CN110585474B

CN110585474B - Preparation method of marine organism polysaccharide-based composite sponge, composite sponge and application

Info

Publication number: CN110585474B
Application number: CN201910930314.4A
Authority: CN
Inventors: 周祺惠; 郝源萍; 袁昌青; 李培峰
Original assignee: Affiliated Hospital of University of Qingdao
Current assignee: Affiliated Hospital of University of Qingdao
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2022-01-04
Anticipated expiration: 2039-09-29
Also published as: CN110585474A

Abstract

The invention provides a preparation method of marine organism polysaccharide-based composite sponge, composite sponge and application, and relates to the technical field of polymer composite materials. The method has the advantages of simple process, convenient operation and wide raw material source, and is suitable for large-scale production and application. The composite sponge material prepared by taking marine organism polysaccharide sodium alginate, chitosan and fucoidin as raw materials through a freeze-drying-crosslinking-freeze-drying method not only integrates a plurality of excellent functions and biological activities of the raw materials, but also can effectively increase the specific surface area of the composite sponge material and improve the porosity and pore connectivity of the composite sponge material, thereby improving nutrient substance transportation and cell response and further accelerating wound healing.

Description

Preparation method of marine organism polysaccharide-based composite sponge, composite sponge and application

Technical Field

The invention relates to the technical field of polymer composite materials, in particular to a preparation method of a composite sponge, the composite sponge and application.

Background

The skin is the largest organ of the human body, can protect various tissues and organs in the body from being invaded by mechanical, physical, chemical and pathogenic microorganisms in the external environment, and has the function of beauty. In various skin injuries, especially large-area wounds easily cause infection diffusion and chronic wounds, so that patients have inconvenient actions and amputation and even death. Therefore, the treatment of large-area wound surfaces is a hot spot of international research. Aiming at the problems of easy bleeding, infection, inflammation, scar remaining and the like of a large-area wound surface, how to prepare a novel dressing for repairing the large-area wound surface has multiple functions of stopping bleeding, inhibiting bacteria, resisting inflammatory reaction, accelerating tissue healing, preserving moisture and removing scars and the like, thereby meeting the continuous requirements of domestic markets for medical dressings for chronic wound surfaces, realizing organic combination of production and research, and being a practical problem to be solved urgently in the market of medical instruments in China.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

In view of the drawbacks of the prior art, a first object of the present invention is to provide a method for preparing a composite sponge, which at least alleviates one of the technical problems of the prior art.

The second purpose of the invention is to provide the composite sponge prepared by the preparation method, and the composite sponge has higher specific surface area, porosity and pore connectivity.

The third purpose of the invention is to provide the preparation method and the application of the prepared composite sponge in preparing skin tissue repair products.

The invention provides a preparation method of a composite sponge, which comprises the following steps:

the acid mixed solution of chitosan, sodium alginate and optional fucoidin is freeze-dried and fixed for molding for the first time, then is put into a calcium chloride-absolute ethyl alcohol-glycerin coagulating bath for crosslinking, and then is freeze-dried for the second time to obtain the composite sponge.

Further, the concentration of the acid mixed solution is 1 to 5 wt%, preferably 2 to 4 wt%, more preferably 2.5 to 3.5 wt%.

Further, the mass ratio of the chitosan to the sodium alginate is 1:1-3, and the mass of the fucoidin is 10% -30% of the total mass of the chitosan and the sodium alginate;

preferably, the mass ratio of the chitosan to the sodium alginate is 1:2, and the mass of the fucoidin is 10% -30% of the total mass of the chitosan and the sodium alginate.

Further, the acid mixed solution is a mixed solution of a weak acid aqueous solution, chitosan, sodium alginate and optional fucoidin;

preferably, the weak acid comprises acetic acid, carbonic acid or dilute hydrochloric acid;

preferably, the concentration of the weak acid aqueous solution is 0.1 to 2 wt%, preferably 0.2 to 1.5 wt%, more preferably 0.5 to 1 wt%.

