Highly water-absorbing hyaluronic acid porous sponge-like hydrogel and method for preparing the same

Abstract

The application provides a high-water-absorption hyaluronic acid porous sponge-like hydrogel and a preparation method thereof, and belongs to the field of biomedical technology. The hyaluronic acid and its salt are protonated and subjected to freeze-thaw treatment to obtain a physically cross-linked hyaluronic acid porous hydrogel, then compression dehydration treatment is carried out under a specific strain (greater than or equal to the strain corresponding to the yield point, less than the critical strain at which the hydrogel is damaged in shape) to obtain the high-water-absorption hyaluronic acid porous sponge-like hydrogel with high swelling and high tensile properties. The preparation process of the high-water-absorption hyaluronic acid porous sponge-like hydrogel is simple, no chemical cross-linking agent is added, the biological safety is good, the hydrogel has the dual characteristics of a porous sponge and a hydrogel, has the high water content of the hydrogel and the high liquid absorption of the porous sponge, and has the characteristics of high tensile property and flexibility, and can be used in the fields of tissue filling, wound dressing, liquid absorption material and hemostatic material and the like in the biomedical field.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to a high water-absorbing hyaluronic acid porous sponge-like hydrogel and a preparation method thereof. BACKGROUND

[0002] Hyaluronic acid hydrogel has excellent moisturizing and lubricating properties, can carry a large amount of water to form a stable three-dimensional structure, and is suitable for skin repair, joint lubrication, eye surgery, etc. Hyaluronic acid hydrogel has good biocompatibility and degradability, can be absorbed and metabolized by the body, is not easy to cause immune rejection or allergic reaction, has high safety, and is suitable as a tissue scaffold and absorbable implant material; hyaluronic acid hydrogel itself can interact with specific receptors on the cell surface to regulate cell proliferation, migration and differentiation, and good permeability allows free diffusion of oxygen, nutrients and metabolic waste, which is conducive to cell survival and growth, which makes it have natural advantages in promoting tissue regeneration and wound healing.

[0003] Although hyaluronic acid hydrogel has unique physical and chemical properties and biological activity and has made great achievements in the medical field, most of the current hyaluronic acid hydrogel is still prepared by chemical crosslinking method. Commonly used chemical crosslinking agents such as BDDE have potential biological toxicity and are difficult to be degraded and metabolized by the body, which may cause allergic reactions, inflammation, etc.

[0004] At present, physical crosslinking is a new idea. By protonation and repeated freezing and thawing, a network structure of hydrogen bond crosslinking is formed, and finally a hydrogel is formed. The physical crosslinking hydrogel prepared by the freezing and thawing method avoids the use of chemical crosslinking agents and reduces the toxic and side effects on the body.

[0005] However, the hydrogel prepared by the freezing and thawing method has poor mechanical strength, low tensile strain, and no ability to absorb liquid, which seriously limits its application. SUMMARY

[0006] In view of the technical problems in the background art, the present application provides a high water-absorbing hyaluronic acid porous sponge-like hydrogel and a preparation method thereof, which solves the problem of poor liquid absorption and no tensile property of traditional physical crosslinking hydrogel, and provides a new idea for the preparation of hyaluronic acid porous sponge-like hydrogel.

[0007] The high water-absorbing hyaluronic acid porous sponge-like hydrogel has high liquid absorption and high tensile properties. Its high water content, high water absorption, high swelling and high tensile properties make it have wide application value in biomedicine.

[0008] In a first aspect, the present application provides a preparation method of a high water-absorbing hyaluronic acid porous sponge-like hydrogel, comprising the following steps:

[0009] S1. Mixing hyaluronic acid or salt thereof powder with deionized water, stirring until fully dissolved to prepare a hyaluronic acid or salt solution;

[0010] S2. Adjusting the pH of the solution to 1.0-2.0 with hydrochloric acid, then repeating the freeze-thaw treatment of the solution by freezing and thawing to prepare a traditional physically cross-linked hyaluronic acid hydrogel;

[0011] S3. Removing acid and salt from the traditional physically cross-linked hyaluronic acid hydrogel, and calculating the yield point corresponding strain and the structural damage critical point strain of the traditional physically cross-linked hyaluronic acid hydrogel by compression test;

[0012] S4. Removing acid and salt from the traditional physically cross-linked hydrogel obtained in step S2, and then performing compression dehydration at a specific strain, i.e. obtaining a high water-absorbing hyaluronic acid porous sponge-like hydrogel; the specific strain is greater than or equal to the yield point corresponding strain and less than the structural damage critical point strain.

[0013] Further, the specific strain is the yield point corresponding strain.

[0014] Further, in step S3, the process of calculating the yield point corresponding strain of the traditional physically cross-linked hyaluronic acid hydrogel by compression test is as follows:

[0015] After the acid- and salt-removed material is saturated with water, its weight is measured as A, and it is placed on a texture analyzer for compression treatment at 0%-90% strain. After each compression to a strain value, the squeezed-out water is absorbed, and then its weight is measured as a, and the weight remaining rate is calculated as a / A x 100%. The above operation is repeated until the critical point where the weight remaining rate is constant and no more water is squeezed out is found, and the strain value at this point is the yield point corresponding strain of the traditional physically cross-linked hyaluronic acid hydrogel.

