Polysaccharide-based hydrogel as well as preparation method and application thereof

Abstract

The invention relates to polysaccharide-based hydrogel as well as a preparation method and application thereof. The polysaccharide-based hydrogel is prepared from the following raw materials: hydroformylation-modified ortho-hydroxyl-containing polysaccharide and an extracellular matrix enzyme digestive juice, the hydroformylation-modified ortho-hydroxyl-containing polysaccharide is obtained by oxidizing hydroxyl in the ortho-hydroxyl-containing polysaccharide into aldehyde groups. The aldehyde group of the hydroformylated modified ortho-hydroxyl-containing polysaccharide in the hydrogel is cross-linked with the amino group of the acellular extracellular matrix enzyme digestive juice, and the aldolization reaction of the hydroformylated modified ortho-hydroxyl-containing polysaccharide is carried out to form a double-cross-linked hydrogel system; the obtained gel has excellent mechanical properties, good biological safety, certain self-repairing performance and relatively high anti-inflammatory, antibacterial and antioxidant properties, and can be applied to skin or mucous membrane tissue wound repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogels, and particularly relates to a polysaccharide-based hydrogel dressing, a preparation method thereof, and an application thereof. Background Art

[0002] Polysaccharide-based hydrogels have many unique physical and chemical properties, such as biocompatibility, biodegradability, and no immune response. Therefore, polysaccharides are widely used in various biomedical applications. There are a large number of functional groups available for modification in the polysaccharide structural unit, and polysaccharide-based hydrogels can be constructed through physical crosslinking, chemical crosslinking, or enzymatic crosslinking. Polysaccharides have many advantages such as relatively low price, wide sources of raw materials, easy industrial extraction, and good biological activity. They have a variety of physiological activities, including antiviral, antioxidant, and anti-inflammatory activities, etc., and have broad application prospects in biomedicine.

[0003] Decellularized extracellular matrix refers to a biomaterial formed from human or animal organs / tissues by removing immunogenic cellular components through decellularization technology. The decellularized extracellular matrix is mainly composed of extracellular matrix, which contains extracellular macromolecules such as collagen, elastin, fibronectin, laminin, and matricellular proteins. The hydrogel prepared by the traditional method of adjusting to neutral and gelling after enzymatic digestion of the single decellularized extracellular matrix has disadvantages such as poor mechanical properties and no self-healing ability when used as a dressing, which limits the practical application of the decellularized extracellular matrix hydrogel. Summary of the Invention

[0004] Aiming at the deficiencies of the above-mentioned existing technologies, the present invention provides a polysaccharide-based hydrogel, a preparation method thereof, and an application thereof. By aldehyde-modifying the polysaccharide containing ortho-hydroxy groups and crosslinking with the decellularized extracellular matrix enzymatic digestion solution, a polysaccharide-based hydrogel with excellent mechanical properties and wound healing promoting ability is obtained. Through the design of the raw materials for preparing the hydrogel, the obtained hydrogel has good biocompatibility, analgesia, adhesiveness, antioxidant property, self-healing ability, and excellent mechanical properties, and can better adapt to various wounds and usage environments.

[0005] The specific technical solution of the present invention is as follows: In the first aspect of the present invention, a polysaccharide-based hydrogel is provided, and its raw materials include: aldehyde-modified polysaccharide containing ortho-hydroxy groups, decellularized extracellular matrix enzymatic digestion solution; the aldehyde-modified polysaccharide containing ortho-hydroxy groups is obtained by oxidizing the hydroxy groups in the polysaccharide containing ortho-hydroxy groups to aldehyde groups.

[0006] The aldehyde-modified polysaccharide containing ortho-hydroxy groups can react with the acellular extracellular matrix enzyme digestion solution through a Schiff base reaction, that is, the aldehyde groups of the aldehyde-modified polysaccharide containing ortho-hydroxy groups crosslink with the amino groups of the acellular extracellular matrix enzyme digestion solution. At the same time, the aldehyde-modified polysaccharide containing ortho-hydroxy groups itself can also undergo an acetal reaction, thereby forming a double-crosslinked hydrogel system. The obtained gel has excellent mechanical properties, good biological safety, certain self-healing properties, and high anti-inflammatory, antibacterial, and antioxidant properties.

[0007] In one implementation of the present invention, in the aldehyde-modified polysaccharide containing ortho-hydroxy groups, the degree of oxidation of the hydroxyl groups is 10% to 50%. For example, it can be 10%, 20%, 30%, 40%, 50%, etc.

