Method for modifying chitosan sponge material and application thereof

Abstract

The application provides a chitosan sponge material modification method and application, relates to the technical field of biological materials, and the alkylated chitosan sponge is prepared according to the following steps: providing a polymer fiber template with a filling rate of 15-75%, pouring chitosan solution into the pores of the polymer fiber template, washing off the polymer fiber template after freeze-drying, obtaining chitosan sponge, modifying the chitosan sponge by using an aldehyde compound in the presence of a reducing agent, and obtaining alkylated chitosan sponge, wherein the aldehyde compound is a linear or branched C8-C20 aldehyde. The alkylated chitosan sponge provided by the application has a highly connected and controllable microchannel structure, excellent mechanical properties, strong water / blood absorption capacity and fast shape recovery performance, and can be used for non-compressive penetrating wound hemostasis and promoting in-situ tissue regeneration.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a method for modifying chitosan sponge materials and its application. Background Technology

[0002] In daily life and war, massive blood loss can trigger serious complications such as hypothermia, acidosis, and multiple organ failure, leading to high mortality rates. Therefore, rapid and effective hemostasis is crucial. Although bleeding can activate the body's own coagulation cascade to control wound bleeding, the body's own hemostatic capacity is insufficient for severe bleeding, especially non-compression penetrating wounds, often requiring the assistance of hemostatic agents with shape memory properties. Therefore, developing a hemostatic agent suitable for non-compression penetrating wounds is highly beneficial.

[0003] Currently, researchers have developed a variety of shape memory hemostatic agents. XStat TM This is a commercially available hemostatic device filled with compressed cellulose sponge. After absorbing blood, the cellulose sponge expands rapidly, filling and compressing the wound to control bleeding. However, cellulose sponge has poor biodegradability; leaving it at the wound site can hinder healing, requiring removal after hemostasis. Furthermore, due to its lack of highly interconnected pore structure, cellulose sponge cannot guide in-situ tissue regeneration. Polymer foams have also shown potential in hemostasis of non-compression penetrating wounds. However, polymer foams typically have poor blood absorption and shape memory properties, making it difficult to compress the wound and control bleeding promptly. Additionally, cryocrystalline gels with high blood absorption and shape memory properties have been applied to hemostasis of non-compression penetrating wounds. However, the pores created by gas foaming and ice crystal removal typically have poor (low) connectivity, which prolongs the time for blood to flow into the hemostatic agent, resulting in slow shape recovery and reduced hemostatic efficiency.

[0004] In view of this, an invention is proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for modifying chitosan sponge materials to prepare a material that not only has good biocompatibility, biodegradability, procoagulant and anti-infection properties, but also has a highly interconnected and controllable microchannel structure, high water / blood adsorption capacity, and fast shape recovery function, which can be used for hemostasis of non-compression penetrating wounds and promote in-situ tissue regeneration.

[0006] The chitosan sponge material modification method provided by the present invention is prepared according to the following steps: a polymer fiber template with a filling rate of 15%-75% is provided, a chitosan solution is injected into the pores of the polymer fiber template, the polymer fiber template is eluted after freeze-drying to obtain a chitosan sponge, and then the chitosan sponge is modified with an aldehyde compound in the presence of a reducing agent to obtain an alkylated chitosan sponge.

[0007] In this invention, typically, but not limitingly, the filler content of the polymer fiber template is 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.

[0008] By controlling the filling rate of the polymer fiber template to 15%-75%, it is possible to avoid excessively high filling rates, which would result in alkylated chitosan with low mechanical strength after subsequent polymer fiber template elution, making it unable to maintain its stable shape. At the same time, it is also possible to avoid excessively low filling rates, which would result in excessively low porosity of the alkylated chitosan, affecting its blood / water adsorption capacity and thus failing to achieve the technical effects of rapid hemostasis and coagulation promotion.

[0009] Chitosan possesses good biocompatibility, biodegradability, hemostatic properties, and anti-infective capabilities. However, its hemostatic and anti-infective abilities are limited, making it difficult to address severe bleeding and bacterial infections. This invention improves the hemostatic and anti-infective capabilities of chitosan by grafting alkyl chains onto it, utilizing the strong hydrophobic interaction between the hydrophobic alkyl chains and the cell membrane.

[0010] This invention integrates a microchannel structure into a chitosan sponge, which not only promotes the transport of nutrients and oxygen needed by the body and the excretion of metabolic products, but also provides a comfortable microenvironment for host cell infiltration, angiogenesis and tissue ingrowth, while also facilitating the rapid infiltration of liquids.

[0011] This invention prepares an alkylated chitosan sponge with a microchannel structure by combining polymer fiber template leaching, freeze drying, and alkylation modification. It not only has good biocompatibility, biodegradability, hemostatic and anti-infective properties, but also has a highly interconnected and controllable microchannel structure, high water / blood adsorption capacity, and fast shape recovery function. It can be used for hemostasis of non-compression penetrating wounds and to promote in-situ tissue regeneration.

[0012] [Polymer fiber template]

[0013] In a preferred embodiment of the present invention, the polymer used to prepare the polymer fiber template includes any one or a mixture of several of the following in any proportion: polycaprolactone (PCL), poly(lactide-caprolactone) copolymer (PLCL), polyurethane (PU), polyglycerol sebate (PGS), polydioxanone (PDS), polyglycolic acid (PGA), polylactide (PLA), poly(lactide-glycolic acid) copolymer (PLGA), polyhydroxyalkanoate (PHA), and polyethylene glycol (PEO). When the raw material for the polymer fiber template is PLA, it is more conducive to subsequent elution operations.

[0014] In a preferred embodiment of the present invention, the filling rate of the polymer fiber template is 20%-60%, so that the alkylated chitosan sponge obtained after subsequent elution of the polymer fiber template has both excellent mechanical strength and can maintain structural stability, and has high porosity, which can promote the rapid adsorption of blood / water and achieve the effects of rapid hemostasis and anticoagulation. In particular, when the filling rate of the polymer fiber template is 40%, the alkylated chitosan sponge prepared subsequently has even better mechanical properties, hemostatic properties and procoagulant function.

[0015] [Chitosan solution]

[0016] In a preferred embodiment of the present invention, the chitosan solution is an aqueous solution of chitosan in acetic acid, and the mass concentration of the chitosan solution is 1-6%, preferably 4%.

[0017] Aqueous solutions of acetic acid are good solvents for chitosan. Using aqueous solutions of acetic acid to dissolve chitosan improves its solubility.

[0018] Preferably, the aqueous solution of acetic acid has a mass concentration of 1-4%, more preferably 2%.

[0019] Typical, but not limiting, chitosan solutions have a mass concentration of 1%, 2%, 4%, or 6%.

[0020] A chitosan concentration of less than 4% results in poor mechanical properties of the formed alkylated chitosan, making it unable to maintain a stable shape. Conversely, a chitosan concentration greater than 4% leads to excessively high viscosity, making it difficult to infuse into the pores of the PLA template. A chitosan concentration of 4% provides a balance between maintaining the strength of the chitosan sponge and keeping the viscosity low, facilitating the infusion process.

[0021] [Aldehydes]

[0022] This invention uses aldehyde compounds with hydrophobic carbon chains to react with amino groups on chitosan, thereby modifying chitosan with hydrophobic carbon chains. The strong hydrophobic interaction between the hydrophobic alkyl chains and the cell membrane enhances the hemostatic and anti-infective capabilities of chitosan.

[0023] In a preferred embodiment of the present invention, the aldehyde compound is a straight-chain or branched C10-C14 aldehyde, which not only ensures the hydrophobic properties of chitosan, but also facilitates the modification of the hydrophobic carbon chain onto chitosan.

