Microspheres of Carboxymethyl Chitosan, Sodium Alginate, and

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Tissue Engineering and Regenerative Medicine

Microspheres of Carboxymethyl Chitosan, Sodium Alginate and Collagen as a Hemostatic Agent In Vivo Jia Jin, Zhixiao Ji, Ming Xu, Chenyu Liu, Xiaoqing Ye, Weiyao Zhang, Si Li, Dan Wang, Wenping Zhang, Jianqing Chen, Fei Ye, and Zhengbing Lv ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00453 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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Microspheres of Carboxymethyl Chitosan, Sodium Alginate and Collagen as a Hemostatic Agent In Vivo Jia Jin 1,2*, Zhixiao Ji 1, Ming Xu1, Chenyu Liu1, Xiaoqing Ye1, Weiyao Zhang1, Si Li1,2, Dan Wang1,2, Wenping Zhang1,2, Jianqing Chen1,2, Fei Ye1*, Zhengbing Lv1,2* 1 College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, 310018, China 2 Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Hangzhou, 310018, China Keywords biomaterials, CSCM, wound healing, biocompatible, biodegradable

Abstract

In the search for biocompatible composite microspheres to be used as a hemostatic agent, in a previous study, we designed a novel biomaterial, consisting of composite microspheres containing three natural biological ingredients, carboxymethyl chitosan, sodium alginate and collagen (CSCM). Furthermore, the chemical and physical properties, hemostatic ability, biocompatibility and cytotoxicity were investigated in vitro. In this work, the in vivo hemostatic

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performance, wound healing, hemocompatibility, histocompatibility, and biodegradability were evaluated by a series of experiments. The results showed that CSCM could both stop bleeding and enhance healing efficiency by accelerating the clotting and the wound closure rate, suggesting that CSCM acts as a hemostat, and enhances wound healing. In addition, the CSCM material had negligible intracutaneous stimulation reactions and no obvious hemolytic reactions. More importantly, CSCM can be degraded in vivo without significant impacts on physiology, biochemistry and organization. Thus, CSCM may be a useful tool to stop bleeding in emergency conditions in both military and civilian settings.

Introduction Hemostats for field administration after traumatic injury are particularly important, as uncontrolled hemorrhage is still the major cause of death in the military and in civilian medicine.1-2 Almost 50% of military deaths, 90% of preventable military battlefield casualties and 33-56% of pre-hospital deaths are due to blood loss.3-4 Emergency and medical surgeries such as cardiovascular, hepatic, and orthopedic surgeries, have a high incidence of severe hemorrhage-related mortality and morbidity, requiring hemostatic intervention.5-6 In recent decades, various materials have been developed to control severe bleeding for external application,7-12 some of which are well known, such as QuikClot zeolite powder (First-generation QuikClot),13 QuikClot kaolin powder (Second-generation QuikClot),14-15 Rapid Deployment Haemostat (RDH) and HemCon dressings.

16-17

Each material has advantages and

disadvantages.18 For instance, QuikClot zeolite power is effective for high-pressure bleeding.19 However, its exothermic reaction causes tissue injuries, and its poor biodegradability leads to foreign body reactions.20 Hence, QuikClot kaolin powder uses kaolin instead of zeolite to avoid exothermic reactions. RDH is effective for minor wounds, but is not suitable for severe wounds.