[0008] In one implementation of the present invention, the polysaccharide containing ortho-hydroxy groups can be at least one of fenugreek gum, guar gum, locust bean gum, gellan gum, sodium alginate, agarose, and konjac glucomannan.

[0009] It should be noted that the aldehyde groups of the aldehyde-modified polysaccharide in the hydrogel of the present application invention are formed by the oxidation of the ortho-hydroxy groups of the polysaccharide. Therefore, in principle, as long as it is a polysaccharide containing ortho-hydroxy groups, it can be applicable to the present application, not limited to fenugreek gum, guar gum, locust bean gum, gellan gum, sodium alginate, agarose, and konjac glucomannan.

[0010] In one implementation of the present invention, the aldehyde-modified polysaccharide containing ortho-hydroxy groups is obtained through an oxidation reaction, reaction termination, and purification steps from the polysaccharide containing ortho-hydroxy groups; the oxidation reaction includes adding an oxidant to react in a solution of the polysaccharide containing hydroxyl groups; the reaction termination includes adding an oxidation termination reagent to terminate the reaction after the reaction with the oxidant is completed; the purification step includes, after oxidation termination, using dialysis and freeze-drying techniques to obtain the purified aldehyde-modified polysaccharide, that is, the aldehyde-modified polysaccharide containing ortho-hydroxy groups of the present application.

[0011] In one implementation of the present invention, the mass ratio of the polysaccharide and the oxidant in the oxidation reaction is 1:(0.01 - 1), and the oxidation reaction time is 1 - 72 h.

[0012] In one implementation of the present invention, the oxidation termination reagent is ethylene glycol, and the reaction termination time is 15 - 60 min.

[0013] In one implementation of the present invention, the dialysis membrane specification used for dialysis is 3500 kDa.

[0014] In one implementation of the present invention, the acellular extracellular matrix enzyme digestion solution is prepared through the following steps: (1) Mix and stir the animal skin tissue and the degreasing solution to obtain the degreased tissue; (2) Mix and stir the degreased tissue with a PBS mixed solution of Triton X-100 and ethylenediaminetetraacetic acid to obtain a pretreated animal tissue matrix; (3) Mix the pretreated animal tissue matrix and a DNA enzyme solution for a DNA enzymatic digestion reaction to obtain an acellular extracellular matrix of animal tissue with DNA removed; (4) Freeze-dry the acellular extracellular matrix of animal tissue with DNA removed, pre-cool it in liquid nitrogen, then grind it into powder and sieve it to obtain acellular extracellular matrix powder; (5) Mix the acellular extracellular matrix powder and a protease solution for an enzymatic digestion reaction to obtain the acellular extracellular matrix enzymatic digestion solution.

[0015] In one implementation of the present invention, in step (1), the animal skin tissue includes one or more of pig skin, cow skin, and fish skin.

[0016] In one implementation of the present invention, in step (2), the mass percentage content of Triton X-100 in the PBS mixed solution is 0.1~1 wt%, and the mass percentage content of ethylenediaminetetraacetic acid in the PBS mixed solution is 0.05~1 wt%; the ratio of the mass of the degreased tissue to the volume of the PBS mixed solution is (0.5~3) g : (100~500) mL.

[0017] In one implementation of the present invention, in step (3), the concentration of the DNA enzyme solution is 10~100 U / mL, and the ratio of the mass of the pretreated animal tissue matrix to the volume of the DNA enzyme solution is (0.2~2) g : (100~500) mL.

[0018] In one implementation of the present invention, in step (4), the protease solution is one or more of pepsin, papain, α-amylase, and collagenase; the mass concentration of the protease solution is 0.1~2 mg / mL, and the enzymatic digestion reaction time is 1~36 h; the ratio of the volume of the acellular extracellular matrix of animal tissue to the protease solution is (0.2~1) g : (50~1000) mL.

[0019] In one implementation of the present invention, the hydrogel contains an aldehyde-modified ortho-hydroxy polysaccharide and an acellular extracellular matrix enzymatic digestion solution.

[0020] In one implementation of the present invention, in the hydrogel, by mass ratio, aldehyde-modified ortho-hydroxy polysaccharide : acellular extracellular matrix in the acellular extracellular matrix enzymatic digestion solution = (0.1~10) : (0.2~2).

