Development of a Rapid-Acting Oxidized Regenerated Cellulose-Based Powder for Hemostatic Applications

Abstract

Local hemostasis is widely used to control intraoperative bleeding. Among these approaches, locally acting hemostatic powders are particularly useful for managing bleeding across large areas in conventional surgery and for controlling bleeding in anatomically challenging sites during laparoscopic surgery, owing to their ability to be delivered using applicators such as powder dispensers. In this study, we developed DMSP (Daedeok plant medical devices surgiguard powder), a novel powder-type hemostatic agent with a porous structure composed of ORC and CMC. DMSP effectively promotes platelet aggregation and concentrates coagulation factors through rapid water absorption, thereby accelerating local clot formation. In our study, DMSP showed excellent clot-forming capacity and mechanical strength, and thromboelastography (TEG) analysis revealed a shorter time to coagulation onset and enhanced clot firmness. Similarly, DMSP also showed a strong hemostatic effect than commercially available products. These findings confirm the potential of DMSP as a next-generation hemostatic solution that improves the limitations of current powder-type products, such as insufficient hemostatic efficacy, delayed initiation of hemostasis, and slow clot formation. In addition, it demonstrates suitability for clinical applications in surgical procedures where significant bleeding control is required.

1. Introduction

Failure to achieve complete hemostasis during surgery can have a serious impact on postoperative recovery. In severe cases, excessive hemorrhage can be life-threatening. , Persistent microscopic oozing during surgery requires caution, as even small amounts can accumulate and lead to significant blood loss. , This type of bleeding often occurs over a wide area and can make hemostasis more difficult when the bleeding occurs at anatomically less accessible locations or where direct pressure cannot be applied. ,

Conventionally, mechanical interventions such as sutures, ligating clips, and staplers have been used to achieve hemostasis. When these methods are impractical or insufficient, locally acting hemostatic agents are used adjunctively. , These agents are classified based on formulation, including gauze, sponge, gel, film, and powder types, and use various materials, including gelatin, collagen, oxidized regenerated cellulose (ORC), plant polysaccharides, synthetic polymers, thrombin, and fibrinogen. They exert a hemostatic effect by providing a physical substrate that promotes coagulation or through direct interaction with blood coagulation pathways through biologically active compounds that promote fibrin clot formation. Hemostatic agents are effective when the site of bleeding is local and clearly exposed. Typically, pressure is applied after using the product to encourage hemostasis. However, formulations such as gauze, sponge, and film-type products present significant application challenges in complex bleeding scenarios, such as wide bleeding surface area, or confined surgical spaces encountered during laparoscopic or other procedures, thereby limiting their deployment and clinical effectiveness. Powder-type hemostatic agents can overcome the limitations of conventional forms by facilitating controlled and efficient delivery through applicators. Accordingly, powder-type hemostatic agents have been developed, some of which are available on the market. ,

For example, hemostatic powders derived from plant polysaccharides have the advantages of rapid absorption and biodegradation; however, several studies have reported that their hemostatic efficacy is lower than ORC-based hemostatic powders. , An ORC-based hemostatic powder penetrates the blood after application to the bleeding site owing to its high density, which is attributed to a nonporous structure formed under physical pressure. During this process, the powder separates into microfibers, which facilitate effective hemostasis. However, the dense structure may restrict the initial contact with blood, and because physical hemostatic agents act by absorbing water from the blood and concentrating nearby coagulation factors, the onset of hemostatic activity may be slightly delayed. In the present study, to improve blood clotting efficiency, we developed a novel powder-type hemostatic agent composed of ORC microfibers and carboxymethyl cellulose (CMC). The ORC used in DMSP is derived from the same raw material as SurgiGuard, which is commercially available in Europe and Korea and has been evaluated for biocompatibility in accordance with ISO 10993 standards. In addition, CMC is widely used in pharmaceutical and biomedical applications and is generally regarded as biocompatible. In order to promote rapid hemostasis, a porous structure with large surface area was adapted that would upon application rapidly absorb water from the blood increasing the local concentration of coagulation factors. ORC was selected as the base hemostatic agent and CMC was incorporated for its excellent water absorption abilities. −

In this study, the hemostatic effects of DMSP are systematically evaluated in comparison with hemostatic powders currently used in the clinic, including an ORC-based SURGICEL Powder and starch-based Arista AH. The clotting ability and mechanical strength of the formed blood clots were evaluated in vitro, and the time to hemostasis was investigated in vivo using rat partial nephrectomy and pig liver punch models.

2. Experimental Section

2.1. Test Materials

The materials used in this study were ORC microfibers (Samyang Biopharm, Corp., Seongnam, Republic of Korea), DMSP (Samyang Biopharm, Corp., Seongnam, Republic of Korea), SURGICEL Powder (Ethicon, Somerville, NJ, USA), and Arista AH (Becton Dickinson, Franklin Lakes, NJ, USA). All powders were dried at room temperature (20–25 °C) for at least 24 h to minimize the effects of ambient moisture and were weighed immediately prior to use.