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HemCon dressings have antimicrobial properties, but they are unfit for deep, narrow or irregularly shaped wounds.21 Moreover, research regarding novel hemostats has focused on physicochemical properties and hemostatic ability. However, the safety and biocompatibility of the materials have not been seriously considered in the early stages of material design.22 Thus, most materials cannot pass preclinical evaluations due to their poor safety and biocompatibility.18 For instance, montmorillonite hemostatic agents may cause thrombosis in vessels.23 In addition, the chemical modification biomaterials could also have safety and biocompatibility problems. Some of these biomaterials induce cytotoxicity, and some cause hemolysis. Furthermore, material degradation is also a remarkable problem. Some materials either cannot biodegrade, or their degradation products are poisonous, which blocks the biosafety and the approval of clinical trials. Thus, an ideal hemostatic material should be effective for compressible or non-compressible severe injuries, even irregularly shaped wounds, easily stored at high temperatures for extended periods of time, and have a good biodegradability and biocompatibility.24-28 Based on their good fibrinogen adsorption, high water absorption, macrophage phagocytosis activation, platelet aggregation promotion, immune activity facilitation, wound infection prevention, and antimicrobial activity, three promising biomaterials from biomass sources (Carboxymethyl chitosan (CMC), sodium alginate (SA) and collagen) are commonly applied to hemostat for external application, tissue engineering and drug delivery systems.29-33 However, a single-component material could not afford all these properties. For instance, CMC is crosslinked with SA to increase the production of spherical microparticles with a porous surface34. Consequently, we designed a hemostatic powder CSCM (carboxymethyl chitosan, sodium alginate and collagen composite microspheres) for external application in a previous study35.

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Compared with the traditional hemostatic materials, these microspheres exhibit a remarkable blood-absorption property with excellent stacking interactions. This property is particularly important for bleeding control.36-39 As shown in Figure 1, when plasma, red blood cells (RBCs) and platelets flow out from the injury site, the specific microspherical shape, colossal surface area and material skeleton structure of CSCM enables this hemostatic agent to adhere to the injury site, absorb the plasma, activate platelets, and form a barrier to accelerate blood clotting. Due to its large capacity to absorb water from blood, CSCM concentrates coagulation factors to activate coagulation cascades and thrombin, to convert fibrinogen to fibrin mesh, a beneficial mechanism for hemostasis. In previous research, we found that CSCM could facilitate platelet performance, exhibited rapid biodegradability and was non-cytotoxic in vitro.35 These achievements encouraged us to further our experiments on hemostasis, wound healing and safety experiments with CSCM in vivo. In this study, the micro and porous structure of CSCM was detected through scanning electron microscopy (SEM) and particle size analysis. Furthermore, we carried out a hemostatic assay and a wound healing assay, which revealed that the material had a suitable hemostatic performance and healing ability in vivo. The biocompatibility of the material was investigated in detail, including a hemolytic assay and an intracutaneous stimulation test in a rabbit model. In addition, an in vivo degradation experiment was performed to assess the comprehensive safety of CSCM by subcutaneous implantation in a rat model. In these experiments, we used standard gauze or commercial compound microporous polysaccharide hemostatic powder (CMPHP) as a comparison group. Materials and methods

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Materials. Carboxymethyl chitosan (deacetylation degree > 60%) was purchased from Zhejiang Aoxing Co., Ltd. (Zhejiang, China). Sodium alginate was obtained from Xian Yuelai Medcine Co., Ltd. (Xian, China). Collagen was obtained from Zhejiang Sanchuang Biomedicine Co., Ltd. (Zhejiang, China). CMPHP was purchased from Shandong Success Pharmaceutical Technology Co., Ltd. (Shandong, China). Composite microspheres consisting of carboxymethyl chitosan, sodium alginate and collagen (CSCM) were prepared following the reported method.35 Briefly, the three-component mixture of CMC, SA and collagen was prepared in an aqueous solution. The mixture was later combined with medicinal paraffin oil and Tween-80 for one hour; then, a CaCl2 solution was added and stirred for another hour. Finally, the sample was precipitated in ethanol, washed with alcohol /petroleum ether, and dried in a vacuum. All reagents and solvents were of analytical grade and used without further purification. All chemical reagents were available from Sinopharm Chemical Reagent (Shanghai, China). Methods. Surface morphology characterization by scanning electron microscopy. Samples of CSCM and CMPHP were directly mounted on a holder with double-sided conductive adhesive tape and sputter coated with gold. The surface morphology of the samples was analyzed with a SEM (Hitachi S-4800, Electron Microscopy Center of East China Normal University, Shanghai, China) Particle size analysis. The particle size distributions of CSCM and CMPHP were measured by Malvern Mastersizer 2000 particle analyzer.

