Repair hydrogel as well as preparation method and application thereof

Abstract

The invention discloses repair hydrogel as well as a preparation method and application thereof. The raw materials of the repairing hydrogel comprise 3D-ECM, 3D-EVs and sodium hyaluronate, the 3D-ECM is an epimatrix extracted by 3D culture of human dermal fibroblasts in a hydrogel scaffold, and the 3D-EVs is an epivesicle extracted by 3D culture of human stem cells in the hydrogel scaffold. The repairing hydrogel provides a good example for the application of the 3D-ECM and the 3D-EVs; the repair hydrogel is rich in more than 80 kinds of protein and polysaccharide substances beneficial to regeneration and repair of the articular cartilage, and has a good effect of promoting repair of the articular cartilage injury.

Description

Technical Field

[0001] The invention relates to the technical field of medical preparations, and in particular to a repair hydrogel and a preparation method and application thereof. Background Art

[0002] Cartilage is a type of connective tissue in the human body that plays a supporting and protective role. It is mainly composed of chondrocytes and intercellular matrix. According to the difference in intercellular matrix, cartilage can be divided into three types, namely hyaline cartilage, elastic cartilage and fibrous cartilage. Since cartilage does not contain blood vessels and lymphatic vessels, it is difficult for the body to repair itself when cartilage is damaged, and clinical intervention treatment is required.

[0003] At present, there are various methods for treating cartilage injuries clinically, and the treatment methods chosen are different according to the different stages of the patient's disease progression. For mild patients, conservative treatment is mainly used, and drugs such as sodium hyaluronate and chondroitin sulfate are injected into the joint cavity; for severe patients, when conservative treatment is ineffective, microfracture surgery and artificial joint replacement are mainly used, but the above methods require surgical intervention, which brings surgical trauma and potential infection risks to patients. Therefore, there is an urgent need to develop a non-invasive, safe, effective, and injectable biological agent for cartilage injury repair in clinical practice.

[0004] The prior art provides some biological preparation composition formulas, such as the compound based on human dermal fibroblast extracellular matrix and hyaluronic acid. However, this technology is limited to the compound of extracellular matrix and hyaluronic acid, and lacks the verification of cell efficacy and safety. Summary of the invention

[0005] The purpose of the present invention is to disclose a repair hydrogel and a preparation method and application thereof, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions: The first aspect of the present invention is to provide a repair hydrogel.

[0007] The second aspect of the present invention is to provide a method for preparing the repair hydrogel described in the first aspect of the present invention.

[0008] The third aspect of the present invention is to provide application directions of the repair hydrogel described in the first aspect of the present invention.

[0009] The raw materials of the repair hydrogel described in the first aspect of the present invention include 3D-ECM, 3D-EVs and sodium hyaluronate. The 3D-ECM is an extracellular matrix extracted by 3D culturing human dermal fibroblasts in a hydrogel scaffold, and the 3D-EVs are extracellular vesicles extracted by 3D culturing human stem cells in a hydrogel scaffold.

[0010] Extracellular Matrix (ECM) is an extracellular microenvironment composed of proteins and polysaccharides secreted by cells into the extracellular matrix, which provides structural and biochemical support for cells and lays the foundation for cell growth and proliferation. ECM is mainly composed of collagen, proteoglycans (also known as proteoglycans), adhesion glycoproteins (such as fibronectin and laminin), and other biomolecules (such as elastin, glycosaminoglycans, etc.). 3D-ECM refers to the extracellular matrix obtained by using 3D printing technology to prepare hydrogel scaffolds and cultivate human dermal fibroblasts in this technical solution.

[0011] Extracellular vesicles (EVs) are small membrane structures secreted and released from various cells and are commonly found in various biological fluids, including blood, urine, and saliva. The 3D-EVs refer to the extracellular vesicles obtained by preparing hydrogel scaffolds and cultivating human stem cells using 3D printing technology in this technical solution.

[0012] The 3D-ECM has been tested to have more than 1,200 proteins, which is richer than the extracellular matrix under 2D culture conditions, and there are more than 1,000 upregulated protein genes. At the same time, the 3D-Evs has a more obvious effect of promoting cell proliferation than the extracellular vesicles under 2D culture conditions. The repair hydrogel of the present invention combines the advantages of the 3D-ECM and the 3D-Evs, and can accelerate the repair of body tissues.

