[System]
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---

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```
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</analysis>

<response>
{
  "has_error": <true|false>,
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      "location": "Section 2.1",
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      "description": "XAxis labeled ‘Time (s)’ but units appear to be milliseconds; scale bar mismatches caption."
    }
    // …more entries…
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}
</response>
```

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Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/carbpol

Self-assembling chitosan based injectable and expandable sponge with antimicrobial property for hemostasis and wound healing

Shujun Caoa, Peihong Jiaa, Lili Haoa, Chao Sunb,*, Hongli Maoa,*, Zhongwei Gua

a Research Institute for Biomaterials, Tech Institute for Advanced Materials, Bio-inspired Biomedical Materials & Devices Center, College of Materials Science and Engineering, Jiangsu Collaborative Innovation Center for Advanced Inorganic Function Composites, Suqian Advanced Materials Industry Technology Innovation Center, Nanjing Tech University, Nanjing 211816, China
b Department of Spine Surgery, The Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing 211100, China

ARTICLE INFO

Keywords:
Chitosan
Injectable
Shape memory
Sponge
Hemostasis
Wound healing

ABSTRACT

Chitosan and chitosan derivative are widely used in hemostasis, antibiosis and wound repair for its good biocompatibility and unique effect. However, the preparation of chitosan based hemostatic materials or wound dressings generally involves chemical crosslinking agent introduction, acid residue or complicated preparation process, which limits its clinical application. In this study, an injectable and expandable chitosan sponge was constructed by chitosan (CS) and quaternized chitosan (QCS) self-assembly without acid retention and chemical crosslinker introduction. In the neutral condition, the hydrogen bond of CS molecules can act as the driving force to form cross-linking network, and the QCS was introduced to regulate the hydrogen bond of CS to avoid the excessive aggregation. The porous QCS/CS sponge was obtained by freeze-drying of the self-assembly QCS/CS hydrogel. The sponge exhibited high expansibility, injectability and water/blood triggered shape memory property. Due to the introduction of QCS, the sponge showed good antibacterial properties, which can protect the wound from bacterial invasion. The convenient and green preparation method of injectable and expandable QCS/CS sponge is a potential method for the treatment of hemostasis and wound healing.

Due to the high physicochemical activity and biocompatibility of chitosan (CS), the CS based expandable sponge exhibits obvious advantages in hemostasis and wound healing promotion (Liu et al., 2022; Zhang et al., 2024). However, there are still some issues in the application of CS based expandable sponge, such as complex preparation process, or adverse stimulation caused by residual acid and chemical crosslinking agents (Cao et al., 2022, 2023a; Liu et al., 2023). In order to reduce the stimulation caused by acid and enhance the stability of the CS based sponge, Jin et al. prepared the acid-removed chitosan sponge by soaking the acidic chitosan sponge in the alkaline solution (Jin et al., 2023). Unfortunately, with the acid removing from CS sponge, the attractive force between the molecular chains of CS increases rapidly, which can lead to the shrinking and entanglement of CS molecular, causing a decline in the water absorption and expandability (Z.T. Li et al., 2024; Wang et al., 2023). In addition, the acidity removal of CS (pH > 6.5) can lead to its antibacterial decrease or disappear (Juan et al., 2022; Mottaghitalab et al., 2024; Priddy-Arrington et al., 2023). For retaining the high expansibility of CS sponge, researchers added glutaraldehyde into CS solution in the preparation process, followed by freeze-drying and soaking in alkaline solution to remove excess acid (Ahmad et al., 2024; Yao et al., 2022). But the volume of prepared CS sponge still reduces uncontrollably (Luo et al., 2022).

Acute hemorrhage caused by accidents and surgery is easy to lead excessive blood loss and bacterial infection, threatening life’s safety (Lu et al., 2022; Zhao et al., 2024). At present, gauze and bandages is the commonly used materials in clinical hemostasis (Peng et al., 2023). However, those hemostatic dressing is difficult to reach deep wounds, and has poor degradability, hemostatic and antibacterial properties. Expandable hemostatic sponge is a kind of hemostatic material with good absorptive capacity and controllable volume (Marani & Ehtesabi, 2023). The sponge can be easily transported into wound and plays a role by rapid expansion. So, the development of expandable sponges with good biodegradability, blood coagulability and antibacterial properties has broad prospect in hemostasis (Zhao et al., 2021).

In order to maintain the high expansion capacity, recent studies have been conducted by neutralizing the acidity of chitosan in solution (Qi

0144-8617/© 2024 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Inspired by previous studies, a novel injectable and expandable QCS/CS sponge with good antibacterial ability was developed for hemostasis and wound healing. The QCS/CS sponge was prepared following Scheme 1. By neutralizing the acidity of CS in solution and introducing QCS to regulate the hydrogen bond of CS to avoid the precipitation, a cross-linking network formed between CS and QCS/CS hydrogel was prepared through hydrogen bond self-assembly. QCS/CS sponge was obtained through freeze-drying. The experimental results showed that the prepared QCS/CS sponge has excellent shape memory properties and high expansion rate. Compared with gauze and gelatin sponge, QCS/CS sponge exhibited better blood cell adhesion and clotting ability. In addition, the prepared QCS/CS sponge can effectively kill S. aureus and E. coli to avoid bacterial infection. The results of rat liver injury model showed that QCS/CS sponge can effectively fill the wound and stop bleeding quickly. All the results demonstrate that the QCS/CS sponge is an appropriate candidate for the treatment of deep acute hemorrhage and wound healing.