All animal procedures were approved by the Animal Care Committee, and the animals were cared for and treated in accordance with the regulations for the administration of affairs

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concerning experimental animals at the Hangzhou Normal University Institute of animal experimental center, and Jiangsu Provincial Institute of Materia Medica. In vivo hemostatic performance (rat tail amputation). Sprague-Dawley (SD) rats (♂, 0.2-0.25 kg, purchased from Hangzhou Normal University Institute of animal experimental center, Hangzhou, China) were anesthetized by an intraperitoneal injection with 10% chloral hydrate (0.03 ml/kg); half the length of the tail was cut with surgical scissors, and the remaining tail was placed in air for 15 s to ensure a normal hemorrhage model. Subsequently, pre-weighed tubes with 100 mg of CSCM, CMPHP, Wet Gauze (the standard gauze 5 × 5 cm saturated with normal saline) or Mixed CSC (CMC/SA/collagen mixed by the weight of 6/2/0.1 without further treatment) were placed to cover the wound with minimal pressure by immersing tails into tubes. The clotting time (s) and blood loss (g) were recorded during the hemostatic process. The tubes with Wet Gauze and CMPHP were used as comparison groups, and the empty tubes were used as a blank group; parallel groups (n ≥ 6) were tested to obtain an average value. Wound healing. Twenty-four SD rats (♀/♂ = 1:1, 0.2-0.25 kg) were acclimated in a cage for 5 days in-house and used for this study. They were divided into two groups, the test group and normal control group. In the experiment, a whole layer ring incision of 2.8 cm in diameter was cut on the back of each SD rat. The normal control group used Wet Gauze to control the bleeding with minor pressure. The test group used a spraying device (designed by our cooperative partner with an authorized Chinese patent); and 100 mg of CSCM was sprayed to control the bleeding. After the operation until the 12th day, the wound size was recorded to evaluate the wound healing at 2 or 3-day intervals. Intracutaneous stimulation test. The hemostatic agent (1 g) was placed into sterile and pyrogen-free PBS (90 ml) for 24 h at 37°C to create a hydrophilic leaching liquor, and the

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hemostatic agent (1 g) was placed into sterile and pyrogen-free olive oil (90 ml) for 24 h at 37°C to create a lipophilic leaching liquor. Four New Zealand white rabbits (♀/♂ = 1:1, 2–2.5 kg) were acclimated in a cage for 5 days in-house and divided into two groups, the hydrophilic leaching liquor group and lipophilic leaching liquor group. Two days before the test, the back spine of the animal was shaved (10 cm × 15 cm) and prepared for injection of the leaching liquor. There were 10 assigned injection points on each side of the spine (0.2 ml for each point, left side for the leaching liquor test group, right side for the blank leaching liquor control group). The observation time was set at 0, 24, 48, and 72 h, and the stimulation was recorded and scored. The intracutaneous stimulation response scoring criteria were as follows: erythema and scab formation (None-0, Bare-1, Clear2, Moderate-3, and Severe-4); and edema formation (None-0, Bare-1, Clear-2, Moderate-3, Severe-4). The higher the score was, the greater the irritation. The primary stimulus index (PII) was calculated by the mean of the stimulus index of each animal. The primary cutaneous stimulus response type (PCSRT) was defined as follows: Bare, PII 0-0.4; Clear, PII 0.5-1.9; Moderate, PII 2.0-4.9; and Severe, PII 5.0-8.0. Hemolysis assay. New Zealand white rabbits (♀/♂ = 1:1, 2-2.5 kg) were acclimated in a cage for 5 days in-house. Subsequently, blood (10 ml) was drawn from the ear vein of the rabbits, placed into a heparin anticoagulant tube and centrifuged (3000 r/min for 10 min). The supernatant was removed, and the remaining RBCs were suspended in an isotonic saline solution (12 ml, containing 2% albumin), sufficiently oscillated and then centrifuged (3000 r/min for 10 min). The whole procedure was repeated 3 times. The test-RBC suspension was obtained by resuspending the RBCs in isotonic saline solution. Hemostat (CSCM, 3 g) was transferred into the sterile, pyrogen-free PBS (270 ml) for 24 h at 37°C. Then, three tubes of the leaching liquor (10

