Preclinical Evaluation of Colisorb: Surfactant-Free Hydrophilic Gelatin Sponge with Rapid Clotting and Enhanced Absorbency without Mechanical Compression

Abstract

Rapid hemostasis is a major challenge in surgery, especially for noncompressible wounds such as liver wounds. Conventional hemostatic products often fall short of achieving the desired level of hemostasis. The current study introduces Colisorb, a novel cross-linked gelatin-based sponge for controlling surgical bleeding. The Colisorb sponge is a superabsorbent, adhesive, biocompatible, biodegradable, mechanically stable gelatin-based sponge with superior swelling kinetics, which allows strong swelling capability up to 70× its weight, thus indicating strong capability for blood absorption. Electron microscopic examination shows that the sponge has an interconnected network structure with pore sizes of 100 to 300 μm. Colisorb sponge also exhibits higher porosity and tensile strength compared to commercial products. Additionally, the Colisorb sponge demonstrated rapid degradation in only 3 days. In vitro examination of the Colisorb sponge demonstrated that it is noncytotoxic on mouse fibroblast and human fibroblast cells. In vivo, coagulation experiments showed that Colisorb promotes hemostasis in a rat liver laceration model and significantly reduces the amount of blood loss (100 mg with Colisorb as compared to 300 mg with Cutanplast). Collectively, the findings of the current study highlight the potential of the Colisorb gelatin hemostatic sponge to drive advancements in clinical applications for managing noncompressible hemorrhage and facilitating subsequent wound closure.

1. Introduction

Major blood loss due to trauma or during surgery is the most feared surgical complication, , as it leads to serious complications and death. ,, With over 300 million surgeries annually, intraoperative bleeding continues to be a significant contributor to postoperative mortality and morbidity. There is a critical need for an effective hemostatic agent worldwide. ,

The natural hemostatic response initiates a cascade of events that aim to control bleeding. This primary hemostasis is followed by a coagulation pathway leading to the fibrin activation that stabilizes the platelet plug with a mesh of fibrin threads.

A wide spectrum of hemostatic materials has been developed to control bleeding, including gauzes, tourniquets, and bandages. , Recently developed hemostatic agents could be classified into three main groups: sealants, hemostats, and adhesives. They are available in multiple forms, such as nanofiber dressings, powders, sponges, or hydrogels. ,, Mechanical hemostasis is the safest, easiest to apply, and most effective in stopping hemorrhage and increasing coagulation. ,

Hemostatic agents are often removed after hemostasis due to their nondegradability, which increases the risk of rebleeding, secondary injuries, and delayed healing. Accordingly, the ideal hemostatic agents should demonstrate biocompatibility and biodegradability in addition to enhanced blood absorption capacity that would allow them to achieve proper hemostasis. Additionally, they should be stable, easy to use, nontoxic or nonirritant, and must effectively promote the healing process without any complications. , One of the most utilized commercially available conventional sponges is made of gelatin, such as Cutanplast, an absorbable gelatin sponge intended for hemostatic use, which is characterized by its porous structure that promotes the coagulation process at the site of application. Gelatin, a water-soluble protein derived from collagen hydrolysis, is a widely used biomaterial in medical applications due to its biodegradable, biocompatible, and biosafe qualities. Utilization of gelatin minimizes intraoperative blood loss and generates a physical network for platelets to aggregate by absorbing blood fluids, leading to thrombin generation as well as protein cross-linking and wound hemostasis. Furthermore, the negative charge on gelatin enhances its ability to recruit coagulation factors to the wound site rapidly and efficiently.

The reported blood absorption capacity of the available commercial gelatin sponges does not exceed 40× their weight. , Additionally, one of the time-consuming and limiting steps in commercially available gelatin sponges is the need for mechanical compression in sterile saline before insertion at the wound site due to their hydrophobic nature.

Contemporary gelatin-based hemostatic materials have evolved significantly beyond conventional hemostatic sponge formulations. Biomimetic design strategies, exemplified by DNA hydrogel systems that mimic natural neutrophil extracellular traps and act as an artificial scaffold to aggregate blood cells, have demonstrated enhanced hemostatic capacity in rat trauma models while maintaining favorable biocompatibility. Furthermore, recent advances in advanced materials engineering have further expanded this field through optimized composite formulations and novel cross-linking strategies, such as a novel hemostatic peptide with methacrylated gelatin triggered by blue laser, establishing new benchmarks for rapid hemostatic performance. Such advancements in hemostasis research collectively demonstrate that rational material design can substantially improve hemostatic efficacy, motivating the present investigation of Colisorb as an optimized cross-linked gelatin-based hemostatic platform with promising favorable outcomes for clinical and surgical applications. ,

In the current study, we introduce Colisorb, a novel gelatin-based sponge made from a proprietary composite of cross-linked gelatin sponge to control intraoperative bleeding. The newly developed Colisorb sponge was developed as a superabsorbent, adhesive, biocompatible, biodegradable, and mechanically stable sponge based on gelatin. The developed gelatin sponge novel cross-linking allowed it to exhibit maximum air entrapment that reached up to 70% of the total sponge volume. Additionally, it allowed the developed sponge to display optimal pore structure, pore number, size, and homogeneous distribution. Aided by high shear mechanical mixing, the reduction of the air bubble size was achieved to only 100–300 μm, which was a significant factor in enhancing its absorption capacity and mechanical stability. Such properties are of huge importance in surgery for efficient bleeding control.

Accordingly, the newly developed Colisorb sponge is proposed to possess a higher hemostatic ability than commercially available sponges due to its unique cross-linking that allows increased absorbency, enhanced biocompatibility, and improved adhesion. Therefore, the presented novel sponge is an ideal hemostatic agent for use in surgical and trauma settings, where effective hemostasis is critical for enhanced recovery outcomes.