et al., 2023a, b). After neutralizing the acidity, a stable and dispersed CS solution can be formed by breaking the aggregation through high-speed homogenizer, and epichlorohydrin (ECH) was further introduced to crosslink the CS to form a stable structure with sufficient chemical crosslinking. The preparation process avoids the appearance of gelation and removes the acidity, preserving the high expansibility of the CS based sponge. Therefore, neutralizing the acidity of CS in solution to prepare expandable sponge is an efficient method. However, there are still great challenges in enhancing antibacterial properties and reducing the use of chemical crosslinkers. Chitosan derivative such as quaternary ammonium (QCS) shows good biocompatibility and antibacterial properties (Liang et al., 2023; Qiao et al., 2023; Yang et al., 2021). The introduction of charged polysaccharide into the CS is expected to play an important role.

Chitosan (CS, Deacetylation ≥95 %, MW = 500,000) and Sodium bicarbonate (NaHCO₃) was purchased from Macklin Co., Ltd. Glycidyl trimethylammonium chloride (GTMAC) were obtained from Macklin Co., Ltd. mouse NIH 3T3 fibroblasts were purchased from Shanghai Institutes for Biological Sciences. All animal research was carried out by the National Research Council’s Guide for the Care and Use of Laboratory Animals and was authorized by Nanjing Tech University’s animal experimentation ethical committee. SD rats (5–6 weeks old, 150–200 g, male) were purchased from the Nanjing Qinglongshan Animal Breeding Co., Ltd.

QCS was prepared according to previous reports (Z.T. Li et al., 2024). A certain amount of CS was dissolved in 15 mL acetic acid aqueous solution to obtain CS solution. Then, 2 wt% QCS solution was added to the CS solution and stirred at room temperature, and the mass ratio of QCS to CS was 1:1, 1:1.25, 1:1.5, 1:1.75. Following, 0.147, 0.147, 0.221 and 0.294 g NaHCO₃ was added to QCS/CS solution. After stirring, the sponge precursor was transferred to the mold and reacted at −20 °C for 24 h. Then, the obtained sponge was thawed at room temperature, and the sponge was soaked in DI water for 2–3 days to remove excess CS, QCS, NaHCO₃. Finally, the QCS/CS sponges were obtained by freeze-dried. The sponges were named QCS/CS1, QCS/CS2, QCS/CS3 and QCS/CS4.

Scanning electron microscopy (SEM, Philips XL 30 FEG, USA) was adopted to observe the morphologies of QCS/CS sponges. Fourier transform infrared spectroscopy (FT-IR, Bruker VERTEX70, Germany) was used to study the chemical functional groups of sponge.

The partial charge of CS and QCS molecule was calculated using Gaussian 16 code and the 6-311 g (d, p) basis functions were applied.

Take a certain volume of blood drops in QCS/CS sponge and incubate at 37 °C for 5 min. Then, the blood cells on the sponge were immobilized with 2.5% glutaraldehyde and dehydrated with graded concentrations of ethanol (25%, 50%, 75%, 95%, and 100%). Finally, after freeze-drying, the adhesion of blood cells on QCS/CS then was observed by SEM.

Ordinary SD rats were randomly divided into three groups as control group, gelatin sponge group and QCS/CS sponge group. A wound with a diameter of 1.5 cm was created and infected with a 10^8 CFU/mL S. aureus suspension (200 μL) to establish an infectious wound model. In the control group without any treatment, and the commercial gelatin sponge group and the QCS/CS sponge group were placed at the wound site and fixed, respectively. Wound photos were taken on 0, 7th, and 12th, and the regenerated tissues were taken out for H&E staining to further observe the tissue growth. The wound closure ratio was obtained by the formula (5):

First, 100 μL bacterial suspension (10^6 CFU/mL) was co-incubated with different group sponge (10 mg) for 4 h, and the bacteria suspension was diluted by a certain time with PBS. Subsequently, 10 μL bacterial suspension was evenly smeared on LB agar plate and placed in the incubator for 24 h. Finally, take photos of the culture plate. S. aureus (ATCC 29213) and E. coli (ATCC 8739) without sponge were used as control groups to evaluate the antibacterial activity of sponge.

The hemostatic ability of QCS/CS sponge was evaluated by the liver prick injury model and liver noncompressible hemorrhage model. SD rats were randomly divided into 3 groups with 5 rats in each group: the control group (no treatment after bleeding), gelatin sponge group, and QCS/CS sponge.