[0016] Further, in step S3, the process of calculating the structural damage critical point strain of the hydrogel by compression test is as follows: the acid- and salt-removed material is placed on a texture analyzer for compression treatment at 100% strain. As the compression strain value increases, the data curve slowly rises. When the data curve appears a sharp peak and sharply decreases, the material suddenly breaks and the test is interrupted. The strain value at this point is the structural damage critical point strain of the traditional physically cross-linked hyaluronic acid hydrogel.

[0017] Further, in step S1, the molecular weight of the hyaluronic acid or salt thereof is greater than 400 kDa.

[0018] Further, in step S2, the number of freeze-thaw cycles is 1-5, and the single freezing time is 2-5 days; the freezing temperature is-15 to-40 DEG C, and the thawing temperature is 4-37 DEG C.

[0019] Further, in step S3, the acid removal refers to soaking the hydrogel in a weak alkaline buffer solution for soaking treatment, and the buffer solution is replaced every 1-3 hours, and the treatment time is 12-48 hours; the buffer solution is one of phosphate buffer, HBSS buffer, Tris-HCl buffer and HEPES buffer.

[0020] Further, in step S3, the salt removal refers to placing the hydrogel soaked in the buffer solution in deionized water, and the water is replaced every 1-3 hours, and the treatment time is 24-48 hours.

[0021] In a second aspect, the application provides a high water-absorbing hyaluronic acid porous sponge-like hydrogel, which is prepared by the above preparation method, and the water content of the high water-absorbing hyaluronic acid porous sponge-like hydrogel is as high as 96%, and the hydrogel can absorb liquid 1.744 times to 2.718 times of its own weight, and has high water absorption and high swelling performance. At the same time, the high water-absorbing hyaluronic acid porous sponge-like hydrogel can also withstand a tensile strain of 189.68%, and the maximum tensile force reaches 49.46 kPa, and has a high tensile property which is not possessed by traditional physical freeze-thaw hydrogel.

[0022] The high water-absorbing hyaluronic acid porous sponge-like hydrogel is used for preparing tissue filling materials, wound dressings, liquid absorbing materials and hemostatic materials.

[0023] The application has the following beneficial effects:

[0024] (1) The preparation method of the high water-absorbing hyaluronic acid porous sponge-like hydrogel combines freeze-thaw treatment and unique dehydration treatment, and obtains a sponge-like hydrogel which has both porous sponge and hydrogel properties. The unique dehydration treatment breaks the conventional cognition of avoiding dehydration of hydrogel, and gives the hydrogel unique properties. The preparation process of the application only needs freeze-thaw treatment and dehydration treatment under specific conditions, and does not involve chemical crosslinking agent, so that excellent tensile properties are obtained.

[0025] (2) The preparation method of the high-water-absorption hyaluronic acid porous sponge-like hydrogel solves the problem of no liquid absorption of traditional physical crosslinking hydrogel, and the prepared hydrogel has the dual characteristics of high liquid absorption of sponge and high water content of hydrogel, can store a large amount of water like hydrogel, and can absorb a large amount of water like sponge, has the characteristics of high water content, high water absorption, high swelling and high tensile performance, and has good medical prospects. Taking wound dressing as an example, the advantages of high water content of hydrogel can provide a moist environment for the wound and accelerate healing; and the advantages of high water absorption of sponge can absorb the exudate of the wound, reduce bacterial growth and reduce the risk of infection.

[0026] (3) The high-water-absorption hyaluronic acid porous sponge-like hydrogel prepared by the application has the tensile performance that traditional physical crosslinking hydrogel does not have, and the tensile capacity can be controlled and adjusted. According to the requirements, suitable freeze-thaw conditions can be selected, and with the increase of the number of freeze-thaw, the tensile performance of the hyaluronic acid porous sponge-like hydrogel is enhanced.

[0027] (4) The high-water-absorption hyaluronic acid porous sponge-like hydrogel prepared by the application is physically crosslinked, the system is stable, and there is no other chemical additive, and the degradation product is polysaccharide or monosaccharide, which has the advantages of safety and harmlessness. Due to the characteristics of high water content, high liquid absorption, high swelling and high tensile performance, in addition to being used as medical dressing, it can also be used for tissue filling, liquid absorption material and hemostatic material.

[0028] (5) The high-water-absorption hyaluronic acid porous sponge-like hydrogel uses hyaluronic acid or its salt and deionized water as raw materials, has no other chemical additives, and the degradation product is polysaccharide or monosaccharide, which has the advantages of safety and harmlessness.