2. Materials and Methods

2.1. Fabrication and Characterization of the Colisorb

The fabrication of Colisorb is currently under patenting. Briefly, cross-linked gelatin and other proprietary components allowed the fabrication of a stable foam that was then poured into plastic molds and left to freeze at −20 °C for 18 h, followed by a 48 h freeze-drying process in a freeze-dryer. The resulting dried sponge is cut into cubic shapes of varying sizes based on intended use.

2.1.1. Water Absorption Rate and Swelling Rate

The fabricated sponge was cut into 1 cm³ cubes. The cubes were weighed, and distilled water was added. The sponge was allowed to swell in the water for 30 s. The sponge was taken out of the water, and any excess water on the surface was gently expelled. The swollen sponge was weighed, and the percentage swelling of the sponge was computed by using the following formula:

$$\mathrm{Percentage}\mathrm{swelling}(\%)=(\mathrm{Wt}-\mathrm{Wd})/\mathrm{Wd}\times 100)$$

where Wt is the weight of the swollen sponge and Wd is the initial weight of the dry sponge cube.

2.1.2. SEM Morphology

In order to closely examine the morphology of the sponge’s surface, a scanning electron microscope (SEM) was utilized. The sponge sample to be examined was prepared by carefully cutting it into small pieces and coating them with a thin layer of gold under vacuum at 15 kV. The sponge pieces were observed under a scanning electron microscope at 50× magnification.

2.1.3. Porosity Analysis

The dried samples underwent a 1 h immersion in alcohol, and their weight was recorded. The porosity of the sponge samples was determined using the following equation:

$$P\mathrm{orosity}(\%)=(W2-W1/\rho \times V)\times 100\%$$

In this equation, W1 is equivalent to the weight of the sample before immersion in alcohol, while W2 is equivalent to the weight of the sponge after immersion. V denotes the volume of the samples, and ρ represents the density of alcohol (0.79 g/cm³ at room temperature).

2.1.4. Mechanical Properties Assessment

The mechanical characteristics of sponges were studied with a universal testing machine (UTM, Shimadzu-AGX-10 kN, Kyoto, Japan). Rectangular sponge samples measuring around 10 × 40 × 5 mm were tested with a cross-head speed of 50 mm/min. During the testing process, the tensile strength of the sponge was recorded.

2.1.5. Contact Angle Measurement

The sponges’ contact angle was measured with a contact angle goniometer (Drop Shape Analyzer DSA25, Krüss, Germany) in order to assess the wettability of the sponge. Water was gently added in a dropwise manner on the top surface of the sponge, and changes in the contact angle over time were measured.

2.1.6. Degradation Assay

To investigate the process of biodegradation, the weight of a cubic sponge was measured and recorded. The sponge was then placed in phosphate-buffered saline (PBS) and kept inside an incubator set at a temperature of 37 °C for 8 days. The weight of the sponge after being dried every day was recorded. To calculate the remaining weight as a percentage, we used the following equation: Remaining Weight (%) = (Wd – Wt)/Wd × 100, where Wt represents the weight of the dried sponge, and Wd represents the initial weight of the dry sponge cube.

2.2. In Vitro Biological Assessment

2.2.1. Blood Uptake Ratio

The sponge with dimensions 1 cm × 1 cm × 1 cm was incubated at 37 °C for 5 min with 1 mL of blood (V0) (citrated rabbit whole blood from rabbits) in 24-well plates. The sponges were removed, and the volume of blood remaining in the wells (V1) was measured. The absorbed blood volume (VB) was calculated using the following formula: VB = V0 – V1. Additionally, the sponges were visually evaluated for their mechanical stability following blood intake after 5 min.

2.2.2. Blood Clotting Time (BCT)

To assess the time required for blood to clot with Colisorb, 1 mL of citrated whole blood was added to the wells of a 24-well plate, followed by 100 μL of 0.2 M CaCl₂, and then incubated at 37 °C for 5 min. Sponge samples (1 cm × 1 cm × 1 cm) were added to the wells, and the time taken for the blood to initiate clotting was recorded. The wells were gently tilted every 10 s to examine the blood.

2.2.3. Blood Clotting Index (BCI)

Sponges were cut into 1 cm³ squares and placed in a prewarmed 24-well plate. 200 μL of 3.2% citrated whole blood was added to the sponges simultaneously with 20 μL of 0.2 M CaCl₂ and incubated for 5 min at 37 °C. Sponges were then immersed in 25 mL of distilled water and incubated for 10 min at 37 °C with 30 rpm shaking. The commercial sponge, Cutanplast (Mascia Brunelli S.p.a., Milan, Italy), was used as a positive control, and citrated whole blood with deionized water was used as a blank. Finally, 500 μL of each sample mixture was measured for absorbance at 545 nm. , BCI was computed using the following formula:

$$\mathrm{BCI}\mathrm{index}=100\times (\mathrm{absorbance}\mathrm{of}\mathrm{the}\mathrm{sample})/(\mathrm{absorbance}\mathrm{of}\mathrm{blank})$$

2.2.4. In Vitro Hemolysis Test

A preweighed sponge sample (20 mg) was submerged in 2 mL of saline and incubated for 30 min at 37 °C. Red blood cells (RBCs) were prepared by mixing 4 mL of whole citrated blood with 5 mL of saline, followed by centrifugation at 2000 rpm for 10 min. One mL of each saline-sponge mixture was added to 20 μL of RBCs. 10 μL of triton + 20 μL of RBCs + 1 mL of saline were used as a positive control. Saline (0.9% NaCl) served as the negative control in this experiment. All sample mixtures were centrifuged at 2000 rpm for 5 min. The absorbance of the supernatant was then measured at 545 nm. The in vitro hemolysis rate (HR %) was determined by using the following formula:

$$\mathrm{HR}\%=(\mathrm{Dsample}-\mathrm{Ddist})/(\mathrm{Ddist}-\mathrm{Dsaline})\times 100$$

