Bioabsorbable Carboxymethyl Starch–Calcium Ionic Assembly Powder as a Hemostatic Agent

In contrast to hemostatic fabrics, foams, and gels, hemostatic spray powders may be conveniently applied on narrow and complex bleeding sites. However, powdered hemostatic agents are easily desorbed from the bleeding surface because of blood flow, which seriously decreases their hemostatic function. In this study, the hemostatic performance of a bioabsorbable powder with decreased desorption was investigated. The proposed hemostatic powder (OOZFIXTM) is an ionic assembly of carboxymethyl starch and calcium. The microstructure and chemical properties of the hemostatic powder were analyzed. The hemostatic performance (blood absorption, blood absorption rate, and coagulation time), thromboelastography (TEG), rheology, adhesion force, and C3a complement activation of the OOZFIXTM were evaluated and compared with those of the carboxymethyl starch-based commercial hemostatic powder (AristaTM AH). The in vivo rat hepatic hemorrhage model for hemostasis time and bioabsorption of the OOZFIXTM showed quick biodegradation (<3 weeks) and a significantly improved hemostasis rate (78 ± 17 s) compared to that of AristaTM AH (182 ± 11) because of the reduced desorption. The bioabsorbable hemostatic powder OOZFIXTM is expected to be a promising hemostatic agent for precise medical surgical treatments.


Introduction
In recent years, the frequency of surgical operations has considerably increased owing to developments in medical technology and operation convenience [1]. Bleeding from local wounds formed during simple surgical procedures is sufficiently controlled by bodily hemostasis. The amount of blood in an average adult is approximately 6-7% of body weight, and blood loss presents a risk to survival only when it accounts for more than 10% of the total blood volume [2]. When the body is wounded, epithelialization proceeds through hemostasis and the inflammatory phase within the first day, followed by the proliferation of epithelial cells for 3 weeks [3,4]. Hemostatic agents are applied at the earliest stage of medical treatment to wound sites to prevent bleeding and blood loss [5][6][7][8][9][10].
In this study, an effective hemostatic agent addressing the above problems is proposed. A powder-type highly absorbent adhesive hemostatic agent containing blood coagulation factors (calcium chloride) and with carboxymethyl starch was designed. Powder-type hemostatic agents are easy to apply even in narrow and complicated areas and generally, do not cause intense foreign body sensations [15,16]. In addition, the proposed hemostatic powder was intended to maintain good hemostasis performance without desorption because of carboxymethyl starch and calcium ionic assembly network. Fine particles have a high specific surface area and coating ability; thus, they are widely used in the medical field [17][18][19][20]. In particular, microparticles have very small diameters and a large specific surface area enabling good blood plasma absorption, thereby maximizing the effect of blood coagulation factors.
Here, we demonstrated blood plasma absorption and blood coagulations of bioabsorbable hemostatic powder composed of carboxymethyl starch and calcium ions. The developed hemostatic powder is highly promising for use in clinical applications and surgery.

Fabrication of Hemostatic Powder
Carboxymethyl starch (50 wt%; JRS PHARMA GmbH, Germany) was dissolved and stirred in a solution of calcium chloride (0.08 wt%; CaCl 2, Daejung, Siheung, Korea) in ethanol (99%, Daejung, Siheung, Korea) for 2 h. The obtained solution containing precipitate was filtered using a filter paper to eliminate unreacted calcium chloride, which was then rinsed with ethanol three times. Subsequently, the filtered solution was dried in an oven (40 • C for 24 h) and pulverized by cryogenic grinding. The ground hemostatic particles were then sieved using a sieve tray (nominal aperture 125 µm, Daihan Scientific, Seoul, Korea) to obtain a homogeneous powder. The powdered sample was sterilized under UV light (254 nm) and kept in a sealed pack for further use.

