Macroporous hydrogel containing micro-pipeline as well as preparation method and biomedical application thereof

Abstract

The invention belongs to the technical field of hydrogel, and discloses macroporous hydrogel containing micro-pipelines and a preparation method and biomedical application thereof, and is characterized in that the sol of the macroporous hydrogel is sodium alginate sol, the gelling agent is calcium chloride, the macroporous hydrogel contains the micro-pipelines which are through up and down or through left and right or through front and back, and the micro-pipelines are uniformly distributed on the surface of the macroporous hydrogel. The pore diameter of the micro-pipeline is 100-550 [mu] m, and the porosity of the macroporous hydrogel is greater than 90%. Compared with the prior art, the hydrogel provided by the invention has the following beneficial technical effects that the hydrogel provided by the invention has a completely through pore structure, the porosity is greater than 90%, the water content is high, the biocompatibility is good, and the hydrogel has a wide application prospect in the biological medicine fields of drug delivery, cell culture, tissue regeneration, wound dressing and the like; the aperture size of the hydrogel provided by the invention is greater than 100 [mu] m, and the aperture size is good in uniformity and easy to regulate and control. The method disclosed by the invention is simple in process and low in cost, and the required 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 of natural or synthetic materials with high water content and high 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 have a softness similar to that of living tissue, making hydrogels ideal materials for biomedical applications. Specific applications include three-dimensional cell culture, drug delivery, acute and chronic skin wound dressings, and tissue regeneration engineering.

[0003] In addition, the application of hydrogels in tissue engineering is not limited to providing physical support. It can also simulate the behavior of natural extracellular matrix (ECM) by adjusting its mechanical properties (such as stress relaxation rate), thereby promoting the function of specific cell types, such as osteogenic differentiation of mesenchymal stem cells. In addition, 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 controllable drug release properties while also having 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. There are the following problems in biomedical applications: 1) Low drug transport efficiency: Due to the high-density three-dimensional polymer network, drug transport in hydrogels is hindered, which is not conducive to the rapid release of drugs; 2) Limited spatial support: small pores cannot provide enough space for cell proliferation and tissue growth; 3) Poor pore connectivity: undesirable pore structure affects cell migration and nutrient delivery. Although a variety of methods for preparing macroporous hydrogels have been reported, such as freeze-drying, template-assisted method, and ion gelation method, 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 complicated 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] In view of the above problems existing in the prior art, the present invention provides a method for preparing a macroporous hydrogel material containing microchannels with completely connected pores and high porosity, and a method for controlling the pore size thereof. Microchannel pore structure is formed by using sodium alginate sol in the process of calcium ion directional diffusion gelation, and a large-size pore is obtained by using a sufficient amount of low-concentration gelling agent, and the pore size can be controlled by changing the concentration of the gelling agent. The pore size obtained by this method is greater than 100 μm, the pore size is uniform and controllable, the pores are completely connected, and the porosity is high.

[0009] Specifically, the present invention provides:

[0010] 1) 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 pass through from top to bottom, from left to right, or from 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) A method for preparing a macroporous hydrogel according to any one of 1) to 3), comprising:

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

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

[0016] (3) Covering the filter paper or filter membrane at the nozzle on the top of the gelling agent pool, so that the filter paper or filter membrane is fully soaked 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 completed, demolding to obtain a hydrogel; and

[0019] (6) Rinse 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 time required for the gelation 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, which is used to contain the gel, and includes 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 tube mouth 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 inside of the gel is completely replaced with the culture medium, and then a cell suspension is evenly dripped along the pore direction of the macroporous hydrogel so that the cells 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; the hydrogel has a high water content and good biocompatibility, and has broad application prospects in biomedical fields such as drug delivery, cell culture, tissue regeneration, and wound dressings;

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

[0035] (3) The method of the present invention has a simple process and low cost, and does not require the use of a directional freezing device, an external template, or a 3D printing device, and 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 creative work.

[0038] Figure 1 This is a 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 pore longitudinal 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.

[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, which is the pore size distribution result of the mercury intrusion method test.

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

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

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

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

[0046] Fig. 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] Fig.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 to 200 μm, as determined by mercury intrusion testing. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is 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 the 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 regulated. The hydrogel prepared by the present invention has completely connected microchannels, a pore size greater than 100 μm, an pore size that is easy to regulate, and a high water content, a water content 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 dressings.

