Macroporous hydrogel containing microchannels and its preparation method and biomedical application

Abstract

The present invention belongs to the field of hydrogel technology, discloses a macroporous hydrogel containing micro-pipeline and its preparation method and biomedical application, it is characterised in that the sol of the macroporous hydrogel is sodium alginate sol, and the gelling agent is calcium chloride, and the macroporous hydrogel contains micro-pipeline that is connected up and down or connected left and right or connected front and back, and the pore size of the micro-pipeline is 100-550 μm, and the porosity of the macroporous hydrogel is greater than 90%. The present invention has the following beneficial technical effects: the hydrogel provided by the present invention has a completely connected pore structure, a porosity greater than 90%, a high water content, good biocompatibility, and has broad application prospects in the biomedical fields such as drug delivery, cell culture, tissue regeneration, wound dressings; the hydrogel pore size provided by the present invention is greater than 100 μm, and the pore size uniformity is good, easy to control. The process of the method of the present invention is simple, cost-effective, and the desired product can be successfully prepared within 12 hours.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogels, and particularly relates to a macroporous hydrogel containing microchannels, a preparation method thereof, and biomedical applications. Background Art

[0002] Hydrogels are three-dimensional polymer networks made from natural or synthetic materials, characterized by high water content and exceptional flexibility. These polymer structures are rich in hydrophilic groups, such as hydroxyl groups (-OH), amide bonds (-CONH-, -CONH2), and sulfonic acid groups (-SO3H). Under physiological conditions, they can absorb large amounts of water or biological fluids and possess a softness similar to that of living tissue, making them ideal materials for biomedical applications. Specific applications include three-dimensional cell culture, drug delivery, dressings for acute and chronic skin wounds, and tissue regeneration.

[0003] Furthermore, the application of hydrogels in tissue engineering is not limited to providing physical support. They can also mimic the behavior of the natural extracellular matrix (ECM) by regulating its mechanical properties (such as stress relaxation rate), thereby promoting the function of specific cell types, such as the osteogenic differentiation of mesenchymal stem cells. Furthermore, collagen hydrogels carrying specific microRNAs can effectively repair articular cartilage damage, and functionalized sodium alginate hydrogels have also shown the potential to promote the proliferation and chondrogenic differentiation of bone marrow mesenchymal stem cells. These studies emphasize the importance of developing new hydrogel materials, especially those that can maintain biocompatibility and controlled drug release properties while also possessing excellent mechanical properties and adjustable pore structures.

[0004] However, the pore size of hydrogels prepared by traditional processes is mostly less than 100 μm, and the pores are isotropic. This leads to the following problems in biomedical applications: 1) Low drug transport efficiency: Due to the high-density three-dimensional polymer network, drug transport in the hydrogel is hindered, which is not conducive to rapid drug release; 2) Limited spatial support: Small pores cannot provide sufficient space for cell proliferation and tissue growth; 3) Poor pore connectivity: Unsatisfactory pore structure affects cell migration and nutrient delivery. Although various methods for preparing macroporous hydrogels have been reported, such as freeze-drying, template-assisted methods, and ion gelation, these technologies still have some limitations.

[0005] For example, patent CN111978588A uses gelatin microspheres as sacrificial templates to construct a macroporous structure with a pore size of 30-600 μm. This process is relatively complex and difficult to control the pore size, and the pore penetration is poor.

[0006] Patent CN116120619A forms a hydrogel by cross-linking sodium alginate and polyvinyl alcohol under the action of calcium chloride. Although the pore size range can reach 100~900 μm, its porosity is only about 85%, and there is a lack of effective means to control the pore morphology and distribution.

[0007] Therefore, developing a macroporous hydrogel with simple preparation process, high porosity, excellent pore connectivity, uniform and controllable pore size is an urgent problem to be solved in the field of biomedical hydrogels. Summary of the Invention

[0008] To address the aforementioned issues with the existing technology, the present invention provides a method for preparing a macroporous hydrogel material containing microchannels with fully interconnected pores and high porosity, and a method for controlling its pore size. This method utilizes a sodium alginate sol to form a microchannel pore structure during the gelation process of directional calcium ion diffusion. Large pore sizes are achieved by using a sufficient amount of a gelling agent at a low concentration, and the pore size can be controlled by varying the gelling agent concentration. The resulting pores are larger than 100 μm, have uniform and controllable pore sizes, are fully interconnected, and have a high porosity.