Microstructure and Chemical Analysis
The morphology of the hemostatic powder was investigated using scanning electron microscopy (SEM, JSM-6380, JEOL Ltd., Tokyo, Japan) with an acceleration voltage range of 11-15 kV after sputter-coating with platinum. The average diameter (D) of the polygonal powder particles was determined as the equivalent area diameter (D 2 = 4 A/π, where A is the cross-sectional particle area) using image analysis software (I-solution Lite, IMT i-solution, Seoul, Korea) and a particle size analyzer (Shimadzu SALD-2300, WingSALD II, Version 3.3.0, Kyoto, Japan). The powder particle size was measured in triplicate for each specimen.
The chemical compositions of hemostatic powders were investigated using an attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy (VERTEX 80V, Bruker, Ettlingen, Germany) with a germanium ATR crystal. All spectra were recorded in absorption mode with a 4.0 cm −1 scan interval in the scanning range of 4000-800 cm −1 .

In Vitro Blood Absorption and Coagulation Time (Lee-White Method)
Quantitative analysis of the blood absorption amount, blood absorption rate, and coagulation ability is essential for evaluating the hemostatic performance. The absorption and coagulation characteristics of the blood treated with the hemostatic powder were analyzed using whole blood (dog, 13% anticoagulant, Korea Animal Blood Bank, Seoul, Korea). CaCl 2 (0.0125 M) was dissolved in the blood prior to the experiment to avoid any anticoagulant effects. In brief, sample powder (0.1 g) was placed in a watch glass, and then the blood was dropped onto it until saturation. At the moment of saturation, the blood volume and rate (elapsed time) were recorded. The blood absorption was calculated using the following equation: Blood absorption (mL/g) = absorbed blood amount (mL)/sample weight (g) Blood coagulation time was measured using the Lee-White method [16,21]. A glass vial was placed in a 37 • C water bath for 10 min. Then, blood (3 mL) and hemostatic powder (0.1 g) were added into the vial, and the vial was tilted every 20 s. The time until blood no longer flowed after contacting the hemostatic agent was recorded.

TEG
The blood-clotting activity of human blood exposed to the hemostatic powder was analyzed using TEG assays. The TEG machine (TEG Hemostasis Analyzer 5000; Haemoscope Corporation, Niles, IL, USA) was calibrated before use according to quality control standards. TEG assays were performed within 2 h of sample collection, in accordance with the manufacturer's instructions. To study the effect of powder dosage on coagulation function, 5 mL of whole blood were collected in a tube containing citrate (3.2%). The specimen (0.01 g), whole blood (340 µL, dog, Korea Animal Blood Bank, Sokcho, Korea), and CaCl 2 (40 µL) were placed in a TEG cup and analyzed using a plastic TEG instrument (39 • C). The reaction time (R, min), clotting time (K, min), angle (α, • ), and maximum amplitude (MA, mm) were recorded. Whole blood (5.4 mL) was collected from other 10 healthy blood donors in 2 tubes (2.7 mL per tube) containing citrate (3.2%); an additional 2 mL of whole blood was collected in a tube containing EDTA-k2. Using these samples, conventional coagulation tests, TEG assays (with 20 mg of each powder), and blood cell counts were performed.

Adhesion Ability
The adhesion force was measured by the falling force between the tissue and the coagulated powder sample. Briefly, a piece of pig skin was attached to the upper surface (40 mm in diameter parallel Peltier steel plate, 37 • C) of the rheometer (ARES, TA instruments, New Castle, DE, USA). The powder (0.2 g) was placed on the bottom plate of the device, mixed with 1 mL of purified water, and allowed to swell. The swollen sample was applied with an upward force to the pig skin attached to the top of the rheometer.