[0029] The above description is only a summary of the technical scheme of the application, in order to more clearly understand the technical means of the application, the specific embodiments of the application can be implemented according to the content of the specification, and in order to make the above and other purposes, characteristics and advantages of the application more obvious and easy to understand, the following will specifically describe the embodiments of the application. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical scheme of the application, the following will briefly introduce the drawings used in the application. Obviously, the drawings described below are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creating laborious work.

[0031] Figure 1 The strain-weight residual rate graph of the dehydrated process of the once freeze-thaw hyaluronic acid porous sponge-like hydrogel prepared for experimental example 1.

[0032] Figure 2Strain-remaining weight ratio graph of the secondary freeze-thawed porous hyaluronic acid sponge-like hydrogel prepared for Experimental Example 2 during the dehydration process.

[0033] Figure 3 Strain-remaining weight ratio graph of the tertiary freeze-thawed porous hyaluronic acid sponge-like hydrogel prepared for Experimental Example 3 during the dehydration process.

[0034] Figure 4 Compression strength graph of the secondary freeze-thawed hyaluronic acid sponge-like hydrogel prepared for Comparative Example 1 at the yield point or more (100% strain).

[0035] Figure 5 Actual appearance display graph of the secondary freeze-thawed hyaluronic acid sponge-like hydrogel prepared for Comparative Example 1 after compression at the yield point or more (100% strain).

[0036] Figure 6 Water content graph of the high water-absorbing hyaluronic acid sponge-like hydrogels prepared for Examples 1-3.

[0037] Figure 7 Water absorption capacity graph of the high water-absorbing hyaluronic acid sponge-like hydrogels prepared for Examples 1-3.

[0038] Figure 8 Tensile capacity comparison graph of the sponge-like hydrogels before and after dehydration at different freeze-thawing times. Among them, (a) is a tensile capacity graph of the sponge-like hydrogels prepared in Examples 2 and Comparative Example 2 before and after dehydration treatment of secondary freeze-thawing, (b) is a tensile capacity graph of the sponge-like hydrogels prepared in Examples 3 and Comparative Example 3 before and after dehydration treatment of tertiary freeze-thawing.

[0039] Figure 9 Rheological graph of the sponge-like hydrogels prepared in Examples 2 and Comparative Example 2 before and after dehydration treatment of secondary freeze-thawing.

[0040] Figure 10 Schematic diagram of the high water-absorbing hyaluronic acid sponge-like hydrogel prepared in Example 2 repeatedly absorbing water and dehydrating.

[0041] Figure 11 Actual appearance display graph of the high water-absorbing hyaluronic acid sponge-like hydrogel prepared in Example 2 used as a dressing.

[0042] Figure 12 Actual appearance display graph of the high water-absorbing hyaluronic acid sponge-like hydrogel prepared in Example 2 absorbing deionized water and absorbing artificial blood.

[0043] Figure 13 Actual appearance display graph of the high water-absorbing hyaluronic acid sponge-like hydrogel prepared in Example 2 before and after stretching.

[0044] Figure 14A photograph of the hydrogel prepared for Comparative Example 4. DETAILED DESCRIPTION

[0045] The embodiments of the technical solutions of the present application are described in detail below. The following examples are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terms "comprising" and "having", and any variations thereof, used herein are only for the purpose of describing the specific embodiments of the present application, and are not intended to limit the present application.

[0047] Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearance of the phrase in various places in the specification does not necessarily all refer to the same embodiment, nor is it necessarily independent of other embodiments. It is explicitly and implicitly understood that the embodiments described herein can be combined with other embodiments.

[0048] In the prior art, the hydrogel prepared by freeze-thaw method has poor mechanical strength, low tensile fracture strain, and no ability to absorb liquid, which seriously limits its application.

[0049] The present application provides a preparation method of a high-water-absorption hyaluronic acid porous sponge-like hydrogel, comprising the following steps:

[0050] S1. Mix hyaluronic acid or its salt powder with deionized water, and stir until fully dissolved to prepare a hyaluronic acid or its salt solution;

[0051] Preferably, the molecular weight of the hyaluronic acid or its salt is 1370 KDa. When the molecular weight is less than 400 kDa, the freeze-thaw treatment cannot form a hyaluronic acid self-crosslinked hydrogel. This is because when the molecular weight is low, the molecular chain of hyaluronic acid is too short to form sufficient entanglement and hydrogen bond crosslinking points, so it cannot build a stable physical crosslinking network.

[0052] The mass fraction of the hyaluronic acid or its salt solution is 1.0-5.0%, preferably 1.5-2.0%.

[0053] S2. Adjust the pH of the solution to 1.0-2.0 with hydrochloric acid, and then perform repeated freeze-thaw treatment of freezing and thawing on the solution to prepare a conventional physically crosslinked hyaluronic acid hydrogel;

[0054] The freeze-thawing times are 1-5 times, and the single freeze time is 2-5 days; the freeze temperature is-15 to-40 DEG C, and the thawing temperature is 4-37 DEG C.

