Method for producing chitosan hydrogel

Abstract

The present invention provides a method for producing a chitosan hydrogel comprising the following steps: (1) A step for adding urea to an acidic solution in which chitosan is dissolved, and (2) a step for subjecting the solution obtained in (1) to autoclave treatment.

Description

Method for producing chitosan hydrogel

[0001] The present invention relates to a novel method for producing chitosan hydrogels and chitosan hydrogels produced by said method.

[0002] Chitosan is a natural polysaccharide with antibacterial and wound-healing properties. Due to these properties, chitosan has been widely used as a wound dressing material. Wound dressings are required to promote wound healing. Hydrogel wound dressings that can provide a moist environment at the wound site have been reported to promote granulation tissue formation and re-epithelialization at the wound site, thereby accelerating wound healing. However, chitosan is poorly water-soluble at physiological pH and can only be dissolved in acidic solutions. Therefore, conventionally produced chitosan hydrogels have an acidic pH. Furthermore, the preparation of hydrogel-type chitosan wound dressings requires the use of chemical crosslinkers such as glutaraldehyde and formaldehyde during gel preparation. However, the residual chemical crosslinkers in the gel make them unsuitable for medical use due to their biotoxicity.

[0003] Recently, chitosan derivatives that exhibit water solubility at physiological pH have been reported, in which chitosan is modified with aldonic acids (Patent Documents 1 and 2). Patent Document 1 reports a method for preparing hydrogels of chitosan derivatives without using additives such as crosslinkers that are harmful to living organisms. Specifically, a non-biologically toxic hydrogel is prepared by freezing and thawing an aqueous solution containing aldonic acid-modified chitosan. However, the method taught in Patent Document 1 requires multiple steps, including preparation of aldonic acid-modified chitosan, freezing and thawing, and sterilization with ethanol. Furthermore, the method taught in Patent Document 2 also requires multiple steps, including preparation of aldonic acid-modified chitosan, pH adjustment of the aldonic acid-modified chitosan, and autoclaving.

[0004] Patent No. 5907489 International Publication No. 2023 / 167123

[0005] In light of this background, an object of the present invention is to provide a method for producing a chitosan hydrogel that is safe for use in living organisms in a simpler manner.

[0006] As a result of extensive research into the above-mentioned problems, the present inventors have found that a sterilized neutral chitosan hydrogel can be produced very simply by adding urea to an acidic solution containing chitosan and subjecting the solution to autoclaving. Based on this finding, further research has led to the completion of the present invention.

[0007] [Item 1] A method for producing a chitosan hydrogel, comprising the following steps: (1) adding urea to an acidic solution in which chitosan has been dissolved, and (2) autoclaving the solution obtained in (1). [Item 2] The method according to Item 1, wherein the acidic solution is a dilute aqueous hydrochloric acid solution or an aqueous acetic acid solution. [Item 3-1] The method according to Item 1 or 2, wherein the final concentration of urea in the acidic solution in (1) is 0.6 to 1.2% (w / v). [Item 3-2] The method according to Item 1 or 2, wherein the final concentration of urea in the acidic solution in (1) is 0.6 to 1.6% (w / v). [Item 4] A chitosan hydrogel containing urea. [Item 5] NH ３ and N.H. ４ ＋ Item 6. The chitosan hydrogel according to Item 4, further comprising either or both of the following: (A) NH per 1 L of chitosan hydrogel ３ and N.H. ４ ＋(Item 7) The chitosan hydrogel of Item 6, further comprising the following characteristics: (B) pH at 20°C: 6.5 to 8.0 and / or (C) ion / molecule concentration: 270 to 350 mmol / L. (Item 8) The chitosan hydrogel of any one of Items 4 to 7, which has been sterilized. (Item 9) The chitosan hydrogel of Item 8, wherein the sterilization is by autoclave sterilization. (Item 10) A wound dressing comprising or consisting of the chitosan hydrogel of any one of Items 4 to 9. (Item 11) A method for treating a wound in a subject, comprising covering a wound site in the subject with the chitosan hydrogel of any one of Items 4 to 9. (Item 12) Use of the chitosan hydrogel of any one of Items 4 to 9 in the manufacture of a wound dressing. (Item 13) The chitosan hydrogel of any one of Items 4 to 9 for use in treating a wound.