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ml), three tubes of the negative control PBS (10 ml) and three tubes of the positive control distilled water (10 ml) were placed in a water bath for 30 min at 37°C. Subsequently, 0.2 ml of the suspended RBCs were added into each tube, and the mixture remained in the bath for another 60 min at 37°C. All the samples were centrifuged, and the optical density (OD) of the supernatant was tested at 545 nm with a UV-vis spectrophotometer to obtain the hemolytic rate. The percent hemolysis was calculated according to the following formula: Hemolysis (%) = (sample abs545 nm – negative control abs545 nm) / (positive control abs545 nm – negative control abs545 nm) × 100%.40 Safety assay in vivo. To evaluate the safety of the materials in vivo, CSCM was implanted on the backs of the rats in a six week experiment. To eliminate the influence of the surgery, a normal group and an operation group were used. The normal group was the control, and the operation group received the same operation as that of the test groups but without receiving the implant. The safety assay was evaluated using 80 SD rats (♀/♂ = 1:1, 0.21-0.25 kg) acclimated in a cage for 5 days in-house. They were divided into ten groups including six test groups, two normal groups and two operation groups. The animals were anesthetized using 5% chloral hydrate, their backs were shaved and disinfected, and they were fixed on the operating table. A 1.5 cm incision was made with a surgical scalpel on the back, and a pocket was created by one-sided blunt-skin isolation. The CSCM powder (0.25 g) was implanted into the pocket, and the detection indices, including the hematological index, biochemical index and immune factor assay, were evaluated during the degradation period.

Results and Discussion.

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Scanning Electron Microscopy. Based on our previous studies, we suspected that CSCM could have a pore structure or large internal spaces, which would facilitate the absorption ability. However, the internal structure was not directly observed. During the process of synthesis and preparation, the microsphere could first swell in base fluid, and then dehydrate to the final shape in the drying process. In this process, it is possible that the microsphere keeps its shape or collapses as shown in Figure 2A. For this reason, we tried to obtain images of some collapsed microspheres or broken microspheres to detect the internal structure by SEM. In Figure 2B, we could observe both ball-shaped and collapse-shaped microspheres. As shown in Figure 2C, a large, deep internal space is presented in the broken microsphere, which could increase the ability to absorb water and decrease the volume expansion ratio. On the other hand, some microspheres collapse and curl into a donut-like shape (Figure 2D). All these skeleton structures contribute to improve blood swelling and reduce tissue compression in material applications. Particle size distribution. The particle size distribution of CSCM is shown in Figure 3A. The average particle size had a volume of 39.33 µm, and the average particle size of surface area was 13.33 µm. The particle size distribution curve indicates that the particle sizes of CSCM are between 0.3 and 160 µm with d(0.1): 8.17 µm, d(0.5): 31.62 µm, and d(0.9): 79.60 µm. This observation means that 10% of CSCM is less than 8.17 µm, 50% of CSCM is less than 31.62 µm and 90% of CSCM is less than 79.60 µm. Since small particles adheres to the larger particles, altering the test results, the actual particle size of CSCM could be smaller. In vivo hemostatic performance. To evaluate the hemostatic effect of CSCM in vivo, the rat tail amputation model was employed by using Wet Gauze, Mixed CSC and commercial hemostatic agent, CMPHP, as comparison groups. The blank group and Mixed CSC group could not stop bleeding over ten minutes as expected. This phenomena explain it is difficult to achieve