2.2. Morphological Analysis

Field emission-scanning electron microscopy (FE-SEM; JEOL Ltd., Tokyo, Japan) was used to examine the morphological differences between the test substances. Prior to FE-SEM observation, the samples were coated with platinum using a sputter coater (Cressington Scientific Instruments Ltd., Watford, United Kingdom). Next, the magnification was set to 300×, with an accelerating voltage of 5 kV and working distance of 25 mm under vacuum conditions.

X-ray microscopy (XRM; Xradia 520 Versa, Carl Zeiss, Oberkochen, Germany) was used to visualize the internal structure of DMSP and SURGICEL Powder. The analysis was conducted at a power of 2 W with an accelerating voltage of 30 kV. A total of 1601 projection images were acquired over a 360° rotation with an exposure time of 15 s per projection. An optical magnification of 4× was applied to achieve an effective pixel size of 0.7 μm. The obtained 3D data sets were processed and reconstructed using Dragonfly software (Object Research Systems, Montreal, Canada). Subsequently, the 3D reconstructed volume of the individual particles was virtually divided into four sections to extract representative 2D cross-sectional images.

2.3. Water Absorption Test

The water absorption behaviors of the hemostatic powders were analyzed using a force tensiometer Sigma-701 (Tensiometer; Biolin Scientific, Gothenburg, Sweden) and the Washburn adsorption method. Briefly, this method determines the contact angle of a powder by measuring the weight of a solution absorbed by capillary action when a powder-filled cylinder is placed in contact with the solution. In this experiment, the constant required to calculate the contact angle was not used, and only the water absorption rate was evaluated.

[Washburn adsorption method]

$$\cos \theta =\frac{m^2}{t}\times \frac{\eta }{{\rho }^2\sigma c}$$ 1

­(η = liquid viscosity, ρ = liquid density, σ = liquid surface tension, c = sample holder geometry, θ = contact angle, m = weight, t = time).

The sample holder was filled at least halfway with sterile physiological saline (JW Pharmaceutical Co., Ltd., Seoul, Republic of Korea) and then mounted on the tensiometer. After precisely measuring 250 mg of ORC microfibers, DMSP, SURGICEL Powder, and Arista AH, the powders were placed in a powder wettability measuring device and attached to the apparatus. The apparatus was brought into contact, and the water absorption behaviors were measured for 5 min. The water absorption properties of the powders were compared based on the water absorption rate (m ²/t) in the first 5 s and the amount of water absorbed at equilibrium. If equilibrium was not reached within 5 min, the amount of water absorbed after 5 min was recorded. All experiments were repeated four times per group.

2.4. In Vitro Blood Clotting Test

The Sprague–Dawley (SD) rats used in this study were purchased from Orient Bio, Inc. (Seongnam, Republic of Korea). All experimental procedures were performed in accordance with the guidelines for the care and use of laboratory animals and were approved by the Samyang Biopharm Corp. Institutional Animal Care and Use Committee (IACUC; approval no. SYAU2502).

2.4.1. In Vitro Blood Clotting Performance Test

An in vitro test was performed to investigate the blood coagulation performance of the test substances. This test was designed as a fixed-time comparative evaluation rather than a time-to-clot measurement. Blood was collected from the abdominal aorta of SD rats under general anesthesia using a sterilized syringe and immediately transferred into 3.2% sodium citrate tubes (BD, Franklin Lakes, NJ, USA) to prevent coagulation. To ensure even mixing, the blood samples were rolled in a roller mixer at room temperature. The collected blood was pooled into a 50 mL conical tube prior to the experiment to minimize variations between samples.

All test substances were weighed to 100 mg immediately before the experiment and stored in an EP tube until use.

After pipetting 1 mL of blood into a preweighed 2 mL glass vial (Lklab, Namyangju, Republic of Korea), 100 mg of each test substance was evenly and simultaneously applied to the blood surface. The blood-test substance mixture was allowed to stand for 2 min at room temperature to induce natural coagulation and enable a direct comparison of initial clotting efficiency under identical conditions. Following this fixed interval, the vial was inverted and tapped at least ten times on filter paper (Whatman, Cytiva, Marlborough, MA, USA) to remove uncongealed blood, unreacted test substance, and the formed clot from the vial.

The coagulated clot was collected, placed back into the same vial, and weighed. To calculate the weight of the pure clot formed by the reaction between blood and the test substance, untreated blood was used as a control group. All experiments were repeated five times per group.

2.4.2. Blood clotting index (BCI) test

An in vitro test was performed to investigate the whole-blood coagulation kinetics of the test substances. The blood and test substances were prepared as described in Section .

