Hydrogel biomaterials, methods of making and uses thereof

Abstract

The present application relates to a kind of hydrogel biomaterials and its preparation method and application.The hydrogel biomaterial has the three-dimensional network structure formed by glycosaminoglycan and methyl methacrylated gelatin crosslinking, the crosslinking between glycosaminoglycan and methyl methacrylated gelatin includes amide bond covalent crosslinking, and the glycosaminoglycan is at least one of glycosaminoglycan, the modifier of glycosaminoglycan and depolymerization product of glycosaminoglycan.The hydrogel biomaterial has better biocompatibility, and can promote wound healing and biodegradable.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a hydrogel biomaterial, its preparation method, and its application. Background Technology

[0002] Wound healing is a complex and highly regulated physiological process, typically involving several overlapping phases: hemostasis, inflammation, proliferation, and remodeling. Clinical and experimental studies have found that a prolonged inflammatory phase makes it difficult to progress to the proliferation and remodeling phases, hindering tissue regeneration and preventing wound healing, leading to chronic wounds. Chronic wounds are particularly prevalent in the elderly, obese individuals, and those with diabetes. Clinically, these chronic wounds exhibit healing times far exceeding those of normal wounds, or even fail to heal altogether, resulting in expensive treatment and generally poor prognoses.

[0003] Diabetic foot, a type of chronic wound caused by diabetes, is one of the most common causes of diabetes-related complications. Globally, diabetic foot ulcers can lead to diabetic foot disease, resulting in amputation and even death, and are the most costly complication of diabetes. Because the body's own repair mechanisms are often ineffective, wounds continuously release exudate and free radicals, stimulating microbial infection and inflammation, severely hindering healing. Therefore, effective surgical treatment is necessary. A range of treatment strategies have been developed, including skin replacements, cell delivery, medical bandages, medical dressings, and adhesives. Among these, medical dressings are the most widely used.

[0004] Medical dressings are used to cover wounds, serving as a physical barrier to prevent external infection and as an induction template to guide skin remodeling and subsequent infiltration and integration of host tissues, thus having a significant therapeutic effect on wound healing. An ideal skin wound dressing should meet the following requirements: (1) good tissue compatibility, without causing toxicity or inflammation; (2) good moisturizing properties, able to keep the wound moist, promote cell hydration, and have a certain absorption effect on wound exudate; (3) sufficient physical and mechanical strength to ensure its integrity and avoid material damage that could lead to the invasion of external bacteria; (4) suitable surface microstructure and biochemical properties to promote cell adhesion, proliferation, and differentiation.

[0005] Traditional medical dressings such as bandages and gauze, while effectively absorbing exudate and keeping wounds dry, tend to adhere to the wound, causing secondary damage during dressing changes and failing to effectively isolate pathogens. To better promote wound healing, several novel medical dressings have been developed, including hydrogels, alginates, hydrocolloids, foam dressings, and transparent dressings. Hydrogels, in particular, are three-dimensional network gels formed from natural and / or synthetic hydrophilic polymers through chemical or physical cross-linking, exhibiting excellent moisture absorption and retention capabilities. Due to the cross-linked network structure of the polymer chains, hydrogels do not dissolve easily, and their mechanical properties and adhesiveness are also improved. Theoretically, hydrogels are structurally and functionally similar to the natural extracellular matrix (ECM), serving as cell scaffolds and signal transduction carriers during the healing process. As medical dressings for wounds, hydrogels not only form a physical barrier to remove excess exudate but also provide moisture to keep the wound moist, facilitating debridement. Furthermore, the high water content of hydrogels can also have a cooling effect, soothing wound pain. However, the safety and efficacy of these novel hydrogel dressings for chronic wounds still require large-scale clinical validation. Some hydrogels are non-biodegradable and can cause inflammatory responses in subcutaneous tissues; some growth factor-loaded hydrogels even pose a carcinogenic risk. Therefore, current hydrogel medical dressings used for acute and chronic wounds of the skin and internal organs are far from meeting the needs of actual clinical applications. Summary of the Invention

[0006] Therefore, it is necessary to provide a hydrogel biomaterial that is biocompatible, promotes wound healing, and is biodegradable, as well as its preparation method and application.

[0007] The present invention is achieved through the following technical solution.

[0008] In one aspect, the present invention provides a hydrogel biomaterial having a three-dimensional network structure formed by crosslinking a glycosaminoglycan and methacrylamide gelatin, wherein the crosslinking between the glycosaminoglycan and methacrylamide gelatin includes covalent crosslinking of amide bonds, and the glycosaminoglycan is at least one of glycosaminoglycan, a glycosaminoglycan modifier, and a glycosaminoglycan depolymerization product.

[0009] In some embodiments, the structural modifiers of the glycosaminoglycan include at least one of alkylation products, acylation products, and sulfation products of the hydroxyl groups on the sugar ring of the glycosaminoglycan; and / or, the depolymerization products of the glycosaminoglycan include depolymerization products prepared by free radical depolymerization, beta-elimination, and / or deamination depolymerization.

[0010] In some embodiments, the alkylation of the hydroxyl group is at least one of methylation, ethylation, isopropylation, allylation, and benzylation, and the acylation of the hydroxyl group is at least one of acetylation, propionylation, benzoylation, and phenylacetylation.

[0011] In some embodiments, the glycosaminoglycan is selected from at least one of snail glycosaminoglycan, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, and fucoidan.

[0012] In some embodiments, the methacrylamide gelatin has a methacrylamide substitution degree of 10% to 100%.

[0013] In some embodiments, the raw materials for preparing the hydrogel biomaterial, by mass parts, include 0.375 to 5 parts of glycosaminoglycans and 0.5 to 10 parts of methacryloyl gelatin.

[0014] In some embodiments, the raw materials for preparing the hydrogel biomaterial include, by mass parts, 0.375 to 1.5 parts of glycosaminoglycans and 2 to 8 parts of methacryloyl gelatin.

[0015] Another aspect of the present invention provides a method for preparing hydrogel biomaterials, comprising the following steps:

[0016] A solution A is obtained by dissolving a glycosaminoglycan in a biocompatible medium and adding a carboxyl activator; the glycosaminoglycan is at least one of glycosaminoglycan, a glycosaminoglycan modifier, and a glycosaminoglycan depolymerization product.

[0017] Methacrylamide gelatin was dissolved in a biocompatible medium and a photoinitiator was added and mixed to obtain solution B;

[0018] The hydrogel precursor solution was obtained by mixing the solution A and the solution B, and then crosslinked by light.

[0019] Another aspect of the present invention provides the application of the hydrogel biomaterials described in any of the above claims in the preparation of wound tissue repair drugs or tissue fluid permeation sealing products, or as carriers for drugs, proteins and / or cells in the preparation of sustained-release formulations.

[0020] In another aspect, the present invention provides a wound tissue repair medicine comprising the hydrogel biomaterial described in any one of the preceding claims.

[0021] In another aspect, the present invention provides a tissue fluid permeation sealing product comprising the hydrogel biomaterial described in any one of the preceding claims.

[0022] In another aspect, the present invention provides a sustained-release formulation using the hydrogel biomaterial described in any of the preceding claims as a carrier for drugs, proteins, and / or cells.

[0023] The aforementioned hydrogel biomaterial has a three-dimensional network structure, which has good water absorption and retention capacity. In addition, the hydrogel biomaterial has good biocompatibility and degradability, and can be completely degraded and absorbed during the wound healing cycle without the need for additional treatment and removal, thus avoiding secondary damage to the wound.

[0024] Furthermore, in the aforementioned hydrogel biomaterial, methacrylamide gelatin serves as the matrix, and glycosaminoglycans act as the framework and bioactive substances. This allows it to exhibit excellent anti-inflammatory effects by binding chemokines and / or cytokines, significantly accelerating wound healing without the need for any other therapeutic agents. Simultaneously, this hydrogel biomaterial can form amide bonds by reacting with amino and / or carboxyl groups on the surface of biological tissues, including human skin, thereby adhering tightly to the wound surface and conforming to irregular wounds, providing comprehensive wound protection and absorbing tissue fluid released from the wound. Thus, the aforementioned hydrogel biomaterial can slowly release pharmacologically functional glycosaminoglycans and bind with high affinity to pro-inflammatory cytokines continuously released from chronic wound tissues, effectively improving the inflammatory environment of the wound, promoting angiogenesis and re-epithelialization, and accelerating wound healing. Attached Figure Description

[0025] Figure 1 The schematic diagrams and adhesion data for the tissue adhesion test of glycosaminoglycan-based hydrogel (AFG-GelMA) are shown in Figures A and B, respectively.

[0026] Figure 2 The adhesion test diagrams and real-time mechanical changes of glycosaminoglycan-based hydrogel (AFG-GelMA) on uncoated and blood-coated pig casings are shown in Figures A, B, and C, respectively.