[0021] In a second aspect of the present invention, there is also provided a method for preparing the polysaccharide-based hydrogel, comprising: mixing and stirring an aldehyde-modified polysaccharide solution containing ortho-hydroxy groups and a decellularized extracellular matrix enzyme digestion solution, adjusting to neutral with a sodium hydroxide solution, and then standing to form a gel.

[0022] In one implementation of the present invention, in the preparation method, the mass percentage of the solute in the aldehyde-modified polysaccharide solution containing ortho-hydroxy groups is 1-5%; the mass percentage of the solute in the decellularized extracellular matrix enzyme digestion solution is 1-5%.

[0023] The present invention has found through research that selecting the mass percentage of the solute in the solution within the above range is beneficial to the gel-forming reaction.

[0024] In one implementation of the present invention, the solvents of the aldehyde-modified polysaccharide solution containing ortho-hydroxy groups and the decellularized extracellular matrix enzyme digestion solution are preferably water.

[0025] In one implementation of the present invention, the mixing and stirring are uniformly carried out by a rapid vortex method until no layering is observed with the naked eye.

[0026] In one implementation of the present invention, the standing time for gel formation is preferably 10-30 min.

[0027] In a third aspect of the present invention, there is also provided the application of the polysaccharide-based hydrogel in the preparation of a wound repair dressing for skin or mucosal tissues.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: The decellularized extracellular matrix has excellent biochemical properties and can provide necessary biological signals for cell physiological activities. However, when used alone, its mechanical properties and adhesion are extremely poor. The cross-linking of the aldehyde-modified polysaccharide containing ortho-hydroxy groups with the decellularized extracellular matrix can significantly improve the mechanical properties of the gel. Specifically, it has good tensile strength, adhesion, and self-healing properties. At the same time, the hydrogel provided by the present invention has an antibacterial effect, and also has the effects of hemostasis, pain relief, anti-inflammatory, and promoting wound healing. Therefore, it can be widely applied to damaged skin or mucosal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is an optical photograph of the hydrogel in the embodiment of the present invention; Figure 2 is a graph showing the results of the cytocompatibility of the hydrogel in the embodiment of the present invention; Figure 3 is a graph showing the results of the blood compatibility of the hydrogel in the embodiment of the present invention; Figure 4 is a graph showing the results of in vivo wound healing of the hydrogel in the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides a polysaccharide-based composite hydrogel with adhesiveness and antibacterial properties, which comprises an aldehyde-modified polysaccharide containing ortho-hydroxy groups and an enzyme digestion solution of acellular extracellular matrix of animal tissues. The aldehyde-modified polysaccharide containing ortho-hydroxy groups is obtained by oxidizing the hydroxy groups in the polysaccharide containing ortho-hydroxy groups into aldehyde groups.

[0031] The hydrogel provided by the present invention comprises an aldehyde-modified polysaccharide containing ortho-hydroxy groups. The aldehyde-modified polysaccharide containing ortho-hydroxy groups is obtained by oxidizing the hydroxy groups in the polysaccharide containing ortho-hydroxy groups into aldehyde groups.

[0032] In the present invention, the oxidation degree of the aldehyde-modified polysaccharide containing ortho-hydroxy groups is preferably 10-50%.

[0033] In the present invention, the preparation method of the aldehyde-modified polysaccharide containing ortho-hydroxy groups preferably comprises the following steps: dissolving the polysaccharide in water to prepare a uniform solution with a concentration of 0.5-5 wt%, adding an oxidant to the oxidant concentration of 0.01-1 wt% in a light-shielded environment, and continuously stirring and oxidizing for 8-40 h, then adding a terminator to the terminator concentration of 0.01-1 wt% and continuously stirring for 20-60 min to terminate the reaction. Then, dialysis is carried out for 72 h using a dialysis bag, and freeze-drying is performed to obtain the aldehyde-modified polysaccharide.

[0034] In the present invention, the polysaccharide is preferably guar gum, and the concentration is preferably 1-3 wt%; the oxidant is preferably sodium periodate, and the final concentration in the reaction system is preferably 0.1-0.5 wt%; the terminator is preferably ethylene glycol, and the concentration is preferably 0.1-0.5 wt%.

[0035] The hydrogel provided by the present invention comprises an enzyme digestion solution of acellular extracellular matrix of animal tissues.