After pipetting 1 mL of whole blood into a 15 mL conical tube, 100 mg of the test substance was added. The mixture was then vortexed for 5 s at 3000 rpm to ensure thorough mixing. Subsequently, the mixture was incubated for 2 min in a shaking incubator set at 30 rpm and 37 °C to induce coagulation. Following coagulation, 10 mL of deionized water was added to each tube and mixed through gentle inversion. Next, the tubes were further incubated for 10 min in a shaking incubator set at 50 rpm and 37 °C to lyse the red blood cells and release free hemoglobin.

After incubation, the samples were centrifuged for 5 min at 300 rpm, and the supernatant was collected. The supernatant was transferred into a 96-well plate, and an ultraviolet–visible (UV–vis) spectrometer (BioTek, Seoul, Republic of Korea) was used to measure the optical density (OD) at 540 nm.

Untreated blood was used as the control group. OD values were measured in triplicate for each sample, and the mean OD (OD_sample) was calculated as the final OD of each sample. The BCI was calculated using the following equation:

$$\mathrm{BCI}(\%)=\left(\frac{{\mathrm{OD}}_{\mathrm{control}}-{\mathrm{OD}}_{\mathrm{sample}}}{{\mathrm{OD}}_{\mathrm{control}}}\right)\times 100$$ 2

All experiments were repeated five times per group.

2.5. Thromboelastography (TEG) Test

To compare the clot formation ability of the hemostatic powders, TEG was performed using a TEG Hemostasis Analyzer 5000 (TEG 5000; Hemonetics Corporation, Boston, MA, USA). The blood used in the analysis was prepared in the same way as described in Section . The temperature of the TEG 5000 was set to 37 °C. Once the device reached the target temperature, 340 μL of blood, 10 mg of the test material, and 20 μL of sterile distilled water (JW Pharmaceutical Co., Ltd., Seoul, Republic of Korea) were added to the TEG 5000 cup, and the reaction time (R, min), kinetics time (K, min), clot formation rate (α, °), and maximum amplitude (MA, mm) were analyzed separately. For the control group, sterile distilled water was replaced with the same amount of 0.2 M CaCl₂ (Hemonetics Corporation, Boston, MA, USA), and all other methods were used as previously explained.

To investigate the blood coagulation response at different powder doses, DMSP and SURGICEL Powder were applied at 7.5 and 12.5 mg, respectively, and the experimental conditions were the same as above. The TEG analysis was performed for each powder until all parameters were recorded. All experiments were repeated three times per group.

2.6. In Vivo Hemostasis Test

2.6.1. Rat Partial Nephrectomy model

All animal experimental procedures were approved by the Samyang Biopharm Corp. IACUC (approval no. SYAU2402). Male SD rats, 7 weeks old, were purchased from Orient Bio, Inc. The rats were housed under conditions of a 12 h light-dark cycle, 24 ± 2 °C, and 50 ± 10% relative humidity, and food and water were provided ad libitum during the experimental period. All rats were acclimated for at least 1 week before the in vivo tests.

Immediately prior to the experiment, 100 mg each of Arista AH and SURGICEL Powder and 50 and 100 mg of DMSP were prepared.

The rats were anesthetized using intramuscular injections of 0.8 mL/kg rompun and alfaxan, and placed in the supine position. After a midline abdominal incision, the left kidney was exposed, and adipose tissue surrounding the cortex, renal artery, and renal vein was removed to minimize factors that could affect hemostasis.

After temporarily occluding the blood supply to the kidney, the upper one-fifth of the kidney was horizontally excised. The preweighed test substance was immediately applied to the cut surface, and blood flow was restored. The time from application of the test substance to completion of hemostasis was recorded and the excised kidney was weighed.

A nontreated group was set as the negative control to evaluate natural hemostasis, and the time to natural hemostasis was recorded. The completion of hemostasis was defined as the absence of additional bleeding for 1 min after bleeding has stopped, as confirmed by three independent observers. All experiments were repeated ten times for each group.

2.6.2. Pig Liver Punch Model

All experimental procedures were approved by the HLB BioStep Co. Ltd. IACUC (approval no. 24-HB-0305). Female white Yucatan minipigs (5 months old, weighing 47.4–50.0 kg, n = 4) were purchased from Optipharm (Cheongju, Republic of Korea). The pigs were housed under conditions of a 12 h light-dark cycle, 23 ± 3 °C, 55 ± 15% relative humidity, ventilation rate of 10–20 times per h, and illuminance of 150–300 lx, with food and water provided ad libitum during the experimental period. After 2 weeks of acclimatization, the pigs were used for in vivo test to investigate the hemostatic effects of the test substances. ,

Anesthesia was induced through intramuscular injection of alfaxalone (3 mg/kg), followed by inhalation of 100% oxygen and 0.5–5% isoflurane to maintain anesthesia. Respiratory anesthesia was maintained throughout the procedure, and pigs were positioned in dorsal recumbency. The abdomen was shaved and disinfected with 2% povidone and 70% ethyl alcohol. Electrocardiogram (ECG) and noninvasive blood pressure (NIBP) monitoring were performed to ensure adequate anesthesia and vital signs monitoring during surgery.