[0008] According to the present invention, a chitosan hydrogel that can be safely applied to living bodies can be prepared in an extremely simple manner without using any modifying agents or the like that are harmful to living bodies.

[0009] Figure 1 shows the appearance of a chitosan aqueous solution before autoclaving (left) and a chitosan hydrogel after autoclaving (right). Figure 2 shows the hemostatic properties of the chitosan hydrogel (Chitosan) of the present invention, gluconic acid-modified chitosan hydrogel (CG), and polyvinyl alcohol hydrogel (PVA) (n=3). Figure 3 shows the results of treating a rat wound model using the chitosan hydrogel (Chitosan hydrogel) or CG hydrogel (CG hydrogel) (Day 0 and Day 2: n=9, Day 4: n=7, Day 6: n=6, Day 8, Day 10, and Day 12: n=5).

[0010] The present invention will be described in detail below. The term "chitosan hydrogel" used in this specification refers to a chitosan that absorbs water, swells, and has no fluidity of its own. The pH value refers to a value measured at room temperature (20°C).

[0011] 1. Method for Producing Chitosan Hydrogel The present invention provides a method for producing a chitosan hydrogel (hereinafter, sometimes referred to as the "production method of the present invention"), which comprises the following steps: (1) adding urea to an acidic solution in which chitosan has been dissolved; and (2) subjecting the solution obtained in (1) to autoclaving.

[0012] The term "chitosan" used in the production method of the present invention refers to a deacetylated chitin. The main sugar units constituting chitin and chitosan are N-acetylglucosamine and glucosamine, respectively. Generally, chitin is characterized by a high N-acetylglucosamine content and is poorly soluble in acidic aqueous solutions, while chitosan is characterized by a high glucosamine content and is soluble in acidic aqueous solutions. The degree of deacetylation of the chitosan used in the production method of the present invention is not particularly limited as long as the desired effect is obtained, but it can usually be 50% or more, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. The upper limit of the degree of deacetylation can be, but is not limited to, 100% or less, 98% or less, 96% or less, 94% or less, 92% or less, 90% or less, 88% or less, 86% or less, 84% or less, or 82% or less. In one embodiment of the present invention, the degree of deacetylation of chitosan is usually 50 to 100%, and may be preferably 60 to 98%, 65 to 94%, or 70 to 90%, but is not limited thereto.

[0013] The chitosan used in the production method of the present invention may be either artificially modified or unmodified. Examples of artificially modified chitosan include, but are not limited to, aldonic acid-modified chitosan (e.g., gluconic acid-modified chitosan). Such artificially modified chitosan and unmodified chitosan can be prepared by methods known per se. Commercially available modified or unmodified chitosan can also be used. In a preferred embodiment, the chitosan used in the production method of the present invention may be unmodified chitosan.

[0014] In the production method of the present invention, any acidic aqueous solution may be used as the aqueous solution for dissolving chitosan as long as it produces the desired effect. Examples of the aqueous acidic solution that can be used in the production method of the present invention include, but are not limited to, a dilute aqueous hydrochloric acid solution and an aqueous acetic acid solution.

[0015] In the production method of the present invention, the pH of the acidic aqueous solution for dissolving chitosan is not particularly limited as long as it is within a range that allows chitosan to be dissolved. The pH of the acidic aqueous solution for dissolving chitosan is usually 1 or higher, preferably 1.5 or higher, 2 or higher, 2.5 or higher, 3 or higher, 3.5 or higher, 4 or higher, or 4.5 or higher, but is not limited to these. The upper limit of the pH may be 6.5 or lower, 6 or lower, 5.9 or lower, 5.8 or lower, 5.7 or lower, or 5.6 or lower, but is not limited to these. In one embodiment, the pH range of the acidic aqueous solution for dissolving chitosan includes, but is not limited to, 1 to 6, 2 to 6, 3 to 6, 4 to 6, or 5 to 6 (e.g., pH 5.55). In a preferred embodiment, the pH of the acidic aqueous solution for dissolving chitosan may be 5 to 6 (e.g., pH 5.55).

