CN113456882A

CN113456882A - Chitosan sponge material modification method and application

Info

Publication number: CN113456882A
Application number: CN202110739835.9A
Authority: CN
Inventors: 王连永; 孔德领; 杜欣辰; 朱美峰
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2021-10-01

Abstract

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

Description

Chitosan sponge material modification method and application

Technical Field

The invention relates to the technical field of biological materials, in particular to a modification method and application of a chitosan sponge material.

Background

In daily life and war, massive blood loss can cause serious complications such as hypothermia, acidosis and multiple organ failure, which can lead to high mortality. Therefore, rapid and effective hemostasis is crucial. Although bleeding activates the body's own coagulation cascade to control wound bleeding, for severe bleeding, especially non-compressive penetrating wound bleeding, the body's own hemostatic ability is insufficient, often requiring the assistance of a hemostatic agent with shape memory. Therefore, it would be beneficial to develop a hemostatic agent that can be used to stop bleeding in non-compressive penetrating wounds.

Currently, researchers have developed a variety of shape memory hemostatic agents. XStat^TMIs a commercial hemostatic device filled with compressed cellulose sponge. After absorbing blood, the cellulose sponge can be rapidly expanded to fill and press the wound, thereby controlling the bleeding of the wound. However, cellulose sponges have poor biodegradability, leaving the wound site to impede wound healing, requiring removal from the wound after hemostasis. In addition, cellulose sponges cannot guide tissue regeneration in situ due to their lack of highly interconnected pore structure. Polymeric foams also show some potential for use in non-compressive through-wound hemostasis. However, polymer foams generally have poor blood absorption capacity and shape recovery properties, making it difficult to timely compress wounds and control bleeding. In addition, cold-frozen cryogels with high blood adsorption and shape memory properties are also used for hemostasis in non-compressive penetrations. However, the pores created by gas foaming and ice crystal removal often have poor (low) connectivity, which can prolong the time for blood to flow into the hemostat, resulting in slow shape recovery of the hemostat and reduced hemostatic efficiency.

In view of the above, the invention is particularly provided.

Disclosure of Invention

One of the purposes of the invention is to provide a chitosan sponge material modification method, so as to prepare a chitosan sponge material which not only has good biocompatibility, biodegradability, procoagulant and anti-infection performances, but also has a highly communicated and controllable microchannel structure, high water/blood adsorption capacity and a fast shape recovery function, and can be used for non-compression transfixion hemostasis and tissue in-situ regeneration promotion.

The chitosan sponge material modification method provided by the invention is prepared according to the following steps: providing a polymer fiber template with the filling rate of 15% -75%, pouring a chitosan solution into pores of the polymer fiber template, eluting the polymer fiber template after freeze drying to obtain chitosan sponge, and modifying the chitosan sponge by adopting an aldehyde compound in the presence of a reducing agent to obtain the alkylated chitosan sponge.

In the present invention. Typically, but not by way of limitation, the polymeric fiber template has a fill factor of, e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.

The filling rate of the polymer fiber template is controlled to be 15% -75%, so that the situation that the filling rate is too high, the mechanical strength of the alkylated chitosan obtained after the subsequent polymer fiber template is eluted is too low, the stable shape of the alkylated chitosan cannot be kept, and the situation that the porosity of the alkylated chitosan is too low and the adsorption capacity of blood/water is influenced due to too low filling rate can be avoided, and the technical effects of rapid hemostasis and procoagulant blood can not be achieved.

Chitosan has good biocompatibility, biodegradability, hemostatic and anti-infective capabilities, however, chitosan has limited hemostatic and anti-infective capabilities, which are difficult to handle in severe bleeding and bacterial infections. According to the invention, the chitosan hemostatic and anti-infection capacity is improved by grafting the alkyl chain on the chitosan and utilizing the strong hydrophobic interaction between the hydrophobic alkyl chain and the cell membrane.

The invention integrates the micro-channel structure into the chitosan sponge, which not only can promote the delivery of nutrient substances and oxygen required by the organism and the discharge of metabolites, but also can provide a comfortable microenvironment for host cell infiltration, blood vessel formation and tissue ingrowth, and is also beneficial to the rapid infiltration of liquid.

The alkylated chitosan sponge with the microchannel structure is prepared by combining polymer fiber template leaching, freeze drying and alkylation modification, has good biocompatibility, biodegradability, procoagulant and anti-infection properties, has a highly communicated and controllable microchannel structure, high water/blood adsorption capacity and a quick shape recovery function, and can be used for non-compressive penetration wound hemostasis and tissue in-situ regeneration promotion.

[ Polymer fiber template ]

In a preferred embodiment of the present invention, the polymer for preparing the polymer fiber template comprises one or more of Polycaprolactone (PCL), poly (lactide-caprolactone) copolymer (PLCL), Polyurethane (PU), poly (glycerol sebacate) (PGS), poly (p-dioxanone) (PDS), polyglycolic acid (PGA), Polylactide (PLA), poly (lactide-glycolic acid) copolymer (PLGA), Polyhydroxyalkanoate (PHA), and polyethylene glycol (PEO) in any ratio. When the raw material of the polymer fiber template is PLA, the subsequent elution operation is more favorably carried out.

In a preferred scheme of the invention, the filling rate of the polymer fiber template is 20% -60%, so that the alkylated chitosan sponge obtained after the subsequent elution of the polymer fiber template has excellent mechanical strength, can maintain the structural stability, has high porosity, can promote the rapid adsorption of blood/water, and achieves the effects of rapid hemostasis and anticoagulation, and especially when the filling rate of the polymer fiber template is 40%, the alkylated chitosan sponge obtained by the subsequent preparation has more excellent mechanical properties, hemostatic performance and procoagulant function.

[ Chitosan solution ]

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

The aqueous solution of acetic acid belongs to a good solvent of chitosan, and the aqueous solution of acetic acid is adopted to dissolve chitosan, so that the chitosan has better dissolving performance.

Preferably, the concentration of acetic acid in the aqueous solution of acetic acid is 1 to 4% by mass, preferably 2% by mass.

Typically, but not by way of limitation, the chitosan solution has a mass concentration of, for example, 1%, 2%, 4%, or 6%.

The mass concentration of the chitosan is less than 4%, so that the formed alkylated chitosan has poor mechanical property and cannot keep a stable shape, and when the mass concentration of the chitosan is more than 4%, the viscosity is too high and the alkylated chitosan is not easy to be poured into pores of a PLA template. When the mass concentration of the chitosan is 4%, the strength of the chitosan sponge can be maintained, the viscosity can be kept low, and the filling operation is easy to perform.

[ aldehyde Compound ]

The invention adopts aldehyde compounds with hydrophobic carbon chains to react with amino on chitosan so as to modify the hydrophobic carbon chains on the chitosan, and utilizes strong hydrophobic interaction between the hydrophobic alkyl chains and cell membranes to improve the hemostasis and anti-infection capacity of the chitosan.

In a preferred embodiment of the invention, the aldehyde compound is a linear or branched C10-C14 aldehyde, which is beneficial to modifying a hydrophobic carbon chain onto chitosan while ensuring the hydrophobic property of chitosan.