[0055] S3. The traditional physically cross-linked hyaluronic acid hydrogel is subjected to acid and salt removal treatment, and the yield point corresponding strain and the structural damage critical point strain of the traditional physically cross-linked hyaluronic acid hydrogel prepared in step S2 are calculated through compression test.

[0056] The acid removal refers to soaking the hydrogel in a weak alkali buffer solution for soaking treatment, and the buffer solution is replaced every 1-3 hours, and the treatment time is 12-48 hours.

[0057] The buffer solution is one of phosphate buffer solution (PBS), HBSS buffer solution, Tris-HCl buffer solution and HEPES buffer solution, and preferably the phosphate buffer solution.

[0058] The salt removal refers to placing the hydrogel soaked in the buffer solution in deionized water, and the water is replaced every 1-3 hours, and the treatment time is 24-48 hours.

[0059] The time requirement for acid removal and salt removal is that the higher the concentration of hyaluronic acid is, the longer the treatment time is.

[0060] Specifically, the process of calculating the yield point corresponding strain of the traditional physically cross-linked hyaluronic acid hydrogel through compression test is as follows:

[0061] After the material after acid and salt removal is saturated with water, the weight A is weighed, and the material is placed on a texture analyzer for compression treatment under 0%-90% strain. After each compression to a strain value, the squeezed-out water is absorbed, and then the weight a is weighed, and the weight remaining rate is calculated as a / A*100%. The above operation is repeated until the critical point of constant weight remaining rate, i.e. no more water, is found, and the strain value at this time is the yield point corresponding strain of the traditional physically cross-linked hyaluronic acid hydrogel.

[0062] The process of calculating the structural damage critical point strain of the hydrogel through compression test is as follows: the material after acid and salt removal is placed on a texture analyzer for compression treatment under 100% strain. With the continuous increase of the compression strain value, the data curve slowly rises. When the data curve appears a sharp peak and sharply decreases, the material suddenly breaks, and the test is interrupted. The strain value at this time is the critical point corresponding strain of the traditional physically cross-linked hyaluronic acid hydrogel.

[0063] S4. The conventional physically cross-linked hydrogel obtained in step S2 is subjected to acid and salt removal treatment and then is compressed and dehydrated under a specific strain to obtain a high water-absorbing hyaluronic acid porous sponge-like hydrogel. The specific strain is greater than or equal to the strain corresponding to the yield point and less than the strain at the structural destruction critical point, otherwise the hydrogel cannot be completely dehydrated or is structurally destroyed.

[0064] Preferably, the specific strain is the strain corresponding to the yield point.

[0065] In the present application, the dehydration conditions of the hydrogel under different freeze-thaw conditions are different.

[0066] With the increase of the number of freeze-thaw cycles, the strain value required for the dehydration of the hydrogel is greater and greater, and the strain value is about 60% for one freeze-thaw cycle, about 70% for two freeze-thaw cycles, and about 75% for three freeze-thaw cycles. The maximum strain value that can be tolerated by the material under all freeze-thaw cycles is 90%. When the strain value is below the strain corresponding to the yield point, the free water in the pores of the material cannot be completely removed, and the dehydration is not complete. When the strain value is greater than or equal to the strain corresponding to the yield point and less than the critical strain at which the shape of the hydrogel is destroyed, the dehydration is complete and the material performance is good. When the strain value is greater than the critical strain at which the shape of the hydrogel is destroyed, the material is structurally destroyed.

[0067] In the present application, hyaluronic acid and its salt are protonated and subjected to freeze-thaw treatment to obtain a physically cross-linked hyaluronic acid porous hydrogel. Then, the hydrogel is subjected to compression and dehydration treatment under a specific strain to obtain a hyaluronic acid porous sponge-like hydrogel with high swelling and high tensile properties.

[0068] The preparation method of the high water-absorbing hyaluronic acid porous sponge-like hydrogel provided by the present application is simple, uses hyaluronic acid or its salt and deionized water as raw materials, does not add a chemical cross-linking agent, and has good biocompatibility with a degradation product of polysaccharide or monosaccharide. The hydrogel has the dual characteristics of a porous sponge and a hydrogel, has high water content and high liquid absorption of the porous sponge, and has high tensile properties and flexibility. The hydrogel can be used for tissue filling, wound dressing, liquid absorption material, and hemostatic material, and has biomedical applications.

[0069] The hyaluronic acid porous sponge-like hydrogel prepared by the foregoing method has greater water absorption capacity, and the water absorption capacity can be controlled and adjusted according to the number of freeze-thaw cycles. Moreover, the sponge-like hydrogel after dehydration has greater tensile properties, and the tensile properties can also be controlled and adjusted. In addition, the sponge-like hydrogel after dehydration has greater storage modulus and loss modulus, i.e., better tensile properties and adhesion.

