Selenium-loaded photo-crosslinking alginate hydrogel and preparation method thereof

Abstract

The invention discloses selenium-loaded photo-crosslinking alginate hydrogel and a preparation method thereof, and relates to the technical field of polymer gel. The preparation method comprises the following steps: dissolving alginate in deionized water, adding sodium chloride and a buffer agent, stirring until the sodium chloride and the buffer agent are completely dissolved, adjusting the pH value to weak acidity, then adding N-hydroxy-thiosuccinimide and a condensing agent, uniformly stirring, and then adding 2-aminoethyl methacrylate hydrochloride to carry out a methacrylic acid esterification reaction, so as to obtain the methacrylic acid esterified alginate. Respectively dissolving the selenomethionine and a photoinitiator in deionized water to prepare a storage solution, and dissolving selenomethionine in the photoinitiator storage solution; and finally, mixing a photoinitiator solution containing selenomethionine with the methacrylic acid esterified alginate storage solution, and initiating a photo-crosslinking reaction through ultraviolet irradiation to prepare the selenium-loaded photo-crosslinked alginate hydrogel. According to the invention, selenomethionine is introduced into the photo-crosslinking alginate hydrogel scaffold, so that the photo-crosslinking alginate hydrogel scaffold has good cell biocompatibility and an osteogenesis promoting effect.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer gels, and specifically to a selenium-loaded photocrosslinkable alginate hydrogel and a preparation method thereof. Background Art

[0002] In the continuous development process of the biomedical materials field, hydrogels have become a research hotspot due to their unique properties. Hydrogels have high hydration properties, can absorb a large amount of water and maintain the stability of their own structure. This property endows them with good biocompatibility and can effectively reduce the irritation to surrounding tissues. Their three-dimensional network structure not only provides a microenvironment similar to the extracellular matrix for cell adhesion, proliferation and differentiation, but also can be used as a carrier to achieve the loading and release of substances such as drugs and bioactive molecules, showing great application potential in many medical fields such as drug delivery, wound dressings, wound repair and tissue engineering.

[0003] Alginate, as a natural polymer material extracted from marine algae, occupies an important position in the preparation of hydrogels due to its outstanding biodegradability and biocompatibility. Alginate hydrogels can be gradually degraded in vivo by enzymatic hydrolysis or hydrolysis, and their degradation products are non-toxic small molecule substances that can be naturally metabolized and excreted by the human body without accumulating in the body and causing adverse effects. At the same time, the abundant active groups on the alginate molecule can interact with the receptors on the cell surface, promote cell adhesion and growth, and provide physical support and bioactive signals for tissue repair.

[0004] In recent years, fruitful results have been achieved in the research of trace element selenium in the biomedical field. Selenium is an essential trace element for the human body and participates in a variety of important physiological processes in the body. From the perspective of antioxidant, selenium is a key component of antioxidant enzymes such as glutathione peroxidase, which can catalyze reduction reactions, scavenge excessive reactive oxygen species and free radicals in the body, protect cells from oxidative damage, and maintain the intracellular redox balance. In terms of immune regulation, selenium has an important impact on the development, maturation and function of immune cells. It can enhance the activities of T lymphocytes, B lymphocytes and macrophages, regulate the synthesis and secretion of immunoglobulins, and improve the body's immune response ability. During the inflammatory reaction process, selenium can inhibit the release of inflammatory cytokines such as tumor necrosis factor and interleukin, reduce the infiltration of inflammatory cells, and thus play an anti-inflammatory role. Especially in bone metabolism, selenium has a regulatory effect on the activities of osteoblasts and osteoclasts, can promote the proliferation and differentiation of osteoblasts, inhibit the excessive resorption of osteoclasts, maintain the normal metabolism and reconstruction of bone tissue, and play an indispensable role in the growth, repair and bone mass maintenance of bone tissue.

[0005] However, despite the certain progress made in the research and application of hydrogels, such as the existing preparation methods of drug-loaded hydrogels opening up new paths for hydrogels in drug delivery. However, in the research of numerous drug-loaded hydrogels, the introduction of selenium has not been effectively studied. Summary of the Invention

[0006] The object of the present invention is to provide a selenium-loaded photo-crosslinked alginate hydrogel and its preparation method to solve the technical problems raised in the above background technology. The present invention introduces selenomethionine into the photo-crosslinked alginate hydrogel scaffold, which is beneficial to the adhesion and spreading of surface cells, improves the osteogenic activity of osteoblasts, and has a good osteogenic promoting effect.