Further, in the calcium chloride-absolute ethyl alcohol-glycerol coagulation bath, the mass ratio of calcium chloride, glycerol and absolute ethyl alcohol is 1-10:1-10:80-100, preferably 5-9:1-5:85-95, and more preferably 7:3: 90;

preferably, the time of crosslinking is 4 to 8 hours, preferably 5 to 7 hours, more preferably 6 hours.

Further, the method also comprises a washing step after crosslinking, and then composite sponge is obtained through secondary freeze-drying;

preferably, the washing is performed using distilled water.

Further, the preparation method comprises the following steps:

(a) respectively providing a sodium alginate aqueous solution and a weak acid solution of chitosan;

(b) optionally, adding fucoidin into the water solution of sodium alginate to obtain a mixed water solution;

(c) dropwise adding weak acid solution of chitosan into the mixed aqueous solution, and uniformly stirring to obtain viscous solution;

(d) injecting the viscous solution into a model, and freeze-drying for one time to obtain a crude product of the composite sponge;

(e) putting the composite sponge crude product into a calcium chloride-absolute ethyl alcohol-glycerin coagulating bath for crosslinking to obtain a crosslinked product;

(f) and washing the cross-linked product, and performing secondary freeze-drying to obtain the composite sponge.

The invention also provides the composite sponge prepared by the preparation method.

In addition, the invention also provides a preparation method of the composite sponge or application of the prepared composite sponge in preparation of skin tissue repair products.

Compared with the prior art, the invention has the following beneficial effects:

the preparation method of the composite sponge comprises the steps of freeze-drying and fixing a mixed solution of chitosan, sodium alginate and optional acid of fucoidin for one time, placing the fixed solution into a calcium chloride-absolute ethyl alcohol-glycerin coagulating bath for cross-linking, and freeze-drying for the second time to obtain the composite sponge. The method has the advantages of simple process, convenient operation and wide raw material source, and is suitable for large-scale production and application. The composite sponge material prepared by taking marine organism polysaccharide sodium alginate, chitosan and fucoidin as raw materials through a freeze-drying-crosslinking-freeze-drying method not only integrates a plurality of excellent functions and biological activities of the raw materials, but also can effectively increase the specific surface area of the composite sponge material and improve the porosity and pore connectivity of the composite sponge material, thereby improving nutrient substance transportation and cell response (adhesion, proliferation, activity and specific functions).

The composite sponge provided by the invention is prepared by adopting the preparation method provided by the invention, integrates a plurality of excellent functions and biological activities of all raw materials, not only can provide an ideal microenvironment for growth, proliferation and differentiation for cells, but also has the effects of resisting bacteria, resisting inflammation, stopping bleeding, debriding, promoting new vessels, lightening scars and the like. In addition, the composite sponge has stronger water absorption and water retention, and has good compression resilience performance in a wet state. Animal experiments show that the composite sponge provided by the invention has repair performance which is obviously superior to that of the conventional sterilized gauze dressing. Therefore, the composite sponge provided by the invention has a very good application prospect in the field of chronic wound repair.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 is a schematic view of a composite sponge provided by an embodiment of the present invention;

FIG. 2 is an electron microscope image of a composite sponge according to an embodiment of the present invention;

FIG. 3 is an infrared spectrum of the composite sponge and components provided by the present invention;

FIG. 4 is an XRD pattern of the composite sponge and components provided by the examples of the present invention;

FIG. 5 is a composite sponge porosity provided by an embodiment of the present invention;

FIG. 6 is a composite sponge cytotoxicity assessment provided by an embodiment of the invention;

FIG. 7 shows the composite sponge provided by the embodiment of the invention used for the repair evaluation of large-area rat full-skin defects.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

According to one aspect of the present invention, there is provided a method of making a composite sponge, the method comprising:

Among them, Chitosan (Chitosan, CS) is an aminopolysaccharide obtained by deacetylation of chitin, and has the characteristics of good biocompatibility, inherent antibacterial property, wide source, and the like. Sodium Alginate (SA) is one of seaweed derivatives, and has the advantages of low cost, wide source, strong hydrophilicity, low immunogenicity, certain biological inertia, and strong ability to absorb wound exudate. Fucoidan (Fucoidan) is a sulfate group-containing polysaccharide extracted from brown algae, and has antibacterial, antiinflammatory, antiviral, antitumor, antioxidant, immunity regulating, free radical scavenging, antiaging, anticoagulant and antithrombotic effects. In addition, fucoidan also adsorbs, stabilizes and activates various heparin-binding cell growth factors in the tissue exudate, thereby inducing neovascularization. Fucoidin can also regulate the secretion of high-level TGF-beta of cells, and has important effects on stimulating cell division and differentiation, reducing scar generation and promoting the formation of typical granulation tissues.

The preparation method of the composite sponge provided by the invention is simple in process, convenient to operate, wide in raw material source and suitable for large-scale production and application. The composite sponge material prepared by taking marine organism polysaccharide sodium alginate, chitosan and fucoidin as raw materials through a freeze-drying-crosslinking-freeze-drying method not only integrates a plurality of excellent functions and biological activities of the raw materials, but also can effectively increase the specific surface area of the composite sponge material and improve the porosity and pore connectivity of the composite sponge material, thereby improving nutrient substance transportation and cell response (adhesion, proliferation, activity and specific functions).

In the present invention, the acid mixed solution refers to a solution having an acid solution as a solvent and chitosan, sodium alginate, and fucoidan as solutes.

In the present invention, the optional fucoidan means that chitosan and sodium alginate can be used as raw materials for preparation, and chitosan, sodium alginate and fucoidan can also be used as raw materials for preparation.

In the present invention, the terms "primary" and "secondary" in the primary lyophilization and the secondary lyophilization are only used for distinguishing and do not necessarily represent the order or the primary and secondary, and the processes of the primary lyophilization and the secondary lyophilization may be the same or different, as long as the lyophilization is performed, and the present invention is not limited thereto.

In some preferred embodiments, the concentration of the acid mixed solution is 1 to 5 wt%, for example, but not limited to, 1 wt%, 2 wt%, 3 wt%, 4 wt%, or 5 wt%.

When the concentration of the acid mixed solution is within the concentration range, the prepared composite sponge has better biological activity.

Preferably, the concentration of the acid mixed solution is 2 to 4 wt%, more preferably 2.5 to 3.5 wt%.

The concentration of the acid mixed solution is further adjusted and optimized, so that the prepared composite sponge has better biological activity.

It should be noted that, in the present invention, the acid mixed solution refers to a solution having an acid solution as a solvent and chitosan, sodium alginate and fucoidan as solutes, and therefore, the concentration of the acid mixed solution refers to the concentration of chitosan, sodium alginate and fucoidan in the acid solution, and does not include the concentration of the acid itself.

In some preferred embodiments, the mass ratio of chitosan to sodium alginate is 1:1-3, such as but not limited to 1:1, 1:2 or 1:3, and the mass of fucoidan is 10% -30% of the total mass of chitosan and sodium alginate, such as but not limited to 10%, 15%, 20%, 25% or 30%.

When the dosage of the chitosan, the sodium alginate and the fucoidin is within the concentration range, the prepared composite sponge has better biological activity.

The biological activity of the prepared composite sponge can be better by further adjusting and optimizing the dosage of chitosan, sodium alginate and fucoidin.

In some preferred embodiments, the acid mixed solution is a mixed solution of a weak acid aqueous solution with chitosan, sodium alginate and fucoidan.

The chitosan is dissolved in an acidic environment, and the weak acid can be used for avoiding the performance damage of the material (such as activity loss and accelerated degradation caused by oxidation) and avoiding the danger in experimental operation and the pollution to the environment.

Preferably, the weak acid comprises acetic acid, carbonic acid or 1N dilute hydrochloric acid.