[0009] Specifically, the present invention provides:

[0010] 1) A macroporous hydrogel containing microchannels, characterized in that the sol of the macroporous hydrogel is a sodium alginate sol, the gelling agent is calcium chloride, the macroporous hydrogel contains microchannels that run vertically, horizontally, or front to back, the pore size of the microchannels is 100-550 μm, and the porosity of the macroporous hydrogel is greater than 90%.

[0011] 2) The macroporous hydrogel according to 1), wherein the pore size distribution of the microchannels is mainly concentrated in 100-300 μm, more preferably 200-300 μm.

[0012] 3) The macroporous hydrogel according to 1) or 2), wherein the porosity of the macroporous hydrogel is 95%-99%, more preferably 97%-99%.

[0013] 4) The method for preparing the macroporous hydrogel according to any one of 1) to 3), comprising:

[0014] (1) adding sodium alginate to deionized water and stirring the solution to completely dissolve the sodium alginate to obtain a sodium alginate sol;

[0015] (2) preparing a calcium chloride solution as a gelling agent, and completely filling the gelling agent pool in the directional gelation device with the calcium chloride solution;

[0016] (3) Covering the nozzle on the top of the gelling agent pool with filter paper or filter membrane to fully soak the filter paper or filter membrane in the calcium chloride solution;

[0017] (4) injecting the sodium alginate sol obtained in step (1) into a sol mold in a directional gelation device so that the bottom of the sol is in full contact with the filter paper or filter membrane soaked in calcium chloride solution to perform gelation;

[0018] (5) after gelation is complete, demolding to obtain the hydrogel; and

[0019] (6) Wash the hydrogel thoroughly with deionized water to remove unreacted calcium ions in the hydrogel.

[0020] 5) The preparation method according to 4), characterized in that the concentration of sodium alginate in the sol in step (1) is 0.1%-5%.

[0021] 6) The preparation method according to 4) or 5), characterized in that the concentration of the calcium chloride solution in step (2) is 0.01-0.1 mol / L.

[0022] 7) The preparation method according to any one of 4) to 6), characterized in that the volume ratio of the calcium chloride solution to the sol in step (4) is (10-100):1.

[0023] 8) The preparation method according to any one of 4) to 7), characterized in that the gelation time in step (4) is 1-12 hours.

[0024] 9) The preparation method according to any one of 4) to 8), characterized in that the pore size of the macroporous hydrogel is regulated by adjusting the concentration of the calcium chloride solution, specifically by increasing the pore size of the hydrogel by reducing the concentration of the calcium chloride solution.

[0025] 10) A directional gelation device comprising:

[0026] a gel tank for containing the gel, comprising a nozzle located at the top of the gel tank;

[0027] filter paper or membrane; and

[0028] a sol mold for containing the sol,

[0029] The area of ​​the filter paper or filter membrane is larger than the area of ​​the nozzle of the gelling agent pool, and the volume ratio of the gelling agent pool to the sol mold is (10-100):1.

[0030] 11) Use of the macroporous hydrogel according to any one of 1) to 3) in drug delivery, cell culture, tissue regeneration, and wound dressing.

[0031] 12) A cell culture method, characterized in that the macroporous hydrogel according to any one of 1) to 3) is used, the macroporous hydrogel is immersed in a culture medium so that the interior of the gel is completely replaced with the culture medium, and then a cell suspension is evenly added dropwise along the pore direction of the macroporous hydrogel to allow the cells to enter the internal pores of the hydrogel for culture.

[0032] Compared with other technologies, the present invention has the following beneficial technical effects:

[0033] (1) The hydrogel provided by the present invention has a completely through-pore structure with a porosity greater than 90%. The path required for the loaded drugs and cells to diffuse into the tissue is the shortest, which is conducive to the rapid diffusion of drugs and cells to the target tissue. The through-pore structure is also conducive to the input of nutrients and oxygen and the discharge of metabolic waste, thereby providing a better environment for cell growth. With high water content and good biocompatibility, it has broad application prospects in biomedical fields such as drug delivery, cell culture, tissue regeneration, and wound dressing.

[0034] (2) The hydrogel provided by the present invention has a pore size greater than 100 μm, and the pore size is uniform and easy to control. The large pore size provides ample space for cell growth;

[0035] (3) The method of the present invention is simple and low-cost, and does not require the use of a directional freezing device, an external template, or a 3D printing device. The desired product can be successfully prepared within 12 hours.

