CN117122721A

CN117122721A - Sponge

Info

Publication number: CN117122721A
Application number: CN202310952221.8A
Authority: CN
Inventors: 韩雪; 吴明开; 刘海; 杨平飞; 张烨珊
Original assignee: GUIZHOU CROP VARIETIES RESOURCE INSTITUTE
Current assignee: GUIZHOU CROP VARIETIES RESOURCE INSTITUTE
Priority date: 2023-07-31
Filing date: 2023-07-31
Publication date: 2023-11-28

Abstract

The invention discloses a sponge, and belongs to the technical field of medical materials. The sponge comprises bletilla polysaccharide and chitosan, wherein the bletilla polysaccharide is crosslinked by a diacyl crosslinking agent to form the sponge, and the mass ratio of the bletilla polysaccharide to the chitosan is 0.3-3:1. The bletilla polysaccharide and the chitosan are matched, so that the effects of stopping bleeding, inhibiting bacteria, resisting inflammation, promoting cell proliferation and the like of the sponge are improved, and the repairing rate of the sponge in the treatment of wound surfaces is improved. And the bletilla striata polysaccharide and the chitosan are crosslinked through diacyl so as to optimize the physical properties of the sponge, such as liquid absorption performance, tensile stress, compressive stress and the like.

Description

Sponge

Technical Field

The invention relates to the technical field of medical materials, in particular to a sponge.

Background

Trauma is a common human injury in life and clinic. Skin acts as the first line of defense for the human body and, when damaged, undergoes healing processes such as hemostasis, inflammation, proliferation and remodeling. However, if the wound is improperly treated during the healing process, there is a risk of infection, and complications such as fever, pain and the like are induced, and even necrosis of tissues and organs, amputation, shock and death are caused. Analysis of medical insurance beneficiaries in 2018 finds that about 820 thousands of wounds with infection or non-infection exist, more than 60 tens of thousands of wounds are serious sequelae and even death caused by untimely or incorrect treatment of wounds each year, and the treatment of wounds is a necessary basic medical guarantee for human beings, so that safe, simple and effective treatment of wounds is a key of treatment. Wound dressing is an important tool for wound treatment, however, the traditional dressing has the problems of single efficacy, poor antibacterial and anti-infection effects, easy adhesion to tissues, secondary injury and the like. Therefore, some novel polymer dressings, such as microspheres, gels, and polymer sponges, have been developed, and the novel dressings have become a research hotspot in recent years.

Rhizoma Bletillae (Bletilla striata (Thunb.) Reichb.f.) is dry tuber of rhizoma Bletillae of Orchidaceae, and has astringent, hemostatic, repercussive, and granulation promoting effects. The rhizoma bletillae is rich in polysaccharide components, is a natural polysaccharide, mainly comprises beta-glucose, alpha-mannose and beta-mannose, can reach the content of more than 25% in the rhizoma bletillae, and has good biocompatibility, biological activity and safety. The bletilla striata polysaccharide not only can be used as a drug carrier, but also can be used as a drug effect active ingredient, and can be singly used or used in combination with other drugs to play a role, so that the bletilla striata polysaccharide is an ideal biological macromolecule material. Is commonly used for preparing wound dressing, vascular embolism material, drug targeting carrier, polymer micelle and the like. The research and development of polysaccharide in rhizoma bletilla can overcome the defects of unstable quality of medicinal materials, difficult control of dosage, unsatisfactory single medicine effect, long and tedious medicine decoction process, inconvenient carrying and emergency use and the like when rhizoma bletillae is used as a traditional Chinese medicine. In order to better exert the treatment characteristics of the rhizoma bletillae such as astringing to stop bleeding, detumescence and promoting granulation, the novel function and the novel use of the rhizoma bletillae as a raw material of a high-molecular sponge material are developed, the high-quality and high-efficiency development of the rhizoma bletillae industry is promoted, and a novel thought is provided for the modern development of the traditional Chinese medicine rhizoma bletillae.

The patent with the application number of CN202110702861.4 discloses a compound sponge dressing of bletilla striata and coptis chinensis and a preparation method thereof, wherein the compound sponge dressing of bletilla striata and coptis chinensis comprises bletilla striata polysaccharide and coptis chinensis total alkaloids, and the bletilla striata polysaccharide and the coptis chinensis total alkaloids form a porous lamellar sponge structure. The bletilla striata-coptis chinensis compound sponge dressing can be prepared by a one-step freeze drying method, and has the advantages of high porosity, good water absorption performance, good moisture retention, excellent elasticity and medicine slow release effect, and the preparation process is simple and the production cost is low.

The sponge auxiliary material obtained by the patent has the advantages of high porosity, good water absorption performance and the like, but still has the following problems: 1. the bletilla striata polysaccharide and the coptis total alkaloids form a porous lamellar sponge structure in a two-dimensional form, so that the water absorption and the elasticity of the sponge are easily reduced;

2. the bletilla polysaccharide and the coptis total alkaloids are only mixed by simple physics, and the mixture of the bletilla polysaccharide and the coptis total alkaloids is obtained, wherein the bletilla polysaccharide and the coptis total alkaloids are independent and do not form new substances through chemical reaction, so that the functions of the bletilla polysaccharide and the coptis total alkaloids are exerted independently even if the bletilla polysaccharide and the coptis total alkaloids are combined, and the functions of the bletilla polysaccharide and the coptis total alkaloids are not changed obviously.

Disclosure of Invention

An object of the present invention is to solve at least one of the problems of the background art and to provide a sponge.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the sponge comprises bletilla polysaccharide and chitosan, wherein the bletilla polysaccharide and the chitosan are crosslinked through diacyl groups to form the sponge with a hole structure, and the mass ratio of the bletilla polysaccharide to the chitosan is 0.3-3:1.

Preferably, the diacyl is one or more of malonyl, oxalyl, methylmalonyl, ethylmalonyl, butylmalonyl, succinyl, 2-methylsuccinyl, 2-dimethylsuccinyl, 2-ethyl-2-methylsuccinyl, 2, 3-dimethylsuccinyl, glutaryl, 2-methylglutaryl, 3-methylglutaryl, 2-dimethylglutaryl, 2, 3-dimethylglutaryl, 3-dimethylglutaryl, adipoyl, pimeloyl, suberoyl, nonylglutaryl, sebacoyl, maleoyl and fumaryl.

Preferably, the diacyl is one or more of malonyl, oxalyl, methylmalonyl, ethylmalonyl, butylmalonyl, succinyl, glutaryl, adipoyl, pimeloyl, suberoyl, nonyldiacyl and sebacoyl.

Preferably, the diacyl is one or more of malonyl, oxalyl, succinyl, glutaryl, adipoyl and pimeloyl.

Preferably, the diacyl groups account for seven to one thousandth of a part per million in the sponge.

Preferably, the molecular weight of the bletilla polysaccharide ranges from 65000kDa to 150000kDa.

Preferably, the porosity of the sponge is 9% -35%.