2.2.4.1. Direct Hemolysis Analysis

The hydrophilic surfactant-free (Colisorb) was examined for its direct hemolysis effect and compared to Cutanplast. The plasma was separated from the RBCs via centrifugation of 8 mL of 3.5% citrated whole blood at 800 × g for 15 min at 4 °C. The RBC pellet was centrifuged again to ensure complete plasma separation. RBCs were then resuspended in sterile CMF-PBS to achieve a 5% dilution. One mL of diluted blood was added to Colisorb and Cutanplast (100 mg each) sponges, and CMF-PBS was added consecutively to reach a total volume of 9 mL. A total of 1 mL of diluted blood was added to 7 mL of water for injection and CMF-PBS to act as the positive and the negative control, respectively. CMF-PBS only was used as a zeroing blank. All samples were incubated for 3 h at 37 °C with periodic shaking every 30 min. The absorbance of the supernatant (optical density OD) was then measured at 540 nm (ISO 10993-4:2017 and ASTM F0756-13, 2017). The blank-corrected % hemolysis was calculated using the following equation:

$$\mathrm{Blank}\mathrm{Corrected}\%\mathrm{Hemolysis}=(\mathrm{OD}\mathrm{Sample}-\mathrm{OD}\mathrm{Blank})/(\mathrm{OD}+\mathrm{positive}\mathrm{control}-\mathrm{OD}\mathrm{Blank})\times 100$$

2.2.5. Prothrombin Time (PT) Analysis

Platelet-poor plasma was collected by centrifugation of citrated whole blood at 2000 g for 15 min at 4 °C. 100 μL of platelet-poor plasma and 100 μL of PT reagent were incubated at 37 °C for 3 min. After incubation, 2 mg of sponge samples was added to 200 μL of plasma and incubated for 5 min at 37 °C. Finally, the PT reagent was added to the plasma-sponge mixture. 100 μL of aPTT reagents and 100 μL of PPP were incubated at 37 °C for 3 min in order to measure aPTT. Following this, 100 μL of 0.025 mol/L CaCl₂ and sponge samples were added. Then, PT and aPTT were analyzed using a semiautomatic coagulation analyzer (TS6000, MD Pacific Biotechnology Co., Ltd., China).

2.2.6. In Vitro RBC and Platelet Aggregation Studies

The hemostatic mechanism of Colisorb compared to Cutanplast was examined by using scanning electron microscopy (SEM) to study the morphology and adhesion of red blood cells (RBCs) and platelets on the sponges. A sponge dimension of 1 × 1 × 0.5 cm was prepared and exposed to 1 mL of citrated whole blood (n = 3 per group). Each sponge sample was incubated at 37 °C for 30–60 min, gently rinsed 2× with phosphate-buffered saline (PBS, pH 7.4), and fixed in 2% glutaraldehyde at 4 °C for 2 h to preserve cellular morphology for SEM analysis. Following fixation, samples were dehydrated through an ascending ethanol series (70%, 80%, 90%, and 100%), each for 2 min, then air-dried for 24 h.

For platelet adhesion, platelet-rich plasma (PRP) was obtained by centrifuging citrated whole blood at 800 rcf for 15 min at 4 °C. A 1 × 1 × 0.5 cm dimension of Colisorb and Cutanplast sponges was placed in a sterile tube, followed by the addition of 1 mL of PRP, and incubated at 37 °C for 1 h. Washing and fixation conditions were performed as the RBCs adhesion testing.

2.2.7. Cell Viability (MTT) Assay

To assess whether the sponge is cytotoxic on normal cells, cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay as per the ISO 10993-5 and ISO 10993-12. , Mouse fibroblast L929 cells, hepatocellular carcinoma HepG2 cells, and human fibroblast (hFB) cells were seeded in 96-well plates at a density of 10,000 cells per well. The plates were incubated at 37 °C 5% CO₂ for 24 h. An extraction of the sponge was prepared by soaking 10 mg of each sponge (Colisorb and Cutanplast) in 2 mL of complete DMEM media at 37 °C for 24 h. The extract was filtered with a 0.22 μm syringe filter. Old media was discarded, the extract was added to the cells, and they were incubated for 24 h. 20 μL of MTT and 100 μL of media were added to each well. The plate was incubated for 2 h at 37 °C. After incubation, the media was discarded, 120 μL of DMSO were added, and the absorbance was measured at 570 nm using a SPECTROstar Nano plate reader.

2.3. In Vivo Biological Assessment

2.3.1. In Vivo Bleeding Assessment

To assess the hemostatic efficacy of the sponge in controlling liver hemorrhage in a rat model, female Sprague-Dawley rats (125–150 g) were obtained from Theodor Bilharz. All surgical procedures and postsurgical care were performed according to the National Research Council’s Guide for the Care and Use of Laboratory Animals. The study was approved by the Institutional Animal Care and Use Committee (IACUC) of Ain Shams University (ASU-SCI-BIOC-2024-1-1).

In order to assess hemostatic time and blood loss in vivo, rats were randomly assigned to three groups (n = 6) for the study. Hemostatic efficacy was assessed using the liver laceration model. Rats were anesthetized with 80–100 mg/kg of Ketamine and 10–12.5 mg/kg of Xylazine via an intraperitoneal injection. Consequently, an anterior abdominal subcostal incision was made, and the rat’s left lateral lobe was gently exposed and exteriorized. Upon exposure of the liver lobe, a preweighed sterile gauze (W₀) was positioned beneath the lobe. Then, using a scalpel blade, a 10 mm transverse incision was made on the liver lobe to simulate hemorrhage, and the sponge was promptly applied to the wound. Bleeding was carefully monitored, with the gauze replaced every minute. Successful hemostasis was defined as the absence of bleeding within 5 min, and the time to hemostasis was recorded using a stopwatch. The preweighed sterile gauze was weighed again after hemostasis was achieved (W_t). The incision was then closed, and the animal was allowed to recover. Nearing completion of the experiment, rats were anesthetized with the same ketamine/xylazine protocol, and blood samples were collected via cardiac puncture. Tissue samples were also harvested for further biochemical and histological analysis. Total blood loss was computed by the following formula: Blood loss = 1/4 W_t – W₀.