Cytotoxicity
Elution test of bioabsorbable hemostatic powder was performed using L929 fibroblasts (connective mouse tissue) to investigate cytotoxicity (ISO 10993-5: 2009, Biological Evaluation of Medical Devices). The test substance and control substance were eluted with minimum essential medium (MEM) culture solution containing 10% fetal bovine serum (FBS), 1% penicillin streptomycin in an incubator (37 • C, 5% CO 2 ) for 24 h. Test materials eluate (test group) in L-929 cells were high-density polyethylene (HDPE) film (Hatano Research Institute, Japan) as a negative control material (negative control group), 0.25% zinc dibuthyldithiocarbamate (ZDBC) polyurethane film (Hatano Research Institute, Japan) as a positive control material (positive control group), blank test solution (solvent control group), and OOZFIX TM powder. Samples were incubated in an incubator for 48 h.
For qualitative evaluation, it was determined under ISO 10993-5 regulation. Eluate of the test substance had no cytotoxicity if it was grade 2 or less. Quantitative evaluation was carried out after qualitative evaluation, and Trypsin-EDTA was treated in each cell culture well and incubated for about 5 min in an incubator. Relative cell count (RCC) was calculated as the following equation.
If the viability decreased to less than 70% (<70%) of the blank group, it was judged to be potentially cytotoxic.

C3a Complement Activation Assay
An enzymatic immunoassay for the quantification of C3a fragments present in the human blood after the interaction with hemostatic powders was performed using the C3a PlusEIA kit from MicroVue (Quidel, San Diego, CA, USA) and immunosorbent assay (PHOmo, Autobio, Zhengzhou, China). The powder samples were incubated at 37 • C for 1 h and subsequently processed according to the manufacturer's instructions. The absorbance was measured at 450 nm using a microplate reader (Anthos HT III, type 12,600, Anthos Mikrosysteme GmbH, Krefeld, Germany). The concentration of C3a was expressed in ng/mL and as a percentage of the activation in the control, which was incubated and treated using the same procedure. Zymosan (Sigma Aldrich, St Louis, MO, USA) was used as the positive control. Measurements were performed in duplicate.

In Vivo Hemostasis and Biodegradation
Outbred Sprague-Dawley rats (male, 230-280 g, 8 weeks old, Hyochang Science, Daegu, Korea) were used as experimental models for evaluating in vivo hemostasis and biodegradation. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the Kyungpook National University (No.: 2020-0105, Date: 24 October 2020). The experimental procedures were approved by the Animal Care Committee and performed as follows. The rats were anesthetized using a 4:1 volume ratio of Zoletil (Virbac, France) to Rompun (Bayer, Germany). Following stabilization of the rats, their livers beside the hilus were opened, and bleeding was induced using punch biopsy (6 mm) with a cross (+) shape (1 × 1 cm). The bleeding site was covered with cotton gauze, and after the removal of the gauze, the powder samples (0.1 g) were applied. Thereafter, a 50-gram weight block was placed onto the site for 60 s. The site was observed every 15 s and rinsed with a saline solution.
The absorbable hemostatic powder does not require the removal of the blood-clot complex material. We performed in vivo experiments to evaluate the biodegradability of OOZFIX TM powder. The powder (0.1 g) was injected intradermally into the backs of the rats (n = 16). The biodegradability of the powder was evaluated at weeks 1, 2, 3, and 4.

Statistical Analysis
All data are presented as mean ± standard deviation. Statistical analyses were performed using KyPlot software version 6.0 (KyensLab, Inc., Tokyo, Japan). Significance levels were calculated using parametric Student's t-tests and one-way ANOVA with a Tukey's post hoc test. Statistical significance was set to p < 0.05.