Sterile QCS/CS sponge was implanted subcutaneously into the back of SD rats and the wound was sutured. The sponge, surrounding tissue, and skin were removed at the 1 week and 3 weeks, respectively. H&E staining was used to observe the degradation degree and histomorphology changes of sponge.

The weighed sponge (Wa) was soaked in blood for 24 h, then the blood was discarded, and the excess blood on the surface of the sponge was gently wiped with filter paper and weighed (Wb). The blood absorption ratio was obtained by the formula (4):

In the rat liver prick injury model, anesthetized rats were fixed to the operating table, and exposed to the liver. A syringe was used to stab the surface of the liver, and the sample was immediately pasted to the wound until the bleeding stopped. Then, the blood loss and hemostasis time were recorded.

In the rat liver noncompressible hemorrhage model, a biopsy instrument with a diameter of 5 mm was used to create the liver hemorrhage, and the sample was immediately injected into the wound until the bleeding stopped. Then, the blood loss and hemostasis time were recorded.

The compression properties of sponges were tested by a rheometer (DHR-1, TA, USA). The compression rate is set to 1 mm/s. Sponge (D = 13 mm) was immersed in PBS and then compressed at 75% deformation. For the cyclic compression test, sponge was immersed in PBS and then cycled 5 times with a compression strain of 75%.

The aggregation behavior of CS and QCS in aqueous solution was studied by molecular dynamics simulation. The details of molecular dynamics simulation are in the supplementary material.

All data are represented as means ± SDs, Statistical analysis was performed in GraphPad Prism 7.0 and by one-way ANOVA, and *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.

The weighed sponge (Wa) was soaked in PBS solution for 24 h, then the PBS was discarded, and the excess liquid on the surface of the sponge was gently wiped with filter paper and weighed (Wb). The Swelling ratio was obtained by the formula (2):

50 μL bacterial solution (10^6 CFU/mL) was dropped on the sponge, incubated at 37 °C for 4 h, and then 950 μL LB liquid medium was added to sponge. The 200 μL bacterial solution was transferred to the 96-well plate and OD600 was measured by Microplate reader. The group containing only bacterial solution and medium was set as the control group. The antibacterial rate is calculated according to formula (3):

The blood clotting index (BCI) of QCS/CS sponge was carried out according to previous reports with some modifications, as detailed in the Supporting Information.

The hemolysis ratio of QCS/CS sponge was carried out according to previous reports with some modifications (Z.T. Li et al., 2024), as detailed in the Supporting Information.

The pre-weighed sponge (W_1) was immersed in ethanol, placed in a vacuum glass drying dish for 10 min, and then taken out and weighed (W_2). The porosity was obtained by the formula (1):

The red blood cell adhesion (RBC) rate of QCS/CS sponge was carried out according to previous reports with some modifications (P.X. Zhao et al., 2023), as detailed in the Supporting Information.

The cell proliferation and cytotoxicity of sponge were carried out according to previous reports with some modifications (Cao et al., 2023b), as detailed in the Supporting Information.

Blood absorption ratio (%) = (W₂ - W₁)/W₁ × 100%                               (4)

The antibacterial rate (%) = (OD_control - OD_sponge)/OD_control × 100%           (3)

Swelling ratio (%) = (W_b - W_a)/W_a × 100%                                    (2)

Wound closure ratio (%) = (Area_0 day - Area_n day)/Area_0 day × 100%          (5)

P (%) = (W₂ - W₁)/(ρ × V₁) × 100%                                              (1)

ρ is the density of ethanol; V₁ is the volume of sponges.

Under acidic conditions, the amino group of CS is protonated and the solubility of CS in aqueous solution increases (Fig. 1A-a). Under neutral or alkaline conditions, CS is deprotonated and the intermolecular hydrogen bond is enhanced, resulting in the aggregation and precipitation of CS molecules (Fig. 1A-b). Based on above studies, QCS was introduced to regulate the hydrogen bond of CS. QCS can form hydrogen bond with CS to reduce the effect of intermolecular hydrogen bond of CS to avoid precipitation (Fig. 1A-c). Hydrogen bond between CS acted as a

It can be further seen from Fig. 1B that with the increase of CS, the color of the QCS/CS hydrogel changed from transparent to white, which is due to the increase of cross-linking sites between CS, increasing the cross-linking density of the QCS/CS hydrogel.

To verify our hypothesis, the state of CS in a neutral aqueous solution was simulated using molecular dynamics. After applying a force field, CS molecules rapidly aggregate in neutral aqueous solution and form CS aggregates (Fig. 2A). The number of CS aggregates formed in the final system is 18 (Fig. 2C) and the largest cluster structure consists of 8 CS molecules (Fig. 2B). Under neutral conditions, there exists strong hydrogen bond effect between CS and they are easy to form aggregates and precipitate (Mottaghitalab et al., 2024), which is reflected in the simulation result. After the introduction of QCS in the system, multiple CS-QCS and CS-CS aggregates form (Fig. 2D), which shows that QCS and CS can form a stable structure through hydrogen bonding. The number of CS-QCS and CS-CS aggregates formed in the final system is 25 (Fig. 2F), with the largest cluster structure consisting of 3 CS molecules and 1 QCS molecule (Fig. 2E). The introduction of QCS can effectively avoid the over aggregation of CS molecules after neutralizing the acidity,

and CS molecule can still form stable crosslinking through hydrogen bond, which verifies the hypothesis proposed in this study.