2.3.2. Biochemical Assays

The drawn blood samples of 1 mL each from the rats were analyzed to evaluate the final concentrations of liver enzymes: Aspartate and Alanine aminotransferases (AST and ALT), as well as the final concentrations of serum urea and blood creatinine, after utilizing Colisorb to control hemorrhage in the rat liver laceration model. The samples’ biochemical analysis was done by utilizing the clinical chemistry autoanalyzer Mindray BS-240, China.

2.3.3. Histological Assessment

After 14 days, the rats were sacrificed, and their tissues were collected for analysis. Extracted tissues were dissected and fixed for 24 h in paraformaldehyde (4%). The skin, liver, and kidney tissue samples were carefully trimmed, washed, and dehydrated through a series of increasing alcohol concentrations. Following dehydration, the tissues were cleared in xylene, embedded in paraffin blocks, and sectioned into thin slices measuring 4–6 μm in thickness. The tissue sections were then deparaffinized with xylol and stained with hematoxylin and eosin (H&E) as well as Masson’s Trichrome for histopathological evaluation. The stained samples were examined under a light microscope (Olympus XC30, Tokyo, Japan) using CellSense Dimensions software.

2.3.4. Scoring of Skin Inflammation

Inflammation was assessed on a 0–5 scale: 0 for no inflammation, 1 for mild inflammation, 2 for mild to moderate inflammation, 3 for moderate inflammation, 4 for moderate to severe inflammation, and 5 for severe inflammation. Skin inflammation was evaluated by two researchers, who scored the stained sections independently and without prior knowledge of sample identities.

2.3.5. Scoring of Liver Laceration

Fibrosis in liver sections was graded according to a specific numeric scale adopted from Park et al. A score of 0 indicated no fibrosis, grade I represented slight fibrosis confined to the central liver lobule, grade II denoted moderate fibrosis with widened central areas, and grade III indicated severe fibrosis.

2.3.6. Semiquantitatively Assessment of Kidney and Liver Toxicity

Lesions in kidney tissue were evaluated using a semiquantitative scoring system. A score of 0 indicated normal histology, while a score of 1 represented tubular epithelial cell degeneration without significant necrosis or apoptosis. Scores that ranged from 2 to 5 were based on increasing severity of tubular epithelial cell necrosis and apoptosis: 2 for <25%, 3 for <50%, 4 for <75%, and 5 for ≥75%.

Similarly, liver lesions were scored on a scale ranging from grade 0 to grade IV. Grade 0 indicated no visible injury, grade I represented hepatocyte swelling, grade II indicated hepatocyte ballooning, grade III signified lipid droplet formation within hepatocytes, and grade IV was characterized by hepatocyte necrosis.

2.4. Statistical Analysis

Data obtained from the experiments in the study were analyzed using GraphPad Prism Software Inc. (version 7.00, San Diego, CA, USA). Data were expressed as the mean ± standard deviation (SD). The significance level was set at 5%; hence, P-values < 0.05 were considered statistically significant. The one-way ANOVA test was followed by Dunnett’s test for multiple comparisons. The two-way ANOVA test was followed by Sidak’s test for multiple comparisons.

3. Results

3.1. Swelling Rate

The swelling kinetics were analyzed based on the sponges’ water uptake and swelling behavior over time. At 30 s, Colisorb demonstrated remarkable swelling kinetics with an increase in size of approximately 15×, whereas Cutanplast exhibited a significantly lower swelling of only 2×. This indicates that Colisorb has a faster initial absorption rate, making it more efficient in controlling bleeding during the critical early stages of a surgical procedure. After 10 min, Colisorb continued to exhibit superior performance, with a swelling capacity of 77×, while Cutanplast reached a swelling capacity of 31×. These results indicate that Colisorb possesses a higher absorption capacity, allowing it to absorb a greater volume of water compared to Cutanplast, which displayed limited absorption ability (Figure ).

1.

The swelling kinetics of the Colisorb sponge as compared to the Cutanplast hemostatic sponge.

3.2. SEM Morphology and Porosity Analysis

Cutanplast was found to display a consistent network structure with various ranges of pore sizes. On the other hand, Colisorb exhibited an interconnected network structure of gelatin and demonstrated a highly porous structure (Figure ). The porosity of both Colisorb and Cutanplast was evaluated. It was observed that the Cutanplast sponge demonstrated larger pore sizes compared to the Colisorb. The pore size varied between 100 and 300 μm, with a mean pore size of 210 and 243 μm for Colisorb and Cutanplast, respectively. This can be attributed to differences in pore wall thickness and structural collapse upon drying, which may give Cutanplast a denser look despite its relatively larger and less interconnected pores. Most importantly, Cutanplast exhibited a porosity of 42%, while Colisorb displayed a higher porosity of 78%. Colisorb exhibits higher overall porosity due to its more interconnected, open-cell structure formed during the foaming process.

2.

Scanning electron microscope (SEM) micrographs showing the internal structure of Colisorb (A) and Cutanplast (B).

3.3. Mechanical Analysis

The results of the mechanical evaluation demonstrated that Colisorb exhibited a higher tensile strength as compared to Cutanplast. The break stress value of 0.155 N/mm² suggests that Colisorb is capable of withstanding higher amounts of force or tension before breaking or tearing. This is a positive attribute, as it indicates that the Colisorb sponge is less likely to rupture during its application compared to Cutanplast, which has a lower break stress value of 0.105 N/mm². Additionally, the optimized unique cross-linking, which allowed for enhanced mechanical integrity, resulted in optimal mechanical strength, hence maintaining an integral structure of the sponge while simultaneously allowing for maximum expansion for enhanced swelling ability and blood absorption (Figure ).

3.

Stress–strain curves of Colisorb and Cutanplast after repeated mechanical compression.