Typically, but not by way of limitation, C10-C14 aldehydes, such as C10 aldehyde, C11 aldehyde, C12 aldehyde, C13 aldehyde, or C14 aldehyde, can be used to graft hydrophobic chains to chitosan, especially when the aldehyde compound is dodecanal, while ensuring the hydrophobic properties of chitosan and facilitating the modification reaction.

In a preferred embodiment of the present invention, the reducing agent includes, but is not limited to, NaCNBH₃。

Aldehyde groups in the aldehyde compounds can react with amino groups on chitosan molecules to form Schiff bases (C ═ N), and the Schiff bases are sensitive to water and easy to break, and are reduced by using a reducing agent to be converted into C-N with high stability.

In a preferred embodiment of the present invention, after the chitosan sponge is modified with the aldehyde compound, the mixed solution of ethanol and water is used to soak and remove the unreacted aldehyde compound and the reducing agent, so as to avoid the influence of the residual unreacted aldehyde compound and the reducing agent on the biocompatibility of the alkylated chitosan.

In a preferred embodiment of the present invention, after the polymer fiber template is eluted, the acetic acid remaining in the chitosan sponge is neutralized with a mixed solution of ethanol and NaOH, so as to prevent the remaining acetic acid from affecting the subsequent modification reaction of the aldehyde compound.

Preferably, the volume ratio of the ethanol to the NaOH mixed solution is 8-9:2-1, and preferably 9: 1.

In a preferred scheme of the invention, liquid nitrogen precooling is carried out before freeze drying, so as to be beneficial to forming a micro-channel structure with uniformly distributed channel pore sizes in the chitosan sponge.

In a preferred embodiment of the present invention, the polymer fiber template is prepared by combining any one or more of 3D printing, casting, phase separation, electrostatic spinning, wet spinning, melt spinning, and particle leaching, and especially when the polymer fiber template is prepared by 3D printing, the control of the filling rate and the improvement of the processing efficiency are facilitated.

In one exemplary embodiment of the invention, the method for modifying the chitosan sponge material comprises the following steps, wherein CS represents chitosan:

(1) printing a PLA fiber template with the filling rate of 20-60% by using a 3D printer;

dissolving CS in an aqueous acetic acid solution (2%, v/v) to obtain a CS solution with a concentration of 1-4% (w/v); filling a PLA fiber template with 1-4% (w/v) of CS solution, precooling by liquid nitrogen, and freeze-drying; wherein the freezing temperature of liquid nitrogen is-196 deg.C, and the freezing time is 5-30 min; the temperature of freeze drying is-40 to-80 ℃; freeze-drying time: 24-72 h;

(2) eluting a PLA fiber template in the CS/PLA complex by using dichloromethane to obtain CS sponge with a microchannel structure;

(3) neutralizing acetic acid remained in the CS sponge by using an ethanol/NaOH (9/1, v/v) mixed solution;

(4) in NaCNBH₃In the presence of the C8-C14 aldehyde compound, performing surface modification on the CS sponge to prepare alkylated CS sponge;

(5) after modification, the alkylated CS sponge is soaked in an ethanol/water mixed solution to remove unreacted aldehyde compounds andNaCNBH₃and obtaining the alkylated chitosan sponge.

The second purpose of the invention is to provide an alkylated chitosan, and the alkylated chitosan adopts the chitosan sponge material modification method provided by the first purpose of the invention.

The chitosan sponge material provided by the invention has a microchannel structure inside, the microchannels are mutually communicated, the porosity is up to more than 65%, the chitosan sponge material has excellent biocompatibility, biodegradability, procoagulant and anti-infection properties, has high water/blood adsorption capacity and a quick shape recovery function, and can be used for stopping bleeding and promoting tissue in-situ regeneration of non-compression new penetration wounds.

The invention also aims to provide application of the chitosan sponge material in the fields of preparing a non-compression penetrating wound hemostatic and promoting tissue in-situ regeneration.

Drawings

FIG. 1 is an FTIR chart of Chitosan (CS) powder, polylactic acid (PLA) fiber template prepared in example 1, CS/PLA composite prepared in example 1, and chitosan sponge having a micro-channel structure provided in comparative example 1;

FIG. 2 is an XPS plot of chitosan sponge (MCS-2) provided in comparative example 1;

FIG. 3 is an XPS plot of an alkylated chitosan sponge (MACS-2) as provided in example 2;

FIG. 4 is a graph of macro and micro structure, mechanical property test, and mechanical property after blood draw for the alkylated chitosan sponge 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;

FIG. 5 is a graph of water/blood adsorption characterization of the macro and micro structure of the alkylated chitosan sponge MACSs provided in examples 1-3 and the alkylated chitosan sponge ACS provided in comparative example 2;

FIG. 6 is a graph depicting the macro and microstructure shape recovery of alkylated chitosan sponge MACSs provided in examples 1-3 and of alkylated chitosan sponge ACS provided in comparative example 2;

FIG. 7 is a histogram of the pore size statistics of alkylated chitosan sponge MACSs as provided in examples 1-3 before and after imbibition of water/blood;

FIG. 8 is a graph depicting the in vitro procoagulant activity of the alkylated chitosan sponge MACSs as provided in examples 1-3;

FIG. 9 shows GS and CELOX^TM、CELOX^TM-G, MCS-2 provided by comparative example 1, ACS provided by comparative example 2 and MACS-2 provided by example 2 in vivo profile of hemostatic ability in rats;

FIG. 10 shows water, PBS, gauze, GS, and CELOX^TMCharacterization plots of the hemocompatibility and cytocompatibility of the chitosan sponge MCS-2 provided in comparative example 1, the alkylated chitosan sponge ACS provided in comparative example 2, and the alkylated chitosan MACS-2 provided in example 2;

FIG. 11 shows TCP, gauze, GS, and CELOX^TM、CELOX^TM-G, in vitro anti-infective performance profiles of the chitosan sponge MCS-2 provided in comparative example 1, of the alkylated chitosan sponge ACS provided in comparative example 2 and of the alkylated chitosan MACS-2 provided in example 2;

FIG. 12 is a graph depicting the tissue regeneration promoting capacity of the alkylated chitosan sponge MACS-2 provided in example 2 and the alkylated chitosan sponge ACS provided in comparative example 2.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This example provides an alkylated chitosan sponge (designated MACS-1) prepared as follows:

(1) printing a PLA fiber template with a filling rate of 20% by using a 3D printer;

(2) dissolving CS (chitosan) in an acetic acid aqueous solution (2%, v/v) to obtain a CS solution with a concentration of 4% (w/v); filling a PLA fiber template with 4% (w/v) of CS solution, precooling by liquid nitrogen, and freeze-drying to obtain a CS/PLA complex; wherein the freezing temperature of liquid nitrogen is-196 deg.C, and the freezing time is 5-30 min; the temperature of freeze drying is-40 to-80 ℃; freeze-drying time: 24-72 h;

(3) eluting a PLA fiber template in the CS/PLA complex by using dichloromethane to obtain CS sponge with a microchannel structure;

(4) neutralizing acetic acid remained in the CS sponge by using an ethanol/NaOH (9/1, v/v) mixed solution;

(5) in NaCNBH₃In the presence of Dodecyl Aldehyde (DA), performing surface modification on the CS sponge to prepare alkylated CS sponge;

(6) after modification, the alkylated CS sponge is soaked in ethanol/water mixed solution to remove unreacted dodecanal and NaCNBH₃And obtaining the alkylated chitosan sponge.