Microstructure of Hemostatic Powders
The particle size and surface morphology are the key parameters for assessing the blood absorption and clotting behavior of hemostatic powders with the same base. This is because the specific surface area of the particles and the volume of voids allowing quick blood absorption are proportional to the particle dimensions and surface structure; thus, a high specific area leads to rapid hemostasis. The OOZFIX TM construct was prepared via ionic assembly of carboxymethyl starch with CaCl 2 . The dried construct was cryogenically ground and sieved to obtain a homogeneous powder. As shown in Figure 1, the particle sizes of the OOZFIX TM and Arista TM AH powders are similar, with the mean diameter ranging from 30 to 150 µm. The OOZFIX TM powder (mean diameter: 64.4 µm) consists of spheroidal particles with a relatively smooth surface. The Arista TM AH (mean diameter: 65.5 µm) powder particles are spherical but have a wrinkled surface. Singh reported that irregularities in non-spherical particles of hemostatic powders contained more voids for blood absorption and clot formation [22,23]. Although the microstructure of both powders is not identical, the mean diameter of globular powders has a similar distribution with negligible voids. Therefore, the results suggest that OOZFIX TM and Arista TM AH powder can absorb blood plasma in a similar time.
All data are presented as mean ± standard deviation. Statistical analyses were performed using KyPlot software version 6.0 (KyensLab, Inc., Tokyo, Japan). Significance levels were calculated using parametric Student's t-tests and one-way ANOVA with a Tukey's post hoc test. Statistical significance was set to p < 0.05.

Microstructure of Hemostatic Powders
The particle size and surface morphology are the key parameters for assessing the blood absorption and clotting behavior of hemostatic powders with the same base. This is because the specific surface area of the particles and the volume of voids allowing quick blood absorption are proportional to the particle dimensions and surface structure; thus, a high specific area leads to rapid hemostasis. The OOZFIX TM construct was prepared via ionic assembly of carboxymethyl starch with CaCl2. The dried construct was cryogenically ground and sieved to obtain a homogeneous powder. As shown in Figure 1, the particle sizes of the OOZFIX TM and Arista TM AH powders are similar, with the mean diameter ranging from 30 to 150 μm. The OOZFIX TM powder (mean diameter: 64.4 μm) consists of spheroidal particles with a relatively smooth surface. The Arista TM AH (mean diameter: 65.5 μm) powder particles are spherical but have a wrinkled surface. Singh reported that irregularities in non-spherical particles of hemostatic powders contained more voids for blood absorption and clot formation [22,23]. Although the microstructure of both powders is not identical, the mean diameter of globular powders has a similar distribution with negligible voids. Therefore, the results suggest that OOZFIX TM and Arista TM AH powder can absorb blood plasma in a similar time.

Chemical Analysis
The functional groups of the hemostatic agent were confirmed using ATR-FTIR analysis ( Figure 2). The base material of both the OOZFIX TM and Arista TM AH powders is carboxymethyl starch. The characteristic spectra of carboxymethyl starch show broad

Chemical Analysis
The functional groups of the hemostatic agent were confirmed using ATR-FTIR analysis ( Figure 2). The base material of both the OOZFIX TM and Arista TM AH powders is carboxymethyl starch. The characteristic spectra of carboxymethyl starch show broad peaks at 3430 and 2930 cm −1 , corresponding to the stretching vibrations of -OH and -CH 2 , respectively. The peaks at 1650 and 1365 cm −1 were assigned to the -OH bending vibration. The peaks at 1000-1200 cm −1 were ascribed to the stretching of the -C-O-C-linkage in the polysaccharide backbone chain. The ATR-FTIR spectrum of the OOZFIX TM powder shows peaks at 1612 and 1425 cm −1 , which are not observed in the spectrum of the Arista TM AH powder. We attributed these peaks in OOZFIX TM to the -COO − asymmetric and symmetric stretching vibrations, respectively, caused by the ionic assembly with Ca 2+ [10].
respectively. The peaks at 1650 and 1365 cm were assigned to the -OH bending vibra-tion. The peaks at 1000-1200 cm −1 were ascribed to the stretching of the -C-O-C-linkage in the polysaccharide backbone chain. The ATR-FTIR spectrum of the OOZFIX TM powder shows peaks at 1612 and 1425 cm −1 , which are not observed in the spectrum of the Arista TM AH powder. We attributed these peaks in OOZFIX TM to the -COO − asymmetric and symmetric stretching vibrations, respectively, caused by the ionic assembly with Ca 2+ [10].

