A gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradability and its preparation method

Abstract

A gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradation and its preparation method. First, through the acetalization reaction of benzaldehyde with different multi-arm polyethylene glycols, water-soluble multi-arm benzaldehyde-modified polyethylene glycol is synthesized. At the same time, adipic dihydrazide is modified onto the gelatin polymer chain to obtain adipic dihydrazide-modified gelatin. Then, the multi-arm benzaldehyde-modified polyethylene glycol and adipic dihydrazide-modified gelatin undergo a Schiff base reaction under physiological conditions to generate dynamic hydrazone bonds and imine bonds, obtaining a series of hydrogels with independently controllable degradation and different cross-linking network structures. The advantages of the present invention are that polyethylene glycol with different multi-arm grid structures endows the hydrogel system with different degradation rates, and hydrogels with different mechanical moduli can be prepared according to the substitution degree of adipic dihydrazide-modified gelatin and the gel-forming mass fraction to match various tissues, while other parameters such as its gel-forming components, polymer concentration, and mechanical properties can be kept unified and unchanged.

Description

Technical Field

[0001] The present invention relates to the technical fields of polymer materials and stem cell biology, and particularly relates to a gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradation and a preparation method thereof. Specifically, a series of hydrogels with different cell-mediated degradabilities are formed by crosslinking poly(ethylene glycol) diacrylate modified with different multi-arm benzaldehydes (multi-arm-PEGDA) and gelatin modified with adipic dihydrazide (GtnADH) polymer chains through dynamic imine bonds and hydrazone bonds. Background Art

[0002] Polymer hydrogels are a class of biomimetic polymer materials with high water content, good biocompatibility, and a three-dimensional mesh structure that mimics the extracellular matrix, and have wide applications in the field of regenerative medicine such as three-dimensional cell scaffolds and tissue engineering. In in vitro three-dimensional stem cell culture, matrix metalloproteinases secreted by stem cells can effectively reshape and degrade the hydrogel network structure, enabling cells to further undergo morphogenesis, migration, proliferation, or enhance cell-cell connections. The design of different degradabilities of the hydrogel itself in response to matrix metalloproteinases can effectively regulate stem cell behavior, including stemness maintenance, proliferation, differentiation, and migration.

[0003] The patent application with the application number 202110477460.3 discloses a preparation method of an injectable self-adaptive natural hydrogel adhesive, which is used for repairing damaged lung tissue but cannot be degraded independently and controllably by cell mediation. Currently, the degradation of hydrogel materials is mainly regulated by two factors, one is the cleavage rate of the chemical bonds used for crosslinking, and the other is its network structure. Adjusting the polymer concentration or crosslinking chemical bonds can effectively change its degradability. However, changes in the polymer composition, concentration, or crosslinking method will also cause changes in other properties of the hydrogel, such as pore size, crosslinking density, content of bioactive sites, and mechanical strength. Therefore, its degradability is difficult to regulate independently. Summary of the Invention

[0004] In order to overcome the defects of the prior art, the object of the present invention is to provide a gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradability and its preparation method. First, through the acetalization reaction of benzaldehyde (DA) with different multi-arm polyethylene glycols, water-soluble multi-arm benzaldehyde polyethylene glycol (multi-arm-PEGDA) is synthesized. At the same time, adipic dihydrazide (ADH) is modified onto the gelatin polymer chain to obtain adipic dihydrazide gelatin (GtnADH). Finally, the multi-arm benzaldehyde polyethylene glycol and adipic dihydrazide gelatin with different acylhydrazide substitution degrees undergo a Schiff base reaction under physiological conditions to form dynamic acylhydrazone bonds and imine bonds, obtaining a series of gelatin-multi-arm polyethylene glycol hydrogels with different cross-linked structures. Due to the cell degradation sites inherent in the gelatin polymer chain and the different multi-arm structures of polyethylene glycol, the hydrogel system has cell-mediated controllable degradability. In addition, the dynamic reversible characteristics of the acylhydrazone bond and the imine bond endow the hydrogel with excellent self-healing and injectability.