A midline skin incision was made from the xiphoid process to the umbilicus. Abdominal organs were kept moist using physiological saline and sterilized saline-soaked gauze.

Immediately prior to the experiment, DMSP and SURGICEL Powder were weighed to 37.5 and 75 mg, respectively. These doses were predetermined to adequately cover the defect area created by the biopsy punch. A nontreated group was included as a control to compare hemostasis time and success rate.

For the liver punch model, defects were created in three lobules (left medial, right medial, and right lateral) using a 6 mm biopsy punch (Kai Medical, Seki City, Gifu, Japan) calibrated to a depth of 3 mm. To minimize variability between lobules and individuals, the two doses of the test substance and the nontreatment control were applied once per lobule in all animals. All experiments were performed 12 times per group.

After the biopsy punch, defect sites were allowed to bleed naturally for a few seconds to enable observation of the initial bleeding. Only mild (capillary, arteriole, or venule oozing) or moderate (flowing venous and/or arterial) bleeding were included; defects with minimal or severe (pulsatile or spurting arterial or high-volume venous hemorrhage) bleeding were excluded. Unassessed or failed sites were treated using the sponge-type hemostatic agent SPONGOSTAN Standard (Ethicon, Somerville, NJ, USA). For ethical protection of the animals and to prevent death, assessment per site was limited to 10 min.

For sites with mild to moderate bleeding, the area was cleaned with sterilized gauze and the preweighed test substance was evenly applied. Next, the wound was covered with Telfa nonadherent wound dressing (Covidien, Mansfield, MA, USA), and pressure was applied for 30 s. The same procedure was followed for the control group without test substance application. After 30 s, the dressing was removed, and bleeding was reassessed. For sites that were still bleeding, the process of covering with the dressing and applying pressure for 30 s was repeated. Once effective hemostasis was achieved, the time to hemostasis (TTH) was recorded; if hemostasis was not achieved, the procedure continued for up to 10 min.

Sites where hemostasis was completed were determined to be successful when hemostasis was maintained for 1 min without additional pressure. Pinpoint bleeding was not considered a failure. TTH was defined as the time until the end of the last application of pressure, and if hemostasis was not achieved within 10 min, the TTH was recorded as >10 min.

The untreated group was assessed twice at the start and once at the end of the experiment to confirm that pressure alone could not achieve hemostasis within 10 min and that bleeding levels remained consistent. The order of test substance application was randomized prior to the experiment. This study was conducted at HLB BioStep Co. Ltd. (Incheon, Republic of Korea).

2.7. Statistical Analysis

All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) in GraphPad Prism 10 (GraphPad Software Inc., Boston, MA, USA). Statistical significance was set at p < 0.05.

3. Results and Discussion

3.1. Characterization of Hemostatic Powders

SEM analysis was performed to investigate the morphological characteristics of the DMSP and to compare it with other commercially approved hemostatic powders (Figure A). DMSP was manufactured using ORC microfibers obtained by pulverizing SurgiGuard Original (Samyang Biopharm Corp., Seongnam, Republic of Korea; Figure A­(a)). These ORC microfibers were agglomerated with CMC through wet granulation. Here, CMC acted as a binder to induce binding between the ORC microfibers, and ultimately, a highly porous spherical agglomerate was formed (Figure A­(b)). Notably, for SURGICEL Powder, an agglomerate was formed using only ORC microfibers, resulting in a dense, compressed structure with very few internal pores (Figure A­(c)). For Arista AH, porous particles were fabricated by cross-linking purified plant starch, forming a spherical particle with a wrinkled surface (Figure A­(d)). Based on the SEM results, DMSP exhibited a clearly porous structure compared to the other hemostatic powders.

1.

Structural and functional analyses of hemostatic powders. (A) SEM images of (a) ORC microfibers, (b) DMSP, (c) SURGICEL Powder (SURGICEL), and (d) Arista AH (Arista). Scale bar = 50 μm. B. XRM images of the ORC-based powders, DMSP and SURGICEL Powder. Scale bar = 50 μm. (C) Time-dependent water absorption profiles over a 5 min period (n = 4). (D) Initial water absorption rates (n = 4). (E) Equilibrium water uptake values (n = 4). The water uptake of Arista AH was measured at the end of the experiment, as equilibrium was not reached. Data are presented as mean ± SD. Statistical analyses were performed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

To further examine the internal pore structures, which are difficult to fully resolve using SEM alone, high-resolution X-ray microscopy (XRM) analysis was performed on individual particles (Figure B and S1). The 2D cross-sectional images extracted from the reconstructed 3D XRM data provided additional insight into their internal architecture. As shown in Figure B­(a), DMSP exhibited a porous internal network, suggesting that the granulation process with CMC contributes to the formation of internal void spaces. In contrast, the cross-sectional XRM image of SURGICEL Powder (Figure B­(b)) revealed a relatively denser and more compact internal structure. Although the submicron scale of the intraparticle pores limited reliable quantitative morphometric analysis, these qualitative XRM observations, together with the SEM results, support that DMSP possesses a more porous internal structure compared to the other hemostatic powders.