Example 2

This example provides an alkylated chitosan sponge (designated MACS-2) which differs from example 1 in that the 3D printing resulted in a filling rate of 40% of PLA fiber template in step 1.

Example 3

This example provides an alkylated chitosan sponge (designated MACS-3) which differs from example 1 in that the 3D printing resulted in a filling rate of 60% of PLA fiber template in step (1).

Example 4

This example provides an alkylated chitosan sponge that differs from example 1 in that the CS solution perfused with the PLA fiber template in step (2) has a mass concentration of 1%.

Example 5

This example provides an alkylated chitosan sponge that differs from example 1 in that the CS solution perfused with the PLA fiber template in step (2) has a mass concentration of 2%.

Comparative example 1

This comparative example provides a chitosan sponge (designated MCS-2) which was prepared in the same manner as in steps (1) to (4) of example 1, except that no alkylation modification was performed.

Comparative example 2

This comparative example provides an alkylated chitosan sponge (named ACS) prepared according to the following steps:

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

(2) precooling a 4% (w/v) CS solution by adopting liquid nitrogen, and freeze-drying to obtain chitosan sponge; wherein the freezing temperature of liquid nitrogen is-196 deg.C, and the freezing time is 5-30 min; the temperature of freeze drying is-40 to-80 ℃; freeze-drying time: 24-72 h;

(3) in NaCNBH₃In the presence of the catalyst, performing surface modification on the CS sponge by using dodecanal to prepare alkylated CS sponge;

(4) after modification, the alkylated CS sponge is soaked in ethanol/water mixed solution to remove unreacted dodecanal and NaCNBH₃And obtaining the alkylated chitosan sponge.

Test example 1

The characterizing functional groups of the CS (chitosan) powder, the PLA fiber template prepared in example 1, the CS/PLA complex prepared in example 1 and the chitosan sponge provided in comparative example 1 were characterized by FTIR testing. As shown in fig. 1, from the FTIR chart of the CS sponge in fig. 1, only characteristic peaks ascribed to CS were observed, and characteristic peaks ascribed to PLA were not observed, indicating that no PLA remained in the CS sponge.

Test example 2

The chemical structures and the element contents of the surfaces of the alkylated chitosan sponge provided in example 2 and the chitosan sponge provided in comparative example 1 were analyzed and detected by XPS test. The XPS profile of chitosan sponge is shown in FIG. 2, and the XPS profile of alkylated CS sponge is shown in FIG. 3. The alkylated chitosan sponge provided in example 2 and the chitosan sponge provided in comparative example 1 had C-N H₂，-N*H-COCH₃And the peak areas of C-N X H-C are shown in Table 1 below.

TABLE 1 peak area of N1s

	C-N*H₂％	-N*H-COCH₃％	C-N*H-C％
				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％

As can be seen from fig. 2 and 3 and table 1, the chemical state of N1s in the chitosan sponge provided in comparative example 1 is C-N × H₂and-N x H-COCH₃. Example 2 provides alkylated chitosan sponge with N1s in the chemical state C-N × H₂，-N*H-COCH₃And C-N H-C. Comparative example 1 chitosan sponge with C-N H₂and-N x H-COCH₃The peak areas of (A) were 86.18. + -. 4.24% and 13.82. + -. 4.24%, respectively. Example 2 provides a C-N H in alkylated chitosan sponge₂，-N*H-COCH₃And the peak areas of C-N H-C were 57.29 + -15.57%, 14.83 + -3.55% and 27.86 + -18.90%, respectively. Appearance of C-N H-C and C-N H₂The decrease in peak area demonstrates successful modification of dodecanal. The grafting rate of the dodecanal was 27.86. + -. 18.90%.

Test example 3

(1) The alkylated chitosan sponges provided in examples 1-5 were characterized using Micro-CT and SEM and comparative example 2 provided the macro and microstructure of the alkylated chitosan sponge, the pore size was measured using Image-J software, and the porosity was measured using Micro-CT.

(2) The compressive stress of the cylindrical MACSs provided in examples 4, 2 and 5 (filling rate of PLA fiber template of 40%, CS concentration of 1, 2 and 4% (w/v)), the cylindrical MACSs provided in examples 1 to 3 (filling rate of PLA fiber template of 20, 40 and 60%, CS concentration of 4% (w/v)) and the cylindrical ACS provided in comparative example 2 were tested using a universal mechanical testing machine. The compressive strain and velocity were 70% and 1mm/min, respectively.

(3) The cylindrical MACSs provided in examples 4, 2 and 5 (filling rate of PLA fiber template 40%,

CS concentration

1, 2 and 4% (w/v)), the cylindrical MACSs provided in examples 1 to 3 (filling rate of

PLA fiber template

20, 40 and 60%, CS concentration 4% (w/v)) and the cylindrical ACS provided in comparative example 2 were immersed in blood for 5 to 10min and then taken out, and then tested for compression pressure, compression strain and speed of 70% and 1mm/min, respectively, using a universal mechanical testing machine.

FIG. 4(A) is a schematic diagram of the preparation of MACSs; FIG. 4(B) is a stereomicroscope image of PLA fiber template, PLA/CS composite, CS sponge, and alkylated CS sponge; FIG. 4(C) is a Micro-CT plot of ACS provided in comparative example 2 and MACSs provided in examples 1-3; FIG. 4(D) is an SEM photograph of ACS provided for comparative example 2 and MACSs provided for examples 1-3; FIG. 4(E) is a statistical histogram of pore sizes across ACS provided in comparative example 2 and MACSs provided in examples 1-3 (n 16); FIG. 4(F) is a histogram of pore size statistics obtained by slitting ACS provided in comparative example 2 and MACSs provided in examples 1-3 (n: 16); FIG. 4(G) is a histogram of the porosity statistics for ACS provided in comparative example 2 and MACSs provided in examples 1-3 (n 25); FIG. 4(H) is a graph showing the compressive stress-strain curves of MACSs with different CS concentrations provided in examples 4, 2 and 5; FIG. 4(I) is a histogram of the compressive stress statistics of MACSs with different CS concentrations provided in examples 4, 2 and 5; FIG. 4(J) is a graph of the compressive stress-strain curves of the ACS provided by comparative example 2, the MACSs provided by comparative example 1 and MCS-2, and examples 1-3; FIG. 4(K) is a histogram of the compressive stress statistics of the ACS provided by comparative example 2, the MACSs provided by comparative example 1, MCS-2, and examples 1-3. FIG. 4(L) is a graph of the compressive stress-strain curves of ACS provided by comparative example 2, MCS-2 provided by comparative example 1, and MACSs provided by examples 1-3 after blood draw; FIG. 4(M) is a histogram of the compressive stress statistics of ACS provided by comparative example 2, MCS-2 provided by comparative example 1, and MACSs provided by examples 1-3 after blood draw; FIG. 4(N) is a histogram of the mechanical fold enhancement statistics of ACS provided by comparative example 2, MCS-2 provided by comparative example 1, and MACSs provided by examples 1-3 after blood draw. The mean number of samples 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.