QCS/CS sponges have interpenetrating pore structure, as shown in Fig. 3B. With the increase of CS content, the concentration of the system increases, resulting in the decrease of the porosity of QCS/CS sponge. Moreover, it can be seen from the macroscopic diagram that the surface of QCS/CS3 and QCS/CS4 sponges is rough, which further proves that the increase of CS would lead to excessive crosslinking of CS in QCS/CS sponge.

It can be seen from the infrared spectrum (Fig. 3C) that compared with the infrared absorption peak of CS, the in-plane N–H vibration absorption peak of NH2 at 1600 cm⁻¹ and the out-of-plane N–H vibration peak of NH2 at 890 cm⁻¹ in the QCS spectrum are significantly shifted, indicating that glycidyl trimethyl ammonium chloride has successfully reacted with NH2. The absorption peak of N–H in the amino group is slightly shifted. After the combination of QCS and CS, it can be seen that the width of N–H absorption peak in the amino group is between QCS and CS, indicating that the N–H absorption peak of the amino group in QCS and CS merged. The ¹H NMR spectrum of QCS (Fig. 1S) showed a peak at 3.10 ppm, corresponding to -N(CH3)3, suggesting that QCS was synthesized successfully.

It has been reported that sponge with porous structure can rapidly absorb tissue fluid to reduce the hydration effect of tissue fluid on the skin (Deng et al., 2023; P.X. Zhao et al., 2023). Meanwhile, the porous structure can promote the absorption of blood and promote the adhesion of blood cells and platelets to achieve rapid hemostasis (Chen et al., 2022; Wang et al., 2024). Wound dressings with high porosity are conducive to the adhesion of cells, blood cells and platelets, thus to promote hemostasis and wound repair. The porosity of QCS/CS sponges decreased with the increase of CS content (Fig. 3D), which is caused by the increase of cross-linking degree of CS, and the result of porosity is consistent with the conclusion of SEM. Wound dressings with swelling properties can condense coagulation factors to enhance the hemostatic ability of the dressings. As can be seen from Fig. 3E, the swelling rate of QCS/CS sponge ranges from 1100% to 1900%, and the swelling rate decreases with the increase of CS content. Furthermore, it can be observed from Fig. 3F that the volume and shape of QCS/CS sponge changes little after water absorption, indicating that the prepared sponge has good stability.

Since the sponge is required to quickly absorb water/blood and restore its original shape while maintaining its structural integrity after transported to the deep wound, mechanical stability and dynamic recoverability are particularly important for the expansibility of sponge (Ahmad et al., 2024; Li et al., 2023; Y.S. Li et al., 2024). As can be seen from Fig. 4A, the QCS/CS1 sponges can recover to its original shape after compression. In addition, as seen in Movies S1-S4, QCS/CS1 and QCS/CS2 sponges immediately returned to their original shape after compression, indicating that the prepared QCS/CS sponges have good shape memory properties. However, QCS/CS3 and QCS/CS4 sponges did not return to their original state. Which is due to the fact that the stable crosslinked structure increases with the CS concentration increase. The stable crosslinked structure is difficult to recover after broking in compression process. Furthermore, compression-strain tests were

performed on QCS/CS sponges to evaluate their mechanical stability with a maximum strain of 75%. It can be seen from Fig. 4B and C that the compression stress of the QCS/CS sponges gradually increase, which is due to the increase of CS, improving the mechanical properties of the sponge. The sponges were compressed for five times under maximum strain of 75%, and the results showed that the recovery of QCS/CS₁ sponge obvious and the sponge exhibits good compression elasticity. In addition, the blood-sucking expansion ability of QCS/CS₁ sponge was tested after compression in vitro (Fig. 4H). The compressed QCS/CS sponge was placed in a syringe and then injected into a container with anticoagulant blood. The results showed that the sponge can quickly absorbed blood and returned to its original shape (Fig. 4I), indicating that the prepared QCS/CS sponge has potential advantages in the treatment of deep wound bleeding.

The prepared QCS/CS sponges were placed in 2 mL blood for 0, 5, 10, and 15 s to observe the ability to absorb blood respectively. As can be seen from Fig. 5A, the blood absorbing rate of QCS/CS₁ and QCS/CS₂ was slightly higher than that of QCS/CS₃ and QCS/CS₄, which is caused by the degree of cross-linking in each group. Furthermore, the hemostatic ability of QCS/CS sponge was evaluated by blood clotting index (BCI) and blood cell adhesion rate (RBC). In the BCI test, after the water was added into the test tube, the uncoagulated red blood cells will burst, exposing hemoglobin to the water and causing the solution turn red. So,

The blood cell adhesion of QCS/CS sponge was further evaluated. Fig. 5F showed the adhesion morphology of blood cells on the surface of the sponge. As the results showed, the blood cell adhesion in gauze is few and increases slightly in gelatin group. The QCS/CS₁ sponge had large number of blood cell aggregate in the porous network, which is due to the large and porous network structure. The QCS/CS sponges can rapidly bind water and blood cells in the blood, and the positively charged QCS can bind to blood cells by electric charge to promote blood cell adhesion. These results indicated that QCS/CS₁ sponges had excellent hemostatic effect.