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hemostasis without hemostatic agent in rat tail amputation model. As shown in Figure 4A, a block of blood clots was formed in the Wet Gauze group after hemostasis, while no apparent clot was observed in the CMPHP and CSCM groups. Additionally, only a small amount of the material powder was stuck. Furthermore, the blood clots that formed on the artery side of the tail were larger than those formed on the vein side (Figure 4A), implying the instantaneous fluid absorption capacity of CSCM. Consequently, the clotting time of the CSCM group was shortest (249.2 ± 44.7 s), followed by CMPHP (370.0 ± 43.1 s), gauze (428.0 ± 83.3 s) (Figure 4B) and the blank control group (over 900 s still bleeding). The mean clotting time of CSCM decreased by nearly 120 s compared to that of the CMPHP group (t-test, p < 0.01, n = 6), and by approximately 180 s compared to that of the Wet Gauze group. Although the blood loss of the CSCM (37.6 ± 8.1 mg) and CMPHP (54.5 ± 24.6 mg) groups was similar (t-test, p > 0.05, n = 6) (Figure 4C), the blood loss in the Wet Gauze group (665.2 ± 51.3 mg) was significantly increased (ten-fold) compared to that of the CMPHP or CSCM groups. These phenomena may be caused by the high fluid absorption of the CMPHP and CSCM materials, and activation of platelets. The combination of CMC, SA and collagen have integrated the advantages of the three materials and provided superior bleeding control through multiple hemostasis performances, as this experiment proved. Due to the microspheric structure and shape, the timely fluid-absorption ability of CSCM is beneficial for concentrating platelet and clotting factors. Hence, CSCM may shorten the time for the intrinsic coagulation cascade. In addition, hemadsorption induced by CMC could lead to the aggregation of RBCs and the formation of a blood clotting barrier. Moreover, the interface stimulated by alginate is able to activate coagulation factors with the

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eggshell skeleton containing Ca2+ ions. Finally, the collagen could strengthen platelet aggregation and activate intrinsic coagulation pathways, further promoting hemostasis. Wound healing. To further evaluate the hemostatic ability of CSCM, we investigated wound healing using a back laceration model in SD rats. CSCM was applied to skin incisions on SD rats as a wound closure material in the test group, and Wet Gauzes was applied on the normal control group. After the wound was formed, a small amount of bleeding appeared. CSCM was sprayed on the wound. CSCM absorbed the weep and blood, and formed a protective film on the wound. The protective film shrank and cured after the second day of surgery, demonstrating the high efficiency of CSCM in the wound-healing process at different time points (Figure 5). No infection was observed in the wounds of the two groups for the duration of the experiment. As shown in Figure 5, the test group showed better wound healing than that of the normal control group over the testing period, and it was particularly obvious during the first four days. The CSCM showed double the healing speed compared with that of the control in the first two days, indicating that it might increase wound healing in the coagulation and inflammation phase. However, the wound size of the control group was close to that of the test group by the end of the experiment. It is possible that the high efficiency of hemostasis, biocompatibility and material skeleton features are beneficial to wound healing and skin cell growth. However, the easy degradation and biocompatible characteristics that elicited these results could not be maintained as long as standard gauze (long-term physical barriers). Hence, the wound healing effect of CSCM was obviously superior to that of the standard gauze in the early stage, and there was no significant difference at the end of the experiment. These results suggest that our material may be more suitable for quick healing with minimally invasive surgery, or that its application form should be changed to prolong the effective use of the material.

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Intracutaneous stimulation test. As mentioned above, CSCM could reduce bleeding and wound healing time. Next, we measured the intracutaneous stimulation of CSCM through an in vivo rabbit model. Over ten observation points, only one bare erythema and a scab was observed after 24 h in the hydrophilic leaching group, while one bare edema was observed after 24 h in the hydrophilic leaching group and the blank control group (Table 1). The PII was only 0.05, which represents bare stimulation. There was no difference between the blank control and the test group, indicating that there was no stimulation in the lipophilic leaching group. The PII was only 0-0.05 in the test group, which showed that the material had very mild intracutaneous stimulation reaction. Hemolysis assay. The in vitro hemolysis assay is a universal method to assess the hemocompatibility of materials.41 The negative control, positive control and CSCM groups were incubated with the RBC solution, and the OD at 545 nm was measured. These results confirmed that CSCM does not show obvious hemolysis at a high dosage (only 0.83% hemolysis was observed with the dosage of the CSCM of 11.11 mg/mL). Therefore, the hemolytic ratio of CSCM is 0.83%, which is far lower than 5%, suggesting that CSCM is not hemolytic (Table 2). Safety assay in vivo. Finally, we carried out in vivo safety assays. All SD rats survived during the experiment period (7-42 days) without any signs of physical impairments or systemic inflammation and exhibited regular somatic growth. All of the normal group rats grew well, without displaying an abnormal diet, drinking, feces or back lesions. Two to three days after the operation, a decrease in the consumption of water and food was easily detected both in the operation and test group rats, but it returned to normal soon after. After surgery, the implanted materials (CSCM) could be easily detected in the subcutaneous pockets of the test group. Infection and abscesses were not observed over the next 42 days. When the experiment was