As shown in FIG. 4(C), MACSs have a highly connected microchannel structure, and the density of microchannels increases with the filling rate of PLA fiber templates. ACS only has a dense microporous structure.

As shown in FIGS. 4(D), (E) and (F), the MACSs have micro-channels of 136.5. + -. 17.8 μm and micro-pore structures of 8.3. + -. 0.8 μm distributed therein. The ACS only has a micropore structure with the diameter of 8.1 +/-1.0 mu m distributed inside. The microchannel structure inside the MACSs is highly connected and controllable. In contrast, the microporous structure inside the ACS has lower connectivity and controllability.

As shown in FIG. 4(G), the porosity of the MACSs increases from 73.2 + -2.9% to 88.8 + -1.6% with the increase of the filling rate of the PLA fiber template. The porosities of MACSs were significantly higher than 32.1. + -. 1.9% of ACS.

As shown in FIGS. 4(H) and (I), the compressive stress of MACSs increased from 0.6. + -. 0.16kPa to 23.3. + -. 1.2kPa as the CS concentration increased from 1% to 4% (w/v).

As shown in FIGS. 4(J) and (K), when the filling rate of the PLA fiber template is increased from 20% to 60%, the compressive stress of the MACSs is reduced from 46.3 + -6.5 kPa to 8.1 + -0.8 kPa. The reduction in compressive stress of MACSs results primarily from a reduction in solids content per unit volume. Furthermore, the compressive stress of MACSs is significantly lower than that of ACS (138.0 + -13.4 kPa), which is attributed to the presence of microchannels.

As shown in fig. 4(L) and (M), the compressive stress of MACSs increases significantly after blood draw due to clot formation. When blood enters the microchannel, it interacts with CS and hydrophobic alkyl chains, and clotting occurs. Blood has a higher enhancing effect on the mechanical strength of MACSs than ACS. Furthermore, the enhancement effect of blood on the mechanical strength of MACSs increases with increasing porosity. The enhancement of the reinforcing effect is closely related to the porosity. The higher the porosity, the larger the specific surface area. The large specific surface area is beneficial to more sufficient contact between blood and the hemostatic agent, and more blood clots can be formed.

Test example 4

The alkylated chitosan sponge MACSs provided in examples 1-3 and comparative example 2 provided the macroscopic and microscopic water/blood adsorption capacity of the alkylated chitosan sponge ACS tested by qualitative and quantitative experiments.

The experimental procedure of the qualitative experiment is as follows:

1) placing MACSs and ACS on the surface of filter paper, compressing and discharging free water in the filter paper;

2) placing the drained MACSs and ACS in blood;

3) the positions of MACSs and ACS in the blood were recorded using a digital camera.

The experimental steps of the quantitative experiment are as follows:

1) measuring the volumes of MACSs and ACS, and marking as V;

2) placing MACSs and ACS on the surface of filter paper, compressing and discharging free water in the filter paper;

3) weighing the weights of the MACSs and the ACS after drainage, and marking as Wd;

4) submerging the drained MACSs and ACS in water/blood;

5) after immersing for a certain time, taking out, weighing and recording the weight as Wt;

6) the water/blood adsorption amounts of MACSs and ACS were calculated according to the formula (water/blood adsorption amount/sponge volume (g/cm3) ═ Wt-Wd)/V. The water/blood adsorption rates (in g/cm3/s) of MACSs and ACS were calculated from the water/blood adsorption amount-time kinetics curves.

FIG. 5(A) is a macroscopic view of ACS and MACSs soaked in blood; FIG. 5(B) is a graph of water adsorption versus time for ACS and MACSs; FIG. 5(C) is a graph showing the blood adsorption amount-time curves of ACS and MACSs; FIG. 5(D) is a histogram of the saturated water uptake of ACS and MACSs; FIG. 5(E) is a histogram of the saturated blood volumes taken by ACS and MACSs; FIG. 5(F) is a histogram of the water uptake rates of ACS and MACSs; FIG. 5(G) is a histogram of blood draw rate statistics for ACS and MACSs; FIG. 5(H) macroscopic pictures of ACS and MACSs in fixed shape before and after imbibition. FIG. 5(I) macroscopic pictures of ACS and MACSs in fixed shape before and after blood aspiration. FIG. 5(J) is a computer simulation image of the fluid adsorption capacity of ACS and MACSs. FIG. 5(K) shows the total fluid velocity of the ACS and MACSs. The mean number of samples 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.

As shown in FIG. 5(A), after absorption of blood, the MACSs sink to the bottom of the container. In contrast, ACS is suspended on the blood surface. As shown in fig. 5(B), fig. 5(C), fig. 5(D), fig. 5(E), fig. 5(F) and fig. 5(G), the saturated water/blood volume of MACSs is significantly higher than that of ACS, and gradually increases as the porosity increases. Furthermore, the water uptake/blood rate of MACSs is also significantly higher than that of ACS and increases with increasing porosity. As shown in fig. 5(H) and 5(I), MACSs are capable of allowing rapid infiltration of water and blood. ACS can allow rapid infiltration of water, however, ACS impedes the infiltration of blood. Next, we further simulated the fluid adsorption behavior of MACSs and ACS by computer. As shown in FIGS. 5(J) and 5(K), the computer simulates the fluid suction diagrams of the MACSs and ACS, wherein the distribution area of the high flow rate fluid inside the MACSs is larger than that inside the ACS. The distribution area of the high flow rate fluid inside the macs increases with the number of channels. Furthermore, the total flow rate of the fluid inside the macs is significantly higher than the total flow rate inside the ACS and gradually increases as the number of microchannels increases. The above results indicate that MACSs have a stronger water/blood adsorption capacity than ACS, which is derived from the presence of microchannels. The microchannel structure allows the MACSs to have high porosity, which in turn allows more liquid to be contained. In addition, the microchannel structure has high connectivity, which facilitates rapid penetration of liquid.

Test example 5

Testing of the alkylated chitosan sponge MACSs provided in examples 1-3 and comparative example 2 provided the shape memory properties of the alkylated chitosan sponge ACS according to the following experimental procedure:

1) placing cylindrical MACSs and ACS on the surface of filter paper, and compressing to drain free water in the filter paper;

2) dripping water/blood on the surfaces of the MACSs and ACS with fixed shapes;

3) recording the shape recovery process of the MACSs and ACS with fixed shapes by using a digital camera;

4) quantitatively evaluating the shape recovery rate and the shape recovery time of the MACSs and the ACS;

5) in addition, SEM was used to observe the change in microstructure of MACSs and ACS during the compression-recovery process;

6) the dimensions of the wells were measured using Image-J software.