[0016] In the production method of the present invention, the concentration of chitosan dissolved in the acidic aqueous solution is also not particularly limited as long as the desired chitosan hydrogel can be produced. The concentration of chitosan dissolved in the acidic aqueous solution can be, for example, usually 1% or more, preferably 2% or more, but is not limited thereto. The upper limit can be usually 5% or less, preferably 3% or less, but is not limited thereto. In one embodiment, the concentration of chitosan dissolved in the acidic aqueous solution can be in the range of usually 1 to 5%, preferably 2 to 3%, but is not limited thereto.

[0017] In the manufacturing method of the present invention, urea (CO(NH) ２ ) ２ ) is added.

[0018] The urea used in the production method of the present invention may be commercially available. Furthermore, in the production method of the present invention, the amount of urea added to the acidic solution containing dissolved chitosan is not particularly limited as long as it allows the production of the desired chitosan hydrogel. The amount of urea to be added can be appropriately determined by those skilled in the art, taking into account the pH of the acidic solution containing dissolved chitosan and the amount of chitosan dissolved. In one embodiment, when the pH of the acidic solution before autoclaving is 5.55, the amount of urea in the acidic solution is typically at a final concentration of 0.6% (w / v) or more, preferably 0.65% (w / v) or more, 0.7% (w / v) or more, or 0.75% (w / v) or more, but is not limited thereto. The upper limit is typically 1.65% (w / v) or less, preferably 1.6% (w / v) or less, 1.55% (w / v) or less, or 1.5% (w / v) or less, but is not limited thereto. In one embodiment, the amount of urea in the acidic solution is generally 0.6 to 1.6% (w / v), preferably 0.6 to 1.5% (w / v), 0.6 to 1.4% (w / v), 0.6 to 1.3% (w / v), 0.6 to 1.2% (w / v), 0.7 to 1.1% (w / v), 0.7 to 1.0% (w / v), or 0.75 to 1.0% (w / v), which makes it possible to prepare a high-quality chitosan hydrogel that is less toxic to living organisms after autoclaving.

[0019] The acidic solution thus obtained, in which chitosan and urea have been added, will not hydrogel as it is. However, by subjecting the acidic solution to autoclaving, the following reactions (1) and (2) will proceed based on the added urea, resulting in the formation of OH. － Ions are generated, neutralizing the solution and allowing chitosan hydrogel to form.

[0020] (NH ２ ) ２ CO → 2NH ３ +CO ２ (1) NH ３ +H ２ O → NH ４ ＋ +OH － (2)

[0021] The autoclave conditions used in the production method of the present invention are not particularly limited as long as they allow the production of the desired chitosan hydrogel. Examples of autoclave conditions include, but are not limited to, a temperature of typically 105 to 135°C, preferably 115 to 130°C, and more preferably 118 to 123°C (e.g., 121±1°C). The pressure is typically, but is not limited to, 0.12 to 0.32 MPa, preferably 0.17 to 0.27 MPa, and more preferably 0.19 to 0.23 MPa (e.g., 0.21±0.1 MPa). The autoclave time is typically, but is not limited to, 10 to 300 minutes, preferably 20 to 240 minutes, and more preferably 20 to 120 minutes.

[0022] In one embodiment, the pH of the chitosan hydrogel produced by the production method of the present invention can be adjusted to a range of 6.5 to 7.6 in order to ensure safe application to living organisms. For the same purpose, the chitosan hydrogel can be produced so that the ion / molecule concentration (osmotic pressure) is in the range of 270 to 350 mmol / L. For the same purpose, the NH per 1 L of chitosan hydrogel can be adjusted to a range of 270 to 350 mmol / L. ３ and N.H. ４ ＋ The total amount (referred to herein as "NH ３ / NH ４ ＋ concentration) is 500 mmol or less (i.e., "NH ３ / NH ４ ＋ The chitosan hydrogel produced by the production method of the present invention can be produced so that its "NH ３ / NH ４ ＋The "NH concentration" is usually 500 mmol / L or less, and may be preferably 450 mmol / L or less, 400 mmol / L or less, 350 mmol / L or less, 300 mmol / L or less, 250 mmol / L or less, 200 mmol / L or less, 150 mmol / L or less, 130 mmol / L or less, or 105 mmol / L or less, but is not limited to these. The lower limit is usually 1 mmol / L or more, preferably 2 mmol / L or more, 3 mmol / L or more, 4 mmol / L or more, 5 mmol / L or more, 6 mmol / L or more, 7 mmol / L or more, 8 mmol / L or more, 9 mmol / L or more, or 10 mmol / L or more, but is not limited to these. In one aspect, NH ３ / NH ４ ＋ The concentration may typically be, but is not limited to, 1 to 500 mmol / L, preferably 1 to 450 mmol / L, 1 to 400 mmol / L, 1 to 350 mmol / L, 1 to 300 mmol / L, 1 to 250 mmol / L, 1 to 200 mmol / L, 1 to 150 mmol / L, 1 to 130 mmol / L, or 1 to 105 mmol / L. ３ / NH ４ ＋ The concentration can be in the range of, but is not limited to, 40 to 102 mmol / L.