[0024] Typical, but not limiting, C10-C14 aldehydes, such as straight-chain or branched C10 aldehydes, straight-chain or branched C11 aldehydes, straight-chain or branched C12 aldehydes, straight-chain or branched C13 aldehydes, or straight-chain or branched C14 aldehydes, especially when the aldehyde compound is dodecaldehyde, can both ensure the hydrophobic properties of chitosan and facilitate modification reactions to graft hydrophobic links onto chitosan.

[0025] In a preferred embodiment of the present invention, the reducing agent includes, but is not limited to, NaCNBH3.

[0026] The aldehyde group in aldehyde compounds can react with the amino group on chitosan molecules to form a Schiff base (C=N). Because Schiff bases are sensitive to water and easily broken, they can be reduced by a reducing agent to convert them into CN, which has high stability.

[0027] In a preferred embodiment of the present invention, after modifying the chitosan sponge with aldehyde compounds, the unreacted aldehyde compounds and reducing agents are removed by soaking in a mixed solution of ethanol and water, so as to avoid the residual unreacted aldehyde compounds and reducing agents affecting the biocompatibility of alkylated chitosan.

[0028] In a preferred embodiment of the present invention, after the polymer fiber template is eluted, the residual acetic acid in the chitosan sponge is neutralized with a mixed solution of ethanol and NaOH to avoid the residual acetic acid affecting the subsequent modification reaction of aldehyde compounds.

[0029] Preferably, in the mixed solution of ethanol and NaOH, the volume ratio of the two is 8-9:2-1, more preferably 9:1.

[0030] In a preferred embodiment of the present invention, liquid nitrogen is used for pre-cooling before freeze-drying to facilitate the formation of a microchannel structure with uniform pore size distribution inside the chitosan sponge.

[0031] In a preferred embodiment of the present invention, the polymer fiber template is prepared by any one or a combination of 3D printing, casting, phase separation, electrospinning, wet spinning, melt spinning or particle leaching. In particular, when the polymer fiber template is prepared by 3D printing, it is more conducive to controlling the filling rate and improving the processing efficiency.

[0032] In a typical embodiment of the present invention, the method for modifying chitosan sponge material includes the following steps, wherein CS represents chitosan:

[0033] (1) Use a 3D printer to print PLA fiber templates with an infill rate of 20-60%;

[0034] CS was dissolved in an aqueous acetic acid solution (2%, v / v) to obtain a CS solution with a concentration of 1-4% (w / v); PLA fiber templates were infused with the 1-4% (w / v) CS solution, pre-cooled with liquid nitrogen, and then freeze-dried; the liquid nitrogen freezing temperature was -196℃, the freezing time was 5-30 min; the freeze-drying temperature was -40 to -80℃; and the freeze-drying time was 24-72 h.

[0035] (2) The PLA fiber template in the CS / PLA composite was eluted with dichloromethane to obtain a CS sponge with a microchannel structure;

[0036] (3) Neutralize the acetic acid remaining in the CS sponge with a mixed solution of ethanol / NaOH (9 / 1, v / v);

[0037] (4) In the presence of NaCNBH3, CS sponge was surface modified with C8-C14 aldehyde compounds to prepare alkylated CS sponge.

[0038] (5) After modification, the alkylated CS sponge was soaked in an ethanol / water mixed solution to remove unreacted aldehydes and NaCNBH3, and alkylated chitosan sponge was obtained.

[0039] A second objective of this invention is to provide an alkylated chitosan, which is modified using the chitosan sponge material modification method provided in one objective of this invention.

[0040] The chitosan sponge material provided by this invention has a microchannel structure inside, with interconnected microchannels and a porosity of over 65%. It not only has excellent biocompatibility, biodegradability, hemostatic and anti-infective properties, but also has high water / blood adsorption capacity and fast shape recovery function. It can be used for hemostasis of non-compression new penetrating wounds and to promote in-situ tissue regeneration.

[0041] The third objective of this invention is to provide the application of the above-mentioned chitosan sponge material in the preparation of non-compression penetrating wound hemostatic agents and in-situ tissue regeneration promotion. Attached Figure Description

[0042] Figure 1 FTIR images of chitosan (CS) powder, polylactic acid (PLA) fiber template prepared in Example 1, CS / PLA composite prepared in Example 1, and chitosan sponge with microchannel structure provided in Comparative Example 1;

[0043] Figure 2 XPS plot of chitosan sponge (MCS-2) provided for Comparative Example 1;

[0044] Figure 3 XPS image of the alkylated chitosan sponge (MACS-2) provided in Example 2;

[0045] Figure 4 Macroscopic and microscopic structural diagrams, mechanical property test diagrams, and mechanical property diagrams after blood absorption are provided for the alkylated chitosan sponges MACSs provided in Examples 1-5, the chitosan sponge MCS-2 provided in Comparative Example 1, and the alkylated chitosan sponge ACS provided in Comparative Example 2.

[0046] Figure 5 Water / blood adsorption characterization diagrams of the macroscopic and microscopic structures of the alkylated chitosan sponges (MACSs) provided in Examples 1-3 and the alkylated chitosan sponges (ACS) provided in Comparative Example 2;

[0047] Figure 6 Macroscopic and microscopic structural shape recovery characterization images of the alkylated chitosan sponges (MACSs) provided in Examples 1-3 and the alkylated chitosan sponges (ACS) provided in Comparative Example 2;

[0048] Figure 7 Statistical histograms of pore sizes of the alkylated chitosan sponges (MACSs) provided in Examples 1-3 before and after absorbing water / blood;

[0049] Figure 8 The in vitro procoagulant activity characterization diagrams of the alkylated chitosan sponge MACSs provided in Examples 1-3 are shown.

[0050] Figure 9 For GS, CELOX TM CELOX TM -Charts of hemostatic ability of MCS-2 provided in Comparative Example 1, ACS provided in Comparative Example 2 and MCS-2 provided in Example 2 in rats.

[0051] Figure 10 For water, PBS, gauze, GS, CELOX TM Blood compatibility and cell compatibility characterization diagrams of chitosan sponge MCS-2 provided in Comparative Example 1, alkylated chitosan sponge ACS provided in Comparative Example 2, and alkylated chitosan MCS-2 provided in Example 2;

[0052] Figure 11 For TCP, gauze, GS, CELOX TM CELOX TM -G. Characterization diagram of the in vitro anti-infection ability of chitosan sponge MCS-2 provided in Comparative Example 1, alkylated chitosan sponge ACS provided in Comparative Example 2, and alkylated chitosan MCS-2 provided in Example 2.

[0053] Figure 12 Characterization diagram of the tissue-promoting in-situ regeneration ability of the alkylated chitosan sponge MACS-2 provided in Example 2 and the alkylated chitosan sponge ACS provided in Comparative Example 2. Detailed Implementation

[0054] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] Example 1

[0056] This embodiment provides an alkylated chitosan sponge (named MACS-1), which is prepared according to the following steps:

[0057] (1) Use a 3D printer to print PLA fiber templates with an infill rate of 20%;

[0058] (2) CS (chitosan) was dissolved in an aqueous acetic acid solution (2%, v / v) to obtain a CS solution with a concentration of 4% (w / v); PLA fiber template was infused with the 4% (w / v) CS solution, pre-cooled with liquid nitrogen, and then freeze-dried to obtain a CS / PLA composite; wherein, the liquid nitrogen freezing temperature was -196℃, the freezing time was 5-30 min; the freeze-drying temperature was -40 to -80℃; and the freeze-drying time was 24-72 h.