[0036] (4) The reactants used in the method of the present invention are harmless to the human body and have good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings used in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 Schematic diagram of the directional gelation device used in the present invention.

[0039] Figure 2 This is a SEM image of the pore cross section of the macroporous hydrogel prepared in Example 1 of the present invention, wherein the pore size distribution is mainly concentrated in the range of 200-300 μm.

[0040] Figure 3 This is a SEM image of the longitudinal section of the pores of the macroporous hydrogel prepared in Example 1 of the present invention, wherein the pore size distribution is mainly concentrated in the range of 200-300 μm.

[0041] Figure 4 The pore size distribution of the macroporous hydrogel prepared in Example 1 of the present invention is mainly concentrated in the range of 200-300 μm, as measured by mercury intrusion porosimetry.

[0042] Figure 5 This is a SEM image of the pore cross section of the small-pore hydrogel prepared in Comparative Example 1 of the present invention, in which the pore size distribution is mainly concentrated in the range of 30-40 μm.

[0043] Figure 6 The figure is a comparison chart of the OD values ​​of cell viability determination of Example 2 of the present invention and Control Example 2.

[0044] Figure 7 2 is a comparison chart of bone formation marker concentrations in Example 2 of the present invention and Control Example 2.

[0045] Figure 8 3 is a comparison chart of the drug release rates of Example 3 of the present invention and Control Example 3.

[0046] Figure 9 This is a SEM image of the pore cross section of the macroporous hydrogel with a pore size of 100-200 μm prepared in Example 4 of the present invention.

[0047] Figure 10 The pore size distribution of the macroporous hydrogel prepared in Example 4 of the present invention is mainly concentrated in the range of 100-200 μm as determined by mercury intrusion porosimetry. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. The following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0049] The present invention provides a macroporous hydrogel containing microchannels and a preparation method thereof. The preparation method is as follows: sodium alginate is dissolved in water to prepare a sol, the sol is transferred to a mold, and the bottom of the sol is contacted with a sufficient low-concentration calcium chloride solution through filter paper to perform directional gelation. After gelation is completed, the hydrogel is washed to remove unreacted calcium ions to obtain a macroporous hydrogel containing microchannels. By adjusting the concentration of the calcium chloride solution, the pore size of the hydrogel can be controlled. The hydrogel prepared by the present invention has completely connected microchannels, a pore size greater than 100 μm, and an easily controllable pore size. It also has a high water content of greater than or equal to 95% and good biocompatibility. It has broad application prospects in biomedical fields such as drug delivery, cell culture, tissue regeneration, and wound dressing.

[0050] More specifically, the present invention provides a macroporous hydrogel containing microchannels, characterized in that the sol of the macroporous hydrogel is sodium alginate sol, the gelling agent is calcium chloride, the macroporous hydrogel contains microchannels that run vertically, horizontally, or front to back, the pore size of the microchannels is 100-550 μm, and the porosity of the macroporous hydrogel is greater than 90%.

[0051] The present inventors have tried various sols and gelling agents and found that only sodium alginate sol and calcium chloride as sol and gelling agents can prepare macroporous hydrogels with the above-mentioned microchannel characteristics.

[0052] In a preferred embodiment, the pore size distribution of the microchannels is mainly concentrated in 100-300 μm, more preferably 200-300 μm.

[0053] In a preferred embodiment, the porosity of the macroporous hydrogel is 95%-99%, more preferably 97%-99%.

[0054] In a preferred embodiment, the water content of the macroporous hydrogel is greater than or equal to 95%, preferably 95%-99%, more preferably 98%-99%.

[0055] Another aspect of the present invention provides a directional gelation device, comprising:

[0056] a gel tank for containing the gel, comprising a nozzle located at the top of the gel tank;

[0057] filter paper or membrane; and

[0058] a sol mold for containing the sol,

[0059] The area of ​​the filter paper or filter membrane is larger than the area of ​​the nozzle of the gelling agent pool, and the volume ratio of the gelling agent pool to the sol mold is (10-100):1.

[0060] In the directional gelation device of the present invention, the filter membrane or filter paper stably separates the gelling agent and the sol. Under the action of the chemical concentration gradient force, the gelling ions (calcium ions) in the gelling agent pool pass through the filter membrane or filter paper and diffuse directionally into the sodium alginate sol to achieve gelation and form microchannels.