3.4. Contact Angle Measurements

The wettability of biomaterial surfaces is a significant characteristic that can influence the absorption ability, proliferation, migration, and viability. Evaluation of the water contact angle is a common method to assess the wettability, as it helps determine their surface interaction with water.

For Colisorb, at 0 s, contact angles were between 107° and 108°, while after 5 s, the contact angles were between 76.1° and 71.6°, and after 10 s, the contact angle was equal to 0°. For the Cutanplast surface, contact angles measured were 112°, 108°, and 89° after 10 s. The observation of the water contact angle on the Colisorb revealed that the water droplet was quickly absorbed by the sponge, resulting in the absence of a contact angle within a mere 10 s. This indicates that Colisorb has a high level of hydrophilicity, allowing it to rapidly and effectively absorb water. In comparison, the Cutanplast demonstrated a contact angle, which indicates a hydrophobic surface. This results in slower water absorption, which requires 12 min for complete absorption of the water droplet (Figure ).

4.

Water contact angle of Colisorb and Cutanplast.

3.5. Degradation Test

The degradation percentage of the Colisorb gelatin sponge was assessed by incubating it in phosphate-buffered saline (PBS) with shaking at 37 °C, using the commercial sponge Cutanplast as the positive control. The Colisorb samples demonstrated a fast degradation rate, where 59% of the whole weight of the sponge was degraded by day 3. Complete degradation of the Colisorb sponge was achieved by the third day (Figure ). On the other hand, Cutanplast samples showed slower degradation, where 50% were degraded by day 5 and complete degradation was achieved after 8 days (Figure ). Such results demonstrate the high degradation rate of the developed Colisorb sponge as compared to Cutanplast.

5.

In vitro degradation rate of Colisorb sponge compared to Cutanplast.

3.6. In Vitro Assessment

3.6.1. Blood Uptake Ratio Test

The blood uptake ratio for Colisorb and Cutanplast was measured after 3 min on blood-sponge surface interaction. Colisorb demonstrated a strong blood-absorbing capability, showing a 2.94× higher uptake ratio than Cutanplast (Figure b). Additionally, Colisorb showed a higher ability to retain its original sponge structure after blood uptake and slight pressure application with forceps. Whereas, Cutanplast demonstrated lower mechanical stability than Colisorb as it loses its (1 cm × 1 cm × 1 cm) shape over time (Figure a), thus resulting in diminished blood absorption and reduced ability to maintain blood within its matrix.

6.

In vitro assessment of blood uptake and blood coagulation. (a) A photograph was taken after the blood uptake ratio test, demonstrating the structural stability of the Colisorb sponge as compared to the commercial Cutanplast. (b) Blood uptake ratio of Colisorb and Cutanplast (****P < 0.0001). The clotting time (c) of Colisorb and Cutanplast was significant compared to the untreated blood (***P < 0.001, ****P < 0.0001). (d) The blood clotting index (BCI) for both Colisorb and Cutanplast was significant compared to the control; there was no difference between Colisorb and Cutanplast. Coagulation (prothrombin time, PT) was not significantly different (e) between all groups. aPTT time (e) was significantly higher in Colisorb and Cutanplast samples compared to the control (untreated blood).

3.6.2. Blood Clotting Time Test

Blood clotting was significantly faster with Colisorb than with Cutanplast, with a blood clotting time of 4 min with Colisorb compared to 5.8 min with Cutanplast. The control group (no sponge) took 13 min to reach successful hemostasis (Figure c).

3.6.3. Blood Clotting Time Index

The whole blood coagulation test measures the coagulation potential of hemostatic devices, where lower values of the Blood Clotting Index (BCI) are an indication of enhancing the coagulation process. BCI values for Colisorb and Cutanplast were measured and compared with the negative control (no sponge). Colisorb exhibited the lowest clotting index, almost 53% lower than the control, while Cutanplast was only 34% lower than the control (Figure c).

3.6.4. Prothrombin Time Analysis

Coagulation was assessed using prothrombin time (PT) in vitro to evaluate the Colisorb sponge and the Cutanplast for their plasma coagulation activity compared with the negative control. Colisorb had a much lower PT time than both the control and Cutanplast by 0.5 and 2.85 s, respectively (Figure d). Activated partial prothrombin time (aPTT) was significantly higher in Colisorb- and Cutanplast-treated samples compared to the untreated control.

3.6.5. In Vitro Hemolysis Test

Colisorb and Cutanplast were compared with negative and positive controls that had no sponge samples. The Colisorb and Cutanplast showed no significant hemolytic activity, and both were lower than the positive control and higher than the negative control (Figure ).

7.

In vitro hemolysis assay. Blood hemolysis was minimal after Colisorb and Cutanplast. Compared to the positive control (10 μL of triton-treated blood), normal saline was used as a negative control.

3.6.5.1. Direct Hemolysis Analysis

Colisorb and Cutanplast sponges’ biocompatibility with blood was assessed by the direct hemolysis assay to evaluate RBCs in contact. Optical density measured at 540 nm, Colisorb value was 0.057, corresponding to a hemolysis percentage of 1.4%, while that of Cutanplast was significantly higher at 0.363, causing 29.77% hemolysis. The negative control demonstrated negligible absorbance (0.048, 0% hemolysis), whereas the positive control induced complete hemolysis (OD = 1.1063, 100%) (Table ).

1. Direct Hemolysis Analysis.

Sample	OD	Mean OD	Blank corrected% Hemolysis
Blank	0.042, 0.0416, 0.043	0.0422	0
Control	0.0486, 0.0461, 0.0493	0.048	0.5
Colisorb	0.0577, 0.0590, 0.0586	0.057	1.4
Cutanplast	0.354, 0.3651, 0.3702	0.3631	30.1
Positive Control	1.07, 1.11, 1.139	1.1063	100

3.6.6. In Vitro RBC and Platelet Aggregation Studies

For RBC adhesion, Colisorb samples displayed a dark red color of concentrated RBCs, while the Cutanplast sponge showed faint red color upon optical observation. For PRP activation, Colisorb appeared to possess a dark yellow color upon platelet adhesion compared to a no-change in color in Cutanplast samples (Figure ).