[0013] In some application embodiments of the first aspect of the present invention, the method for preparing the 3D-ECM comprises the steps of: (1) preparing a methacryloylated hyaluronic acid (HAMA) solution containing human dermal fibroblasts as printing ink; (2) using an extrusion 3D printing device to print the printing ink and then photocuring it; (3) The solidified hydrogel scaffold was immersed in culture medium, dextran sulfate was added to induce cells to secrete extracellular matrix, and cultured for 5 to 7 days; (4) removing the hydrogel scaffold, adding hyaluronidase to lyse it, and centrifuging to remove the lysate and collect the precipitate; (5) Add cell lysis buffer and collect the precipitate by centrifugation, which is 3D-ECM.

[0014] In some application embodiments of the first aspect of the present invention, the methacrylated hyaluronic acid solution further contains an initiator, which is a photoinitiator 2,4,6-trimethylbenzoyldiphenylphosphine oxide (LAP).

[0015] In some application embodiments of the first aspect of the present invention, the concentration of dextran sulfate (DS) is 20 μg / mL.

[0016] In some application embodiments of the first aspect of the present invention, the 3D-ECM is further freeze-dried and / or ground.

[0017] In some application embodiments of the first aspect of the present invention, the method for preparing 3D-EVs comprises the steps of: (a) preparing a gelatin solution and a sodium alginate solution, and adding human stem cells to prepare a printing ink; (b) Using an extrusion 3D printing device to print the printing ink and using CaCl 2 Solution and transglutaminase for solidification; (c) The solidified hydrogel scaffold is immersed in culture medium, and the old culture medium is collected and replaced with new culture medium every two days; (d) The collected old culture medium was subjected to gradient centrifugation, and the precipitate was resuspended and filtered through a 0.22 μm membrane to obtain the 3D-EVs.

[0018] In some application embodiments of the first aspect of the present invention, the printing ink in step (a) contains fibrin and / or transglutaminase.

[0019] In some application embodiments of the first aspect of the present invention, the raw materials of the repair hydrogel include, by mass volume ratio: 1-5% of the 3D-ECM, 0.1-1% of the 3D-EVs and 5-10% of the sodium hyaluronate.

[0020] The preparation method described in the second aspect of the present invention is to mix the 3D-ECM, the 3D-EVs and the sodium hyaluronate, add a cross-linking agent, stir under an alkaline environment until cross-linking is formed, and then adjust the pH value of the reaction solution to neutral to terminate the cross-linking.

[0021] The application direction described in the third aspect of the present invention refers to the use of the repair hydrogel for preparing cartilage repair drugs.

[0022] Compared with the prior art, the above technical solution of the present invention has at least the following advantages or beneficial effects: 1) Preparation of repair hydrogels with 3D-ECM and 3D-EVs as the main active ingredients, providing a good example for the application of 3D-ECM and 3D-EVs; 2) After testing, the repair hydrogel is rich in more than 80 kinds of proteins and polysaccharides that are beneficial to the regeneration and repair of articular cartilage, such as collagen I, collagen III, collagen VI, fibronectin, tenascin, laminin, proteoglycan, nidogen, vimentin, talin, emilin, periostin, etc., and has a good effect on promoting the repair of articular cartilage damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a bar graph of proteomic analysis of different extracellular matrices in Example 2; Figure 2 is a bar graph of cell activity detection using different extracellular vesicles in Example 4; Figure 3 are photos of cartilage tissue samples of each experimental group in Example 5; Figure 4 3 is the HE staining picture of cartilage tissue sections of each experimental group in Example 5. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0025] The reagents used in the examples were mainly prepared by the laboratory according to standard procedures, and the information of the other purchased reagents is as follows: Commercialized human extracellular matrix gel, trade name: Mevitia, model: WD-II, produced by Hunan Meibai Biotechnology Co., Ltd. (Xiang Medical Device Registration No.: 20222140972; Batch No.: C003-231202).

[0026] Hyaluronic acid methacryloyl (HAMA): EFL brand company (Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; model: EFL-HAMA-150K).

[0027] Lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, photoinitiator (LAP): EFL brand company (Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; Cas number: 85073-19-4).

[0028] Hyaluronidase: Merck, (CAS No.: 37326-33-3).

[0029] Triton-NH 4 OH cell lysis buffer: Regen Biotechnology (lot number: PS0005-100 mL).

[0030] Sodium hyaluronate (HA): Shandong Zhongshan Biotechnology Co., Ltd. (Batch No.: SH02230505-19).

[0031] Sodium hydroxide (NaOH): Shanghai Aladdin Biochemical Technology Co., Ltd. (Cas No.: 1310-73-2).

[0032] Hydrochloric acid (HCl): Shanghai Aladdin Biochemical Technology Co., Ltd. (Cas No.: 7647-01-0).