FIG. 6(A) is a macroscopic view of MACSs and ACS before and after water absorption; FIG. 6(B) is a macroscopic view of MACSs and ACS before and after blood suction; FIG. 6(C) is a histogram of the shape recovery rate of ACS and MACSs after water absorption; FIG. 6(D) is a histogram of the shape recovery rate of ACS and MACSs after blood draw; FIG. 6(E) is a histogram of the shape recovery times of ACS and MACSs after water absorption; FIG. 6(F) is a histogram of the shape recovery times of ACS and MACSs after blood draw; FIG. 6(G) is an SEM image of ACS and MACSs before and after water/blood absorption. The arrows indicate the deformed tunnels. Table 2 is a table of comparative shape recovery times for MACS-2 and reported hemostats. The mean number of samples 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.

TABLE 2 comparison of shape recovery time data for MACS-2 and reported hemostatic agents

Hemostats

XSat^TM

GT25/DA8

Peanut

TRAP/Sp

QCS/PDA4

MACS-2

Water (W)

4.2±0.2s

～10s

10s

2.4s

2.1±0.1s

Blood, blood-enriching agent and method for producing the same

25s

23.4±2.4s

～10s

19.8±4.9s

2.5±0.5s

Note: XSatTM is a commercial hemostatic agent. The rest of the hemostatic agents are reported as the study hemostatic agents

FIG. 7(A) is a histogram of the pore size statistics of MACS-1 before and after imbibition/blood; FIG. 7(B) is a histogram of the pore size statistics of MACS-2 before and after imbibition/blood; FIG. 7(C) is a histogram of the pore size of MACS-3 before and after imbibition/blood. Mean of samples 6, data are expressed as mean ± standard deviation, ns indicates no significant difference between groups, P <0.05, P <0.01, P <0.001

As shown in FIGS. 6(A) and 6(B), the MACSs and ACS can achieve shape fixation after compression of the drainage water. Upon imbibition of water, the fixed-shape MACSs and ACS are able to return to their original shape. After blood draw, the fixed-shape MACSs are able to return to the original shape, whereas the fixed-shape ACS is unable to return to the original shape.

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

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

Furthermore, as can be seen from Table 2, the shape recovery times of MACSs are significantly shorter than those reported for hemostatic agents. The shape recovery time of MACSs is not affected by the viscosity of body fluids (blood). In contrast, it has been reported in part that the shape recovery time of a hemostatic agent is prolonged as the viscosity of the body fluid increases. The above results indicate that MACSs have a stronger shape-recovering ability than ACS and reported hemostats, mainly due to the presence of microchannels inside MACSs. Microchannels have a high degree of connectivity, allowing water/blood to rapidly permeate into MACSs. In contrast, the pore structure inside ACS and reported hemostats has poor connectivity, which would hinder rapid water/blood infiltration.

Further observation of the changes in pore structures within the MACSs and ACS was made using SEM, as shown in FIG. 6(G), and the MACSs had a circular microchannel structure inside thereof which was connected to each other before compression. The ACS has a dense microporous structure inside. After compression, the circular microchannels inside the MACSs collapse into flat channels. The dense microporous structure inside the ACS also collapses and becomes more dense. Upon absorption of water, the deformed microchannels within the MACSs return to their original state and the dimensions of the microchannels substantially correspond to the dimensions of the microchannels prior to compression (as shown in figure 7). The deformed micro-holes inside the ACS also recover the original shape. The deformed microchannels inside MACSs can also return to their original shape after blood draw, and large numbers of packed RBCs can be observed inside the microchannels. In contrast, deformed micropores inside ACS cannot be restored to the original state, and RBCs are hardly observed inside the deformed micropores because the microporous structure is dense, limiting the infiltration of blood.

Test example 6

The Blood Clotting Index (BCI) was measured to evaluate the procoagulant capacity of the alkylated chitosan sponge MACSs provided in examples 1-3. Gauze, GS, CELOX^TM、CELOX^TMG, Chitosan sponge MCS-2 provided in comparative example 1 and alkylated Chitosan sponge ACS provided in comparative example 2 as controls, wherein gauze, GS, CELOX^TM、CELOX^TM-G was purchased commercially and the experimental procedure was as follows:

1) placing MACSs on the surface of filter paper, and compressing to discharge free water in the MACSs;

2) placing the drained MACSs in an EP tube;

3) dripping 50 mu L of sodium citrate anticoagulated whole blood on the surfaces of the MACSs with fixed shapes;

4)4) after incubation at 37 ℃ for a period of time, 3mL of deionized water was added to the EP tube to immerse the MACSs;

5) OD measurement of supernatant using microplate reader_540nmA value;

6) measurement of OD of anticoagulated whole blood/deionized water solution using microplate reader_540nmA value, and noted as a reference value;

7) according to the formula (BCI (%)) -. OD_{Hemostatic agent}/OD_{Reference value}X 100%) the BCI values of the MACSs were calculated.

FIG. 8(A) shows gauze, GS, and CELOX^TM、CELOX^TM-BCI-time curves for the groups G, ACS, MCS-2 and MACSs; FIG. 8(B) shows RBC in gauze, GS, CELOX^TM、CELOX^TM-histogram of percentage of adhesions of G, ACS, MCS-2 and MACSs surfaces; FIG. 8(C) is a histogram of the percentage of platelet adhesion on the surface of gauze, GS, CELOXTM-G, ACS, MCS-2, and MACSs; FIG. 8(D) is a graph showing the adhesion to gauze, GS, CELOXTM-G, ACS, MCS-2 and MACSs surface SEM images of RBCs; FIG. 8(E) is an SEM image of platelets adhering to the surface of gauze, GS, CELOXTM-G, ACS, MCS-2, and MACSs; FIG. 8(F) is a photograph of immunofluorescent staining of platelets adhered to the surface of a hemostatic agent; FIG. 8(G) is a schematic diagram of the procoagulant mechanism of MACSs in vitro. Mean 3 samples, data expressed as mean ± standard deviation, ns indicates no significant difference between groups,. P<0.05，**P<0.01，***P<0.001。

As shown in FIG. 8(A), the BCI values of the MACSs decreased gradually with the increase of the incubation time, indicating that the procoagulant activities of the MACSs increased gradually. At the same time point, the BCI values for the MACSs' panel decreased with increasing porosity, indicating that the procoagulant capacity of the MACSs was positively correlated with porosity. The BCI values for the MACSs group were significantly lower than those for the ACS group, indicating that the MACSs were more procoagulant than ACS, which resulted from increased porosity. The higher the porosity, the larger the specific surface area, which is favorable for a more adequate contact of the blood with the hemostatic agent. The BCI values for the MACSs group were also significantly lower than those for the MCS-2 group, indicating that the procoagulant capacity of MACSs was greater than that of MCS-2, which was mainly due to the introduction of hydrophobic alkyl chains. Hydrophobic alkyl chains can promote RBC adhesion and aggregation. Furthermore, macs also exhibited a stronger procoagulant capacity than gauze, GS, CELOXTM, and CELOXTM-G, which resulted from the synergistic effect of CS and hydrophobic alkyl chains.