8.

Color observation of sponge upon RBCs and platelet adhesion test preparation for SEM imaging.

SEM imaging demonstrated dense red blood cell aggregation to the Colisorb sponge distributed along its interconnected porous structure, with many cells exhibiting close contact with the sponge fibrils. Morphologically, the majority of adhered RBCs maintained their characteristic biconcave discoid shape, along with star-shaped RBCs adhering to Colisorb’s porous microarchitecture. Cutanplast, on the other hand, showed less uniform RBC adhesion. Major areas of the surface showed fewer RBC clusters. Many RBCs were loosely attached and displayed more spherical morphologies (Figures and ).

9.

Scanning electron microscope (SEM) micrographs of RBC adhesion density and behavior in Colisorb and Cutanplast sponges. Multiple magnification powers were used to elucidate RBCs adhesion: (a and b) at ×240 and (c and d) at ×500 for Colisorb and Cutanplast, respectively.

10.

Scanning electron microscope micrographs of platelet adhesion morphology in (a) Colisorb and (b) Cutanplast sponges.

3.6.7. Cytocompatibility Assessment

In vitro cell viability assessment was conducted by performing an MTT cytotoxicity assay. The results showed that Colisorb did not significantly affect the cell viability of L929 cells as compared to the control, with a significant difference observed between Colisorb and Cutanplast (P < 0.001). The cell viability of L929 mouse fibroblast cells with Colisorb was 98%, while with Cutanplast, the viability was 80% (Figure a). In addition, complementary assessments utilizing HepG2 and hFB cell lines revealed the following results. The cell viability of HepG2 cells with Colisorb was 95.62%, while with Cutanplast, cell viability decreased significantly, reaching 75.74%, compared to the control and Colisorb. A similar pattern was observed with the hFB (human fibroblast) cell line, where Colisorb resulted in a mean cell viability of 97.07%, with no significant difference compared to the control. Conversely, Cutanplast resulted in a cell viability of 72.25%, showing a significantly lower cell viability when compared to the control and Colisorb (Figure a,b).

11.

Cytocompatibility of the Colisorb. Cell viability of (a) L929 cells, (b) HepG2 cells, and (c) hFB cells upon treatment with Colisorb and Cutanplast (**** P < 0.0001, *** P < 0.001).

3.7. In Vivo Assessment of Hemostasis

3.7.1. Blood Loss

Blood loss was measured in vivo in a liver laceration injury model in the rat. Colisorb and Cutanplast were assessed in comparison with the negative control. Colisorb showed the lowest blood loss (100 mg) as compared to the untreated control and Cutanplast, with 450 and 300 mg amounts of blood loss, respectively (Figure a). Colisorb demonstrated significantly lower blood loss than the untreated control by 78%, while Cutanplast was lower than the control by only 33%.

12.

The amount of blood loss from the liver in vivo (a) was significantly lower compared to that in the untreated animals. Blood Clotting Time (BCT) (b) was also significantly lower after using Colisorb and Cutanplast compared to the untreated control. Blood loss (a) and clotting time (b) were significantly lower after using Colisorb compared to Cutanplast.

3.7.2. Blood Clotting Time

Colisorb and Cutanplast sponges (1 cm × 1 cm × 1 cm) were placed on the liver incision, and time was recorded until the bleeding stopped. Colisorb exhibited enhanced clotting ability and had the highest blood clotting rate, where the time to hemostasis was only 40 s as compared to the Cutanplast, which achieved hemostasis in 90 s. Therefore, Colisorb exhibited a significantly higher rate of blood clotting compared to Cutanplast and untreated control samples (Figure b).

3.8. In Vivo Assessment

3.8.1. Assessment of Liver Tissue

The liver tissue section of the untreated control showed dense fibrous connective tissue proliferation at the line of incision with moderate inflammatory cell infiltration, mainly lymphocytes and macrophages, with few numbers of polymorphonuclear cells. The underlying hepatocytes showed mild swelling with a granular cytoplasm. Examination of the liver treated with Colisorb revealed fibrous connective tissue, in addition to vacuolar degeneration of the hepatocytes and a few numbers of apoptotic bodies. The liver tissue section of Cutanplast-treated liver showed a relatively thin layer of fibrous tissue mixed with inflammatory cells and embedded sponge material (Figure ).

13.

Histological examination of the liver following laceration and hemostasis. Normal liver (a,f) showed normal histology of the hepatic lobules and hepatic cords. Nontreated control (b,g) liver samples showed dense fibrous connective tissue proliferation at the line of the incision (arrow). Gauze-treated (c,h) samples showed Colisorb-treated (d,i) samples showing fibrous tissue with vacuolar degeneration of hepatocytes and few numbers of apoptotic bodies (arrow). Cutanplast-treated samples (e,j) showed a thin layer of fibrous tissue mixed with inflammatory cells and embedded sponge material (arrow).

3.8.2. Assessment of Skin and Subcutaneous Tissue

Histological analysis of skin tissue from normal samples, Colisorb-treated samples, and Cutanplast-treated samples revealed a well-organized epidermis with a stratified squamous epithelium composed of four keratinocyte layers. The basal cell layer was covered by a thin stratum granulosum and an eosinophilic keratin thin layer. The dermis contained dense fibrous connective tissue, subcutaneous adipose tissue, and sparse blood capillaries. No signs of inflammation were observed, resulting in a score of 0 (Figure ).

14.

Histological assessment of skin and subcutaneous tissue. Photomicrographs of skin tissue sections showing normal (a) histological structure of epidermal and dermal layers (arrows). Colisorb-treated (b) and Cutanplast-treated (c) samples showed a normal, well-organized histological appearance of the epidermis with layers of stratified squamous epithelium covered with layers of keratinocytes. The dermis shows dense fibrous connective tissue, subcutaneous adipose tissue, and few blood capillaries without any inflammatory reaction.