[0036] In the present invention, the preparation method of the enzyme digestion solution of acellular extracellular matrix of animal tissues preferably comprises the following steps: Mixing and stirring animal skin tissue and a degreasing solution to obtain degreased tissue; Mixing and stirring the degreased tissue with a PBS mixed solution of Triton X-100 and ethylenediaminetetraacetic acid to obtain a pretreated animal tissue matrix; Mixing the pretreated animal tissue matrix and a DNA enzyme solution to carry out a DNA enzymatic hydrolysis reaction to obtain an acellular extracellular matrix of animal tissues with DNA removed; Freeze-drying the acellular extracellular matrix of animal tissues with DNA removed, pre-cooling it in liquid nitrogen, then grinding it into powder and sieving it to obtain acellular extracellular matrix powder; Mixing the acellular extracellular matrix powder and an enzyme solution to carry out an enzymatic hydrolysis reaction to obtain the enzyme digestion solution of acellular extracellular matrix of animal tissues.

[0037] In the present invention, animal skin tissue and a degreasing solution are mixed and continuously stirred for a period of time to obtain degreased tissue. In the present invention, the animal skin preferably includes one or more of pig skin, cow skin, and fish skin, and the degreasing solution is an organic solvent, preferably an equimolar mixture solution of ethyl acetate and methanol.

[0038] In the present invention, the degreased tissue is placed in a mixed solution of Triton X-100, ethylenediaminetetraacetic acid, and PBS, and mixed for a period of time to obtain a pretreated animal tissue matrix. In the present invention, the mass percentage content of Triton X-100 in the PBS mixed solution is preferably 0.1-1 wt%. In the present invention, the mass percentage content of ethylenediaminetetraacetic acid in the PBS mixed solution is preferably 0.05-1 wt%. The ratio of the mass of the degreased tissue to the volume of the PBS mixed solution is preferably (0.5-3) g:(100-500) mL. In the present invention, the temperature of the stirring is preferably 37 °C, and the time of the stirring is preferably 6-72 h.

[0039] After obtaining the pretreated animal tissue matrix, in the present invention, the pretreated animal tissue matrix and a DNA enzyme solution are mixed for a DNA enzymatic digestion reaction to obtain an acellular extracellular matrix of animal tissue.

[0040] In the present invention, the concentration of the DNA enzyme solution is preferably 10-100 U / mL. The ratio of the mass of the pretreated animal tissue matrix to the volume of the DNA enzyme solution is preferably (0.2-2) g:(100-500) mL. In the present invention, the temperature of the DNA enzymatic digestion reaction is preferably 37 °C, and the time of the DNA enzymatic digestion reaction is preferably 1-8 h.

[0041] After obtaining the acellular extracellular matrix, in the present invention, the acellular extracellular matrix is freeze-dried, pre-cooled in liquid nitrogen, and then ground into a powder and sieved to obtain an acellular extracellular matrix powder.

[0042] After obtaining the acellular extracellular matrix powder, in the present invention, the acellular extracellular matrix powder and a protease solution are mixed for a proteolytic reaction to obtain the acellular extracellular matrix enzymatic digestion solution of animal tissue.

[0043] In the present invention, the protease solution is one or more of pepsin, papain, α-amylase, and collagenase, preferably a pepsin solution. The mass concentration of the protease solution is preferably 0.1-2 mg / mL, and the enzymatic reaction time is 1-36 h. The volume ratio of the acellular extracellular matrix of animal tissue to the protease solution is preferably (0.2-1) g:(50-1000) mL. The temperature of the proteolytic reaction is preferably 37 °C.

[0044] The present invention provides a method for preparing the hydrogel described in the above technical solution, comprising the following steps: Mix the aldehyde-modified polysaccharide with ortho-hydroxy groups and the animal acellular extracellular matrix evenly, and adjust to neutral with a sodium hydroxide solution to obtain a hydrogel. In the present invention, the mixing is preferably carried out at 37 °C.