[0005] In order to achieve the above object, the technical solution of the present invention is realized as follows:

[0006] A gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradability is composed of equal volumes of different multi-arm benzaldehyde polyethylene glycol solutions and adipic dihydrazide gelatin solutions;

[0007] The mass fractions of the benzaldehyde polyethylene glycol solution and the adipic dihydrazide gelatin solution are both 1% - 20%, and the solvent is PBS solution;

[0008] The benzaldehyde polyethylene glycol includes: multi-arm polyethylene glycol, 4-formylbenzoic acid p-CBA, 4-dimethylaminopyridine DMAP, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC . HCL, controlling the molar ratio of the hydroxyl group of multi-arm polyethylene glycol, the carboxyl group of 4-formylbenzoic acid, EDC . HCL, DMAP to be 1:2:2:0.5;

[0009] The multi-arm polyethylene glycol is selected from: 2-arm-PEG, 3-arm-PEG, 4-arm-PEG, 5-arm-PEG, 6-arm-PEG, 7-arm-PEG, 8-arm-PEG, 9-arm-PEG, 10-arm-PEG;

[0010] The adipic dihydrazide gelatin includes gelatin, adipic dihydrazide (ADH), 1-hydroxybenzotriazole hydrate HOBT, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDC .HCL, in this reaction system, based on 1 g of gelatin, and the molar ratios of the remaining ADH, HOBT, and EDC . to HCL are 1:0.5 - 2:0.4 - 0.8.

[0011] A preparation method of a gelatin - multi - arm polyethylene glycol hydrogel system with cell - mediated degradability independently controllable, comprising the following steps:

[0012] Step 1: Add 100 - 250 mL of dichloromethane into a 500 mL round - bottom flask. Under the protection of nitrogen, successively add multi - arm polyethylene glycol with a molecular weight between 500 and 20000 Da, 4 - formylbenzoic acid p - CBA, 4 - dimethylaminopyridine DMAP, 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide hydrochloride EDC . HCL, and control the molar ratios of the hydroxyl group of multi - arm polyethylene glycol, the carboxyl group of 4 - formylbenzoic acid, EDC . HCL, and DMAP to be 1:2:2:0.5. React at room temperature for 18 - 36 h; After the reaction is completed, remove the reaction flask, extract the solution with saturated brine 2 - 5 times, dry it with anhydrous magnesium sulfate, and then concentrate it with a rotary evaporator; Then precipitate the upper layer liquid in excess ice - ether, repeat 2 - 5 times; The obtained product is dialyzed with distilled water, changing the water once a day, dialyze for one week, and finally lyophilize it with a freeze - dryer to obtain different multi - armed benzaldehyde polyethylene glycols;

[0013] The multi - arm polyethylene glycol described is selected from: 2 - arm - PEG, 3 - arm - PEG, 4 - arm - PEG, 5 - arm - PEG, 6 - arm - PEG, 7 - arm - PEG, 8 - arm - PEG, 9 - arm - PEG, 10 - arm - PEG;

[0014] Step 2: Add 40 - 160 mL of PBS with pH 5.2 - 5.5 into a 250 mL round - bottom flask, then add 1 g of type A pigskin gelatin, and under the heating condition of 50 - 60 °C, magnetically rotate and stir for 1 - 1.5 h to fully dissolve it; Then add adipic dihydrazide (ADH) and stir evenly; Add 1 - hydroxybenzotriazole hydrate (HOBT) dissolved in dimethyl sulfoxide (DMSO) into the reaction flask and stir evenly; Finally, add N - (3 - dimethylaminopropyl) - N'-ethylcarbodiimide hydrochloride EDC . HCL, in this reaction system, ADH, HOBT, EDC .The molar ratio of HCl is 1:0.5 - 2:0.4 - 0.8, and adipic dihydrazide gelatin with a substitution degree of 20 - 80% is finally obtained; at the same time, the pH is adjusted to 5.0 - 5.3 with sodium hydroxide NaOH solution; the mixture is stirred at 50 - 60 °C for 24 - 36 h, and after the reaction, it is dialyzed with distilled water, changing the water once a day for one week, and then freeze-dried to obtain adipic dihydrazide gelatin;

[0015] Step 3: Dissolve the products of step 1 with different multi-arm benzaldehyde-functionalized polyethylene glycols in PBS solution with a mass fraction of 1% - 20%; dissolve the product of step 2, adipic dihydrazide gelatin with amino and hydrazide groups, in PBS solution with a mass fraction of 1% - 20%. Then, take equal volumes of the solutions of different multi-arm benzaldehyde-functionalized polyethylene glycols and the solution of adipic dihydrazide gelatin, vortex and mix them evenly at 37 °C, and let them stand to obtain hydrogel systems with different degradabilities.