[0059] (3) Use dichloromethane to elute the PLA fiber template in the CS / PLA composite to obtain a CS sponge with a microchannel structure;

[0060] (4) Neutralize the residual acetic acid in the CS sponge with a mixed solution of ethanol / NaOH (9 / 1, v / v);

[0061] (5) In the presence of NaCNBH3, CS sponge was surface modified with dodecyl aldehyde (DA) to prepare alkylated CS sponge;

[0062] (6) After modification, the alkylated CS sponge was immersed in an ethanol / water mixed solution to remove unreacted dodecaldehyde and NaCNBH3, and alkylated chitosan sponge was obtained.

[0063] Example 2

[0064] This embodiment provides an alkylated chitosan sponge (named MACS-2), which differs from Embodiment 1 in that, in step 1, the PLA fiber template obtained by 3D printing has a fill rate of 40%.

[0065] Example 3

[0066] This embodiment provides an alkylated chitosan sponge (named MACS-3), which differs from Embodiment 1 in that, in step (1), the PLA fiber template obtained by 3D printing has a filling rate of 60%.

[0067] Example 4

[0068] This embodiment provides an alkylated chitosan sponge, which differs from Embodiment 1 in that the mass concentration of the CS solution used to infuse the PLA fiber template in step (2) is 1%.

[0069] Example 5

[0070] This embodiment provides an alkylated chitosan sponge, which differs from Embodiment 1 in that the mass concentration of the CS solution used to infuse the PLA fiber template in step (2) is 2%.

[0071] Comparative Example 1

[0072] This comparative example provides a chitosan sponge (named MCS-2), which is prepared by the same method as steps (1)-(4) in Example 1. The difference between it and Example 1 is that no alkylation modification is performed.

[0073] Comparative Example 2

[0074] This comparative example provides an alkylated chitosan sponge (named ACS), which is prepared according to the following steps:

[0075] (1) Dissolve CS in an aqueous acetic acid solution (2%, v / v) to obtain a CS solution with a concentration of 4% (w / v);

[0076] (2) A 4% (w / v) CS solution was pre-cooled with liquid nitrogen and then freeze-dried to obtain chitosan sponge; wherein the liquid nitrogen freezing temperature was -196℃ and the freezing time was 5-30 min; the freeze-drying temperature was -40 to -80℃; and the freeze-drying time was 24-72 h.

[0077] (3) In the presence of NaCNBH3, CS sponge was surface modified with dodecyl aldehyde to prepare alkylated CS sponge;

[0078] (4) After modification, the alkylated CS sponge was soaked in an ethanol / water mixed solution to remove unreacted dodecylaldehyde and NaCNBH3 to obtain alkylated chitosan sponge.

[0079] Experimental Example 1

[0080] The characteristic functional groups of CS (chitosan) powder, the PLA fiber template prepared in Example 1, the CS / PLA composite prepared in Example 1, and the chitosan sponge provided in Comparative Example 1 were characterized by FTIR testing. The results are as follows: Figure 1 As shown, from Figure 1 The FTIR spectrum of the CS sponge showed only characteristic peaks belonging to CS and no characteristic peaks belonging to PLA, indicating that there is no PLA residue in the CS sponge.

[0081] Experimental Example 2

[0082] The chemical structure and elemental content of the surface of the alkylated chitosan sponge provided in Example 2 and the chitosan sponge provided in Comparative Example 1 were analyzed and detected by XPS testing. The XPS images of the chitosan sponges are shown below. Figure 2 As shown, the XPS plot of the alkylated CS sponge is as follows. Figure 3 As shown in Table 1, the peak areas of CN*H2,-N*H-COCH3 and CN*HC in the alkylated chitosan sponge provided in Example 2 and the chitosan sponge provided in Comparative Example 1 are shown in Table 1 below.

[0083] Table 1. N1s peak area

[0084] Comparative Example 1 (CS Sponge) 86.18±4.24％ 13.82±4.24％ 0.00±0.00％ Example 2 (Alkylated CS Sponge) 57.29±15.57％ 14.83±3.55％ 27.86±18.90％

[0085] Combination Figure 2 , Figure 3 As shown in Table 1, the chemical states of N1s in the chitosan sponge provided in Comparative Example 1 are CN*H2 and -N*H-COCH3. The chemical states of N1s in the alkylated chitosan sponge provided in Example 2 are CN*H2, -N*H-COCH3, and CN*HC. The peak areas of CN*H2 and -N*H-COCH3 in the chitosan sponge provided in Comparative Example 1 are 86.18±4.24% and 13.82±4.24%, respectively. The peak areas of CN*H2, -N*H-COCH3, and CN*HC in the alkylated chitosan sponge provided in Example 2 are 57.29±15.57%, 14.83±3.55%, and 27.86±18.90%, respectively. The presence of CN*HC and the decrease in the peak area of ​​CN*H2 demonstrate the successful modification with dodecylaldehyde. The grafting rate of dodecylaldehyde is 27.86±18.90%.

[0086] Experimental Example 3

[0087] (1) The macro and micro structures of the alkylated chitosan sponges provided in Examples 1-5 and Comparative Example 2 were characterized using Micro-CT and SEM. The pore size was measured using Image-J software and the porosity was measured using Micro-CT.

[0088] (2) The compressive stress of the cylindrical MACSs (PLA fiber template filling rate of 40%, CS concentration of 1, 2 and 4% (w / v)) provided in Examples 4, 2 and 5, the cylindrical MACSs (PLA fiber template filling rate of 20, 40 and 60%, CS concentration of 4% (w / v)) provided in Examples 1-3, and the cylindrical ACS provided in Comparative Example 2 were tested using a universal testing machine. The compressive strain and velocity were 70% and 1 mm / min, respectively.

[0089] (3) The cylindrical MACSs (PLA fiber template filling rate of 40%, CS concentration of 1, 2 and 4% (w / v)) provided in Examples 4, 2 and 5, the cylindrical MACSs (PLA fiber template filling rate of 20, 40 and 60%, CS concentration of 4% (w / v)) provided in Examples 1-3, and the cylindrical ACS provided in Comparative Example 2 were immersed in blood for 5-10 minutes and then removed. The compression pressure, compression strain and speed were tested using a universal testing machine, respectively, at 70% and 1 mm / min.

[0090] Figure 4 (A) is a schematic diagram of the preparation process of MACSs; Figure 4 (B) is a stereomicroscopic image of the PLA fiber template, PLA / CS composite, CS sponge and alkylated CS sponge; Figure 4 (C) Micro-CT images of ACS and MACSs provided in Examples 1-3 are provided for Comparative Example 2; Figure 4 (D) SEM images of ACS and MACSs provided in Examples 1-3 are provided for Comparative Example 2; Figure 4 (E) Provides a statistical histogram of hole dimensions (n=16) for the ACS and MACSs provided in Examples 1-3 for cross-section comparative example 2; Figure 4 (F) Provides a statistical histogram of hole dimensions (n=16) for the ACS and MACSs provided in Examples 1-3 for longitudinal section comparative example 2; Figure 4 (G) Provides porosity statistical histograms (n=25) of ACS and MACSs provided in Comparative Example 2 and Examples 1-3; Figure 4 (H) shows the compressive stress-strain curves of MACSs with different CS concentrations provided in Examples 4, 2 and 5; Figure 4 (I) Statistical histogram of compressive stress for MACSs with different CS concentrations provided in Examples 4, 2 and 5; Figure 4 (J) is the ACS provided for Comparative Example 2, the MCS-2 provided for Comparative Example 1, and the MCSs provided for Examples 1-3; Figure 4(K) is the ACS provided in Comparative Example 2, the MCS-2 provided in Comparative Example 1, and the MCSs provided in Examples 1-3. Figure 4 (L) represents the compressive stress-strain curves of the ACS provided in Comparative Example 2, the MCS-2 provided in Comparative Example 1, and the MCSs provided in Examples 1-3 after blood absorption. Figure 4 (M) is a statistical histogram of compressive stress of the ACS provided by Comparative Example 2, the MCS-2 provided by Comparative Example 1, and the MCSs provided by Examples 1-3 after blood absorption. Figure 4 (N) is a bar chart showing the mechanical enhancement factor of ACS provided by Comparative Example 2, MCS-2 provided by Comparative Example 1, and MACSs provided by Examples 1-3 after blood absorption. The sample mean was 3, and the data are expressed as mean ± standard deviation. ns indicates no significant difference between groups. *P<0.05, **P<0.01, ***P<0.001.