[0045] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Unless otherwise specified, the reagents, methods, and equipment used in the implementation of the present invention are conventional reagents, methods, and equipment in the technical field.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0048] Example 1 This example provides a hydrogel, and its preparation method is as follows: 1) Preparation of acellular extracellular matrix enzyme digestion solution: Cut fresh pig skin into small pieces of 1 cm × 1 cm in size, and wash it repeatedly; place the cut small pieces of pig skin tissue in a blue-capped bottle, add a solution with a volume ratio of ethyl acetate to methanol of 1:1 to completely immerse the pig skin tissue, and stir for a period of time to obtain defatted pig skin tissue; At 37 °C, add a mixed solution of PBS containing 0.5 wt% of Triton X-100 and 0.2 wt% of ethylenediaminetetraacetic acid to the defatted pig skin tissue, stir continuously for 8 h, filter, and wash the solid matter with water to obtain a pretreated animal tissue matrix; At 37 °C, add a DNA enzyme solution of 50 U / mL to the pretreated animal tissue matrix, where the mass ratio of the pretreated animal tissue matrix to the volume of the DNA enzyme solution is 1 g:200 mL, stir continuously for 8 h, filter, and wash the solid matter with water to obtain an acellular extracellular matrix; Freeze-dry the acellular extracellular matrix, pre-cool it in liquid nitrogen, grind it into particles with a grinder, and pass through a 100-mesh sieve to obtain acellular extracellular matrix powder; Add the acellular extracellular matrix powder to a pepsin solution of 0.5 mg / mL for treatment, where the mass ratio of the acellular extracellular matrix powder to the volume of the protease solution is 1 g:200 mL, to obtain a pig skin acellular extracellular matrix enzyme digestion solution.

[0049] 2) Preparation of aldehyde-modified polysaccharide with ortho-hydroxy groups: Weigh 5 g of guar gum powder, stir and dissolve the raw material powder in 250 mL of water to prepare a polysaccharide solution with a concentration of 2%.

[0050] Weigh 1 g of sodium periodate as an oxidant and add it to the polysaccharide solution. Stir magnetically at room temperature in the dark for 12 h.

[0051] After the reaction is completed, pipette 2 mL of ethylene glycol as an oxidation termination reagent into the reaction vessel and terminate the reaction under magnetic stirring for 40 min.

[0052] After the reaction is terminated, transfer the reaction solution into a dialysis bag with a molecular weight cut-off of 3500 kDa and dialyze it with ultrapure water for 3 days, changing the water 2 times a day.

[0053] After dialysis, freeze it at -20 °C for 24 h. Then freeze-dry it in a freeze dryer to obtain oxidized guar gum. The oxidation degree of the oxidized guar gum prepared in this example is determined by the hydroxylamine hydrochloride titration method, and the oxidation degree is 15.27%.

[0054] 3) Preparation of hydrogel: Weigh 0.1 g of the oxidized guar gum obtained in step 2) and dissolve it in 2 mL of water to obtain an oxidized guar gum solution with a concentration of 5%. At 37 °C, mix the oxidized guar gum solution with the acellular extracellular matrix enzyme digestion solution obtained in step 1) at a volume ratio of 1:1. Use a rapid vortex method to mix until no layering is observed visually, and let it stand for 10 min to form a gel to obtain a hydrogel.

[0055] Example 2 The hydrogel provided by the embodiment of the present invention is the same as that in Example 1 except that: in the preparation process of 3) the hydrogel, the addition volumes of the aldehyde-modified polysaccharide containing ortho-hydroxyl groups and the acellular extracellular matrix enzyme digestion solution are changed, while keeping their total volume unchanged, and the volume ratio of the aldehyde-modified polysaccharide containing ortho-hydroxyl groups to the acellular extracellular matrix enzyme digestion solution is 2:1.

[0056] Example 3 The hydrogel provided in this example is the same as that in Example 1 except that: in the preparation process of 3) the hydrogel, weigh 0.1 g of the oxidized guar gum obtained in step 2) and dissolve it in 1.5 mL of water, and the volume of the acellular extracellular matrix enzyme digestion solution is 0.5 mL.

[0057] Example 4 The hydrogel provided in this example is the same as that in Example 1 except that the guar gum in step 2 is changed to konjac glucomannan.

[0058] Example 5 The hydrogel provided in this example is the same as that in Example 1 except that: the guar gum in step 2) is changed to sodium alginate.

[0059] Comparative Example 1 This example provides a hydrogel, which is different from that of Example 1 in that no guar gum oxide solution is added.

[0060] Comparative Example 2 This example provides a hydrogel, which is different from that of Example 1 in that no acellular extracellular matrix enzyme digestion solution is added.