[0016] The beneficial effects of the present invention:

[0017] 1. The amino and hydrazide groups on adipic dihydrazide gelatin react with the aldehyde groups on multi-arm benzaldehyde-functionalized polyethylene glycol to prepare a hydrogel system with independently controllable cell-mediated degradation through the formation of reversible imine bonds and hydrazone bonds. Among them, the amino and hydrazide groups on the adipic dihydrazide gelatin macromolecule can react with the aldehyde groups on the multi-arm benzaldehyde-functionalized polyethylene glycol to form dynamic imine bonds and hydrazone bonds respectively through Schiff base reaction. These two dynamic covalent bonds both have the characteristics of dynamic reversibility. The present invention selects different multi-arm benzaldehyde-functionalized polyethylene glycols to synthesize hydrogels with different cross-linked network structures with adipic dihydrazide gelatin, and utilizes the cell degradability of gelatin and the different multi-arm network structures of polyethylene glycol to endow the hydrogel system with independently controllable cell degradability.

[0018] 2. The synthesis process of the present invention is simple and green, with mild reaction conditions. The obtained hydrogel has good application prospects in biomedical fields such as tissue engineering, three-dimensional cell culture, stem cell transplantation, and drug sustained release. Due to the cell degradation sites inherently present on the gelatin polymer chain and the different multi-arm structures of polyethylene glycol, the hydrogel system exhibits cell-mediated controllable degradability. At the same time, different degrees of substitution can regulate the mechanical properties of the hydrogel. In addition, the dynamic reversible characteristics of the hydrazone bond and imine bond endow the hydrogel with excellent self-healing and injectability. Matrix metalloproteinases secreted by cells can break and degrade the gelatin polymer chain, and polyethylene glycol with different multi-arm structures endows the hydrogel system with different degradation rates. That is, the cell degradability of the hydrogel can be independently regulated by adjusting the different multi-arm grid structures of polyethylene glycol, while keeping other parameters such as its gel-forming components, polymer concentration, and mechanical properties unchanged. In addition, the reversible cross-linking mode of the dynamic imine bond and hydrazone bond also endows the hydrogel system with excellent self-healing performance and injectability. This hydrogel system can be widely used in the three-dimensional culture, expansion, and differentiation of different stem cells in vitro, and can be used for stem cell transplantation therapy. Brief Description of the Drawings

[0019] Figure 1 Schematic diagram of a gelatin-multi-arm polyethylene glycol hydrogel system (2, 4, 8 arms) with independently controllable cell-mediated degradability. Detailed Embodiments

[0020] The present invention will be described in more detail below with reference to the embodiments.

[0021] Example 1

[0022] This example includes the following steps:

[0023] Step 1: Prepare nine 500 mL round-bottom flasks, each containing 50 mL of dichloromethane. Under the protection of nitrogen, add 1 g of 2-arm polyethylene glycol (2-arm-PEG) (molecular weight 5000 Da), 1 g of 3-arm polyethylene glycol (3-arm-PEG) (molecular weight 7500 Da), 1 g of 4-arm polyethylene glycol (4-arm-PEG) (molecular weight 10000 Da), 1 g of 5-arm polyethylene glycol (5-arm-PEG) (molecular weight 12500 Da), 1 g of 6-arm polyethylene glycol (6-arm-PEG) (molecular weight 15000 Da), 1 g of 7-arm polyethylene glycol (7-arm-PEG) (molecular weight 17500 Da), 1 g of 8-arm polyethylene glycol (8-arm-PEG) (molecular weight 20000 Da), 1 g of 9-arm polyethylene glycol (9-arm-PEG) (molecular weight 22500 Da), and 1 g of 10-arm polyethylene glycol (10-arm-PEG) (molecular weight 25000 Da) respectively. After stirring and mixing evenly, add 0.1201 g of 4-formylbenzoic acid (p-CBA), 0.0244 g of 4-dimethylaminopyridine (DMAP), and 0.1534 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC . HCL), and react at room temperature for 24 h. After the reaction is completed, remove the reaction flask, extract the solution with about 100 ml of saturated brine three times, dry it with anhydrous magnesium sulfate, and then concentrate it with a rotary evaporator. Then, add it to 100 ml of pre-cooled ether to precipitate, repeat three times, and dialyze the obtained product with distilled water. For 2 and 3-arm-PEG, use a 5 kDa dialysis bag; for 4 and 5-arm-PEG, use a 10 kDa dialysis bag; for 6 and 7-arm-PEG, use a 15 kDa dialysis bag; for 8, 9, and 10-arm-PEG, use a 20 kDa dialysis bag. Change the water once a day and dialyze for about one week. Finally, lyophilize it with a freeze dryer to obtain benzaldehyde polyethylene glycol with two, three, four, five, six, seven, eight, nine, and ten arms.

[0024] Step 2: Add 40 mL of PBS with a pH of 5.5 to a 250 mL round-bottom flask, add 1.0 g of type A porcine skin gelatin, and heat it at 50 °C and magnetically stir for about 1 hour to dissolve it completely. Then add 1.392 g of adipic dihydrazide and stir evenly. Then add 0.308 g of 1-hydroxybenzotriazole hydrate (HOBT) dissolved in 5 ml of DMSO to the reaction flask and stir evenly. Finally, add 0.308 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ((EDC .HCL)). At the same time, 1M NaOH was used to quickly adjust the pH to 5.0-5.3; the mixture was stirred at 50°C for 24 hours. After the reaction, the mixture was dialyzed with distilled water (8kDA dialysis bag), and the water was changed once a day for about one week, followed by freeze drying to obtain adipic acid dihydrazide gelatin with a substitution degree of about 38%.

[0025] Step 3: According to the source of stem cells, a precursor substance of appropriate concentration is selected for dissolution. For example, the product of step 1 having an aldehyde group, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nine-, or ten-arm benzaldehyde polyethylene glycol (2-arm-PEGDA, 3-arm-PEGDA, 4-arm-PEGDA, 5-arm-PEGDA, 6-arm-PEGDA, 7-arm-PEGDA, 8-arm-PEGDA, 9-arm-PEGDA, and 10-arm-PEGDA) is dissolved in a phosphate buffer solution at a mass fraction of 2% to obtain precursor solutions of 2% 2-arm-PEGDA, 2% 3-arm-PEGDA, 2% 4-arm-PEGDA, 2% 5-arm-PEGDA, 2% 6-arm-PEGDA, 2% 7-arm-PEGDA, 2% 8-arm-PEGDA, 2% 9-arm-PEGDA, and 2% 10-arm-PEGDA, respectively; the product of step 2, adipic acid dihydrazide gelatin having an amino group and a hydrazide group, is dissolved in a phosphate buffer solution at a mass fraction of 1 0% was dissolved in a phosphate buffer solution, and after being thoroughly vortexed, a precursor solution of 10% GtnADH was obtained; an equal volume of 100 μL of di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, or deca-arm benzaldehyde polyethylene glycol solution and an equal volume of 100 μL of adipic acid dihydrazide gelatin solution were taken, and the two were shaken evenly at 37°C, and the two were also allowed to stand at 37°C to obtain a gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradation, i.e., 1% 2-arm-PEGDA / 5% GtnADH, 1% 3- arm-PEGDA / 5%GtnADH, 1%4-arm-PEGDA / 5%GtnADH, 1%5-arm-PEGDA / 5%GtnADH, 1%6-arm-PEGDA / 5%GtnADH, 1% 7-arm-PEGDA / 5%GtnADH, 1%8-arm-PEGDA / 5%GtnADH, 1%9-arm-PEGDA / 5%GtnADH, 1%10-arm-PEGDA / 5%GtnADH.

