AT cryogels might be attributed to the highly porous and flexible network of cryogels.

3.5. Shape recovery and injectable properties

During practical applications, hemostatic materials may encounter diverse wounds, necessitating their adaptation to various configurations. For critical injuries at junctional extremities, it is strongly advised to pursue hemostats that possess injectable properties and robust shape-memory capability. Given their excellent mechanical properties, we proceeded to investigate the water/blood-induced shape memory capability and injectability of the AT cryogels (Fig. 4). The dry/wet samples of AAm and AT cryogels underwent structural compactness upon 80 % compression to attain a fixed state and rapidly recovered their original shape upon contacting water/blood (Fig. 4a). The shape recovery time observed in AAm and AT cryogels was remarkably shorter (water, 4.5 ± 1 s and blood, 6 ± 1 s), contrary to XStat® (water, 8 ± 1.5 s and blood, 13 ± 1 s) (Figs. S6a–d). In addition, all cryogels and XStat® achieved a 100 % recovery ratio when exposed to water and blood.

Fig. 4.

Shape fixing and recovery properties of pAAm-based cryogels. (a) The digital images of the pAAm-based cryogels in the original, compressed, and shape recovery states triggered by water/blood. (b) Digital images and SEM images show the morphological properties of the cryogels in the original, compressed, and shape recovery states. (c) The schematic representation shows the effect of applied force and shape recovery of the cryogel. (d) Digital images of the cryogels before/after compression and subsequent complete shape recovery of all the samples.

All the samples were compressed to 80 % of the initial length for 5 min, and the digital images of respective shapes of the AAm and AT cryogels (AT-1, AT-2 and AT-3) before and after compression were shown in the first column of Fig. 4b. The morphological and structural changes in the cryogels in compressed and recovery states were further analyzed by SEM (Fig. 4b). The SEM images of the shape memory cryogels showed closed pores in a compressed state. In the shape-recovery state, the cryogels restored a uniform porous structure comparable to the original state. Fig. 4c shows a schematic illustrating the effects of applied forces in various directions and subsequent water/blood-triggered shape recovery. The AT cryogels exhibited rapid shape recovery from compressed states in the forms of discs, bent shapes, and flat sheets to their original shape once the applied force was released upon contact with water (Fig. 4d).

The consistent shape and strength retention highlight a resilient and flexible cryogel network with robust fatigue resistance. These characteristics enable the compression of the cryogels into a syringe, followed by rapid water/blood absorption and shape recovery after injection. This effectively creates a sealing barrier at the injection site, filling the cavity. Fig. 5 illustrates the compressed AT-2 cryogels packed in an injecting device (a modified plastic syringe). The demonstration revealed the ease of injecting the compressed cryogels and their instant ability to re-expand to the initial state within 4 s (Fig. 5candd and Movie S2). Additionally, XStat®, available in the form of compact discs, expands only in the longitudinal direction, exerting pressure primarily in that direction and may pose challenges in effectively compressing the arteries, particularly in cases of narrow and deep wounds [33]. In the case of AT cryogels, the ability to recover shape from distinct types of compressed forms makes them promising hemostats to fill narrow, irregular and deep wounds. The results elucidated the remarkable compressibility, resistance to fatigue, and swift shape recovery of our AT cryogels. This balance between mechanical strength and compressibility positions the AT cryogels as highly promising for contemporary hemostatic applications, particularly in the context of junctional hemorrhage.

Fig. 5.

Packaging of the developed cryogels. (a) AT shape memory cryogels in expanded form. (b) Cryogels were compressed and packed in a syringe. (c)–(e) Injecting compressed cryogels into the water at 0 s, 1 s and 4 s, respectively.