Preferably, the sponge has a thin film structure, and the thin film structures are interwoven to form a three-dimensional hole-like structure; the film structure has a size of 10 μm to 70 μm.

Preferably, the thickness of the film is 0.1 μm to 2 μm, and the diameter of the holes of the film is 1 μm to 90 μm.

The invention has the following beneficial effects:

1. the sponge is based on bletilla polysaccharide and chitosan to carry out free radical chemical crosslinking under the action of a crosslinking agent, so that the high molecular sponge material with good biocompatibility and good antibacterial and anti-infective properties is obtained, and the sponge is applied to wound repair in life and clinic. The wound dressing has high-density pores, good liquid absorption capacity, stable mechanical properties, certain antibacterial property and biological activity, can promote benign healing of wound surfaces, avoid complications and rejection caused by traditional materials, further reduce pain of patients and increase use compliance of the patients.

2. When the sponge is in the wound bleeding period, the bletilla striata polysaccharide can absorb wound blood, gather platelets and accelerate coagulation while absorbing wound bleeding. In the wound repair period, the bletilla striata polysaccharide can exert the bacteriostasis and reduce the wound infection risk. In addition, the good biological activity of the polysaccharide of bletilla striata allows it to regulate cytokines (e.g. coagulation factors, platelet-derived growth factors, transforming growth factors, etc.) and inflammatory cells (e.g. leukocytes, platelets, fibroblasts, etc.) and a variety of coagulation-related enzymes (prothrombin, thrombin, plasmin). Therefore, the sponge can accelerate hemostasis and promote wound healing through the mechanism.

Drawings

FIG. 1 shows a schematic flow chart of the present invention showing that polysaccharide and chitosan are crosslinked to give crosslinked products of both under oxalyl chloride;

FIG. 2 shows an SEM image of a sponge of the present invention;

FIG. 3a shows a tensile strain curve of an understanding of the present invention and a polysaccharide sponge;

FIG. 3b shows a graph of compressive strain of a polysaccharide sponge as understood by the present invention;

FIG. 4 shows SEM images of different sponges with different amounts of polysaccharide to chitosan apparent in the present invention;

FIG. 5 shows a plot of porosity versus wicking ratio for different amounts of bletilla striata polysaccharide to chitosan ratios of the present invention;

FIG. 6a shows a graph of tensile strain for different amounts of bletilla striata polysaccharide to chitosan according to the invention;

FIG. 6b shows a graph of compressive strain for different amounts of bletilla striata polysaccharide to chitosan in accordance with the present invention;

FIG. 7 shows SEM images of different lyophilized volumes of sponge of the present invention;

FIG. 8 shows a line graph of porosity and imbibition ratio for different freeze-dried volumes of sponge according to the invention;

FIG. 9a shows a graph of tensile strain of different lyophilization volumes of bletilla polysaccharide sponges of the present invention;

FIG. 9b shows a graph of compressive strain of different lyophilization volumes of bletilla polysaccharide sponges of the present invention;

FIG. 10 shows SEM images of sponges prepared with different oxalyl chloride concentrations according to the invention;

FIG. 11 shows a line graph of the sponge containing bletilla polysaccharide prepared at different oxalyl chloride concentrations according to the invention with different porosities and imbibition ratios;

FIG. 12a shows graphs of different tensile strains of bletilla polysaccharide sponges prepared with different oxalyl chloride concentrations according to the invention;

FIG. 12b shows graphs of different compressive strains of bletilla polysaccharide sponges prepared with different oxalyl chloride concentrations according to the invention;

FIG. 13 shows SEM images of sponges of the present invention taken in different reaction sequences of polysaccharide, chitosan, DMF, oxalyl chloride;

FIG. 14 shows a line graph of the present invention showing that sponges with different porosities and imbibition ratios for different reaction sequences of polysaccharide, chitosan, DMF, oxalyl chloride;

FIG. 15a shows a graph of different tensile strains of bletilla striata polysaccharide sponges according to the present invention for different reaction sequences of polysaccharide, chitosan, DMF, oxalyl chloride;

FIG. 15b shows a graph of the present invention showing that bletilla striata polysaccharide sponges have different compressive strains for different reaction sequences of polysaccharide, chitosan, DMF, oxalyl chloride;

FIG. 16 shows FTIR spectra of BSP, CS-BSP-CK, CS-BSP-1, CS-BSP-2 in the present invention;

FIG. 17a shows hydrogen spectra of BSP, CS-BSP-CK, CS-BSP-1, CS-BSP-2 in the present invention;

FIG. 17b shows a hydrogen spectrum of the delta 8.3 magnification of FIG. 17a in accordance with the present invention;

FIG. 18 shows X-ray diffraction patterns of BSP, CS-BSP-CK, CS-BSP-1, CS-BSP-2 in accordance with the present invention;

FIG. 19a shows a line graph of OD values of CS-BSP-1 according to the present invention;

FIG. 19b shows a line graph of OD values of CS-BSP-2 according to the present invention;

FIG. 20 is a line graph showing weight change in 21 days after molding mice with CK, CS-BSP-1 and CS-BSP-2, respectively, in accordance with the present invention;

FIG. 21 shows a diagram of a bacteriostatic media according to the invention obtained by a paper diffusion method;

FIG. 22 is a bar chart showing the status of the zone of inhibition obtained by the co-cultivation method of the paper sheet of the present invention;

FIG. 23 shows a histogram of blood loss after hemostasis using CK, CS-BSP-1 and CS-BSP-2, respectively, in accordance with the present invention;

FIG. 24 is a diagram showing bleeding after hemostasis using CK, CS-BSP-1, CS-BSP-2, respectively, in accordance with the present invention;

FIG. 25 is a graph showing the hemostatic index of a sponge in accordance with the present invention;

FIG. 26 is a view showing the healing of a wound in the present invention;

fig. 27 shows a simulated view of the wound healing area in the present invention;

FIG. 28 shows a graph of wound healing rate in the present invention;

FIG. 29 is a graph showing the recovery of inflammatory cell infiltration of the wound surface according to the present invention;

FIG. 30 is a view showing recovery of collagen in the present invention;

FIG. 31a is a bar graph showing the reduction of TNF-alpha by polysaccharide sponges as understood by the present invention;

FIG. 31b is a bar graph showing that the present invention recognizes and polysaccharide sponges reduce IL-6 inflammatory factors;

FIG. 31c is a bar graph showing the effect of polysaccharide sponges on VEGF content as understood by the present invention;

FIG. 32 shows a schematic diagram of a test procedure for a mouse wound model of the present invention.

Detailed Description

The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention.

The embodiment provides a sponge, which comprises bletilla striata polysaccharide and chitosan, wherein the bletilla striata polysaccharide and the chitosan form a structure with holes through diacyl crosslinking, and the mass ratio of the bletilla striata polysaccharide to the chitosan is 0.3-3:1.