[0070] In the technical scheme of the embodiment of the present application, the prepared high-water-absorption hyaluronic acid porous sponge-like hydrogel has the dual characteristics of sponge and hydrogel, can store a large amount of water like a hydrogel, can reabsorb a large amount of water like a sponge, and has the characteristics of high water content, high water absorption, high swelling and high tensile performance. In addition, the hyaluronic acid porous sponge-like hydrogel uses hyaluronic acid or a salt thereof and deionized water as raw materials, has no other chemical additives, and has the advantage of safe and harmless degradation products of polysaccharides or monosaccharides.

[0071] In the technical scheme of the embodiment of the present application, the prepared high-water-absorption hyaluronic acid porous sponge-like hydrogel can be used for biomedical applications such as tissue filling, wound dressing, liquid absorption material and hemostatic material.

[0072] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are used to explain the present application, and cannot be understood as a limitation of the present application. If the specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art or according to the product manual is used. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0073] Experimental Example 1

[0074] The present experimental example provides a preparation method of a hyaluronic acid sponge-like hydrogel, which specifically comprises the following steps:

[0075] S1. Dissolve hyaluronic acid or a salt thereof with a molecular weight of 1370 kDa in deionized water to prepare a hyaluronic acid solution with a concentration of 1.5%.

[0076] S2. Adjust the pH of the hyaluronic acid solution to 1.5 with hydrochloric acid, then pour the solution into a suitable mold, and then transfer it to a-20℃ refrigerator for freezing for 3 days. After 3 days, take out the sponge-like hydrogel and place it in a 25℃ oven for thawing for 4 h to complete one freeze-thaw cycle.

[0077] S3. Bubble the thawed sponge-like hydrogel with PBS and water to remove excess hydrochloric acid and salt; use a compression strength tester to test the yield point of the sponge-like hydrogel, and use a compression strain value of 0% to 90% (increased by 5% units) as a parameter for the experiment.

[0078] The yield point strain of the conventional hyaluronic acid porous sponge-like hydrogel after one freeze-thaw cycle is 60%, as shown in Figure 1 .

[0079] Experimental Example 2

[0080] The difference between Experimental Example 2 and Experimental Example 1 is that the freeze-thaw cycle of Experimental Example 2 is twice, and the other experimental parameters and conditions are basically the same as those of Experimental Example 1, which will not be repeated here.

[0081] Experimental Example 3

[0082] The difference between Experimental Example 3 and Experimental Example 1 is that the freeze-thawing number of Experimental Example 3 is three times, and other experimental parameters and conditions are basically the same as those of Experimental Example 1, which will not be repeated here.

[0083] The purpose of Experimental Examples 1-3 is to explore the compression strain value required for the dehydration treatment of the hydrogel under different freeze-thawing numbers, that is, to find the corresponding yield point, so as to lay a foundation for the subsequent examples.

[0084] Figures 1 to 3 The strain-remaining weight ratio graph of the dehydration process of the hyaluronic acid porous sponge-like hydrogel prepared by one freeze-thawing, two freeze-thawings and three freeze-thawings in Experimental Examples 1-3 respectively, shows the yield point of the hyaluronic acid hydrogel under different freeze-thawing numbers.

[0085] Specific operation: after the material after acid and salt removal is saturated with water, its weight is weighed as A, and it is placed on the texture meter for compression treatment under 0%-90% strain (5% increment). After each compression to a strain value, the squeezed-out water is absorbed, and then its weight is weighed as a, and the weight remaining rate is calculated as a / A x 100%. Repeat the above operation until the critical point of constant weight remaining rate is found, that is, no more water is discharged, and the strain value at this time is the yield point required for the dehydration treatment of the traditional physical freeze-thawing hydrogel.

[0086] From the above experimental results, it can be seen that the yield point of the hydrogel material after one freeze-thawing is about 60%, the yield point of the hydrogel material after two freeze-thawings is about 70%, and the yield point of the hydrogel material after three freeze-thawings is about 75%. Figures 1 to 3 It can be seen that the weight remaining rate of the hydrogel material after one freeze-thawing remains at about 55% at 60% compression strain and does not change any more; the weight remaining rate of the hydrogel material after two freeze-thawings remains at about 45% at 70% compression strain and does not change any more, and the weight remaining rate of the hydrogel material after three freeze-thawings remains at about 35% at 75% compression strain and does not change any more.

[0087] Therefore, it is concluded that the yield point of the hydrogel material after one freeze-thawing is about 60%, the yield point of the hydrogel material after two freeze-thawings is about 70%, and the yield point of the hydrogel material after three freeze-thawings is about 75%.

[0088] Example 1

[0089] The present embodiment provides a preparation method of a high-water-absorption hyaluronic acid sponge-like hydrogel, which specifically comprises the following steps:

[0090] S1. Dissolve hyaluronic acid or its salt powder with a molecular weight of 1370 kDa in deionized water to prepare a hyaluronic acid solution with a concentration of 1.5%.

[0091] S2. Adjust the pH of the hyaluronic acid solution to 1.5 with hydrochloric acid, then pour the solution into a suitable mold, and then transfer into a -20 ℃ refrigerator to freeze for 3 days. After 3 days, take out the sponge-like hydrogel and place it in a 25 ℃ oven to thaw for 4 h, complete a freeze-thaw cycle, and prepare a traditional physically cross-linked hyaluronic acid hydrogel (sponge-like hydrogel after thawing).