[0023] 2. Chitosan Hydrogel The present invention also provides a chitosan hydrogel containing urea (hereinafter, sometimes referred to as "the gel of the present invention").

[0024] The gel of the present invention is a chitosan hydrogel produced by the above-mentioned production method of the present invention. In the production method of the present invention, urea is added to an acidic solution in which chitosan is dissolved, and even after autoclaving, a portion of the urea remains unreacted and is included in the chitosan hydrogel. In addition, as described above, NH ３ and NH ４ ＋ In one embodiment, the NH of the chitosan hydrogel is ３ / NH ４ ＋The concentration is usually 500 mmol / L or less, and may be preferably 450 mmol / L or less, 400 mmol / L or less, 350 mmol / L or less, 300 mmol / L or less, 250 mmol / L or less, 200 mmol / L or less, 150 mmol / L or less, 130 mmol / L or less, or 105 mmol / L or less, but is not limited thereto. The lower limit is usually 1 mmol / L or more, preferably 2 mmol / L or more, 3 mmol / L or more, 4 mmol / L or more, 5 mmol / L or more, 6 mmol / L or more, 7 mmol / L or more, 8 mmol / L or more, 9 mmol / L or more, or 10 mmol / L or more, but is not limited thereto. In one aspect, NH ３ / NH ４ ＋ The concentration may be, but is not limited to, typically 1 to 500 mmol / L, preferably 1 to 450 mmol / L, 1 to 400 mmol / L, 1 to 350 mmol / L, 1 to 300 mmol / L, 1 to 250 mmol / L, 1 to 200 mmol / L, 1 to 150 mmol / L, 1 to 130 mmol / L, or 1 to 105 mmol / L. ３ / NH ４ ＋ The concentration can be in the range of 40 to 102 mmol, but is not limited thereto. ３ / NH ４ ＋ When the "concentration" is in this range, the chitosan hydrogel can be applied to a living body very safely.

[0025] When the chitosan used in the gel of the present invention is unmodified, no chemical crosslinking agents, such as glutaraldehyde or formaldehyde, which may be harmful to living organisms, are used. Therefore, the produced chitosan hydrogel does not require a washing step, such as immersing it in physiological saline to remove the chemical crosslinking agent, which is preferable not only from the viewpoint of safety but also from the viewpoint of production cost and labor.

[0026] Furthermore, the gel of the present invention is sterilized by undergoing autoclave treatment during its manufacturing process, and therefore can be directly applied to a living body without sterilization using ethanol or the like.

[0027] In a preferred embodiment of the gel of the present invention, the gel of the present invention can be used alone or in combination with other pharmaceutically acceptable components as a wound dressing (sometimes referred to as the "wound dressing of the present invention"). The wound dressing of the present invention can also be referred to as the wound dressing composition of the present invention. The other pharmaceutically acceptable components that can be contained in the wound dressing of the present invention are not particularly limited and can be appropriately adjusted depending on the type of wound (e.g., surgical wound, burn, cut, gunshot wound, etc.). Examples of other pharmaceutically acceptable additional ingredients include, but are not limited to, moisturizers (e.g., glycerin, propylene glycol), anti-infective agents (e.g., antibacterial agents, antiviral agents, antifungal agents, antiparasitic agents), analgesics (e.g., anesthetics, nonsteroidal anti-inflammatory drugs (NSAIDs)), anti-inflammatory agents (e.g., steroids, NSAIDs, antihistamines), antiproliferative agents (e.g., glycerin), keratolytic agents (e.g., toll-like receptor 7 (TLR7) agonists, TLR2 agonists, TLR4 agonists), extracellular matrix modifying agents (e.g., proteinases, elastases, matriptase), intercellular junction modifying agents (e.g., adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3)), biological lubricants (e.g., glycerin), pigmentation modifying agents (e.g., depigmenting agents, pigmentation promoting agents), and combinations thereof.

