Preparation method of enzyme photo-dual crosslinking hydrogel and method for loading cells by using enzyme photo-dual crosslinking hydrogel

Abstract

The application belongs to the technical field of biomedical hydrogel, and particularly relates to a preparation method of enzyme-light double-crosslinked hydrogel and a method for loading cells by using the enzyme-light double-crosslinked hydrogel. The preparation method of the enzyme-light double-crosslinked hydrogel comprises the following steps: modifying a gelatin solution to perform enzyme crosslinking under the condition of (a) hydrogen peroxide and horseradish peroxidase, and performing light crosslinking under the condition of (b) a light initiator; the modified gelatin in the modified gelatin solution is p-hydroxyphenylpropionic acid-methyl methacrylic acid anhydrous glyceryl ester mixed graft modified gelatin, or is a mixed gelatin composed of p-hydroxyphenylpropionic acid-methyl methacrylic acid anhydrous glyceryl ester mixed graft modified gelatin and p-hydroxyphenylpropionic acid graft modified gelatin. The obtained hydrogel has dynamic mechanical properties, adjustable hardness and good stability, and has wide application prospects in the fields of tissue engineering, regenerative medicine and drug controlled release.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical hydrogel, and particularly relates to a preparation method of enzyme-light double cross-linked hydrogel and a method for loading cells by using the enzyme-light double cross-linked hydrogel. BACKGROUND

[0002] With the development of the field of tissue engineering and the progress of regenerative medicine, the demand for biomedical materials with excellent mechanical properties and good biocompatibility with biological tissues is also increasing. The extracellular matrix plays a key role in controlling various activities of cells, and the swelling process of hydrogel as a cross-linked network similar to the cytoplasmic matrix can rapidly absorb and contain a large number of water molecules, and in terms of structure and function, it can meet the needs of various dynamic biological processes.

[0003] The hardening of the cytoplasmic matrix plays a decisive role in development, wound healing, and disease processes, and greatly affects the repair and regeneration of damaged organs and tissues. Biomedical hydrogels, as a separate repair matrix or in combination with certain cells and growth factors, have been widely used in the field of tissue engineering and regenerative medicine.

[0004] Gelatin is a product formed by partial hydrolysis of animal collagen chains, which has a wide source, good biocompatibility, excellent mechanical properties and controllable degradation performance, and therefore has a broad application prospect in the field of biological tissue engineering. However, the mechanical properties of the hydrogel material prepared by simply using gelatin solution are poor, and the degradation is rapid and not easy to control, thereby limiting its practical application in certain biological material fields, such as the repair of pathological sites of bones and cartilages with heavy load in the human body, which are easily degraded at 37℃. In view of this problem, some studies report that gelatin is modified, such as grafting a certain molecule on the gelatin molecular chain, so that the modified gelatin molecular chain has stronger mechanical properties, for example, grafting p-hydroxyphenylpropionic acid on the gelatin molecular chain, and the obtained GelHPA can be subjected to an enzyme cross-linking reaction under the action of horseradish peroxidase. This chemical cross-linking method can effectively improve the mechanical properties of the obtained hydrogel, but it is difficult to realize the dynamic hardening of the hydrogel, which limits the study of dynamic biological processes. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of enzyme-light double cross-linked hydrogel, and the obtained hydrogel has dynamic mechanical properties, adjustable hardness and good stability.

[0006] The second purpose of the present application is to provide a method for loading cells by using enzyme-light double cross-linked hydrogel, which can realize the dynamic hardening of the hydrogel matrix.

[0007] To achieve the above purpose, the technical scheme of the preparation method of enzyme-light double cross-linked hydrogel of the present application is:

[0008] A preparation method of an enzyme-light double crosslinking hydrogel, comprising the following steps: modifying a gelatin solution to be a modified gelatin solution, and then performing enzyme crosslinking under the condition of (a) hydrogen peroxide and horseradish peroxidase, and performing light crosslinking under the condition of (b) a photoinitiator; the modified gelatin in the modified gelatin solution is a p-hydroxyphenylpropionic acid-methacrylic anhydrglycerol mixed graft modified gelatin, or a mixed gelatin composed of a p-hydroxyphenylpropionic acid-methacrylic anhydrglycerol mixed graft modified gelatin and a p-hydroxyphenylpropionic acid graft modified gelatin;

[0009] The enzyme crosslinking under the condition of (a) is performed first, and then the light crosslinking under the condition of (b) is performed; or the light crosslinking under the condition of (b) is performed first, and then the enzyme crosslinking under the condition of (a) is performed; or the enzyme crosslinking under the condition of (a) and the light crosslinking under the condition of (b) are simultaneously performed.

[0010] The preparation method of the enzyme-light double crosslinking hydrogel of the present application grafts two polymer groups of p-hydroxyphenylpropionic acid and methacrylic anhydrglycerol on the molecular chain of gelatin, so that the gelatin can be both enzyme-crosslinked and light-crosslinked. By adjusting the concentrations of horseradish peroxidase and hydrogen peroxide, the hardness of the hydrogel obtained by enzyme crosslinking can be adjusted, and by adjusting the time of UV light irradiation, the hardness of the hydrogel obtained by light crosslinking can be adjusted. The enzyme-light double crosslinking hydrogel obtained by double modification of gelatin can realize dynamic hardening of the hydrogel matrix, has adjustable dynamic mechanical properties and controllable degradation performance, and has good biocompatibility. In addition, the raw materials for preparation are abundant, the method is simple and easy to operate, and has good repeatability, can meet the needs of various life activities of cells in two-dimensional and three-dimensional biological environment, and has wide application prospects in the fields of tissue engineering, regenerative medicine and drug controlled release.

[0011] Preferably, in the modified gelatin solution, the mass fraction of the modified gelatin is 5-30%; and in the mixed gelatin, the mass fraction of the p-hydroxyphenylpropionic acid graft modified gelatin is not more than 90%. More preferably, the mass fraction of the modified gelatin is 10%-20%, and more preferably 10-15%. In the mixed gelatin, the mass fraction of the p-hydroxyphenylpropionic acid graft modified gelatin is 30-70%.