[0091] like Figure 4 As shown in (C), MACSs have a highly interconnected microchannel structure, and the microchannel density increases with the increase of PLA fiber template filling rate. ACS, on the other hand, only have a dense microporous structure.

[0092] like Figure 4 As shown in (D), (E), and (F), MACSs contain both microchannels of 136.5 ± 17.8 μm and micropores of 8.3 ± 0.8 μm. ACS, on the other hand, contain only micropores of 8.1 ± 1.0 μm. The microchannel structure within MACSs is highly interconnected and controllable. Conversely, the micropore structure within ACS exhibits lower connectivity and controllability.

[0093] like Figure 4 As shown in (G), with the increase of PLA fiber template filling rate, the porosity of MACSs increased from 73.2±2.9% to 88.8±1.6%. The porosity of MACSs was significantly higher than that of ACS (32.1±1.9%).

[0094] like Figure 4 As shown in (H) and (I), when the CS concentration increased from 1% to 4% (w / v), the compressive stress of MACSs increased from 0.6 ± 0.16 kPa to 23.3 ± 1.2 kPa.

[0095] like Figure 4As shown in (J) and (K), when the PLA fiber template filling rate increased from 20% to 60%, the compressive stress of MACSs decreased from 46.3 ± 6.5 kPa to 8.1 ± 0.8 kPa. The decrease in compressive stress of MACSs is mainly due to the reduction in solid content per unit volume. Furthermore, the compressive stress of MACSs was significantly lower than that of ACS (138.0 ± 13.4 kPa), which is attributed to the presence of microchannels.

[0096] like Figure 4 As shown in (L) and (M), the compressive stress of MACSs significantly increases after blood absorption, which is attributed to clot formation. When blood enters the microchannels, it interacts with CS and hydrophobic alkyl chains, resulting in coagulation. Compared to ACS, blood has a higher enhancing effect on the mechanical strength of MACSs. ​​Furthermore, the enhancing effect of blood on the mechanical strength of MACSs increases with increasing porosity. The increase in enhancing effect is closely related to porosity. Higher porosity results in a larger specific surface area. A larger specific surface area facilitates more thorough contact between blood and hemostatic agents, thereby enabling the formation of more clots.

[0097] Test Example 4

[0098] The water / blood adsorption capacity of the macroscopic and microscopic structures of the alkylated chitosan sponges (MACSs) provided in Examples 1-3 and the alkylated chitosan sponges (ACS) provided in Comparative Example 2 were tested using qualitative and quantitative experiments.

[0099] (I) The experimental steps of the qualitative experiment are as follows:

[0100] 1) Place MACSs and ACS on the surface of filter paper and compress to expel the free water inside;

[0101] 2) Place drained MACS and ACS into the bloodstream;

[0102] 3) Use a digital camera to record the location of MACS and ACS in the blood.

[0103] (II) The experimental steps for quantitative experiments are as follows:

[0104] 1) Measure the volume of MACSs and ACS, denoted as V;

[0105] 2) Place MACSs and ACS on the surface of filter paper and compress to expel the free water inside;

[0106] 3) Weigh the MACSs and ACS after drainage, and record the weight as Wd;

[0107] 4) Immerse drained MACS and ACS in water / blood;

[0108] 5) After immersion for a certain period of time, remove the item, weigh it, and record the weight as Wt;

[0109] 6) Calculate the water / blood adsorption capacity of MACSs and ACS using the formula (water / blood adsorption capacity / sponge volume (g / cm3) = (Wt - Wd) / V). Calculate the water / blood adsorption rate of MACSs and ACS (unit: g / cm3 / s) based on the water / blood adsorption capacity-time kinetic curve.

[0110] Figure 5 (A) is a macroscopic view of ACS and MACS immersed in blood; Figure 5 (B) shows the water adsorption capacity-time curves of ACS and MACSs; Figure 5 (C) shows the blood adsorption-time curves of ACS and MACSs; Figure 5 (D) is a bar chart showing the saturated water absorption of ACS and MACSs; Figure 5 (E) is a bar chart showing the saturation blood absorption of ACS and MACSs; Figure 5 (F) is a bar chart showing the water absorption rates of ACS and MACSs; Figure 5 (G) is a bar chart showing the blood-sucking rate of ACS and MACSs; Figure 5 (H) Macroscopic images of ACS and MACS with fixed shapes before and after water absorption. Figure 5 (I) Macroscopic images of ACS and MACS with fixed shapes before and after blood aspiration. Figure 5 (J) is a computer simulation image of the fluid adsorption capacity of ACS and MACSs. Figure 5 (K) represents the total fluid velocity of ACS and MACSs. ​​The sample mean was 3, and the data are expressed as mean ± standard deviation. ns indicates no significant difference between groups. *P<0.05, **P<0.01, ***P<0.001.

[0111] like Figure 5 As shown in (A), after absorbing blood, MACSs sink to the bottom of the container. Conversely, ACSs remain suspended on the surface of the blood. Figure 5 (B) Figure 5 (C) Figure 5 (D) Figure 5 (E) Figure 5 (F) and Figure 5 As shown in (G), the saturated water absorption / blood volume of MACS is significantly higher than that of ACS, and gradually increases with increasing porosity. Furthermore, the water absorption / blood volume rate of MACS is also significantly higher than that of ACS, and increases with increasing porosity. Figure 5 (H) and Figure 5(I) MACSs allow for rapid penetration of water and blood. ACSs allow for rapid penetration of water, but hinder blood penetration. Next, we further simulated the fluid adsorption behavior of MACSs and ACSs using computer simulations. Figure 5 (J) and Figure 5 As shown in (K), in the computer simulation of fluid adsorption diagrams for MACS and ACS, the distribution area of ​​high-velocity fluid inside MACS is larger than that inside ACS. The distribution area of ​​high-velocity fluid inside MACS increases with the increase of the number of channels. Furthermore, the total flow velocity inside MACS is significantly higher than that inside ACS, and gradually increases with the increase of the number of microchannels. These results indicate that MACS has a stronger water / blood adsorption capacity than ACS, which stems from the presence of microchannels. The microchannel structure gives MACS high porosity, thus enabling it to hold more liquid. In addition, the high connectivity of the microchannel structure facilitates rapid liquid penetration.

[0112] Experimental Example 5

[0113] The shape memory properties of the alkylated chitosan sponges (MACSs) provided in Examples 1-3 and the alkylated chitosan sponges (ACS) provided in Comparative Example 2 were tested. The experimental steps are as follows:

[0114] 1) Place cylindrical MACSs and ACS on the surface of filter paper and compress them to expel the free water inside;

[0115] 2) Add water / blood to the surfaces of fixed-shape MACSs and ACS;

[0116] 3) Use a digital camera to record the shape restoration process of fixed-shape MACSs and ACS;

[0117] 4) Quantitatively evaluate the shape recovery rate and shape recovery time of MACSs and ACS;

[0118] 5) In addition, SEM was used to observe the changes in the microstructure of MACSs and ACS during the compression-recovery process;

[0119] 6) Use Image-J software to measure the size of the hole.