The body's active coagulation cascade depends mainly on the aggregation of RBCs and the adhesion and activation of platelets. As shown in FIGS. 8(B) and 8(C), the RBC adhesion percentages for gauze, GS, CELOXTM-G, ACS, MCS-2, MACS-1, MACS-2, and MACS-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 percent of platelet adhesion in the gauze, GS, CELOXTM-G, ACS, MCS-2, MACS-1, MACS-2, and MACS-3 groups was 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.

As shown in fig. 8(D) and 8(E), more RBCs and platelets adhere to MACSs than other hemostatic agents. In addition, aggregated RBCs and activated platelets were observed on the surfaces of MACSs in higher numbers than in other groups (fig. 8 (F)). The above experimental results indicate that MACSs have strong procoagulant ability in vitro, which is mainly derived from the synergistic effects of CS, microchannel structure and hydrophobic alkyl chains (FIG. 8 (G)). The microchannel structure can accelerate blood permeation, thereby promoting RBC and platelet to fully contact with hemostatic. CS promotes RBC aggregation and platelet adhesion, aggregation and activation. Hydrophobic alkyl chains can actively trap and aggregate RBCs and platelets through hydrophobic interactions.

Test example 7

The in vivo hemostatic capacity of the alkylated chitosan sponge MACS-2 provided in example 2 was evaluated by a lethal liver-penetrating bleeding model in rats. Gauze, GS, CELOXTM, CELOXTM-G, chitosan sponge MCS-2 as provided in comparative example 1 and alkylated chitosan sponge ACS as provided in comparative example 2 and as a control. Animal experiments were performed with approval from the ethical committee of animal experiments at the university of south kayaking. The experimental procedure was as follows:

1) rats were anesthetized with 10% (w/v) chloral hydrate and fixed on a surgical plate. The dosage of the anesthetic is 300g/1 mL;

2) after hair on the abdomen of the rat is scraped, the abdomen of the rat is cut, the liver is taken out and placed on the surface of filter paper which is weighed in advance;

3) creating a penetrating wound with a diameter of 6mm on the surface of the liver by using a tissue biopsy device;

4) compressed drained MACS-2 (original diameter 8mm) was filled into the wound;

5) recording the hemostatic process using a digital camera;

6) recording the hemostasis time using a timer;

7) by the formula (blood loss (g) ═ W_{Blood + filter paper}+W_{Blood and hemostatic agent})－(W_{Filter paper}+W_{Hemostatic agent}) Calculate total blood loss.

FIG. 9(A) is a schematic diagram of hemostasis of a liver penetrating wound of a normal rat; FIG. 9(B) shows untreated wounds with gauze, GS, CELOX^TM、CELOX^TMMacroscopic map of G, ACS, MCS-2 and MACS-2 treatment wounds, arrows anddashed lines indicate wound and liver margin, respectively; FIG. 9(C) shows untreated group and gauze, GS, CELOX^TM、CELOX^TM-histogram of total blood loss for the G, ACS, MCS-2 and MACS-2 treatment groups; FIG. 9(D) shows untreated group and gauze, GS, CELOX^TM、CELOX^TMHistogram of hemostasis time for the G, ACS, MCS-2 and MACS-2 treatment groups. Mean 3 for samples, data expressed as mean ± standard deviation, ns indicates no significant difference between groups, P<0.05，**P<0.01，***P<0.001。

As shown in FIG. 9(B), untreated wound, gauze, GS, CELOX^TM-G、CELOX^TMACS and MCS-2 treated wounds developed significant blood leakage, with a large amount of blood being dispersed on the filter paper surface. In contrast, MACS-2 treated wounds did not develop significant blood leakage, with only a small amount of blood being dispersed on the filter paper surface. As shown in FIGS. 9(C) and 9(D), the total blood loss in the MACS-2 group was 0.5. + -. 0.1g, which is significantly lower than that in the other groups. The hemostasis time for the MACS-2 group was 12.7. + -. 2.5s, which is significantly shorter than the hemostasis time for the other groups. The above results indicate that MACS-2 has a better hemostatic effect than other hemostatic agents.

Test example 10

(ii) blood compatibility test

The blood compatibility of the alkylated chitosan sponge MACS-2 provided in example 2 was evaluated by observing and quantifying the release of hemoglobin. DIW, PBS, gauze, GS, CELOX^TMThe chitosan sponge MCS-2 provided in comparative example 1 and the alkylated chitosan sponge ACS provided in comparative example 2 served as controls. The experimental procedure was as follows:

1) placing the anticoagulated whole blood filled into a centrifuge tube into a centrifuge, and centrifuging at 1000rpm for 15min to obtain concentrated RBC;

2) washing the concentrated RBCs repeatedly with PBS;

3) RBC suspensions were obtained at 2% (v/v) concentration by diluting RBCs with PBS;

4) placing MACS-2 on the surface of filter paper, compressing to discharge free water inside, and placing into a 1.5mL centrifuge tube;

5) adding the diluted RBC suspension into a centrifuge tube, and immersing MACS-2;

6)6) after incubation at 37 ℃ for 1h, the MACS-2/RBC mixture was centrifuged. The centrifugation speed and the centrifugation time are respectively 1000rpm and 15 min;

7) using a digital camera to obtain a macroscopic picture of the centrifuged MACS-2/RBC mixture;

8) 100 μ L of the supernatant was collected and OD was measured using a microplate reader_540nmA value;

9) according to the formula (hemolysis ratio (%) - (OD)_{Hemostatic agent}－OD_PBS)/(OD_DIW－OD_PBS) X 100%) the rate of hemolysis of MACS-2 was calculated.

(II) cell compatibility test

The alkylated chitosan MACS-2 provided in example 2 was evaluated for its cytocompatibility with 3T3 fibroblasts by CCK-8 and live/dead staining experiments, with GS as a control group.

(1) The experimental procedure for the CCK-8 experiment was as follows:

1) placing the sterilized MACS-2 into a 48-well cell culture plate;

2) dripping 3T3 fibroblast cell suspension on the surface of MACS-2;

3) incubate at 37 ℃ for 1, 3 and 5 days;

4) after the incubation is finished, adding a CCK-8 reagent into the hole, and continuing the incubation for 4 hours;

5) 100 mu L of supernatant is dripped into a 96-hole cell culture plate;

6) OD measurement of supernatant using microplate reader_450nmThe value is obtained. This was used to evaluate the proliferation of 3T3 fibroblasts.

(2) The experimental procedure for the live/dead staining experiment was as follows:

1) placing the sterilized MACS-2 into a 48-well cell culture plate;

2) dripping 3T3 fibroblast cell suspension on the surface of MACS-2;

3) after incubation at 37 ℃ for 1, 3 and 5 days, live/dead staining reagent was added to the wells;

4) after 30min, the stained cells were observed using a confocal laser microscope and corresponding images were obtained.