[0027] Figure 3 The schematic diagram, physical image, and real-time mechanical change graph of the shear force test of glycosaminoglycan-based hydrogel (AFG-GelMA) are shown in Figures A, B, and C, respectively; where B in AFG-GelMA-B / Fibrin glue-B represents Bloody (with blood on it); and C in AFG-GelMA-C / Fibrin glue-C represents Clear (without blood on it).

[0028] Figure 4 Cell viability assessment data for glycosaminoglycan-based hydrogel raw materials;

[0029] Figure 5The anticoagulant activity of glycosaminoglycan-based hydrogel and the wound bleeding caused by heparin-based hydrogel (HEP-G30) are shown in Figures A and B, respectively.

[0030] Figure 6 H&E staining image of glycosaminoglycan-based hydrogel (AFG-G30) degrading in rat skin, with medical 504 (CA) as a positive control;

[0031] Figure 7 A visual representation of the effect of glycosaminoglycan-based hydrogels as a cell matrix for cell culture.

[0032] Figure 8 A fluorescence confocal image of mouse macrophages M1 to M2 phenotypic polarization induced by glycosaminoglycan-based hydrogel.

[0033] Figure 9 The images show the effect of glycosaminoglycan-based hydrogel (AFG-G30) on promoting wound healing in normal rats and the statistical graph of the healing rate, as shown in Figures A and B, respectively. The saline treatment group was used as the control. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0034] Figure 10 The effects of glycosaminoglycan-based hydrogels (AFG-G30 and HA-G30, 1% / 4%) on promoting skin wound healing in diabetic rats are shown in Figures A and B, respectively. The saline-treated group served as the control; AFG-G30 and HA-G30 were the experimental groups; and G30 was the gel blank group. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0035] Figure 11 H&E staining image of wound tissue section on the 10th day after surgery. The length of the arrow indicates the length of reepithelialization of the wound tissue.

[0036] Figure 12 Immunofluorescence staining of α-SMA and CD31 in wound tissue of diabetic rats on postoperative day 10, and statistical results of α-SMA and CD31 are shown in Figures A, B, and C, respectively; in B and C, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0037] Figure 13 The changes in the expression levels of pro-inflammatory cytokine mRNA in wound tissues of diabetic rats on days 3 and 10 were detected by RT-qPCR; where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0038] Figure 14To detect the levels of inflammatory factors in wound tissue of diabetic rats on days 3 and 10 post-surgery using Bio-Plex suspension chip technology; where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0039] Figure 15 To detect the levels of inflammatory factors in wound tissue and the expression levels of pro-inflammatory cytokine proteins in untraumatized skin tissue of diabetic rats on day 14 post-surgery using Bio-Plex suspension chip technology; where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Detailed Implementation

[0040] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. It should be understood that these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0041] 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 invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0042] One embodiment of the present invention provides a hydrogel biomaterial having a three-dimensional network structure formed by crosslinking glycosaminoglycans (GAG) and methacrylated gelatin (GelMA), with the GAG ​​and methacrylated gelatin covalently crosslinked via amide bonds. Specifically, after the carboxyl groups of the GAG ​​are activated, it can react with the free amino groups in the methacrylated gelatin to form amide bonds for covalent crosslinking, and can further form non-covalent crosslinks through hydrogen bonds, ionic bonds, etc.; on the other hand, it can react with the amino and / or carboxyl groups in biological tissues to form amide bonds and strongly adhere to the tissue surface through interactions such as hydrogen bonds and ionic bonds. In other words, the crosslinking between the GAG ​​and methacrylated gelatin includes covalent crosslinking via amide bonds, and may further include non-covalent crosslinking via at least one of hydrogen bonds, ionic bonds, etc.

[0043] The glycosaminoglycans are at least one of glycosaminoglycans, glycosaminoglycan modifiers, and glycosaminoglycan depolymerization products. The structural modifiers of the aforementioned glycosaminoglycans include, but are not limited to, at least one of alkylation products, acylation products, and sulfation products of hydroxyl groups on the sugar ring of the glycosaminoglycan. Further, alkylation of hydroxyl groups includes, but is not limited to, at least one of methylation, ethylation, isopropylation, allylation, and benzylation; further, acylation of hydroxyl groups includes, but is not limited to, at least one of acetylation, propionylation, benzoylation, and phenylacetylation.

[0044] The depolymerization products of the above-mentioned glycosaminoglycans include, but are not limited to, depolymerization products prepared by free radical depolymerization, beta-elimination and / or deamination depolymerization.

[0045] It is easy to understand that the above-mentioned hydrogel biomaterial is a glycosaminoglycan-based hydrogel.

[0046] The aforementioned hydrogel biomaterial has a three-dimensional network structure, which has good water absorption and retention capacity. In addition, the hydrogel biomaterial has good biocompatibility and degradability, and can be completely degraded and absorbed during the wound healing cycle without the need for additional treatment and removal, thus avoiding secondary damage to the wound.

[0047] Furthermore, in the aforementioned hydrogel biomaterial, methacrylamide gelatin serves as the matrix, and glycosaminoglycans act as the framework and bioactive substances. This allows it to exhibit excellent anti-inflammatory effects by binding chemokines and / or cytokines, significantly accelerating wound healing without the need for any other therapeutic agents. Simultaneously, this hydrogel biomaterial can form amide bonds by reacting with amino and / or carboxyl groups on the surface of biological tissues, including human skin, thereby adhering tightly to the wound surface and conforming to irregular wounds, providing comprehensive wound protection and absorbing tissue fluid released from the wound. Thus, the aforementioned hydrogel biomaterial can slowly release pharmacologically functional glycosaminoglycans and bind with high affinity to pro-inflammatory cytokines continuously released from chronic wound tissues, effectively improving the inflammatory environment of the wound, promoting angiogenesis and re-epithelialization, and accelerating wound healing.

[0048] A hydrogel biomaterial is prepared by crosslinking pharmacologically functional glycosaminoglycans with methacrylamide gelatin. This hydrogel biomaterial has good biocompatibility and degradability, and can be completely degraded and absorbed during the wound healing cycle without the need for additional treatment or removal, thus avoiding secondary damage to the wound. This greatly improves the convenience of use and overcomes the disadvantages of applying glycosaminoglycans alone to acute and chronic wounds, such as inconvenience of administration, large dosage, and the need for continuous administration.

[0049] Compared to hydrogel formulations made from glycosaminoglycans using ordinary gelatin as an excipient, the aforementioned hydrogel biomaterial with a cross-linked three-dimensional network structure is a high-molecular cross-linked material that can adhere tightly to the wound surface and conform to irregular wounds, providing comprehensive wound protection.

[0050] In some embodiments, the glycosaminoglycans include, but are not limited to, at least one of the following: snail glycosaminoglycan (GAG from snail, SGAG), hyaluronic acid (HA), heparin (HEP), heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and fucosylate glycosaminoglycan (FG).

[0051] For example, the inventors have discovered that a novel snail glycosaminoglycan SGAG (GAG from snail) and its depolymerization derivatives can bind to chemokines with high affinity, promoting the healing of diabetic skin tissue wounds. The inventors have also disclosed that the novel snail glycosaminoglycan and its depolymerization derivatives have novel uses for treating and / or preventing diabetic complications such as diabetic foot or other skin tissue wound ulcers or slow-healing lesions. Furthermore, unlike glycosaminoglycans such as heparin and chondroitin sulfate, which have strong anticoagulant activity, snail glycosaminoglycans do not pose a risk of causing bleeding from wounds due to their lack of anticoagulant activity.

[0052] Furthermore, glycosaminoglycans that do not pose a risk of anticoagulation and bleeding are preferred; for example, snail glycosaminoglycans, hyaluronic acid, heparin sulfate, chondroitin sulfate, dermatan sulfate, and keratin sulfate are preferred, among one or more.

[0053] In some embodiments, crosslinking is photo-triggered crosslinking, where methacrylamide gelatin and glycosaminoglycans can rapidly gel under phototriggered conditions.

[0054] Furthermore, the aforementioned hydrogel biomaterial can be a wet gel or a dry gel. Wet gel and dry gel are relative terms; a wet gel refers to the state in which the aforementioned hydrogel biomaterial adsorbs solvents such as biocompatible media, while a dry gel refers to the state in which the hydrogel biomaterial adsorbing solvents such as biocompatible media has been dried to remove the solvents. Specifically, the drying is preferably freeze-drying, and the resulting dry gel is a freeze-dried gel, which is a freeze-dried sponge material with a porous sponge-like structure.

[0055] In some embodiments, the preparation method of the above-mentioned hydrogel biomaterial includes the following steps S10 to S50.

[0056] Step S10: Dissolve glycosaminoglycans in a biocompatible medium to obtain a glycosaminoglycan solution.

[0057] Step S20: Add a carboxyl activating reagent to the glycosaminoglycan solution, mix well, and obtain solution A.