[0012] Preferably, in the enzyme crosslinking reaction, the final concentration of horseradish peroxidase in the system is 0.1-10 U / mL, and the final concentration of H2O2 in the system is 1-10 mM; and the enzyme crosslinking reaction time is 5 s-60 min. More preferably, the final concentration of horseradish peroxidase in the system is 0.4-0.6 U / mL, and the final concentration of H2O2 in the system is 2-3 mM; and the enzyme crosslinking reaction time is 5 s-30 min.

[0013] The photo-crosslinking reaction is carried out by using a photo-initiator selected from Irgacure 2959 or LAP, and the concentration of the photo-initiator in the system is 0.1-50 mg / mL, and the photo-crosslinking reaction is carried out for 1 s-60 min. Preferably, the concentration of the photo-initiator in the system is 1-10 mg / mL, more preferably 4-6 mg / mL, and the photo-crosslinking reaction is carried out for 5 s-30 min. The photo-initiators are all commercially available, and can be irradiated according to the recommended wavelength, for example, Irgacure 2959 can be crosslinked under the condition of 365 nm UV light, and the photo-initiator LAP (lithium acylphosphinat) can be crosslinked under the condition of 405 nm visible light.

[0014] Preferably, the preparation method of the p-hydroxyphenylpropionic acid grafted and modified gelatin comprises the following steps: the gelatin and the p-hydroxyphenylpropionic acid are subjected to a grafting reaction in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and then subjected to dialysis treatment to obtain a p-hydroxyphenylpropionic acid grafted and modified gelatin solution, and then subjected to freeze-drying. More preferably, the mass ratio of the gelatin to the p-hydroxyphenylpropionic acid is 100:1-1:100. More preferably, the mass ratio is 5:1-1:2. During the grafting reaction, the pH of the reaction system is maintained at 3-6, and the reaction is stirred at room temperature overnight.

[0015] Preferably, the preparation method of the p-hydroxyphenylpropionic acid-methacrylic acid glycidyl ester mixed grafted and modified gelatin comprises the following steps: the methacrylic acid glycidyl ester is added to the p-hydroxyphenylpropionic acid grafted and modified gelatin solution, and then subjected to a grafting reaction under the condition of pH=3-4 and temperature of 45-60℃, and then subjected to dialysis and freeze-drying treatment. More preferably, for every 3-4 g of the gelatin, the amount of the p-hydroxyphenylpropionic acid is 0.6-8 g, and the amount of the methacrylic acid glycidyl ester is 0.01-100 g, preferably 1-10 g.

[0016] The technical scheme of the method for loading cells by using the enzyme-light double crosslinking hydrogel according to the present application is as follows:

[0017] The method for loading cells by using the enzyme-light double crosslinking hydrogel comprises the following steps:

[0018] 1) The modified gelatin solution and the cells are uniformly mixed to obtain a cell premix; the modified gelatin in the modified gelatin solution is a p-hydroxyphenylpropionic acid-methacrylic acid glycidyl ester mixed grafted and modified gelatin, or a mixed gelatin composed of a p-hydroxyphenylpropionic acid-methacrylic acid glycidyl ester mixed grafted and modified gelatin and a p-hydroxyphenylpropionic acid grafted and modified gelatin;

[0019] 2) The cell premix is subjected to enzyme crosslinking under the condition of hydrogen peroxide and horseradish peroxidase to obtain an enzyme crosslinking hydrogel;

[0020] The enzyme crosslinking hydrogel is photo-crosslinked under the condition of a photo-initiator to obtain the enzyme photo-double crosslinking hydrogel loaded with cells; or the enzyme crosslinking hydrogel is soaked in a buffer or cell culture medium for a predetermined time, and then photo-crosslinked under the condition of a photo-initiator to obtain the dynamically hardened enzyme photo-double crosslinking hydrogel loaded with cells. The buffer is PBS or the like.

[0021] A method for loading cells by using enzyme photo-double crosslinking hydrogel, comprising the following steps:

[0022] 1) uniformly mixing a modified gelatin solution and cells to obtain a cell premix; the modified gelatin in the modified gelatin solution is p-hydroxyphenyl propionic acid-methyl methacrylic acid glycerol ester mixed graft modified gelatin, or is a mixed gelatin composed of p-hydroxyphenyl propionic acid-methyl methacrylic acid glycerol ester mixed graft modified gelatin and p-hydroxyphenyl propionic acid graft modified gelatin;

[0023] 2) photo-crosslinking the cell premix under a condition to obtain a photo-crosslinking hydrogel;

[0024] The photo-crosslinking hydrogel is enzyme-crosslinked under the condition of hydrogen peroxide and horseradish peroxidase to obtain the enzyme photo-double crosslinking hydrogel loaded with cells; or the photo-crosslinking hydrogel is soaked in a buffer or cell culture medium for a predetermined time, and then enzyme-crosslinked under the condition of hydrogen peroxide and horseradish peroxidase to obtain the dynamically hardened enzyme photo-double crosslinking hydrogel loaded with cells.

[0025] The method for loading cells by using enzyme photo-double crosslinking hydrogel of the application adopts the enzyme photo-double crosslinking hydrogel to perform dynamic culture on cells in two dimensions or three dimensions. The method is simple and easy to operate, rapid in reaction and adjustable, and can be used for preparing various tissue engineering scaffolds and replacement and repair hydrogels for damaged or diseased bone and cartilage tissues.

[0026] In the technical scheme for dynamically culturing cells by using enzyme photo-double crosslinking hydrogel, the preferred scheme in the preparation process of the hydrogel is the same as the preferred scheme of the enzyme photo-double crosslinking hydrogel, and will not be repeated here.

[0027] In the application, the dynamic mechanical property means that two hardenings can be performed in time sequence, for example, the second crosslinking (photo-crosslinking) can be performed after swelling for 1 day or 3 days or 7 days after enzyme crosslinking. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a structure schematic diagram of GelHPA-GMA. 1 NMR schematic diagram.