Preferably, the concentration of the weak acid aqueous solution is 0.1 to 2 wt%, and for example, may be, but is not limited to, 0.1 wt%, 0.2 wt%, 0.5 wt%, 0.8 wt%, 1 wt%, 1.2 wt%, 1.5 wt%, 1.8 wt%, or 2 wt%, preferably 0.2 to 1.5 wt%, and more preferably 0.5 to 1 wt%.

When the concentration of the weak acid aqueous solution is within the concentration range, the prepared composite sponge has better biological activity.

In some preferred embodiments, the mass ratio of calcium chloride, glycerol and absolute ethanol in the calcium chloride-absolute ethanol-glycerol coagulation bath is 1-10:1-10:80-100, and may be, for example, but not limited to, 1:1:80, 10:10:100, 1:10:80, 10:1:100, 5:5:90 or 9:3: 90.

When the mass ratio of the calcium chloride, the glycerol and the absolute ethyl alcohol is within the concentration range, the prepared composite sponge has better biological activity.

Preferably, the mass ratio of the calcium chloride to the glycerol to the absolute ethanol in the calcium chloride-absolute ethanol-glycerol coagulation bath is 5-9:1-5:85-95, more preferably 7:3: 90.

The biological activity of the prepared composite sponge can be better by further adjusting and optimizing the mass ratio of the calcium chloride, the glycerol and the absolute ethyl alcohol.

In some preferred embodiments, the time for crosslinking is 4 to 8 hours, such as but not limited to 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours, preferably 5 to 7 hours, more preferably 6 hours.

When the crosslinking time is in the range, the effectiveness of crosslinking can be ensured in the shortest time, so that the prepared composite sponge has better bioactivity.

In some preferred embodiments, the method further comprises a step of washing after crosslinking, and then performing secondary freeze-drying to obtain a composite sponge;

preferably, the washing is performed using distilled water.

The crosslinked product is washed, so that calcium chloride-absolute ethyl alcohol-glycerol can be effectively removed, and the purity of the final product is higher.

Based on the above preparation conditions, in some specific embodiments, the preparation method of the composite sponge provided by the invention comprises the following steps:

(b) adding fucoidin into the water solution of the sodium alginate to obtain a mixed water solution;

The composite sponge provided by the invention integrates a plurality of excellent functions and biological activities of three raw materials, not only can provide an ideal microenvironment for growth, proliferation and differentiation for cells, but also has the effects of resisting bacteria, resisting inflammation, stopping bleeding, debriding, promoting new vessels, lightening scars and the like. In addition, the composite sponge has stronger water absorption and water retention, and has good compression resilience performance in a wet state. Animal experiments show that the composite sponge provided by the invention has repair performance which is obviously superior to that of the conventional sterilized gauze dressing. Therefore, the composite sponge provided by the invention has a very good application prospect in the field of chronic wound repair.

The present invention will be further described with reference to specific examples and comparative examples.

Example 1

The preparation method of the composite sponge provided by the embodiment comprises the following steps:

(a) dissolving sodium alginate (3% w/v, Mingyue seaweed) in 100mL of distilled water, and mechanically stirring until the sodium alginate is completely dissolved to obtain an aqueous solution of the sodium alginate; dissolving chitosan (3% w/v, 300kDa, 95%, Zhejiang Chitosan pharmaceutical industry) in 50mL of 1% acetic acid solution to obtain acetic acid solution of chitosan; wherein the mass ratio of the chitosan to the sodium alginate is 1: 2;

(b) dropwise adding an acetic acid solution of chitosan into the aqueous solution of sodium alginate to obtain an acid mixed solution with the concentration of 3 wt%, and mixing and stirring the acid mixed solution at room temperature for 2 hours to obtain a uniform viscous solution;

(c) the viscous solution was poured into a cylindrical mold, pre-frozen at-20 ℃ and placed in a freeze dryer (Alpha 1-2 plus;

germany) to obtain a crude product of the composite sponge;

(d) putting the composite sponge crude product into a calcium chloride-absolute ethyl alcohol-glycerin coagulating bath for crosslinking for 6 hours to obtain a crosslinked product; wherein the mass ratio of the calcium chloride to the glycerol to the absolute ethyl alcohol is 7:3: 90;

(e) the crosslinked product was washed with distilled water, pre-frozen at-20 ℃ and placed in a lyophilizer (Alpha 1-2 plus;

germany) ofThe second lyophilization was performed to obtain the complex sponge (AC, as shown in fig. 1).