[0061] Another aspect of the present invention provides a method for preparing the macroporous hydrogel, which comprises:

[0062] (1) adding sodium alginate to deionized water and stirring the solution to completely dissolve the sodium alginate to obtain a sodium alginate sol;

[0063] (2) preparing a calcium chloride solution as a gelling agent, and completely filling the gelling agent pool in the directional gelation device with the calcium chloride solution;

[0064] (3) Covering the filter paper or filter membrane at the top of the gelling agent pool at the nozzle, so that the filter paper or filter membrane is fully soaked in the calcium chloride solution;

[0065] (4) injecting the sodium alginate sol obtained in step (1) into a sol mold in a directional gelation device so that the bottom of the sol is in full contact with the filter paper or filter membrane soaked in calcium chloride solution to perform gelation;

[0066] (5) After gelation is complete, demolding to obtain the hydrogel;

[0067] (6) Wash the hydrogel thoroughly with deionized water to remove unreacted calcium ions in the hydrogel.

[0068] In a preferred embodiment, the concentration of sodium alginate in the sol in step (1) is 0.1%-5%.

[0069] In a preferred embodiment, the concentration of the calcium chloride solution in step (2) is 0.01-0.1 mol / L.

[0070] In a preferred embodiment, the volume ratio of the calcium chloride solution to the sol in step (4) is (10-100):1.

[0071] In a preferred embodiment, the time required for the gelation in step (4) is 1-12 hours.

[0072] In a preferred embodiment, the pore size of the macroporous hydrogel is controlled by adjusting the concentration of the calcium chloride solution, specifically by increasing the pore size of the hydrogel by reducing the concentration of the calcium chloride solution.

[0073] Another aspect of the present invention provides uses of the macroporous hydrogel in drug delivery, cell culture, tissue regeneration, and wound dressing.

[0074] Another aspect of the present invention provides a cell culture method, characterized in that the macroporous hydrogel is used, immersed in a culture medium so that the interior of the gel is completely replaced with the culture medium, and then a cell suspension is evenly added dropwise along the pore direction of the macroporous hydrogel so that the cells enter the internal pores of the hydrogel for culture.

[0075] The specific embodiments of the present invention will be described in detail below. It should be noted that the technical features or combinations of technical features described in the following embodiments should not be considered isolated, and they can be combined with each other to achieve better technical effects.

[0076] Example 1

[0077] (1) Prepare sodium alginate sol: Add 0.1 g of sodium alginate to 10 mL of deionized water at room temperature and stir for 1 h to completely dissolve the sodium alginate.

[0078] (2) Prepare gelling agent: Prepare 1 L of 0.01 mol / L calcium chloride solution as gelling agent at room temperature;

[0079] (3) Gelation reaction: Fill the gelling agent pool with calcium chloride solution, place a filter paper with an area larger than the diameter of the gelling agent pool tube on the gelling agent pool, and make the filter paper fully soaked in calcium chloride solution. Then place a cylindrical through-hole silicone mold on the filter paper, pour sodium alginate sol into the mold, let it stand for 3 h, and demold after gelation is completed;

[0080] (4) Washing the hydrogel: The hydrogel was thoroughly washed with deionized water to remove unreacted calcium ions and obtain a macroporous hydrogel;

[0081] (5) Water content test: The weight of the hydrogel was weighed as M1 using an electronic balance. The hydrogel was completely frozen in a refrigerator and then placed in a freeze dryer. The water was completely removed by freeze drying at -50 °C and a vacuum of less than 1 Pa for 24 h. The weight of the hydrogel was weighed as M2. The water content of the hydrogel was calculated as w = (M1-M2) / M1×100%;

[0082] (6) Pore morphology characterization: The pore morphology of the above-mentioned dehydrated samples was observed using a scanning electron microscope (Zeiss, Supra 55);

[0083] (7) Porosity test: The porosity and pore size of the samples after dehydration were tested using a mercury intrusion instrument (Micromeritics, AutoPore V9600).