[0007] To achieve the above object, the present invention provides the following technical solutions: A preparation method of a selenium-loaded photo-crosslinked alginate hydrogel, comprising the following steps: S1. Dissolve alginate in deionized water, add sodium chloride and a buffer, stir until completely dissolved, adjust the pH value of the solution to weakly acidic, then add N-hydroxy-thiobutanimide and a condensing agent, stir evenly, add 2-aminoethyl methacrylate hydrochloride, and carry out methacrylation reaction to obtain methacrylated alginate; S2. Dissolve methacrylated alginate in deionized water to obtain a methacrylated alginate storage solution; S3. Dissolve a photoinitiator in deionized water to obtain a photoinitiator storage solution, dissolve selenomethionine in the photoinitiator storage solution to obtain a photoinitiator solution containing selenomethionine; S4. Mix the photoinitiator solution containing selenomethionine with the methacrylated alginate storage solution to obtain a precursor solution, and irradiate the precursor solution with ultraviolet light to initiate a photo-crosslinking reaction to prepare a selenium-loaded photo-crosslinked alginate hydrogel.

[0008] Preferably, in step S1, the alginate is selected from one or more of sodium alginate, potassium alginate, and ammonium alginate.

[0009] Preferably, in step S1, the buffer is 2-(N-morpholino)ethanesulfonic acid.

[0010] Preferably, in step S1, the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0011] Preferably, in step S1, the mass ratio of alginate to 2-aminoethyl methacrylate hydrochloride is 6:2-4.

[0012] Preferably, in step S3, the photoinitiator is 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone.

[0013] In the technical solution of the present invention, alginate is used as the basic raw material to construct the basic framework structure of the hydrogel. Utilizing its good biodegradability and biocompatibility, the basic framework of the hydrogel is constructed. In vivo, the hydrogel can gradually degrade, and the degradation products are non-toxic and harmless, can be metabolized and excreted by the human body, and will not have long-term adverse effects on the body. At the same time, it has high affinity with surrounding tissues, which is beneficial to tissue repair and regeneration and reduces the occurrence of immune rejection reactions. Sodium chloride is used to adjust the ionic strength of the solution and maintain the stability of the reaction system. The buffer 2-(N-morpholino)ethanesulfonic acid maintains the pH value of the solution in the weakly acidic range to ensure that the subsequent carboxyl activation reaction can proceed under suitable acidity and alkalinity. N-hydroxysulfosuccinimide and the condensing agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride act synergistically to activate the carboxyl groups on the alginate molecules, improve their reaction activity, facilitate the reaction with 2-aminoethyl methacrylate hydrochloride, and introduce methacrylate groups into the alginate molecular chain, endowing it with photocrosslinking ability. A photoinitiator such as 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone decomposes under ultraviolet irradiation to generate free radicals, initiating the polymerization reaction of the double bonds on the methacrylated alginate molecular chain to achieve photocrosslinking. Selenomethionine is a selenium-containing amino acid with various biological activities such as antioxidant, immunomodulatory, and osteogenic promotion. Through the crosslinking reaction of methacrylated alginate, selenomethionine is loaded inside the crosslinked alginate hydrogel to obtain a selenium-loaded photocrosslinked alginate hydrogel. The reaction flow chart for the preparation of the selenium-loaded photocrosslinked alginate hydrogel is shown in Figure 1 .

[0014] Introducing selenomethionine into the hydrogel changes the surface properties of the hydrogel. Selenomethionine not only has biological activity itself but also can act synergistically with other components to promote the adhesion and spreading of cells on the hydrogel surface. The lack of osteoinductivity is the key problem that traditional photocrosslinked alginate hydrogels are difficult to meet the clinical requirements for bone defect repair. The present invention hopes to effectively solve this problem by virtue of the biological activity of selenomethionine. Selenomethionine can enhance the antioxidant and anti-inflammatory responses of osteoblasts through signal pathways, and then promote the osteogenic differentiation and mineralization ability of osteoblasts. In vitro and in vivo experiments have both confirmed that the selenium-loaded methacrylated alginate hydrogel can significantly improve the osteogenic activity of osteoblasts. In the rat calvarial defect experiment, when the hydrogel of the present invention was implanted, it was found by Micro-CT detection that the bone density at the bone defect site increased significantly, and the amount of newly formed bone tissue was significantly more than that of the control group, fully demonstrating its excellent effect in promoting osteogenesis.

[0015] Preferably, in step S4, the mass of selenomethionine is 1-6% of the mass of methacrylated alginate.

[0016] In the technical solution of the present invention, as described above, by introducing selenomethionine into the alginate hydrogel, the mechanical strength of the hydrogel and the osteogenic promotion effect of the hydrogel can be significantly improved. To achieve the above technical effects, the alginate hydrogel must bind a sufficient amount of selenomethionine. Therefore, the present invention controls the mass of selenomethionine to be more than 1% of the mass of methacrylated alginate. However, with the continuous increase in the amount of selenomethionine used, the research team of the present invention unexpectedly found that when an excessive amount of selenomethionine binds to the alginate hydrogel, that is, when the mass of selenomethionine is more than 6% of the mass of methacrylated alginate, the mechanical properties (tensile strength) of the alginate hydrogel scaffold suddenly decrease significantly, and then it is difficult to withstand the physiological stress during bone tissue repair, and it is prone to deformation and rupture in vivo, and cannot provide a stable support structure for bone tissue regeneration. This may be because selenomethionine has a certain molecular structure and size, and too much selenomethionine will occupy a large space in the reaction system. In the cross-linking reaction, the molecules participating in the reaction need to approach each other and reach a suitable spatial orientation to react. Too many selenomethionine molecules will spatially hinder other substances participating in the cross-linking reaction from approaching each other, increasing the steric hindrance of the reaction, and then affecting the normal formation of the cross-linking network, resulting in a decrease in its mechanical properties. Therefore, the present invention also controls the mass of selenomethionine to be less than 6% of the mass of methacrylated alginate. By controlling the addition amount of selenomethionine, the osteogenic promotion effect and good mechanical properties of the alginate hydrogel scaffold are balanced.

[0017] A selenium-loaded photocrosslinkable alginate hydrogel is prepared by the above method.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention introduces selenomethionine into the photocrosslinkable alginate hydrogel, effectively solving the problem that traditional gels lack osteoinductivity. Selenomethionine enhances the antioxidant and anti-inflammatory responses of osteoblasts through specific signal pathways, significantly promotes the differentiation and mineralization of osteoblasts, and has a good osteogenic effect.

[0019] 2. Using alginate as the basic raw material, a hydrogel basic framework is constructed by utilizing its good biodegradability and biocompatibility. In vivo, the hydrogel can gradually degrade, and the degradation products are non-toxic and harmless, can be metabolized and excreted by the human body, will not have long-term adverse effects on the body, and at the same time have high affinity with surrounding tissues, which is conducive to tissue repair and regeneration and reduces the occurrence of immune rejection reactions.

[0020] 3. By precisely controlling the addition amount of selenomethionine, the osteogenic promotion effect and mechanical properties of the hydrogel scaffold are effectively balanced. It can not only ensure the biological activity of selenomethionine in promoting osteogenesis, but also ensure that the hydrogel has sufficient mechanical strength to withstand the physiological stress during bone tissue repair, providing a stable support structure for bone tissue regeneration, meeting different clinical needs, and improving the adaptability and effectiveness of the hydrogel in bone tissue engineering applications. Brief Description of the Drawings

[0021] Figure 1 It is a reaction flow chart for the preparation of the selenium-loaded photo-crosslinked alginate hydrogel of the present invention.

[0022] Figure 2 It is a microscopic image of ALP staining solution staining of rat bone marrow stromal cells encapsulated in the hydrogel of Comparative Example 1 after 14 days of in vitro culture.

[0023] Figure 3 It is a microscopic image of ALP staining solution staining of rat bone marrow stromal cells encapsulated in the hydrogel of Example 1 after 14 days of in vitro culture.

[0024] Figure 4 It is a microscopic image of ARS staining solution staining of rat bone marrow stromal cells encapsulated in the hydrogel of Comparative Example 1 after 21 days of in vitro culture.

[0025] Figure 5 It is a microscopic image of ARS staining solution staining of rat bone marrow stromal cells encapsulated in the hydrogel of Example 1 after 21 days of in vitro culture.

[0026] Figure 6 It is a Micro-CT image of the rat skull repair by the hydrogel of the blank control group.

[0027] Figure 7 It is a Micro-CT image of the rat skull repair by the hydrogel of Comparative Example 1.

[0028] Figure 8 It is a Micro-CT image of the rat skull repair by the hydrogel of Example 1. Detailed Embodiments

[0029] The technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1 A preparation method of a selenium-loaded photo-crosslinked alginate hydrogel, comprising the following steps: Step S1: At room temperature, dissolve 4 g of sodium alginate in 400 mL of deionized water. Add 11.69 g of sodium chloride and 3.91 g of 2-(N-morpholino)ethanesulfonic acid buffer. After magnetic stirring overnight until completely dissolved, titrate with 5 mol / L sodium hydroxide to make the solution pH = 6.5. Then add 1.06 g of N-hydroxysuccinimide and 3.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride condensing agent respectively. After stirring for 5 min, add 2.28 g of 2-aminoethyl methacrylate hydrochloride, and react in the dark for 24 h. Then pour the reaction solution into 2000 mL of acetone to precipitate the product methacrylated alginate, and filter it through a Buchner funnel. The product is redissolved in 400 ml of deionized water. After the product is completely dissolved, aliquot the solution into dialysis bags (molecular weight 3500 Da), and dialyze with deionized water for 3 days, changing the deionized water every 12 hours. After dialysis, filter twice with a filter membrane (pore size 0.22 μm), and then freeze-dry with a freeze dryer to obtain methacrylated alginate.

[0031] Step S2: Weigh 12 g of methacrylated alginate, add it to 480 mL of deionized water, and stir until completely dissolved to obtain a methacrylated alginate stock solution.

[0032] Step S3: Dissolve 0.5 g of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in 100 mL of deionized water, and stir until completely dissolved to obtain a photoinitiator stock solution. Dissolve 0.63 g of selenomethionine in the above photoinitiator stock solution, and stir evenly to obtain a photoinitiator solution containing selenomethionine.

[0033] Step S4: Mix the photoinitiator solution containing selenomethionine with the methacrylated alginate stock solution to obtain a precursor solution. Pour the precursor solution into a specific mold, and irradiate it with an ultraviolet lamp (wavelength 365 nm, intensity 1.7 mW / cm²) for 15 min to initiate a photocrosslinking reaction, thereby preparing a selenium-loaded photocrosslinked alginate hydrogel.

[0034] Example 2 A method for preparing a selenium-loaded photocrosslinked alginate hydrogel, comprising the following steps: Step S1: At room temperature, dissolve 4 g of sodium alginate in 400 mL of deionized water. Add 11.69 g of sodium chloride and 3.91 g of 2-(N-morpholino)ethanesulfonic acid buffer. Stir magnetically overnight until completely dissolved. Then, titrate with 5 mol / L sodium hydroxide to make the solution pH = 6.5. Subsequently, add 1.06 g of N-hydroxysuccinimide and 3.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride condensing agent. After stirring for 5 min, add 2.28 g of 2-aminoethyl methacrylate hydrochloride. React in the dark for 24 h. Then, pour the reaction solution into 2000 mL of acetone to precipitate the product methacrylated alginate, and filter it through a Buchner funnel. The product is redissolved in 400 ml of deionized water. After the product is completely dissolved, dispense the solution into dialysis bags (molecular weight 3500 Da) and dialyze with deionized water for 3 days, changing the deionized water every 12 hours. After dialysis, filter twice with a filter membrane (pore size 0.22 μm), and then lyophilize with a freeze dryer to obtain methacrylated alginate.

[0035] Step S2: Weigh 12 g of methacrylated alginate and add it to 480 mL of deionized water. Stir until completely dissolved to obtain a methacrylated alginate stock solution.

[0036] Step S3: Dissolve 0.5 g of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in 100 mL of deionized water. Stir until completely dissolved to obtain a photoinitiator stock solution. Dissolve 0.26 g of selenomethionine in the above photoinitiator stock solution and stir evenly to obtain a photoinitiator solution containing selenomethionine.

[0037] Step S4: Mix the photoinitiator solution containing selenomethionine with the methacrylated alginate stock solution to obtain a precursor solution. Pour the precursor solution into a specific mold and irradiate it with an ultraviolet lamp (wavelength 365 nm, intensity 1.7 mW / cm²) for 15 min to initiate a photocrosslinking reaction to prepare a selenium-loaded photocrosslinked alginate hydrogel.

[0038] Example 3 A method for preparing a selenium-loaded photocrosslinked alginate hydrogel, comprising the following steps: Step S1: At room temperature, dissolve 4 g of sodium alginate in 400 mL of deionized water. Add 11.69 g of sodium chloride and 3.91 g of 2-(N-morpholino)ethanesulfonic acid buffer. Stir magnetically overnight until completely dissolved. Then titrate with 5 mol / L sodium hydroxide to make the solution pH = 6.5. Subsequently, add 1.06 g of N-hydroxysuccinimide and 3.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride condensing agent. After stirring for 5 min, add 2.28 g of 2-aminoethyl methacrylate hydrochloride. React under light protection for 24 h. Then pour the reaction solution into 2000 mL of acetone to precipitate the product methacrylated alginate, and filter it through a Buchner funnel. The product is redissolved in 400 ml of deionized water. After the product is completely dissolved, divide the solution into dialysis bags (molecular weight 3500 Da) and dialyze with deionized water for 3 days, changing the deionized water every 12 hours. After dialysis, filter twice with a filter membrane (pore size 0.22 μm), and then freeze-dry with a freeze dryer to obtain methacrylated alginate.

[0039] Step S2: Weigh 12 g of methacrylated alginate and add it to 480 mL of deionized water. Stir until completely dissolved to obtain a methacrylated alginate stock solution.

[0040] Step S3: Dissolve 0.5 g of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in 100 mL of deionized water. Stir until completely dissolved to obtain a photoinitiator stock solution. Dissolve 0.53 g of selenomethionine in the above photoinitiator stock solution and stir evenly to obtain a photoinitiator solution containing selenomethionine.

[0041] Step S4: Mix the photoinitiator solution containing selenomethionine with the methacrylated alginate stock solution to obtain a precursor solution. Pour the precursor solution into a specific mold and irradiate it with an ultraviolet lamp (wavelength 365 nm, intensity 1.7 mW / cm²) for 15 min to initiate a photocrosslinking reaction to prepare a selenium-loaded photocrosslinked alginate hydrogel.

[0042] Example 4 A method for preparing a selenium-loaded photocrosslinked alginate hydrogel, comprising the following steps: Step S1: At room temperature, dissolve 4 g of sodium alginate in 400 mL of deionized water. Add 11.69 g of sodium chloride and 3.91 g of 2-(N-morpholino)ethanesulfonic acid buffer. After magnetic stirring overnight until completely dissolved, titrate with 5 mol / L sodium hydroxide to make the solution pH = 6.5. Then add 1.06 g of N-hydroxysuccinimide and 3.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride condensing agent respectively. After stirring for 5 min, add 2.28 g of 2-aminoethyl methacrylate hydrochloride. React in the dark for 24 h. Then pour the reaction solution into 2000 mL of acetone to precipitate the product methacrylated alginate, and filter it through a Buchner funnel. The product is redissolved in 400 ml of deionized water. After the product is completely dissolved, aliquot the solution into dialysis bags (molecular weight 3500 Da) and dialyze with deionized water for 3 days, changing the deionized water every 12 hours. After dialysis, filter twice with a filter membrane (pore size 0.22 μm), and then freeze-dry with a freeze dryer to obtain methacrylated alginate.

[0043] Step S2: Weigh 12 g of methacrylated alginate and add it to 480 mL of deionized water. Stir until completely dissolved to obtain a methacrylated alginate stock solution.

[0044] Step S3: Dissolve 0.5 g of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in 100 mL of deionized water. Stir until completely dissolved to obtain a photoinitiator stock solution. Dissolve 0.72 g of selenomethionine in the above photoinitiator stock solution and stir evenly to obtain a photoinitiator solution containing selenomethionine.

[0045] Step S4: Mix the photoinitiator solution containing selenomethionine with the methacrylated alginate stock solution to obtain a precursor solution. Pour the precursor solution into a specific mold and irradiate it with an ultraviolet lamp (wavelength 365 nm, intensity 1.7 mW / cm²) for 15 min to initiate a photocrosslinking reaction to prepare a selenium-loaded photocrosslinked alginate hydrogel.

[0046] Example 5 A preparation method of a selenium-loaded photocrosslinked alginate hydrogel, comprising the following steps: Step S1: At room temperature, dissolve 4 g of sodium alginate in 400 mL of deionized water. Add 11.69 g of sodium chloride and 3.91 g of 2-(N-morpholino)ethanesulfonic acid buffer. Stir magnetically overnight until completely dissolved. Then titrate with 5 mol / L sodium hydroxide to make the solution pH = 6.5. Next, add 1.06 g of N-hydroxysulfosuccinimide and 3.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride condensing agent. After stirring for 5 min, add 2.28 g of 2-aminoethyl methacrylate hydrochloride. React in the dark for 24 h. Then pour the reaction solution into 2000 mL of acetone to precipitate the product methacrylated alginate, and filter it through a Buchner funnel. The product is redissolved in 400 ml of deionized water. After the product is completely dissolved, aliquot the solution into dialysis bags (molecular weight 3500 Da) and dialyze with deionized water for 3 days, changing the deionized water every 12 hours. After dialysis, filter twice with a filter membrane (pore size 0.22 μm), and then lyophilize with a freeze dryer to obtain methacrylated alginate.

[0047] Step S2: Weigh 12 g of methacrylated alginate and add it to 480 mL of deionized water. Stir until completely dissolved to obtain a methacrylated alginate stock solution.

[0048] Step S3: Dissolve 0.5 g of the photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone in 100 mL of deionized water. Stir until completely dissolved to obtain a photoinitiator stock solution. Dissolve 0.12 g of selenomethionine in the above photoinitiator stock solution and stir evenly to obtain a photoinitiator solution containing selenomethionine.

[0049] Step S4: Mix the photoinitiator solution containing selenomethionine with the methacrylated alginate stock solution to obtain a precursor solution. Pour the precursor solution into a specific mold and irradiate it with a UV lamp (wavelength 365 nm, intensity 1.7 mW / cm²) for 15 min to initiate a photocrosslinking reaction to prepare a selenium-loaded photocrosslinked alginate hydrogel.

[0050] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that selenomethionine is not loaded in the crosslinked alginate hydrogel, and the remaining steps are the same.

[0051] Comparative Example 2 The difference between Comparative Example 2 and Example 5 is that the mass of selenomethionine is 0.5% of the mass of methacrylated alginate, and the remaining steps are the same.

[0052] Comparative Example 3 The difference between Comparative Example 3 and Example 4 is that the mass of selenomethionine is 7% of the mass of methacrylated alginate, and the remaining steps are the same.

[0053] Comparative Example 4 The difference between Comparative Example 4 and Example 4 lies in that the mass of selenomethionine is 8% of the mass of methacrylated alginate, and the remaining steps are the same.

[0054] Performance test: 1. Osteogenic promotion performance test: (1) Wrap the hydrogels prepared in Example 1 and Comparative Example 1 with rat bone marrow stromal cells. After culturing in vitro for 14 days, stain with ALP staining solution for 2 h, observe and record under a microscope. After culturing in vitro for 21 days, stain with ARS staining solution for 30 min, gently rinse with pure water twice, and observe and record under a microscope. The microscope observation images are shown in Figure 2-5 . From Figure 2-5 it can be seen that the ALP and ARS staining areas and numbers in Example 1 are significantly more than those in Comparative Example 1, proving that the hydrogel of the present invention has good osteogenic promotion performance.

[0055] (2) Use a 5 mm dental trephine to create a bone defect in the rat skull. Flush the created bone defect site with normal saline, implant the hydrogels in Example 1 and Comparative Example 1 respectively, and set up a group without implanting hydrogel as a blank control group. Sacrifice the experimental animals after breeding for 8 weeks, and obtain the skull specimens. Use Micro-CT to detect the bone density at the bone defect of each group to evaluate and compare the repair effects of the defect sites. The Micro-CT images are shown in Figure 6-8 . From Figure 6-8 it can be seen that the repair area of the hydrogel in Example 1 for the rat skull is significantly larger than that in Comparative Example 1 and the blank control group, proving that the hydrogel of the present invention has good osteogenic promotion performance.

[0056] 2. Mechanical property test: To accurately evaluate the mechanical properties of the selenium-loaded photocrosslinked alginate hydrogel, 5 rectangular specimens with dimensions of 20 mm in length, 5 mm in width, and 2 mm in thickness are respectively cut from the hydrogels of Examples 1-5 and Comparative Examples 1-4. Use an electronic universal material testing machine with a precision of 0.01 N for testing. Install the specimen on the testing machine fixture, adjust the fixture spacing to 10 mm, and set the tensile speed to 10 mm / min. During the tensile process, the testing machine automatically records the force-displacement data until the specimen breaks. Each sample is tested 5 times and the average value is taken. The tensile strength is calculated by the formula σ = Fmax / S (where σ is the tensile strength (MPa), Fmax is the maximum force (N) when the specimen breaks, and S is the initial cross-sectional area of the specimen. In this experiment, S = 5 mm × 2 mm = 10 mm 2 ). Calculate the tensile strength (MPa) of the specimen according to the above formula. The calculation results are shown in Table 1.

[0057] Table 1: As can be seen from the data, in Examples 1-5, the mass of selenomethionine was 1%-6% of the mass of methacrylated alginate, and the tensile strength of the hydrogel was between 1.50-1.68 MPa, which was relatively stable and higher than 1.41 MPa of Comparative Example 1 (without loaded selenomethionine), indicating that appropriate addition of selenomethionine helped to improve the mechanical strength of the hydrogel. In Comparative Example 3 and Comparative Example 4, the mass ratios of selenomethionine were 7% and 8% respectively, and the tensile strength decreased significantly to 1.03 MPa and 0.82 MPa, proving that when the mass of selenomethionine exceeded 6% of the mass of methacrylated alginate, the mechanical properties of the hydrogel would be significantly reduced.

[0058] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of selenium-loaded photocrosslinkable alginate hydrogel, characterized in that, It includes the following steps: S1. Dissolve alginate in deionized water, add sodium chloride and a buffer, stir until completely dissolved, adjust the pH value of the solution to weakly acidic, then add N-hydroxy-succinimide and a condensing agent, stir evenly, add 2-aminoethyl methacrylate hydrochloride, and carry out methacrylation reaction to obtain methacrylated alginate; S2. Dissolve methacrylated alginate in deionized water to obtain a methacrylated alginate storage solution; S3. Dissolve a photoinitiator in deionized water to obtain a photoinitiator storage solution, dissolve selenomethionine in the photoinitiator storage solution to obtain a photoinitiator solution containing selenomethionine; S4. Mix the photoinitiator solution containing selenomethionine with the methacrylated alginate storage solution to obtain a precursor solution, and irradiate the precursor solution with ultraviolet light to initiate a photocrosslinking reaction to prepare a selenium-loaded photocrosslinked alginate hydrogel.

2. The preparation method of a selenium-loaded photocrosslinkable alginate hydrogel according to claim 1, characterized in that, In the step S1, the alginate is selected from one or more of sodium alginate, potassium alginate, and ammonium alginate.

3. The preparation method of a selenium-loaded photocrosslinkable alginate hydrogel according to claim 1, characterized in that, In the step S1, the buffer is 2-(N-morpholino)ethanesulfonic acid.

4. The preparation method of a selenium-loaded photocrosslinkable alginate hydrogel according to claim 1, characterized in that, In the step S1, the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

5. The preparation method of a selenium-loaded photocrosslinkable alginate hydrogel according to claim 1, characterized in that, In the step S1, the mass ratio of alginate to 2-aminoethyl methacrylate hydrochloride is 6:2-4.

6. The preparation method of a selenium-loaded photocrosslinked alginate hydrogel according to claim 1, characterized in that, In the step S3, the photoinitiator is 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone.

7. The preparation method of a selenium-loaded photocrosslinked alginate hydrogel according to claim 1, wherein, In the step S4, the mass of selenomethionine is 1-6% of the mass of methacrylated alginate.

8. A selenium-loaded photocrosslinked alginate hydrogel, characterized in that, Prepared by the method according to any one of claims 1-7.

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