[0029] Figure 2 is a structure schematic diagram of GelHPA-GMA.

[0030] Figure 3 Compression modulus of enzyme crosslinked hydrogel and enzyme photo dual crosslinked hydrogel prepared from 12% mass fraction of GelHPA-GMA and GelHPA mixed solution in Example 1;

[0031] Figure 4 SEM image of enzyme crosslinked hydrogel prepared from 12% mass fraction of GelHPA-GMA and GelHPA mixed solution in Example 1;

[0032] Figure 5 SEM image of enzyme photo dual crosslinked hydrogel prepared from 12% mass fraction of GelHPA-GMA and GelHPA mixed solution in Example 1;

[0033] Figure 6 Rheological diagram of enzyme crosslinked hydrogel and enzyme photo dual crosslinked hydrogel prepared from 12% mass fraction of GelHPA-GMA and GelHPA mixed solution in Example 1;

[0034] Figure 7 Swelling diagram of enzyme crosslinked hydrogel and enzyme photo dual crosslinked hydrogel prepared from 12% mass fraction of GelHPA-GMA and GelHPA mixed solution in Example 1;

[0035] Figure 8 Cell viability of human umbilical cord stem cells cultured in enzyme photo dual crosslinked hydrogel prepared from 12% mass fraction of GelHPA-GMA and GelHPA mixed solution in Example 5 for 7 days;

[0036] Figure 9 Compression modulus of enzyme crosslinked hydrogel prepared from 10%-15% mass fraction of GelHPA-GMA and GelHPA mixed solution;

[0037] Figure 10 Compression modulus of enzyme photo dual crosslinked hydrogel prepared from 10%-15% mass fraction of GelHPA-GMA and GelHPA mixed solution;

[0038] Figure 11 Compression modulus of enzyme photo dual crosslinked hydrogel (first photo crosslinking and then enzyme crosslinking) prepared from 10% mass fraction of GelHPA-GMA and GelHPA mixed solution. DETAILED DESCRIPTION

[0039] The present application uses two chemical cross-linking methods of enzyme and light to obtain a hydrogel that can be dynamically hardened, and simultaneously provides a method for three-dimensional culture of cells loaded in the enzyme-light double cross-linked hydrogel. The present application uses horseradish peroxidase (HRP) to catalyze cross-linking to form an enzyme cross-linking network; and in the presence of a photoinitiator, UV light is irradiated to generate free radicals to induce cross-linking to form a light cross-linking network. The enzyme cross-linking network and the light cross-linking network are interpenetrated and do not affect each other. This method can adjust the ratio of GelHPA-GMA and GelHPA, the mass fraction of the mixed solution of GelHPA-GMA and GelHPA, the amount of photoinitiator, the concentration of horseradish peroxidase, the concentration of hydrogen peroxide and the time of UV irradiation to regulate the mechanical properties of the enzyme-light double cross-linked hydrogel, so as to achieve the purpose of dynamic hardening.

[0040] 1. Synthesis of GelHPA-GMA, comprising the following steps:

[0041] (1) 1-2 g of p-hydroxyphenylpropionic acid (HPA) is weighed and dissolved in 40 mL of dimethyl sulfoxide (DMSO) at room temperature, and the solution is sealed and stirred until completely dissolved, and then 60 mL of ultrapure water is added to the completely dissolved solution.

[0042] (2) 1-2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-2 g of N-hydroxysuccinimide (NHS) are weighed respectively, and after being added to the above solution, the pH value is adjusted to 3-6, and the solution is stirred at room temperature for 2-5 hours.

[0043] (3) 60 mL of a pure gel solution with a mass fraction of 5-8% is prepared, heated and stirred in a 50°C water bath for 1-3 hours, and after complete dissolution, it is poured into the above solution containing HPA and EDC / NHS, and the pH of the reaction system is maintained at 3-6, and the reaction is stirred at high speed at room temperature overnight.

[0044] (4) After the reaction is completed, it is transferred to a dialysis bag (MWCO=12-14K), and 100 mM NaCl solution, 25% ethanol solution and pure water are used for dialysis to remove the reaction substances and by-products.

[0045] (5) 7-9 ml of glycidyl methacrylate is added to the solution after dialysis (about 250 ml), the pH of the solution is adjusted to 3-4, and the solution is heated at 45-60°C and stirred at high speed overnight.

[0046] (6) The solution after the reaction is completed is transferred to a dialysis bag (MWCO=12-14K), and pure water is used for dialysis to remove the reaction substances and by-products.

[0047] (7) After dialysis is completed, the water is extracted using a freeze dryer, and the product is stored at low temperature and in the dark for later use.

[0048] As a further preference, in step (1), the mass of p-hydroxyphenylpropionic acid is 1.32 g.

[0049] As a further preference, in step (2), the mass of EDC is 1.52 g. The mass of NHS is 1.28 g. The pH value of the solution is adjusted to 4.7. The solution is stirred at room temperature for 3.5 hours.

[0050] As a further preference, in step (3), the mass fraction of the pure gelatin solution is 6.6%. The stirring time of the pure gelatin solution is 2.5 hours. The pH value of the reaction system after adding the pure gelatin solution is 4.7.

[0051] As a further preference, in step (5), the amount of added methyl methacrylate dehydrated glycerol is 8 mL, and the dropping rate is preferably 1 drop per second. The pH value of the solution is adjusted to 3.5. The reaction temperature is 50°C.

[0052] 2. The method for synthesizing the enzyme photo double cross-linked hydrogel comprises the following steps: preparing a solution with a mass fraction of 10-15% (preferably in the range of 5-30%) by mixing the single GelHPA-GMA or the mixed solution of the synthesized GelHPA-GMA and GelHPA according to a certain proportion, adding a certain amount of photo initiator and horseradish peroxidase, heating and stirring at 30-50°C until completely dissolved to obtain a precursor solution, uniformly mixing with hydrogen peroxide, and obtaining the enzyme cross-linked hydrogel after gelation; and irradiating the obtained enzyme cross-linked hydrogel with UV light for an appropriate length of time to obtain the enzyme photo double cross-linked hydrogel.