3.2. Water Absorption Characteristics of Hemostatic Powders

To evaluate the effects of differences in structural properties and composition on the water absorption ability of the powders, a tensiometer was used to analyze the water absorption characteristics of each powder (Supplementary Figure 2). Tensiometers are typically used to quantitatively analyze the interactions between liquids and solids. In this experiment, a tensiometer was used to measure both the initial water absorption rate and the cumulative amount of water absorbed until equilibrium was reached, as water was first adsorbed to and subsequently absorbed by the powder surface. Figure C shows the water absorption behavior of DMSP, SURGICEL Powder, and Arista AH over time. First, to examine the effects of ORC microfiber-based granulation and CMC addition, the initial water absorption rates of ORC microfibers, DMSP, and Surgicel Powder were compared. The initial water absorption rates were 0.002 ± 0.000, 0.056 ± 0.003, and 0.010 ± 0.001 g²/s, respectively (Figure D). Particularly, the initial water absorption rate of DMSP was 28 times faster than that of the ORC microfibers and five times faster than that of SURGICEL Powder. This was attributed to the formation of a porous structure through granulation and the presence of CMC, which has excellent water-absorption properties. , Next, to compare the characteristics of the ORC-based and starch-based hemostatic agents, the amount of water absorbed at equilibrium was compared. The amount of water absorbed was 0.623 ± 0.012 g for DMSP and 0.582 ± 0.010 g for SURGICEL Powder. While the incorporation of CMC in DMSP accelerates the initial rate of water uptake, the maximum absorption capacity appears to be primarily determined by the inherent properties of the ORC matrix, leading to a convergence in the final uptake values among ORC-based materials. Notably, as shown in Figure C, Arista AH did not reach equilibrium by the end of the experiment but had absorbed 1.304 ± 0.020 g of water by this time (Figure E). These differences in the amount of water absorbed are likely attributable to the high water retention capacity of starch and the differences in the physical properties of the materials. , Overall, the addition of CMC was associated with acceleration of the water absorption rate but did not significantly affect the amount of water absorbed.

In summary, although DMSP exhibited a rapid initial absorption rate owing to the addition of CMC, surprisingly the cumulative amount of absorbed water was lower than that of Arista AH, a starch-based hemostatic agent, and similar to that of SURGICEL Powder, another ORC-based hemostatic agent. The effects of water absorption characteristics on blood clotting were further investigated in subsequent experiments.

3.3. In Vitro Blood Clotting Assay

3.3.1. In Vitro Blood Clotting Performance Test

The blood coagulation performance and clot durability of the test substances were compared in vitro. In the nontreated group, blood immediately flowed out of the tube upon inversion. For the Arista AH and SURGICEL Powder groups, blood clot and some residual blood flowed out after tapping the tube. In contrast, the DMSP group showed no change even after five taps, revealing the formation of the strongest and most durable clot (Figure A). After a 2 min reaction under identical conditions, the tubes were inverted, and the clots were collected and weighed. DMSP produced significantly larger clots than Arista AH and SURGICEL Powder (Figure B). Specifically, the clot mass was approximately 11 and five times larger than each powder, respectively (Arista AH, 0.092 ± 0.048 g; SURGICEL Powder, 0.195 ± 0.055 g; DMSP, 1.056 ± 0.048 g). These results indicate that most of the test substances in the DMSP group effectively reacted with the blood, forming a significantly greater clot mass compared to other hemostatic powders.

2.

In vitro hemostatic analysis of the hemostatic powders. (A) Images of the in vitro blood clotting performance test using hemostatic powders. Images were captured (a) before powder application, (b) immediately after powder application, and (c) after 2 min incubation. Clot durability was evaluated by (d) tube inversion and tapping (e) two and (f) five times. (B) Quantification of clot weight (n = 5). (C) Images of the BCI test using hemostatic powders. (D) Quantitative analysis of BCI (n = 5). BCI values of test samples were normalized to those of the nontreated group. Data are presented as mean ± SD. Statistical analyses were performed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. The test materials used were as follows: (1) nontreated; (2) Arista AH; (3) SURGICEL Powder; (4) DMSP.