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complete, there were no observable differences between the test and normal groups. On day 28, the CSCM particles had integrated with the surrounding tissue based on the tissue anatomy and pathological section observations. Taken together, these observations indicate that CSCM predominantly degraded within 42 days following implantation (Figure 6). During the degradation period, the majority of the hematological indices were evaluated and recorded (Figure 7 and Figure 8). The index NEUT% and MONO% were increased in both the test and operation groups compared to those in the normal group. For the test group, the index was higher than that of the operation group in the first week, showing that a mild immune reaction was induced by the surgery and implanted material. These indices returned to a normal level in the next week, and there was no significant difference from the levels of the normal group by the end of the experiment (Figure 7(b), (d)). The index LYMPH% declined in both the test and operation group in the first week, but returned to normal the next week. This result suggests that the changes were due to the operation and not an infection (Figure 7(c)). The other indices, WBC, EOS%, BASO%, LUC%, RBC, and HGB, were not significantly different from those of the normal group in the experiment. The hemorrhage at the time of surgery may be the reason for the minor imperceptible decline in HCT% in the test group during the first week, but the value soon recovered (Figure 8(a)). In the first week after the operation, the indices PLT and PCT% were increased in the test and operation groups. This result was due to the activation of the hemostatic mechanism by the operation and the implanted material, which has been proven to activate platelets in vitro. The indices PLT and PCT% decreased as time passed, and the material degraded and returned to normal levels by the end of the experiment (Figure 8(f) and (g)). The other indices of the test group, MCV, MCH, MCHC, RDW%, and MPV, were not significantly different from those of

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the normal group in the experiment. The only index that declined at the end of the experiment was PDW%. However, this result shows that the platelets were more uniform than those in the normal group (Figure 8(i)). Most of the biochemical indices for the test group showed no significant differences compared to those of the normal group, including ALT, ALP, TP, CHO, and Na+. Some indices slightly declined in the first week and then recovered, such as AST, ALB, CREA, and K+. After the sixth week, the indices GLU and TG in the test and operation groups declined slightly compared to those of the normal group, but they were within a reasonable range (Figure 10(b), (c)). The TBIL of the test group increased in the first 5 weeks and started to decline in the sixth week. The TBIL was still higher than that of the normal group and operation group at the end of the experiment, indicating that the degradable biomaterial might be metabolized, slightly enhancing the load of the liver function (Figure 9(d)). Since the raw CSCM material contains proteins, an immune reaction could occur. Therefore, we performed an immune factor assay. Implants of CSCM may have induced a mild, locally restricted inflammatory reaction, as indicated by the immune index IgA, in the first week, but it disappeared in the following week (Figure 11(a)). The immune factor assay indicated that there was not a statistically significant difference between the normal group and test group at the end of experiment, suggesting that CSCM had no effect on the immune system. Overall, the test groups were safe compared to the normal and operation groups, (Figure 6 – 11). Moreover, in the test groups, wound healing and complete degradation of CSCM were detected around the implants at day 42, which indicated that the CSCM is safe and does not have side effects.

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Conclusion In summary, we investigated the composite microsphere, CSCM (based on the material of CMC, SA and collagen), which we previously designed in vivo. These results demonstrate that this novel hemostatic material exhibits the potential to reduce hemorrhage, promote coagulation and heal wound, In addition, the material has a good biodegradability, biocompatibility and safety. Hence, CSCM might be applicable to stop bleeding in military and civilian emergency situations. Future research will evaluate the long-term risks associated with hemostatic material administration and further assess their efficacy in large animal models where the hemodynamic flow conditions may be similar to those seen in clinical situations.