[0026] By injecting the hydrogel solutions of di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, and deca-armed polyethylene glycol into a mold, taking them out after gelation, and measuring the modulus and degradability of the hydrogels, it can be found that their moduli are consistent. At the same time, by measuring the degradability with a matrix metalloproteinase solution, it can be found that as the number of polyethylene glycol arms in the hydrogel system increases, the degradability becomes slower, confirming that this type of hydrogel has the same stiffness but different degradabilities.

[0027] Referring to Figure 1 , due to the cell degradation sites inherently present on the gelatin polymer chains and the different multi-arm structures of polyethylene glycol, this hydrogel system exhibits cell-mediated controllable degradability.

[0028] Example 2:

[0029] This example includes the following steps:

[0030] Step 1: Prepare 9 500 mL round-bottom flasks, each adding 50 mL of dichloromethane. Under the protection of nitrogen, add 1 g of 2-arm polyethylene glycol (2-arm-PEG) (molecular weight 5000 Da), 1 g of 3-arm polyethylene glycol (3-arm-PEG) (molecular weight 7500 Da), 1 g of 4-arm polyethylene glycol (4-arm-PEG) (molecular weight 10000 Da), 1 g of 5-arm polyethylene glycol (5-arm-PEG) (molecular weight 12500 Da), 1 g of 6-arm polyethylene glycol (6-arm-PEG) (molecular weight 15000 Da), 1 g of 7-arm polyethylene glycol (7-arm-PEG) (molecular weight 17500 Da), 1 g of 8-arm polyethylene glycol (8-arm-PEG) (molecular weight 20000 Da), 1 g of 9-arm polyethylene glycol (9-arm-PEG) (molecular weight 22500 Da), and 1 g of 10-arm polyethylene glycol (10-arm-PEG) (molecular weight 25000 Da). After stirring and mixing evenly, add 0.1201 g of 4-formylbenzoic acid (p-CBA), 0.0244 g of 4-dimethylaminopyridine (DMAP), and 0.1534 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC .(HCL), react at room temperature for 36 h; After the reaction, remove the reaction flask and extract the solution 4 times with about 100 ml of saturated brine, dry it with anhydrous magnesium sulfate, and then concentrate it with a rotary evaporator; Then add it to 100 ml of pre-cooled diethyl ether to precipitate in the upper layer of the liquid, repeat 4 times, and dialyze the obtained product with distilled water. For 2, 3-arm-PEG, use a 5 kDa dialysis bag, for 4, 5-arm-PEG, use a 10 kDa dialysis bag, for 6, 7-arm-PEG, use a 15 kDa dialysis bag, and for 8, 9, 10-arm-PEG, use a 20 kDa dialysis bag. Change the water once a day, dialyze for about one week, and finally freeze-dry with a freeze dryer to obtain benzaldehyde polyethyleneglycol with two, three, four, five, six, seven, eight, nine, and ten arms.

[0031] Step 2: Add 40 mL of PBS with a pH of 5.5 to a 250 mL round-bottom flask, add 1.0 g of type A porcine skin gelatin, and heat it at 50 °C and magnetically stir for about 1 hour to dissolve it completely; Then add 1.74 g of adipic dihydrazide and stir well; Then add 0.77 g of 1-hydroxybenzotriazole hydrate (HOBT) dissolved in 5 ml of DMSO to the reaction flask and stir well; Finally, add 0.77 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ((EDC . HCL)). At the same time, quickly adjust the pH to 5.0 - 5.3 with 1 M NaOH; Stir the mixture at 55 °C for 36 h. After the reaction, dialyze with distilled water (8 kDa dialysis bag), change the water once a day, dialyze for about one week, and then freeze-dry to obtain adipic dihydrazide gelatin with a substitution degree of about 56%.