the higher the number of uncoagulated red blood cells, the darker the solution. The clotting ability of the sponge is judged by the color of the solution. And darker color means worse clotting ability. As can be seen from Fig. 5B, the color of the QCS/CS group is lighter compared with gauze group and gelatin group, and the color of the QCS/CS₁ group is the lightest. Furthermore, it can also be seen from the results of the BCI (Fig. 5D) that QCS/CS₁ group has low BCI, which indicates that QCS/CS₁ group has excellent coagulation effect. In addition, the RBC (Fig. 5E) was above 40%, which was significantly higher than gauze and gelatin sponge.

3.6. Antibacterial, antioxidant property and biocompatibility of QCS/CS sponge

After damaged, the skin loses its function of barrier protection, which makes the wound vulnerable to bacterial attack (Wang et al., 2024). Therefore, the hemostatic sponge should have good antibacterial property to protect the wound from external bacterial infection, accelerating the wound healing. It can be seen from Fig. 6A that compared with control group, QCS/CS sponges showed good antibacterial properties (Fig. 6B). The bacteriostatic rate of each sponge was further tested. As shown in Fig. 6C and D, the results indicate that all the QCS/CS sponges had great antibacterial property. Due to the introduction of QCS, the positive charge on the surface of QCS/CS sponges interacted with the negative charge on the surface of the bacteria, resulting in the death of the bacteria.

Damaged wound would produce a large number of oxygen free radicals, which can lead to sustained oxidative stress and damage cells, hindering the process of wound healing (Liu et al., 2024; Pranantyo et al., 2024). Therefore, hemostatic sponge also needs free radical scavenging ability to accelerate wound healing. The free radical scavenging capacity of QCS/CS sponges was evaluated by DPPH scavenging test. As shown in Fig. 6E, compared with the control group, all the QCS/CS groups were slightly lighter in color, indicating that the QCS/CS sponges had free radical scavenging ability. The DPPH scavenging rates of the QCS/CS sponges were (15.62 ± 5.59)%, (15.55 ± 5.55)%, (19.64 ± 1.83)% and (21.43 ± 1.99)%, respectively. The results showed that QCS/CS sponges have antioxidant properties because quaternary ammonium salts can interfere with the hydrogen bond network between hydroxyl and amine groups and thus have the ability to scour free radicals (Andreica et al., 2023).

The hemolysis rate of QCS/CS sponge with different concentration (5 and 10 mg/mL) was tested (Fig. 6F). The hemolysis rate of all the QCS/CS sponge group at different concentrations was < 5% (Fig. 6G), indicating that the QCS/CS sponge has good blood compatibility and meets the requirement as hemostatic agents. In addition, the cytocompatibility of QCS/CS sponges was further evaluated. As shown in Fig. 6H, the extract liquid of QCS/CS sponge with different concentration was co-cultured with cell. The results show that the cell proliferation rate of the prepared sponge with concentration less than or equal to 0.5 mg/mL is > 70%, showing good cytocompatibility. In addition, live/dead staining was also used to evaluate the cytotoxicity of the sponge. The positive charge of the QCS would react with the negative charge on the cell surface, resulting in a small amount of cell death. Therefore, when the concentration of the sponge extract was 0.5 mg/mL, most of the cells are alive and only small amount of dead cells exist (Fig. 6H). It also can be seen from the Fig. 6J that the number of cells in QCS/CS₃ and QCS/CS₄ groups decreased compared with other groups. The aggregation of CS in QCS/CS₃ and QCS/CS₄ was greater than other groups, weakening the cross-linking between CS and QCS. The instability of the prepared sponge results in the leaching of QCS, further leading to the cell death (Xu et al., 2023; Zhao, Tian, et al., 2023). In conclusion, the prepared QCS/CS₁ sponge had good biocompatibility and can satisfy the application of hemostasis.