[0092] S3. Bubble the sponge-like hydrogel after thawing with PBS and water to remove excess hydrochloric acid and salt; according to Experimental Example 1, the yield point strain of the traditional hyaluronic acid porous sponge-like hydrogel after one freeze-thaw cycle is 60%;

[0093] S4. Perform compression dewatering on the sponge-like hydrogel after thawing and acid and salt removal at a strain of 60% to obtain a high water absorption hyaluronic acid porous sponge-like hydrogel.

[0094] Example 2

[0095] The main difference between Example 2 and Example 1 is that two freeze-thaw cycles are performed in step S2; according to Experimental Example 2, compression dewatering is performed at a strain of 70% in step S4. Other aspects are substantially the same as those of Example 1, and are not described here again.

[0096] Example 3

[0097] The main difference between Example 3 and Example 1 is that three freeze-thaw cycles are performed in step S2; according to Experimental Example 3, compression dewatering is performed at a strain of 75% in step S4. Other aspects are substantially the same as those of Example 1, and are not described here again.

[0098] Comparative Example 1

[0099] The main difference between Comparative Example 1 and Example 2 is that compression dewatering is performed at a strain of 100% in step S4. Other aspects are substantially the same as those of Example 2, and are not described here again.

[0100] Comparative Example 2

[0101] The main difference between Comparative Example 2 and Example 2 is that the compression dewatering treatment in step S4 is not performed, i.e., the traditional physically cross-linked hyaluronic acid hydrogel after two freeze-thaw cycles is subjected to acid and salt removal to obtain a sponge-like hydrogel. Other aspects are substantially the same as those of Example 2, and are not described here again.

[0102] Comparative Example 3

[0103] The main difference between Comparative Example 3 and Example 3 is that the compression dewatering treatment in step S4 is not performed, i.e., the traditional physically cross-linked hyaluronic acid hydrogel after three freeze-thaw cycles is subjected to acid and salt removal to obtain a sponge-like hydrogel. Other aspects are substantially the same as those of Example 3, and are not described here again.

[0104] For ease of comparison, the main differences between Examples 1-3 and Comparative Examples 1-3 are summarized in Table 1 below.

[0105] Table 1. Process Comparison of Examples 1-3 and Comparative Examples 1-3

[0106]

[0107] Figure 4 The image shows the compressive strength of the hyaluronic acid sponge-like hydrogel prepared in Comparative Example 1 at 100% strain. It can be seen that when the strain value is less than 90% (above the 70% strain of the second freeze-thaw yield point), the material properties are intact; when the strain value is greater than 90% (100% strain), the material is crushed, as shown below. Figure 5 As shown, 90% strain is the critical point at which the hydrogel undergoes shape failure.

[0108] Therefore, the strain threshold at which the hydrogel will break down during dehydration must not be exceeded.

[0109] Depend on Figures 1 to 5 It is known that the preparation of highly absorbent hyaluronic acid porous sponge-like hydrogels requires specific dehydration treatment. For single-freeze-thaw hydrogels, a strain of 60%–90% can be selected; for double-freeze-thaw hydrogels, 70%–90%; and for triple-freeze-thaw hydrogels, 75%–90%. These compressive strain values ​​are precisely calculated to ensure that the strain used during dehydration is greater than or equal to the yield point strain, but less than the critical strain at which the hydrogel undergoes shape failure. If the strain value is less than the yield point, the material cannot be completely dehydrated; if the strain value is greater than 90%, the material will rupture due to its inability to withstand high pressure.

[0110] Figure 6 The water content diagram shows the water content of the highly absorbent hyaluronic acid sponge-like hydrogels prepared in Examples 1-3.

[0111] Given that the material contains bound water that cannot be squeezed out, weigh the sponge-like hydrogel after dehydration treatment (M), then place it in a 60 ℃ oven to dry to constant weight and weigh it again (m). The water content of the material after dehydration treatment is (Mm) / M×100%.

[0112] Depend on Figure 6 It can be seen that the water content of the highly absorbent hyaluronic acid sponge-like hydrogels prepared in Examples 1-3 is all around 96%, indicating that the highly absorbent hyaluronic acid sponge-like hydrogels prepared in Examples 1-3 all have extremely high water content.

[0113] Figure 7 This is a graph showing the water absorption capacity of the highly absorbent hyaluronic acid sponge-like hydrogels prepared in Examples 1-3. Figures 1 to 3Corresponding to the yield point diagram, as the number of freeze-thaw cycles increases, the reabsorption capacity of the dehydrated sponge-like hydrogel gradually increases.