[0028] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0029] Example 1 Preparation of Chitosan Hydrogels Using Urea 0.25 g of chitosan was dissolved in 10 mL of dilute hydrochloric acid to prepare a 2.5% (w / v) chitosan solution (initial pH: 5.55 (room temperature)). Urea was then dissolved in this chitosan solution to give final concentrations of 0.25% (w / v), 0.50% (w / v), 0.75% (w / v), 1.0% (w / v), 1.25% (w / v), or 1.50% (w / v). The six dilute hydrochloric acid solutions containing chitosan and urea thus prepared were designated Samples No. 1 to 6, respectively.

[0030] The prepared aqueous solutions Nos. 1 to 6 were autoclaved at 121°C and 2 atm (0.202 MPa) for 20 minutes. After autoclaving, the samples were analyzed for (1) pH, (2) urea decomposition rate, (3) presence or absence of gelation, (4) ion / molecule concentration, and (5) NH ３ / NH ４ ＋ The results are shown in Table 1. The appearance of the chitosan aqueous solution before autoclaving and the chitosan hydrogel after autoclaving are shown in Figure 1.

[0031]

[0032] As shown in Table 1, gelation was confirmed under conditions No. 3 to 6. In particular, under conditions No. 3 or 4, the ion / molecule concentration (osmotic pressure) and NH ３ / NH ４ ＋ It was shown that a chitosan hydrogel that is safe for use in the body can be obtained in terms of concentration.

[0033] In this example, the hemostatic properties of the chitosan hydrogel of the present invention were evaluated by a blood coagulation test. For comparison, gluconic acid-modified chitosan (CG) hydrogel and polyvinyl alcohol (PVA) hydrogel were used.

[0034] Chitosan was dissolved in dilute hydrochloric acid to a concentration of 2.5% (w / v) (pH 5.5). Urea was dissolved in the chitosan aqueous solution to a concentration of 1.0% (w / v). This solution was autoclaved (121°C, 20 min) to prepare chitosan hydrogels. Gluconic acid-modified chitosan (CG) was dissolved in dilute hydrochloric acid to a concentration of 2.0% (w / v) (pH 4.0). The solution was neutralized (pH 7.0) by adding 6 M NaOH solution dropwise while vigorously stirring. This neutral CG aqueous solution was autoclaved (121°C, 20 min) to prepare CG hydrogels. Polyvinyl alcohol (PVA) was dissolved in heated pure water to a concentration of 5% (w / v). This PVA aqueous solution was frozen at -30°C for 6 hours and then thawed at room temperature to prepare PVA hydrogels. These three hydrogels (1 mL) were flash-frozen in liquid nitrogen (-196°C) and vacuum-dried to form dry sponges. Blood was collected from the abdominal aorta of male Wistar rats (8 weeks old) under gas anesthesia with isoflurane. Whole blood was prepared by mixing blood with 109 mM sodium citrate aqueous solution at a 9:1 (v / v) ratio at 37°C to prevent blood coagulation. 50 μL of whole blood was dropped onto a dry sponge and allowed to absorb. 5 μL of 0.2 M calcium chloride aqueous solution was then added and incubated at 37°C for 5 minutes. Erythrocytes not trapped in the thrombus were hemolyzed in water. The concentration of hemoglobin released from hemolyzed erythrocytes into water was measured by measuring the absorbance at 540 nm. As a reference, the absorbance of pure water added directly to an equal volume of whole blood was measured. The blood coagulation index (BCI) was determined by the following equation:

[0035]