3.6. Thrombin release from AT cryogels

To augment the hemostatic effectiveness of cryogels, thrombin, a clotting factor commonly used in clinical settings, was incorporated. Fig. S7a illustrates the release kinetics of thrombin from the AT-2 cryogel. The results revealed a slower release of thrombin, around ∼20 % within the initial 4 h, and increased up to 60 % after 72 h, which may be ascribed to the fabrication process and non-covalent intermolecular interaction. The released thrombin plays a central role directly in the final step of the blood coagulation process by facilitating the insoluble fibrin conversion from fibrinogen. Therefore, incorporating thrombin would notably enhance the hemostatic efficiency of the cryogel, particularly in cases involving coagulation disorders that are characterized by thrombin deficiency.

3.7. Hemocompatibility study

Hemocompatibility, a crucial factor in determining the potential translational application of the cryogels, was assessed by a hemolysis test using isolated human erythrocytes. This test signifies higher cell lysis at elevated absorbance of hemoglobin (Hb) and minimal to no lysis of erythrocytes at lower absorbance. Fig. S7b shows the hemolytic effect of the cryogels and other control groups. The AAm and AT cryogel (AT-1, AT-2 and AT-3) groups and XStat® exhibited negligible hemolysis (<2 ± 1 %), showing a non-significant effect on erythrocytes. As expected, no hemolysis was observed in negative PBS control, in contrast to the Triton X-100 (positive control) group, which induced 100 % hemolysis of RBCs. The results illustrated that the hemolytic effect of approximately 2 % falls below the safe range of 5 %, aligning with findings reported in the literature [34]. Therefore, the developed cryogels demonstrate good hemocompatibility and are deemed safe for use as hemostats.

3.8. Cell viability study

As an ideal hemostat and its direct contact with injured tissue, the material should be cytocompatible. The cell viability of cryogels, both with and without the incorporation of thrombin, was assessed in vitro using the CCK-8 assay over a three-day period. Figs. S7c and d show the effect of the cryogels with/out thrombin on HDFs and HUVEC, respectively. The cryogel group's AAm, AT-1, AT-2 and AT-3 showed similar cell growth as observed in the 2D control group after 24 h. On days 2 and 3, the cryogels showed a slight decrease in HDFs growth compared to the 2D group; however, the difference was statistically non-significant. Similarly, the cell viability of HUVEC cells in the cryogel-treated groups was comparable to that of the 2D group throughout the experiment. In both HDFs and HUVECs, cell proliferation generally exhibited an upward trend upon treatment with AAm, AT-1, AT-2, and AT-3 cryogel groups. In addition, the results showed that incorporating thrombin into the cryogels did not induce any cell toxicity. Overall, the results indicated that all cryogel groups displayed good cytocompatibility.

3.9. Hemostatic efficacy in vitro

The blood clotting ability of the cryogels was determined by assessing the BCI. The BCI determines the non-clotting RBCs by measuring the absorbance of Hb. The BCI value is inversely related to the clotting ability, where a low value indicates a higher clotting ability of the material [35]. The results demonstrated the Ctr and XStat® groups had a clotting time of approximately 605 s, whereas AT-2 and AT-3 exhibited faster clotting (60 ± 5 s), followed by AT-1 (∼200 s) and AAm (∼380 s) groups (Fig. 6a). As expected, the BCI value was the highest in the Ctr group (100 %) due to the absence of any coagulation ability (Fig. 6b). The BCI value in the AT-2 and AT-3 groups was ∼22 % after 180 s which was significantly lower compared to the AT-1 group and other groups. However, the clotting ability of AT-1 gradually improved, and eventually, the difference in BCI values became negligible compared to that in the AT-2 and AT-3 groups. The BCI value in the AAm group (∼40 %) indicated lower clotting ability than AT cryogel groups but was higher compared to the ones in the XStat® (∼65 %) and control groups. Notably, the XStat® group showed a higher BCI value than all cryogel groups, indicating its poor clotting ability.

Fig. 6.

Blood clotting and material–cell interaction of pAAm-based cryogels and XStat®. (a) and (b) Blood clotting time and blood clotting index of pAAm-based cryogels and XStat®. (c) Digital image shows the blood clotting ability of acrylamide-based cryogels and XStat®. (d)–(g) Digital images of the AT-2 cryogel and XStat® before and after blood absorption. (h) SEM images show the enhanced adhesion of RBCs and platelets on thrombin-loaded cryogels (AT-1, AT-2 and AT-3) in contrast to groups AAm cryogel and XStat®. (i) Schematic illustration of blood clotting formation in the AT cryogels with interconnected pores.