[0084] Result analysis:

[0085] (1) The water content test results showed that the water content w of the hydrogel was 98.6%, indicating that the hydrogel had excellent water carrying capacity;

[0086] (2) The cross-sectional and longitudinal morphologies of the macroporous hydrogel after freeze-drying are shown in Figure 2. Figure 2 and Figure 3 As shown, the prepared calcium alginate hydrogel has completely through-hole tubular macropores with a pore size greater than 200 μm;

[0087] (3) The porosity measured by mercury intrusion is 97.4%, and the pore size distribution is as follows: Figure 4 As shown, it was confirmed that the hydrogel had a pore size greater than 200 μm.

[0088] Comparative Example 1

[0089] (1) Prepare sodium alginate sol: Add 0.1 g of sodium alginate to 10 mL of deionized water at room temperature and stir for 1 h to completely dissolve the sodium alginate.

[0090] (2) Prepare the gelling agent: Prepare 100 mL of 6.0 mol / L calcium chloride solution at room temperature as the gelling agent;

[0091] (3) Gelation reaction: Sodium alginate sol was injected into a cylindrical silicone mold, and the gelling agent was evenly sprayed onto the top of the sol using a spray bottle. The sol was allowed to stand for 1 h, and then demolded after gelation was completed.

[0092] (4) Cleaning the hydrogel: The hydrogel was thoroughly cleaned with deionized water to remove unreacted calcium ions and obtain a small-pore hydrogel;

[0093] (5) Pore morphology characterization: The water in the sample was removed by vacuum freeze drying, and the pore morphology was observed under a scanning electron microscope.

[0094] Result analysis: The cross-sectional morphology of the small-pore hydrogel after freeze-drying is as follows: Figure 5 As shown in the figure, the prepared calcium alginate hydrogel has small pores, with a pore size of about 30~40 μm.

[0095] Example 2

[0096] Steps (1) to (4) are the same as in Example 1;

[0097] (5) Prepare complete cell culture medium: Add 10% fetal bovine serum to MEMα medium to prepare complete cell culture medium;

[0098] (6) MC3T3-E1 osteoblast culture: MC3T3-E1 osteoblasts (mouse embryonic cranial fibroblasts) were cultured using the complete medium prepared in step (5) in a 37°C, 5% CO2 cell culture incubator. The medium was changed every 2-3 days. When the cell density reached approximately 80-90%, the cells were passaged at a 1:3 ratio using trypsin digestion.

[0099] (7) Soaking the hydrogel in culture medium: The macroporous hydrogel sample obtained in step (4) was sterilized by ultraviolet light, placed in a sterile 48-well plate, and then soaked in the cell culture medium prepared in step (5). The culture medium was replaced every 6 hours. After soaking for 12 hours, the interior of the gel was completely filled with culture medium and reached equilibrium;

[0100] (8) Culturing cells on macroporous hydrogels: The MC3T3-E1 cells cultured in step (6) were digested with trypsin, and fresh culture medium was added. The cells were resuspended to 2×10 4 Cells were then evenly added to the macroporous hydrogel using a pipette. After 10 minutes of stabilization, the suspension was shaken in a figure-eight pattern. The stabilization and shaking process was repeated three times to allow the cells to evenly penetrate the pores of the hydrogel and adhere to the surface for growth. The replacement of the culture medium was consistent with traditional cell culture methods and no special treatment was required.

[0101] (9) Cell viability assay: After 1, 4, and 7 days of culture, the cell morphology inside the gel was observed under an optical microscope. The cell viability was determined by the CCK-8 assay to measure the OD value and evaluate the cell growth activity.

[0102] (10) Analysis of bone formation markers: After the culture, samples were collected and the alkaline phosphatase (ALP) activity and osteocalcin (OCN) level were measured to evaluate osteogenic differentiation.

[0103] Comparative Example 2

[0104] Steps (1) to (4) are the same as those in Control Example 1;

[0105] (5) Prepare complete cell culture medium: Add 10% fetal bovine serum to MEMα medium to prepare complete cell culture medium;

[0106] (6) MC3T3-E1 osteoblast culture: MC3T3-E1 osteoblasts were cultured using the complete medium prepared in step (5) in a 37°C, 5% CO2 cell culture incubator. The medium was changed every 2-3 days. When the cell density reached approximately 80-90%, the cells were digested with trypsin at a 1:3 ratio and passaged.