[0114] The highly absorbent hyaluronic acid sponge-like hydrogel prepared in Example 1 can absorb approximately 1.744 times its own weight in liquid, the highly absorbent hyaluronic acid sponge-like hydrogel prepared in Example 2 can absorb approximately 2.188 times its own weight in liquid, and the highly absorbent hyaluronic acid sponge-like hydrogel prepared in Example 3 can absorb approximately 2.718 times its own weight in liquid. It is evident that the highly absorbent hyaluronic acid sponge-like hydrogel prepared by this invention has high liquid absorption capacity, and this capacity is adjustable, allowing for the selection of appropriate freeze-thaw conditions according to requirements.

[0115] Figure 8 This image shows a comparison of the tensile strength of sponge-like hydrogels before and after dehydration under different freeze-thaw cycles. Figure 8 (a) in the figure shows the tensile strength of the sponge-like hydrogels prepared in Example 2 and Comparative Example 2 before and after the secondary freeze-thaw dehydration treatment. Figure 8 (b) in the figure shows the tensile strength of the sponge-like hydrogels prepared in Example 3 and Comparative Example 3 before and after three freeze-thaw dehydration treatments.

[0116] Depend on Figure 8 As can be seen from (a), the tensile strength at break of the sponge-like hydrogel before dehydration was 4.65 kPa, and it broke at 47.371% strain. The tensile strength at break of the sponge-like hydrogel after dehydration was 19.22 kPa, and it broke at 103.066% strain. Its tensile strength, elongation at break and modulus were significantly improved.

[0117] Figure 8 As shown in (b), the tensile strength at break of the sponge-like hydrogel before the three freeze-thaw dehydration treatments was 10.8 kPa, and it fractured at 109.39% strain. After dehydration, the tensile strength at break of the sponge-like hydrogel was 49.46 kPa, and it fractured at 189.68% strain. Its tensile strength, elongation at break, and modulus were significantly improved. These results demonstrate that the hyaluronic acid hydrogel prepared by dehydration treatment exhibits significantly improved tensile strength, elongation at break, and modulus, indicating that the porous sponge-like hyaluronic acid hydrogel prepared in this invention differs fundamentally in structure and properties from the hyaluronic acid hydrogel prepared by the conventional freeze-thaw method.

[0118] Figure 9 The rheological diagrams are shown for the sponge-like hydrogels prepared in Example 2 and Comparative Example 2 before and after the secondary freeze-thaw dehydration treatment.

[0119] It can be seen that, first, the storage modulus G' of the sponge-like hydrogel is greater than the loss modulus G'' regardless of whether the dehydration treatment is performed, proving that the material has gel properties. Second, the data shows that the storage modulus and loss modulus of the sponge-like hydrogel after dehydration treatment are greater than those of the material without dehydration treatment, proving that the material after dehydration has good elasticity and viscosity, and thus can be well attached to the skin when used as a dressing, and has a certain compliance to the skin.

[0120] Figure 10 The schematic diagram of the high water-absorbing hyaluronic acid sponge-like hydrogel prepared in Example 2 repeatedly absorbing water and dehydrating. It can be seen that, on the one hand, the sponge-like hydrogel after dehydration treatment has very high water-absorbing and swelling properties, and on the other hand, the high water-absorbing hyaluronic acid sponge-like hydrogel can be repeatedly treated by absorbing water and dehydrating. That is, the high water-absorbing hyaluronic acid sponge-like hydrogel prepared by the present application has excellent fatigue resistance.

[0121] Figure 11 The physical display diagram of the high water-absorbing hyaluronic acid sponge-like hydrogel prepared in Example 2 used as a dressing. It can be seen that the sponge-like hydrogel dressing after dehydration treatment has good ductility and adhesion, and at the same time, the dressing can not only take advantage of the high water content of the hydrogel to provide a moist environment for the wound and accelerate healing, but also can take advantage of the high water-absorbing property of the sponge to absorb the exudate of the wound, reduce bacterial growth and reduce the risk of infection.

[0122] Figure 12 The physical display diagram of the high water-absorbing hyaluronic acid sponge-like hydrogel prepared in Example 2 absorbing deionized water and absorbing artificial blood. It can be seen that the sponge-like hydrogel after dehydration treatment has high liquid absorption and high swelling properties which the conventional freeze-thaw method prepared hyaluronic acid hydrogel does not have, and can be used as a tissue filling material and a hemostatic material, which is safe and harmless.

[0123] The hydrogels prepared in Examples 2-3 and Comparative Examples 2-3 were tested for tensile properties, and the results are shown in Table 2 below.

[0124] Table 2 Comparison of tensile properties of Examples 2-3 and Comparative Examples 2-3

[0125]

[0126] Figure 13 The physical display diagram of the high water-absorbing hyaluronic acid sponge-like hydrogel prepared in Example 2 before and after stretching.

[0127] From Table 2 and Figure 13 It can be seen that the sponge-like hydrogel after dehydration treatment has high tensile property which the conventional freeze-thaw method prepared hyaluronic acid hydrogel does not have.