[0053] The precursor solution and the hydrogen peroxide mixed pre-mixed solution can also be irradiated with UV light before gelation to obtain the enzyme photo double cross-linked hydrogel.

[0054] The precursor solution and the hydrogen peroxide mixed pre-mixed solution can also be irradiated with UV light before gelation to obtain the enzyme photo double cross-linked hydrogel.

[0055] The synthesized GelHPA-GMA and GelHPA can also be prepared into a solution with a mass fraction of 10-15% according to a certain proportion, heated and stirred at 30-50°C until completely dissolved to obtain a precursor solution, irradiated with UV light for an appropriate length of time to obtain the photo cross-linked hydrogel; and the obtained photo cross-linked hydrogel is soaked in a HRP enzyme solution and a hydrogen peroxide solution with an appropriate concentration to obtain the enzyme photo double cross-linked hydrogel.

[0056] In a specific implementation, the mixing of the above-mentioned solutions can be carried out in an EP tube. Preferably, the GelHPA-GMA and the GelHPA are formulated into a solution with a mass fraction of 10-15% in a certain proportion, a certain amount of a photoinitiator is added, the obtained solution is divided into two parts, one part is added with horseradish peroxidase and mixed uniformly (horseradish peroxidase premixed solution), and the other part is added with hydrogen peroxide and mixed uniformly (hydrogen peroxide premixed solution), the horseradish peroxidase premixed solution and the hydrogen peroxide premixed solution are respectively added into two barrels of a Y-shaped syringe for mixing injection, and are left in a gel mold to wait for the formation of an enzyme crosslinked hydrogel; and then the enzyme crosslinked hydrogel is irradiated with UV light for an appropriate length of time to obtain an enzyme photo-dual crosslinked hydrogel.

[0057] The gel mold can be selected from a polytetrafluoroethylene template, and the mold through which UV light is irradiated is a thin quartz plate.

[0058] 3. Dynamic culture of cells loaded in the enzyme photo-dual crosslinked hydrogel

[0059] The main process is as follows:

[0060] The synthesized GelHPA-GMA and GelHPA are formulated into a solution with a mass fraction of 5-30% in a certain proportion, a certain amount of a photoinitiator is added, and the solution is heated and stirred until completely dissolved to obtain a precursor solution. The cell clusters after centrifugation and removal of supernatant are dispersed into the precursor solution to obtain a cell premix solution.

[0061] The concentration of the horseradish peroxidase and the hydrogen peroxide is appropriately adjusted. The cell premix solution is mixed with the horseradish peroxidase and the hydrogen peroxide, and is left to gel, and then is irradiated with UV light for an appropriate length of time to obtain a three-dimensional enzyme photo-dual crosslinked hydrogel loaded with cells. The hydrogel is soaked in a cell culture medium and is cultured in a 37°C and 5% CO2 incubator.

[0062] Alternatively, the cell premix solution is mixed with the horseradish peroxidase and the hydrogen peroxide, and the obtained enzyme crosslinked hydrogel after gelation is soaked in a cell culture medium and is cultured in a 37°C and 5% CO2 incubator for 1D-7D; the enzyme crosslinked hydrogel is soaked in a PBS solution containing a certain amount of a photoinitiator for 30min-1h, and is irradiated with UV light for an appropriate length of time to obtain a dynamically hardened enzyme photo-dual crosslinked hydrogel for three-dimensional culture of cells.

[0063] The embodiments of the present application are further described below in combination with specific examples.

[0064] I. Specific examples of the preparation method of the enzyme photo-dual crosslinked hydrogel of the present application

[0065] Example 1

[0066] The preparation method of the enzyme photo-dual crosslinked hydrogel of the present application comprises the following steps:

[0067] (1) Synthesis of p-hydroxyphenylpropionic acid and glycidyl methacrylate modified gelatin (GelHPA-GMA):

[0068] 1) Dissolve gelatin in pure water at room temperature, and heat to 50°C for 2.5h with magnetic stirring to obtain a 6.6% gelatin solution by mass fraction, 60mL.

[0069] 2) Dissolve 1.32g of p-hydroxyphenylpropionic acid in 40mL of dimethyl sulfoxide (DMSO) at room temperature, and continue to stir until completely dissolved at room temperature 25°C. Then add 60mL of pure water and continue to stir. Add 1.52g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1.28g of N-hydroxysuccinimide (NHS) to the solution, adjust the pH to 4.7, and then stir at room temperature 25°C for 3.5h. Slowly pour the gelatin solution in 1) into the solution in 2) at room temperature, and stir at high speed. Adjust the pH of the mixed solution to 4.7, and then measure the pH of the mixed solution after 1h. Stir at room temperature 25°C and high speed overnight. Dialyze with 100mM NaCl solution for three days, and then dialyze with 25% ethanol solution and pure water for one day each to obtain solution A.

[0070] 3) Slowly add 8mL of glycidyl methacrylate to solution A (about 250mL), and control the dropwise addition rate so that it is added dropwise to solution A at a rate of 1 drop / s. Adjust the pH of the solution to 3.5, and then measure the pH of the solution again after 1h. Stir at high speed with a magnetic stirrer and heat to 50°C overnight. After sufficient reaction, dialyze with deionized water to remove unreacted reactants and reaction byproducts, and then freeze-dry to obtain p-hydroxyphenylpropionic acid and glycidyl methacrylate modified gelatin (GelHPA-GMA).

[0071] Freeze-dry solution A to obtain p-hydroxyphenylpropionic acid grafted modified gelatin (GelHPA).

[0072] (2) Preparation of enzyme photo-crosslinking hydrogel

[0073] a) Prepare a mixed solution of GelHPA and GelHPA-GMA with a mass fraction of 12% in PBS solution, 1mL, wherein the ratio of GelHPA and GelHPA-GMA is 3:7. Add 5mg of a photo initiator (2-hydroxy-4'-2-(hydroxyethoxy)-2-methylphenyl ethanone (Irgacure 2959)) to the solution, heat to 45°C, and stir with a magnetic stirrer for 3h. After complete dissolution, store at 37°C for standby use.