In Vitro Blood Absorption and Coagulation Time
During in vitro evaluation, the amount and rate of blood absorption until maximum absorption are measured by contacting the blood with a hemostatic powder. As shown in Figure 3A, OOZFIX TM and Arista TM AH powders exhibit similar blood absorption amounts and both show a Grade 1 blood absorption rate (less than 5 s). The amount and rate of blood absorption are proportional to the source materials and specific surface area [3][4][5]. As shown in Figure 1, the powders have the same base material and similar particle sizes. Therefore, the amount and rate of blood absorption do not differ significantly.
Shorter coagulation time indicates the effective activation of blood coagulation factors and stronger adhesion of platelets. As shown in Figure 3B, the blood coagulation time in the OOZFIX TM group is significantly lower than that in the Arista TM AH group. Even though the blood absorption amounts of both hemostatic powders are similar, the OOZFIX TM group shows faster blood coagulation than the Arista TM AH group. After blood absorption, OOZFIX TM maintained its network structure because of ionic assembly with Ca 2+ that promoted blood coagulation. Therefore, we concluded that Ca 2+ on the carboxymethyl starch network structure facilitated hydrophilicity and blood coagulation with gelation at later steps of hemostasis.

In Vitro Blood Absorption and Coagulation Time
During in vitro evaluation, the amount and rate of blood absorption until maximum absorption are measured by contacting the blood with a hemostatic powder. As shown in Figure 3A, OOZFIX TM and Arista TM AH powders exhibit similar blood absorption amounts and both show a Grade 1 blood absorption rate (less than 5 s). The amount and rate of blood absorption are proportional to the source materials and specific surface area [3][4][5]. As shown in Figure 1, the powders have the same base material and similar particle sizes. Therefore, the amount and rate of blood absorption do not differ significantly.

TEG
The TEG parameters (R, K, α, and MA) of the OOZFIX TM and Arista TM AH powders are summarized in Table 1. The fibrin and clot formation profiles (TEG tracings) of the blood samples treated with hemostatic powders are shown in Figure 4. The R and K values for the OOZFIX TM group (R = 3.6, K = 0.8) are lower than those for the Arista TM AH group (R = 4.6, K = 1.6). Clotting parameters α and MA for the OOZFIX TM group (α = 82.4, MA = 83.1) are much higher than those for the Arista TM AH group (α = 59.2, MA = 75.1). The TEG data support the results of the blood coagulation time test (Figure 3B), indicating lower initial fibrin coagulation and clot formation times in the OOZFIX TM group. In addition, the maximum clot strength (MA) in the OOZFIX TM group is higher than that in the Arista TM AH group.  Shorter coagulation time indicates the effective activation of blood coagulation factors and stronger adhesion of platelets. As shown in Figure 3B, the blood coagulation time in the OOZFIX TM group is significantly lower than that in the Arista TM AH group. Even though the blood absorption amounts of both hemostatic powders are similar, the OOZFIX TM group shows faster blood coagulation than the Arista TM AH group. After blood absorption, OOZFIX TM maintained its network structure because of ionic assembly with Ca 2+ that promoted blood coagulation. Therefore, we concluded that Ca 2+ on the carboxymethyl starch network structure facilitated hydrophilicity and blood coagulation with gelation at later steps of hemostasis.

TEG
The TEG parameters (R, K, α, and MA) of the OOZFIX TM and Arista TM AH powders are summarized in Table 1. The fibrin and clot formation profiles (TEG tracings) of the blood samples treated with hemostatic powders are shown in Figure 4. The R and K values for the OOZFIX TM group (R = 3.6, K = 0.8) are lower than those for the Arista TM AH group (R = 4.6, K = 1.6). Clotting parameters α and MA for the OOZFIX TM group (α = 82.4, MA = 83.1) are much higher than those for the Arista TM AH group (α = 59.2, MA = 75.1). The TEG data support the results of the blood coagulation time test (Figure 3B), indicating lower initial fibrin coagulation and clot formation times in the OOZFIX TM group. In addition, the maximum clot strength (MA) in the OOZFIX TM group is higher than that in the Arista TM AH group.