In the embodiment, chitosan is used as a natural polymer material with rich sources and low manufacturing cost, and has good performances of moisture preservation, adsorption, biocompatibility, antibiosis, hemostasis and the like. The bletilla polysaccharide and the chitosan are matched for use, so that the sponge containing the bletilla polysaccharide in the embodiment simultaneously has the treatment effects of the chitosan containing the bletilla polysaccharide, and the treatment effects of the sponge containing the bletilla polysaccharide such as hemostasis, antibiosis, cell growth promotion, wound healing promotion and the like are enhanced.

The porosity of the sponge was obtained by setting the mass ratio of bletilla polysaccharide to chitosan material, as shown in figure 5. By setting the proportion of the bletilla polysaccharide to the chitosan, the porosity is 17.5% -27%, and when the component amount of the bletilla polysaccharide is higher than that of the chitosan, the porosity is lower than 23.5%; when the component amount of bletilla polysaccharide is lower than that of chitosan, the porosity is higher than 20.5%; when the component amount of bletilla polysaccharide is equal to that of chitosan, the porosity is lower than 18.5%.

In some embodiments, oxalyl chloride is used as a cross-linking agent, and when the bletilla polysaccharide, chitosan, and oxalyl chloride are mixed, one end of the oxalyl chloride radical attacks the-OH in the chemical structure of the bletilla polysaccharide, so that the oxalyl chloride is connected with the chemical structure of the bletilla polysaccharide to form a chemical structure, and the other end of the oxalyl chloride radical attacks the-NH in the chemical structure of the chitosan ₂ Thus, the chemical structure of bletilla striata polysaccharide is synthesized into a chemical substance through the chemical structure of oxalyl chloride and chitosan, as shown in figure 1. Since in the chemical structure of bletilla polysaccharide, there are a plurality of structural sites of-OH, and in the chemical structure of chitosan, there are a plurality of structural sites of-NH ₂ So that when one end of the oxalyl chloride is free to be connected with the-OH of one of the sites, the other end of the oxalyl chloride is free to be connected with the-NH of one of the sites ₂ And the chemical structures of the bletilla striata and the chitosan are connected, so that the chemical structures of the bletilla striata and the chitosan have various connection modes, namely different combination modes of the chemical structures of the bletilla striata and the chitosan exist in the same reactant, and further the bletilla striata and the chitosan are connected through oxalyl chloride to form a film-shaped structure, the films are mutually interwoven to form a three-dimensional hole structure, and an SEM image of the obtained powder sponge is displayed as a network interpenetrating structure, as shown in figure 3.

In some embodiments, the diacyl is one or more of malonyl, oxalyl, methylmalonyl, ethylmalonyl, butylmalonyl, succinyl, 2-methylsuccinyl, 2-dimethylsuccinyl, 2-ethyl-2-methyl-succinyl, 2, 3-dimethylsuccinyl, glutaryl, 2-methylglutaryl, 3-methylglutaryl, 2-dimethylglutaryl, 2, 3-dimethylglutaryl, 3-dimethylglutaryl, adipoyl, pimeloyl, suberoyl, nonylglutaryl, sebacoyl, maleoyl, fumaryl.

The malonyl, oxalyl, methylmalonyl, ethylmalonyl, butylmalonyl, succinyl, 2-methylsuccinyl, 2-dimethylsuccinyl, 2-ethyl-2-methyl-succinyl, 2, 3-dimethylsuccinyl, glutaryl, 2-methylglutaryl, 3-methylglutaryl, 2-dimethylglutaryl, 2, 3-dimethylglutaryl, 3-dimethylglutaryl, adipoyl, pimeloyl, maleyl, fumaryl, can be malonyl chloride, oxalyl chloride, methylmalonyl chloride, oxalyl chloride, ding Lvbing diacid chloride, succinyl chloride, 2-methylsuccinyl chloride, 2-dimethylsuccinyl chloride, 2-ethylchloro-2-methylsuccinyl chloride, 2, 3-dimethylsuccinyl chloride, glutaryl chloride, 2-methylglutaryl chloride, 3-methylglutaryl chloride, 2-dimethylglutaryl chloride, 2, 3-dimethylglutaryl chloride, 3-dimethylglutaryl chloride, adipoyl chloride, pimeloyl chloride, maleic chloride, fumaroyl chloride.

In some embodiments, the diacyl is one or more of malonyl, oxalyl, methylmalonyl, ethylmalonyl, butylmalonyl, succinyl, glutaryl, adipoyl, pimeloyl, suberoyl, nonyldiacyl, sebacoyl.

In some embodiments, the diacyl is one or more of malonyl, oxalyl, succinyl, glutaryl, adipoyl, pimeloyl.

In some embodiments, chitosan may be replaced with other polymeric polysaccharides.

According to one embodiment of the invention, the cross-linking agent is present in the sponge in a proportion of seven to one thousandth.

According to one embodiment of the invention, the molecular weight of the bletilla polysaccharide is 65000kDa to 150000kDa.

In some embodiments, the molecular weight of chitosan is 50000kDa to 60000kDa.

According to one embodiment of the present invention, as shown in fig. 2, 5, 8, 11 and 14, the porosity of the sponge is 9% -35%. The sponge has better liquid absorption, moisture retention and stress property, and provides a good moisture environment for the wound surface.

According to one embodiment of the invention, the sponge has a thin film structure, as shown in fig. 2, 4, 7, 10 and 13, and the thin film structures are interwoven to form a three-dimensional porous structure; the size of the film structure is 10-70 μm. Improves the deformability and plasticity of the sponge, and further enables the sponge to be applied to different wound surfaces of different parts.

The sponge with good deformability can be applied to the situation that the wound surface is not on the same plane, the sponge can adapt according to the radian of the wound surface, and then the sponge can be attached to the wound surface to play a role.

The sponge with good plasticity can be used for the wound surface with low requirement on the deformation capability of the sponge. Thereby increasing the application range of the sponge.

According to one embodiment of the present invention, as shown in fig. 2, 5, 8, 11, and 14, the thickness of the film is 0.1 μm to 2 μm and the diameter of the holes of the film is 1 μm to 90 μm.

Harvesting rhizoma bletilla, cleaning, cutting off fibrous roots and overground parts, drying at 65deg.C for 48 hr, pulverizing into powder, and sieving with 60 mesh sieve.

Weighing rhizoma bletilla powder (according to mass ratio of rhizoma bletilla to ultrapure water=1:12), adding ultrapure water, leaching at 90 ℃ for 24 hours, centrifuging, taking supernatant, adding absolute ethanol to a final concentration of 80%, centrifuging, collecting precipitate, freeze-drying, and crushing to obtain rhizoma bletilla polysaccharide freeze-dried powder.

The preparation method of the sponge comprises the following steps:

precisely weighing 100mg of bletilla polysaccharide freeze-dried powder and 150mg of chitosan, dissolving in 10mL of DMF, continuously stirring, adding 0.1mL of oxalyl chloride, continuously reacting for 15min, centrifuging, discarding supernatant to obtain a product, adding 20mL of distilled water to swell uniformly, and freeze-drying at-60-80 ℃ for 12-18 h to obtain CS-BSP-1.