FIG. 10(A) shows water, PBS, gauze, GS, and CELOX^TMDissolution of ACS, MCS-2 and MACS-2A blood macro map; FIG. 10(B) is a histogram of hemolysis rate statistics for the water, PBS, gauze, GS, CELOXTM, ACS, MCS-2 and MACS-2 groups; FIG. 10(C) is the OD of the cell suspension in the GS and MACS-2 groups_450nmA value statistics histogram; FIG. 10(D) is a fluorescent photograph of live/dead staining of 3T3 fibroblasts after co-culture with GS and MACS-2. Mean 3 samples, data expressed as mean ± standard deviation, ns indicates no significant difference between groups,. P<0.05，**P<0.01，***P<0.001。

As shown in fig. 10(a), the color of the supernatant in the water group was bright red due to the release of hemoglobin after RBC rupture. In contrast, the supernatant in MACS-2 was light pink in color, similar to the color of the supernatants in the other groups. As shown in FIG. 10(B), the hemolysis rate of the MACS-2 group was significantly lower than that of the water group. Furthermore, the hemolysis rate of the MACS-2 group was 5% lower than the hemolysis rate safety standard for biomaterials. The above results indicate that MACS-2 has good hemocompatibility. We evaluated MACS-2 for its cytocompatibility with 3T3 fibroblasts by CCK-8 and live/dead staining experiments. GS served as control group. As shown in FIG. 10(C), OD of MACS-2 group_450nmThe value gradually increased with the increase of the culture time and the OD of GS group_450nmThere was no significant difference between the values. As shown in FIG. 10(D), almost no dead 3T3 fibroblasts were observed in the MACS-2 group. The above results indicate that MACS-2 has good cytocompatibility with 3T3 fibroblasts.

Test example 11

The alkylated chitosan sponge MACS-2 provided in example 2 was evaluated for its in vitro anti-infective activity against staphylococcus aureus and escherichia coli by a contact sterilization experiment. Tissue Culture Plate (TCP), gauze, GS, CELOX^TM、CELOX^TM-G, alkylated chitosan sponge ACS as provided in comparative example 2 and chitosan sponge MCS-2 as provided in example 1 as controls. The experimental procedure was as follows:

1) placing the fixed-shape MACS-2 into a 48-well cell culture plate;

2) after UV irradiation, 10. mu.L (10) was added dropwise⁸CFUs/mL) bacterial suspension on the surface of MACS-2;

3) after incubation for 2h at 37 ℃, 200 μ L PBS resuspended viable bacteria was added to each well;

4) taking 20 mu L of the heavy suspension bacterial liquid, and diluting for six times ten times to obtain final diluted bacterial liquid;

5) taking 20 mu L of final diluted bacterial liquid, and dropwise adding the final diluted bacterial liquid on the surface of an LB agar plate;

6) after overnight culture at 37 ℃, counting the number of colonies formed on an LB agar plate;

7) the bactericidal rate of MACS-2 against staphylococcus aureus and escherichia coli was calculated according to the formula (CFUs of the CFUs-LogTCP group of the logincreate ═ Log haemostatic group).

FIG. 11(A) shows the interaction with TCP, gauze, GS, and CELOX^TM、CELOX^TM-macroscopic picture of results of plating of staphylococcus aureus after G, ACS, MCS-2 and MACS-2 contact; FIG. 11(B) is a graph showing a graph relating to TCP, gauze, GS, and CELOX^TM、CELOX^TMMacroscopic view of the results of the plating of E.coli after contact with G, ACS, MCS-2 and MACS-2; FIG. 11(C) shows gauze, GS, and CELOX^TM、CELOX^TMHistogram of the bactericidal rate of G, ACS, MCS-2 and MACS-2 against Staphylococcus aureus; FIG. 11(D) is a histogram showing the bactericidal activity against E.coli of gauze, GS, CELOXTM-G, ACS, MCS-2 and MACS-2. Mean 3 samples, data expressed as mean ± standard deviation, ns indicates no significant difference between groups,. P<0.05，**P<0.01，***P<0.001。

As shown in FIG. 11(A), the number of Staphylococcus aureus colonies in the MACS-2 group was significantly less than those in the gauze, GS and ACS groups. The number of colonies of Staphylococcus aureus in MACS-2 group was not significantly different from the number of colonies of Staphylococcus aureus in CELOXTM-G, CELOXTM and MCS-2 group. As shown in FIG. 11(B), the number of E.coli colonies in the MACS-2 group was significantly smaller than those in the other groups. As shown in fig. 11(C), the LogIncrease value of the MACS-2 group was significantly lower than the LogIncrease value of the gauze and GS groups. There was no significant difference between the LogIncreate values of the MACS-2 group and the LogIncreate values of the CELOXTM-G, CELOXTM and MCS-2 groups. As shown in FIG. 11(D), the LogIncrase value of the MACS-2 group was significantly lower than that of the other groups. The above results show that MACS-2 has a stronger anti-infective ability against Staphylococcus aureus than gauze, GS and ACS. The anti-infective ability of MACS-2 against Staphylococcus aureus is comparable to that of CELOXTM-G, CELOXTM and MCS-2 against Staphylococcus aureus. MACS-2 has a stronger ability to resist infection of E.coli than other hemostatic agents. The anti-infective ability of MACS-2 is attributed to the synergistic effect of the microchannel structure, the grafted hydrophobic alkyl chain and the CS itself. The micro-channel structure enables the bacteria to be in sufficient contact with MACS-2. During the culture of bacteria in contact with MACS-2, bacterial respiration produces a large amount of acidic substances (lactic acid and carbonic acid) which induce local acidification of MACS-2 and protonate the amino group in the CS molecule. The protonated amino groups can electrostatically interact with the biofilm, thereby inducing deformation and rupture of the biofilm, resulting in intracellular nutrient loss and bacterial death. The protonated amino groups remaining in MACS-2 are also capable of exerting an antibacterial effect. The grafted hydrophobic alkyl chains can be embedded inside the bacterial membrane through hydrophobic interactions, disrupting the cell membrane, resulting in loss of intracellular nutrients, which in turn induces bacterial death. In addition, amino and hydroxyl groups in the CS molecule can perform chelation with metal ions required for maintaining the stability of cell membranes and normal metabolism of cells, interfere with the metabolism of bacteria, destroy the stability of the cell membranes, cause permeability change and inhibit the growth of the bacteria.