[0058] Furthermore, the carboxyl activating agent includes, but is not limited to, one or more of the following: carbodiimide activating agents such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); triazine activating agents such as 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM); ononium salt activating agents such as 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU); and carbonyl diimidazole activating agents such as N,N'-carbonyldiimidazole (CDI). More specifically, EDC and NHS are preferred as the carboxyl activating agents.

[0059] Step S30: Dissolve methacrylamide gelatin in a biocompatible medium to obtain a methacrylamide gelatin solution.

[0060] Furthermore, the biocompatible media in steps S10 and S30 can be selected from one or more of water, cell culture medium, and physiological buffer; wherein the physiological buffer can be selected from one or more of physiological saline, phosphate buffer, Tris buffer, citrate buffer, blood, plasma, and platelet-rich plasma. The water can be distilled water or deionized water.

[0061] Step S40: Add a photoinitiator to the methacrylamide gelatin solution and mix well to obtain solution B.

[0062] Furthermore, the photoinitiator is typically from the Irgacure series, such as 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (Irgacure 2959), 2-hydroxy-2-methylphenylpropanone (Irgacure 1173); one or more of phenyl-(2,4,6-trimethylbenzoyl-lithium phosphate (LAP), 2',4',5',7'-tetrabromofluorescein disodium salt (eosin Y) and riboflavin (vitamin B2), preferably LAP.

[0063] Step S50: Mix solution A and solution B to obtain a hydrogel precursor solution, and then crosslink it under light to obtain the above-mentioned hydrogel material.

[0064] Furthermore, the wavelength of light used is 320–600 nm.

[0065] Furthermore, in some embodiments, step S60 is included, in which the hydrogel biomaterial is dried by means of freeze-drying or other methods to form a dry gel.

[0066] The above-mentioned method for preparing hydrogel biomaterials is low in cost, simple to operate, highly practical, and has a wide range of applications. The hydrogel precursor solution can rapidly gel in situ under humid conditions, exhibiting excellent gel rheological and biomechanical properties, safety, non-toxicity, good biocompatibility, and complete biodegradability.

[0067] The three-dimensional structure of the aforementioned hydrogel biomaterials is similar to that of the extracellular matrix. Rheological tests of the hydrogels show that their energy storage and loss moduli are close to those of human soft tissue. Furthermore, by adjusting the composition ratio of glycosaminoglycans and methacrylamide gelatin, hydrogels with different rheological properties can be prepared.

[0068] In some embodiments, the raw materials for preparing the above-mentioned hydrogel biomaterials, by mass parts, comprise 0.375 to 5 parts of glycosaminoglycans and 0.5 to 10 parts of methacrylamide gelatin. Further, the raw materials for preparing the above-mentioned hydrogel biomaterials comprise 0.35 to 1.5 parts or 0.75 to 1.5 parts of glycosaminoglycans; further still, the methacrylamide gelatin comprises 2 to 8 parts by mass, more preferably 4 to 8 parts.

[0069] In some of these embodiments, the degree of methacrylation substitution of the methacrylamide gelatin is 10% to 100%.

[0070] In some embodiments, the mass-to-volume ratio of glycosaminoglycans in the hydrogel precursor solution is 0.37% to 5% (g / mL); and / or, the mass-to-volume ratio of methacrylamide gelatin in the hydrogel precursor solution is 0.5% to 10% (g / mL), more specifically 2% to 8% (g / mL).

[0071] Furthermore, the mass-to-volume ratio of glycosaminoglycans in the hydrogel precursor solution can be 0.375% (g / mL), 0.5% (g / mL), 0.7% (g / mL), 1% (g / mL), 1.2% (g / mL), 1.5% (g / mL), 2% (g / mL), 3% (g / mL), or 5% (g / mL), preferably 0.75% to 1.5% (g / mL).

[0072] Furthermore, the mass-to-volume ratio of methacrylamide gelatin in the hydrogel precursor solution can be 0.5% (g / mL), 1% (g / mL), 2% (g / mL), 3% (g / mL), 4% (g / mL), 5% (g / mL), 6% (g / mL), 7% (g / mL), 8% (g / mL), 9% (g / mL), or 10% (g / mL), preferably 4% to 8% (g / mL).

[0073] Taking the adhesion of the above-mentioned hydrogel biomaterial to pig casings or pig skin as an example, mechanical parameters such as tensile force, compressive force and peel force were tested respectively. With or without water, plasma or blood, it was proved that the above-mentioned hydrogel biomaterial forms a strong adhesion to biological tissues including casings, skin and so on, and the presence of water and blood does not affect this adhesion force.

[0074] Cell culture experiments were conducted using the raw materials glycosaminoglycans and methacrylamide gelatin as cell culture media for the aforementioned hydrogel biomaterials. Cell viability was consistently above 80% for all hydrogel components at different concentrations and time points, demonstrating no cytotoxicity. Subcutaneous embedding of glycosaminoglycan-based hydrogels in animals and observation of the hydrogel degradation process, along with photographic observation and hematoxylin and eosin (H&E) staining results, showed that the aforementioned hydrogel biomaterials did not induce subcutaneous tissue inflammation and were completely degraded within 14–21 days, a degradation process that closely coincides with wound healing time. Therefore, cell and animal experiments confirm that the aforementioned hydrogel biomaterials possess excellent biocompatibility and degradability, exhibiting significant advantages compared to clinically used fibrin glue and cyanoacrylates (such as medical 504 and 508).

[0075] Through multi-level pharmacological and pharmacodynamic studies at the cellular, tissue, and animal levels, the inventors of this invention have surprisingly discovered that the aforementioned hydrogel biomaterial not only effectively closes wounds but also exhibits significant anti-inflammatory activity and accelerates vascular remodeling, collagen deposition, and epidermal regeneration in wound tissues, including chronic diabetic wounds, thus enabling sustained and effective wound healing. For example, cell and animal experiments have confirmed that the aforementioned hydrogel biomaterial accelerates wound healing by regulating macrophage polarization without the presence of any other therapeutic drugs. This hydrogel biomaterial adheres tightly to the wound surface and conforms to irregular wounds, providing comprehensive wound protection and absorbing tissue fluid released from the wound. It slowly releases pharmacologically functional glycosaminoglycans and binds with high affinity to pro-inflammatory cytokines continuously released from chronic wound tissues, thereby effectively improving the inflammatory environment of the wound, promoting angiogenesis and re-epithelialization, and accelerating wound healing. Simultaneously, it possesses good biocompatibility and degradability, being completely degraded and absorbed during the wound healing cycle without requiring additional treatment or removal to avoid secondary damage to the wound.

[0076] Diabetic skin ulcers, including diabetic foot, are a common high-risk complication in diabetic patients. These ulcers affect patients to varying degrees, and in severe cases, can lead to amputation, posing a serious health threat and imposing a heavy socioeconomic burden. However, due to the complexity of their pathological mechanisms, there is an urgent clinical need for safe and effective treatments. The hydrogel biomaterial described in this invention can significantly promote the healing of skin wounds and / or lesions and / or ulcers, including diabetic foot, thus possessing significant potential for the prevention and / or treatment of diabetic skin ulcers.

[0077] In other words, one embodiment of the present invention provides the application of the above-mentioned hydrogel biomaterial in the preparation of wound tissue repair drugs. Various wound healing and wound closure agents described herein can be used to promote the healing and closure of various wounds.

[0078] In some embodiments, wound tissue repair may be for skin trauma repair; further, wound tissue repair may be for diabetic foot and / or other non-healing wounds of the skin tissue.

[0079] The aforementioned hydrogel biomaterials can absorb tissue fluid permeation and can be further used to prepare tissue fluid permeation sealing products.

[0080] Furthermore, wound tissue repair drugs or tissue fluid penetration sealing products can be used as medical dressings. The aforementioned hydrogel biomaterials can be formulated into medical dressings, using them as the active ingredient. In other words, this invention also provides a medical dressing whose active ingredient includes the aforementioned hydrogel biomaterials. Medical dressings made from the aforementioned hydrogel biomaterials are convenient to administer, and these hydrogel biomaterials have good biocompatibility and degradability, allowing them to be completely degraded and absorbed during the wound healing cycle without requiring additional treatment or removal, thus avoiding secondary damage to the wound. This greatly improves ease of use and overcomes the disadvantages of using glycosaminoglycans alone in acute and chronic wounds, such as inconvenient administration, large dosage, and the need for continuous administration.

[0081] The hydrogel biomaterial in the aforementioned medical dressing can be a wet gel or a dry gel. The hydrogel biomaterial in the aforementioned medical dressing is a lyophilized gel. Further, the aforementioned medical dressing is a hydrogel patch; even further, it is a lyophilized patch.

[0082] The applications of the aforementioned hydrogel biomaterials are not limited to these. They can also be used as carriers for drugs, proteins, and / or cells to prepare sustained-release formulations, thereby promoting wound healing. They can be widely used in tissue engineering and regenerative medicine, such as in cosmetic applications.