QCS/CS₁ sponge presented good antibacterial ability, blood coagulation property and biocompatibility through in vitro experiments, showing its potential as an ideal material for rapid hemostasis and wound healing promotion. Therefore, in vivo evaluation of the hemostatic performance of the cryogenic solution was further performed using rat liver prick injury model and rat liver defect model. The hemostatic capacity of QCS/CS sponge in vivo was assessed by blood loss in rat liver prick injury model (Fig. 7A and B). As shown in Fig. 7C and D, the blood loss of QCS/CS₁ sponge group (0.23 ± 0.057) g was lower than that of blank group (0.91 ± 0.18) g and gelatin sponge group (0.31 ± 0.11) g, indicating that the hemostatic capacity of QCS/CS₁ sponge was higher than that of commercial gelatin hemostatic sponge. In addition, QCS/CS₁ sponge also has the shortest hemostatic time with only 21 s, while gelatin sponge is 42 s. A similar result was observed in rat liver defect model (Fig. 7E). As shown in Fig. 7F, the hemostatic effect of QCS/CS₁ sponge was better than that of gauze and gelatin sponge. The blood loss (Fig. 7G) in the control group (1.01 ± 0.34 g) was higher than that in the gelatin (0.57 ± 0.10 g) group and QCS/CS (0.40 ± 0.09 g) group. As shown in Fig. 7H, the maximum hemostatic time of the control

group is (124 ± 17.96) s, while the time of QCS/CS₁ sponge shortens to (48 ± 10.23) s. The hemostatic mechanism of QCS/CS sponge was shown in Fig. 7I. Firstly, the porous structure of sponge can rapidly absorb blood and expand to fill the wound after transported into the wound, forming a physical barrier to promote hemostasis (Zhang et al., 2022). In addition, the positively charged QCS can promote the adhesion and enrichment of the negative charged erythrocyte and platelets (Liang et al., 2023; P. Zhao et al., 2023). The porous structure of QCS/CS sponges increases the retention time of erythrocyte and platelets, and further activates platelets to promote the formation of fibrin, thus promoting the rapid coagulation of blood (Lee et al., 2024). In summary, QCS/CS sponge has great potential in the application of deep wound bleeding and is a promising hemostatic material.

Shujun Cao: Conceptualization, Investigation, Data curation, Formal analysis, Writing – original draft, Funding acquisition. Peihong Ji: Investigation, Data curation, Formal analysis. Lili Hao: Software, Validation, Funding acquisition. Chao Sun: Resources, Funding acquisition, Writing - review & editing. Hongli Mao: Conceptualization, Resources, Funding acquisition, Project administration, Supervision, Writing - review & editing. Zhongwei Gu: Supervision, Writing - review & editing.

This work was financially supported by the National Natural Science Foundation of China (32271467, 32301246), the Natural Science Foundation of Jiangsu Province (BK20230315), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Postdoctoral Fellowship Program of CPSF (GZC20240697), and the Jiangsu Funding Program for Excellent Postdoctoral Talent.

In summary, an injectable and expandable QCS/CS sponge was prepared for the hemostasis of deep wound and wound healing. Compared with traditional preparation method, the QCS/CS sponge was constructed by CS and QCS self-assembly without acid retention and chemical crosslinker introduction. QCS can form hydrogen bond with CS to reduce the effect of intermolecular hydrogen bond of CS to avoid precipitation. By regulating the ratio of QCS to CS, the stable QCS/CS1 sponge was selected. The QCS/CS1 sponge exhibits expansibility and water/blood triggered shape memory property. Meanwhile, it exhibits good antibacterial property, which can protect the wound from bacterial invasion. The simple and green preparation method is an efficient way to prepare expandable chitosan-based sponge for hemostasis and wound healing.

The wound regeneration effect was further evaluated by H&E staining. As shown in Fig. 8D, there was only a small amount of new granulation tissue existing, and a large number of inflammatory cells can be seen in the blank group on the 7th day. The fibroblasts are sparse and a few new capillaries are disorganized. QCS/CS1 group had fewer inflammatory cells and denser granulation tissue formation. On the 12th day, compared with the control group, complete collagen fibers began to appear in the QCS/CS1 group, and the tissue formation was dense and orderly.

QCS/CS1 sponge was implanted into the subcutaneous tissue in the back of rats to observe the degradation. As can be seen in Fig. 8E, there was a large amount of undegraded sponge (green dashed ellipse) and inflammatory cells (yellow arrow) appearing in the surrounding tissue after implantation for 7th day. On the 21th day, it can be observed from H&E staining that the sponge had been basically degraded, and no obvious pathological reactions appeared in the tissue, indicating that the QCS/CS1 sponge had good biodegradability. Therefore, QCS/CS sponge can promote wound repair as well as good biocompatibility and biodegradability, which is a promising material for wound repair.