Example 2

(a) dissolving sodium alginate (3% w/v, Qingdao Mingyue seaweed group) in 100mL of distilled water, and mechanically stirring until the sodium alginate is completely dissolved to obtain a sodium alginate aqueous solution; dissolving chitosan (3% w/v, 300kDa, 95%, Zhejiang Chitosan pharmaceutical industry) in 50mL of 1% acetic acid solution to obtain acetic acid solution of chitosan; wherein the mass ratio of the chitosan to the sodium alginate is 1: 2;

(b) adding fucoidin (Qingdao Mingyue seaweed group) into the sodium alginate aqueous solution, and stirring for 1h to obtain a mixed aqueous solution; wherein the mass of the fucoidin is 20% of the total mass of the chitosan and the sodium alginate;

(c) dropwise adding an acetic acid solution of chitosan into the mixed aqueous solution to obtain an acid mixed solution with the concentration of 3 wt%, and mixing and stirring the acid mixed solution at room temperature for 2 hours to obtain a uniform viscous solution;

(d) the viscous solution was poured into a cylindrical mold, pre-frozen at-20 ℃ and placed in a freeze dryer (Alpha 1-2 plus;

germany) to obtain a crude product of the composite sponge;

(e) putting the composite sponge crude product into a calcium chloride-absolute ethyl alcohol-glycerin coagulating bath for crosslinking for 6 hours to obtain a crosslinked product; wherein the mass ratio of the calcium chloride to the glycerol to the absolute ethyl alcohol is 7:3: 90;

(f) the crosslinked product was washed with distilled water, pre-frozen at-20 ℃ and placed in a lyophilizer (Alpha 1-2 plus;

germany) to obtain the complex sponge (ACF-2, shown in fig. 1).

Example 3

This example differs from example 1 in that the mass of fucoidan is 10% of the total mass of chitosan and sodium alginate (ACF-1, shown in fig. 1).

Example 4

This example differs from example 1 in that the mass of fucoidan is 30% of the total mass of chitosan and sodium alginate (ACF-3, shown in fig. 1).

Example 5

(a) dissolving sodium alginate (3% w/v, Qingdao Mingyue seaweed group) in 100mL of distilled water, and mechanically stirring until the sodium alginate is completely dissolved to obtain a sodium alginate aqueous solution; dissolving chitosan (3% w/v, 300kDa, 95%, Zhejiang Chitosan pharmaceutical industry) in 50mL of 0.1% acetic acid solution to obtain acetic acid solution of chitosan; wherein the mass ratio of the chitosan to the sodium alginate is 1: 3;

(c) dropwise adding an acetic acid solution of chitosan into the mixed aqueous solution to obtain an acid mixed solution with the concentration of 1 wt%, and mixing and stirring the acid mixed solution at room temperature for 2 hours to obtain a uniform viscous solution;

germany) to obtain a crude product of the composite sponge;

(e) putting the composite sponge crude product into a calcium chloride-absolute ethyl alcohol-glycerin coagulating bath for crosslinking for 8 hours to obtain a crosslinked product; wherein the mass ratio of the calcium chloride to the glycerol to the absolute ethyl alcohol is 1:1: 80;

germany) to obtain the composite sponge.