Fig. 6c shows the photos of blood in the vials after different treatments, clearly indicating better clotting ability of the AT cryogel groups than other control groups. Particularly, AT-2 and AT-3 groups showed rapid and stable clot formation, followed by AT-1 and AAm groups. The XStat® group exhibited slow coagulation with blood cells diffusing from a weak clot, and no clotting was observed in the Ctr group. In addition, for the intended application, AT-2 cryogel and XStat® were used in the compressed states to evaluate their clotting ability while quickly returning to their original shapes (Fig. 6d–g). The remarkable clotting property of AT cryogel groups may be primarily endorsed to thrombin incorporation. The clotting effect in AT cryogel and AAm groups could be attributed to the small pore size and interconnected porous architecture. This property significantly aids in absorbing the blood quickly and amasses RBCs, platelets, and clotting factors inside the cryogels, leading to rapid clot formation. The poor clotting ability in XStat® may be ascribed to its large pore size, absence of an interconnected porous structure, and lack of clotting factors [5,16,18].

3.10. Material-cell interaction: in vitro adhesion of platelets and RBCs

To better understand the effect of AT cryogels on blood coagulation and determine the clotting mechanism, the influence of material interface on RBCs/platelets was evaluated. The adhesion of RBCs and platelets interacting with the material surface and the subsequent changes in their shape and morphology are shown in Fig. 6h. SEM images showed the adhesion of RBCs in all cryogel and XStat® groups. The number of adhered RBCs varied substantially among the groups. A higher number of aggregated RBCs were observed in the AT-2 and AT-3 groups, followed by the AT-1 group, than in the AAm and XStat® groups. Interestingly, the AAm cryogels exhibited a larger number of adhered RBCs than XStat®. The platelet aggregation and activation play a central role during the initial steps of the coagulation cascade in forming a clot [28]. The results demonstrated a higher number of adherent platelets in AT-2 and AT-3 groups than in AT-1, AAm and XStat® groups. The AT-1 and AAm groups demonstrated a similar number of adherent platelets, which were significantly higher than those observed in the XStat® group. In the XStat® group, only a sparse number of platelets adhered, and no aggregation was observed. In addition, the SEM images of AT-1, AT-2 and AT-3 groups revealed an irregular shape and pseudopodia stretching, indicating a distinctive platelet morphology that endorses the activated forms of platelets. In the case of the AAm group, a certain number of platelets showed morphological changes, while the XStat® group failed to promote sufficient platelet adhesion and activation.

These results validated excellent adhesion and aggregation of RBCs/platelets and platelet activation in the AT cryogels, particularly AT-2 and AT-3 cryogels, aligning well with their rapid blood clotting ability. Moreover, we speculate that the synergistic effect of thrombin incorporation, small pore size, and a highly interconnected porous architecture could contribute to the rapid blood clotting observed in injectable and shape-adaptable AT cryogels (Fig. 6i).