Precisely weighing 100mg of bletilla polysaccharide freeze-dried powder and 30mg of chitosan, dissolving in 10mL of DMF, continuously stirring, adding 0.2mL of oxalyl chloride, continuously reacting for 15min, centrifuging, discarding the supernatant to obtain a product, adding 25mL of distilled water to swell uniformly, and freeze-drying at-60-80 ℃ for 12-18 h to obtain CS-BSP-2.

Among them, DMF may also be other solvents including but not limited to DMSO, pyridine, furans, etc.

Wherein BSP represents bletilla polysaccharide and CS represents chitosan.

The water can be alkaline, neutral or acidic, and when the water is alkaline, the prepared sponge has better looseness. When the sponge is acidic, the prepared sponge has better viscosity. And the sugar sponge prepared from neutral water has better comprehensiveness.

As shown in fig. 1, a schematic flow chart of a cross-linked product of bletilla striata polysaccharide and chitosan obtained by cross-linking of oxalyl chloride is shown.

Test examples

1. Test preparation

The chitosan has the viscosity of 100-200 mPas, the deacetylation degree of more than or equal to 98 percent and is available from Shanghai Ala Biochemical technology Co., ltd; DMF, anhuizhen technologies inc; oxalyl chloride, shanghai microphone Lin Biochemical technology Co., ltd; PBS, siemens technologies Co., ltd; chloral hydrate, shanghai Michelia Biochemical technology Co., ltd; normal saline, wuhansai wile biotechnology limited; 4% paraformaldehyde, blue Ke Jie technologies; 4% sodium citrate anticoagulant, italian biotechnology Limited company; a number 0.5 tube manufactured by the Pinctada martensii Biotechnology Co., ltd; nutrient agar, produced by Beijing Obocin Biotechnology Limited liability company; LB broth, produced by Qingdao high technology Industrial Yuan Haibo Biotechnology Co., ltd; ELISA kit (ELISA, IL-6, TNF-alpha, VEGF), whan Seville Biotechnology Co., ltd; mouse embryo cells (NIH-3T 3), wohplaunorace life technologies Co., ltd; coli (Yersinia enterocoLitica, lot 20220420), staphylococcus aureus (StaphyLococcus aureus, lot 20220420), pseudomonas aeruginosa (p. Aeromonas, lot 20220420) were all purchased from the Shanghai collection biotechnology center.

2. Characterization of sponge

2.1 scanning electron microscope, taking a trace amount of sponge to directly adhere to the conductive adhesive, and spraying metal for 45s by using an Oxford quick SC7620 sputter coating instrument, wherein the metal spraying is 10mA; then a scanning electron microscope (SEM, TESCAN MIRA LMS) is used for shooting the appearance of the sponge, the accelerating voltage is 15kV during shooting, and the detector is an SE2 secondary electron detector.

By scanning electron microscopy of the sponge CS-BSP-1 and CS-BSP-2, as shown in FIG. 2, in the SEM images of CS-BSP-1 and CS-BSP-2, the sponge has a membranous layered structure and rich holes, and with some fibrous structures, the surfaces of the films are smooth and have high concavo-convex, the films extend in the horizontal direction and the vertical direction, the films extending in the horizontal direction are interwoven with each other, and the films extending in the vertical direction are interwoven with each other to form holes; or the films extending along the horizontal direction are interwoven with the films extending along the vertical direction to form holes; the film is provided with fibrous films which are mutually interwoven to form holes; or the fibrous thin film is interwoven with the thin film extending along the horizontal direction to form holes, or the fibrous thin film is interwoven with the thin film extending along the vertical direction to form holes; the holes are extended along the horizontal direction and also extended along the vertical direction, the films extended along different directions are interwoven with the fibrous films, and the fibrous films are interwoven with each other to form a three-dimensional hole-shaped structure. From the figure, it can be seen that the size of the film structure is 10 μm to 70 μm, the thickness of the film is 0.1 μm to 2 μm, and the diameter of the hole is 1 μm to 90 μm. The three-dimensional cellular structure has a crucial influence on the physical properties of the sponge.

In addition, CS-BSP-1 and CS-BSP-2 were subjected to tensile strain and compressive strain tests.

As shown in FIG. 3a, the sponge still has good recovery property when the tensile force is continuously applied to the CS-BSP-1 sponge, the tensile strain rate of the sponge is close to 13% when the tensile force applied to the CS-BSP-1 sponge reaches approximately 0.07MPa, then the tensile strain rate of the sponge is higher than 10% when the tensile force is reduced to approximately 0.03MPa, and the tensile strain rate of the sponge is still higher than 10% when the tensile force is reduced. When the tensile force is continuously applied to the CS-BSP-2 sponge to be close to 0.04Mpa, the tensile strain rate of the sponge exceeds 13 percent, and then the tensile strain rate of the sponge reaches 17.5 percent in the process of reducing the tensile force. The sponge has better recovery capability after stretching.

As shown in FIG. 3b, the compressive strain rate of the sponge exceeded 80% as the compressive forces were continuously applied to CS-BSP-1 and CS-BSP-2 approaching 0.3 MPa. The sponge of the invention has good recovery ability after compression.

From the results, the bletilla striata polysaccharide and the chitosan are crosslinked through oxalyl chloride, so that the physical properties of the sponge are improved to a certain extent, and the physical properties are related to the three-dimensional porous structure of the sponge, the size, the thickness, the hole diameter and the like of the film.

2.1.1 different ratios of bletilla polysaccharide to chitosan with different SEM images of sponges

TABLE 1 different bletilla polysaccharide and chitosan content ratios

As shown in fig. 4, when the ratio of bletilla polysaccharide to chitosan is 1:1, the sponge SEM image shows a distinct layered structure, and the arrangement of the membranous structure and the porous structure is more compact. As the chitosan content increases, the porosity of the sponge increases. As the number of BSPs increases, the sponge-like structure and the porous structure decrease, gradually forming lumps. As the number of BSPs increases, the phenomenon of forming lumps becomes more pronounced. Therefore, the proportion of the bletilla polysaccharide to the chitosan is properly controlled, the membranous structure and the porous structure of the sponge can be adjusted, the quantity of the chitosan is properly higher than that of the bletilla polysaccharide, and a better loose and porous structure is generated in the sponge.

2.1.1.1 different ratios of bletilla striata polysaccharide to chitosan have different porosities and imbibition ratios

As shown in fig. 5, when the amount of chitosan was gradually increased as compared to the amount of bletilla polysaccharide, the porosity and the liquid absorption ratio of the sponge were increased accordingly. Ratio when the ratio of bletilla polysaccharide to chitosan was 1:2, 1:3, the sponge exhibited better porosity and liquid absorption. When the amount of bletilla polysaccharide is gradually increased compared with the amount of chitosan, the porosity and the liquid absorption ratio of the sponge are reduced. The sponge has better porosity and liquid absorption due to the fact that the chitosan amount is higher than the bletilla polysaccharide amount. It is known that chitosan plays a critical role in the porosity and the liquid absorption ratio of the sponge.

It can also be derived from fig. 5 that the porosity and the liquid absorption have a substantially positive correlation.