[0036] The results are shown in Figure 2. A higher blood coagulation index (BCI) indicates hemolysis of red blood cells not trapped in the hemolyzed thrombus, while a lower BCI indicates red blood cells trapped in the thrombus, i.e., coagulation. Compared to polyvinyl alcohol (PVA) hydrogel, which is the main material used in commercially available wound dressings, the chitosan hydrogel and gluconic acid-modified chitosan (CG) hydrogel of the present invention exhibited lower BCIs, demonstrating successful hemostasis. Chitosan, a positively charged polymer, is known to electrostatically attract negatively charged red blood cells and platelets. The high BCI of the chitosan hydrogel of the present invention is believed to be due to the immobilization of red blood cells and platelets through electrostatic interactions with chitosan, resulting in the formation of a thrombus. Furthermore, the CG hydrogel has been shown to have hemostatic properties equivalent to or superior to those of commercially available wound dressings (alginate), which are known to have excellent hemostatic properties. The chitosan hydrogel of the present invention exhibits hemostatic properties equivalent to those of the CG hydrogel, demonstrating its excellent hemostatic properties.

[0037] Example 3 Wound Treatment in a Rat Wound Model In this example, the chitosan hydrogel of the present invention was used to treat a rat wound model in which a circular full-thickness skin defect was created, demonstrating the usefulness of the chitosan hydrogel of the present invention as a wound dressing. CG hydrogel was used as a control.

[0038] Ten male Wistar rats were used in this experiment. While the rats were anesthetized under a triple-dose anesthesia (medetomidine hydrochloride, midazolam, and butorphal tartrate), the dorsal skin was disinfected with 70% ethanol and the back was shaved. Two 10 mm diameter circular full-thickness skin defects were created on the rats' dorsal skin using scissors. Each wound was covered with the chitosan hydrogel or CG hydrogel of the present invention. An adhesive waterproof film (BFR, Nichiban) was then applied as a secondary dressing. The wounds were then securely fixed with elastic bandages (Skinagate Gachit, Nichiban). The shape of the wound was traced on a transparent film every other day. The wound area was determined from the trace using image analysis software (ImageJ). Each rat was euthanized by an overdose of isoflurane every four days, and the entire wound, including the adjacent intact skin, was excised for histological analysis.

[0039] The results are shown in Figure 3, which shows the progress of wound area after treatment with the chitosan hydrogel or CG hydrogel of the present invention. The chitosan hydrogel and CG hydrogel demonstrated comparable wound healing processes. Four days after wounding, the wounds treated with the chitosan hydrogel and CG hydrogel of the present invention had contracted to 38.6 and 53.6% of their initial wound area, respectively. Both wounds had fully epithelialized by 12 days after wounding. These results demonstrate that the hydrogel composed of pure chitosan prepared by urea hydrolysis with autoclave sterilization of the present invention is as effective as the CG hydrogel, which has already been shown to have excellent wound healing promoting effects. Therefore, it was demonstrated that the chitosan hydrogel of the present invention can be used as a highly biosafe wound dressing that can replace hydrogels composed of chitosan derivatives, such as CG hydrogel.

[0040] According to the production method of the present invention, a chitosan hydrogel that can be safely applied to living bodies can be produced more easily, and therefore the present invention is extremely useful in the medical field.

[0041] This application is based on patent application No. 2024-067578 filed in Japan (filing date: April 18, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing chitosan hydrogel, comprising the steps of: (1) adding urea to an acidic solution in which chitosan has been dissolved; and (2) subjecting the solution obtained in (1) to autoclaving.

2. The method of claim 1, wherein the acidic solution is a dilute aqueous hydrochloric acid solution or an aqueous acetic acid solution.

3. The method according to claim 1 or 2, wherein the final concentration of urea in the acidic solution in (1) is 0.6 to 1.6% (w / v).

4. Chitosan hydrogel containing urea.

5. NH ３ and N.H. ４ ＋ The chitosan hydrogel of claim 4, further comprising either or both of:

6. The chitosan hydrogel of claim 5, further characterized by: (A) NH per liter of chitosan hydrogel ３ and N.H. ４ ＋ Total amount: 40 to 102 mmol.

7. The chitosan hydrogel according to claim 6, further characterized by: (B) a pH at 20°C of 6.5 to 8.0; and / or (C) an ion / molecule concentration of 270 to 350 mmol / L.

8. The chitosan hydrogel according to any one of claims 4 to 7, which is sterilized.

9. The chitosan hydrogel according to claim 8, wherein the sterilization is by autoclave sterilization.

10. A wound dressing comprising or consisting of the chitosan hydrogel according to any one of claims 4 to 7.

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