Figure 1. Simplified diagram of the blood clotting process with the CSCM.

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Figure 2. Micrographs of CSCM. (A) The formation mechanism of CSCM. (B-D) SEM images of the structures of CSCM: (B) a stack of ball-shaped and collapse-shaped CSCM (scale bar = 100 µm), (C) a broken CSCM (scale bar = 5.00 µm), (D) a collapsed CSCM (scale bar = 1.00 µm). Note the red arrow indicates the magnified structure area of CSCM.

Figure 3. Macro structure and particle size distribution of CSCM.

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Figure 4. The rat tail amputation model. (A) Photographs of the hemostatic effects of the three material (Wet Gauze, commercial hemostat CMPHP and CSCM). (B) Clotting time. (C) Blood loss. Values correspond to the mean ± SD, n = 6: *p < 0.05, **p < 0.01, ***p < 0.001 denotes significant differences compared with control groups.

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Figure 5. Wound healing assay. The wound areas were treated with Wet Gauze (normal control group) or CSCM (test group) for 12 days in a back laceration model on SD rats. The data represent the mean ± SD (n = 4, *p < 0.05, compared to the control group).

Figure 6. In vivo safety evaluation of CSCM. (a) Subcutaneous implantation of CSCM in SD rats. (b) When the experiment ended, the wound healed completely with no observable differences between the normal and operation groups. (c) Seven days after operation, the CSCM could be easily detected in the subcutaneous pockets, but the volume was already lower than that

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at implantation. (d) Twenty-eight days after operation, volume of the CSCM was significant decreased. (e) CSCM completely degraded within 42 days.

Figure 7. Hematological indices of the CSCM group measured in comparison with the normal and operation groups. (a) white blood cell count (WBC), (b) neutrophilic granulocyte percentage (NEUT%), (c) lymphocyte percentage (LYMPH%), (d) mononuclear cell percentage (MONO%), (e) eosinophil percentage (EOS%), (f) basophil percentage (BASO%), (g) large unstained cell percentage (LUC%), (h) red blood cell count (RBC), and (i) hemoglobin (HGB). (n = 8, mean ± SD, *p < 0.05 relative to the normal group in the first week, #p < 0.05 relative to the normal group in the sixth week, $p < 0.05 relative to the operation group in the sixth week).

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Figure 8. Hematological indices. (a) hematocrit (HCT), (b) mean corpuscular volume (MCV), (c) mean corpuscular hemoglobin (MCH), (d) mean corpuscular hemoglobin concentration (MCHC), (e) red cell volume distribution width (RDW), (f) platelet count (PLT), (g) plateletcrit (PCT), (h) mean platelet volume (MPV), and (i) platelet distribution width (PDW). (n = 8, mean ± SD, *p < 0.05 relative to the normal group in the first week, #p < 0.05 relative to the normal group in the sixth week, $p < 0.05 relative to the operation group in the sixth week).

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Figure

9.

Biochemical indices. (a) aspartate aminotransferase (AST), (b) alanine

aminotransferase (ALT), (c) alkaline phosphatase (ALP), (d) total bilirubin (TBIL), (e) total protein (TP) and (f) albumin (ALB). (n = 8, mean ± SD, *p < 0.05 relative to the normal group in the first week, #p < 0.05 relative to the normal group in the sixth week, $p < 0.05 relative to the operation group in the sixth week).

Figure 10. Biochemical indices. (a) creatinine (CREA), (b) glucose (GLU), (c) triglyceride (TG), (d) total cholesterol (CHO), (e) Na+, and (f) K+. (n = 8, mean ± SD, *p < 0.05 relative to

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the normal group in the first week, #p < 0.05 relative to the normal group in the sixth week, $p < 0.05 relative to the operation group in the sixth week).

Figure 11. Immune factor assay. (a) immune globulin A content analysis, (b) immune globulin B content analysis, (c) immune globulin C content analysis, (d) immune globulin D content analysis, and (e) immune globulin E content analysis. (n = 8, mean ± SD, *p < 0.05 relative to the normal group in the first week, #p < 0.05 relative to the normal group in the sixth week, $p < 0.05 relative to the operation group in the sixth week).