2.1.1.2 different ratios of bletilla striata polysaccharide to chitosan have different tensile and compressive strain curves

As shown in fig. 6b, the strain rate of the sponge after compression of BL1-BL5 reaches 80%, and when the applied pressure is continuously increased to the sponge of BL1-BL5, the strain rate of the sponge is also increased accordingly, and then when the pressure is decreased, the strain rate of the sponge is also increased. Among the quantitative ratios of BL1 to BL5, BL1 exhibits the best strain capacity compared to other BL.

As shown in FIG. 6b, when a tensile force of not more than 0.13MPa is applied only to the sponge, the strain rate can reach 12%. When the tensile force is reduced, the strain rate exceeds 17%.

2.1.2 different lyophilized volumes of sponge with different SEM images

TABLE 2 different lyophilization volumes

As shown in fig. 7, the porosity of the sponge shows a biphasic response to the freeze-dried volume of the sponge, the pore structure increases first, and the three-dimensional pore shape is more pronounced, and then the pore structure decreases, and the three-dimensional pore structure also decreases gradually. These observations indicate that too small or too large a lyophilization volume is not the best choice for achieving the desired pore structure of the sponge. The lyophilized volume of the sponge has a crucial effect on the pore structure formation of the sponge.

2.1.2.1 sponges of different freeze-dried volumes have different porosities and imbibition ratios

As shown in fig. 8, at 10mL in V2, the sponge had better porosity and liquid absorption, which decreased with increasing volume. From this, it is clear that the lyophilization volume has an effect on the porosity and liquid absorption of the sponge.

2.1.2.2 sponges of different freeze-dried volumes have different tensile and compressive strain curves

As shown in fig. 9a and 9b, different lyophilization volumes of sponges have different tensile and compressive strain curves. From this, it was found that the freeze-dried volume had an effect on the deformation of the sponge.

2.1.3 sponges with different oxalyl chloride concentrations have different SEM images

TABLE 3 oxalyl chloride at different concentrations

As shown in fig. 10, the sponge of C1 was an SEM image without oxalyl chloride, and it can be seen that the sponge had a dense lamellar structure, and that the lamellar structure was similar to the arrangement, and no apparent pore structure was seen. As oxalyl chloride concentration increases, the morphology and structure of the sponge changes. The sponge with low concentration of oxalyl chloride has a block structure which is scattered and is complicated to interweave, and interweaving is firm. As oxalyl chloride concentration increases, the sponge gradually converts into a membranous structure, resulting in a looser, more organized internal structure, and eventually a three-dimensional porous structure. As the concentration of oxygen and chlorine is further increased, the sponge maintains its porous structure, but the membranous structure becomes less obvious, resulting in tighter internal structure, increased thickness of the membrane, and affecting the strength of the sponge. From this, it was found that the spatial morphology of the sponge after the addition of oxalyl chloride was different from that of the sponge without the addition of oxalyl chloride, and the sponge with the addition of oxalyl chloride was entangled through the membrane to form a three-dimensional pore structure, whereas the sponge with the addition of oxalyl chloride did not exhibit the characteristic of the three-dimensional pore structure. From this, oxalyl chloride is a key factor in the three-dimensional pore structure of the sponge.

2.1.3.1 sponges with different oxalyl chloride concentrations have different porosities and imbibition ratios

As shown in fig. 11, the sponge without oxalyl chloride added exhibited lower porosity and liquid absorption, and the sponge with low concentration of oxalyl chloride also had lower liquid absorption but higher porosity. From this, oxalyl chloride is a key factor affecting the porosity of the sponge; oxalyl chloride concentration is a key factor affecting the sponge imbibition ratio. While too high a concentration of oxalyl chloride results in a decrease in porosity and liquid absorption of the sponge. Thus, proper oxalyl chloride concentration can increase the porosity and liquid absorption of the sponge.

2.1.3.2 sponges with different oxalyl chloride concentrations have different tensile and compressive strain curves

As can be seen in fig. 12a and 12b, sponges of different oxalyl chloride concentrations have different tensile and compressive strain curves. From this, it was found that the oxalyl chloride concentration had an effect on the deformation of the sponge.

2.1.4 sponges with different charging sequences have different SEM images

TABLE 4 different charging sequences

As shown in FIG. 13, the sponge of S1 has a three-dimensional film-like porous structure, the film structure is loose, and the holes and the films are arranged regularly. The sponge prepared by the other three reactants still has a certain porous structure, but the arrangement of the films is tighter, and the concave-convex degree is reduced; thus, the conditions of S1 are more favorable for the formation of a sponge structure.

2.1.4.1 sponges of different charging sequences have different porosities and imbibition ratios

As shown in fig. 14, the porosity and liquid absorption of the sponge of S1 is higher than those of the sponges of S2 to S4. Thus, the order of addition affects the porosity and the liquid absorption ratio of the resulting sponge.

2.1.2.2 sponges of different order of addition have different tensile and compressive strain curves

As shown in fig. 15a and 15b, sponges of different order of addition have different tensile strain curves and compressive strain curves. It follows that different feeding sequences have an influence on the deformation of the sponge.

2.2FTIR spectral characterization

By KBr tabletting technique, the tablet is compressed in the middle infrared (400 cm) ^-1 ～4000cm ^-1 ) FTIR spectroscopic analysis was performed in the range. 10mg of the sample was homogeneously mixed with 300mg of KBr. A sample sphere of diameter 7 mm was pressed at a pressure of 2 tons for 3 minutes. An infrared spectrum of the sample was obtained in the mid-infrared range using a Thermo Scientific Nicolet 6700 spectrophotometer. For each sample, 32 scans were recorded, with a resolution of 4cm ^-1 。

As shown in FIG. 16, at 3436cm ^-1 The absorption peak of the physical mixture (CS-BSP-CK) of bletilla polysaccharide (BSP), BSP and Chitosan (CS) sponge is most obvious, which shows that the hydroxyl of BSP in CS-BSP-CK is not consumed at the moment, while the-OH of BSP is consumed by the addition of oxalyl chloride containing diacyl, and the absorption of CS-BSP-1 and CS-BSP-2 is reduced at the moment and moves to 3350cm ^-1 In the vicinity, it is presumed that this is due to the effect of hydrogen bonding on amide bond formation after the reaction of oxalyl chloride and CS. At 1524cm ^-1 Here, the sponges of BSP and CS-BSP-CK are gentle, and the C=O stretching vibration peaks of secondary amide appear in CS-BSP-1 and CS-BSP-2, and at the same time, 1418cm ^-1 Here, C-N stretching peaks of the primary amide appear, all of which confirm the presence of the amide bond.

2.3 characterization of nuclear magnetic resonance hydrogen spectrum

Using D ₂ O is used as a solvent, and the lyophilized BSP, CS-BSP-CK, CS-BSP-1, CS-BSP-2 and deuterium are exchanged for three times. Then measured by NMR spectrometer (Bruker AVANCE III HD MHz) ¹ H NMR spectrum.