3.8.3. Assessment of Liver and Renal Tissues Following Systemic Administration

Liver tissue from the normal group exhibited a typical histological structure with well-defined hepatic lobules, hepatic cords, and central hepatic veins. Hepatic cells were polygonal, arranged in anastomosing plates, and bordered by sinusoids or adjacent hepatocytes. In the Colisorb group, liver sections showed mild disruptions in hepatic cord organization, an increased number of Kupffer cells, and narrowed sinusoids (grade I). Conversely, liver sections from the Cutanplast group displayed normal hepatic parenchyma without notable pathological changes (grade 0) (Figure ).

15.

Histological assessment of liver and kidney tissues. Assessment of the systemic toxicity following the use of Colisorb (b,e) and Cutanplast (c,f) showed that liver tissues in the Colisorb-treated group revealed mild disorganization of hepatic cords, an increased number of Kupffer cells, and narrowing of hepatic sinusoids. On the other side, Cutanplast-treated animals showed a normal histological structure similar to the normal group (a), which showed the normal histological structure of hepatic lobules and hepatic cords. The kidney tissue in all animals (d–f) exhibited a normal histological architecture, with intact glomeruli, well-defined capillary tufts, and Bowman’s capsule. Both proximal and distal convoluted tubules displayed an intact epithelial lining and a regular structural arrangement.

Normal histology of kidney tissues was observed across all of the rat groups. Glomeruli were intact with regular capillary tufts and Bowman’s capsules, while the epithelial lining of both proximal and distal convoluted renal tubules appeared normal, intact, and well-organized, resulting in a score of 0 (Figure ).

3.8.4. Assessment of Liver and Renal Functions

Liver function enzymes Alanine transaminase (ALT) and Aspartate transaminase (AST) (Figure ) showed no significant changes in rats receiving either Colisorb or Cutanplast treatment compared to normal rats. Similarly, kidney function tests showed no significant variations in serum creatinine and urea levels across all groups (Figure ).

16.

Biochemical assessment of renal and liver functions. Assessment of renal (Creatinine (a) and Urea (b)) and liver functions (ALT (c) and AST (d)) showed no significant difference between normal, Colisorb, and Cutanplast-treated animals.

4. Discussion

Uncontrolled hemorrhage is the most dangerous surgical complication. Following bleeding, the rapid depletion of platelets and the weak fibrin network lead to the formation of fragile blood clots that are inadequate for stopping bleeding. , Pathological complications resulting from continuous bleeding can lead to cellular and metabolic dysfunction, acidosis, and hemorrhagic shock. Therefore, it is critical to introduce new hemostatic agents to achieve successful hemostasis and prevent excessive blood loss.

In the current study, Colisorb, a novel gelatin hemostatic sponge, is proposed. The developed gelatin sponge novel cross-linking allowed it to exhibit maximum air entrapment that reached up to 77% of the total sponge volume, allowing it to reach 15× its weight absorption capacity after only 30 s. Most importantly, it is the interplay of very high porosity and optimal mechanical integrity that allowed the sponge to swell while maintaining mechanical integrity and reach up to 77× absorption capacity at 10 min, unlike Cutanplast, which reached a swelling capacity of only 31× its weight at the same time point and demonstrated mechanical collapse, not allowing it to swell efficiently and maintain blood absorption. Such results indicate that the newly developed Colisorb sponge can absorb and retain larger amounts of blood. Additionally, the hydrophilic surface of the newly developed Colisorb sponge contributes to its superior efficiency in absorbing and retaining blood. Most importantly, it allows the Colisorb sponge to directly absorb blood without the need for a prewetting step that is usually mandated for current commercially available gelatin sponges, thus saving time during surgical operations.

The strong absorption capacity as well as the optimal mechanical property of the developed Colisorb sponge was reflected in the assessment of the blood intake, clotting time, and blood clotting index. The blood intake of the developed Colisorb sponges demonstrated its high ability for blood absorption, reaching a blood intake of 735 μL after 3 min as compared to 250 μL for Cutanplast. Such strong absorption ability is reached due to the hydrophilic nature of the developed sponge and its high porosity as well as its optimal mechanical properties, allowing it to maintain a well-structured matrix even after blood absorption for a few minutes. Thus, this allowed the sponge to maintain optimal mechanical strength while allowing maximum expansion for enhanced swelling ability and blood absorption.

Such high absorption capacity leads to a fast concentration of the clotting factors, resulting in a shorter clotting time of only 4 min as compared to the commercial product Cutanplast, which exhibited a blood clotting time of 12 min. In addition, Colisorb exhibited the lowest clotting index, almost 53% lower than the control, while Cutanplast was only 34% lower than the control. This is possibly due to the unique cross-linking of the Colisorb sponge that allowed maximum air entrapment that reached up to 70% of the total sponge volume. Prothrombin time (PT) measurements showed that the Colisorb sponge had a lower PT time compared to the commercial product. Prothrombin time (PT) is an assay that measures the time that it takes for the liquid portion (plasma) of blood to coagulate. PT is correlated with the extrinsic coagulation pathway. , Previous studies showed that the gelatin sponge demonstrated the lowest PT values compared to other samples made with either keratin or collagen. On the other hand, other studies reported that the difference was not significant.

Ideal hemostatic materials should have a hemolysis rate that is less than 5%. According to ISO 10993-4 and ASTM F756 standards, the acceptance criteria for the hemolytic index is below 2%, which is considered nonhemolytic. However, those between 2–5% are slightly hemolytic, and those exceeding 5% are classified as hemolytic. , Assessment of the hemolysis test is essential to assess the biocompatibility of the biomaterials. The results of the in vitro hemolysis assay of the Colisorb sponge demonstrated no significant difference in the hemolytic activity compared to the commercial product Cutanplast. Yan et al. reported that gelatin sponges showed the lowest hemolysis rate when compared with keratin and chitosan sponges. Colisorb’s hemolysis percentage (1.4%) falls within the nonhemolytic range, reflecting biocompatibility with RBCs and safe membrane adhesion. For Cutanplast, its significantly high hemolysis rate (30.1%) exceeds the acceptance criteria by almost 15%. This highlights its incompatibility with direct RBC membrane adhesion. According to Atef et al. (2025), Colisorb’s surfactant-free formulation, high hydrophilicity, and smoother porous structure support rapid plasma absorption without inducing shear or chemical stress on RBC membranes.