The wound healing promotion of QCS/CS sponge was tested by establishing a full-thickness skin defect model in rat. As shown in Fig. 8A, the wound repair in blank, gelatin sponge and QCS/CS1 sponge groups were observed by taking macroscopic images on the 0, 7th and 12th day, respectively. On the 7th day, significant defect was observed in the control group, and the damaged area in all groups reduced. QCS/CS1 sponge showed the most distinguished contraction in wound area on the 7th and 12th day. The recovery rate of gelatin sponge group (62.04 ± 4.83)% and QCS/CS group (69.63 ± 10.48)% was significantly improved compared to the control group (50.11 ± 8.30)% after 7 days. On the 12th day, the wound healing rate of QCS/CS1 group was over 90%. These results indicate that the QCS/CS1 sponge has better effect in promoting skin wound healing. Most sponge dressings tend to adhere to the wound after long-term contact with the wound, which is easy to cause secondary damage when removed. The prepared QCS/CS1 sponge in this study has the advantage of on-demand stripping (Fig. S2). The QCS/CS1 sponge was prepared by physical cross-linking induced by hydrogen bond. Under the action of acetic acid in low concentration, the amino group in the QCS/CS1 sponge network was protonated, and the hydrogen bond effect was reduced greatly. So the surface of the sponge dissolved quickly, and the sponge can be easily detached from the wound. QCS/CS sponge with porous structure can rapidly absorb tissue fluid to reduce the hydration effect of tissue fluid on the skin. In addition, the QCS/CS sponges exhibit good hemostatic properties and anti-bacterial capabilities, offering a significant advantage in wound care and hemostasis. The QCS/CS sponges are physically crosslinked and can be dissolved quickly under the action of acetic acid of low concentration, which allows the sponge to be peeled off on demand. Therefore, the prepared QCS/CS sponges hold promising prospects for application in preventing wound infection and rapid hemostasis.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.carbpol.2024.122699.

Data will be made available on request.

The authors declare no conflict of interest.

- Li, Y. S., Chu, C. N., Chen, C. T., et al. (2024). Quaternized chitosan/oxidized bacterial cellulose cryogels with shape recovery for noncompressible hemorrhage and wound healing [J]. Carbohydrate Polymers, 327, Article 121679. https://doi.org/10.1016/j.carbpol.2023.121679

- Li, Z. T., Xing, X. L., Zhao, C. R., et al. (2024). A rapid interactive chitosan-based medium with antioxidant and pro-vascularization properties for infected burn wound healing [J]. Carbohydrate Polymers, 333, Article 121991. https://doi.org/10.1016/j.carbpol.2024.121991

- Liang, Z., Luo, J. L., Liu, S. M., et al. (2023). Injectable, antibacterial, ROS scavenging and pro-angiogenic hydrogel adhesives promote chronic wound healing in diabetes via synergistic release of NMN and Mg²⁺ [J]. Chemical Engineering Journal, 475, Article 146092. https://doi.org/10.1016/j.cej.2023.146092

- Liu, X., Sun, Y., Wang, J., et al. (2024). A tough, antibacterial and antioxidant hydrogel dressing accelerates wound healing and suppresses hypertrophic scar formation in infected wounds [J]. Bioactive Materials, 34, 269–281. https://doi.org/10.1016/j.bioactmat.2023.12.019

- Liu, Y., Niu, H., Wang, C., et al. (2022). Bio-inspired, bio-degradable adenosine 5′-diphosphate-modified hyaluronic acid coordinated hydrophobic undecanal-modified chitosan for hemostasis and wound healing [J]. Bioactive Materials, 17, 162–177. https://doi.org/10.1016/j.bioactmat.2022.01.025

- Liu, Z. C., Xu, Y. Z., Su, H. N., et al. (2023). Chitosan-based hemostatic sponges as new generation hemostatic materials for uncontrolled bleeding emergency: Modification, composition, and applications [J]. Carbohydrate Polymers, 311, Article 120780. https://doi.org/10.1016/j.carbpol.2023.120780

- Lu, B. T., Hu, E. L., Xie, R. Q., et al. (2022). Microcluster colloidosomes for hemostat delivery into complex wounds: A platform inspired by the attack action of torpedoes [J]. Bioactive Materials, 16, 372–387. https://doi.org/10.1016/j.bioactmat.2022.01.002

- Luo, Y., Cui, L., Zou, L., et al. (2022). Mechanically strong and on-demand dissoluble chitosan hydrogels for wound dressing applications [J]. Carbohydrate Polymers, 294, Article 119774. https://doi.org/10.1016/j.carbpol.2022.119774

- Marani, R. S., & Ehtesabi, H. (2023). A flexible and hemostatic chitosan, polyvinyl alcohol, carbon dot nanocomposite sponge for wound dressing application [J]. International Journal of Biological Macromolecules, 224, 831–839. https://doi.org/10.1016/j.ijbiomac.2022.10.169

- Mottaghitalab, F., Yazdi, M. K., Saeb, M. R., et al. (2024). Green and sustainable hydrogels based on quaternized chitosan to enhance wound healing [J]. Chemical Engineering Journal, 492, Article 152288. https://doi.org/10.1016/j.cej.2024.152288