[0120] Figure 6 (A) is a macroscopic view of MACSs and ACS before and after water absorption; Figure 6 (B) is a macroscopic view of MACSs and ACS before and after blood absorption; Figure 6 (C) is a bar chart showing the shape recovery rate of ACS and MACSs after water absorption; Figure 6 (D) is a bar chart showing the shape recovery rate of ACS and MACSs after blood absorption; Figure 6(E) is a bar chart showing the shape recovery time of ACS and MACSs after water absorption; Figure 6 (F) is a bar chart showing the shape recovery time of ACS and MACSs after blood absorption; Figure 6 (G) SEM images of ACS and MACSs before and after water / blood absorption. Arrows indicate deformed channels. Table 2 shows the shape recovery time comparison data of MACS-2 and reported hemostatic agents. The sample mean was 3, and the data are expressed as mean ± standard deviation. ns indicates no significant difference between groups, *P<0.05, **P<0.01, ***P<0.001.

[0121] Table 2 Comparison of shape recovery time between MACS-2 and reported hemostatic agents

[0122] water 4.2±0.2s ～10s 10s 2.4s 2.1±0.1s blood 25s 23.4±2.4s ～10s 19.8±4.9s 2.5±0.5s

[0123] Note: XSat™ is a commercially available hemostatic agent. The other hemostatic agents are research reports.

[0124] Figure 7 (A) is a bar chart showing the pore size statistics of MACS-1 before and after absorbing water / blood; Figure 7 (B) is a bar chart showing the pore size statistics of MACS-2 before and after water / blood absorption; Figure 7 (C) is a bar chart showing the pore size statistics of MACS-3 before and after water / blood absorption. The sample mean was 6. Data are expressed as mean ± standard deviation. ns indicates no significant difference between groups. *P<0.05, **P<0.01, ***P<0.001

[0125] like Figure 6 (A) and Figure 6 As shown in (B), after compression and drainage, MACSs and ACS can maintain their shape. After absorbing water, the shape-fixed MACSs and ACS can return to their initial shape. After absorbing blood, the shape-fixed MACSs can return to their initial shape, while the shape-fixed ACS cannot return to their initial shape.

[0126] like Figure 6 (C) and Figure 6 As shown in (D), after water absorption, the shape recovery rates of MACS-1, MACS-2, MACS-3, and ACS were 100±0%, 99.6±0.6%, 99.6±0.6%, and 98.3±1.5%, respectively. After blood absorption, the shape recovery rates of MACS-1, MACS-2, MACS-3, and ACS were 100±0%, 99.6±0.6%, 99.6±0.6%, and 0.0±0.0%, respectively.

[0127] like Figure 6 (E) and Figure 6As shown in (F), after water absorption, the shape recovery times of MACS-1, MACS-2, MACS-3, and ACS were 3.3±0.6s, 2.1±0.1s, 1.7±0.6s, and 41±3.6s, respectively. After blood absorption, the shape recovery times of MACS-1, MACS-2, and MACS-3 were 4.0±1.0s, 2.5±0.5s, and 2.1±0.1s, respectively.

[0128] Furthermore, as shown in Table 2, the shape recovery time of MACSs is significantly shorter than that of previously reported hemostatic agents. The shape recovery time of MACSs is not affected by body fluid (blood) viscosity. Conversely, the shape recovery time of some previously reported hemostatic agents increases with increasing body fluid viscosity. These results indicate that MACSs possess stronger shape recovery capabilities than ACSs and previously reported hemostatic agents, primarily due to the presence of microchannels within MACSs. ​​These microchannels exhibit high connectivity, allowing for rapid water / blood penetration into MACSs. ​​Conversely, the poor connectivity of the pore structures within ACSs and previously reported hemostatic agents hinders rapid water / blood penetration.

[0129] The changes in the internal pore structure of MACSs and ACS were further observed using SEM, such as Figure 6 As shown in (G), before compression, MACSs have an interconnected circular microchannel structure. ACS have a dense microporous structure. After compression, the circular microchannels inside MACSs collapse and become flat channels. The dense microporous structure inside ACS also collapses, becoming even denser. After water absorption, the deformed microchannels inside MACSs return to their original state, and the size of the micropores is basically the same as before compression (e.g., ...). Figure 7 (As shown). The deformed micropores inside the ACS also return to their initial shape. After blood absorption, the deformed microchannels inside the MACS can also return to their original state, and a large number of aggregated RBCs can be observed inside the microchannels. In contrast, the deformed micropores inside the ACS cannot return to their original state, and almost no RBCs can be observed inside the deformed micropores. This is because the micropore structure is relatively dense, which restricts the penetration of blood.

[0130] Experimental Example 6

[0131] The blood coagulation index (BCI) was measured to evaluate the procoagulant activity of the alkylated chitosan sponges (MACSs) provided in Examples 1-3. Gauze, GS, CELOX TM CELOX TM -G, chitosan sponge MCS-2 provided in Comparative Example 1, and alkylated chitosan sponge ACS provided in Comparative Example 2 were used as control groups. Among them, gauze, GS, and CELOX were used as control groups. TM CELOX TM-G was purchased commercially available. The experimental steps are as follows:

[0132] 1) Place MACSs on the surface of filter paper and compress to expel the free water inside;

[0133] 2) Place the draining MACSs in the EP pipe;

[0134] 3) Add 50 μL of sodium citrate anticoagulated whole blood to the surface of the fixed-shape MACSs;

[0135] 4) After incubating at 37℃ for a period of time, add 3 mL of deionized water to the EP tube to submerge the MACSs;

[0136] 5) Measure the OD of the supernatant using an ELISA reader. 540nm value;

[0137] 6) Measure the OD of anticoagulated whole blood / deionized water solution using an ELISA reader. 540nm The value is recorded as a reference value.

[0138] 7) According to the formula (BCI(%)=OD 止血剂 / OD 参考值 Calculate the BCI value of MACSs by (×100%).

[0139] Figure 8 (A) is gauze, GS, CELOX TM CELOX TM BCI-time curves for the G, ACS, MCS-2 and MACS groups; Figure 8 (B) RBC in gauze, GS, CELOX TM CELOX TM - Histogram of adhesion percentages on G, ACS, MCS-2 and MACS surfaces; Figure 8 (C) is a bar chart showing the percentage of platelets adhering to the surfaces of gauze, GS, CELOXTM, CELOXTM-G, ACS, MCS-2 and MACSs. Figure 8 (D) is a SEM image of RBCs adhered to the surfaces of gauze, GS, CELOX™, CELOX™-G, ACS, MCS-2 and MACSs. Figure 8 (E) is a SEM image of platelets adhering to the surfaces of gauze, GS, CELOX™, CELOX™-G, ACS, MCS-2 and MACSs. Figure 8 (F) is an immunofluorescence staining image of platelets adhering to the surface of the hemostatic agent; Figure 8(G) is a schematic diagram of the in vitro procoagulant mechanism of MACSs. ​​The sample mean was 3, and the data are expressed as mean ± standard deviation. ns indicates no significant difference between groups. *P<0.05, **P<0.01, ***P<0.001.