[0083] Furthermore, the aforementioned drugs include, but are not limited to, vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF) drug molecules.

[0084] One embodiment of the present invention also provides a method for treating or cosmetically pleasing wounds or lesions, which uses the above-mentioned hydrogel biomaterial.

[0085] The observation results of culturing mouse fibroblasts using the above-mentioned glycosaminoglycan-based hydrogel biomaterial as a matrix showed that the cells grew and survived well, indicating that photocrosslinked glycosaminoglycan-based hydrogels can be used as a cell culture matrix. Furthermore, the above-mentioned glycosaminoglycan-based hydrogel biomaterial contains glycosaminoglycans, and because glycosaminoglycans are rich in functional groups such as hydroxyl, carboxyl, and sulfate groups, they can form hydrogen bonds, ionic bonds, and other interactions with small molecule drugs, proteins, nucleic acids, and cells. Therefore, glycosaminoglycan-based hydrogel biomaterials can serve as carriers for loading functional components such as drugs, proteins, nucleic acids, and cells. These chemical bonds, being reversible, facilitate slow release.

[0086] Therefore, the above-mentioned glycosaminoglycan-based hydrogel biomaterials have applications in the preparation of wound tissue repair drugs, tissue fluid permeation sealing products, and as carriers of drugs, proteins and / or cells in tissue engineering and regenerative medicine.

[0087] In summary, the glycosaminoglycan-based hydrogel biomaterials of this invention have the following innovations compared with the prior art:

[0088] (1) The above-mentioned glycosaminoglycan-based hydrogel biomaterials are prepared by photochemical cross-linking of glycosaminoglycans and methacrylamide gelatin. Their three-dimensional structure is similar to that of extracellular matrix, their biomechanical properties are close to those of human soft tissue, and they have suitable rigidity, elasticity, deformability and strong adhesion, as well as good biocompatibility and biodegradability.

[0089] (2) The chemical structure, composition, degradation time, adhesion strength, thickness, and shape of the above-mentioned glycosaminoglycan-based hydrogel biomaterials are all adjustable, allowing for flexible adjustment of the composition and properties of the gel material according to different applications. Moreover, because it can rapidly form gel in situ on the wound surface, it is suitable for the closure and healing of wounds with irregular shapes.

[0090] (3) The above-mentioned glycosaminoglycan-based hydrogel biomaterials have significant anti-inflammatory activity and promote vascular reconstruction and epidermal regeneration of chronic wound tissues. They can continuously and effectively heal chronic wounds and gradually degrade during the wound healing period to avoid secondary damage when changing medications.

[0091] (4) The above-mentioned glycosaminoglycan-based hydrogel biomaterials can significantly promote the healing of chronic wounds by effectively guiding the polarization of macrophages to the M2 mode. They can accelerate wound healing and angiogenesis at the wound site without the presence of any drugs, exogenous growth factors, or seed cells, and have extremely high practical application value. At the same time, the above-mentioned hydrogel biomaterials can also be used as carriers for drugs, proteins, and cells in biomedicine, including but not limited to soft tissue engineering and regeneration.

[0092] To make the objectives, technical solutions, and advantages of this invention clearer and more concise, the invention is described using the following specific embodiments, but the invention is by no means limited to these embodiments. The embodiments described below are merely preferred embodiments of the invention and can be used to describe the invention, but should not be construed as limiting the scope of the invention. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the protection scope of this invention.

[0093] To better illustrate the present invention, the following embodiments are provided for further explanation. The specific embodiments are as follows.

[0094]

Example 1

[0095] 1.1 Experimental Materials

[0096] 30% methacryloyl-substituted gelatin (GelMA30) and 100% methacryloyl-substituted gelatin (GelMA100), Suzhou Yongqinquan Intelligent Equipment Co., Ltd. GelMA retains the advantages of ordinary gelatin, such as good biocompatibility and stable properties, while being able to rapidly form hydrogel materials by initiating a cross-linking reaction with a phototriggered agent under ultraviolet and / or visible light. Furthermore, the mechanical properties and degradation rate of the formed hydrogel can be adjusted with changes in its degree of substitution and solid content, and it has been widely used in the preparation of bioengineering materials. Snail glycosaminoglycan, with a weight-average molecular weight of 366 kDa, was isolated and purified from the white jade snail by the applicant and can be called white jade snail glycosaminoglycan (the snail Achatina). Frucica (GAG, AFG) is a monosaccharide composed of D-acetylglucosamine and L-iduronic acid in a molar ratio of 1:(1±0.3). The monosaccharides are linked by alternating α(1→4) glycosidic bonds to form a polysaccharide chain. Heparin (HEP), from Sigma-Aldrich, has a weight-average molecular weight of 16.2 kDa. Hyaluronic acid (HA), from Bloomage Freda Biopharmaceutical Co., Ltd., has a weight-average molecular weight of 200-400 kDa.

[0097] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), analytical grade, Shanghai Yuanye Biotechnology Co., Ltd.; phenyl-(2,4,6-trimethylbenzoyl)-lithium phosphate (LAP), chemically pure, Suzhou Yongqinquan Intelligent Equipment Co., Ltd.

[0098] 1.2 Preparation process of glycosaminoglycan-based hydrogels

[0099] Glycosaminoglycans, including snail glycosaminoglycan AFG, hyaluronic acid HA, and heparin HEP ​​(denoted as A1, A2, and A3 respectively), and gel matrices represented by gelatin with 30% and 100% methacryloyl substitution (GelMA30, B1 and GelMA100, B2), were used to prepare glycosaminoglycan-based hydrogels by photochemical crosslinking.

[0100] Solution A and solution B were prepared according to the parameters of glycosaminoglycans and methacrylamide gelatin in Table 1. Taking A1B1 in Table 1 as an example, which is 0.75% snail glycosaminoglycans and 30% substituted GelMA (8%), the specific experimental steps are described as follows: Weigh 15 mg of glycosaminoglycans and dissolve them in 1 mL of PBS; then add 17.18 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 9.73 mg of N-hydroxysuccinimide (NHS) as carboxyl activating agents, mix well, and obtain solution A. Weigh 160 mg of methacrylamide gelatin (GelMA) and dissolve it in 1 mL of PBS; add 3.2 mg of photoinitiator LAP to the resulting solution, mix well in a 40°C water bath, and obtain solution B. To prepare a photo-triggered crosslinked glycosaminoglycan-based hydrogel material, mix solutions A and B in a 1:1 ratio. Irradiate the solution with light at 405 nm.

[0101] The light-triggered crosslinked glycosaminoglycan-based hydrogel material prepared according to the above method can be freeze-dried into freeze-dried patches or freeze-dried sponge materials after freeze-drying.

[0102] Table 1. Preparation of photocrosslinked glycosaminoglycan-based hydrogels

[0103]

[0104] Note: The table uses A1 and B1 as examples of 0.75% / 2%, which means that the mass-volume ratio of A1 in the hydrogel precursor solution is 0.75% (g / mL), i.e., 7.5 mg / mL; and the mass-volume ratio of B1 is 2% (g / mL), i.e., 2 mg / mL.

[0105] The above 18 groups of hydrogel precursor solutions were respectively subjected to 405 nm (20 mW / cm).2 Hydrogels with different chemical compositions can be obtained by irradiating them for a certain period of time under certain conditions.

[0106] The results showed that gelation time was related to the solid content of GelMA; the higher the solid content of GelMA, the shorter the gelation time. However, when GelMA was present alone, the gelation time was relatively long at solid contents of 4% and 2%, approximately 50 minutes or longer. When glycosaminoglycans were added to GelMA, the gelation time was significantly shortened. At a solid content of 8%, a solid hydrogel was formed within 10 seconds and adhered strongly to the soft tissue surface; at a solid content of 4%, it was formed within 35 seconds; and at a solid content of 2%, it was formed within 260 seconds. Furthermore, different gel materials exhibited different biological effects, allowing for the targeted selection of gel material composition based on specific applications.

[0107] [Example 2] Rheological properties of glycosaminoglycan-based hydrogels

[0108] Glycosaminoglycan-based hydrogels with compositions shown in Table 2 were prepared, including AFG-GelMA30, HA-GelMA30, HEP-GelMA30, AFG-GelMA100, HA-GelMA100, and HEP-GelMA100. Oscillatory shear experiments were conducted on an MCR302 rheometer (Anton Paar, Austria) to measure the storage modulus (G′) and loss modulus (G″) of hydrogels at different concentrations. Parallel plates were used for the experiments, and the gap was controlled automatically at 25°C. Amplitude scanning was performed first to define the linear viscoelastic region, and an appropriate strain amplitude was selected based on this region. The frequency range for frequency scanning was 10 Hz. +2 -10 -1 rad·s -1 At least three independent measurements should be performed for each concentration. Calculate 10. +1 -10 -1 rad·s -1 The average value of storage modulus and loss modulus over the frequency range.