[0032] Step 3: According to the source of stem cells, select the appropriate concentration of precursor material for dissolution, such as dissolving the product of step 1 with aldehyde groups of di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nine-, or ten-arm benzaldehyde polyethylene glycol (2-arm-PEGDA, 3-arm-PEGDA, 4-arm-PEGDA, 5-arm-PEGDA, 6-arm-PEGDA, 7-arm-PEGDA, 8-arm-PEGDA, 9-arm-PEGDA, 10-arm-PEGDA) at a mass fraction of 3% The precursor solutions were 3% 2-arm-PEGDA, 3% 3-arm-PEGDA, 3% 4-arm-PEGDA, 3% 5-arm-PEGDA, 3% 6-arm-PEGDA, 3% 7-arm-PEGDA, 3% 8-arm-PEGDA, 3% 9-arm-PEGDA and 3% 10-arm-PEGDA respectively; the product of step 2, adipic acid dihydrazide gelatin with amino and hydrazine groups, was dissolved in phosphate buffer solution at a mass fraction of 12%. The precursor solution was vortexed thoroughly to obtain a 12% GtnADH precursor solution; an equal volume of 100 μL of di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, and deca-arm benzaldehyde polyethylene glycol solution and an equal volume of 100 μL of adipic acid dihydrazide gelatin solution were taken and shaken evenly at 37°C, and the gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradation was obtained, i.e., 1.5% 2-arm-PEGDA / 6% GtnADH, 1.5% 3-arm-PEGDA / 6%GtnADH, 1.5%4-arm-PEGDA / 6%GtnADH, 1.5%5-arm-PEGDA / 6%GtnADH, 1.5%6-arm-PEGDA / 6%GtnADH, 1.5%7-ar m-PEGDA / 6%GtnADH, 1.5%8-arm-PEGDA / 6%GtnADH, 1.5%9-arm-PEGDA / 6%GtnADH, 1.5%10-arm-PEGDA / 6%GtnADH.

[0033] By injecting the hydrogel solution of two, three, four, five, six, seven, eight, nine, and ten-arm polyethylene glycol into the mold, taking out the hydrogel after gelation and measuring the modulus and degradability of the hydrogel, it can be found that their moduli are consistent. At the same time, the degradation is measured with a matrix metalloproteinase solution, and it can be found that as the number of polyethylene glycol arms in the hydrogel system increases, the degradation is slower, which proves that the hydrogel has the same stiffness but different degradability.

[0034] Embodiment three:

[0035] This embodiment includes the following steps:

[0036] Step 1: Prepare nine 500 mL round-bottom flasks, each containing 50 mL of dichloromethane. Under the protection of nitrogen, add 1 g of 2-arm polyethylene glycol (2-arm-PEG) (molecular weight 5000 Da), 1 g of 3-arm polyethylene glycol (3-arm-PEG) (molecular weight 7500 Da), 1 g of 4-arm polyethylene glycol (4-arm-PEG) (molecular weight 10000 Da), 1 g of 5-arm polyethylene glycol (5-arm-PEG) (molecular weight 12500 Da), 1 g of 6-arm polyethylene glycol (6-arm-PEG) (molecular weight 15000 Da), 1 g of 7-arm polyethylene glycol (7-arm-PEG) (molecular weight 17500 Da), 1 g of 8-arm polyethylene glycol (8-arm-PEG) (molecular weight 20000 Da), 1 g of 9-arm polyethylene glycol (9-arm-PEG) (molecular weight 22500 Da), and 1 g of 10-arm polyethylene glycol (10-arm-PEG) (molecular weight 25000 Da). After stirring and mixing evenly, add 0.1201 g of 4-formylbenzoic acid (p-CBA), 0.0244 g of 4-dimethylaminopyridine (DMAP), and 0.1534 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC . HCl), and react at room temperature for 18 h. After the reaction is completed, remove the reaction flask, extract the solution with about 100 ml of saturated brine 5 times, dry it with anhydrous magnesium sulfate, and then concentrate it with a rotary evaporator. Then, add the supernatant to 100 ml of pre-cooled ether to precipitate, repeat 5 times, and dialyze the obtained product with distilled water. Use a 5 kDa dialysis bag for 2 and 3-arm-PEG, a 10 kDa dialysis bag for 4 and 5-arm-PEG, a 15 kDa dialysis bag for 6 and 7-arm-PEG, and a 20 kDa dialysis bag for 8, 9, and 10-arm-PEG. Change the water once a day and dialyze for about one week. Finally, lyophilize with a freeze dryer to obtain benzaldehyde polyethylene glycol with two, three, four, five, six, seven, eight, nine, and ten arms.