Rheology
Viscoelasticity of hemostatic powders reflects the physical strength of the gelated powder on wound skin site after blood absorption and hemostasis. As shown in Figure 5, the OOZFIX TM and Arista TM AH gels exhibit the rubbery plateau region storage (G', elasticity) of 3000 and 1000 Pa, and loss moduli (G'', viscosity) of 500 and 100 Pa, respectively. The viscoelastic gel properties on plateau region and maximum delta values (δ, damping behavior) of the OOZFIX TM gels maintain higher values than those of the Arista TM AH gels. This means that the OOZFIX TM gels are expected to maintain on the wound skin despite increasing strain after blood absorption and coagulation.

Rheology
Viscoelasticity of hemostatic powders reflects the physical strength of the gelated powder on wound skin site after blood absorption and hemostasis. As shown in Figure 5, the OOZFIX TM and Arista TM AH gels exhibit the rubbery plateau region storage (G', elasticity) of 3000 and 1000 Pa, and loss moduli (G", viscosity) of 500 and 100 Pa, respectively. The viscoelastic gel properties on plateau region and maximum delta values (δ, damping behavior) of the OOZFIX TM gels maintain higher values than those of the Arista TM AH gels. This means that the OOZFIX TM gels are expected to maintain on the wound skin despite increasing strain after blood absorption and coagulation. The viscoelastic gel properties on plateau region and maximum delta values (δ, damping behavior) of the OOZFIX TM gels maintain higher values than those of the Arista TM AH gels. This means that the OOZFIX TM gels are expected to maintain on the wound skin despite increasing strain after blood absorption and coagulation.

Adhesion
Bioabsorbable hemostatic powders have been widely adopted for the convenience of their use in narrow and complicated sites, particularly during laparoscopy. However, their efficacy is limited because of partial desorption at the bleeding site, which is not observed with other types (fabric, sponge, gel) of hemostatic agents [16,17]. A tissue-adhesive hemostatic powder would solve this issue and show fair hemostatic ability without serious loss of hemostatic powder. As shown in Figure 6, the adhesive strength of the OOZFIX TM group is −0.45 N, which is twice that of the Arista TM AH group. The OOZFIX TM powder is a carboxymethyl starch-calcium ion assembly [15], and its hydrophilicity increases with hydrogelation through blood plasma absorption, thus enabling strong adhesion of the OOZFIX TM gels.

Adhesion
Bioabsorbable hemostatic powders have been widely adopted for the convenience of their use in narrow and complicated sites, particularly during laparoscopy. However, their efficacy is limited because of partial desorption at the bleeding site, which is not observed with other types (fabric, sponge, gel) of hemostatic agents [16,17]. A tissue-adhesive hemostatic powder would solve this issue and show fair hemostatic ability without serious loss of hemostatic powder. As shown in Figure 6, the adhesive strength of the OOZFIX TM group is −0.45 N, which is twice that of the Arista TM AH group. The OOZFIX TM powder is a carboxymethyl starch-calcium ion assembly [15], and its hydrophilicity increases with hydrogelation through blood plasma absorption, thus enabling strong adhesion of the OOZFIX TM gels.