Example 6

(a) dissolving sodium alginate (3% w/v, Qingdao Mingyue seaweed group) in 100mL of distilled water, and mechanically stirring until the sodium alginate is completely dissolved to obtain a sodium alginate aqueous solution; dissolving chitosan (3% w/v, 300kDa, 95%, Zhejiang Chitosan pharmaceutical industry) in 50mL of 0.2% acetic acid solution to obtain acetic acid solution of chitosan; wherein the mass ratio of the chitosan to the sodium alginate is 1: 1;

(c) dropwise adding an acetic acid solution of chitosan into the mixed aqueous solution to obtain an acid mixed solution with the concentration of 5 wt%, and mixing and stirring the acid mixed solution at room temperature for 2 hours to obtain a uniform viscous solution;

germany) to obtain a crude product of the composite sponge;

(e) putting the composite sponge crude product into a calcium chloride-absolute ethyl alcohol-glycerin coagulating bath for crosslinking for 4 hours to obtain a crosslinked product; wherein the mass ratio of the calcium chloride to the glycerol to the absolute ethyl alcohol is 10:10: 100;

germany) to obtain the composite sponge.

Example 7

This example differs from example 1 in that acetic acid is replaced by hypochlorous acid.

Comparative example 1

This comparative example differs from example 1 in that chitosan was not used.

Comparative example 2

This comparative example differs from example 1 in that sodium alginate is not used.

Comparative example 3

This comparative example differs from example 1 in that acetic acid is replaced by hydrochloric acid.

Comparative example 4

This comparative example differs from example 1 in that it is placed in a calcium chloride-absolute ethanol coagulation bath for crosslinking.

Comparative example 5

This comparative example differs from example 1 in that it is placed in a calcium chloride-glycerol coagulation bath for crosslinking.

Comparative example 6

This comparative example differs from example 1 in that no secondary lyophilization is included.

Experimental example 1 measurement of porosity of composite sponge

The composite sponge samples prepared in examples 1-7 and comparative examples 1-6 are accurately weighed, immersed into sample bottles filled with a small amount of absolute ethyl alcohol, then the containers and the samples are put into a numerical control ultrasonic cleaning machine for cleaning treatment, finally, the containers and the samples are filled with the absolute ethyl alcohol and are kept still for 30min, then the samples are carefully taken out, and the porosity P of the samples is calculated to be (W2-W1)/rho V, wherein W1 and W2 are the mass of the samples before and after the samples are immersed into the ethyl alcohol, V is the volume of the samples before the samples are immersed into the ethyl alcohol, and rho is the density of the ethyl alcohol. Three replicates of each sample gave the following porosity as shown in the table:

the results show that the composite sponges prepared by the preparation method provided by the invention have good porosity, and the composite sponges prepared by the preparation method under the optimal preparation conditions are good in porosity.

In order to save costs, the following experiments were carried out using the composite sponges provided in examples 1-4 of the present invention, and the results of the porosity experiments are graphically shown in fig. 5.

Experimental example 2 measurement of Water vapor Transmission Rate of composite sponge

The composite sponge samples prepared in examples 1 to 7 and comparative examples 1 to 6 were cut into 12mm × 12mm diameter samples, and 10mL of deionized water was injected into a 12mm screw-top seed bottle having an inner diameter D measured. Covering the sponge on the bottle mouth of the strain bottle, sealing the gap between the sponge and the bottle mouth with a sealing film, standing for a period of time until the weight is constant, precisely weighing the sponge sample, the deionized water and the screw-mouth strain bottle to obtain the total mass m0, placing the sponge sample, the deionized water and the screw-mouth strain bottle into a constant-temperature drying oven, keeping the temperature of the drying oven at 37 ℃, taking out the screw-mouth bottle after 24 hours, and precisely measuring the total mass m1 of the sponge sample, the deionized water and the strain bottle. The sponge has a water vapor transmission rate Wv of 4(m0-m 1)/pi D2, wherein Wv is the water vapor transmission rate and has the unit of g/(24h · m 2); m0 is the initial total mass of the sponge sample, deionized water and the strain bottle; m1 is the total mass of the sponge sample, deionized water and the strain bottle after being dried for 24 hours; d is the inner diameter of the screw-mouth strain bottle; each sample was measured 3 times and averaged to give the water vapor transmission rates shown in the table below:

the water vapor transmission rate of the ideal wound dressing is in the range of 2000-2500g/(24 h.m 2), so that the wound dressing can prevent the wound from drying and losing water and the wound exudate from forming. The results show that the composite sponge prepared by the preparation method provided by the invention has better water vapor permeability, and the water vapor permeability of the composite sponge prepared by the preparation method under the optimal preparation condition is more suitable for wound repair.