3.11. Hemostatic efficacy in a lethal swine junctional hemorrhage model

The pre-clinical hemostatic efficacy of the injectable and rapid shape memory AT cryogels was examined in a lethal swine junctional hemorrhage model. A lethal swine model was established through the complete transection of the femoral artery and vein to replicate penetrating junctional injuries, encompassing scenarios related to the axillary and subclavian arteries (Fig. 7). These injuries are associated with considerable rates of mortality in both military and civilian environments [16]. To more closely emulate human physiology, a midline abdominal laparotomy followed by splenectomy was conducted in this porcine hemorrhage model. The animals were randomly distributed into 5 groups, including the control group without treatment (Ctr), AAm, AT-2, XStat® and CG. Among the AT cryogel groups, the AT-2 group was selected and further validated for potential pre-clinical hemostasis because of its rapid clotting ability and enhanced adhesion and activation of RBCs/platelets. The XStat® and CG, used for junctional hemorrhage in prehospital and hospital scenarios, were used as positive control groups. Fig. 7a presents a schematic illustrating the surgical intervention and subsequent treatment steps followed until the end of the experiment, which lasted 180 min (Movie S3). Briefly, the swine femoral artery and vein were transected, followed by free blood blow for 30 s to mimic the injury. For the control group, no treatment was applied. For the treatment groups, the materials were applied through either injection (AAm, AT-2, XStat®) or packing (CG), and cotton gauze was placed on the top and compressed manually for 3 min. Then, the animals were monitored for 180 min.

Fig. 7.

Hemostatic performance of cryogels in managing lethal junctional hemorrhage in swine. (a) Schematic illustrating the procedure for developing a swine lethal groin injury model and treatment with injectable and expandable cryogels. (b) Digital images show hemostasis of the bleeding site and after treatment with the AAm and AT-2 cryogels, XStat®, and combat gauze (CG), while no hemostasis was observed in the control group. (c) The time taken to apply the treatment at the injury site. (d) Total blood loss after 30 s of post-injury and after applying the treatment.

Fig. 7b shows the representative images of the treated injured site immediately after releasing manual compression and at the end of the experiment, indicating the order of rapid and effective blood coagulation was AT-2, AAm, XStat®, and then other groups. The treatment with injectable and rapid shape-memory AAm and AT-2 cryogels showed rapid blood absorption and filling of the injury site instantaneously to stop massive bleeding. Further, the AAm and AT-2 groups exhibited high resilience properties, quickly re-expanding while maintaining their structural integrity. As expected, the control group showed a quick and massive blood loss after the injury and failed to attain any hemostasis on its own. It is worth mentioning that only a single applicator containing AT-2 cryogel samples (∼14 g) was sufficient to stop the lethal junctional hemorrhage compared to the groups treated with two syringes of each AAm cryogel (∼40 g) and XStat®. Hence, the time to inject the AT-2 cryogels into the injured site was significantly shorter (3 s) than AAm and XStat® treated groups (∼8 s) (Fig. 7c). The time duration for applying the CG treatment was the longest (20 s) among all the treated groups. The hemostatic efficiency was further evaluated by quantifying the total blood loss post-treatment (Fig. 7d). As anticipated, due to continuous bleeding, the total blood loss in the control group was the highest (2200 ± 330 ml) among all the groups. Remarkably, the AT-2 treated group showed significantly less blood loss (110 ± 90 ml) compared to the AAm (476 ± 280 ml), XStat® (770 ± 140 ml), and CG (1540 ± 240 ml) groups. Obviously, the CG group had a large amount of blood loss, indicating its poor clotting ability. The blood loss in the AAm and XStat® groups varied slightly but was statistically non-significant. Surprisingly, some of the pouch-packed pellets in the XStat® product failed to expand, which could be attributed to the hindrance of the mesh pouch itself and potential quality issues of those pellets (Fig. S8).

Next, we examined the effect of treatment groups on the survival time and survivability of the animals (Fig. 8). The post-treatment results manifested an interesting outcome and revealed that all the subjects in the control group died within 15 ± 6 min, followed by the CG group with a survival time of 70 ± 50 min. Notably, in both the AAm and AT-2 groups, the animals survived until the end of the experiment, i.e., 180 min, demonstrating a 100 % survival rate, unlike the XStat® and CG groups. In the case of the XStat® treated group, an average of 70 % of the pigs survived until 140 ± 60 min, while the remaining 30 % died after 30 min (Fig. 8a and b). In the CG group, 50 % of the animals died within 1 h, and approximately 70 % succumbed around the 2-h mark. The final mean artery pressure (MAP, mmHg) monitored in the AT-2 group was higher, at approximately 62 mmHg, compared to the group's AAm cryogels, XStat® and CG, recorded at approximately 32 mmHg. Moreover, to achieve proficient hemostasis in junctional hemorrhage in clinical settings, hemostats should not only demonstrate initial clotting but also be effective in preventing post-treatment rebleeding. No rebleeding was observed in the AT-2 group except in one pig that only lasted for approximately 4 min. This was in contrast to the AAm groups, which also experienced rebleeding but for a shorter duration (∼7 min), compared to the XStat® and CG groups, where rebleeding lasted for 15 min (Fig. 8c). Fig. 8d shows a schematic illustrating the procedure of AT-2 treatment and possible clotting mechanism induced by blood-triggered rapid expansion and accumulation/activation of blood cells and clotting factors. In summary, the AT-2 cryogels show significantly higher hemostatic efficacy than XStat® and CG in the lethal and marginally compressible swine junctional hemorrhage model.