[0037] Step 2: Add 40 mL of PBS with a pH of 5.5 to a 250 mL round-bottom flask, add 1.0 g of type A porcine skin gelatin, and heat it at 50 °C and magnetically stir for about 1 hour to dissolve it completely. Then add 3.915 g of adipic dihydrazide and stir evenly. Then add 1.8943 g of 1-hydroxybenzotriazole hydrate (HOBT) dissolved in 5 ml of DMSO to the reaction flask and stir evenly. Finally, add 1.7325 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride ((EDC .HCL)). At the same time, the pH is quickly adjusted to 5.0-5.3 using 1M NaOH; the mixture is stirred at 60°C for 30 hours. After the reaction, the mixture is dialyzed with distilled water (8kDA dialysis bag), the water is changed once a day, and the dialysis is performed for about one week, followed by freeze drying to obtain adipic acid dihydrazide gelatin with a substitution degree of about 75%.

[0038] Step 3: According to the source of stem cells, a precursor substance of appropriate concentration is selected for dissolution. For example, the product of step 1 having an aldehyde group of two, three, four, five, six, seven, eight, nine, or ten-arm benzaldehyde polyethylene glycol (2-arm-PEGDA, 3-arm-PEGDA, 4-arm-PEGDA, 5-arm-PEGDA, 6-arm-PEGDA, 7-arm-PEGDA, 8-arm-PEGDA, 9-arm-PEGDA, and 10-arm-PEGDA) is dissolved in a phosphate buffer solution at a mass fraction of 4% to obtain precursor solutions of 4% 2-arm-PEGDA, 4% 3-arm-PEGDA, 4% 4-arm-PEGDA, 4% 5-arm-PEGDA, 4% 6-arm-PEGDA, 4% 7-arm-PEGDA, 4% 8-arm-PEGDA, 4% 9-arm-PEGDA, and 4% 10-arm-PEGDA, respectively; the product of step 2 having an amino group and a hydrazine group of adipic acid dihydrazide gelatin is dissolved in a mass fraction of 4%. 8% was dissolved in a phosphate buffer solution, and after being thoroughly vortexed, a precursor solution of 8% GtnADH was obtained; an equal volume of 100 μL of di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, or deca-arm benzaldehyde polyethylene glycol solution and an equal volume of 100 μL of adipic acid dihydrazide gelatin solution were taken, and the two were shaken evenly at 37°C, and the two were also allowed to stand at 37°C to obtain a gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradation, i.e., 2% 2-arm-PEGDA / 4% GtnADH, 2% 3-a rm-PEGDA / 4%GtnADH, 2%4-arm-PEGDA / 4%GtnADH, 2%5-arm-PEGDA / 4%GtnADH, 2%6-arm-PEGDA / 4%GtnADH, 2%7 -arm-PEGDA / 4%GtnADH, 2%8-arm-PEGDA / 4%GtnADH, 2%9-arm-PEGDA / 4%GtnADH, 2%10-arm-PEGDA / 4%GtnADH.

[0039] By injecting the hydrogel solutions of two-, three-, four-, five-, six-, seven-, eight-, nine-, and ten-arm polyethylene glycol into a mold, taking out the hydrogel after gelation and measuring the modulus and degradability of the hydrogel, it can be found that their modulus is consistent, and as the number of arms increases, the degradation becomes slower, confirming that the hydrogel has the same stiffness but different degradability.