[0140] like Figure 8 As shown in (A), the BCI value of the MACSs group gradually decreased with increasing incubation time, indicating that the procoagulant activity of MACSs gradually increased. At the same time point, the BCI value of the MACSs group decreased with increasing porosity, indicating that the procoagulant ability of MACSs was positively correlated with porosity. The BCI value of the MACSs group was significantly lower than that of the ACS group, indicating that the procoagulant ability of MACSs was stronger than that of ACS, which was due to the increased porosity. Higher porosity means a larger specific surface area, which is conducive to more complete contact between blood and hemostatic agents. The BCI value of the MACSs group was also significantly lower than that of the MCS-2 group, indicating that the procoagulant ability of MACSs was stronger than that of MCS-2, which was mainly attributed to the introduction of hydrophobic alkyl chains. Hydrophobic alkyl chains can promote RBC adhesion and aggregation. In addition, compared with gauze, GS, CELOXTM, and CELOXTM-G, MACSs also showed stronger procoagulant ability, which was due to the synergistic effect of CS and hydrophobic alkyl chains.

[0141] The body's active coagulation cascade primarily depends on RBC aggregation and platelet adhesion and activation. For example... Figure 8 As shown in (B) and 8(C), the percentages of RBC adhesion for the gauze, GS, CELOX™, CELOX™-G, ACS, MCS-2, MCS-1, MCS-2, and MCS-3 groups were 7.1±1.4%, 9.8±1.2%, 26.2±1.9%, 25.3±2.1%, 16.8±1.6%, 20.4±1.6%, 32.8±2.3%, 41.0±2.0%, and 48.2±2.2%, respectively. The percentages of platelet adhesion in the gauze, GS, CELOX™, CELOX™-G, ACS, MCS-2, MACS-1, MACS-2, and MACS-3 groups were 14.4±1.1%, 11.9±1.5%, 30.8±2.3%, 28.9±2.6%, 18.7±1.6%, 20.8±2.2%, 38.0±2.3%, 44.7±2.2%, and 49.8±2.5%, respectively.

[0142] like Figure 8 As shown in (D) and 8(E), more RBCs and platelets adhered to MACSs compared to other hemostatic agents. Furthermore, aggregated RBCs and activated platelets were observed on the surface of MACSs in greater numbers than in other groups. Figure 8(F)). The above experimental results indicate that MACSs possess strong in vitro procoagulant activity, primarily derived from the synergistic effect of CS, microchannel structure, and hydrophobic alkyl chains. Figure 8 (G)). The microchannel structure accelerates blood penetration, thereby promoting full contact between RBCs and platelets and the hemostatic agent. CS can promote RBC aggregation and platelet adhesion, aggregation, and activation. The hydrophobic alkyl chains can actively capture and aggregate RBCs and platelets through hydrophobic interactions.

[0143] Experimental Example 7

[0144] The in vivo hemostatic ability of the alkylated chitosan sponge MACS-2 provided in Example 2 was evaluated using a lethal penetrating liver hemorrhage model in rats. Gauze, GS, CELOX™, CELOX™-G, the chitosan sponge MACS-2 provided in Comparative Example 1, and the alkylated chitosan sponge ACS provided in Comparative Example 2 were used as controls. The animal experiments were approved by the Animal Experiment Ethics Committee of Nankai University. The experimental procedures are as follows:

[0145] 1) Anesthetize rats with 10% (w / v) chloral hydrate and fix them on a surgical board. The dosage of the anesthetic is 300 g / mL;

[0146] 2) After scraping off the hair on the rat's abdomen, cut open the rat's abdomen, remove the liver, and place it on the surface of pre-weighed filter paper;

[0147] 3) Using a tissue biopsy device, create a penetrating wound with a diameter of 6 mm on the surface of the liver;

[0148] 4) Fill the wound with a compressed drainage MACS-2 (original diameter 8mm);

[0149] 5) Use a digital camera to record the hemostasis process;

[0150] 6) Use a timer to record the time it takes for the bleeding to stop;

[0151] 7) Using the formula (blood loss (g) = (W) 血液+滤纸 +W 血液+止血剂 )-(W 滤纸 +W 止血剂 )) Calculate the total blood loss.

[0152] Figure 9 (A) is a schematic diagram of hemostasis after a penetrating liver injury in a normal rat. Figure 9 (B) Untreated wound with gauze, GS, CELOX TM CELOX TM - Macroscopic diagram of wound treatment using G, ACS, MCS-2 and MCS-2, with arrows and dashed lines indicating the wound and liver edge, respectively; Figure 9(C) represents the untreated group with gauze, GS, and CELOX. TM CELOX TM -Bar chart showing total blood loss in the G, ACS, MCS-2 and MCS-2 treatment groups; Figure 9 (D) represents the untreated group and gauze, GS, CELOX. TM CELOX TM - Bar chart showing hemostasis time statistics for the G, ACS, MCS-2, and MCS-2 treatment groups. The sample mean was 3. Data are expressed as mean ± standard deviation. ns indicates no significant difference between groups. *P<0.05, **P<0.01, ***P<0.001.

[0153] like Figure 9 (B) shows the untreated wound, gauze, GS, and CELOX. TM -G、CELOX TM Wounds treated with ACS and MCS-2 showed significant blood leakage, with a large amount of blood dispersed on the filter paper surface. Conversely, wounds treated with MCS-2 did not show significant blood leakage, with only a small amount of blood dispersed on the filter paper surface. Figure 9 (C) and Figure 9 As shown in (D), the total blood loss in the MACS-2 group was 0.5 ± 0.1 g, which was significantly lower than that in other groups. The hemostasis time in the MACS-2 group was 12.7 ± 2.5 s, which was significantly shorter than that in other groups. These results indicate that MACS-2 has a better hemostatic effect than other hemostatic agents.

[0154] Experimental Example 10

[0155] (I) Blood compatibility test

[0156] The blood compatibility of the alkylated chitosan sponge MACS-2 provided in Example 2 was evaluated by observing and quantifying hemoglobin release. DIW, PBS, gauze, GS, CELOX TM The chitosan sponge MCS-2 provided in Comparative Example 1 and the alkylated chitosan sponge ACS provided in Comparative Example 2 served as control groups. The experimental procedures are as follows:

[0157] 1) Place the anticoagulated whole blood in the centrifuge tube into the centrifuge and centrifuge at 1000 rpm for 15 min to obtain concentrated RBC;

[0158] 2) Wash the concentrated RBCs repeatedly with PBS;

[0159] 3) Dilute RBCs with PBS to obtain an RBC suspension with a concentration of 2% (v / v);

[0160] 4) Place MACS-2 on the surface of filter paper, compress to remove the internal free water, and put it into a 1.5 mL centrifuge tube;

[0161] 5) Add diluted RBC suspension to the centrifuge tube to submerge MACS-2;

[0162] 6) After incubating at 37℃ for 1 hour, centrifuge the MACS-2 / RBC mixture. The centrifugation speed and time are 1000 rpm and 15 min, respectively.

[0163] 7) Use a digital camera to obtain macroscopic photographs of the centrifuged MACS-2 / RBC mixture;

[0164] 8) Take 100 μL of supernatant and measure its OD using an ELISA reader. 540nm value;

[0165] 9) According to the formula (hemolysis rate (%) = (OD) 止血剂 -OD PBS ) / (OD DIW -OD PBS The hemolysis rate of MACS-2 was calculated by multiplying the result by 100%.

[0166] (II) Cell compatibility test

[0167] The cytocompatibility of the alkylated chitosan MACS-2 provided in Example 2 with 3T3 fibroblasts was evaluated using CCK-8 and live / dead staining assays, with GS serving as the control group.