Cytotoxicity for a Bioabsorbable Hemostatic Agent
Quantitative cytotoxicity of powder-type hemostatic agents was evaluated by counting live cells cultured in the eluate of the sample (Figure 7). Compared with the positive control, the negative control and OOZFIX TM showed cell viability of 96.64% and

Cytotoxicity for a Bioabsorbable Hemostatic Agent
Quantitative cytotoxicity of powder-type hemostatic agents was evaluated by counting live cells cultured in the eluate of the sample (Figure 7). Compared with the positive control, the negative control and OOZFIX TM showed cell viability of 96.64% and 94.62%, respectively. In addition, qualitative analysis was performed through microscopic observation of a cell monolayer. Compared with the positive control, the negative control and OOZFIX TM were hardly observed to separate the extracellular matrix and inhibit the growth of cells, and showed grades 0 and 1, respectively. Although foreign body reactions of natural carbohydrates are rare, investigating cytotoxicity of a new powder product made of polysaccharides (OOZFIX TM ) is important. In both quantitative and qualitative results, the OOZFIX TM powder-type hemostatic agent was judged to be safe to apply as a medical material.

C3a Complement Activation
Complement system activation is a major parameter for evaluating the inflamm tory reaction to hemostatic agents. The complement system is critical for inducing th immunogenic response to bacteria, viruses, and other foreign bodies. Activation of th system can proceed through three possible pathways, which all involve multistep prote cleavage, but differ by the nature of the triggering element. These three enzymatic ca cades have a common enzyme, C3 convertase, which cleaves the C3 protein in an phylatoxin C3a. The level of C3a detected in the blood incubated with the OOZFIX TM an Arista TM AH powders reflects the inflammation reaction. As shown in Figure 8, th Arista TM AH powder exhibits significant activation.

C3a Complement Activation
Complement system activation is a major parameter for evaluating the inflammatory reaction to hemostatic agents. The complement system is critical for inducing the immunogenic response to bacteria, viruses, and other foreign bodies. Activation of this system can proceed through three possible pathways, which all involve multistep protein cleavage, but differ by the nature of the triggering element. These three enzymatic cascades have a common enzyme, C3 convertase, which cleaves the C3 protein in anaphylatoxin C3a. The level of C3a detected in the blood incubated with the OOZFIX TM and Arista TM AH powders reflects the inflammation reaction. As shown in Figure 8, the Arista TM AH powder exhibits significant activation.

In Vivo Hemostasis Time
Hemostatic evaluation through animal model experiments is the most important method for analyzing the performance of hemostatic agents. As shown in Figure 9, the average hemostasis times in groups treated with the OOZFIX TM and Arista TM AH powders are 78 ± 17 and 182 ± 11 s, respectively. The OOZFIX TM group showed a significant reduction in hemostasis time. Several hemostatic powders contain both blood absorption materials and blood-clotting reagents, such as thrombin and CaCl 2 , to facilitate clotting [12,15]. Therefore, the excellent hemostatic performance of the OOZFIX TM powder was attributed to the synergistic effects of carboxymethyl starch and CaCl 2 .
cleavage, but differ by the nature of the triggering element. These three enzymati cades have a common enzyme, C3 convertase, which cleaves the C3 protein in phylatoxin C3a. The level of C3a detected in the blood incubated with the OOZFIX T Arista TM AH powders reflects the inflammation reaction. As shown in Figure Arista TM AH powder exhibits significant activation.

In Vivo Hemostasis Time
Hemostatic evaluation through animal model experiments is the most imp method for analyzing the performance of hemostatic agents. As shown in Figure  average hemostasis times in groups treated with the OOZFIX TM and Arista TM AH ders are 78 ± 17 and 182 ± 11 s, respectively. The OOZFIX TM group showed a signi reduction in hemostasis time. Several hemostatic powders contain both blood abso materials and blood-clotting reagents, such as thrombin and CaCl2, to facilitate cl Polymers 2022, 14, x FOR PEER REVIEW [12,15]. Therefore, the excellent hemostatic performance of the OOZFIX TM powd attributed to the synergistic effects of carboxymethyl starch and CaCl2.