Fig. 8.

In vivo efficacy of the cryogels on survivability and rebleeding time. (a) Post-treatment survival time for all the treatment groups. (b) Survival rate for all the treatment groups. (c) Rebleeding time for all the treatment groups. (d) Schematic illustrating the treatment and hemostatic mechanism of the AT-2 cryogels for efficient hemostasis.

4. Discussion

The majority of bleeding casualties occur within the initial 30 min following a severe injury, such as junctional wounds, contributing to high patient mortality rates [5,36]. These fatalities can be mitigated by promptly employing effective hemostats. Despite considerable progress in material science that has yielded several promising hemostats, only a select few have successfully transitioned into clinical application. Therefore, effectively achieving hemostasis for severe bleeding still poses a significant challenge. Here, we have presented a straightforward, easy, and cost-effective method to fabricate the injectable and rapidly expandable AT cryogels for use as a propitious hemostat, efficiently managing marginally compressible junctional hemorrhage.

The ability of any hemostat to stop excessive bleeding rapidly and effectively has been primarily shown to depend on various key parameters, including fabrication technology, pore size, porosity, and the incorporation of desired hemostatic agents [11,18,37]. In this study, we used cryogelation technology to fabricate AT cryogels with a unique interconnected porous architecture, desired pore size, and rapid shape memory properties. During the optimization, it was observed that the pAAm cryogels with either smaller or larger pore sizes were less effective in blood clotting and also exhibited compromised mechanical strength. Subsequently, pAAm-3, which demonstrated the required ideal properties, was selected and used to prepare AT cryogels for further in vitro and in vivo validation. One of the essential properties of hemostats is their significant liquid absorptive capacity, which aids in speeding up the blood coagulation cascade [38,39]. Because of high porosity as well as a uniform interconnected porous architecture, the AT cryogels showed a higher fluid absorption rate and capacity, like XStat®.

In prehospital settings, persistent cases of extensive bleeding from deep and irregular wound sites present significant challenges, especially junctional hemorrhage. To mitigate these complications, it is strongly recommended to employ hemostats possessing both injectable and rapid expandable properties. The AT cryogels, after being compressed into various shapes, showed remarkable water/blood-triggered shape memory and can rapidly restore their original shape in a few seconds. The mechanical study proved that the cryogels withstand high stress without experiencing mechanical fractures, even after 80 % compression. The outstanding mechanical stability and shape memory capability of the cryogels can be attributed to its interconnected porous network, which allows for reversible pore deformation, ensuring the unimpeded penetration of water/blood [40]. Additionally, its high flexibility supports effective recovery, and its polarity facilitates water uptake [5,11]. Overall, these properties indicate the potential application of our cryogels in the injectable form for managing junctional hemorrhage and possibly addressing noncompressible torso hemorrhage, a leading cause of potentially preventable trauma mortality [1].