As shown in FIGS. 17a and 17b, the hydrogen of CS-BSP-CK and BSP is mainly concentrated in the high field region, which is hydrogen on hydrocarbon groups, and there is almost no hydrogen signal in the low field region. While CS-BSP-1 and CS-BSP-2 have hydrogen signals near the low field region delta 8.3, and the chemical shift value of the amide bond is generally near delta 5-8.5, the hydrogen signals near delta 8.3 can be judged as the hydrogen signals in the amide bond generated by the reaction. The above results all indicate that a sponge of CS-crosslinked BSP was successfully prepared.

2.4X-ray diffraction characterization

The BSP and BSP sponges were characterized by X-ray diffraction (XRD) using a p-analytical X' Pert powder diffractometer at a voltage of 40kV and a current of 40 mA. XRD diffraction spectrum is in the range of 10-70 deg. and scanning speed is 2 deg. min ^-1 The step size is 0.02 °.

As shown in fig. 18, BSP and CK have a broad peak around 2θ=20°, and the peak of CS-BSP-CK is strong and sharp corresponding to the crystalline region, whereas CS-BSP-1, CS-BSP-2 have a weak peak around 2θ=23°, indicating a decrease in crystallinity. The lower the crystallinity of the composite, the better the miscibility of the material components in the composite sponge. The bletilla striata polysaccharide and the chitosan have good miscibility, and the bletilla striata polysaccharide is crosslinked with the chitosan through oxalyl chloride, so that the miscibility is increased.

3. In vitro test of sponge

3.1 cell growth promoting Performance test

After 1mL of 0.2% pancreatin was used to digest 3min for logarithmic growth phase cells using mouse fibroblasts (NIH-3T 3), the digestion was stopped by adding an equal amount of DMEM medium, centrifuging at 1000rpm for 4min, removing the supernatant, adding fresh medium, gently beating the cells to be suspended in the medium.

Inoculating NIH-3T3 cells with density of 104/mL into 96-well culture dish, each well having volume of 100 μl, setting one circle around the well plate as blank group, adding PBS solution, adding only culture solution into negative control group without adding cells, and adding CO at 37deg.C and 5% ₂ Incubation was carried out for 24h to allow cell attachment.

The culture medium was replaced with the prepared CS-BSP-1 and CS-BSP-2 extracts (100. Mu.L), and after co-culturing for 24 hours, 48 hours and 72 hours, the original culture medium was aspirated, 100. Mu.L of fresh culture medium was added to each well, CCK-8 (10. Mu.L) was added to each well and culturing was continued at 37℃for 4 hours. The absorbance at 450nm was measured by microplate reader.

As a result of toxicity analysis of CS-BSP-1 and CS-BSP-2 by CCK-8 method, as shown in FIGS. 19a and 19b, the OD values of CS-BSP-1 and CS-BSP-2 were higher than that of DMEM group from 0.04g to 0.16g in 72h, showing that CS-BSP-1 and CS-BSP-2 were not only non-cytotoxic but also had some growth promoting effect on cells, especially evident at 0.04g, and after 72h, the OD value of CS-BSP-1 was 1.95 times that of DMEM, and the OD value of CS-BSP-2 was 1.71 times that of DMEM, with significant difference (P < 0.0001). It is presumed that at low concentrations, white polysaccharide and sponge have a strong promoting effect on cell growth and proliferation.

By analyzing the body weight of the mice after molding, whether the mice are toxic or not can be estimated after sponge treatment, the body weight change of the mice in the treatment process is shown in fig. 20, and the body weight of each group of mice is basically and stably increased within 21 days, and no statistical difference exists among groups, so that the sponge has no obvious toxicity to the treatment of the mice.

3.2 antibacterial Property test of sponge

The antibacterial activity of the sponges of the present invention was measured by a filter paper agar diffusion method and a co-culture method.

10mgCS-BSP-1 and CS-BSP-2 sponge samples are weighed and immersed in 1mL PBS for 24 hours to prepare sample lixivium. Respectively soaking sterile filter paper sheets (with the diameter of 6 mm) in a sample leaching solution for 30min, absorbing 200 mu L of 3 bacterial suspensions (such as escherichia coli, staphylococcus aureus and pseudomonas aeruginosa) with the concentration of about 1 multiplied by 108cfu/mL, uniformly coating on a nutrient agar plate, placing the soaked sterile filter paper sheets on the agar plate at equal intervals, taking PBS as a negative control, then inversely incubating the plate for 24h at the temperature of 37 ℃, and recording the diameter of a bacteriostasis ring.

mu.L of bacterial suspension diluted to 1X 108CFU/mL with PBS was added to a 96-well plate. Next, 180 μl of sample leaching solution and PBS solution (CK) were added to each well. The 96-well plate is placed in a 37 ℃ incubator for 4 hours, then taken out, the solution in the well plate is blown uniformly by a pipette, then bacterial suspension in the well plate is sucked, 20 mu L of each well is sucked, then 1mLPBS solution is used for dilution, 60 mu L of the diluted bacterial suspension is dripped on an agar plate, and finally all agar plates are placed in the 37 ℃ incubator for 24 hours after being uniformly coated by a completely sterilized triangular glass rod. Bacterial growth was observed.

Bacteria are a major cause of many infections in humans, as they are often found in the mucous membranes or skin of humans, leading to inflammation and exacerbation of wounds. Therefore, the CS-BSP-1 and CS-BSP-2 were tested for their resistance to E.coli, staphylococcus aureus and Pseudomonas aeruginosa by both the paper diffusion method and co-culture method.

As shown in FIG. 21, the sheet diffusion method showed that CS-BSP-1 and CS-BSP-2 had a distinct zone of inhibition, whereas CK did not have a zone of inhibition due to lack of antimicrobial.

As shown in FIG. 22, the co-culture method shows that after 24 hours of culture, CS-BSP-1 and CS-BSP-2 have obvious antibacterial effects, and CS-BSP-1 has stronger resistance to staphylococcus aureus and pseudomonas aeruginosa, and has better inhibition effects on escherichia coli, CS-BSP-1 and CS-BSP-2.

3.3 sponge hemostatic Performance test

The tail-breaking model test of mice, the test mice are SPF-grade healthy male ICR mice, and the weight is about 18 g. The mice are housed in cages at room temperature, water and food can be freely obtained, 5 mice in each cage, and the total number of the mice is 3. The water and wood chip pad were changed 2 times a week, fed daily, and entered the test 2-3 weeks after laboratory feeding.

The anesthetized mice were quantitatively intraperitoneally injected with 4% chloral hydrate and fixed on a surgical cork plate, and the tail length of the mice was cut off 2/3 with surgical scissors. Pre-weighed filter paper is placed under the wound to accurately estimate the blood loss, the tail of the mouse is suspended for 5s after cutting to ensure the normal blood loss, a hemostatic sponge is applied to the wound to stop bleeding, the hemostatic time and the weight of the filter paper (blood loss) are recorded, each test is repeated three times, and a tail-breaking model which is not treated by the sponge is used as a Control (CK).