[0050] More specifically, on one hand, 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 pass through up and down, left and right, or front and back, the pore size of the microchannels is 100-550 μm, and the porosity of the macroporous hydrogel is greater than 90%.

[0051] The inventors have tried various sols and gelling agents and found that only sodium alginate sol and calcium chloride can be used as sol and gelling agents to prepare macroporous hydrogels having 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, which is used to contain the gel, and includes 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 tube mouth 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 from 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 into deionized water, stirring the solution to completely dissolve the sodium alginate, and obtaining a sodium alginate sol;

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

[0064] (3) Covering the filter paper or filter membrane at the tube opening at the top of the gelling agent pool, 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 completed, demolding to obtain the hydrogel;

[0067] (6) Rinse 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 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.

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

[0074] On the other hand, the present invention provides a cell culture method, characterized in that the macroporous hydrogel is used, immersed in a culture medium so that the inside of the gel is completely replaced with the culture medium, and then a cell suspension is evenly dripped 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 pool with calcium chloride solution, place a filter paper with an area larger than the diameter of the gelling pool tube on top of the gelling pool to allow the filter paper to be fully soaked in the calcium chloride solution, then place a cylindrical through-type silicone mold on top of 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: thoroughly washing the hydrogel 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 degree 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 samples after water removal 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 show that the water content w of the hydrogel is 98.6%, indicating that the hydrogel has 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-going tubular macropores with a pore size greater than 200 μm;

[0087] (3) The porosity measured by mercury intrusion injection 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) Preparation of gelling agent: Prepare 100 mL of 6.0 mol / L calcium chloride solution as gelling agent at room temperature;

[0091] (3) Gelation reaction: inject sodium alginate sol into a cylindrical silicone mold, use a spray bottle to evenly spray the gelling agent onto the top of the sol, let it stand for 1 h, and demold after gelation is complete;

[0092] (4) Cleaning the hydrogel: using deionized water to thoroughly clean the hydrogel 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, 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 those in Example 1;

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

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

[0099] (7) Soaking the hydrogel in culture medium: sterilize the macroporous hydrogel sample obtained in step (4) with ultraviolet light, place it in a sterile 48-well plate, and then soak it in the cell culture medium prepared in step (5). Replace the culture medium every 6 hours. After soaking for 12 hours, the interior of the gel is completely filled with culture medium and reaches equilibrium.

[0100] (8) Culturing cells on macroporous hydrogels: The MC3T3-E1 cells cultured in step (6) were digested with trypsin, fresh culture medium was added, and the cells were resuspended to 2×10 4 / mL, then use a pipette to evenly add 1 mL of cell suspension to the macroporous hydrogel, let it stand for 10 minutes, and then shake it in an 8-shaped manner. Repeat the standing and shaking process 3 times to allow the cells to evenly enter the internal pores of the hydrogel and grow on the wall. The replacement of the culture medium is consistent with the traditional cell culture method, and no special treatment is 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, and the cell viability was measured using the CCK-8 method to measure the OD value and evaluate the cell growth activity.

[0102] (10) Analysis of bone formation markers: After the culture, samples were collected to measure the alkaline phosphatase (ALP) activity and osteocalcin (OCN) level 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 cell culture: MC3T3-E1 osteoblast cells were cultured using the complete medium prepared in step (5) in a cell culture incubator at 37°C and 5% CO2. The medium was replaced every 2-3 days. When the cell density reached about 80-90%, the cells were digested with trypsin and passaged at a ratio of 1:3.

[0107] (7) Soaking the hydrogel in culture medium: sterilize the small-pore hydrogel sample obtained in step (4) with ultraviolet light, place it in a sterile 48-well plate, and then soak it in the cell culture medium prepared in step (5). Replace the culture medium every 6 hours. After soaking for 24 hours, the interior of the gel is completely filled with culture medium and reaches equilibrium.