- Peng, W., Liu, C., Lai, Y. J., et al. (2023). An adhesive/anti-adhesive Janus tissue patch for efficient closure of bleeding tissue with inhibited postoperative adhesion [J]. Advanced Science, 10(21), Article 2301427. https://doi.org/10.1002/advs.202301427

- Pranantyo, D., Yeo, C. K., Wu, Y., et al. (2024). Hydrogel dressings with intrinsic antibiofilm and antioxidative dual functionalities accelerate infected diabetic wound healing [J]. Nature Communications, 15(1), 954. https://doi.org/10.1038/s41467-024-44968-y

- Priddy-Arrington, T. R., Edwards, R. E., Colley, C. E., et al. (2023). Characterization and optimization of injectable in situ crosslinked chitosan-genipin hydrogels [J]. Macromolecular Bioscience, 23(6), Article 202200505. https://doi.org/10.1002/mabi.202200505

- Qi, L. H., Mu, L. X., Guo, X. J., et al. (2023a). Fast expandable chitosan-fibers cryogel from ambient drying for noncompressible bleeding control and in situ tissue regeneration [J]. Advanced Functional Materials, 33(16), 202212231. https://doi.org/10.1002/adfm.202212231

- Qi, L. H., Wang, S., Chen, L., et al. (2023b). Bioinspired multiscale micro-/nanofiber network design enabling extremely compressible, fatigue-resistant, and rapidly shape-recoverable cryogels [J]. ACS Nano, 17(7), 6317–6329. https://doi.org/10.1021/acsnano.2c10462

- Qiao, L. P., Liang, Y. P., Chen, J. Y., et al. (2023). Antibacterial conductive self-healing hydrogel wound dressing with dual dynamic bonds promotes infected wound healing [J]. Bioactive Materials, 30, 129–141. https://doi.org/10.1016/j.bioactmat.2023.07.015

- Wang, J. X., He, J. H., Yang, Y. T., et al. (2024). Hemostatic, antibacterial, conductive and vascular regenerative integrated cryogel for accelerating the whole wound healing process [J]. Chemical Engineering Journal, 479, Article 147577. https://doi.org/10.1016/j.cej.2023.147577

- Wang, X. Y., Wang, J., Zhao, C. X., et al. (2023). Facile fabrication of chitosan colloidal films with pH-tunable surface hydrophobicity and mechanical properties [J]. Food Hydrocolloids, 137, Article 108429. https://doi.org/10.1016/j.foodhyd.2022.108429

- Xu, X., Zeng, Y. B., Chen, Z., et al. (2023). Chitosan-based multifunctional hydrogel for sequential wound inflammation elimination, infection inhibition, and wound healing [J]. International Journal of Biological Macromolecules, 235, Article 123847. https://doi.org/10.1016/j.ijbiomac.2023.123847

- Yang, X. X., Wang, C. W., Liu, Y. H., et al. (2021). Inherent antibacterial and instant swelling ε-poly-lysine/poly(ethylene glycol) diglycidyl ether superabsorbent for rapid hemostasis and bacterially infected wound healing [J]. ACS Applied Materials & Interfaces, 13(31), 36709–36721. https://doi.org/10.1021/acsami.1c02421

- Yao, L. T., Gao, H. C., Lin, Z. F., et al. (2022). A shape memory and antibacterial cryogel with rapid hemostasis for noncompressible hemorrhage and wound healing [J]. Chemical Engineering Journal, 428, Article 131005. https://doi.org/10.1016/j.cej.2021.131005

- Zhang, S. X., Lei, X. X., Lv, Y. L., et al. (2024). Recent advances of chitosan as a hemostatic material: Hemostatic mechanism, material design and prospective application [J]. Carbohydrate Polymers, 327, Article 121673. https://doi.org/10.1016/j.carbpol.2023.121673

- Zhang, Y. P., Wang, Y., Chen, L., et al. (2022). An injectable antibacterial chitosan-based cryogel with high absorbency and rapid shape recovery for noncompressible hemorrhage and wound healing [J]. Biomaterials, 285, Article 121546. https://doi.org/10.1016/j.biomaterials.2022.121546

- Zhao, P., Zhang, Y., Chen, X., et al. (2023). Versatile hydrogel dressing with skin adaptiveness and mild photothermal antibacterial activity for methicillin-resistant Staphylococcus aureus-infected dynamic wound healing [J]. Advanced Science, 10(11), Article 202206585. https://doi.org/10.1002/advs.202206585

- Zhao, P. X., Feng, Y., Zhou, Y. Q., et al. (2023). Gold@halloysite nanotubes-chitin composite hydrogel with antibacterial and hemostatic activity for wound healing [J]. Bioactive Materials, 20, 355–367. https://doi.org/10.1016/j.bioactmat.2022.05.035

- Zhao, X., Huang, Y., Li, Z. L., et al. (2024). Injectable self-expanding/self-propelling hydrogel adhesive with procoagulant activity and rapid gelation for lethal massive hemorrhage management [J]. Advanced Materials, 2308701. https://doi.org/10.1002/adma.202308701

- Zhao, X., Liang, Y. P., Guo, B. L., et al. (2021). Injectable dry cryogels with excellent blood-sucking expansion and blood clotting to cease hemorrhage for lethal deep-wounds, coagulopathy and tissue regeneration [J]. Chemical Engineering Journal, 403, Article 126329. https://doi.org/10.1016/j.cej.2020.126329

- Zhao, Y. N., Tian, C., Liu, Y. M., et al. (2023). All-in-one bioactive properties of photothermal nanofibers for accelerating diabetic wound healing [J]. Biomaterials, 295, Article 122029. https://doi.org/10.1016/j.biomaterials.2023.122029