Composite frozen gel capable of resisting oxidation and promoting osteogenesis as well as preparation method and application of composite frozen gel

Abstract

The invention relates to an antioxidant osteogenesis-promoting composite frozen gel as well as a preparation method and application thereof, polyvinyl alcohol (PVA) is used as a matrix material, the PVA and sulfated curdlan (CRs) are subjected to circulating freeze thawing and physical crosslinking to form hydrogel, meanwhile, selenium-containing bioglass (SeBG) is uniformly dispersed in a hydrogel matrix, and the composite frozen gel SeBG-coated CRs / PVA is prepared. The prepared composite frozen gel has excellent mechanical properties and oxidation resistance, and can promote the growth and differentiation of bone marrow mesenchymal stem cells and the regeneration of new bones; and the material is safe in components, green and environment-friendly, is beneficial to mass production, has very high conversion value, and has potential application prospects in bone tissue engineering scaffolds and drug sustained release systems.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and particularly relates to an antioxidant osteogenic composite cryogel and its preparation method and application. Background Art

[0002] Limited osteogenic potential poses a major challenge to the spontaneous healing of bone defects. The reasons for poor bone regeneration are complex and multifaceted, among which excessive reactive oxygen species (ROS) are the key factors leading to this phenomenon. After bone injury, excessive ROS will be generated. Moreover, the insufficient oxygen supply caused by blood vessel rupture after injury will cause continuous increase in ROS, directly or indirectly inhibiting osteoblast differentiation, and then causing secondary damage to tissues, which is not conducive to the rapid repair of bone tissue (Advanced Functional Materials, 2022, 32(10): 2111208). Therefore, developing a multifunctional biomaterial with excellent mechanical properties and good ROS scavenging ability has important clinical significance and application value.

[0003] Polyvinyl alcohol (PVA) is an important water-soluble polymer, which can form hydrogels through multiple freeze-thaw cycles, and its mechanical strength and degradation rate can be precisely controlled by regulating the cross-linking degree. However, PVA lacks effective bioactivity and is difficult to meet the requirements of osteogenic induction and angiogenesis in bone defect repair. Therefore, it is often used in combination with other bioactive materials.

[0004] Curdlan (CR) is a natural polysaccharide produced by microbial fermentation, which has good biocompatibility and biodegradability. By introducing sulfate groups through chemical modification, the water-insolubility of CR is improved and its bioactivity is enhanced, such as promoting osteoblast differentiation and angiogenesis (Carbohydrate Polymers, 2022, 281: 119059.). Sulfated curdlan (CRs) also has antioxidant and anti-inflammatory properties, which can effectively reduce the infection risk at the bone defect site.

[0005] Bioactive glass is a class of inorganic materials with excellent bioactivity and osteoconductivity, which can form chemical bonds with bone tissue and promote new bone formation. By incorporating selenium elements, bioactive glass not only retains its original bioactivity, but also endows the material with functions such as antioxidant property and osteoblast-promoting activity. In addition, selenium-doped bioactive glass can release calcium, silicon, phosphorus and selenium bioactive ions, promote osteoblast activity, and thus accelerate the bone defect repair process. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an antioxidant osteogenic composite cryogel and its preparation method and application to promote the growth and osteogenic differentiation of bone marrow mesenchymal stem cells, and then efficiently promote the repair and regeneration of bone tissue.

[0007] The present invention provides an antioxidant osteogenic composite cryogel. Using polyvinyl alcohol (PVA) as the matrix material, it undergoes physical crosslinking with sulfated curdlan (CRs) through cyclic freezing and thawing to form a hydrogel. Meanwhile, selenium-containing bioactive glass (SeBG) is uniformly dispersed in the hydrogel matrix to prepare the composite cryogel SeBG@CRs / PVA.

[0008] The present invention provides a method for preparing an antioxidant osteogenic composite cryogel, comprising the following steps:

[0009] S1. Preparation of sulfated curdlan (CRs): Prepare a pyridine sulfur trioxide solution, add curdlan (CR) and stir evenly to obtain a suspension solution; add an alkali solution to adjust the pH to neutral, perform alcohol precipitation, filtration, and obtain a precipitate. After dissolving the precipitate in deionized water, perform centrifugation, dialysis, and freeze-drying to obtain sulfated curdlan (CRs);

[0010] S2. Prepare selenium-containing bioactive glass (SeBG) according to the sol-gel method, with its chemical composition being SiO2-CaO-P2O5-SeO2;

[0011] S3. Prepare an aqueous solution of polyvinyl alcohol (PVA), add CRs from step S1, stir to obtain an aqueous CRs / PVA solution, and perform ultrasonic defoaming;

[0012] S4. Prepare a CRs / PVA hydrogel loaded with SeBG: Add the SeBG powder from step S2 to the aqueous CRs / PVA solution, stir until completely dispersed, perform ultrasonic defoaming, and perform freeze-thaw treatment to obtain the composite cryogel SeBG@CRs / PVA.

[0013] Preferably, in step S1, the concentration of the pyridine sulfur trioxide solution is 5.0 - 10.0 wt%, and the solvent used includes one of N,N-dimethylformamide (DMF) or anhydrous dimethyl sulfoxide (DMSO); the concentration of CR in the pyridine sulfur trioxide solution is 3.0 - 5.0 wt%; the alkali solution includes one of NaOH solution or NaHCO3 solution, and the concentration of the alkali solution is 2.0 - 10.0 wt%; the degree of substitution of CRs is 1.0 - 3.0.

[0014] Further, the concentration of the pyridine sulfur trioxide solution is 8.0 wt%, the concentration of CR in the pyridine sulfur trioxide solution is 4.0 wt%; the alkali solution is a 5.0 wt% NaOH solution; the degree of substitution of CRs is 2.07.

[0015] Preferably, in step S1, the reaction conditions are an oil bath reaction at 30 - 50 °C for 2 - 5 h.

[0016] Further, the reaction conditions in step S1 are an oil bath reaction at 30 °C for 3 h.

[0017] Preferably, the specific process for preparing selenium-containing bioactive glass SeBG by the sol-gel method in step S2 is as follows: Add 18 - 20 mL of 2 M HNO3 to 200 mL of deionized water, slowly add TEOS (tetraethyl orthosilicate), stir at room temperature until complete hydrolysis, then sequentially add Ca(NO3)2·4H2O, TEP (triethyl phosphate), and Na2SeO3, and stir until completely dissolved; let stand at room temperature for 24 - 48 h until a gel appears, then dry at 120 °C, and after thorough drying, ball mill; heat the powder by high-temperature calcination to 600 - 800 °C at a rate of 2 °C / min, hold for 4 h to form a porous glass network; grind the calcined sample with a ball mill and pass through a sieve to obtain a uniform powder.

[0018] Preferably, the theoretical molar ratio of SiO2, CaO, P2O5, and SeO2 in the SeBG in step S2 is (40 - 58):(20 - 36):(4 - 6):(0.1 - 5); the particle size diameter of the SeBG is 1.0 - 5.0 μm.

[0019] More preferably, the theoretical molar ratio of SiO2, CaO, P2O5, and SeO2 in the SeBG is 58:35:6:1.

[0020] Preferably, the molecular weight of the polyvinyl alcohol PVA in step S3 is 70000 - 200000, and the degree of alcoholysis is 98.0 - 99.0%; the concentration of the PVA aqueous solution is 5.0 - 10.0 wt%; the concentration of the CRs in the PVA aqueous solution is 0.5 - 5 wt%.

[0021] More preferably, the concentration of the CRs in the PVA aqueous solution is 0.5 - 2 wt%.

[0022] Preferably, the stirring speed in step S3 is 150 - 300 rpm / min, and the stirring time is 2 - 5 h; the ultrasonic defoaming time in steps S3 and S4 is 20 - 40 min.

[0023] Preferably, the concentration of the SeBG in the CRs / PVA aqueous solution in step S4 is 0.05 - 0.5 wt%.

[0024] Preferably, the freezing temperature in step S4 is -40 - -10 °C, the freezing time is 12 - 48 h, the thawing time is 2 - 6 h; the number of freeze-thaw cycles is 1 - 3 times.

[0025] More preferably, the freezing temperature is -20 °C, the freezing time is 24 h, the thawing time is 2 h; the number of freeze-thaw cycles is 2 - 3 times.

[0026] The present invention also provides an application of the antioxidant osteoinductive composite cryogel in bone tissue engineering scaffolds, drug sustained release and other aspects.

[0027] Beneficial effects

[0028] (1) The composite cryogel prepared by the present invention can obtain an implantable active scaffold with a porous structure by dissolving and mixing three components and crosslinking at low temperature. It has safe ingredients, is green and environmentally friendly, has simple operation, is conducive to mass production, and has high transformation value.

[0029] (2) The composite cryogel prepared by the present invention has good compression and rebound performance and can basically rebound to the initial position after ten cycles of compression.

[0030] (3) The composite cryogel prepared by the present invention improves the crosslinking degree of the network under the dual effects of CRs and SeBG, thereby improving the antioxidant performance.

[0031] (4) The molecular chains of the composite cryogel prepared by the present invention can effectively encapsulate SeBG, realize the sustained and slow release of active ions (Ca, Si, P, Se), reduce its toxicity, and promote the growth of bone marrow mesenchymal stem cells and new bone regeneration.

[0032] (5) The composite cryogel prepared by the present invention is conducive to cell adhesion, proliferation and migration, and can effectively synergize the advantages of various components to provide a suitable microenvironment for the growth of bone marrow mesenchymal stem cells.

[0033] (6) The present invention has potential application prospects in bone tissue engineering scaffolds and drug sustained release systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a macroscopic view of cryogels (PVA, CRs / PVA, 0.5SeBG@CRs / PVA, 1SeBG@CRs / PVA, 2SeBG@CRs / PVA) containing different concentrations of SeBG prepared in Examples 1-3 of the present invention.

[0035] Figure 2 It is a compressive stress-strain curve graph (A); (B) cyclic compressive stress-strain curve graph; (C) Young's modulus graph of the mechanical properties of cryogels (PVA, CRs / PVA, 0.5SeBG@CRs / PVA, 1SeBG@CRs / PVA, 2SeBG@CRs / PVA) containing different concentrations of SeBG prepared in Examples 1-3 of the present invention.

[0036] Figure 3Antioxidant capacity (A) ABTS radical scavenging capacity graph; (B) hydroxyl radical scavenging capacity graph of cryogels (PVA, CRs / PVA, 0.5SeBG@CRs / PVA, 1SeBG@CRs / PVA, 2SeBG@CRs / PVA) containing different concentrations of SeBG prepared in Examples 1-3 of the present invention.

[0037] Figure 4 Cell proliferation status of cryogels (PVA, CRs / PVA, 0.5SeBG@CRs / PVA, 1SeBG@CRs / PVA, 2SeBG@CRs / PVA) containing different concentrations of SeBG prepared in Examples 1-3 of the present invention co-cultured with rBMSCs.

[0038] Figure 5 Repair and regeneration of rat skull defects by cryogels (PVA, CRs / PVA, 1SeBG@CRs / PVA) containing different concentrations of SeBG prepared in Example 2 of the present invention: (A) CT image of rat skull defect at 8W; (B) bone volume fraction graph at 8W; (C) bone surface area graph at 8W; (D) bone mineral density graph at 8W. Detailed implementation manners

[0039] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0040] Example 1

[0041] This example provides a composite cryogel with antioxidant and osteogenic promotion properties, and its preparation method includes the following steps:

[0042] S1. Preparation of sulfated curdlan CRs

[0043] Dissolve 6.0 g of pyridine sulfur trioxide in 75 mL of DMF, stir evenly, then add 3.0 g of CR, stir at 30 °C for 3 h, after the reaction is completed, slowly add 5.0 wt% NaOH solution to adjust the pH to neutral, add three times the volume of 95.0 wt% ethanol for alcohol precipitation for 2 h, filter to obtain the crude product, dissolve it in deionized water, centrifuge at 8000 rpm for 15 min, take the supernatant and dialyze for 3 days, and finally freeze-dry to obtain CRs;

[0044] S2. Preparation of selenium-containing bioactive glass SeBG according to the sol-gel method

[0045] Add 20 mL of 2 M HNO3 to 200 mL of deionized water, slowly add TEOS (tetraethyl orthosilicate), stir at room temperature until complete hydrolysis, then add Ca(NO3)2·4H2O, TEP (triethyl phosphate), and Na2SeO3 in sequence, and stir until completely dissolved; let stand at room temperature for 48 h, until a gel appears, then dry at 120 °C, and ball mill after thorough drying; calcine the powder at a rate of 2 °C / min to 800 °C and hold for 4 h to form a porous glass network; grind the calcined sample with a ball mill and sieve to obtain a uniform SeBG powder. The chemical composition of SeBG is SiO2-CaO-P2O5-SeO2, and the theoretical molar ratio of SiO2, CaO, P2O5, and SeO2 is 58:35:6:1;

[0046] S3. Preparation of CRs / PVA aqueous solution

[0047] Dissolve 5.0 g of PVA in 100 mL of deionized water, place it in an oil bath at 95 °C and stir magnetically for 2 h to completely dissolve PVA to obtain a PVA aqueous solution with a concentration of 5.0 wt%; add 0.2 g of CRs to 10 mL of the PVA aqueous solution, and stir at 200 rpm at room temperature to obtain a 2.0 wt% CRs / PVA aqueous solution;

[0048] S4. Preparation of CRs / PVA composite cryogel loaded with SeBG

[0049] Add 5.0 mg of SeBG to the CRs / PVA aqueous solution prepared in step S3 to make the SeBG concentration 0.5 mg / mL, stir at 200 rpm at room temperature for 3 h, and defoam by ultrasonic treatment for 30 min; dispense the obtained solution into molds, 1 mL per well, place it in a -20 °C refrigerator, freeze for 24 h, then thaw at room temperature for 2 h, and perform 2 cycles of freezing-thawing to obtain a composite cryogel (0.5SeBG@CRs / PVA).

[0050] Example 2

[0051] This example provides an antioxidant osteoinductive composite cryogel, and its preparation method includes the following steps:

[0052] S1. Preparation of sulfated curdlan CRs, and the preparation process is the same as that in step S1 of Example 1;

[0053] S2. Preparation of selenium-containing bioglass SeBG according to the sol-gel method, and the preparation process is the same as that in step S2 of Example 1;

[0054] S3. Preparation of CRs / PVA aqueous solution

[0055] Dissolve 5.0 g of PVA in 100 mL of deionized water, place it in an oil bath at 95 °C, and stir magnetically for 2 h to completely dissolve PVA to obtain a PVA aqueous solution with a concentration of 5.0 wt%. Add 0.2 g of CRs to 10 mL of the PVA aqueous solution and stir uniformly at 200 rpm at room temperature to obtain a 2.0 wt% CRs / PVA aqueous solution;

[0056] S4. Preparation of SeBG-loaded CRs / PVA composite cryogel

[0057] Add 10.0 mg of SeBG to the CRs / PVA aqueous solution prepared in step S3 to make the SeBG concentration 1.0 mg / mL, stir uniformly at 200 rpm at room temperature for 3 h, and defoam by ultrasonic treatment for 30 min; Dispense the obtained solution into molds, 1 mL per well, place it in a -20 °C refrigerator, freeze for 24 h, then thaw at room temperature for 2 h, and perform 2 cycles of freezing-thawing to obtain a composite cryogel (1SeBG@CRs / PVA).

[0058] Example 3

[0059] This example provides an antioxidant osteoinductive composite cryogel, and its preparation method includes the following steps:

[0060] S1. Prepare sulfated curdlan CRs, and the preparation process is the same as step S1 in Example 1;

[0061] S2. Prepare selenium-containing bioactive glass SeBG according to the sol-gel method, and the preparation process is the same as step S2 in Example 1;

[0062] S3. Prepare CRs / PVA aqueous solution

[0063] Dissolve 5.0 g of PVA in 100 mL of deionized water, place it in an oil bath at 95 °C, and stir magnetically for 2 h to completely dissolve PVA to obtain a PVA aqueous solution with a concentration of 5.0 wt%. Add 0.2 g of CRs to 10 mL of the PVA aqueous solution and stir uniformly at 200 rpm at room temperature to obtain a 2.0 wt% CRs / PVA aqueous solution;

[0064] S4. Preparation of SeBG-loaded CRs / PVA composite cryogel

[0065] Add 20.0 mg of SeBG to the CRs / PVA aqueous solution prepared in step S3 to make the SeBG concentration 2.0 mg / mL, stir uniformly at 200 rpm at room temperature for 3 h, and defoam by ultrasonic treatment for 30 min; Dispense the obtained solution into molds, 1 mL per well, place it in a -20 °C refrigerator, freeze for 24 h, then thaw at room temperature for 2 h, and perform 2 cycles of freezing-thawing to obtain a composite cryogel (2SeBG@CRs / PVA).

[0066] Example 4

[0067] This example provides an antioxidant and osteogenic composite cryogel, and its preparation method includes the following steps:

[0068] S1. Prepare sulfated curdlan CRs, and the preparation process is the same as step S1 in Example 1;

[0069] S2. Prepare selenium-containing bioactive glass SeBG according to the sol-gel method, and the preparation process is the same as step S2 in Example 1;

[0070] S3. Prepare CRs / PVA aqueous solution

[0071] Take 10.0 g of PVA and dissolve it in 100 mL of deionized water. Place it in an oil bath at 95 °C and stir magnetically for 2 h to completely dissolve PVA to obtain a PVA aqueous solution with a concentration of 10.0 wt%. Add 0.05 g of CRs to 10 mL of the PVA aqueous solution and stir at 200 rpm at room temperature to obtain a 0.5 wt% CRs / PVA aqueous solution;

[0072] S4. Preparation of SeBG-loaded CRs / PVA composite cryogel

[0073] Add 20.0 mg of SeBG to the CRs / PVA aqueous solution prepared in step S3 to make the SeBG concentration 2.0 mg / mL. Stir at 200 rpm at room temperature for 3 h and defoam by ultrasonic treatment for 30 min. Dispense the obtained solution into molds, 1 mL per well, place it in a -20 °C refrigerator, freeze for 24 h, thaw at room temperature for 2 h, and perform 3 cycles of freezing-thawing to obtain the composite cryogel.

[0074] Example 5

[0075] This example provides an antioxidant and osteogenic composite cryogel, and its preparation method includes the following steps:

[0076] S1. Prepare sulfated curdlan CRs, and the preparation process is the same as step S1 in Example 1;

[0077] S2. Prepare selenium-containing bioactive glass SeBG according to the sol-gel method, and the preparation process is the same as step S2 in Example 1;

[0078] S3. Prepare CRs / PVA aqueous solution

[0079] Take 5.0 g of PVA and dissolve it in 100 mL of deionized water. Place it in an oil bath at 95 °C and stir magnetically for 2 h to completely dissolve PVA to obtain a PVA aqueous solution with a concentration of 5.0 wt%. Add 0.2 g of CRs to 10 mL of the PVA aqueous solution and stir at 200 rpm at room temperature to obtain a 2 wt% CRs / PVA aqueous solution;

[0080] S4. Preparation of CRs / PVA Composite Cryogels Loaded with SeBG

[0081] Add 20.0 mg of SeBG to the CRs / PVA aqueous solution prepared in step S3 to make the SeBG concentration 2.0 mg / mL. Stir uniformly at 200 rpm for 3 h at room temperature and defoam by ultrasonic treatment for 30 min. Dispense the obtained solution into molds, 1 mL per well, place it in a -20 °C refrigerator, freeze for 24 h, thaw at room temperature for 2 h, and perform 3 cycles of freezing-thawing to obtain the composite cryogel.

[0082] Perform performance tests on the cryogels (PVA, CRs / PVA, SeBG@CRs / PVA) containing different concentrations of SeBG prepared in the above examples.

[0083] Test 1: Macroscopically observe PVA, CRs / PVA, 0.5SeBG@CRs / PVA prepared in Example 1, 1SeBG@CRs / PVA prepared in Example 2, and 2SeBG@CRs / PVA prepared in Example 3, where the SeBG concentrations in 0.5SeBG@CRs / PVA, 1SeBG@CRs / PVA, and 2SeBG@CRs / PVA are 0.5, 1, and 2 mg / mL respectively, the CRs concentration is 20 mg / mL for all, and the PVA concentration is 50 mg / mL for all, and perform two cycles of freezing-thawing.

[0084] Figure 1 Macroscopic images of cryogels containing different concentrations of SeBG, scale bar: 5 mm.

[0085] Test 2: Perform mechanical property tests on PVA, CRs / PVA, 0.5SeBG@CRs / PVA prepared in Example 1, 1SeBG@CRs / PVA prepared in Example 2, and 2SeBG@CRs / PVA prepared in Example 3. Specifically:

[0086] (1) Compression property test: Make the hydrogel sample into a cylinder with a diameter of 10 mm and a thickness of 6.0 mm, use a universal material testing machine, and perform compression tests on the sample at a standard constant compression speed (5.0 mm / min) and a standard load sensing (20.0 N) until the strain of the sample reaches 70%.

[0087] (2) Cyclic compression test: Perform 10 compression cycles on the sample at a constant compression speed of 5.0 mm / min and a load sensing of 20.0 N (after the sample is compressed and deformed by 60%, stop compression, wait for the sample to recover to before compression, which is 1 compression cycle, and then perform the next cycle after each cycle recovers for 1 min). Other parameters are the same as those in the infinite compression test.

[0088] As Figure 2 shown in A, as the concentration of SeBG increases, the compressive strength of the hydrogel increases. When the strain is 70%, 2SeBG@CRs / PVA shows the best compressive effect.

[0089] As Figure 2 shown in B, after ten cycles of compression, when the strain is 60%, all hydrogels have good resilience.

[0090] As Figure 2 shown in C, as the concentration of SeBG increases, the Young's modulus of the hydrogel increases, and the Young's modulus of 2SeBG@CRs / PVA hydrogel reaches the maximum (117.78 ± 5.81 kPa).

[0091] Test 3: Antioxidant tests were carried out on PVA, CRs / PVA, 0.5SeBG@CRs / PVA prepared in Example 1, 1SeBG@CRs / PVA prepared in Example 2, and 2SeBG@CRs / PVA prepared in Example 3. Specifically:

[0092] (1) Determination of ABTS cation radical:

[0093] Step 1: First, prepare an ABTS cation radical stock solution. Mix the prepared 7.0 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution evenly in a volume ratio of 1:1, and let it stand in the dark at room temperature for 16 h to obtain the ABTS cation radical stock solution.

[0094] Step 2: Dilute the stock solution with deionized water so that the absorbance of the ABTS cation radical stock solution at a wavelength of 734 nm is 0.70 ± 0.02.

[0095] Step 3: Add 1 mL of different hydrogels to 3 mL of the diluted ABTS solution, shake well to make the reaction complete, and measure its absorbance at 734 nm after standing in the dark for 12 h.

[0096] ABTS cation radical scavenging ability / % = [A0 - (A1 - A2)] / A0 × 100;

[0097] In the formula: A1 is the absorbance of different hydrogel samples and the ABTS solution; A0 is the absorbance of the ABTS solution; A2 is the absorbance using deionized water instead of ABTS.

[0098] (2) Determination of hydroxyl radical (·OH):

[0099] Step 1: Add 1 mL of different hydrogel samples, 1 mL of 9.0 mmol / L ferrous sulfate solution, 1 mL of 9.0 mmol / L salicylic acid - ethanol solution, and 1 mL of 8.8 mmol / L hydrogen peroxide solution into a test tube respectively. After mixing well, react in a 37°C water bath for 12 h and then measure the absorbance at 510 nm.

[0100] Hydroxyl radical scavenging ability / % = [A0 - (A1 - A2)] / A0 × 100;

[0101] Where: A1 is the absorbance value of the mixed solution with different hydrogel samples added; A0 is the absorbance value of the mixed solution; A2 is the absorbance value with deionized water replacing the mixed solution.

[0102] As Figure 3 shown in

[0103] A, after adding SeBG, the ABTS radical scavenging ability of this hydrogel basically reaches 100%, and there is no significant difference. Figure 3 As

[0104] shown in

[0105] B, as the concentration of SeBG increases, the hydroxyl radical scavenging ability of this hydrogel gradually increases. The 2SeBG@CRs / PVA hydrogel has the strongest antioxidant ability (79.95 ± 1.22%), so it can be shown that the hydrogel loaded with SeBG has excellent antioxidant performance.

[0106] Test four: Detect the activity and proliferation ability of rat bone marrow mesenchymal stem cells (rBMSCs) for PVA, CRs / PVA prepared in Example 1, 0.5SeBG@CRs / PVA, 1SeBG@CRs / PVA prepared in Example 2, and 2SeBG@CRs / PVA prepared in Example 3. Specifically: 3 Step 1: Sterilize 1 mL of different hydrogel materials by ultraviolet light for 2 h for both front and back sides. After sterilization, place the samples in 10 mL of basal medium and let them stand in a 37°C cell incubator for 24 h. Set the normal medium without hydrogel materials as the control group (Control group).

[0107] Step 2: Inoculate the cells at a cell density of 3×10

[0108] viable cells / well into a 96 - well plate, and add 100 μL of culture medium to each well and culture overnight.

[0109] The cell proliferation situation is as follows Figure 4 shown. The results indicate that all hydrogels containing CRs and SeBG have a promoting effect on the growth of bone marrow mesenchymal stem cells.

[0110] Test Five: An animal experiment on the repair and regeneration of rat cranial bone defects was conducted on the hydrogels (PVA, CRs / PVA, 1SeBG@CRs / PVA) prepared in Example 2. Specifically:

[0111] Step 1: Prepare hydrogels with a diameter of 5.0 mm and a thickness of 1.0 mm, and perform ultraviolet sterilization for 2 hours on each side.

[0112] Step 2: Prepare male SD rats (200 g), with 3 rats in a group. Inject 10% chloral hydrate solution into the abdominal cavity of the rats at a ratio of 300 mg / kg to make the rats enter deep anesthesia, and use surgical scissors to cut open the scalp to expose the skull. Drill two holes with a diameter of about 5 mm in the critical-sized defects of the bilateral skulls, implant the hydrogels of the same group as the experimental group, and use the untreated ones as the control group (Control group) after the operation. Suture the wound to end the operation.

[0113] Step 3: Sacrifice all rats 8 weeks after the operation and collect the skulls. Fix them with 4% paraformaldehyde for 24 hours, wash them clean with PBS, image the collected skull samples with Micro-CT, and perform detection according to the analysis software.

[0114] As Figure 5 shown in A, the CT of the rat skulls after 8 weeks of treatment shows that the 1SeBG@CRs / PVA group has excellent bone repair and regeneration functions.

[0115] As Figure 5 shown in B, the bone volume fractions of all material groups are higher than those of the Control group (3.74 ± 2.32%), and the 1SeBG@CRs / PVA group is as high as 23.32 ± 1.66%.

[0116] As Figure 5 shown in C, the bone surface areas of all material groups are higher than those of the Control group (19.73 ± 12.08 mm 2 ), and the 1SeBG@CRs / PVA group is as high as 72.88 ± 18.60 mm 2 .

[0117] As Figure 5 shown in D, the bone mineral densities of all material groups are higher than those of the Control group (1.03 ± 0.05 g / cm 3 ), and the 1SeBG@CRs / PVA group is as high as 1.14 ± 0.01 g / cm 3 .

[0118] In summary, compared with the prior art, the present invention uses PVA as the main material, forms a gel by physical crosslinking with CRs and freezing, and simultaneously loads SeBG, which improves the crosslinking degree of the network and has good antioxidant properties; the composite cryogel can slowly release active ions (Ca, Si, P, Se) over a long period of time, promoting the growth of bone marrow mesenchymal stem cells and new bone regeneration.

Claims

1. An antioxidant osteoinductive composite cryogel, characterized in that, Using polyvinyl alcohol (PVA) as the matrix material, physical crosslinking occurs through cyclic freezing and thawing with sulfated curdlan (CRs) to form a hydrogel. Meanwhile, selenium-containing bioactive glass (SeBG) is uniformly dispersed in the hydrogel matrix to prepare the composite cryogel SeBG@CRs / PVA.

2. A method for preparing an antioxidant osteoinductive composite cryogel, comprising the following steps: S1. Preparation of sulfated curdlan (CRs): Prepare a pyridine sulfur trioxide solution, add curdlan (CR) and stir evenly to obtain a suspension solution; add an alkali solution to adjust the pH to neutral, perform alcohol precipitation, filtration, and obtain a precipitate. After dissolving the precipitate in deionized water, centrifuge, dialyze, and freeze-dry to obtain sulfated curdlan (CRs); S2. Prepare selenium-containing bioactive glass (SeBG) according to the sol-gel method, and its chemical composition is SiO2-CaO-P2O5-SeO2; S3. Prepare an aqueous solution of polyvinyl alcohol (PVA), add the CRs from step S1, stir to obtain an aqueous CRs / PVA solution, and remove bubbles by ultrasonic treatment; S4. Prepare a CRs / PVA hydrogel loaded with SeBG: Add the SeBG powder from step S2 to the aqueous CRs / PVA solution, stir until completely dispersed, remove bubbles by ultrasonic treatment, and perform freeze-thaw treatment to obtain the composite cryogel SeBG@CRs / PVA.

3. The preparation method of the antioxidant osteoinductive composite cryogel according to claim 2, wherein, In step S1, the concentration of the pyridine sulfur trioxide solution is 5.0 - 10.0 wt%, and the solvent used includes one of N,N-dimethylformamide or anhydrous dimethyl sulfoxide; the concentration of CR in the pyridine sulfur trioxide solution is 3.0 - 5.0 wt%; the alkali solution includes one of NaOH solution or NaHCO3 solution, and the concentration of the alkali solution is 2.0 - 10.0 wt%; the degree of substitution of the CRs is 1.0 - 3.

0.

4. The preparation method of the antioxidant osteogenic composite cryogel according to claim 2, characterized in that, In step S1, the reaction conditions are an oil bath reaction at 30 - 50 °C for 2 - 5 h.

5. The preparation method of the antioxidant bone-promoting composite cryogel according to claim 2, characterized in that, In step S2, the theoretical molar ratio of SiO2, CaO, P2O5, and SeO2 in the SeBG is (40 - 58):(20 - 36):(4 - 6):(0.1 - 5); the particle size diameter of the SeBG is 1.0 - 5.0 μm.

6. The preparation method of the antioxidant osteoinductive composite cryogel according to claim 2, characterized in that, In step S3, the molecular weight of the polyvinyl alcohol (PVA) is 70000 - 200000, and the degree of alcoholysis is 98.0 - 99.0%; the concentration of the PVA aqueous solution is 5.0 - 10.0 wt%; the concentration of the CRs in the PVA aqueous solution is 0.5 - 5 wt%.

7. The preparation method of the antioxidant bone-forming composite cryogel according to claim 2, characterized in that, In step S3, the stirring speed is 150 - 300 rpm / min, and the stirring time is 2 - 5 h; the ultrasonic degassing time in steps S3 and S4 is 20 - 40 min.

8. The preparation method of the antioxidant bone-promoting composite cryogel according to claim 2, characterized in that, In step S4, the concentration of SeBG in the aqueous CRs / PVA solution is 0.05 - 0.5 wt%.

9. The preparation method of the antioxidant osteoinductive composite cryogel according to claim 2, characterized in that, In step S4, the freezing temperature is -40 - -10 °C, the freezing time is 12 - 48 h, the thawing time is 2 - 6 h; the number of freeze-thaw cycles is 1 - 3 times.

10. Use of the antioxidant osteoinductive composite cryogel as described in claim 1 in bone tissue engineering scaffolds and drug sustained release.

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