[0168] (1) The experimental steps of the CCK-8 experiment are as follows:

[0169] 1) Place the sterilized MACS-2 into a 48-well cell culture plate;

[0170] 2) Add a suspension of 3T3 fibroblasts to the surface of MACS-2 cells;

[0171] 3) Incubate at 37°C for 1, 3, and 5 days;

[0172] 4) After incubation, add CCK-8 reagent to the wells and continue incubation for 4 hours;

[0173] 5) Add 100 μL of supernatant to a 96-well cell culture plate;

[0174] 6) Measure the OD of the supernatant using an ELISA reader. 450nm Value. This is used to evaluate the proliferation of 3T3 fibroblasts.

[0175] (2) The experimental steps for the live / dead staining experiment are as follows:

[0176] 1) Place the sterilized MACS-2 into a 48-well cell culture plate;

[0177] 2) Add a suspension of 3T3 fibroblasts to the surface of MACS-2 cells;

[0178] 3) After incubation at 37°C for 1, 3, and 5 days, add live / dead staining reagent to the wells;

[0179] 4) After 30 minutes, observe the stained cells and obtain the corresponding images using a laser confocal microscope.

[0180] Figure 10 (A) contains water, PBS, gauze, GS, and CELOX. TM Macroscopic images of hemolysis in ACS, MCS-2, and MCS-2; Figure 10 (B) is a bar chart showing the hemolysis rate of the water, PBS, gauze, GS, CELOX™, ACS, MCS-2 and MCS-2 groups; Figure 10 (C) represents the OD of cell suspensions in the GS and MACS-2 groups. 450nm Value statistics bar chart; Figure 10 (D) Images of live / dead stained fluorescence of 3T3 fibroblasts after co-culturing with GS and MACS-2. The sample mean was 3. Data are expressed as mean ± standard deviation. ns indicates no significant difference between groups. *P<0.05, **P<0.01, ***P<0.001.

[0181] like Figure 10 As shown in (A), the supernatant in the water group was bright red, attributed to the release of hemoglobin after RBC rupture. Conversely, the supernatant in the MACS-2 group was light pink, similar in color to the supernatants in the other groups. Figure 10 As shown in (B), the hemolysis rate in the MACS-2 group was significantly lower than that in the water group. Furthermore, the hemolysis rate in the MACS-2 group was below the 5% safety standard for hemolysis of biological materials. These results indicate that MACS-2 has good blood compatibility. We evaluated the cell compatibility of MACS-2 with 3T3 fibroblasts using the CCK-8 assay and live / dead staining assay. GS was used as the control group. Figure 10 As shown in (C), the OD of the MACS-2 group 450nm The value gradually increased with the extension of culture time, and was similar to the OD value of the GS group. 450nm There was no significant difference between the values. Figure 10 As shown in (D), almost no dead 3T3 fibroblasts were observed in the MACS-2 group. These results indicate that MACS-2 has good cytocompatibility with 3T3 fibroblasts.

[0182] Experimental Example 11

[0183] The in vitro anti-infective activity of the alkylated chitosan sponge MACS-2 provided in Example 2 against Staphylococcus aureus and Escherichia coli was evaluated by contact sterilization experiments. Tissue culture plates (TCP), gauze, GS, and CELOX were used. TM CELOX TM -G, the alkylated chitosan sponge ACS provided in Comparative Example 2, and the chitosan sponge MCS-2 provided in Example 1 were used as control groups. The experimental procedure is as follows:

[0184] 1) Place the fixed-shape MACS-2 cells into a 48-well cell culture plate;

[0185] 2) After UV irradiation, add 10 μL (10 8 CFUs / mL) bacterial suspension on the surface of MACS-2;

[0186] 3) After incubation at 37℃ for 2 hours, add 200 μL of PBS to each well to resuspend the live bacteria;

[0187] 4) Take 20 μL of the resuspended bacterial solution and dilute it 10-fold six times to obtain the final diluted bacterial solution;

[0188] 5) Take 20 μL of the final diluted bacterial solution and drop it onto the surface of an LB agar plate;

[0189] 6) After overnight incubation at 37°C, count the number of colonies formed on LB agar plates;

[0190] 7) Calculate the bactericidal rate of MACS-2 against Staphylococcus aureus and Escherichia coli according to the formula (LogIncrease = Log CFUs of hemostatic agent group - Log CFUs of TCP group).

[0191] Figure 11 (A) For use with TCP, gauze, GS, CELOX TM CELOX TM Macroscopic image of Staphylococcus aureus smear results after contact with G, ACS, MCS-2 and MCS-2; Figure 11 (B) For use with TCP, gauze, GS, CELOX TM CELOX TM Macroscopic image of E. coli smear results after contact with G, ACS, MCS-2 and MCS-2; Figure 11 (C) is gauze, GS, CELOX TM CELOX TM -Bar chart showing the bactericidal rates of G, ACS, MCS-2 and MCS-2 against Staphylococcus aureus; Figure 11(D) is a bar chart showing the bactericidal rates of gauze, GS, CELOXTM, CELOXTM-G, ACS, MCS-2, and MCS-2 against Escherichia coli. The sample mean was 3. Data are expressed as mean ± standard deviation. ns indicates no significant difference between groups. *P<0.05, **P<0.01, ***P<0.001.

[0192] like Figure 11 As shown in (A), the number of Staphylococcus aureus colonies in the MACS-2 group was significantly lower than that in the gauze, GS, and ACS groups. The number of Staphylococcus aureus colonies in the MACS-2 group was not significantly different from that in the CELOXTM-G, CELOXTM, and MACS-2 groups. Figure 11 As shown in (B), the number of E. coli colonies in the MACS-2 group was significantly lower than that in other groups. Figure 11 As shown in (C), the LogIncrease value of the MACS-2 group was significantly lower than that of the gauze and GS groups. There was no significant difference in LogIncrease value between the MACS-2 group and the CELOXTM-G, CELOXTM, and MACS-2 groups. Figure 11 As shown in (D), the LogIncrease value of the MACS-2 group was significantly lower than that of the other groups. These results indicate that MACS-2 has a stronger anti-infective ability against Staphylococcus aureus compared to gauze, GS, and ACS. The anti-infective ability of MACS-2 against Staphylococcus aureus is comparable to that of CELOXTM-G, CELOXTM, and MACS-2 against Staphylococcus aureus. MACS-2's anti-infective ability against Escherichia coli is stronger than that of other hemostatic agents against Escherichia coli. The anti-infective ability of MACS-2 is attributed to the synergistic effect of its microchannel structure, grafted hydrophobic alkyl chains, and CS itself. The microchannel structure allows for sufficient contact between bacteria and MACS-2. During bacterial culture in contact with MACS-2, bacterial respiration produces a large amount of acidic substances (lactic acid and carbonic acid), which can induce local acidification of MACS-2, causing protonation of the amino groups in the CS molecule. Protonated amino groups can interact electrostatically with bacterial membranes, inducing membrane deformation and rupture, leading to loss of intracellular nutrients and bacterial death. Residual protonated amino groups in MACS-2 also exert antibacterial effects. Grafted hydrophobic alkyl chains can embed themselves into the bacterial membrane through hydrophobic interactions, disrupting the cell membrane, causing loss of intracellular nutrients, and ultimately inducing bacterial death. Furthermore, the amino and hydroxyl groups in the CS molecule can chelate with metal ions necessary for maintaining cell membrane stability and normal cellular metabolism, interfering with bacterial metabolism, disrupting cell membrane stability, causing changes in permeability, and inhibiting bacterial growth.