Experimental example 3 characterization and identification of composite sponge

The physicochemical properties of the composite sponge material prepared iN example 2 were determined by scanning electron microscopy (Vega 3, Tescan), Fourier Infrared Spectroscopy (Thermo Scientific Nicolet iN10) and X-ray diffraction (D8 Advance, Bruker AXS GmbH), as shown iN FIG. 2, FIG. 3 and FIG. 4. Fig. 2 is a scanning electron microscope photograph of the composite sponge, from which it can be seen that the composite sponge presents an irregular 3D porous microstructure, and comparing the electron microscope images of the two-component sponge and the three-component sponge, it can be easily found that as the addition amount of fucoidan increases, the pore size gradually decreases and the porosity increases. FIG. 3 is FTIR spectra of sodium alginate, chitosan, fucoidin, AC and three-component ACF complex sponge, wherein 1590cm-1 and 1409cm-1 are absorption peaks of asymmetric and symmetric stretching vibration characteristics of carboxyl in sodium alginate; the absorption peaks of chitosan at 1645cm-1 and 1585cm-1 wave numbers are assigned to-C ═ O (amide I) and-NH (amide II); for the sodium alginate and chitosan two-component composite sponge AC, a strong absorption peak appears at 1605cm-1, and the peak is an overlapped peak of carboxyl of the sodium alginate and amino of chitosan, which shows that under the acidic condition, -NH 2 of the chitosan is converted into-NH 3+, and interacts with-COO-of the sodium alginate through electrostatic force. The infrared characteristic peaks of fucoidan are S ═ O bond at 1215cm-1 and sulfate group at 828 cm-1. And comparing the infrared spectrogram of the three-component ACF composite sponge, the characteristic peak of S ═ O is subjected to blue shift, which shows that fucoidin and chitosan have electrostatic interaction. As can be seen from the XRD pattern of fig. 4, the characteristic diffraction peak of the two-component composite sponge almost disappears in comparison with the diffraction peak of the raw material, indicating that the interaction of sodium alginate with the chitosan acid solution significantly reduces its crystallinity. However, when fucoidin is added, the three-component ACF spectrum shows wide and weak diffraction peaks, which shows that electrostatic force between fucoidin and sodium alginate and chitosan acts and intermolecular hydrogen bonding force is enhanced.

Experimental example 4 Compound sponge cytotoxicity assay

HGF (human gingival fibroblasts) was cultured in MEM medium containing 10% fetal bovine serum and 1% double antibody for determination of cytotoxicity of complex sponges. The composite sponge AC, ACF-1, ACF-2 and ACF-3 samples with the same size are placed into a 24-hole plate for sterilization, HGF is inoculated on the sponge material at the density of 1 × 104 cells/hole, the sponge material is placed in an incubator after cell inoculation for 1 and 4 days respectively (the culture medium is replaced every two days), CCK8 kit treatment is adopted, the absorbance is measured at 450nm by an enzyme labeling instrument, and each group of samples is performed in parallel for three times. The cytotoxicity results are shown in fig. 6, from which it can be seen that the presence of the complex sponge has no effect on the cell activity compared to the control, and the AC group is even better than the control. This means that none of the materials used are cytotoxic and suitable for subsequent in vivo animal experiments.