[0109] Table 2 shows that, taking methacryloylated gelatin GelMA30 (30% methacryloyl substitution) and GelMA100 (100% methacryloyl substitution) as examples, the degree of substitution has a relatively small impact on the storage modulus and loss modulus. However, increasing the solid content of GelMA leads to a gradual increase in G′ and G″, indicating that the content of methacryloylated gelatin is a key factor affecting the storage modulus and loss modulus. At a specific solid content of GelMA, the addition of GAG can also change G′ and G″, but the magnitude of the change is smaller than that of the change in the solid content of GelMA. When the solid content of GelMA is 2% and 4%, the storage modulus and loss modulus of GAG-GelMA100 are smaller than those of GAG-GelMA30.

[0110] Table 2. Storage modulus and loss modulus of photocrosslinked glycosaminoglycan-based hydrogels

[0111]

[0112]

[0113] Regarding the GelMA solid content, the GelMA solid content is further preferably 4% to 8%. The transparent hydrogel formed by glycosaminoglycans (GAG) and methacrylamide gelatin (GelMA) has good elasticity and toughness, and its storage modulus ranges from 10 Pa (2% solid content GelMA) to 2550 Pa (8% solid content GelMA), corresponding to a Young's modulus between 0.03 and 7.5 kPa, which is close to the Young's modulus of skin (4.5 to 8 kPa), making it particularly suitable for adhesion to soft tissue surfaces, including skin.

[0114] [Example 3] Mechanical property testing of glycosaminoglycan-based hydrogels

[0115] 3.1 Adhesion strength test of glycosaminoglycan-based hydrogels

[0116] (1) Processing of pig intestines

[0117] Rinse the salted pig casings with clean water to remove the salt particles adhering to the surface, then soak them in water for 30 minutes. Cut the soaked pig casings into appropriate sizes, lay them flat on plastic wrap with the outside facing up, and dry them in an oven at 40℃. Peel the pig casings off the paper, secure them with transparent tape to the part that is stuck to the plastic wrap, and cut them into 3 cm × 1 cm strips for later use.

[0118] (2) Experimental procedure without blood smear

[0119] A pre-prepared hydrogel, consisting of A1 (0.75% by mass, g / mL), B1 (GelMA30, 2%, 4%, 8%, g / mL), and B2 (GelMA100, 2%, 4%, 8%, g / mL), prepared as described in Example 1, was added at a 1cm x 1cm position on the end of one section of the hog casing. The same section of the hog casing was overlapped in the opposite direction at the same position. The gel was then cured and bonded together using a UV curing lamp (405nm hv). The bonded hog casing was then stretched using a tensile-compression testing machine, with a force F applied. Figure 1 The tissue adhesion test shown in Figure A records the maximum tensile force required to separate pig casings.

[0120] Each gel was tested three times, and the data were statistically analyzed. For example... Figure 1 As shown in Figure B, the results of the tissue adhesion test are presented. As can be seen from Figure B, the adhesion of the AFG-GelMA hydrogel increases with the increase of the solid content of GelMA.

[0121] Using porcine fibrin glue (Guangzhou Beixiu Biotechnology Co., Ltd.) as a positive control, the adhesion of hydrogels with a solid content of 4% or higher was significantly greater than that of the positive control fibrin glue. The adhesion changes of HA-GelMA and HEP-GelMA were similar to those of AFG-GelMA and higher than those of GelMA hydrogel.

[0122] (3) The blood smear experiment

[0123] Fresh rat blood was evenly applied to the 1cm x 1cm portion of the end of a 3cm length pig casing secured with Scotch tape. Then, pre-made hydrogel containing AFG-GelMA (A1 / B1, 0.75% / 8%, g / mL) was added. The other section of pig casing was then treated as follows... Figure 1 The tissue adhesion test shown in Figure A involved overlapping the two pieces of pig casing in opposite directions at the sites where hydrogel was added. The gel was then cured by UV curing and bonded together. Figure 2 As shown in B.

[0124] A tensile-compression tester was used to stretch the bonded pig casings, and the maximum tensile force required to separate the casings was recorded. Three tests were performed.

[0125] The pre-formed hydrogel without blood and the pre-formed hydrogel with blood have the same composition and are used as a comparison. Tissue adhesion testing is performed as follows. Figure 2 As shown in A, it is related to Figure 1 Similar to A. The comparison results are as follows: Figure 2As shown in Figure C, there was no significant difference in the maximum adhesion force between the hydrogel with and without blood, indicating that the blood component does not affect the adhesion between the hydrogel and the casing.

[0126] 3.2 Peel strength test of glycosaminoglycan-based hydrogels

[0127] (1) Processing of pig intestines

[0128] Peel the pig casing off the paper, secure it with transparent tape to the part that is stuck to the plastic wrap, and cut it into 5cm × 1cm strips for later use.

[0129] (2) The blood smear experiment

[0130] Fresh rat blood was evenly applied to the end (3cm x 1cm) of one section of pig casing, followed by the application of pre-prepared hydrogel containing AFG-GelMA (A1 / B1, 0.75% / 8%, g / mL). The same section of pig casing was then overlapped in the same direction at the same location where hydrogel was added. The gel was then cured using a UV curing lamp, causing the two sections of pig casing to bond together. Figure 3 The leftmost attached image shows two pieces of sausage casing bonded together with hydrogel and coated with blood; then the casing is removed.

[0131] The bonded pig casings were stretched using a tensile and compressive strength testing machine. Figure 3 As shown in Figure B, data was recorded and analyzed using a computer after stripping, and the results are as follows. Figure 3 As shown in Figure C, the shear force of the hydrogels in the blood-smeared and un-blood-smeared environments was not significantly different, and both were greater than that of the positive control porcine fibrin glue. Among them, AFG-GelMA-B and Fibrin glue-B were blood-smeared, while AFG-GelMA-C and Fibrin glue-C were un-blood-smeared.

[0132] [Example 4] Comparison of glycosaminoglycan-binding cytokine activity

[0133] 4.1 Materials

[0134] (1) Glycosamine samples: snail glycosamine, with a weight-average molecular weight of 366 kDa, which was isolated and purified from white jade snail by the inventors and can be called white jade snail glycosamine (the snail Achatina fulica GAG, AFG); heparin HEP, Sigma-Aldrich, USA, with a weight-average molecular weight of 16.2 kDa; low molecular weight hyaluronic acid HA, Bloomage Freda Biopharmaceutical Co., Ltd., with a weight-average molecular weight of 10 kDa.

[0135] (2) Reagents and instruments: Human interleukin-6 (IL-6), Platelet factor 4 (PF4), Interferon gamma-induced protein 10 (IP-10), Human interleukin-8 (IL-8), from Abcam, USA.

[0136] Macromolecular interaction instrument, Biacore S200, from GE Healthcare, USA; CM5 chip, from GE Healthcare, USA.

[0137] 4.2 Methods

[0138] Couple the above proteins (IL-6, IL-8, PF4 and IP-10) to the CM5 chip according to the standard operation procedure, use HBS-EP buffer (0.01M HEPES, 0.15M NaCl, 3mM EDTA, and 0.5% surfactant P20 at pH 7.4) as the running buffer, detect different glycosaminoglycan samples, and analyze the results to obtain the K D value.

[0139] 4.3 Results

[0140] Table 3 Affinity of different glycosaminoglycans binding cytokines

[0141]

[0142] Table 3 results show that three representative glycosaminoglycans can all bind to four cytokines with high affinity, and the affinity constant K D Data shows that the binding ability of snail glycosaminoglycan AFG to cytokines is higher than that of heparin HEP and hyaluronic acid HA.

[0143]

Example 5

[0144] 5.1 Materials

[0145] (1) Mouse fibroblasts: NIH / 3T3, purchased from the cell bank of Kunming Institute of Zoology, Chinese Academy of Sciences.

[0146] (2) Experimental animals: All procedures of the animal experiment comply with the provisions of the "Regulations on the Administration of Laboratory Animals (Revised in 2017)". Male SD rats, 180 - 220 g, from Hunan Slack Jingda Experimental Animal Co., Ltd., license number: SCXK(Xiang)2019 - 0004. Before the experiment, the rats were placed under constant temperature and humidity conditions with a 12h light-dark cycle and adapted to the environment for at least one week.

[0147] (3) Reagents and instruments: CCK8 kit; microplate reader: Thermo Scientific Multiskan FC, Thermo Scientific, USA; MC-4000 blood coagulation analyzer, TICO GmbH, Germany.

[0148] 5.2 Methods

[0149] (1) Cytotoxicity test experiment

[0150] After suspending NIH / 3T3 cells, administer 1×10 4 Cells were densely seeded in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. Then, fresh culture medium containing different concentrations (2.0, 1.0, and 0.10 mg / mL) of single-component hydrogels (snail glycosaminoglycan AFG, hyaluronic acid HA, or methacrylamide gelatin GelMA) was added. Cells cultured normally without any treatment served as the control group.