As shown in FIGS. 23 and 24, the blood loss in the model of the tail-breaking of the CS-BSP-1 treated mice was significantly reduced, and the hemostatic time was the shortest, the blood loss was (17.80.+ -. 0.95 mg), the hemostatic time was (277.33.+ -. 5.51 s), and the blood loss in the model of the tail-breaking of the mice without any treatment (CK) was (30.83.+ -. 0.85 mg), the hemostatic time was (428.67.+ -. 8.02) (P < 0.05), and CK was nearly 2-fold higher than CS-BSP-1 treatment, both in blood loss and hemostatic time, as expressed by CK > CS-BSP-2 > CS-BSP-1. As shown in FIG. 25, the BCI is a clinical common blood coagulation judgment index, the lower the BCI is, the better the blood coagulation effect is, which shows that the better the hemostatic effect is, the hemostatic index is expressed as CS-BSP-CK > CS-BSP-2 > CS-BSP-1, and the hemostatic coefficient of CS-BSP-CK is obviously higher than CS-BSP-1 and CS-BSP-2. As can be seen from FIGS. 23-25, CS-BSP-1 has excellent hemostatic effect, which is closely related to the ratio of chitosan to bletilla gum in CS-BSP-1, and in addition, CS-BSP-1 has good tissue adhesion and mechanical strength, providing a physical barrier for the wound to accelerate the clotting rate of the wound.

4. In vivo test of sponge

4.1 mouse wound model test

4.1.1 wound model establishment

The full thickness skin defect model was used to assess the wound healing capacity of the sponge. The test material and the mice in the earlier stage were fed in the same manner as 2.5.3, 5 mice in each cage, and a total of 12 cages were randomly divided into 3 groups of 20 mice each. 60 mice were anesthetized using intraperitoneal injection of 4% chloral hydrate. After the mice were anesthetized, the back hair of the mice was shaved off by a shaver, and a full-thickness skin defect wound having a diameter of 10mm was formed in the back region of the mice, and molding was completed.

4.1.2 mice weight change and wound healing Condition

After the molding was completed, the mice were randomly divided into 3 groups of 20 mice each, and the wounds of the mice were treated with CS-BSP-1 and CS-BSP-2, respectively, and medical gauze treatment was used as a negative Control (CK), and the mice were taken out of the dressing every 7d to record the degree of wound healing during the treatment starting period, and at the same time, the weight change of the mice was recorded every 3d and the dressing was replaced, and the test was continued for 28d as shown in FIGS. 20, 26 and 32.

In order to study the influence of the compound sponge on wound tissue regeneration, the wound healing area and the wound healing rate in the mouse body are measured. The wound healing rate was calculated using Image J software, and from the simulated figures of the wound healing rate and the healing area, as shown in fig. 27 and 28, the CS-BSP-1 and CS-BSP-2 had no significant difference in healing effect, but significant difference from CK.

On day 7 of treatment, CS-BSP-1 and CS-BSP-2 substantially scab, CS-BSP-1 wound area was significantly reduced, but CK had not yet scab completely, inflammation was the heaviest, and exudates were present. This is because BSP itself can enhance wound healing efficiency by inducing proliferation and migration of vascular endothelial cells. And the composite CS can adjust the mechanical force, water absorption, antibacterial property and other capacities of the sponge prepared by the BSP, and further improve the healing degree of the wound surface.

On treatment day 14, the wound surfaces of CS-BSP-1 and CS-BSP-2 are obviously reduced, and the healing degree is higher. This is due to the porous structure and excellent wicking ability of the sponge, which can remove any excess exudates, create a suitable fluid environment, and promote wound healing rates. In addition, the sponge based on the composite CS has good antibacterial property, promotes cell growth performance, avoids secondary injury caused by external bacterial infection, and promotes wound surface to heal rapidly.

On treatment day 21, the CS-BSP-1 and CS-BSP-2 wounds healed completely, the mice had healthy hair growth, and CK was also visible as an unrepeated wound, indicating that CS-BSP-1 and CS-BSP-2 could heal the wounds faster.

4.2H & E staining and Masson staining

Immediately after the mice are sacrificed, the wound skin is excised, 4% chloral hydrate is used for intraperitoneal injection to anesthetize the mice, and wound tissues are cut off along the wound surface of about 2-3mm, so that the materials are obtained. The wound tissue is immersed in 4% paraformaldehyde for fixation, and the steps of flushing, dewatering, transparentizing, waxing, embedding, repairing, slicing and spreading are sequentially carried out, and then H & E dyeing and Masson dyeing are carried out.

To gain a better understanding of how sponges promote wound healing, mice from the 4.1 mouse wound model test described above were sacrificed at different time intervals and the state of regenerating skin tissue was analyzed by H & E staining and Masson staining. H & E staining and Masson staining can identify inflammatory cell infiltration and collagen recovery of the wound.

As shown in fig. 29, in 7d, a large number of inflammatory cells including lymphocytes, plasma cells and macrophages can be infiltrated and aggregated under the wound surface mirror of the CK group, the hair follicle structure is less, the cell arrangement is more disordered, and the new blood vessels are not obviously increased; the wound surfaces of the CS-BSP-1 and the CS-BSP-2 are accompanied by infiltration of scattered inflammatory cells while granulation tissues are generated, and the CS-BSP-1 has more newly generated capillaries, so that the dermis of a treatment group is thinned, and skin appendages are increased; all 3 groups showed epithelial shedding and no epithelial regeneration was seen.

After 14d, CK granulation tissue is still mainly inflammatory cells, collagen and fibroblasts are less common; obvious epidermis regeneration occurs in the wounds of CS-BSP-1 and CS-BSP-2, collagen in the dermis layer is orderly arranged, granulation tissues are obviously increased, and in addition, compared with the wounds of CS-BSP-1 and CS-BSP-2, skin appendages of CS-BSP-1 such as hair follicles, sweat glands and the like are more proliferated.

After 21d, each group has obvious epidermic regeneration, CS-BSP-1 and CS-BSP-2 epidermic tissues tend to be complete, the keratinized layer is more obvious, the accessory glands are increased, the connective tissue recombination is good, and a large number of new blood vessels appear; CK still has massive inflammatory cell infiltration, few accessory glands in dermis, few fibroblasts in granulation tissue, disordered and discontinuous collagen arrangement, incomplete connective tissue recombination in dermis, and massive gaps.

After 28d, the epidermis of the negative group tended to be intact, the keratinized layer was evident, all groups of wounds had healed, there was apparent appearance of skin appendages, collagen was abundant and neat, similar to normal skin, and the healing process had been substantially completed.

Collagen deposition is an important phenomenon for wound healing, as shown in FIG. 30, and after 7d of treatment, CS-BSP-1, CS-BSP-2 and CK all have small amounts of collagen,

by 14d, collagen deposition was increased for each group relative to 7d, but the CK arrangement was sparse, the collagen deposition amounts of CS-BSP-1 and CS-BSP-2 were slightly larger than CK, and the skin appendages such as hair follicles, sweat glands, etc. of CS-BSP-1 were more proliferated.