Claims

1. A gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradability, characterized in that, It is composed of equal volumes of different multi-arm benzaldehyde-modified polyethylene glycol solutions and adipic dihydrazide-modified gelatin solutions; For the benzaldehyde-modified polyethylene glycol solution and the adipic dihydrazide-modified gelatin solution described above, the mass fraction of both is 1% - 20%, and the solvent is PBS solution; The benzaldehyde-functionalized polyethylene glycol includes: multi-arm polyethylene glycol, 4-formylbenzoic acid (p-CBA), 4-dimethylaminopyridine (DMAP), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). . HCL, controlling the molar ratio of the hydroxyl group of the multi-arm polyethylene glycol, the carboxyl group of 4-formylbenzoic acid, EDC . HCL, and DMAP to be 1:2:2:0.5; The adipic dihydrazide gelatin described above includes gelatin, adipic dihydrazide (ADH), 1-hydroxybenzotriazole hydrate HOBT, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDC . HCL. In this reaction system, based on 1 g of gelatin, the molar ratios of the remaining ADH, HOBT, and EDC . HCL are 1: 0.5 - 2: 0.4 - 0.

8.

2. The gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradability according to claim 1, characterized in that, The multi-arm polyethylene glycol described is selected from: 2-arm-PEG, 3-arm-PEG, 4-arm-PEG, 5-arm-PEG, 6-arm-PEG, 7-arm-PEG, 8-arm-PEG, 9-arm-PEG, 10-arm-PEG.

3. A preparation method of a gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradability, characterized in that, It includes the following steps: Step 1: Add 100 - 250 mL of dichloromethane into a 500 mL round-bottom flask. Under the protection of nitrogen, sequentially add multi-armed polyethylene glycol with a molecular weight between 500 and 20,000 Da, 4-formylbenzoic acid (p-CBA), 4-dimethylaminopyridine (DMAP), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). . HCl, and control the molar ratio of the hydroxyl group of multi-armed polyethylene glycol, the carboxyl group of 4-formylbenzoic acid, EDC . HCl, and DMAP to be 1:2:2:0.

5. React at room temperature for 18 - 36 h; after the reaction is completed, remove the reaction flask, extract the solution with saturated brine 2 - 5 times, dry it with anhydrous magnesium sulfate, and then concentrate it with a rotary evaporator; then precipitate the upper liquid in excess ice ether, repeat 2 - 5 times; dialyze the obtained product with distilled water, change the water once a day, dialyze for one week, and finally freeze-dry it with a freeze dryer to obtain differently multi-armed benzaldehyde polyethylene glycol; Step 2: Add 40 - 160 mL of PBS with a pH of 5.2 - 5.5 into a 250 mL round-bottom flask, then add 1 g of type A porcine skin gelatin. Under the heating condition of 50 - 60 °C, magnetically rotate and stir for 1 - 1.5 h to fully dissolve it; then add adipic dihydrazide (ADH) and stir evenly; add 1-hydroxybenzotriazole hydrate (HOBT) dissolved in dimethyl sulfoxide (DMSO) into the reaction flask and stir evenly; finally, add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDC . HCl. In this reaction system, the molar ratio of ADH, HOBT, EDC . HCl is 1:0.5 - 2:0.4 - 0.8, and finally adipic dihydrazide gelatin with a substitution degree of 20 - 80% is obtained; at the same time, adjust the pH to 5.0 - 5.3 with sodium hydroxide NaOH solution; stir the mixture at 50 - 60 °C for 24 - 36 h, after the reaction, dialyze with distilled water, change the water once a day, dialyze for one week, and then lyophilize to obtain adipic dihydrazide gelatin; Step 3: Dissolve the products of Step 1 with different multi-arm benzaldehyde-modified polyethylene glycols in PBS solution respectively, with a mass fraction of 1% - 20%; Dissolve the product of Step 2, adipic dihydrazide-modified gelatin with amino and hydrazide groups, in PBS solution with a mass fraction of 1% - 20%. Then take equal volumes of different multi-arm benzaldehyde-modified polyethylene glycol solutions and vortex and mix them evenly with the adipic dihydrazide-modified gelatin solution at 37°C, and let it stand to obtain hydrogel systems with different degradabilities.

4. The preparation method of a gelatin-multi-arm polyethylene glycol hydrogel system with independently controllable cell-mediated degradability according to claim 3, characterized in that, The multi-arm polyethylene glycol described is selected from: 2-arm-PEG, 3-arm-PEG, 4-arm-PEG, 5-arm-PEG, 6-arm-PEG, 7-arm-PEG, 8-arm-PEG, 9-arm-PEG, 10-arm-PEG.

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