[0061] Effect verification: 1. Comparison of adhesion performance The hydrogel of Example 1, the hydrogels of Comparative Example 1 and Comparative Example 2 were adhered to the same piece of pigskin. The pigskin was straightened horizontally and the pigskin and the hydrogel faced downwards. The falling time of the hydrogel was observed by timing. The final result was that the hydrogel of Example 1 fell off after 5 hours of adhesion, the hydrogel of Comparative Example 1 could not adhere, and the hydrogel of Comparative Example 2 fell off after 3 hours of adhesion. The results show that the introduction of dialdehyde guar gum can endow the acellular extracellular matrix with good adhesion performance.

[0062] 2. Comparison of self-healing performance The hydrogel of Example 1, the hydrogels of Comparative Example 1 and Comparative Example 2 were respectively cut in half, and then pieced together. After waiting for a period of time, it was observed whether the two hydrogels recovered into a whole. The final result was that the hydrogel of Example 1 self-healed within 20 minutes, the hydrogel of Comparative Example 2 self-healed within 25 minutes, and the hydrogel of Comparative Example 1 could not self-heal. The results show that the introduction of dialdehyde guar gum can endow the acellular extracellular matrix with good self-repair performance.

[0063] 3. Comparison of gelation speed By timing and comparing the gelation times of the hydrogels prepared in Examples 1-5 and Comparative Examples 1-2, the results are shown in Table 1. The results show that the hydrogels of each example and the hydrogels of each comparative example can form gels, but both the type of polysaccharide and the volume ratio of the acellular extracellular matrix enzyme digestion solution to the volume of the polysaccharide solution have an impact on the gelation speed of the hydrogel. In particular, the hydrogel of Comparative Example 1 forms a gel only by self-assembly, with small steric hindrance of itself and a relatively fast gelation speed. The hydrogel of Comparative Example 2 forms a gel only by self-acetalization reaction, lacking the Schiff base reaction with the acellular extracellular matrix enzyme digestion solution, and has a slower gelation speed.

[0064] Table 1 Summary of hydrogel gelation times

[0065] 4. Biocompatibility test The freeze-dried hydrogel dressing of Example 1 was extracted with high-glucose complete medium at 37 °C for 24 h. After the extraction was completed, the concentration of the extract was diluted to 1 mg / mL, 0.1 mg / mL, and 0.01 mg / mL. Mouse fibroblasts NIH3T3 were inoculated on a culture plate containing complete medium and cultured in a cell incubator at 37 °C and a carbon dioxide concentration of 5% for 24 h. Then, the complete medium was discarded and replaced with the hydrogel extract, and the cells were cultured in a cell incubator at 37 °C and a carbon dioxide concentration of 5% for 24 h. The cells were treated with CCK-8 reagent, and the absorbance at a wavelength of 450 nm was measured with an enzyme-labeled instrument. The cell viability was calculated based on the absorbance values of each well.

[0066] The calculation formula is: Cell activity (%) = (A1 - A c / A0 - A c ) × 100%. Where A1 is the absorbance value of the experimental group, A0 is the absorbance value of the control group, and A c is the absorbance of the blank group.

[0067] Figure 2 The results showed that the cell viability of the co-culture of the hydrogel and cells measured according to the above method was above 85%, with good cell compatibility, meeting the cell biosafety requirements of GB / T 16886.

[0068] 5. Blood compatibility experiment of the hydrogel The hydrogel dressing of Example 1 was placed in a 2 mL centrifuge tube, and diluted red blood cells were added. Red blood cells treated with PBS and ultrapure water were used as the negative group and the positive group. After static incubation at 37 °C for 1 h, the supernatant was removed by centrifugation, and the absorbance at 540 nm was measured using an enzyme-labeled instrument. The calculation method of the hemolysis rate is as follows: HR (%) = (OD gel - OD blank ) / (OD control - OD blank ) × 100% OD gel : The absorbance at 540 nm of the supernatant of red blood cells treated with the hydrogel OD control : The absorbance at 540 nm of the supernatant of red blood cells treated with ultrapure water OD blank : The absorbance at 540 nm of the supernatant of red blood cells treated with PBS The test results are as Figure 3 shown. The hydrogel of Example 1 has good blood compatibility, with a hemolysis rate of less than 5%, meeting the blood biosafety requirements of GB / T 16886.

[0069] 6. Experiment on the Wound Healing Promoting Ability of Hydrogel The back hair of BALB / c mice was shaved and a wound was created. 20 μL of a Staphylococcus aureus suspension at a concentration of 1×10 8 CFU / mL was dropped onto the wound area. The hydrogel dressing substrate and the hydrogel dressing of Example 1 were completely coated on the wound surface; mice without adding hydrogel were used as the control group. Finally, a layer of 3M™ Tegaderm™ transparent dressing was used to protect the wound surface in all groups. After the anesthesia recovery, the animals were monitored for any discomfort and provided with water and feed. The hydrogel dressing was changed daily and the body weight was measured.