Test example 12

The alkylated chitosan sponge MACS-2 provided in example 2 and the alkylated chitosan sponge ACS provided in comparative example 2 were evaluated for their tissue regeneration promoting ability in situ using a rat liver defect model. The experimental procedure was as follows:

1) rats were anesthetized with chloral hydrate and fixed on a surgical plate;

2) the hair on the abdomen of the rat is scraped, the abdomen of the rat is cut, the liver is taken out and placed on the surface of the filter paper;

4) the wound was filled with a fixed shape of MACS-2 (8 mm original diameter). ACS 8mm in diameter was directly filled into the wound because compressed ACS could not recover its original shape upon contact with blood;

5) after hemostasis, suturing the abdomen and normally feeding the rat;

6) one month after the operation, the rat is anesthetized, and liver tissues are taken out;

7) the degree of tissue ingrowth in MACS-2 and ACS was assessed by H & E staining;

8) detection of liver glycogen synthesis in MACS-2 and ACS by glycogen staining (PAS);

9) the degree of infiltration of host cells in MACS-2 and ACS was assessed by DAPI staining;

10) the extent of vascularization in MACS-2 and ACS was assessed by immunofluorescent staining for von Willebrand factor (vWF);

11) assessing the extent of infiltration of hepatocytes in MACS-2 and ACS by immunofluorescent staining for Albumin (ALB);

12) the expression of liver factor in MACS-2 and ACS was assessed by immunofluorescent staining for hepatocyte nuclear factor (HNF-4. alpha.).

FIG. 12(A) is a DAPI stained image, H & E stained image, vWF/DAPI immunofluorescent stained image and ALB/DAPI immunofluorescent stained image of natural liver, ACS and MACS-2. Asterisks, bonds and arrows indicate alkylated CS, vascular and hepatic parenchymal cells (LPC), respectively. FIG. 12(B) is a histogram of the number of cells in native liver, ACS and MACS-2 (n-7). Fig. 12(C) is a histogram of the area of tissue in-growth in native liver, ACS and MACS-2 (n-3). Fig. 12(D) is a histogram of the number of blood vessels in native liver, ACS and MACS-2 (n-7). Fig. 12(E) is a histogram of the LPC number in native liver, ACS and MACS-2 (n-7). FIG. 12(F) is a photograph of PAS staining and HNF-4. alpha./DAPI immunofluorescence staining of native liver, ACS and MACS-2. Asterisks and arrows indicate alkylated CS and hepatocyte nuclear factors, respectively. FIG. 12(G) is a schematic representation of ACS and MACS-2 directed regeneration of liver tissue in situ. The mean number of samples was 3 or 7 and data are expressed as mean ± standard deviation, # P <0.05, # P < 0.001.

As shown in FIGS. 12(A) and 12(B), the host cell is able to migrate into the interior of MACS-2. In contrast, host cells cannot migrate into the ACS and only distribute at the ACS margin. After infiltration, the host cells will secrete large amounts of extracellular matrix, forming new tissue.

As shown in FIGS. 12(A) and 12(C), large areas of neogenetic tissue are visible inside MACS-2. In contrast, there is little new tissue inside the ACS. The tissue ingrowth areas for the MACS-2 and ACS groups were 44.8. + -. 5.6% and 0.0. + -. 0.0%, respectively. The survival of the new tissue often requires a supply of oxygen and nutrients. Capillaries are capable of transporting oxygen and nutrients required for tissue survival.

As shown in FIGS. 12(A) and 12(D), MACS-2 has a high density of capillaries distributed within it. In contrast, little capillary blood vessels are present inside the ACS. Furthermore, we further evaluated the degree of integration of MACS-2 with liver tissue by staining liver-specific markers (albumin, glycogen and hepatocyte nuclear factor).

As shown in FIGS. 12(A), 12(E) and 12(F), MACS-2 has a large amount of infiltrated ALB-positive cells as well as glycogen and hepatocyte nuclear factors inside. In contrast, ALB positive cells and glycogen and hepatocyte nuclear factor are hardly found inside ACS. The above experimental results show that MACS-2 has a stronger tissue regeneration promoting ability in situ than ACS, and is mainly derived from a highly interconnected micro-channel structure, high porosity and good biocompatibility. The microchannels can promote cellular infiltration, vascularization and tissue ingrowth (fig. 12 (G)). In contrast, the dense microporous structure limits cellular infiltration.

MACS-2 can simultaneously realize hemostasis and promote tissue in-situ regeneration, which not only expands the application range of the hemostatic, but also provides a new idea for the design and construction of the hemostatic. Furthermore, the use of MACS-2 will alleviate patient discomfort, simplify treatment procedures and potentially reduce medical costs.

In summary, the present invention prepares an alkylated chitosan sponge MACSs by a combination of fiber template washing, freeze-drying and surface active modification. MACSs have highly connected and controllable microchannel structures, excellent mechanical properties, strong water/blood adsorption capacity and fast shape recovery. With gauze, GS, CELOX^TMAnd CELOX^TMMACSs have stronger procoagulant activity than G. Meanwhile, MACSs exhibit stronger hemostatic ability in the rat liver penetrating wound bleeding model. MACSs fund pairsStaphylococcus aureus and Escherichia coli have strong anti-infective activity. More importantly, after hemostasis, MACSs can remain at the wound site, directing liver tissue regeneration in situ by promoting infiltration of liver parenchymal cells, angiogenesis, and liver tissue ingrowth. The results show that the MACSs have great application potential and clinical transformation potential in controlling non-compression penetrating injury bleeding and guiding tissue regeneration in situ.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chitosan sponge material modification method is characterized by comprising the following steps: providing a polymer fiber template with the filling rate of 15-75%, pouring a chitosan solution into pores of the polymer fiber template, eluting the polymer fiber template after freeze drying to obtain chitosan sponge, and modifying the chitosan sponge by adopting an aldehyde compound in the presence of a reducing agent to obtain alkylated chitosan sponge, wherein the aldehyde compound is linear or branched C8-C20 aldehyde.

2. The method for modifying a chitosan sponge material as claimed in claim 1, wherein the raw material of the polymer fiber template comprises at least one of PCL, PLCL, PLA, PDS, PGA, PLGA, PHA, PEO and PU, preferably PLA.

3. The method for modifying the chitosan sponge material as claimed in claim 1, wherein the filling rate of the polymer fiber template is 20% -60%, preferably 40%.

4. The method for modifying the chitosan sponge material as claimed in claim 1, wherein the chitosan solution is acetic acid aqueous solution of chitosan, and the mass concentration of the chitosan solution is 1-6%, preferably 4%.

5. The method for modifying a chitosan sponge material as claimed in claim 1, wherein said aldehyde compound is a linear or branched C10-C14 aldehyde, preferably dodecanal.

6. The method of claim 1, wherein the reducing agent comprises NaCNBH, and wherein the reducing agent comprises NaCNBH₃。

7. The method for modifying the chitosan sponge material as claimed in claim 1, wherein the alkylated chitosan sponge is obtained by modifying chitosan sponge with aldehyde compound, and then removing unreacted aldehyde compound and reducing agent by soaking in mixed solution of ethanol and water.

8. The method for modifying a chitosan sponge material according to any one of claims 1-7, wherein the polymer fiber template is prepared by a method selected from one of 3D printing, casting, phase separation, electrospinning, wet spinning, melt spinning, or particle leaching;

preferably, the polymer fiber template is prepared by a 3D printing method.

9. An alkylated chitosan sponge produced according to the method of any of claims 1-8.

10. Use of the alkylated chitosan sponge material of claim 9 in the preparation of non-compressive transfixion hemostats and in promoting tissue regeneration in situ.