[0108] (8) Cell culture on small-pore hydrogels: The MC3T3-E1 cells cultured in step (6) were digested with trypsin, fresh culture medium was added, and the cells were resuspended to 2×10 4 / mL, then use a pipette to evenly add 1 mL of cell suspension to the small-pore hydrogel, let it stand for 10 minutes, and then shake it in an 8-shaped manner. Repeat the standing and shaking process 3 times to allow the cells to evenly enter the internal pores of the hydrogel and grow on the wall. The replacement of the culture medium is consistent with the traditional cell culture method and no special treatment is 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 measured using the CCK-8 method to measure the OD value and evaluate the cell growth activity;

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

[0111] Result analysis:

[0112] Attached Figure 6 The cell activities of cells cultured in different hydrogels at 1, 4, and 7 days, respectively. The cell activity of the macroporous hydrogel group was significantly higher than that of the small-pore hydrogel group after seven days of culture, indicating that the macroporous hydrogel can provide a more suitable growth environment for osteoblasts and promote cell growth. In addition, the ALP activity and OCN level of the macroporous hydrogel group were also significantly higher than those of the small-pore 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 those 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 a 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 a 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 rotation 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 for 3 times, 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 an equal volume of fresh PBS was added. The bortezomib concentration in the release medium was determined by HPLC. 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 a 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 a 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 rotation speed set at 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 3 times, 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 an equal volume of fresh PBS was added. The bortezomib concentration in the release medium was determined by HPLC. 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 macroporous drug-loaded hydrogel within 1 hour was 13.70%, showing a higher initial release rate; the cumulative release rate of bortezomib from small-pore drug-loaded hydrogel within 1 hour was 5.93%, and the initial release rate was significantly lower than that of macroporous drug-loaded hydrogel. The cumulative release rates of macroporous drug-loaded hydrogel at 24 hours and 48 hours were 37.21% and 59.33%, respectively, showing a higher mid-term release rate; the cumulative release rates of small-pore drug-loaded hydrogel at 24 hours and 48 hours were 19.72% and 33.02%, respectively, and the mid-term release rate was significantly lower than that of macroporous drug-loaded hydrogel. The cumulative release rates of macroporous drug-loaded hydrogel at 72 hours and 96 hours were 77.18% and 87.08%, respectively, showing a higher late release rate; the cumulative release rates of small-pore drug-loaded hydrogel at 72 hours and 96 hours were 41.38% and 44.44%, respectively, and the late release rate was significantly lower than that of 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 relatively 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 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 pool with calcium chloride solution, place a filter paper with an area larger than the diameter of the gelling pool tube on top of the gelling pool to allow the filter paper to be fully soaked in the calcium chloride solution, then place a cylindrical through-type silicone mold on top of 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: thoroughly washing the hydrogel 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 degree 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 show that the water content w of the hydrogel is 98.1%, indicating that the hydrogel has excellent water carrying capacity;

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

[0140] (3) The porosity measured by mercury intrusion injection is 96.7%, and the pore size distribution is as follows: Fig.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: Fig. 9 As shown, 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 given herein, those skilled in the art should understand that the embodiments of the present invention may be modified without departing from the spirit of the present invention. The above embodiments are merely exemplary and should not be used as a limitation on the scope of the present invention.

Claims

1. 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 penetrate vertically, horizontally, or frontally, the pore size of the microchannels is 100-550 μm, and the porosity of the macroporous hydrogel is greater than 90%.

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

3. The macroporous hydrogel according to claim 1 or 2, wherein: The porosity of the macroporous hydrogel is 95%-99%, more preferably 97%-99%.

4. The method for preparing the macroporous hydrogel according to any one of claims 1 to 3, comprising: (1) adding sodium alginate into deionized water, stirring the solution to completely dissolve the sodium alginate, and obtaining a sodium alginate sol; (2) preparing a calcium chloride solution as a gelling agent, and completely filling a gelling agent pool in a directional gelation device with the calcium chloride solution; (3) Covering the filter paper or filter membrane at the nozzle on the top of the gelling agent pool, so that the filter paper or filter membrane is fully soaked 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 completed, demolding to obtain the hydrogel; as well as (6) Rinse the hydrogel thoroughly with deionized water to remove unreacted calcium ions in the hydrogel.

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

7. The preparation method according to claim 4 or 5, characterized in that: In step (4), the volume ratio of the calcium chloride solution to the sol is (10-100):

1.

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

9. The preparation method according to claim 4 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.

10. A directional gelation device, comprising: A gel tank, which is used to contain the gel, and includes 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 tube mouth of the gelling agent pool, and the volume ratio of the gelling agent pool to the sol mold is (10-100):

1.

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

12. A cell culture method, characterized in that: Using the macroporous hydrogel according to any one of claims 1 to 3, 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 dripped along the pore direction of the macroporous hydrogel so that the cells enter the internal pores of the hydrogel for culture.

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