[0070] The test results are as Figure 4 shown. The hydrogel dressing has an obvious antibacterial effect compared with the 3M™ Tegaderm™ transparent dressing group, and can significantly promote wound healing at the same time.

[0071] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A polysaccharide-based hydrogel, characterized in that: The raw materials include: aldehyde-modified polysaccharide containing ortho-hydroxyl groups and decellularized extracellular matrix enzyme digestion solution; the aldehyde-modified polysaccharide containing ortho-hydroxyl groups is obtained by oxidizing the hydroxyl groups in the polysaccharide containing ortho-hydroxyl groups into aldehyde groups.

2. The polysaccharide-based hydrogel according to claim 1, characterized in that: According to the mass ratio, the aldehyde-modified polysaccharide containing ortho-hydroxyl groups: the decellularized extracellular matrix in the decellularized extracellular matrix enzyme digestion solution = (0.1~10): (0.2~2).

3. The polysaccharide-based hydrogel according to claim 1, characterized in that: In the aldehyde-modified polysaccharide containing ortho-hydroxyl groups, the degree of oxidation of hydroxyl groups is 10-50%.

4. The polysaccharide-based hydrogel according to claim 1, characterized in that The decellularized extracellular matrix enzyme digestion solution is prepared by the following steps: (1) mixing and stirring the animal skin tissue and the defatting solution to obtain defatted tissue; (2) mixing and stirring the defatted tissue with a mixed solution of Triton X-100 and ethylenediaminetetraacetic acid in PBS to obtain a pretreated animal tissue matrix; (3) mixing the pretreated animal tissue matrix and the DNA enzyme solution to perform a DNA enzymatic hydrolysis reaction to obtain an animal tissue decellularized extracellular matrix from which DNA has been removed; (4) freeze-drying the DNA-removed animal tissue decellularized extracellular matrix, precooling it in liquid nitrogen, grinding it into powder, and sieving it to obtain decellularized extracellular matrix powder; (5) The decellularized extracellular matrix powder and the protease solution are mixed to perform an enzymatic hydrolysis reaction to obtain the decellularized extracellular matrix enzyme digestion solution.

5. The polysaccharide-based hydrogel according to claim 4, characterized in that: In step (1), the animal skin tissue includes one or more of pig skin, cow skin and fish skin.

6. The polysaccharide-based hydrogel according to claim 4, characterized in that: In step (2), the mass percentage of Triton X-100 in the PBS mixed solution is 0.1-1wt%, and the mass percentage of ethylenediaminetetraacetic acid in the PBS mixed solution is 0.05-1wt%; the ratio of the mass of the defatted tissue to the volume of the PBS mixed solution is (0.5-3) g: (100-500) mL.

7. The polysaccharide-based hydrogel according to claim 4, characterized in that: In step (3), the concentration of the DNA enzyme solution is 10-100 U / mL, and the ratio of the mass of the pretreated animal tissue matrix to the volume of the DNA enzyme solution is (0.2-2) g: (100-500) mL.

8. The polysaccharide-based hydrogel according to claim 4, characterized in that: In step (4), the protease solution is one or more of pepsin, papain, α-amylase and collagenase; the mass concentration of the protease solution is 0.1-2 mg / mL, and the enzymatic reaction time is 1-36 h; the volume ratio of the animal tissue decellularized extracellular matrix to the protease solution is (0.2-1) g: (50-1000) mL.

9. The method for preparing a polysaccharide-based hydrogel according to any one of claims 1 to 8, characterized in that: include: The polysaccharide solution containing aldehyde-modified ortho-hydroxyl groups and the decellularized extracellular matrix enzyme digestion solution are mixed and stirred, and then allowed to stand to form a gel; Preferably, the mass percentage of the solute in the hydroformylation-modified polysaccharide containing ortho-hydroxyl groups is 1-5%; the mass percentage of the solute in the decellularized extracellular matrix enzyme digestion solution is 1-5%.

10. Use of the polysaccharide-based hydrogel according to any one of claims 1 to 8 or the polysaccharide-based hydrogel prepared by the preparation method according to claim 9 in preparing a wound repair dressing for skin or mucosal tissue.

Images