In Vivo Biodegradation
OOZFIX TM hemostatic powder was designed as a biodegradable agent for he sis without removal [24]. Thus, the biodegradation behavior of OOZFIX TM powd investigated in an in vivo environment (intradermal injection into rat scapula) serving the morphology after a set period. As shown in Figure 10, the OOZFIX TM p remained after 1 week, while most of the powder was desorbed with partial debris weeks. No powder traces were observed in the intradermal areas after 3 and 4 wee biodegradation rates of hemostatic agents vary depending on the base material an (fabric, foam, gel, or powder). The OOZFIX TM powder is based on carboxymethyl which is a plant-based bioabsorbable material. Simultaneously, because of the lar cific surface area, the hemostatic powder is quickly biodegraded after inducing stasis, thus relieving the uncomfortable feelings after laparoscopic surgery in narro complicated areas.

In Vivo Biodegradation
OOZFIX TM hemostatic powder was designed as a biodegradable agent for hemostasis without removal [24]. Thus, the biodegradation behavior of OOZFIX TM powder was investigated in an in vivo environment (intradermal injection into rat scapula) by observing the morphology after a set period. As shown in Figure 10, the OOZFIX TM powder remained after 1 week, while most of the powder was desorbed with partial debris after 2 weeks. No powder traces were observed in the intradermal areas after 3 and 4 weeks. The biodegradation rates of hemostatic agents vary depending on the base material and type (fabric, foam, gel, or powder). The OOZFIX TM powder is based on carboxymethyl starch, which is a plant-based bioabsorbable material. Simultaneously, because of the large specific surface area, the hemostatic powder is quickly biodegraded after inducing hemostasis, thus relieving the uncomfortable feelings after laparoscopic surgery in narrow and complicated areas.

Conclusions
A hemostatic powder (OOZFIX TM ) composed of carboxymethyl sta ions was fabricated via ionic assembly, drying, cryogrinding, and sieving powder consisted of spheroidal particles. The in vitro blood absorption a in the OOZFIX TM group were similar to those in the Arista TM AH group. blood coagulation rate and clot strength in the OOZFIX TM group were sig than in the Arista TM AH group. The OOZFIX TM group showed lower lev plement activity than the Arista TM AH group. In vivo rat hepatic hemorr periment showed remarkable hemostatic ability in the OOZFIX TM grou duced desorption. The biocompatibility and hemostatic properties of the starch-calcium powder (OOZFIX TM ) developed in this study were supe commercialized products. Moreover, incorporating active blood coag thrombin, in powder-type hemostatic agents is a challengeable agenda fo In order to expand the application area of powder-type hemostatic ag hemostatic properties are required based on this study. ′ ′ Figure 10. Photographs of in vivo biodegradation of the OOZFIX TM hemostatic powder (0.2 g) that was intradermally injected into the rat scapula (n = 16); (A1-A4) 1, (B1-B4) 2, (C1-C4) 3, and (D1-D4) 4 weeks after the injection.

Conclusions
A hemostatic powder (OOZFIX TM ) composed of carboxymethyl starch and calcium ions was fabricated via ionic assembly, drying, cryogrinding, and sieving. The OOZFIX TM powder consisted of spheroidal particles. The in vitro blood absorption amount and rate in the OOZFIX TM group were similar to those in the Arista TM AH group. In contrast, the blood coagulation rate and clot strength in the OOZFIX TM group were significantly better than in the Arista TM AH group. The OOZFIX TM group showed lower levels of C3a complement activity than the Arista TM AH group. In vivo rat hepatic hemorrhage model experiment showed remarkable hemostatic ability in the OOZFIX TM group owing to reduced desorption. The biocompatibility and hemostatic properties of the carboxymethyl starch-calcium powder (OOZFIX TM ) developed in this study were superior to those of commercialized products. Moreover, incorporating active blood coagulants, such as thrombin, in powder-type hemostatic agents is a challengeable agenda for future works. In order to expand the application area of powder-type hemostatic agents, functional hemostatic properties are required based on this study.

Conflicts of Interest:
The authors declare no conflict of interest.