[0074] b) Each 0.5 mL of the solution is mixed with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) respectively, and then added into 1 mL Y-shaped double barrel syringe to mix the two solutions and inject. The final concentration of HRP in the solution obtained by mixing the solution with HRP and the solution with H2O2 is 0.4 units / mL, and the final concentration of H2O2 is 2 mM.

[0075] c) The final solution is shaped on a gel mold, and the time required for the completion of the gel is recorded by a timer. The gel mold includes a polytetrafluoroethylene bottom plate, a quartz glass top plate, and a silica gel pad sandwiched between the bottom plate and the top plate. The middle part of the silica gel pad is hollowed out, and the hollow space forms a shaping space for the hydrogel. The shaping thickness of the hydrogel is the thickness of the silica gel pad (2 mm). The polytetrafluoroethylene plate and the quartz glass surface are flat and horizontal, which is beneficial for the characterization of the performance of the shaped hydrogel.

[0076] The final solution forms a 1 mL droplet on the plate, and the droplet on the polytetrafluoroethylene plate is not in contact with the surrounding air to avoid affecting the next photo-crosslinking effect. The time for gel shaping is less than 30 s, and the completion of the gel requires 30 min.

[0077] d) After the completion of enzyme crosslinking, the droplet is irradiated with UV through the quartz glass for 10 min, and the whole process is operated in the dark. The enzyme crosslinked hydrogel is transparent, and the middle part of the photo-crosslinked hydrogel becomes white, surrounded by a transparent band, indicating that the place in contact with air has not completed photo-crosslinking. The width of the transparent band is not more than 1 mm.

[0078] On the basis of this embodiment, in step c), UV light can also be used to irradiate the gel during enzyme crosslinking, and the effect is not significantly different from the above steps. Enzyme and photo dual crosslinked hydrogel can also be obtained by this way.

[0079] Example 2

[0080] The preparation method of the enzyme and photo dual crosslinked hydrogel of this embodiment is basically the same as that of Example 1, except that in step (2)-(a), the mass fraction of the mixed solution is 10%.

[0081] Example 3

[0082] The preparation method of the enzyme and photo dual crosslinked hydrogel of this embodiment is basically the same as that of Example 1, except that in step (2)-(a), the mass fraction of the mixed solution is 15%.

[0083] Example 4

[0084] The preparation method of the enzyme and photo dual crosslinked hydrogel of this embodiment is basically the same as that of Example 1, except that in step (2), the enzyme and photo dual crosslinked hydrogel is prepared by photo-crosslinking first and then enzyme crosslinking:

[0085] a) Prepare a mixed solution of 1 mL of GelHPA and GelHPA-GMA with a mass fraction of 10% using a PBS solution, wherein the ratio of GelHPA and GelHPA-GMA is 3:7, weigh 5 mg of a photoinitiator (named 2-hydroxy-4'-2-(hydroxyethoxy)-2-methylphenylacetophenone ((Irgacure 2959)) and add it to the solution, heat to 45°C and magnetically stir for 3 h, and after complete dissolution, store at 37°C.

[0086] b) Take 1 mL of the solution and place it on a gel mold, use UV light to irradiate for 10 min to shape the hydrogel. Then place it in a mixed solution of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) for enzyme crosslinking, and the enzyme crosslinking time is 20 min. In the mixed solution of horseradish peroxidase and hydrogen peroxide, the final concentration of horseradish peroxidase is 0.4 units / mL, and the final concentration of hydrogen peroxide is 2 mM. The final solution forms a 1 mL droplet on the plate, and the operation is carried out in the dark to avoid affecting the enzyme crosslinking effect in the next step.

[0087] The final solution is shaped on the gel mold. The gel mold includes a polytetrafluoroethylene bottom plate, a quartz glass top plate, and a silica gel pad sandwiched between the bottom plate and the top plate. The middle part of the silica gel pad is hollowed out, and the hollowed-out space forms the shaping space of the hydrogel. The shaping thickness of the hydrogel is the thickness of the silica gel pad (2 mm).

[0088] On the basis of Example 1, according to the following proportions of the relative amounts of gelatin, p-hydroxyphenylpropionic acid, and glycidyl methacrylate, the corresponding double-modified gelatin GelHPA-GMA can be obtained:

[0089] 0.6 g of p-hydroxyphenylpropionic acid and 1 g of glycidyl methacrylate are used per 3 g of gelatin;

[0090] 8 g of p-hydroxyphenylpropionic acid and 10 g of glycidyl methacrylate are used per 3 g of gelatin;

[0091] 0.6 g of p-hydroxyphenylpropionic acid and 10 g of glycidyl methacrylate are used per 3 g of gelatin;

[0092] 8 g of p-hydroxyphenylpropionic acid and 1 g of glycidyl methacrylate are used per 4 g of gelatin;

[0093] 5 g of p-hydroxyphenylpropionic acid and 5 g of glycidyl methacrylate are used per 4 g of gelatin.

[0094] On the basis of Example 1, in step (2), the composition of the mixed solution and the conditions of enzyme crosslinking and photocrosslinking can be adjusted as follows:

[0095] The mixed solution is a 20% mass fraction GelHPA-GMA solution without GelHPA. The controllable final concentration of horseradish peroxidase is 0.5 U / mL, and the final concentration of hydrogen peroxide is 2 mM. After enzyme crosslinking for 5 s, light irradiation for 50 min, the preparation of enzyme / light dual crosslinking hydrogel can be completed.

[0096] The mixed solution is a 20% mass fraction GelHPA-GMA solution without GelHPA. The controllable final concentration of horseradish peroxidase is 0.5 U / mL, and the final concentration of hydrogen peroxide is 2 mM. After enzyme crosslinking for 5 s, light irradiation for 50 min, the preparation of enzyme / light dual crosslinking hydrogel can be completed.