[0128] Comparative Example 4

[0129] Comparative Example 4 provides a preparation method of a high water-absorbing hyaluronic acid sponge-like hydrogel, which differs from Example 3 in that a hyaluronic acid solution and a salt solution with a molecular weight of 400 kDa are used as raw materials to prepare the hydrogel. That is, the freeze-thawing is performed for 3 times without subsequent dehydration treatment.

[0130] Figure 14 A physical map of the hydrogel prepared for Comparative Example 4.

[0131] Experiments show that after three freeze-thawing, the system still does not form a hydrogel. This indicates that when the molecular weight of hyaluronic acid or its salt is too low, it is not possible to prepare a hydrogel by freeze-thawing method. This is because when the molecular weight is low, the molecular chain of hyaluronic acid is too short to form sufficient entanglement and hydrogen bond crosslinking points, so it is not possible to build a stable physical crosslinking network.

[0132] In summary, the present application provides a preparation method of a high water-absorbing hyaluronic acid porous sponge-like hydrogel. The method compresses water under the strain condition corresponding to a specific yield point on the basis of the hydrogel prepared by the traditional physical freeze-thawing method, so as to obtain the required high water-absorbing hyaluronic acid sponge-like hydrogel.

[0133] The hyaluronic acid porous sponge-like hydrogel prepared by the present application has the dual characteristics of porous sponge and hydrogel, and has high water content of hydrogel and high liquid absorption of porous sponge. The hyaluronic acid porous sponge-like hydrogel prepared by the present application has the characteristics of simple process, no addition of chemical crosslinking agent, good biological safety, high water retention, high water absorption, high swelling and high tensile properties, and can be used for tissue filling, wound dressing, liquid absorbing material and hemostatic material and other biomedical applications. At the same time, the hyaluronic acid porous sponge-like hydrogel prepared by the present application has adjustable liquid absorption capacity and tensile capacity, and can select appropriate freeze-thawing conditions according to requirements.

[0134] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and embodiments having the same technical idea and playing the same role within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, within the scope of the main idea of the present application, various modifications of the embodiments that can be thought of by those skilled in the art, and other ways constructed by combining part of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for preparing a high water-absorbing hyaluronic acid porous sponge-like hydrogel, characterized by, The method comprises the following steps: S1. mixing hyaluronic acid or salt thereof powder with deionized water, stirring until fully dissolved to prepare a hyaluronic acid or salt solution; wherein the molecular weight of the hyaluronic acid or salt is greater than 400 kDa; S2. adjusting the pH of the solution to 1.0-2.0 with hydrochloric acid, and then subjecting the solution to repeated freeze-thaw treatment by freezing and thawing to prepare a traditional physically cross-linked hyaluronic acid hydrogel; wherein the number of freeze-thaw cycles is 1-5, and the single freezing time is 2-5 days; the freezing temperature is -15 to -40 DEG C, and the thawing temperature is 4-37 DEG C; S3. removing acid and salt from the traditional physically cross-linked hyaluronic acid hydrogel, and calculating the yield point corresponding strain of the traditional physically cross-linked hyaluronic acid hydrogel by compression test; wherein the process of calculating the yield point corresponding strain of the traditional physically cross-linked hyaluronic acid hydrogel by compression test is as follows: After the acid and salt are removed, the material is saturated with water, and its weight is A, and it is placed on a texture analyzer for compression treatment under 0%-90% strain. After each compression to a strain value, the squeezed water is removed, and then its weight is a, and the weight remaining rate is calculated as a / A*100%. Repeat the above operation until the critical point where the weight remaining rate is constant and no water is discharged is found. At this point, the strain value is the yield point corresponding strain of the traditional physically cross-linked hyaluronic acid hydrogel; S4. removing acid and salt from the traditional physically cross-linked hydrogel obtained in step S2, and then performing compression dehydration under a specific strain to obtain a high water absorption hyaluronic acid porous sponge-like hydrogel; the specific strain is the yield point corresponding strain.

2. The method for preparing the highly absorbent hyaluronic acid porous sponge-like hydrogel according to claim 1, characterized in that, In step S3, the acid removal refers to soaking the hydrogel in a weak alkaline buffer solution for soaking treatment, and the buffer solution is replaced every 1-3 hours, and the treatment time is 12-48 hours; the buffer solution is one of phosphate buffer, HBSS buffer, Tris-HCl buffer and HEPES buffer.

3. The method for preparing the highly absorbent hyaluronic acid porous sponge-like hydrogel according to claim 1, characterized in that, In step S3, the salt removal refers to placing the hydrogel soaked in the buffer solution in deionized water, and the water is replaced every 1-3 hours, and the treatment time is 24-48 hours.

4. A highly water-absorbing hyaluronic acid porous sponge-like hydrogel, characterized by, The high water absorption hyaluronic acid porous sponge-like hydrogel prepared by the preparation method of any one of claims 1-3 has a water content of 96%, can absorb 1.744-2.718 times its own weight of liquid, and has a tensile strain of 189.68% and a maximum tensile force of 49.46 kPa.

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