While various material properties, such as wettability and powder density, may influence the in vitro outcomes, the observed differences in clotting performance are primarily attributed to the initial liquid uptake kinetics. Owing to its rapid absorption behavior, DMSP promotes clot formation by facilitating localized concentration of coagulation factors and the formation of a physical scaffold. This rapid absorption and localized hemoconcentration may also contribute to the darker brownish-red coloration observed in the DMSP group (Figure A), potentially in combination with optical effects arising from the dense clot matrix. In addition, the mildly acidic microenvironment generated by oxidized regenerated cellulose (ORC) may induce the chemical transformation of hemoglobin into acid hematin. A similar coloration trend was observed for SURGICEL Powder, suggesting that this appearance is characteristic of ORC–blood interactions. Furthermore, clots formed by DMSP remained intact after repeated tapping, indicating high resistance to mechanical disruption. This property may be beneficial for maintaining clot stability under external stress conditions.

3.3.2. In Vitro Blood Clotting Index (BCI)

BCI analysis was evaluated to quantitatively assess the in vitro coagulation performance of the test substances (Figure C). Recent studies have revealed BCI analysis as an effective method for evaluating the coagulation performance of various biomaterials and as an important index to assess clot durability, with higher values indicating superior coagulation performance. The BCI values for Arista AH, SURGICEL Powder, and DMSP were 24.0 ± 6.6%, 62.6 ± 3.1%, and 92.0 ± 2.4%, respectively, revealing that DMSP exhibited a significantly higher BCI than the other test substances (p < 0.001; Figure D). This behavior is attributed to the rapid liquid absorption and the formation of a physical scaffold, which promote blood concentration and facilitate fibrin network formation. These results confirm that DMSP as a strong hemostatic agent and highlight its excellent clot-forming capability.

Based on the BCI results, DMSP shows strong potential as a hemostatic agent capable of forming stable clots, while minimizing hemolysis, revealing its applicability across diverse bleeding models in future clinical and preclinical settings.

3.4. Kinetic Evaluation of Hemostatic Powders via TEG

TEG is widely used as a diagnostic technique in clinical settings and for in vitro experimental studies. TEG provides real-time measurements of temporal changes throughout the blood coagulation process, enabling the analysis of dynamic characteristics that differentiate it from conventional static blood coagulation tests. This technique can provide quantitative information on various properties of hemostasis, including the initiation of coagulation, rate of coagulation, and clot firmness. Because the in vitro test (Section ) evaluated clot mass at a fixed time point, the reaction time (R), defined as the time to initial fibrin formation, was utilized here as a clinically relevant surrogate for conventional clotting time. After adding the test substances, the time to initial fibrin formation (reaction time, R), time to reach a specific clot firmness of 20 mm (kinetics time, K), clot formation rate (alpha angle, α), and maximum fibrin firmness (maximum amplitude, MA) were measured (Figure ). For Arsita AH, coagulation initiation was not observed, irrespective of the time allowed for reaction with blood, indicating that 10 mg was insufficient for clot formation. When SURGICEL Powder or DMSP was added to the TEG cup, the reaction times were 0.6 ± 0.3 and 0.2 ± 0.0 min, the kinetics times (K) were 9.2 ± 2.9 and 0.8 ± 0.0 min, the clotting rates were 50.5 ± 3.0° and 84.8 ± 0.8°, and the maximum firmness measurements were 33.5 ± 8.1 and 86.3 ± 0.5 mm, respectively (Table ). Notably, the significantly shortened kinetics time and the steeper alpha-angle of DMSP demonstrate an accelerated propagation of the fibrin network following initial coagulation. This suggests that DMSP reacts more rapidly with blood to form clots composed of a stronger fibrin network when compared with SURGICEL Powder. Consistent with the water absorption experiment (Figure C), these results indicate that rapid water absorption increases the local concentration of platelets, erythrocytes, and coagulation proteins, which promotes the rapid initiation of coagulation and accelerates subsequent clot development. , Furthermore, the shortened TEG reaction time (R) of DMSP, which reflects rapid coagulation initiation, is consistent with the enhanced clot formation observed at the 2 min time point in the in vitro assay, supporting its rapid hemostatic performance. In addition, when combined with the previous BCI results, this indicates that DMSP rapidly stimulates and forms more stable blood clots than SURGICEL Powder, resulting in a more effective hemostatic process.

3.

Thromboelastographic assessment of hemostatic powder. Thromboelastographic profiles of Arista AH (Arista), SURGICEL Powder (SURGICEL), and DMSP.

1. Blood Coagulation Parameters Measured Using TEG after Application of Hemostatic Powders .

TEG	reaction time (min)	kinetics time (min)	alpha angle (°)	maximum amplitude (mm)
Arista	n.d.	n.d.	n.d.	n.d.
SURGICEL	0.6 ± 0.3	9.2 ± 2.9	50.5 ± 3.0	33.5 ± 8.1
DMSP	0.2 ± 0.0	0.8 ± 0.0	84.8 ± 0.8	86.3 ± 0.5

^aQuantitative comparison of key TEG parameters, namely reaction time (R), kinetics time (K), alpha angle (α), and maximum amplitude (MA), for each hemostatic powder (n = 3). R and K were n.d. because the amplitude failed to reach the required thresholds of 2 and 20 mm, respectively; hence, α is also undeterminable. MA n.d. as the amplitude was present on the graph but remained below the detectable limit of the device. Data are presented as mean ± SD.