Table 1. Intracutaneous stimulation test

Time Index

(h)

Posterythe operation ma and 24 h scab 48 h

Hydrophilic leaching group

Lipophilic leaching group

1

2

1

2

Blank Test

Blank Test

Blank Test

Blank Test

0

0.1

0

0

0

0

0

0

0.1

0.1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

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edem a

72 h

0

0

0

0

0

0

0

0

Postoperation

0.1

0.1

0

0

0.2

0.2

0

0

24 h

0.1

0.1

0

0

0.2

0.2

0

0

48 h

0

0

0

0

0

0

0

0

72 h

0

0

0

0

0

0

0

0

Animal primary 0.1 stimulus score

0

0

PII

0.05

0

Reaction type

Bare

None

0

Table 2. Hemolysis assay of CSCM Group

Absorbance (OD)

Mean ± SD

Negative

0.049

0.037

0.052

0.046 ± 0.008

Positive

0.643

0.656

0.649

0.649 ± 0.007

Experiment

0.053

0.042

0.057

0.051 ± 0.008

AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected] *Email: [email protected] Author Contributions

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Haemolysis (%)

0.83

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Funding Sources This study was supported by grants from Public Projects of Zhejiang Province (2016C31017, 2016C32012), Natural Science Foundation of Zhejiang Province (LY18H300008) and Zhejiang Provincial Top Key Discipline of Biology, Science Foundation of Zhejiang Sci-Tech University (13042163-Y, 13042159-Y) and the 521 Talent Cultivation Plan of Zhejiang Sci-Tech University. Acknowledgements We would like to thank Jiangsu Experimental Animal Center of the Drug Research Institute and Hangzhou Normal University Institute of animal experimental center for experiment supports. Abbreviations used CSCM, composite microspheres consisting of carboxymethyl chitosan, sodium alginate and collagen; RDH, Rapid Deployment Haemostat; CMC, carboxymethyl chitosan; SA, sodium alginate; RBCs, red blood cells; SEM, scanning electron microscope; CMPHP, Compound Microporous Polysaccharide Hemostatic Powder; PCSRT, primary cutaneous stimulus response type; PBS, Phosphate Buffered Saline; SD, Sprague Dawley; OD, optical density; WBC, white blood cell count; NEUT, neutrophilic granulocyte; LYMPH, lymphocyte; MONO, mononuclear cell; EOS, eosinophil; BASO, basophil; LUC, large unstained cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell volume distribution width; PLT, platelet count; PCT, plateletocrit; MPV, mean platelet volume; PDW, platelet distribution width; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase;

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TBIL, total bilirubin; TP, total protein; ALB, albumin; CREA, creatinine; GLU, glucose; TG, triglyceride; CHO, total cholesterol. References 1.

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For Table of Contents use only

Microspheres of Carboxymethyl Chitosan, Sodium Alginate and Collagen as a Hemostatic Agent In Vivo Jia Jin 1,2*, Zhixiao Ji 1, Ming Xu1, Chenyu Liu1, Xiaoqing Ye1, Weiyao Zhang1, Si Li1,2, Dan Wang1,2, Wenping Zhang1,2, Jianqing Chen1,2, Fei Ye1*, Zhengbing Lv1,2*

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Figure 1. 316x92mm (300 x 300 DPI)

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Figure 2. 350x289mm (300 x 300 DPI)

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Figure 3. 350x289mm (300 x 300 DPI)

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Figure 4. 170x143mm (600 x 600 DPI)

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Figure 5. 149x80mm (300 x 300 DPI)

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Figure 6. 188x118mm (300 x 300 DPI)

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Figure 7. 163x122mm (300 x 300 DPI)

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Figure 8. 176x131mm (300 x 300 DPI)

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Figure 9. 173x86mm (300 x 300 DPI)

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 10. 175x86mm (300 x 300 DPI)

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ACS Biomaterials Science & Engineering

Figure 11. 179x91mm (300 x 300 DPI)

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Table of Contents (TOC) Graphic 84x47mm (300 x 300 DPI)

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