Scanning electron microscopy (SEM) analysis showed distinct and safe interactions between Colisorb and RBCs compared to Cutanplast. The porous and hydrophilic structure of Colisorb promotes the efficient entrapment of both RBCs and platelets. RBC adhesion accommodates the accelerated hemostatic performance of Colisorb as observed by the increased RBC entrapment and activated platelets.

In vitro, cytotoxicity assessment using mouse fibroblasts (L929) showed that the Colisorb enhanced cell viability compared to the commercial product Cutanplast. According to ISO 10993-5, cell viability below 70% of the blank is considered an indication of a cytotoxic effect. Ding et al. showed that gelatin hydrogel showed no cytotoxicity on both human fetal hepatocytes and mouse embryonic fibroblast cells. Complementary testing with HepG2 and hFB cell lines confirmed these trends, consistently indicating that Colisorb exerts minimal cytotoxic effects. The hemolysis and cytotoxicity results confirm the safety of the developed Colisorb sponge.

In vivo experiments demonstrated that the Colisorb sponge reduced blood loss volume and increased the rate of blood clotting compared with the control. For example, in the rat liver laceration model, Colisorb significantly promoted hemostasis, resulting in a blood loss of only 100 mg compared to 300 mg with Cutanplast. A previous study also reported that the use of gelatin sponges in wound healing was associated with enhanced hemostasis and a high level of patient satisfaction. Histopathological analysis revealed no significant differences in liver tissue between the Colisorb and control groups, indicating that Colisorb did not induce undesirable tissue inflammation. These findings support the biocompatibility and safety of Colisorb as a hemostatic material, which aligns with previous reports. , The current study’s findings were compared against published hemostatic agents; comparative in vivo clotting parameters from the literature are presented in Table . The referenced studies have all employed a liver laceration model in rats, followed by hemostatic sponge application directly to the wound site. Colisorb’s in vivo performance metrics are comparable with advanced hemostatic formulations such as starch-based macroporous sponges (Kr-Sps1-4: 30.4 s) and natural collagen sponges (31.62 s), while substantially exceeding standard gelatin sponge performance (90–125 s), which are commonly used commercial hemostatic agents. Such results position Colisorb within the advanced performance tier of hemostatic agents because it combines rapid hemostasis with favorable material characteristics suitable for clinical applications. −

2. Comparative Table of In Vivo Clotting Parameters.

Hemostatic Sponge	Clotting Time (s)	Blood Loss (mg)	Reference
Colisorb	40	100
Cutanplast	90	300
Gelatin Sponge	90	1000
Starch-Based Macroporous Sponges (Kr-Sps1-4)	30.4	37
Gelatin Sponge	125	150
TEMPO-oxidized nanocellulose/gelatin(G) spongeTOCN 2.5G-Th	82.2	-
Natural Collagen Sponge	31.62	-
Chitosan/gelatin (CG) sponge	-	5.5
Collagen Fiber (CF) Sponge	50.33	35.67
Oxidized bacterial cellulose (OBC) and chitosan (CS) with collagen (COL) sponge	86	-

Potential limitations include individuals with known gelatin allergies, which may be unsuitable for patients with religious or dietary restrictions regarding animal-derived materials. In surgical applications, gelatin sponges can swell upon fluid absorption, which may limit their use in confined anatomical spaces due to the risk of compression on adjacent structures. In addition, while our use of the rat liver injury model allowed for reproducible and ethically manageable assessment of Colisorb’s hemostatic performance in early preclinical evaluation, we acknowledge its limitations in simulating human-scale bleeding dynamics, vessel caliber, and tissue perfusion pressures. In this regard, we highlight that an extended phase of this project is already underway, involving established large-animal liver injury models. Such experiments will evaluate Colisorb’s efficacy in controlling hemorrhage under clinically relevant conditions, assess its handling properties, and generate translational data to support future clinical application. We believe that this addition will significantly enhance the clinical relevance of our findings.

5. Conclusion

In conclusion, the current study presents a comprehensive characterization and experimental investigation of the newly developed Colisorb hemostatic device. The mechanical characterization demonstrated that Colisorb is a mechanically stable, highly hydrophilic gelatin sponge with remarkable swelling capacity, reaching 77 times its weight in 10 min. Moreover, biocompatibility evaluation through in vitro assessments revealed a high degree of biocompatibility, evidenced by the material’s noncytotoxic effects on three distinct cell lines (hepatocytes, human fibroblasts, and mouse fibroblasts), indicating its safety and suitability for biomedical applications. Furthermore, in vitro hemostatic evaluation demonstrated enhanced blood-absorbing capacity and BCI of Colisorb, indicating its favorable properties for effective blood clotting and reduction of blood loss. Such in vitro observations were confirmed in an in vivo efficacy study in a rat liver laceration model, where Colisorb demonstrated superior hemostatic performance, exemplified in a hemostasis time of 40 s and minimal blood loss (100 mg). Taken together, this study proposes a promising hemostatic gelatin sponge that can reduce operative bleeding and promote hemostasis in surgical settings to enhance patient outcomes.

The raw experimental data and detailed methodology used in this study are available from the corresponding author upon reasonable request.

∥.

M.H., E.A.K., and S.A.H. contributed equally to this manuscript.

The authors declare that no external funds, grants, or other financial support were received during the preparation of this manuscript. This research was conducted as part of the research and development activities at Incura Inc.

The authors declare no competing financial interest.