[0193] Experimental Example 12

[0194] The in situ tissue regeneration capacity of the alkylated chitosan sponge MACS-2 provided in Example 2 and the alkylated chitosan sponge ACS provided in Comparative Example 2 was evaluated using a rat liver defect model. The experimental procedures are as follows:

[0195] 1) Anesthetize rats with chloral hydrate and fix them on a surgical board;

[0196] 2) Scrape off the hair on the rat's abdomen, cut open the rat's abdomen, remove the liver, and place it on the surface of filter paper;

[0197] 3) Using a tissue biopsy device, create a penetrating wound with a diameter of 6 mm on the surface of the liver;

[0198] 4) Fill the wound with a fixed-shape MACS-2 (original diameter 8mm). The 8mm diameter ACS is directly filled into the wound because compressed ACS cannot return to its original shape upon contact with blood;

[0199] 5) After hemostasis, suture the abdomen and feed the rats normally;

[0200] 6) One month after the operation, the rats were anesthetized and their liver tissue was removed;

[0201] 7) Evaluate the ingrowth of tissues in MACS-2 and ACS by H&E staining;

[0202] 8) Detection of liver glycogen synthesis in MACS-2 and ACS by glycogen staining (PAS);

[0203] 9) Evaluate the degree of host cell infiltration in MACS-2 and ACS by DAPI staining;

[0204] 10) The degree of vascularization in MACS and ACS was evaluated by immunofluorescence staining of von Willebrand factor (vWF);

[0205] 11) The degree of hepatocyte infiltration in MACS-2 and ACS was evaluated by immunofluorescence staining of albumin (ALB);

[0206] 12) The expression of hepatic factors in MACS-2 and ACS was evaluated by immunofluorescence staining of hepatocyte nuclear factor (HNF-4α).

[0207] Figure 12(A) Images of natural liver, including DAPI staining for ACS and MACS-2, H&E staining, vWF / DAPI immunofluorescence staining, and ALB / DAPI immunofluorescence staining. Asterisks, hash symbols, and arrows indicate alkylated CS, blood vessels, and hepatocytes (LPC), respectively. Figure 12 (B) is a bar chart of cell counts in the natural liver, ACS and MACS-2 (n=7). Figure 12 (C) is a bar chart of tissue ingrowth area in natural liver, ACS and MACS-2 (n=3). Figure 12 (D) is a bar chart of the number of blood vessels in the natural liver, ACS and MACS-2 (n=7). Figure 12 (E) is a bar chart of LPC counts in natural livers, ACS, and MACS-2 (n=7). Figure 12 (F) shows a native liver, PAS staining images of ACS and MACS-2, and HNF-4α / DAPI immunofluorescence staining images. Asterisks and arrows indicate alkylated CS and hepatocyte nuclear factors, respectively. Figure 12 (G) is a schematic diagram of in situ liver tissue regeneration guided by ACS and MACS-2. The sample mean was 3 or 7, and the data are expressed as mean ± standard deviation. *P<0.05, ***P<0.001.

[0208] like Figure 12 (A) and Figure 12 As shown in (B), host cells can migrate into the interior of MACS-2. Conversely, host cells cannot migrate into the interior of ACS and are only distributed at the periphery of ACS. After infiltration, host cells secrete a large amount of extracellular matrix, forming new tissue.

[0209] like Figure 12 (A) and Figure 12 As shown in (C), a large area of ​​new tissue is visible inside MACS-2. Conversely, there is almost no new tissue inside ACS. The tissue ingrowth areas in the MACS-2 and ACS groups were 44.8±5.6% and 0.0±0.0%, respectively. The survival of new tissue often requires the supply of oxygen and nutrients. Capillaries can transport the oxygen and nutrients necessary for tissue survival.

[0210] like Figure 12 (A) and Figure 12 As shown in (D), MACS-2 is characterized by a high density of capillaries. In contrast, ACS contains almost no capillaries. Furthermore, we further evaluated the degree of integration of MACS-2 with liver tissue by staining for liver-specific markers (albumin, glycogen, and hepatocyte nuclear factor).

[0211] like Figure 12 (A) Figure 12 (E) and Figure 12 As shown in (F), MACS-2 cells contained a large number of infiltrating ALB-positive cells, as well as glycogen and hepatocyte nuclear factor. In contrast, ACS cells contained almost no ALB-positive cells, glycogen, or hepatocyte nuclear factor. These experimental results indicate that MACS-2 has a stronger ability to promote in situ tissue regeneration than ACS, primarily due to its highly interconnected microchannel structure, high porosity, and good biocompatibility. Microchannels can promote cell infiltration, angiogenesis, and tissue ingrowth. Figure 12 (G)). Conversely, a dense microporous structure restricts cell infiltration.

[0212] MACS-2 can simultaneously achieve hemostasis and promote in-situ tissue regeneration, which not only expands the application range of hemostatic agents but also provides new ideas for the design and construction of hemostatic agents. Furthermore, the application of MACS-2 will reduce patient discomfort, simplify treatment procedures, and potentially lower medical costs.

[0213] In summary, this invention prepares an alkylated chitosan sponge (MACS) using a combination of fiber template leaching, freeze-drying, and surface-active modification. The MACS possesses a highly interconnected and controllable microchannel structure, excellent mechanical properties, strong water / blood adsorption capacity, and rapid shape recovery. It is comparable to gauze, GS, and CELOX. TM and CELOX TM Compared to -G, MACSs exhibit stronger procoagulant activity. Furthermore, MACSs demonstrated superior hemostatic ability in a rat model of penetrating liver injury. MACSs also exhibited strong anti-infective activity against Staphylococcus aureus and Escherichia coli. More importantly, after hemostasis, MACSs remained at the wound site, guiding in situ liver regeneration by promoting hepatocyte infiltration, angiogenesis, and liver tissue ingrowth. These results indicate that MACSs have significant application and clinical translational potential in controlling non-compression penetrating injury bleeding and guiding in situ tissue regeneration.

[0214] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for modifying chitosan sponge material, characterized in that, Includes the following steps: A polymer fiber template with a fill rate of 40% is provided. Chitosan solution is injected into the pores of the polymer fiber template. After freeze-drying, the polymer fiber template is eluted to obtain a chitosan sponge. In the presence of a reducing agent, the chitosan sponge is modified with an aldehyde compound to obtain an alkylated chitosan sponge, wherein the aldehyde compound is dodecaldehyde. After eluting the polymer fiber template, the residual acetic acid in the chitosan sponge is neutralized with a mixed solution of ethanol and NaOH, wherein the volume ratio of ethanol and NaOH in the mixed solution is 8-9:2-1. The chitosan solution is an aqueous solution of chitosan and acetic acid, and the mass concentration of the chitosan solution is 4%-6%. After modifying the chitosan sponge with an aldehyde compound, the unreacted aldehyde compound and reducing agent are removed by soaking in a mixed solution of ethanol and water to obtain an alkylated chitosan sponge. The reducing agent includes NaCNBH3.

2. The method for modifying chitosan sponge material according to claim 1, characterized in that, The raw materials for the polymer fiber template include at least one of PCL, PLCL, PLA, PDS, PGA, PLGA, PHA, PEO, and PU.

3. The method for modifying chitosan sponge material according to claim 1, characterized in that, The raw material for the polymer fiber template is PLA.

4. The method for modifying chitosan sponge material according to any one of claims 1-3, characterized in that, The preparation method of the polymer fiber template is selected from one of 3D printing, casting, phase separation, electrospinning, wet spinning, melt spinning or particle leaching.

5. The method for modifying chitosan sponge material according to claim 4, characterized in that, The polymer fiber template was prepared using a 3D printing method.

6. An alkylated chitosan sponge material, characterized in that, It is prepared by the improved method of alkylated chitosan according to any one of claims 1-5.

7. The application of the alkylated chitosan sponge material according to claim 6 in the preparation of non-compression penetrating wound hemostatic agents and formulations that promote in-situ tissue regeneration.

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