After 21d, the collagen deposition of the wound dermis layers of the CS-BSP-1 and the CS-BSP-2 is still more than that of CK, the arrangement is more compact and ordered, the skin appendages at the wound of the CS-BSP-2 are more obviously diffused, and the protein deposition is also increased.

By 28 days, all groups of wound collagen deposition became dense and ordered, appearance of wound skin appendages, apparent wound healing and apparent collagen deposition were similar to normal skin.

In conclusion, CS-BSP-1 and CS-BSP-2 can promote wound healing by regulating the inflammation level of the wound, promoting collagen deposition and epithelial cell regeneration, and the promoting effect of CS-BSP-1 is better and more obvious.

4.3 enzyme-linked immunosorbent assay (ELISA)

At the beginning of the treatment, one cage of mice was sacrificed every 7d for each treatment, and mouse whole blood was collected, i.e., 7d, 14d, 21d, 28d. The collected whole blood was subjected to 6000 rpm Zhong Lixin min at room temperature, serum was collected in a frozen tube, labeled and stored at 80℃under zero. The ELISA kit is used for determining the content of inflammatory factors IL-6, TNF-alpha and VEGF in each serum sample, and specific test steps are referred to the kit instruction.

Symptomatic cells are the basis of wound healing, especially macrophages and neutrophils. However, a large amount of data suggests that the prolongation and overexpression of various inflammatory cells eventually prevent the healing process. In the inflammatory phase, important inflammatory factors tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) are selected in the test.

As a result, as shown in FIGS. 31a, 31b and 31c, the IL-6 content of CK was 31.35% higher than CS-BSP-1 and 19.41% higher than CS-BSP-2 on day 7 of the treatment; the TNF-alpha content of CK is 40.18% higher than CS-BSP-1 and 33.49% higher than CS-BSP-2.

On day 14, the IL-6 content of CK was 37.02% higher than CS-BSP-1 and 24.32% higher than CS-BSP-2; the TNF-alpha content of CK is 62.08% higher than CS-BSP-1 and 43.28% higher than CS-BSP-2.

From the treatment day 7 to 21, the TNF-alpha and IL-6 in CS-BSP-1 and CS-BSP-2 are obviously lower than CK, which indicates that the sponge can reduce the over-expression of inflammatory factors such as TNF-alpha and IL-6, so that the wound inflammation period is faster, and the wound healing speed of mice is accelerated. In the wound healing process, angiogenesis is important in tissue formation induced by various growth factors such as Vascular Endothelial Growth Factor (VEGF).

And after CS-BSP-1 and CS-BSP-2 treatment, the VEGF content of CK was 76.69%,65.95% and 87.77% of CS-BSP-1, respectively, from day 7 to day 21; the content of the test groups is 84.65%,79.56% and 94.78 of CS-BSP-2, which are all obviously higher than CK, which shows that the sponge can improve the expression of VEGF, thereby inducing the formation of tissues of the wound surface and leading the wound surface to heal rapidly.

5. Statistical analysis

Statistical analysis was performed using social science statistical program 22.0 software (SPSS, IBM, USA). Data are expressed as mean ± Standard Deviation (SD) of continuous variables, mean analyzed by independent sample t-test and one-way anova. A value of p <0.05 is considered statistically significant.

6. Conclusion(s)

The bletilla striata polysaccharide is used as a natural high molecular polysaccharide extracted from rare and rare traditional Chinese medicine bletilla striata, and a cross-linking technology is used for successfully preparing the sponge with the network interpenetrating structure. It not only has obvious hemostatic effect, but also can be used as a high-quality medicine carrier. Through crosslinking of oxalyl chloride and chitosan, not only the mechanical property and microstructure of the sponge can be better regulated, but also the biological activities of hemostasis, antibiosis and the like of the sponge can be enhanced. By comparing CS-BSP-1 with CS-BSP-2, the CS-BSP-1 has better hemostatic, antibacterial, cell growth promoting, wound healing promoting effects, etc., which may be the reason for higher content of bletilla striata polysaccharide in CS-BSP-1. The cck-8 test results of CS-BSP-1 and CS-BSP-2 sponges show that the sponges have remarkable promotion effect on NIH-3T3 cells, and are also one of the advantages of natural polysaccharide materials.

In sum, the sponge not only has better biocompatibility and good hemostatic, antibacterial and anti-inflammatory capabilities, but also has the effects of promoting cell proliferation and accelerating wound healing, and is beneficial to preventing wound infection in the treatment process. And can be used for treating wound caused by different factors, such as incised wound, scald, etc.

When the requirements for using the sponge are different, the sponge with different physical properties and therapeutic effects is obtained by adjusting the quantity ratio of the bletilla striata polysaccharide and the chitosan, the concentration of oxalyl chloride, the sequence of feeding reaction and the freeze-drying volume of the sponge, so that the sponge has the characteristics of wide application prospect and targeted use.

The above examples merely illustrate specific embodiments of the application, which are described in more detail and are not to be construed as limiting the scope of the application. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the technical idea of the application, which fall within the scope of protection of the application.

Claims

1. The sponge is characterized by comprising bletilla polysaccharide and chitosan, wherein the bletilla polysaccharide and the chitosan are crosslinked through diacyl groups to form the sponge, and the mass ratio of the bletilla polysaccharide to the chitosan is 0.3-3:1.

2. A sponge according to claim 1, wherein said diacyl is one or more of malonyl, oxalyl, methylmalonyl, ethylmalonyl, butylmalonyl, succinyl, 2-methylsuccinyl, 2-dimethylsuccinyl, 2-ethyl-2-methyl-succinyl, 2, 3-dimethylsuccinyl, glutaryl, 2-methylglutaryl, 3-methylglutaryl, 2-dimethylglutaryl, 2, 3-dimethylglutaryl, 3-dimethylglutaryl, adipoyl, pimeloyl, suberoyl, nonylsebacoyl, maleoyl and fumaryl.

3. A sponge according to claim 2 wherein said diacyl is one or more of malonyl, oxalyl, methylmalonyl, ethylmalonyl, butylmalonyl, succinyl, glutaryl, adipoyl, pimeloyl, suberoyl, nonyldiacyl and sebacoyl.

4. A sponge according to claim 3 wherein said diacyl is one or more of malonyl, oxalyl, succinyl, glutaryl, adipoyl and pimeloyl.

5. A sponge according to claim 1 wherein said diacyl groups are present in said sponge in a proportion of from seven to one thousandth.

6. A sponge according to claim 1, wherein said bletilla polysaccharide has a molecular weight of 65000kDa to 150000kDa.

7. A sponge according to claim 1, wherein the porosity of the sponge is from 9% to 35%.

8. A sponge according to claim 1, wherein said sponge has a film structure and said film structures are interwoven to form a three-dimensional cellular structure; the film structure has a size of 10 μm to 70 μm.

9. A sponge according to claim 8, wherein the film has a thickness of 0.1 μm to 2 μm and the pores of the sponge have a diameter of 1 μm to 90 μm.