[0107] (7) Soaking the hydrogel in culture medium: The small-pore hydrogel sample obtained in step (4) was sterilized by ultraviolet light, placed in a sterile 48-well plate, and then soaked in the cell culture medium prepared in step (5). The culture medium was replaced every 6 hours. After soaking for 24 hours, the interior of the gel was completely filled with culture medium and reached equilibrium;

[0108] (8) Cell culture on small-pore hydrogels: The MC3T3-E1 cells cultured in step (6) were digested with trypsin, and fresh culture medium was added to resuspend the cells to 2×10 4 Cells were then evenly added to the hydrogel using a pipette. After 10 minutes of stabilization, the cells were shaken in a figure-eight pattern. The stabilization and shaking process was repeated three times to allow the cells to evenly penetrate the pores of the hydrogel and adhere to the surface for growth. The replacement of the culture medium was consistent with traditional cell culture methods and no special treatment was required.

[0109] (9) Cell viability assay: After 1, 4, and 7 days of culture, the cell morphology inside the gel was observed under an optical microscope, and the cell viability was determined by the CCK-8 assay to measure the OD value and evaluate the cell growth activity;

[0110] (10) Analysis of bone formation markers: After the culture, samples were collected and the alkaline phosphatase (ALP) activity and osteocalcin (OCN) level were measured to evaluate osteogenic differentiation.

[0111] Result analysis:

[0112] Attachment Figure 6 The cell activities of cells cultured in different hydrogels at 1, 4, and 7 days were significantly higher after culturing in macroporous hydrogels than in the microporous hydrogel group after 7 days of culture. This indicates that macroporous hydrogels can provide a more suitable growth environment for osteoblasts and promote cell growth. In addition, the ALP activity and OCN level in the macroporous hydrogel group were significantly higher than those in the microporous hydrogel group ( Figure 7 ), indicating that the macroporous structure of the hydrogel is more conducive to osteoblast differentiation and bone formation.

[0113] Example 3

[0114] Steps (1) to (4) are the same as in Example 1;

[0115] (5) Preparation of drug solution: Weigh 1 mg of bortezomib and dissolve it in 1 mL of sterile PBS to prepare a 1 mg / mL bortezomib solution, which is then diluted to 0.1 mg / mL according to the experimental dosage.

[0116] (6) Hydrogel pretreatment: The macroporous hydrogel sample obtained in step (4) was sterilized by ultraviolet light and then immersed in PBS solution. The PBS solution was replaced every 6 h and equilibrium was reached after 12 h.

[0117] (7) Drug loading: The pretreated macroporous hydrogel obtained in step (6) was transferred to a container containing 0.1 mg / mL bortezomib solution, ensuring that the macroporous hydrogel was completely immersed in the drug solution. The macroporous hydrogel was then placed on a shaker with a speed of 100 rpm and incubated at 37°C for 12 h. The drug solution was replaced every 6 h to ensure that the drug could evenly penetrate into the hydrogel. After the incubation, the drug-loaded macroporous hydrogel was removed from the drug solution and gently rinsed with sterile PBS three times for 5 min each time to remove the drug that was not adsorbed on the surface.

[0118] (8) Drug release experiment: Prepare a PBS solution with a pH of 7.4 as the release medium and ensure that its temperature is maintained at 37°C; place the drug-loaded macroporous hydrogel obtained in step (7) into a dialysis bag containing 5 mL of PBS, and place the dialysis bag in a constant temperature oscillator at 37°C with an oscillation speed set at 100 rpm;

[0119] (9) Determination of cumulative drug release rate: 1 mL of release medium was removed from the dialysis bag at regular intervals (1 h, 24 h, 48 h, 72 h, and 96 h) and replaced with an equal volume of fresh PBS. The bortezomib concentration in the release medium was determined by high-performance liquid chromatography. The drug concentration at each time point was recorded and the cumulative release rate was calculated.

[0120] Comparative Example 3

[0121] Steps (1) to (4) are the same as those in Control Example 1;

[0122] (5) Preparation of drug solution: Weigh 1 mg of bortezomib and dissolve it in 1 mL of sterile PBS to prepare a 1 mg / mL bortezomib solution, which is then diluted to 0.1 mg / mL according to the experimental dosage.

[0123] (6) Hydrogel pretreatment: The small-pore hydrogel sample obtained in step (4) was sterilized by ultraviolet light and then immersed in PBS solution. The PBS solution was replaced every 6 h and equilibrium was reached after 24 h.