Swift cessation of bleeding by forming a stable clot has been established as a critical factor in intervening effectively in cases of massive hemorrhages [41]. We performed in vitro blood clotting assay to investigate the potential impact of AT cryogels in promoting blood clotting. The incorporation of thrombin into cryogels, specifically AT-2, indicated better blood clotting capability in a shorter time than the XStat® and other groups. Interestingly, the clot formed in the AT-2 group was firm and stable. In contrast, XStat® showed poor clotting ability with continuous diffusion of erythrocytes from the unstable clot. Previous studies have reported the key role of RBCs in activating platelets [16]. Upon activation, platelets have the ability to transport numerous other clotting factors, aggregate on a wound surface to form a platelet plug and initiate the blood clotting cascade by facilitating a rapid generation of thrombin [15,16,28,37,42]. The improvement of this process mainly relies on the interaction between the material surface and blood cells. A sparse number of RBCs adhered to XStat®, which could be due to the lack of an interconnected porous network and the presence of larger pore size. On the contrary, AT-2 and AT-3 attracted abundant RBCs and platelets on their surfaces and favored their activation compared to other groups. This may be attributed to an interconnected porous architecture and the small pore size of cryogels that are crucial for high fluid absorption, thereby allowing cryogels to concentrate RBCs/platelets and plasma proteins [35,43]. Moreover, thrombin plays a vital role in accelerating the coagulation cascade and establishes a stable and firm clot by augmenting localized activation of platelets as well as amplifying the fibrin generation from fibrinogen [10,14,28].

The AAm and AT-2 cryogels can be compressed and packed into an applicator (e.g., a syringe device) to deliver them into narrow and irregular wounds (e.g., junctional, truncal, and gunshot wounds). Next, the preclinical efficacy testing of the AT-2, AAm, XStat®, and CG was assessed in a lethal swine junctional hemorrhage model. Among all groups, AT-2 demonstrated a remarkable hemostatic effect in a shorter time with a 100 % survival rate. Although AAm and XStat® demonstrated comparable hemostatic effects, considering the obtained results, AAm outperformed XStat® in terms of the survival rate, which was 100 % in the former and 70 % for the latter. The CG, another commercial hemostat, exhibited poor hemostatic effectiveness with high blood loss, along with a significantly lower survival rate of 30 %. The rapid blood clotting ability, contributing to the excellent hemostatic efficacy of AT-2 cryogels, can be attributed to a combination of cryogelation technology, a hydrophilic polymer, small pore size, a uniformly interconnected porous architecture, and the incorporation of thrombin as an active hemostatic agent.

While our AT-2 cryogels lack antibacterial property, a key characteristic of a hemostat, it is important to note that these cryogels are intended to remain at the injury site for a short period of time until surgical intervention begins. If needed, we can incorporate antimicrobial agents into the cryogels. Our cryogels were comprehensively assessed for physiochemical characteristics and evaluated in a lethal and marginally compressible swine junctional hemorrhage model. To further expand their potential applications in clinics, a swine noncompressible torso hemorrhagic model will be used for further hemostatic efficacy testing. The storage and shelf life of the cryogels were examined for several weeks. A prolonged period, up to two years, will be used for further examination. Some of the major advantages and disadvantages of the developed cryogels and XStat are summarized in Table S1.

5. Conclusions

In conclusion, we have fabricated the AAm and AT cryogels with injectable and rapidly expandable properties for managing lethal junctional hemorrhage in swine. In contrast to commercial hemostats XStat® and CG, the AT-2 cryogels showed significantly less blood loss and a higher survival rate (100 %) in a lethal swine junctional hemorrhage model, indicating their rapid and efficient clotting ability to control massive bleeding. Overall, these results demonstrate the AT-2 cryogels as a promising hemostat for effectively managing marginally compressible junctional hemorrhage.

Ethic approval and consent to participate

The in vivo studies were conducted in strict adherence to the protocol approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC) (Protocol No.: 22-051-08-EP).

CRediT authorship contribution statement

Syed Muntazir Andrabi: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. S.M. Shatil Shahriar: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Al-Murtadha Al-Gahmi: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Benjamin L. Wilczewski: Writing – review & editing, Methodology, Investigation, Data curation. Mark A. Carlson: Writing – review & editing, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Jingwei Xie: Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

The following are the Supplementary data to this article: