Oxidized hyaluronic acid-aminated polypeptide-polyacrylamide hydrogel and microcarrier as well as preparation method and application of oxidized hyaluronic acid-aminated polypeptide-polyacrylamide hydrogel and microcarrier

Abstract

The invention discloses oxidized hyaluronic acid-aminated polypeptide-polyacrylamide hydrogel, a microcarrier and a preparation method and application of the oxidized hyaluronic acid-aminated polypeptide-polyacrylamide hydrogel and the microcarrier, and belongs to the technical field of polymer gels. The polymer is prepared from N, N '-methylene bisacrylamide, tetramethylethylenediamine, aminated polypeptide and ammonium persulfate; the oxidation degree of the oxidized hyaluronic acid is 12-45%; the amino content of the aminated polypeptide is (3.59 + / -0.13 to 4.72 + / -0.18) * 10 <-4 > mol / g. The hydrogel prepared by the invention has injectability and excellent biocompatibility, biodegradability and mechanical properties, can be applied to the fields of regeneration and repair of skin wounds, cartilage and bones and the like, and can be used as a microcarrier to provide a suitable attachment and proliferation surface for serum-free culture of cells; wide clinical application prospects and market requirements are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer gels, and particularly relates to an oxidized hyaluronic acid - aminated gelatin - polyacrylamide hydrogel and microcarriers, a preparation method thereof, and an application thereof. Background Art

[0002] With the aggravation of population aging, injuries, necrosis, and defects of tissues such as skin, cartilage, and bone are common diseases in clinical medicine. However, due to reasons such as large wound areas, severe abrasion, excessive defects, and aging, the defective tissues cannot be completely regenerated and repaired. In recent years, the use of biomaterials to replace and repair defective tissues has made some good progress, but there are still some limitations, such as poor biocompatibility, insufficient mechanical properties, mismatched degradation rates, and lack of biological activity. Therefore, the development of new regenerative repair materials has broad clinical application prospects.

[0003] Natural polymer hydrogels are a class of extremely hydrophilic three - dimensional network structure gels. They rapidly swell in water and can maintain a large volume of water in this swollen state without dissolving (the water content can be as high as 99%). Due to the high water content, their mechanical properties are often poor and cannot yet meet clinical requirements. Hydrogels made by compounding natural polymers and synthetic polymers can not only make up for the deficiency of mechanical properties, but also maintain high biological activity, and at the same time achieve mechanical support and promote tissue regeneration. They are the research hotspots and development directions of clinical applied tissue engineering materials.

[0004] CN105086001A discloses a hyaluronic acid - gelatin / acrylamide double - network hydrogel and a preparation method thereof, which is composed of the interpenetration of a first - network hydrogel and a second - network hydrogel. The first network is a hydrogel formed by cross - linking modified hyaluronic acid and gelatin through Michael addition reaction, and the second network is a hydrogel formed by chemical cross - linking of acrylamide. It modifies hyaluronic acid through its modifiable sites and then combines with gelatin to form the first - network hydrogel. At the same time, acrylamide is introduced as the second network into the hydrogel, so that the hydrogel has good mechanical properties while having good biocompatibility, and broadens the application field of the hydrogel. However, this invention needs ultraviolet light irradiation to cure and cannot be used by injection, which is not conducive to popularization and application.

[0005] In the field of cell therapy, cell culture microcarriers can provide a large growth surface for adherent cells such as stem cells and improve the production efficiency of cell products. Most microcarriers on the market are prepared from polystyrene or dextran modified with positive charges, and these materials are not suitable for the adhesion of cells under serum - free culture conditions. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides an oxidized hyaluronic acid - amino - modified polypeptide - polyacrylamide hydrogel and a microcarrier, as well as a preparation method and application thereof. The oxidized hyaluronic acid - amino - modified polypeptide - polyacrylamide hydrogel of the present invention has injectability, good mechanical properties, biocompatibility and biodegradability, can load drugs or growth factors, and promote the regeneration and repair of tissues such as skin, cartilage and bone. Moreover, the oxidized hyaluronic acid - amino - modified polypeptide - polyacrylamide of the present invention can be used as a microcarrier to provide a suitable surface for the attachment and proliferation of cells in serum - free culture.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides an oxidized hyaluronic acid - amino - modified polypeptide - polyacrylamide hydrogel, which is made of oxidized hyaluronic acid, acrylamide, N,N'-methylenebisacrylamide, tetramethylethylenediamine, amino - modified polypeptide and ammonium persulfate.

[0009] In the present invention, the oxidation degree of the oxidized hyaluronic acid is 12 - 45%, and the measurement method of the oxidation degree is the hydroxylamine hydrochloride method.

[0010] In some embodiments, the preparation method of the oxidized hyaluronic acid is: dissolving hyaluronic acid in water, adding sodium periodate, reacting in the dark, and subjecting to dialysis and freeze - drying to obtain the oxidized hyaluronic acid.

[0011] Preferably, the molecular weight of the hyaluronic acid is 800 - 1500 kDa.

[0012] Preferably, the molar ratio of sodium periodate to disaccharide units of hyaluronic acid is 0.5 - 2:1.

[0013] Preferably, the reaction time in the dark is 2 - 24 h.

[0014] Preferably, the molecular cut - off of the dialysis is 8 - 14 kDa.

[0015] In the present invention, the amino content of the amino - modified polypeptide is (3.59 ± 0.13 - 4.72 ± 0.18)×10 -4 mol / g, and the measurement method of the amino content is the trinitrobenzenesulfonic acid method.

[0016] In some embodiments, the preparation method of the amino - modified polypeptide is: dissolving the polypeptide in water, adding an amino modifier and a catalyst, adjusting the pH value to 5.0 - 5.3, reacting, and subjecting to dialysis and freeze - drying to obtain the amino - modified polypeptide.

[0017] Preferably, the polypeptide is at least one of recombinant collagen, fibronectin, vitronectin and gelatin.

[0018] Preferably, the amino modifier is adipic dihydrazide (ADH) or carbohydrazide (CDH).

[0019] Preferably, the mass ratio of the polypeptide to the amino modifier is 0.5 - 1.5:1.

[0020] Preferably, the catalyst consists of catalyst 1 and catalyst 2, wherein catalyst 1 is 1-hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (NHS); catalyst 2 is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0021] Preferably, the molar ratio of catalyst 1, catalyst 2 to the amino modifier is 1 - 1.5:1:2.

[0022] In a second aspect, the present invention provides a method for preparing the above-mentioned oxidized hyaluronic acid - amino-functionalized polypeptide - polyacrylamide hydrogel, comprising the following steps:

[0023] (1) Add water to oxidized hyaluronic acid and acrylamide to form a mixed solution;

[0024] (2) Mix the mixed solution, N,N'-methylenebisacrylamide solution and tetramethylethylenediamine to obtain a first solution;

[0025] (3) Mix the amino-functionalized polypeptide solution and ammonium persulfate solution to obtain a second solution;

[0026] (4) Mix the first solution obtained in step (2) and the second solution obtained in step (3) for gelation to obtain the oxidized hyaluronic acid - amino-functionalized polypeptide - polyacrylamide hydrogel.

[0027] In some embodiments, the concentration of oxidized hyaluronic acid in the mixed solution in step (1) is 10 - 90 mg / mL; preferably 20 - 80 mg / mL; more preferably 30 - 50 mg / mL; the concentration of acrylamide is 15 - 55 wt%; preferably 20 - 50 wt%; more preferably 35 - 45 wt%.

[0028] In some embodiments, the concentration of the N,N'-methylenebisacrylamide solution in step (2) is 2 - 3 wt%, preferably 2.5 wt%.

[0029] In some embodiments, the volume ratio of the mixed solution, N,N'-methylenebisacrylamide solution and tetramethylethylenediamine in step (2) is 1 mL:20 - 150 μL:0.3 - 6 μL; preferably 1 mL:40 - 120 μL:0.5 - 4 μL; more preferably 1 mL:60 - 100 μL:1 - 3 μL.

[0030] In some embodiments, the concentration of the aminoated polypeptide solution in step (3) is 40 - 200 mg / mL; preferably 60 - 180 mg / mL; more preferably 80 - 160 mg / mL; the concentration of the ammonium persulfate solution is 4 - 6%; preferably 5 wt%.

[0031] In some embodiments, the volume ratio of the aminoated polypeptide solution to the ammonium persulfate solution in step (3) is 1 mL : 20 - 150 μL; preferably 1 mL : 40 - 120 μL; more preferably 1 mL : 60 - 100 μL.

[0032] In a third aspect, the present invention provides the use of the above-mentioned oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide hydrogel in the preparation of composite materials in the field of tissue engineering and composite materials in the field of biomedicine.

[0033] Preferably, the use is in the fields of skin wound regeneration repair, cartilage and bone regeneration repair.

[0034] In a fourth aspect, the present invention also provides the use of the above-mentioned oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide hydrogel in serum-free cell culture.

[0035] In a fifth aspect, the present invention provides an oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide microcarrier made from the aforementioned oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide hydrogel.

[0036] In a sixth aspect, the present invention provides a method for preparing an oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide microcarrier, comprising the following steps: injecting the oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide hydrogel into a castor oil mixture to form a water-in-oil emulsion, washing, and sieving to obtain the microcarrier.

[0037] In some embodiments, the castor oil mixture comprises castor oil and polyoxyethylene castor oil. Preferably, the volume ratio of castor oil to polyoxyethylene castor oil is 1 : 0.01 - 0.05; more preferably 1 : 0.02.

[0038] In some embodiments, the washing is sequentially performed with acetone, absolute ethanol, and pure water.

[0039] In some embodiments, the particle size of the oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide microcarrier is 120 - 200 μm.

[0040] The beneficial effects of the present invention are as follows:

[0041] The oxidized hyaluronic acid - aminated polypeptide glue - polyacrylamide hydrogel prepared by the present invention has a double - network structure: the aldehyde group of oxidized hyaluronic acid and the amino group of aminated gelatin form a Schiff base to constitute the first network structure, and acrylamide and N,N'-methylenebisacrylamide form the second network structure through free - radical polymerization. This hydrogel has injectability, excellent biocompatibility, biodegradability and mechanical properties, and can be applied to fields such as skin wound, cartilage and bone regeneration and repair. At the same time, the aminated polypeptides of the present invention, such as recombinant collagen, fibronectin, vitronectin or gelatin, can promote cell adhesion under serum - free culture conditions. Therefore, the oxidized hyaluronic acid - aminated polypeptide glue - polyacrylamide hydrogel of the present invention can also be used as a micro - carrier to provide a suitable surface for cell adhesion and proliferation in serum - free culture, and has broad clinical application prospects and market demand. Description of the Drawings

[0042] Figure 1 It is the infrared spectrum and nuclear magnetic resonance spectrum of oxidized hyaluronic acid, where HA is hyaluronic acid and oHA is oxidized hyaluronic acid.

[0043] Figure 2 It is the infrared spectrum and nuclear magnetic resonance spectrum of aminated gelatin, where Gel is gelatin, Gel - CDH is gelatin modified with carbohydrazide, and Gel - ADH is gelatin modified with adipic dihydrazide.

[0044] Figure 3 It is the gel - forming property (A) and injectability (B) of the oxidized hyaluronic acid - aminated polypeptide - polyacrylamide hydrogel prepared in Example 6.

[0045] Figure 4 It is the rheological property diagram of the oxidized hyaluronic acid - aminated polypeptide - polyacrylamide hydrogel prepared in Example 6.

[0046] Figure 5 It is the scanning electron microscope image of the oxidized hyaluronic acid - aminated polypeptide - polyacrylamide hydrogel prepared in Example 6.

[0047] Figure 6 It is the live - dead cell staining image of the oxidized hyaluronic acid - aminated polypeptide - polyacrylamide hydrogel prepared in Example 6.

[0048] Figure 7 It is the cell adhesion of the micro - carrier prepared in Example 10.

[0049] Figure 8 It is the cell adhesion of a common micro - carrier (dextran microspheres). Detailed Description of the Invention

[0050] The following describes the embodiments of the present invention in conjunction with specific examples, and those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0051] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments, rather than limiting the protection scope of the present invention.

[0052] The present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all ordinary commercially available products in this technical field. The solvents for solution preparation in the present invention are all high-purity water, and the temperature is room temperature (20 - 25°C). It is worth noting that the purity of gelatin in the present invention is of biotechnological grade and is purchased from Shanghai Macklin Biochemical Co., Ltd.; recombinant collagen (micoreCol.III-CP5-SMART) is purchased from Shanxi Jinbo Biopharmaceutical Co., Ltd.; fibronectin is purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; vitronectin is purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0053] Example 1 Oxidized Hyaluronic Acid

[0054] The preparation method is as follows:

[0055] 1) Weigh 2 g of hyaluronic acid (molecular weight 800 - 1500 kDa) and dissolve it in 200 mL of high-purity water, and continuously stir overnight;

[0056] 2) Weigh 1.1294 g of sodium periodate, dissolve it in 10 mL of high-purity water (protected from light), and then slowly add it dropwise to the hyaluronic acid solution in step 1);

[0057] 3) Continuously stir, protected from light, and react at room temperature for 4 h;

[0058] 4) Collect the obtained reaction mixture, put it into a dialysis bag with a diameter of 44 mm (molecular cut-off 8 - 14 kDa), and dialyze it in a water bath for 3 days, changing the water once a day;

[0059] 5) Collect the liquid obtained by dialysis into a glass petri dish with a diameter of 15 cm and freeze it in a -80°C refrigerator;

[0060] 6) Lyophilize for 5 days to obtain oxidized hyaluronic acid.

[0061] Figure 1 In, the infrared spectrum shows that oxidized hyaluronic acid is at 1732 cm-1 The characteristic peak of aldehyde group appears at this position, while no characteristic peak appears for hyaluronic acid here; the nuclear magnetic resonance spectrum shows that the characteristic peak of aldehyde group appears at 5 ppm for oxidized hyaluronic acid, while no characteristic peak appears for hyaluronic acid here. These results indicate that hyaluronic acid is oxidized to generate aldehyde group, and oxidized hyaluronic acid is obtained.

[0062] Example 2: Aminated gelatin

[0063] The preparation method is as follows:

[0064] 1) Weigh 1 g of gelatin, add it to 100 mL of high-purity water, heat to 50 °C, stir continuously, and cool to room temperature after the gelatin is dissolved;

[0065] 2) Weigh the amino modifier (ADH, 1.0452 g, 6 mmol or CDH, 0.5405 g, 6 mmol), and add it to the gelatin solution;

[0066] 3) Weigh catalyst 1 (HOBt, 0.4054 g, 3 mmol), add it to the gelatin solution, and stir continuously until completely dissolved;

[0067] 4) Weigh catalyst 2 (EDC, 0.5751 g, 3 mmol), and add it to the gelatin solution;

[0068] 5) Adjust the pH value of the reaction solution to 5.0 - 5.3, and stir the reaction for 24 h;

[0069] 6) Collect the obtained reaction mixture, put it into a dialysis bag with a diameter of 44 mm (molecular cut-off volume 8 - 14 kDa), place it in a water bath for dialysis for 3 days, and change the water once a day;

[0070] 7) Collect the liquid obtained by dialysis into a glass petri dish with a diameter of 15 cm, and freeze it in a -80 °C refrigerator;

[0071] 8) Perform freeze-drying treatment for 5 days to obtain aminated gelatin.

[0072] Example 3: Aminated recombinant collagen

[0073] The preparation method of the present invention is the same as that of Example 2, except that recombinant collagen is used instead of gelatin to obtain aminated collagen.

[0074] Example 4: Aminated fibronectin

[0075] The preparation method of the present invention is the same as that of Example 2, except that fibronectin is used instead of gelatin to obtain aminated fibronectin.

[0076] Example 5: Aminated vitronectin

[0077] The preparation method of the present invention is the same as that of Example 2, except that vitronectin is used instead of gelatin to obtain amino-functionalized vitronectin.

[0078] Taking the amino-functionalized gelatin prepared in Example 2 as an example, Figure 2 in which, the infrared spectrum shows that the amino characteristic peaks of adipic dihydrazide-modified gelatin (Gel-ADH) and carbohydrazide-modified gelatin (Gel-CDH) are further enhanced; the nuclear magnetic resonance spectrum shows that both Gel-ADH and Gel-CDH have the characteristic peak of NH in hydrazide at 7.8 ppm, while Gelatin does not have a characteristic peak here. These results indicate that the hydrazide group has been successfully modified onto the gelatin molecular chain and the number of its amino groups has increased.

[0079] The infrared spectrum shows that the hydrazide group can be successfully modified onto the molecular chains of gelatin, recombinant collagen, fibronectin and vitronectin.

[0080] Example 6 Oxidized Hyaluronic Acid-Amino-Functionalized Polypeptide-Polyacrylamide Hydrogel

[0081] The preparation method is as follows:

[0082] 1) Weigh 1.24 g of oxidized hyaluronic acid and 12.4 g of acrylamide, add 20 mL of high-purity water, and continuously stir. The solution swells to about 31 mL to prepare a mixed solution of 40 mg / mL oxidized hyaluronic acid and 40 wt% acrylamide;

[0083] 2) Weigh 3.2 g of amino-functionalized gelatin, add 20 mL of high-purity water, and continuously stir to prepare a 120 mg / mL amino-functionalized gelatin solution;

[0084] 3) Weigh 100 mg of N,N'-methylenebisacrylamide, add 4 mL of high-purity water, and vortex to prepare a 2.5 wt% N,N'-methylenebisacrylamide solution;

[0085] 4) Weigh 150 mg of ammonium persulfate, add 3 mL of high-purity water, and vortex to prepare a 5 wt% ammonium persulfate solution;

[0086] 5) Take 1 mL of the oxidized hyaluronic acid-acrylamide mixed solution, 80 μL of the N,N'-methylenebisacrylamide solution, and 2 μL of tetramethylethylenediamine into syringe ①;

[0087] Take 1 mL of the amino-functionalized gelatin solution and 80 μL of the ammonium persulfate solution into syringe ②;

[0088] 6) Mix syringe ① and ②, inject back and forth at least 10 times, inject the mixed solution into the mold, and the hydrogel can be obtained after sufficient curing. Store it at 4 °C for later use.

[0089] Example 7 Oxidized Hyaluronic Acid - Aminated Polypeptide - Polyacrylamide Hydrogel

[0090] This example is roughly the same as Example 6, with the differences being as follows:

[0091] Aminated collagen is used instead of aminated gelatin;

[0092] During the preparation process,

[0093] The concentration of oxidized hyaluronic acid in the mixed solution is 20 mg / mL, and the concentration of acrylamide is 20 wt%;

[0094] The volume of N,N'-methylenebisacrylamide solution is 40 μL;

[0095] The volume of tetramethylethylenediamine is 0.5 μL;

[0096] The concentration of aminated collagen solution is 60 mg / mL;

[0097] The volume of ammonium persulfate solution is 40 μL.

[0098] Example 8 Oxidized Hyaluronic Acid - Aminated Polypeptide - Polyacrylamide Hydrogel

[0099] This example is roughly the same as Example 6, with the differences being as follows:

[0100] Aminated fibronectin is used instead of aminated gelatin;

[0101] During the preparation process,

[0102] The concentration of oxidized hyaluronic acid is 80 mg / mL, and the concentration of acrylamide is 50 wt%;

[0103] The volume of N,N'-methylenebisacrylamide solution is 120 μL;

[0104] The volume of tetramethylethylenediamine is 4 μL;

[0105] The concentration of aminated fibronectin solution is 180 mg / mL;

[0106] The volume of ammonium persulfate solution is 120 μL.

[0107] Comparative Example 1 Oxidized Hyaluronic Acid - Aminated Polypeptide - Polyacrylamide Hydrogel

[0108] This example is roughly the same as Example 6, with the differences being as follows: Oxidized hyaluronic acid with an oxidation reaction time of 1 h (oxidation degree of 10%, hydroxylamine hydrochloride method) is used; the amino content of aminated gelatin (trinitrobenzenesulfonic acid method) is 3.1×10 -4 mol / g.

[0109] The aldehyde group and amino group concentrations in Comparative Example 1 were too low to form the first network, and the hydrogel had poor gel-forming performance.

[0110] Comparative Example 2 Oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide hydrogel

[0111] This example is roughly the same as Example 6, the difference being that oxidized hyaluronic acid with an oxidation reaction time of 30 h (oxidation degree of 53%, hydroxylamine hydrochloride method) was used; the amino group content of aminoated gelatin (trinitrobenzenesulfonic acid method) was 5.2×10 - 4 mol / g.

[0112] The molecular chain of the oxidized hyaluronic acid in Comparative Example 2 was short, the first network structure was unstable, and the hydrogel had poor gel-forming performance.

[0113] Comparative Example 3 Oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide hydrogel

[0114] This example is roughly the same as Example 6, the difference being that:

[0115] The volume of N,N'-methylenebisacrylamide solution was 10 μL;

[0116] The volume of tetramethylethylenediamine was 0.2 μL;

[0117] The volume of ammonium persulfate solution was 10 μL.

[0118] The contents of the crosslinking agent, initiator, and accelerator in Comparative Example 3 were too low to form the second network, and the hydrogel had poor gel-forming performance.

[0119] Comparative Example 4 Oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide hydrogel

[0120] This example is roughly the same as Example 6, the difference being that:

[0121] The volume of N,N'-methylenebisacrylamide solution was 160 μL;

[0122] The volume of tetramethylethylenediamine was 8 μL;

[0123] The volume of ammonium persulfate solution was 160 μL.

[0124] The contents of the crosslinking agent, initiator, and accelerator in Comparative Example 4 were too high, and the speed of forming the second network was too fast, resulting in poor injectability of the hydrogel.

[0125] Example 9

[0126] This example verified the performance of the above-mentioned oxidized hyaluronic acid - aminoated polypeptide - polyacrylamide hydrogel (hereinafter referred to as hydrogel) as follows:

[0127] 1. Gel-forming property and injectability

[0128] In Examples 6 - 8, before the solution gelled, syringes ① and ② were simultaneously injected into a vial, and the gel-forming property of the hydrogel was observed laterally, with ultrapure water selected as the reference group. In Examples 6 - 8, before the syringe mixture was injected into the mold, a syringe needle was attached to the syringe, and the hydrogel was injected into a glass petri dish to characterize its injection ability.

[0129] The results are as Figure 3 shown. Figure 3 In A, it shows that the hydrogel forms a hydrogel within seconds to minutes; Figure 3 In B, it shows that the hydrogel prepared by this method has good injectability.

[0130] 2. Rheological mechanical properties

[0131] In Examples 6 - 8, before the syringe mixture was injected into the mold, the syringe liquid was injected into the rheometer test platform with a platform diameter of 2.5 cm and a gap height set at 1 mm; the oscillatory amplitude sweep test was performed on the hydrogel using a rheometer with a frequency set at 1 Hz and an amplitude set at 0.1% - 1000%; the flow sweep test was performed on the hydrogel using a rheometer with a linear shear rate set at 1 to 50 s -1 ⁻¹; the self-healing property test was performed on the hydrogel using a rheometer with an oscillatory amplitude set at a small amplitude of 1.25% for 120 seconds and a large amplitude of 500% for 120 seconds, and 4 cycles were performed; the temperature sweep test was performed on the hydrogel using a rheometer with an amplitude set at 1.25%, a frequency set at 1 Hz, a temperature range set at 25°C to 45°C, and a heating rate of 1°C / min.

[0132] The results are as Figure 4 shown. Figure 4 In A, in the amplitude range of 0.1% to 50%, the storage modulus and loss modulus of the hydrogel always remained within a stable range, and the storage modulus was much greater than the loss modulus, showing the characteristics of a solid hydrogel; when the amplitude was approximately 230%, the storage modulus = loss modulus, and the hydrogel was at the critical point between a colloid and a fluid. Figure 4 In B, with the increase of the shear rate, the viscosity of the hydrogel decreased rapidly, showing shear thinning characteristics. Figure 4In the self-healing performance test, under small amplitude conditions, the storage modulus is much larger than the loss modulus, showing the characteristics of a solid hydrogel; under large amplitude conditions, the storage modulus decreases, the loss modulus increases, and the storage modulus is smaller than the loss modulus, showing the characteristics of a liquid fluid; when the amplitude changes back to a small amplitude or a large amplitude, the storage modulus and the loss modulus both return to their original values, and remain consistent for 4 cycles, indicating that the hydrogel has good self-healing properties. Figure 4 In the temperature scanning test, the storage modulus and loss modulus of the hydrogel are always maintained in a stable range, showing that the strength of the hydrogel does not change with temperature.

[0133] 3. Internal structure and biocompatibility

[0134] In Examples 6-8, the hydrogel was cut into blocks of 10*10*2 mm and freeze-dried, and the internal structure of the hydrogel was observed using a scanning electron microscope.

[0135] The results are as follows Figure 5 As shown, it can be observed that the hydrogel scaffold presents a three-dimensional porous double network structure, the pores are interconnected, the pore size of the first network is about 50 μm, and the pore size of the second network is about 5 μm.

[0136] The hydrogel scaffolds were sterilized by ultraviolet irradiation for 24 h and soaked in cell culture medium for 8 h before being used for cell culture to detect their biocompatibility.

[0137] Human bone marrow mesenchymal stem cells were 1*10 4 The cells were seeded on the hydrogel surface at a density of 100 / well. Blank wells were selected as the control group, and the cell viability was detected using the Calcein-AM / PI Live-Dead cell staining kit after 1 and 3 days of culture.

[0138] The results are as follows Figure 6 As shown, after 1 day and 3 days of culture, most of the human bone marrow mesenchymal stem cells on the hydrogel survived (green), and there were almost no dead cells (red). Compared with the blank control group, there was no significant difference in the hydrogel group, indicating that the hydrogel has good cell compatibility and can be used in the field of tissue repair and regenerative medicine.

[0139] Example 10 Oxidized hyaluronic acid-amino polypeptide-polyacrylamide microcarrier

[0140] The preparation method is:

[0141] 1) Weigh 1.24 g of oxidized hyaluronic acid and 12.4 g of acrylamide, add 20 mL of high-purity water, and continue stirring until the solution expands to about 31 mL to prepare a mixed solution of 40 mg / mL oxidized hyaluronic acid and 40 wt% acrylamide;

[0142] 2) Weigh 3.2 g of aminoated collagen, add 20 mL of high-purity water, and continuously stir to prepare a 120 mg / mL aminoated collagen solution;

[0143] 3) Weigh 100 mg of N,N'-methylenebisacrylamide, add 4 mL of high-purity water, and vortex to prepare a 2.5 wt% N,N'-methylenebisacrylamide solution;

[0144] 4) Weigh 150 mg of ammonium persulfate, add 3 mL of high-purity water, and vortex to prepare a 5 wt% ammonium persulfate solution;

[0145] 5) Take 1 mL of oxidized hyaluronic acid-acrylamide mixed solution, 80 μL of N,N'-methylenebisacrylamide solution, and 2 μL of tetramethylethylenediamine into syringe ①;

[0146] Take 1 mL of aminoated collagen solution and 80 μL of ammonium persulfate solution into syringe ②;

[0147] 6) Add 4 mL of castor oil and 80 μL of polyoxyethylene castor oil to a 20 mL glass container, and mix well with a magnetic stirrer;

[0148] 7) Mix syringe ① and ②, inject back and forth at least 10 times, inject the mixed solution into the above castor oil, quickly stir for 8 hours to form a water-in-oil emulsion, then wash it successively with acetone, ethanol, and pure water to obtain hydrogel microspheres, and sieve them with a screening device to obtain microcarriers with a particle size of 120 - 200 microns.

[0149] Comparing the cell adhesion diagrams of conventional microcarriers (such as Figure 8 ) shows that the microcarriers prepared by the present invention have better cell spreading and adhesion effects in serum-free culture (such as Figure 7 ).

[0150] It should be emphasized that the embodiments described in the present invention are illustrative rather than restrictive. Therefore, the present invention includes but is not limited to the embodiments described in the specific embodiments. Those skilled in the art can make changes according to the technology of the present invention.

Claims

1. An oxidized hyaluronic acid-amino polypeptide-polyacrylamide hydrogel, characterized in that: Made from oxidized hyaluronic acid, acrylamide, N,N'-methylenebisacrylamide, tetramethylethylenediamine, amino peptides, and ammonium persulfate; The oxidation degree of the oxidized hyaluronic acid is 12-45%; the amino content of the amino polypeptide is (3.59±0.13-4.72±0.18)×10 -4 mol / g.

2. The oxidized hyaluronic acid-amino polypeptide-polyacrylamide hydrogel according to claim 1, characterized in that: The preparation method of the oxidized hyaluronic acid is as follows: dissolving hyaluronic acid in water, adding sodium periodate, reacting in the dark, and dialysis and freeze drying to obtain the oxidized hyaluronic acid.

3. The oxidized hyaluronic acid-amino polypeptide-polyacrylamide hydrogel according to claim 2, characterized in that: The molecular weight of the hyaluronic acid is 800-1500 kDa; and / or, the molar ratio of sodium periodate to hyaluronic acid disaccharide unit is 0.5-2:1; And / or, the light-proof reaction time is 2-24h; And / or, the molecular cut-off of the dialysis is 8-14 kDa.

4. The oxidized hyaluronic acid-amino polypeptide-polyacrylamide hydrogel according to claim 1, characterized in that: The preparation method of the amino polypeptide is as follows: dissolving the polypeptide in water, adding an amino modifier and a catalyst, adjusting the pH value to 5.0-5.3, reacting, dialysis and freeze drying to obtain the amino polypeptide, wherein the polypeptide is at least one of recombinant collagen, fibronectin, vitronectin and gelatin.

5. The oxidized hyaluronic acid-amino polypeptide-polyacrylamide hydrogel according to claim 4, characterized in that: The amino modifier is adipic acid dihydrazide or carbohydrazide; and / or, the mass ratio of the polypeptide to the amino modifier is 0.5-1.5:1; And / or, the catalyst is composed of catalyst 1 and catalyst 2, wherein catalyst 1 is 1-hydroxybenzotriazole or N-hydroxysuccinimide; and catalyst 2 is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; And / or, the molar ratio of the catalyst 1, the catalyst 2 and the amino modifier is 1-1.5:1:

2.

6. The method for preparing the oxidized hyaluronic acid-amino polypeptide-polyacrylamide hydrogel according to any one of claims 1 to 5, characterized in that: The steps include: (1) adding water to oxidized hyaluronic acid and acrylamide to prepare a mixed solution; (2) mixing the mixed solution, the N,N'-methylenebisacrylamide solution and tetramethylethylenediamine to obtain a first solution; (3) mixing the amino polypeptide solution and the ammonium persulfate solution to obtain a second solution; (4) The first solution obtained in step (2) and the second solution obtained in step (3) are mixed and gelled to obtain the oxidized hyaluronic acid-amino polypeptide-polyacrylamide hydrogel.

7. The preparation method according to claim 6, characterized in that: The concentration of oxidized hyaluronic acid in the mixed solution in step (1) is 10-90 mg / mL; the concentration of acrylamide is 15-55 wt%; the concentration of N,N'-methylenebisacrylamide solution in step (2) is 2-3 wt%; the volume ratio of the mixed solution, N,N'-methylenebisacrylamide solution and tetramethylethylenediamine is 1 mL:20-150 μL:0.3-6 μL; the concentration of the amino polypeptide solution in step (3) is 40-200 mg / mL, and the concentration of the ammonium persulfate solution is 4-6 wt%; the volume ratio of the amino polypeptide solution to the ammonium persulfate solution is 1 mL:20-150 μL.

8. An oxidized hyaluronic acid-amino polypeptide-polyacrylamide microcarrier, characterized in that: The method is prepared from the oxidized hyaluronic acid-amino polypeptide-polyacrylamide hydrogel according to any one of claims 1 to 6 or the oxidized hyaluronic acid-amino polypeptide-polyacrylamide hydrogel prepared by the preparation method according to any one of claim 7.

9. The method for preparing the oxidized hyaluronic acid-amino polypeptide-polyacrylamide microcarrier according to claim 8, characterized in that: The method comprises the following steps: injecting oxidized hyaluronic acid-amino polypeptide-polyacrylamide hydrogel into a castor oil mixture to form a water-in-oil emulsion, washing, and sieving to obtain the product; The castor oil mixture comprises castor oil and polyoxyethylene castor oil.

10. Use of the oxidized hyaluronic acid-aminated gelatin-polyacrylamide hydrogel according to any one of claims 1 to 5 or the oxidized hyaluronic acid-aminated gelatin-polyacrylamide hydrogel prepared by the preparation method according to any one of claims 6 to 9 and microcarriers in preparing composite materials in the field of tissue engineering and composite materials in the field of biomedicine.

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