[0097] The total mass fraction of GelHPA and GelHPA-GMA in the mixed solution is 10%, and the mass ratio of GelHPA to GelHPA-GMA is 3:7. The controllable final concentration of horseradish peroxidase is 0.6 U / mL, and the final concentration of hydrogen peroxide is 2 mM. After enzyme crosslinking for 15 min, light irradiation for 30 min using I2959 photoinitiator, the preparation of enzyme / light dual crosslinking hydrogel can be completed.

[0098] On the basis of Example 4, in step (2), the composition of the mixed solution and the conditions of enzyme crosslinking and light crosslinking can be adjusted as follows: the total mass fraction of GelHPA and GelHPA-GMA in the mixed solution is 15%, and the mass ratio of GelHPA to GelHPA-GMA is 7:3. Light crosslinking is performed using I2959 photoinitiator for 15 min, and then enzyme crosslinking is performed by immersing horseradish peroxidase 0.4 U / mL and hydrogen peroxide 2 mM solution. After 15 min of enzyme crosslinking, the preparation of light enzyme / dual crosslinking hydrogel can be completed.

[0099] Second, specific embodiments of the method for loading cells in the enzyme / light dual crosslinking hydrogel of the present application

[0100] Example 5

[0101] The method for loading cells in the enzyme / light dual crosslinking hydrogel of the present application comprises the following steps:

[0102] (1) The synthesis of GelHPA and GelHPA-GMA is the same as in Example 1.

[0103] (2) Dynamic hardening of enzyme / light dual crosslinking hydrogel for loading human umbilical cord stem cells in three-dimensional culture:

[0104] a) 2 mL of a mixture solution a of Gel HPA and Gel HPA-GMA with a mass fraction of 12% was prepared with a sterile PBS buffer solution, 10 mg of a photoinitiator was weighed and added to the solution, heated to 45°C and magnetically stirred for 2-4 h, and after complete dissolution, sterilized in a biological safety cabinet and then placed in a 2 mL centrifuge tube for storage at 37°C in the dark.

[0105] b) A 10 units / mL solution of horseradish peroxidase was prepared (the solvent used in the experiment was not specially specified, and was phosphate buffer (PBS)), sterilized in a biological safety cabinet and stored at -20°C in the dark. A 100 mM solution of hydrogen peroxide was prepared, sterilized in a biological safety cabinet and stored at room temperature in the dark.

[0106] c) 0.5 mL of the mixture solution a of step a) was extracted and mixed with human umbilical cord stem cells after centrifugation and removal of the supernatant, and the cell clumps were blown apart, horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) were added respectively to obtain a mixed human umbilical cord stem cell precursor solution b, which was quickly blown and mixed uniformly. The solution was transferred to a sterilized polytetrafluoroethylene plate in a state of solution, and the air was isolated and the light was avoided to wait for the completion of the gel. In the precursor solution b, the final concentration of horseradish peroxidase (HRP) was 0.6 units / mL, and the final concentration of hydrogen peroxide (H2O2) was 3 mM. The precursor solution b was shaped on the gel mold, and the time required for the completion of the gel was recorded with a timer (about 30 min). The gel mold was a polytetrafluoroethylene plate, and the final solution formed a 0.5 mL droplet on the plate. A 2 mm thick silicone pad with a hollow center was used to isolate the air around the droplet before it was shaped, and a quartz glass was placed on top to prevent the droplet on the polytetrafluoroethylene plate from contacting the surrounding air, so as not to affect the effect of the next photo-crosslinking. All items were subjected to strict sterilization treatment.

[0107] d) The enzyme-crosslinked hydrogel was cut into a cylindrical block with a diameter of 6 mm and a height of 2 mm using a knife (biopsy punch) and placed in a 24-well plate, 1 mL of prepared culture medium was added to each well, and the plate was placed in a 37°C 5% CO2 incubator for culture, and the medium was changed every two days. After 3 days, the culture medium was washed clean with sterile PBS solution, and then soaked in a sterile PBS buffer containing a photoinitiator, and irradiated with UV light with a wavelength of 365 nm for 10 min. At this time, the photo-crosslinked hydrogel had no transparent zone. In this way, a dynamic hardened enzyme-light double-crosslinked hydrogel for three-dimensional cell culture was obtained. The hydrogel was further placed in the required culture medium and placed in a 37°C 5% CO2 incubator for culture.

[0108] In other embodiments of the method of the present application for loading cells in enzyme photo-dual crosslinked hydrogels, hydrogels with different elastic modulus and hardness can be prepared by different crosslinking methods (using enzyme or light or enzyme photo crosslinking), controlling the time of the second crosslinking (such as swelling for 1 day or 3 days or 7 days after enzyme crosslinking and then photo crosslinking), changing the mass fraction of the hydrogel precursor solution (5%, 25%, etc.), changing the concentration of HRP and H2O2, changing the time of UV light irradiation, etc.

[0109] According to different needs of specific tissue engineering, the softness and hardness of the obtained enzyme photo-dual crosslinked hydrogel can be adjusted, and the compression modulus can be adjusted in a large range of several k to several hundred kPa, so that the obtained hydrogel is more consistent with the actual biological process.

[0110] III. Experimental Examples

[0111] Experimental Example 1

[0112] NMR analysis was performed on the p-hydroxyphenylpropionic acid modified gelatin obtained in step (1) of Example 1, the p-hydroxyphenylpropionic acid and methyl methacrylic acid glycerol ester modified gelatin, and the unmodified gelatin. The H 1 The NMR schematic diagram is shown in Figure 1 .

[0113] As can be seen from Figure 1 , step (1) achieves the graft modification of p-hydroxyphenylpropionic acid and methyl methacrylic acid glycerol ester to gelatin. The structural formula of the modified gelatin GelHPA-GMA after modification is shown in Figure 2 .

[0114] Experimental Example 2

[0115] In Example 1, the compression modulus of the enzyme crosslinked hydrogel obtained in step c) and the enzyme photo-dual crosslinked hydrogel obtained in step d) is compared as shown in Figure 3 . Compression test was performed using a universal testing machine (UTM 2203, Shenzhen Systerm Technology Co., Ltd.). The hydrogel sample with a diameter of 6 mm and a thickness of 2 mm was placed on the test bench, and a mechanical sensor was used to apply pressure to it. According to the stress-strain curve, linear fitting was performed in the strain range of 5%-10%, and the slope obtained was the compression modulus.