Experimental example 5 evaluation of Total cortical Defect repair Effect of Compound sponge rat

Healthy SD rats 12 were divided into 3 groups for complex sponge AC group, ACF-1, ACF-3 and medical sterile gauze control group, respectively. Before surgery, rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium solution at a dose of 50 mg/kg. The rats were fixed on the frame, the back was prepared and the surgical site was disinfected. A circular area with the diameter of 20mm is circled on the back of a rat, the circular area is cut through the whole skin layer to the depth of a fascia layer along the line by using a sterilized tissue scissors, the wound surface is sterilized by using a 75% alcohol cotton ball, and each group of sponge dressing is flatly paved on the wound surface on the back of the rat and then is bound. Feeding in a single cage, and changing the dressing regularly. Wound repair is shown in fig. 7, and after 14 days of operation, the skin wounds of rats treated with different materials have different repair conditions. It can be seen that both ACF-1 and ACF-3 are better than the control group, while the AC group without fucoidan is slightly inferior to the control group in terms of repair effect, which indicates that the addition of fucoidan is beneficial to wound healing. When the 21 st day after the operation, the ACF-1 group is completely recovered, and the ACF-3 is slightly inferior to the control group, which shows that the physical structure of the dressing is also important for wound repair, and the ACF-3 contains more fucoidin, so that the pore diameter is relatively small, the water absorption is strong, the water retention is poor, the air permeability of the dressing is poor in the long-term application process, and the wound repair is influenced.

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; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims

1. A method of making a composite sponge, comprising:

freeze-drying and fixing the acid mixed solution of chitosan, sodium alginate and fucoidin for the first time, placing the acid mixed solution into a calcium chloride-absolute ethyl alcohol-glycerin coagulating bath for crosslinking, and then freeze-drying for the second time to obtain the composite sponge;

the concentration of the acid mixed solution is 1-5 wt%;

the mass ratio of the chitosan to the sodium alginate is 1:1-3, and the mass of the fucoidin is 10% of the total mass of the chitosan and the sodium alginate;

in the calcium chloride-absolute ethyl alcohol-glycerin coagulating bath, the mass ratio of calcium chloride, glycerin and absolute ethyl alcohol is 1-10:1-10: 80-100.

2. The method according to claim 1, wherein the concentration of the acid mixed solution is 2 to 4 wt%.

3. The production method according to claim 1, wherein the concentration of the acid mixed solution is 2.5 to 3.5 wt%.

4. The preparation method of claim 1, wherein the mass ratio of chitosan to sodium alginate is 1: 2.

5. The method according to claim 4, wherein the acid mixed solution is a mixed solution of a weak acid aqueous solution and chitosan, sodium alginate and fucoidan.

6. The method of claim 5, wherein the weak acid comprises acetic acid, carbonic acid, or dilute hydrochloric acid.

7. The method as claimed in claim 5, wherein the concentration of the weak acid aqueous solution is 0.1-2 wt%.

8. The method as claimed in claim 5, wherein the concentration of the weak acid aqueous solution is 0.2-1.5 wt%.

9. The method as claimed in claim 5, wherein the concentration of the weak acid aqueous solution is 0.5-1 wt%.

10. The preparation method according to claim 1, wherein the mass ratio of calcium chloride, glycerol and absolute ethanol in the calcium chloride-absolute ethanol-glycerol coagulation bath is 5-9:1-5: 85-95.

11. The preparation method according to claim 1, wherein the mass ratio of calcium chloride, glycerol and absolute ethanol in the calcium chloride-absolute ethanol-glycerol coagulation bath is 7:3: 90.

12. The method of claim 1, wherein the crosslinking time is 4 to 8 hours.

13. The method of claim 1, wherein the crosslinking time is 5 to 7 hours.

14. The method of claim 1, wherein the crosslinking time is 6 hours.

15. The method for preparing the sponge according to claim 1, wherein the method further comprises a step of washing after crosslinking, and then performing secondary freeze-drying to obtain the sponge.

16. The method of claim 1, further comprising a step of washing with distilled water after the crosslinking, and then performing secondary lyophilization to obtain the composite.

17. The production method according to any one of claims 1 to 6, characterized by comprising the steps of:

18. A composite sponge prepared by the method of any one of claims 1 to 17.

19. A method of making a composite sponge according to any one of claims 1 to 17 or the use of a composite sponge according to claim 18 in the manufacture of a skin tissue repair product.