[0151] Cell viability was assessed using a CCK8 assay and a microplate reader after 1, 2, 3, 5, and 7 days of culture. Cell viability was calculated as follows:

[0152] Cell viability (%) = (average absorbance of experimental group / average absorbance of control group) × 100%.

[0153] (2) Assessment of anticoagulant activity and bleeding risk

[0154] Four types of hydrogels, namely AFG-GelMA, HA-GelMA, HEP-GelMA and GelMA (GelMA is 30% methacrylic acid, abbreviated as G30; 1% / 4%, g / mL unless otherwise specified), were ground in a mortar and dissolved in 1 mL of physiological saline. After centrifugation at 12000g for 5 minutes, the supernatant was collected, and the activated partial thromboplastin time (APTT) activity was detected according to the standard operating procedure.

[0155] (3) Subcutaneous embedding degradation experiment

[0156] SD rats were randomly divided into 6 groups of 8 rats each: sham-operated group (control group), AFG-G30 group, HA-G30 group, HEP-G30 group, G30 group, and medical 504 glue (α-cyanoacrylate, abbreviated as CA, Beijing Kangpaite Medical Devices Co., Ltd.) control group. Hydrogels (1.5 cm in diameter) were embedded subcutaneously in each group. The wounds were opened on days 3, 7, 14, and 18 to observe and photograph the residual hydrogel. Wound tissue was paraffin-embedded, sectioned, and analyzed using hematoxylin-eosin (H&E) staining.

[0157] 5.3 Results

[0158] The results are as follows Figure 4 As shown, the horizontal axis represents time (in days), and the vertical axis represents cell viability. The nine adjacent bars, from left to right, represent AFG at concentrations of 2.0 mg / mL, 1.0 mg / mL, and 0.10 mg / mL (labeled AFG-2, AFG-1, and AFG-0.1), HA at concentrations of 2.0 mg / mL, 1.0 mg / mL, and 0.10 mg / mL (labeled HA-2, HA-1, and HA-0.1), and G30 at concentrations of 2.0 mg / mL, 1.0 mg / mL, and 0.10 mg / mL (labeled G30-2, G30-1, and G30-0.1).

[0159] The cell survival rate of all hydrogel components at different concentrations and time points was greater than 80%, indicating that they were not cytotoxic and confirming that the hydrogel described in this invention has good cell compatibility.

[0160] Figure 5 The anticoagulant activity of glycosaminoglycan-based hydrogels and the wound bleeding induced by heparin-based hydrogels (HEP-G30) are shown in Figures A and B, respectively. In Figure A, the vertical axis represents the coagulation time (ex vivo, min).

[0161] As shown in A, HEP-G30 hydrogel has strong anticoagulant activity and significantly prolongs APTT time (P<0.001). However, the APTT time of HA-G30 and AFG-G30 hydrogels is not significantly different from that of the negative control (using the saline treatment group as a control) and G30, indicating that HA-G30 and AFG-G30 hydrogels have no anticoagulant activity and no risk of bleeding.

[0162] The above in vitro coagulation results are consistent with the application to animal wounds, such as... Figure 5As shown in Figure B, HEP-G30 hydrogel (HEP-GelMA) applied to the wound surface and subcutaneous embedding caused severe wound bleeding within a short period of time (less than 24 hours), while other glycosaminoglycan-based hydrogels such as AFG-G30 (AFG-GelMA), HA-G30, and G30 did not cause bleeding at the wound site.

[0163] Taking AFG-G30 (0.75% / 4%, g / mL) prepared in Example 1 as an example, and medical 504 (CA) as a positive control, it was implanted subcutaneously into rats. Histological sections were analyzed using H&E staining to observe the inflammatory response of the two biomaterials in the subcutaneous tissue at different time points. The results are as follows... Figure 6 As shown, the photocrosslinked glycosaminoglycan-based biofunctional hydrogel can be completely degraded within 14 days, leaving no residue in the body after degradation. Only very mild inflammatory reactions are occasionally observed in the early stages, with no tissue fibrosis or non-physiological hyperplasia observed. In contrast, CA was almost undegraded after 14 days, and tissues in contact with CA consistently exhibited severe inflammatory reactions.

[0164] [Example 6] Glycosaminoglycan-based hydrogels as a matrix for cell culture

[0165] 6.1 Materials

[0166] (1) Various components of photocrosslinked biofunctional hydrogels: AFG, HA and G30.

[0167] (2) Mouse fibroblasts: NIH / 3T3, purchased from the cell bank of Kunming Institute of Zoology, Chinese Academy of Sciences.

[0168] (3) Reagents and instruments: Live / dead cytotoxicity staining kit, Invitrogen, USA; LEICA TCS SP8 X confocal fluorescence microscope, Leica, Germany.

[0169] 6.2 Methods

[0170] The hydrogel precursor solution was prepared according to the method described in this invention, wherein GAG was prepared using 0.75% AFG and HA, and G30 (8% by mass, g / mL) without GAG, and DMEM (Dulbecco's modified eagle medium) containing 10% fetal bovine serum as the biocompatibility medium. NIH / 3T3 cells were suspended and then injected at 4 × 10⁻⁶. 6The solution was added to the hydrogel precursor solution at a specific density, spread onto a 96-well plate, and irradiated with 405 nm light to form a hydrogel. After incubation at 37°C in a 5% CO2 incubator for 24 h, the culture medium was discarded, the cells were washed with DuPont phosphate buffer, stained with a live / dead cell kit, and observed and photographed under a confocal microscope.

[0171] 6.3 Results

[0172] The results are as follows Figure 7 As shown, the vast majority of cells in the microscope field of view are alive (green), with only a very small number of dead cells (red). The cells are growing well, indicating that the photocrosslinked bioactive hydrogel can be used as a matrix carrier for cell culture.

[0173] [Example 7] Glycosaminoglycan-based hydrogels promote macrophage polarization toward the M2 phenotype.

[0174] 7.1 Materials

[0175] (1) Components of photocrosslinked glycosaminoglycan-based hydrogel: AFG, HA, G30, EDC and NHS, etc.

[0176] (2) Mouse macrophage cell line RAW264.7 was purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0177] (3) Reagents and instruments: Lipopolysaccharide (LPS), Sigma-Aldrich, USA; Mouse Mannose Receptor (MR, CD206) primary antibody, Abcam, UK; Alera Fluor 488 secondary antibody, Jackson, USA; 4',6-diamidinyl-2-phenylindole (DAPI) dye, Wuhan Sewell, China; LEICA TCS SP8 X confocal fluorescence microscope, Leica, Germany.

[0178] 7.2 Methods

[0179] Hydrogel precursor solutions were prepared according to the method described in this invention, using AFG and HA at a final concentration of 1% (g / mL) as an example, and G30 (8% mass concentration) without GAG as a control. The hydrogel preform was formed under 405nm light irradiation. RAW264.7 cells were suspended and then injected at 1×10⁻⁶... 5The cells were seeded onto 24-well plates at a density suitable for M2 macrophages and incubated overnight at 37°C in a 5% CO2 incubator. After incubation, the culture medium was discarded, and the cells were washed with DPBS. Then, normal culture medium (blank control, Control), culture medium containing 40 ng / mL IL-4 (positive control, IL-4), culture medium containing 100 ng / mL LPS (negative control, LPS), and culture medium containing 100 ng / mL LPS were added, along with 1 / 10 volume of pre-prepared hydrogel (experimental group). Cells were immunostained with CD206 fluorescent antibody as a marker for M2 macrophages, and observed and photographed using a confocal fluorescence microscope.

[0180] 7.3 Results

[0181] See results Figure 8 Compared with the blank control, IL4 significantly induced macrophages to convert to the M2 phenotype (CD206 marker, green; nucleus stained with DAPI, blue), while LPS significantly inhibited the M2 phenotype. G30 hydrogel without GAG had no significant inhibitory effect on LPS, while hydrogel containing GAG significantly increased the expression of CD206 on the macrophage surface and the proportion of the M2 phenotype, with AFG-G30 hydrogel showing the most significant effect. This indicates that glycosaminoglycan-based hydrogels without any anti-inflammatory drugs can significantly promote macrophage polarization towards the M2 phenotype.

[0182] During wound healing, the transformation of macrophages from the M1 phenotype to the M2 phenotype is a crucial process in the transition from the inflammatory phase to the proliferative phase. Blockage of this process can lead to chronic inflammation, hindering wound healing. The photocrosslinked glycosaminoglycan-based hydrogel prepared in this invention can effectively bind or remove inflammatory cytokines at chronic wound sites, inducing macrophages in the wound microenvironment to polarize towards the M2 phenotype, thereby significantly promoting wound healing.