To evaluate the dose-dependent effects on blood coagulation, the coagulation reactions of SURGICEL Powder and DMSP, which had shown coagulation at a quantity of 10 mg, were further evaluated using the same methods at 7.5 and 12.5 mg (Supplementary Figure 3). At a dose of 7.5 mg, the respective reaction times of the two powders were 0.9 ± 0.4 and 0.2 ± 0.0 min, the coagulation rates were 26.7 ± 7.1° and 79.5 ± 5.2°, and the maximum fibrin firmness was 18.4 ± 4.5 and 61.8 ± 29.5 mm, respectively (Supplementary Table 1). At a 12.5 mg, SURGICEL Powder showed a reaction time of 0.2 ± 0.1 min, coagulation rate of 60.5 ± 7.1°, and maximum fibrin firmness of 72.5 ± 10.6 mm, whereas the results for DMSP exceeded the measurement range due to its rapid reaction with blood and high fibrin firmness (Supplementary Table 1). These results reveal that DMSP can rapidly form stable clots, even at low doses, which is likely due to the excellent water absorption properties observed in previous experiments.

In summary, DMSP exhibited a faster clot formation rate than SURGICEL Powder and Arista AH, and the resulting clots exhibited better structural stability. Effective hemostasis was achieved with relatively smaller amounts of the powder highlighting the effectiveness of DMSP.

3.5. In Vivo Evaluation of Hemostatic Efficacy

3.5.1. Hemostatic Efficacy in the Rat Partial Nephrectomy Model

To investigate the hemostatic effects of the test substances in vivo, a partial nephrectomy model was used in SD rats (Figure A).

4.

In vivo hemostatic efficacy analysis of the hemostatic powders. (A) Schematic illustration of the rat partial nephrectomy model and hemostasis procedure using hemostatic powders. (B) Time to hemostasis of hemostatic powders in a SD rat partial nephrectomy model (n = 10). (C) Images of the pig liver punch model (D) probability of remaining in bleeding in the pig liver punch model after application of hemostatic powders (n = 12).

To evaluate the practical hemostatic performance of DMSP, its efficacy was directly compared with commercially available hemostatic powders, including Arista AH and SURGICEL Powder, under identical experimental conditions.

For DMSP, both full (100 mg) and half (50 mg) doses were assessed against the commercial controls. The nontreated group showed natural hemostasis after 226.2 ± 59.6 s, whereas Arista AH showed a TTH of 219.1 ± 52.0 s, indicating no effect on hemostasis. SURGICEL Powder 100 mg reduced TTH to 173.7 ± 54.5 s, approximately 76.8% of the nontreated group. In contrast, DMSP 100 mg achieved hemostasis in 94.4 ± 32.3 s, corresponding to 41.7% of the nontreated group, and DMSP 50 mg dose achieved hemostasis in 120.4 ± 40.0 s, 53.2% of the time of the nontreated group. DMSP significantly reduced TTH compared to Arista AH and SURGICEL Powder. The mean weight of the excised kidneys was 0.11–0.12 g across all groups, thus confirming equivalent bleeding conditions.

These findings revealed that DMSP provided superior hemostatic effects at both full (100 mg) and half (50 mg) doses, revealing its dose efficiency. In contrast, Arista AH and SURGICEL Powder did not significantly reduce the TTH compared to the nontreated group and exhibited significantly longer TTH than DMSP.

3.5.2. Hemostatic Efficacy in Pig Liver Punch Model

To investigate the hemostatic effects of the test substances in a larger animal model with more clinically relevant bleeding conditions, an in vivo experiment was performed using a pig liver punch model (Figure C). The liver is highly vascularized and presents a challenge for achieving hemostasis, making this model clinically relevant for assessment. ,,

Following a protocol of repeated 30 s compression and reassessment for up to 10 min after hemorrhage induction, the nontreated control group, which received only compression, failed to achieve significant hemostasis, resulting in a 0% success rate. Because bleeding status was strictly evaluated at these fixed 30 s intervals, the recorded hemostasis times presented in Figure D are discretized, leading to an apparent step-like pattern in the data. This discretized measurement protocol, combined with the rapid fluid absorption and threshold-type clot stabilization of DMSP, likely contributes to the higher proportion of sites achieving hemostasis at the first measurable 30 s time point. This indicated that natural hemostasis does not occur in this bleeding model within 10 min. In contrast, all experimental groups achieved a 100% success rate within 10 min. The proportion of bleeding sites achieving hemostasis within 30 s was 90.9% for the DMSP 75 mg group and 66.7% for the SURGICEL Powder 75 mg group. At a half-dose of 37.5 mg, DMSP completed hemostasis within 30 s in 75.0% of sites, compared to only 18.2% for SURGICEL Powder, showing a marked decline in performance for the latter (Table ). The mean TTH was shorter in the DMSP 75 mg group (40.5 ± 34.7 s), followed by the SURGICEL Powder 75 mg group (50.2 ± 32.8 s), with no statistically significant difference between the two groups. However, at half dose of 37.5 mg, DMSP maintained rapid hemostasis (44.0 ± 26.4 s), whereas SURGICEL Powder showed a more than 2-fold increase in TTH (124.3 ± 78.8 s), representing a significant reduction in efficacy. These findings revealed that DMSP exhibits excellent hemostatic performance in a large animal compression model, even at a low dose. Compared to SURGICEL Powder, DMSP achieved faster hemostasis at equivalent doses and similar times at half doses, thus revealing its dose efficiency. Bleeding level was comparable across groups, with seven to ten mild and two to four moderate sites per group, thereby confirming equivalent bleeding conditions for comparison (Table ).