[0116] As can be seen from Figure 3 , the compression modulus of the enzyme photo-dual crosslinked hydrogel is about 4 times that of the enzyme crosslinked hydrogel.

[0117] Experimental Example 3

[0118] In Example 1, SEM detection was performed on the enzyme crosslinked hydrogel obtained in step c) and the enzyme photo-dual crosslinked hydrogel obtained in step d), and the results are shown in Figure 4 and Figure 5 .

[0119] Figure 4 SEM images of hydrogels that have undergone only enzyme cross-linking. Figure 5 The images show SEM images of the enzyme-photochemically cross-linked hydrogels. Both hydrogels have a uniform porous structure, but the pore size varies due to differences in cross-linking density. The enzyme-photochemically cross-linked hydrogel has a higher cross-linking density, resulting in a smaller pore size.

[0120] Experiment Example 4

[0121] For Example 1, this experimental example tested the rheological properties of enzyme-crosslinked hydrogels and enzyme-photocrosslinked hydrogels, and the results are as follows: Figure 6 As shown. The storage modulus (G') and loss modulus (G”) of enzyme-crosslinked hydrogels and enzyme-photo-crosslinked hydrogels were measured using a rotational rheometer fitted with a 40 mm diameter plate. Hydrogel samples with a diameter of 40 mm and a thickness of 1 mm were immersed in PBS solution at 37 °C for one day until swelling equilibrium was reached. After swelling equilibrium, the hydrogel samples were measured using a dynamic oscillation mode under constant temperature conditions of 37 °C. The constant deformation of the dynamic oscillation mode was set to 1%, and the frequency was 1–100 Hz.

[0122] As shown in the figure, the storage modulus (G') of the enzyme-photocrosslinked hydrogel is greater than that of the enzyme-crosslinked hydrogel, exhibiting different rheological properties. The dynamic hardening method of the embodiment can significantly change the rheological properties of the hydrogel, thus leaving a large adjustment space for different tissue engineering needs.

[0123] Experimental Example 5

[0124] In Example 1, this experimental example tested the swelling properties of the enzyme-crosslinked hydrogel obtained in step c) and the enzyme photocrosslinked hydrogel obtained in step d). The results are as follows: Figure 7 As shown.

[0125] The swelling performance was tested under the following conditions: Enzyme-crosslinked hydrogel samples with a diameter of 6 mm and a thickness of 2 mm and enzyme-photocrosslinked hydrogel samples were immersed in PBS solution and placed in a 37°C incubator for swelling. When all samples reached swelling equilibrium, they were carefully removed with a weighing spatula and blotted dry with lint-free paper to remove any residual liquid. They were then weighed on an analytical balance to obtain the equilibrium wet weight (W) at a specified time point. s After each hydrogel sample swelled for 7 days, excess PBS solution was removed, and the samples were frozen overnight at -80°C. Then, the hydrogel samples were freeze-dried for 2 days using a freeze dryer. The dry weight (W) of each sample was measured. d And record it.

[0126] Depend on Figure 7 It is known that enzyme-crosslinked hydrogels have greater swelling and water absorption properties.

[0127] Experimental Example 6

[0128] Example 5 produced a dynamic stiffening enzyme-photocrosslinking hydrogel, and cells were cultured in the 3D carrier thereof. Figure 8 Cell viability (green for live cells, red for dead cells) of human umbilical cord stem cells cultured in the enzyme-photocrosslinking hydrogel for 7 days.

[0129] As can be seen from the figure, the three-dimensional hydrogel carrier has good biocompatibility, and after dynamic stiffening, it can induce human umbilical cord stem cells to differentiate into osteoblasts or chondrocytes. The cells have good biocompatibility during the mixing process, gelation process, and culture process.

[0130] Experimental Example 7

[0131] This experimental example illustrates the performance adjustment of the enzyme-photocrosslinking hydrogel, as shown in Figures 9-11 .

[0132] Figures 9-10 In Example 2-3, the mass fraction of the mixed solution is increased from 10% to 15%, and the compression modulus of the enzyme crosslinking hydrogel is increased accordingly, and the compression modulus of the enzyme-photocrosslinking hydrogel is increased accordingly.

[0133] Figure 11 In Example 4, the compression modulus of the hydrogel is increased after the enzyme crosslinking after the photocrosslinking.

[0134] In the present application, p-hydroxyphenylpropionic acid and methyl methacrylic acid dehydrated glycerol ester are grafted onto gelatin, and the two grafting groups do not affect each other. The modified gelatin can be both enzyme crosslinked and photocrosslinked, and the two crosslinking methods do not affect each other, that is, there is no obvious difference between the results of first enzyme crosslinking and then photocrosslinking and first photocrosslinking and then enzyme crosslinking, and the enzyme crosslinking can be performed simultaneously with photocrosslinking; or the enzyme crosslinking can be directly followed by photocrosslinking, or the enzyme crosslinking can be followed by swelling for 1-7 days before photocrosslinking.

[0135] Enzyme-photocrosslinking increases the crosslinking degree (crosslinking site density) and enhances the mechanical properties of the hydrogel. By changing one or more of the gel precursor solution concentration, the concentration of horseradish peroxidase, the concentration of hydrogen peroxide, the concentration of the photoinitiator, the enzyme crosslinking time, and the UV light irradiation time, the elastic modulus and hardness of the formed hydrogel can be controlled, and the enzyme-photocrosslinking hydrogel with adjustable mechanical properties can be obtained.

[0136] The enzyme cross-linking uses horseradish peroxidase to catalyze cross-linking, has no toxic side effect on cells and is easy to control in concentration; the photo-cross-linking operation is simple and easy to implement, the gel is rapid and the water gel hardness can be regulated by UV light irradiation time. The use of Y type syringe improves the simplicity of enzyme cross-linking, and can make the gel precursor solution fully mixed. The photo initiator is easy to obtain from the surrounding solution as a small molecule, so that dynamic hardening is feasible. In addition, the present application loads human umbilical cord stem cells in the enzyme and photo dual cross-linking water gel to carry out three-dimensional culture, provides a three-dimensional cell differentiation water gel carrier constructed in vitro, and experiments prove that the water gel carrier has good biocompatibility.