[0183] [Example 8] Application of glycosaminoglycan-based hydrogels in loading and releasing protein drugs

[0184] Hydrogels are cross-linked polymer networks that swell but do not dissolve in water. Because hydrogels are mostly composed of water and possess excellent biocompatibility, they are particularly suitable as carriers for drugs and bioactive macromolecules. Drugs or bioactive macromolecules encapsulated in hydrogel materials achieve sustained drug release through molecular diffusion and material degradation. The following is a specific example of drug encapsulation and release: AFG-G30 (1% / 8%) prepared in Example 1 was dissolved in physiological saline to prepare a hydrogel precursor solution of a certain mass concentration. An appropriate amount of recombinant vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF) was added. 200 μL of the above solution was placed in a circular mold and irradiated to form a hydrogel. This hydrogel was then placed in a 24-well cell culture plate, and a certain amount of physiological saline was added for drug release experiments. The amount of drug released in the solution was analyzed by ultraviolet light to evaluate the drug release effect of the material.

[0185] The results showed that AFG-G30 hydrogel could significantly load VEGF or PDGF, and could slowly release the generating factors over time. The mechanism by which AFG-G30 hydrogel can load these generating factors is that snail glycosaminoglycan AFG is rich in carboxyl and sulfate groups. These anions can form ionic bonds with the amino groups in VEGF or PDGF, and the carboxyl or amino groups in methacrylamide gelatin can also form ionic bonds with the amino or carboxyl groups in VEGF or PDGF. Both types of ionic bonds are reversible non-covalent bonds, so it can both load VEGF or PDGF and slowly release these generating factors.

[0186] Hydrogel systems composed of other materials are also photocrosslinked biofunctional hydrogels and can be used for drug encapsulation and release.

[0187] [Example 9] Application of glycosaminoglycan-based hydrogels in the healing and repair of acute skin wounds

[0188] 9.1 Instruments

[0189] ZS-MV-IV Portable Small Animal Anesthesia Machine, Beijing Zhongshidichuang Technology Development Co., Ltd.

[0190] 9.2 Reagents and Materials

[0191] Main reagents: Isoflurane, povidone-iodine, and medical alcohol are commercially available biological or medical grade reagents.

[0192] Experimental animals: The entire process of the animal experiment complied with the regulations of the "Regulations on the Administration of Laboratory Animals (Revised in 2017)". Male Sprague-Dawley (SD) rats, weighing 180 - 220 g, were provided by Hunan Slack Jingda Experimental Animal Co., Ltd., with the license number: SCXK(Xiang)2019 - 0004. Before the experiment, the rats were placed under constant temperature and humidity conditions with a 12-hour light-dark cycle and acclimated to the environment for at least one week.

[0193] Hydrogel material: The AFG-G30(1% / 4%) hydrogel precursor solution synthesized according to the method in Example 1 of the present invention.

[0194] 9.3 Method

[0195] (1) Experimental grouping and method

[0196] Male SD rats, weighing 180 - 220 g, were evenly and randomly divided into 3 groups of 8 rats each according to body weight. Another 5 normal animals were set up for behavioral and routine physiological observations.

[0197] Blank control: Normal saline group, 90 μL / wound;

[0198] Test groups:

[0199] G30 (4% solid content) group: 90 μL / wound. After adding it to the wound, it was immediately irradiated with 405 nm light for 2 minutes to solidify the precursor solution into a hydrogel and firmly adhere it to the wound.

[0200] AFG-G30(1% / 4%) group: 90 μL / wound. After adding it to the wound, it was immediately irradiated with 405 nm light for 2 minutes to solidify the precursor solution into a hydrogel and firmly adhere it to the wound.

[0201] (2) Full-thickness dorsal skin defect model

[0202] The SD rats were anesthetized by inhaling isoflurane, the dorsal hair was removed, and two symmetrical circular wounds with a diameter of 10 mm were made 5 cm behind the posterior edge of the rat's ear. After disinfection with iodophor, the silicone washer was sutured at the wound with surgical sutures to fix the wound. 90 μL of the hydrogel precursor solution was added to the wound, irradiated with light for curing, and photographed for recording. After the operation, the rats were housed individually in cages and allowed free access to water and food.

[0203] (3) Data recording

[0204] ① From the day when the dorsal wound model was established, the behavior and routine physiological indexes of the animals were observed every day.

[0205] ② After the operation, on the 3rd, 6th, 10th, and 14th days, the wound healing and surrounding inflammation conditions of the rats were observed and recorded. The wound area was measured using Image J software and the healing rate was calculated:

[0206] Healing rate (%) = (Initial wound area – Current wound area) / Initial wound area × 100%

[0207] (4) Statistical Analysis

[0208] Wound healing rate: Data are expressed as mean ± standard deviation (x ± s). One-way ANOVA was performed on the statistical data using SPSS 17.0 software. *p<0.05, **p<0.01 and ***p<0.001 were considered to be statistically significant.

[0209] 9.4 Results

[0210] Animal behavior and physiological condition: From the date of establishment of the back wound model, the rats were observed daily. Compared with normal animals, no obvious abnormalities were found in the postoperative behavioral (activity frequency and behavioral status, etc.) and physiological (body weight, water intake, food intake, excrement, etc.) indicators of the rats.

[0211] Wound healing rate: Wounds were photographed on days 0, 3, 6, 10, and 14, and the wound healing rate was calculated. The results are shown in [the table below]. Figure 9 A and B.

[0212] Compared with the control group, the wound healing rate of the AFG-G30 experimental group was significantly higher than that of the control group (G30 group and blank Control group) starting from the 3rd day after surgery. After 14 days, the wound was completely repaired with abundant hair and no significant scars were observed, indicating that AFG hydrogel has a significant effect on promoting the healing and repair of acute wounds.

[0213] [Example 10] Application of glycosaminoglycan-based hydrogels in chronic wound healing and repair

[0214] 10.1 Instruments

[0215] ZS-MV-IV portable small animal anesthesia machine, Beijing Zhongshidichuang Technology Development Co., Ltd.; Rapid tissue pathology morphology section staining system, including paraffin embedding machine, microtome and curing machine, Thermo Scientific, USA.

[0216] 10.2 Reagents and Materials

[0217] Main reagents: streptozotocin (STZ), Sigma-Aldrich, USA; hematoxylin-eosin staining solution, neutral resin and anti-detachment slides, etc., Changsha Aijia Biotechnology Co., Ltd.; isoflurane, povidone-iodine, chloral hydrate, xylene and anhydrous ethanol, etc., are commercially available biological or medical grade reagents.

[0218] Experimental animals: All procedures of the animal experiments complied with the regulations of the "Regulations on the Administration of Laboratory Animals (Revised in 2017)". SD rats, male, 300 - 350 g, Hunan Slack Jingda Experimental Animal Co., Ltd., license number: SCXK(Xiang)2019 - 0004. Before the experiment, the rats were placed under constant temperature and humidity conditions with a 12 - hour light - dark cycle and were allowed to acclimatize to the environment for at least one week.

[0219] Hydrogel materials: According to the preparation method of the glycosaminoglycan - based hydrogel described in the present invention, three glycosaminoglycan - based hydrogels, AFG - G30, HA - G30 and HEP - G30 (1% / 4%), and hydrogel G30 prepared only with methacrylated gelatin without the presence of glycosaminoglycan were prepared respectively.

[0220] 10.3 Methods

[0221] (1) Establishment of diabetic rat model

[0222] Rats were intraperitoneally injected with STZ at a dose of 55 mg / kg. One week later, blood was collected from the tail vein to detect the blood glucose of the rats. If the random blood glucose ≥ 16.7 mmol / L, the establishment of the diabetic model was successful.

[0223] (2) Experimental grouping and methods

[0224] The diabetic rats were evenly and randomly divided into 5 groups with 8 rats in each group. Another 5 normal animals were set for behavioral and routine physiological observations.

[0225] Blank control (control): Normal saline group, 90 μL / wound (90 μL for each wound).

[0226] Test groups:

[0227] AFG - G30 (1% / 4%, g / mL) group: 90 μL / wound. After adding it to the wound, it was immediately irradiated with 405 nm light for 2 min to solidify the precursor solution into a hydrogel and firmly adhere it to the wound;

[0228] HA - G30 (1% / 4%, g / mL) group: 90 μL / wound. After adding it to the wound, it was immediately irradiated with 405 nm light for 2 min to solidify the precursor solution into a hydrogel and firmly adhere it to the wound;

[0229] HEP - G30 (1% / 4%, g / mL) group: 90 μL / wound. After adding it to the wound, it was immediately irradiated with 405 nm light for 2 min to solidify the precursor solution into a hydrogel and firmly adhere it to the wound;

[0230] G30 (4% solids content, g / mL) group: 90 μL / wound. After being added to the wound, it was immediately irradiated with 405 nm light for 2 min to solidify the precursor solution into a hydrogel and firmly adhere it to the wound.