[0124] (7) Drug loading: The pretreated small-pore hydrogel obtained in step (6) was transferred to a container containing 0.1 mg / mL bortezomib solution, ensuring that the small-pore hydrogel was completely immersed in the drug solution. The small-pore hydrogel was then placed on a shaker with a speed of 100 rpm and incubated at 37°C for 24 h. The drug solution was replaced every 6 h to ensure that the drug could evenly penetrate into the hydrogel. After the incubation, the drug-loaded small-pore hydrogel was removed from the drug solution and gently rinsed with sterile PBS three times for 5 min each time to remove the drug that was not adsorbed on the surface.

[0125] (8) Drug release experiment: Prepare a PBS solution with a pH of 7.4 as the release medium and ensure that its temperature is maintained at 37°C; place the drug-loaded small-pore hydrogel obtained in step (7) into a dialysis bag containing 5 mL of PBS, and place the dialysis bag in a constant temperature oscillator at 37°C with an oscillation speed set at 100 rpm;

[0126] (9) Determination of cumulative drug release rate: 1 mL of release medium was removed from the dialysis bag at regular intervals (1 h, 24 h, 48 h, 72 h, and 96 h) and replaced with an equal volume of fresh PBS. The bortezomib concentration in the release medium was determined by high-performance liquid chromatography. The drug concentration at each time point was recorded and the cumulative release rate was calculated.

[0127] Result analysis:

[0128] The cumulative release rate of bortezomib from the macroporous drug-loaded hydrogel within 1 hour was 13.70%, indicating a high initial release rate. The cumulative release rate from the small-porous drug-loaded hydrogel within 1 hour was 5.93%, significantly lower than that of the macroporous drug-loaded hydrogel. The cumulative release rates of the macroporous drug-loaded hydrogel over 24 and 48 hours were 37.21% and 59.33%, respectively, indicating a high intermediate release rate. The cumulative release rates of the small-porous drug-loaded hydrogel over 24 and 48 hours were 19.72% and 33.02%, respectively, significantly lower than that of the macroporous drug-loaded hydrogel. The cumulative release rates of the macroporous drug-loaded hydrogel over 72 and 96 hours were 77.18% and 87.08%, respectively, indicating a high late release rate. The cumulative release rates of the small-porous drug-loaded hydrogel over 72 and 96 hours were 41.38% and 44.44%, respectively, significantly lower than that of the macroporous drug-loaded hydrogel. The results showed that the macroporous drug-loaded hydrogel has the characteristics of rapid and sustained drug release. Its initial and intermediate release rates are high, and it can release a large amount of drugs in a short period of time and quickly reach therapeutic concentrations. The later release rate still remains at a high level, and it can continuously provide drugs for a long time, ensuring the durability of the therapeutic effect.

[0129] Example 4

[0130] (1) Prepare sodium alginate sol: Add 0.1 g of sodium alginate to 10 mL of deionized water at room temperature and stir for 1 h to completely dissolve the sodium alginate.

[0131] (2) Prepare gelling agent: Prepare 1 L of 0.08 mol / L calcium chloride solution as gelling agent at room temperature;

[0132] (3) Gelation reaction: Fill the gelling agent pool with calcium chloride solution, place a filter paper with an area larger than the diameter of the gelling agent pool tube on the gelling agent pool, and make the filter paper fully soaked in calcium chloride solution. Then place a cylindrical through-hole silicone mold on the filter paper, pour sodium alginate sol into the mold, let it stand for 3 h, and demold after gelation is completed;

[0133] (4) Washing the hydrogel: The hydrogel was thoroughly washed with deionized water to remove unreacted calcium ions and obtain a macroporous hydrogel;

[0134] (5) Water content test: The weight of the hydrogel was weighed as M1 using an electronic balance. The hydrogel was completely frozen in a refrigerator and then placed in a freeze dryer. The water was completely removed by freeze drying at -50 °C and a vacuum of less than 1 Pa for 24 h. The weight of the hydrogel was weighed as M2. The water content of the hydrogel was calculated as w = (M1-M2) / M1×100%;

[0135] (6) Pore morphology characterization: The water in the sample was removed by vacuum freeze drying, and the pore morphology was observed under a scanning electron microscope.

[0136] (7) Porosity test: The porosity and pore size of the samples after dehydration were tested using a mercury intrusion instrument (Micromeritics, AutoPore V9600).