2. Hemostatic Success Rate within 30 s in Pig Liver Punch Model.

		SURGICEL Powder		DMSP
	nontreated	37.5 mg	75 mg	37.5 mg	75 mg
<30 s hemostasis	0.0% (0/12)	18.2% (2/11)	66.7% (8/12)	75.0% (9/12)	90.9% (10/11)
bleeding grade	mild 8 + moderate 4	mild 8 + moderate 3	mild 9 + moderate 3	mild 10 + moderate 2	mild 7 + moderate 4

Collectively, the hemostatic effects of the test substances were assessed in both small (SD rats) and large (pigs) animal models. Combining results from the partial nephrectomy model without compression and the liver punch model with compression, DMSP showed excellent hemostatic effects across diverse conditions. Importantly, DMSP demonstrated comparable or improved performance relative to widely used commercial hemostatic powders. Notably, its ability to achieve effective hemostasis with reduced doses emphasized its economic potential and clinical advantages.

4. Conclusions

In this study, the physical properties and hemostatic performance of DMSP, a novel powder-type hemostatic agent composed of ORC microfibers with CMC, are presented. The porous structure formed during granulation, combined with the unique water absorption properties of CMC, enabled the newly developed hemostat to exhibit faster water absorption than existing ORC-based powder products on the market. This rapid water absorption plays a key role in accelerating clot formation by increasing the concentration of platelets and coagulation factors when the hemostatic agent comes into contact with blood at a bleeding site. Notably, DMSP formed larger clots within the same time in the in vitro blood clotting performance test and, in the BCI analysis, exhibited better performance than existing commercial products in terms of the mechanical strength and structural stability of clots. Moreover, in the TEG analysis, DMSP reduced the time to coagulation, increased the clot formation rate, and improved clot firmness, demonstrating excellent dynamic properties throughout the coagulation reaction. This indicates that DMSP will likely provide a more robust and sustained hemostatic environment in actual bleeding scenarios. Thus, DMSP is expected to have high clinical utility, particularly in surgical settings where a high risk of rebleeding exists.

DMSP exhibited superior hemostatic performance in both small and large animal bleeding models compared to existing commercial products and maintained equivalent hemostatic effects even at lower doses. This suggests that the application of DMSP can provide additional cost-saving benefits.

In conclusion, DMSP exhibits rapid coagulation response, high clot firmness, excellent hemostatic stability, and outstanding dose efficiency. These findings highlight the potential of DMSP as a robust hemostatic solution fit for diverse surgical challenges.

Glossary

Abbreviations

α

alpha angle

BCI

blood clotting index

CMC

carboxymethyl cellulose

ECG

electrocardiogram

FE-SEM

field emission-scanning electron microscopy

IACUC

Institutional Animal Care and Use Committee

MA

maximum amplitude

NIBP

noninvasive blood pressure

OD

optical density

ORC

oxidized regenerated cellulose

R

reaction time

TEG

thromboelastography

TTH

time to hemostasis

XRM

X-ray microscopy

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c01249.

• Representative 2D cross-sectional XRM images; schematic illustration of the experimental setup, incorporating a force tensiometer, for evaluating the water absorption behavior of hemostatic powders using the Washburn method; dose-dependent thromboelastographic profiles of the hemostatic powders; quantitative parameters corresponding to the thromboelastographic profiles (PDF)

Conceptualization: H.H.K. and J.H.L.; data curation, formal analysis, investigation, and validation: H.H.K., J.H.L., J.E.P., J.H.S., and J.H.H.; visualization, writingoriginal draft, and writingreview and editing: H.H.K., J.H.L., J.E.P., and J.H.S.; Supervision and project administration: H.C. and H.S.Y. All authors have made contributions to the writing of the manuscript, and all authors have read and approved the final version of the manuscript.

This study did not receive any specific grants from any funding organization. DMSP was manufactured by Samyang Biopharm Corp. for research purposes.

The authors declare no competing financial interest.