Claims

1. A method for preparing an enzyme photo-crosslinked hydrogel, characterized by, The method comprises the following steps: The modified gelatin solution is subjected to enzyme crosslinking under the condition of (a) hydrogen peroxide and horseradish peroxidase, and is subjected to photo crosslinking under the condition of (b) a photo initiator; the modified gelatin in the modified gelatin solution is p-hydroxyphenylpropionic acid-glycidyl methacrylate mixed graft modified gelatin, or is a mixed gelatin composed of p-hydroxyphenylpropionic acid-glycidyl methacrylate mixed graft modified gelatin and p-hydroxyphenylpropionic acid graft modified gelatin; The preparation method of the p-hydroxyphenylpropionic acid-glycidyl methacrylate mixed graft modified gelatin comprises the following steps: glycidyl methacrylate is added into a p-hydroxyphenylpropionic acid graft modified gelatin solution, a graft reaction is carried out under the condition of pH = 3-4 and temperature of 45-60 DEG C, and then dialysis and freeze drying treatment are carried out; The enzyme crosslinking under the condition of (a) is carried out first, and then the photo crosslinking under the condition of (b) is carried out; or the photo crosslinking under the condition of (b) is carried out first, and then the enzyme crosslinking under the condition of (a) is carried out; or the enzyme crosslinking under the condition of (a) and the photo crosslinking under the condition of (b) are simultaneously carried out.

2. The method for preparing an enzyme photo-crosslinking hydrogel according to claim 1, wherein The mass fraction of the modified gelatin in the modified gelatin solution is 5-30%; and the mass proportion of the p-hydroxyphenylpropionic acid graft modified gelatin in the mixed gelatin is not more than 90%.

3. The method for preparing an enzyme photo-crosslinking hydrogel according to claim 1, wherein In the enzyme crosslinking reaction, the final concentration of the horseradish peroxidase in the system is 0.1-10 U / mL, and the final concentration of H2O2 in the system is 1-10 mM; and the time of the enzyme crosslinking reaction is 5 s-60 min.

4. The method for preparing an enzyme photo-crosslinking hydrogel according to claim 1, wherein In the photo crosslinking reaction, the photo initiator is selected from Irgacure 2959 or LAP, the concentration of the photo initiator in the system is 0.1-50 mg / mL, and the time of the photo crosslinking reaction is 1 s-60 min.

5. The method for preparing an enzyme photo-crosslinking hydrogel according to any one of claims 1 to 4, wherein The preparation method of the p-hydroxyphenylpropionic acid graft modified gelatin comprises the following steps: gelatin and p-hydroxyphenylpropionic acid are subjected to a graft reaction in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, a p-hydroxyphenylpropionic acid graft modified gelatin solution is obtained through dialysis treatment, and freeze drying is carried out.

6. The method for preparing the enzyme-photocrosslinked hydrogel as described in claim 5, characterized in that, The mass ratio of the gelatin to the p-hydroxyphenylpropionic acid is 100:1-1:

100.

7. The method for preparing the enzyme-photocrosslinked hydrogel as described in claim 5, characterized in that, In the preparation of the p-hydroxyphenylpropionic acid-glycidyl methacrylate mixed graft modified gelatin, the amount of the p-hydroxyphenylpropionic acid corresponding to 3-4 g of the gelatin is 0.6-8 g, and the amount of the glycidyl methacrylate corresponding to 3-4 g of the gelatin is 0.01-100 g.

8. A method for loading cells in the enzyme photo-dual cross-linked hydrogel obtained by the method according to any one of claims 1 to 7, characterized in that, The method comprises the following steps: 1) The modified gelatin solution and cells are uniformly mixed to obtain a cell premix; the modified gelatin in the modified gelatin solution is p-hydroxyphenylpropionic acid-glycidyl methacrylate mixed graft modified gelatin, or is a mixed gelatin composed of p-hydroxyphenylpropionic acid-glycidyl methacrylate mixed graft modified gelatin and p-hydroxyphenylpropionic acid graft modified gelatin; 2) The cell premix is subjected to enzyme crosslinking under the condition of hydrogen peroxide and horseradish peroxidase to obtain an enzyme crosslinking hydrogel; The enzyme crosslinking hydrogel is photo-crosslinked under the condition of a photo-initiator to obtain an enzyme photo-double crosslinking hydrogel loaded with cells; or the enzyme crosslinking hydrogel is soaked in a buffer or a cell culture medium for a predetermined time, and then photo-crosslinked under the condition of a photo-initiator to obtain a dynamically hardened enzyme photo-double crosslinking hydrogel loaded with cells.

9. A method for loading cells in the enzyme photo-dual cross-linked hydrogel obtained by the method according to any one of claims 1 to 7, characterized in that, The method comprises the following steps: 1) uniformly mixing a modified gelatin solution and cells to obtain a cell premix; the modified gelatin in the modified gelatin solution is p-hydroxyphenylpropionic acid-glycidyl methacrylate mixed graft modified gelatin, or is a mixed gelatin composed of p-hydroxyphenylpropionic acid-glycidyl methacrylate mixed graft modified gelatin and p-hydroxyphenylpropionic acid graft modified gelatin; 2) photo-crosslinking the cell premix under the condition of a photo-initiator to obtain a photo-crosslinking hydrogel; The photo-crosslinking hydrogel is enzyme-crosslinked under the condition of hydrogen peroxide and horseradish peroxidase to obtain an enzyme photo-double crosslinking hydrogel loaded with cells; or the photo-crosslinking hydrogel is soaked in a buffer or a cell culture medium for a predetermined time, and then enzyme-crosslinked under the condition of hydrogen peroxide and horseradish peroxidase to obtain a dynamically hardened enzyme photo-double crosslinking hydrogel loaded with cells.

Images