[0137] Result analysis:

[0138] (1) The water content test results showed that the water content w of the hydrogel was 98.1%, indicating that the hydrogel had excellent water carrying capacity;

[0139] (2) The cross-sectional morphologies of the macroporous hydrogel after freeze-drying are as follows: Figure 9 As shown, the prepared calcium alginate hydrogel has completely through-hole tubular macropores with a pore size greater than 100 μm;

[0140] (3) The porosity measured by mercury intrusion porosity is 96.7%, and the pore size distribution is as follows: Figure 10 As shown, it was confirmed that the hydrogel had a pore size greater than 100 μm.

[0141] The cross-sectional morphologies of the macroporous hydrogels after freeze-drying are as follows: Figure 9 As shown in the figure, the prepared calcium alginate hydrogel has completely through-hole tubular macropores with a pore size of 100~200 μm.

[0142] Although several embodiments of the present invention have been described herein, those skilled in the art will appreciate that modifications may be made to the embodiments herein without departing from the spirit of the present invention. The above embodiments are merely exemplary and should not be used as limitations on the scope of the present invention.

Claims

1. A method for preparing a macroporous hydrogel containing completely connected microchannels, wherein: The sol of the macroporous hydrogel is sodium alginate sol, the gelling agent is calcium chloride, the macroporous hydrogel contains microchannels that penetrate vertically, horizontally, or front to back, the pore size of the microchannels is 100-550 μm, and the porosity of the macroporous hydrogel is 96.7%-99%. The preparation method comprises: (1) adding sodium alginate to deionized water and stirring the solution to completely dissolve the sodium alginate to obtain a sodium alginate sol; (2) preparing a calcium chloride solution as a gelling agent, and completely filling the gelling agent pool in the directional gelation device with the calcium chloride solution; (3) Covering the nozzle on the top of the gelling agent pool with filter paper or filter membrane to fully soak the filter paper or filter membrane in the calcium chloride solution; (4) injecting the sodium alginate sol obtained in step (1) into a sol mold in a directional gelation device so that the bottom of the sol is in full contact with the filter paper or filter membrane soaked in calcium chloride solution to perform gelation; (5) after gelation is complete, demolding to obtain the hydrogel; and (6) Wash the hydrogel thoroughly with deionized water to remove unreacted calcium ions in the hydrogel. Wherein, the concentration of the calcium chloride solution in step (2) is 0.01-0.1 mol / L; The volume ratio of the calcium chloride solution in step (4) to the sol is (10-100):

1.

2. The preparation method according to claim 1, wherein The pore size distribution of the microchannels of the macroporous hydrogel is mainly concentrated in the range of 100-300 μm.

3. The preparation method according to claim 1, wherein The pore size distribution of the microchannels of the macroporous hydrogel is mainly concentrated in the range of 200-300 μm.

4. The preparation method according to claim 1 or 2, characterized in that The porosity of the macroporous hydrogel is 97%-99%.

5. The preparation method according to claim 1, characterized in that The concentration of sodium alginate in the sol in step (1) is 0.1%-5%.

6. The preparation method according to claim 1 or 5, characterized in that The time required for gelation in step (4) is 1-12 hours.

7. The preparation method according to claim 1 or 5, characterized in that The pore size of the macroporous hydrogel is regulated by adjusting the concentration of the calcium chloride solution. Specifically, the pore size of the hydrogel is increased by reducing the concentration of the calcium chloride solution.

8. The preparation method according to claim 1 or 5, characterized in that The directional gelation device comprises: a gel tank for containing the gel, comprising a nozzle located at the top of the gel tank; filter paper or membrane; and a sol mold for containing the sol, The area of ​​the filter paper or filter membrane is larger than the area of ​​the nozzle of the gelling agent pool, and the volume ratio of the gelling agent pool to the sol mold is (10-100):

1.

9. Use of the preparation method according to any one of claims 1 to 8 in preparing a material for drug delivery, a material for cell culture, a material for tissue regeneration or a wound dressing.

10. A cell culture method, characterized in that: The macroporous hydrogel is prepared using the preparation method according to any one of claims 1 to 8, and the macroporous hydrogel is immersed in a culture medium so that the interior of the gel is completely replaced with the culture medium. Then, a cell suspension is evenly added dropwise along the pore direction of the macroporous hydrogel to allow the cells to enter the internal pores of the hydrogel for culture.

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