[0231] (3) Diabetic ulcer modeling

[0232] Diabetic rats were anesthetized with isoflurane inhalation. The fur on their backs was shaved, and two symmetrical circular incisions, each 10 mm in diameter, were made 5 cm posterior to the ear. The wounds were disinfected with povidone-iodine, and silicone gaskets were sutured to the wounds with surgical sutures to fix them in place. 90 μL of hydrogel precursor solution was applied to the wounds, and the wounds were cured by light and photographed. Post-operatively, the rats were housed individually with free access to water and food.

[0233] (4) Data Recording

[0234] ①From the date the diabetic ulcer model was established, the animals' behavior and routine physiological indicators were observed daily;

[0235] ② Postoperatively, wound healing and surrounding inflammation in rats were observed and recorded on days 3, 6, 10, and 14. The wound area and healing rate were calculated using ImageJ software.

[0236] ③ On days 3, 6, 10 and 14 after treatment, the patients were euthanized under isoflurane anesthesia, and wound tissue specimens were collected. Some specimens were fixed in 4% paraformaldehyde solution, routinely embedded in paraffin, sectioned and stained with H&E. CD31 and α-SMA were used as markers of neovascularization for immunofluorescence staining to assess edema, inflammation, reepithelialization and angiogenesis in the wound tissue.

[0237] (5) Statistical Analysis

[0238] Wound healing rate: Data are expressed as mean ± standard deviation. The data were analyzed using SPSS 17.0 software. One-way ANOVA was performed, with *p<0.05, **p<0.01 and ***p<0.001 considered as significant differences.

[0239] Histopathological scores: Data are expressed as mean ± standard deviation. KW rank and nonparametric tests were used, and *p<0.05 was considered statistically significant.

[0240] 10.4 Results

[0241] Animal behavioral changes: Since the establishment of the diabetes model, daily observations have shown no obvious abnormalities in behavioral (activity frequency and behavioral status, etc.) or physiological (body weight, water intake, food intake, excrement, etc.) indicators in diabetic rats.

[0242] Peri-wound inflammation and wound healing rate: Peri-wound inflammation in all groups of rats was significantly reduced on day 3. Figure 9 and Figure 10 Wound healing photographs and healing rates for each group are displayed separately. The AFG-G30 and HA-G30 groups showed significant healing-promoting effects starting on day 10, with significant differences compared to the control group and the G30 group, and the AFG-G30 group showing a highly significant difference (P<0.001). The G30 group showed a promoting effect compared to the control group, but its healing rate was significantly lower than that of the AFG-G30 and HA-G30 groups. HEP-G30 caused severe bleeding in the wound shortly after treatment and is not suitable for use on traumatic wounds.

[0243] Pathological section analysis: Immunofluorescence staining of H&E, CD31, and α-SMA was performed on the skin wounds and surrounding tissues of rats on postoperative days 6, 10, and 14. Histopathological blind scoring was performed based on epidermal regeneration and angiogenesis. Results are shown in […]. Figure 11 and Figure 12 Compared with the G30 group, AFG-G30 hydrogel significantly promoted reepithelialization of wound tissue. Figure 11 ), can significantly promote angiogenesis after day 6. Figure 12 (P<0.05) HA-G30 hydrogel can also significantly promote angiogenesis.

[0244] Figure 12 In A, red fluorescence indicates α-SMA positivity, green fluorescence indicates CD31 positivity, blue fluorescence indicates DAPI positivity, and both red and green fluorescence (Merge) represent neovascularization.

[0245] The above results confirm that glycosaminoglycan-based hydrogels, represented by AFG and HA, have a significant function in promoting the healing and repair of diabetic wounds.

[0246] [Example 11] Glycosaminoglycan-based hydrogels significantly reduced the expression of inflammatory factors in wound tissue of diabetic rats.

[0247] 11.1 Materials

[0248] (1) Reagents and instruments: ABI QuantStudio 7 Flex real-time PCR instrument, ABI Corporation, USA; Bio-Plex 200 suspension chip system, Bio-Rad Corporation, USA; qPCR real-time reagent kit, Nanjing Novizan Biotechnology Co., Ltd.; Factor 8 detection kit, including cytokines such as IL-6, TNF-α, IP-10, and IL-1β, R&D Corporation, USA.

[0249] (2) Biological samples: wound tissues of diabetic rats in each group of experiments in Example 10 of this invention.

[0250] 11.2 Methods

[0251] Wound tissues from diabetic rats were homogenized on postoperative days 3, 10, and 14. RNA and protein were extracted according to standard procedures. Gene expression levels were analyzed using a qPCR quantitative PCR kit, and cytokine protein expression levels were detected using an eight-factor detection kit.

[0252] 11.3 Results

[0253] The results are as follows Figure 13 , Figure 14 and Figure 15 As shown, compared with the blank control group and the G30 group, the glycosaminoglycan-based hydrogel treatment group significantly inhibited the gene and protein expression levels of pro-inflammatory factors IL-6, TNF-α, IP-10 and IL-1β, with the most significant inhibition on days 3 and 10 (P<0.05); on day 14, the expression level of pro-inflammatory cytokines in the AFG-G30 hydrogel treatment group was close to that in normal skin tissue (the expression level of pro-inflammatory cytokines protein in unwound skin tissue, Unwound), indicating that the wound in this group had completely healed without inflammatory response.

[0254] The technical features of the above embodiments can be combined in any way. For the sake of brevity, 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, they should be considered to be within the scope of this specification.

[0255] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A hydrogel biomaterial, characterized in that, The hydrogel biomaterial has a three-dimensional network structure formed by crosslinking carboxyl-activated glycosaminoglycans and methacrylamide gelatin. The crosslinking is photo-triggered crosslinking. The crosslinking between the glycosaminoglycans and methacrylamide gelatin includes amide covalent crosslinking. The glycosaminoglycans are at least one of glycosaminoglycans, structural modifiers of glycosaminoglycans, and depolymerization products of glycosaminoglycans. The glycosaminoglycans are selected from at least one of snail glycosaminoglycans and hyaluronic acid.

2. The hydrogel biomaterial as described in claim 1, characterized in that, The structural modifiers of the glycosaminoglycan include at least one of the following: alkylated products, acylated products, and sulfated products of the hydroxyl groups on the sugar ring of the glycosaminoglycan.

3. The hydrogel biomaterial as described in claim 1, characterized in that, The depolymerization products of the glycosaminoglycans include those prepared by free radical depolymerization, beta-elimination, and / or deamination depolymerization.

4. The hydrogel biomaterial as described in claim 2, characterized in that, The alkylation of the hydroxyl group is at least one of methylation, ethylation, isopropylation, allylation, and benzylation, and the acylation of the hydroxyl group is at least one of acetylation, propionylation, benzoylation, and phenylacetylation.

5. The hydrogel biomaterial according to any one of claims 1 to 4, characterized in that, The degree of methacrylation substitution of the methacrylated gelatin is 10% to 100%.

6. The hydrogel biomaterial according to any one of claims 1 to 4, characterized in that, In the raw materials for preparing the hydrogel biomaterial, the glycosaminoglycans account for 0.375 to 5 parts by mass, and the methacrylamide gelatin accounts for 0.5 to 10 parts by mass.

7. The hydrogel biomaterial according to claim 6, characterized in that, In the raw materials for preparing the hydrogel biomaterial, the glycosaminoglycans account for 0.375 to 1.5 parts by mass, and the methacrylamide gelatin accounts for 2 to 8 parts by mass.

8. The method for preparing hydrogel biomaterials as described in claim 1, characterized in that, Includes the following steps: A solution A is obtained by dissolving a glycosaminoglycan in a biocompatible medium and adding a carboxyl activator; the glycosaminoglycan is at least one of glycosaminoglycan, a structural modifier of glycosaminoglycan, and a depolymerization product of glycosaminoglycan, wherein the glycosaminoglycan is selected from at least one of snail glycosaminoglycan and hyaluronic acid; Methacrylamide gelatin was dissolved in a biocompatible medium and a photoinitiator was added and mixed to obtain solution B; The hydrogel precursor solution was obtained by mixing the solution A and the solution B, and then crosslinked by light.

9. The application of the hydrogel biomaterial as described in any one of claims 1 to 7 in the preparation of wound tissue repair drugs or tissue fluid permeation sealing products, or as a carrier for drugs, proteins and / or cells in the preparation of sustained-release formulations.

10. A wound tissue repair drug, characterized in that, It comprises the hydrogel biomaterial as described in any one of claims 1 to 7.

11. A tissue fluid permeation sealing product, characterized in that, It comprises the hydrogel biomaterial as described in any one of claims 1 to 7.

12. A sustained-release formulation, characterized in that, The hydrogel biomaterials described in any one of claims 1 to 7 are used as carriers for drugs, proteins and / or cells.

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