[0033] 1,4-Butanediol diglycidyl ether crosslinker (BDDE): Shanghai MacLean Biochemical Technology Co., Ltd. (Cas No.: 2425-79-8).

[0034] Cell Counting Kit-8 (CCK-8 kit): Biotime Biotechnology, product number: C0038.

[0035] Example 1. Preparation of 3D-ECM.

[0036] (1) Preparation of printing ink: (1-1) Take 10 mL of PBS and add it to a brown bottle containing 0.025 g of photoinitiator LAP. Heat and dissolve it in a water bath at 40-50°C for 15 min, shaking several times during the process. (1-2) Place HAMA in a glass bottle / beaker, add LAP solution, and stir to dissolve at room temperature for 0.5 to 1 hour; (1-3) Use centrifugation to remove bubbles in the system (3000~5000 rpm) and sterilize using a 0.22 μm sterile syringe filter; (1-4) Human dermal fibroblasts were added to a 5% (w / v) HAMA solution to prepare 5×10 6 cells / mL of HAMA printing ink.

[0037] (2) 3D printed hydrogel scaffolds: (2-1) Using extrusion 3D printing to print the printing ink into a 150 mm culture dish; (2-2) Irradiate with a light source with a wavelength of 405 nm for 10 to 30 seconds to achieve gelation.

[0038] (3) Use induction culture medium to promote extracellular matrix secretion: (3-1) After solidification, add 35 mL of fibroblast basal medium into the culture dish to completely immerse the hydrogel scaffold; (3-2) Add 20 μg / mL of dextran sulfate to the fibroblast basal medium to induce extracellular matrix secretion. Change the induction medium every 2 days for a total of 7 days. (3-3) Collect the hydrogel scaffold and culture medium in the culture dish, remove the culture medium by centrifugation, add hyaluronidase, and react under the conditions of substrate concentration of 10 g / L, enzyme concentration of 150,000 U / L, pH of 5.0, temperature of 50°C for 1 hour; (3-4) Remove the lysate by centrifugation and collect the precipitate.

[0039] (4) Decellularization: (4-1) Add Triton-NH 4 OH cell lysis buffer, add 5 mL of the collected precipitate to each 150 mm culture dish and place at 37°C for 3 minutes; (4-2) Collect the precipitate by centrifugation, and wash it with PBS three times, each time for 5 minutes. The obtained precipitate is the 3D-ECM.

[0040] For the convenience of storage and use, the 3D-ECM can be further prepared into freeze-dried powder.

[0041] (5) Preparation of extracellular matrix powder: (5-1) freezing the 3D-ECM obtained in step (4-2) at -80°C and transferring it to a freeze dryer for freeze drying for 24 hours; (5-2) The freeze-dried 3D-ECM is quenched in liquid nitrogen and then ground into powder.

[0042] Example 2: Proteomic detection of extracellular matrix obtained under 3D-ECM and 2D culture conditions.

[0043] The extracellular matrix in the commercially available human extracellular matrix gel (Hunan Meibai Biopharmaceutical Co., Ltd.; batch number: C003-231202) was obtained under 2D culture conditions.

[0044] The 3D-ECM and the commercially available extracellular matrix were subjected to proteomic analysis. The 3D-ECM contained more than 1,200 protein types, of which 1,009 genes were upregulated compared to the protein genes in the commercially available extracellular matrix (e.g. Figure 1 as shown).

[0045] Example 3. Preparation of 3D-EVs.

[0046] (1) Preparation of printing ink: (1-1) Dissolve 0.1 g of fibrin and 0.1 g of transglutaminase in 2.5 mL of PBS to prepare a mixed solution containing 4% (w / v) fibrin and 4% (w / v) transglutaminase; (1-2) Dissolve 1 g of gelatin in 5 mL of PBS to prepare a 20% (w / v) gelatin solution; (1-3) Dissolve 0.1 g of sodium alginate in 2.5 mL of PBS to prepare a 4% (w / v) sodium alginate solution; (1-4) The above mixed solution, gelatin solution and sodium alginate solution were mixed evenly, and the fifth generation human umbilical cord mesenchymal stem cells were added to prepare 5×10 6 cells / mL of printing ink.

[0047] (2) 3D printed hydrogel scaffolds: (2-1) Using extrusion 3D printing, the above printing ink is printed into a 2% CaCl 2 Solution and transglutaminase were placed in a 150 mm culture dish and the solidification solution was removed after solidification.

[0048] (3) Extracellular vesicle extraction: (3-1) Add 35 mL of mesenchymal stem cell basal medium to the culture dish to completely immerse the hydrogel scaffold; (3-2) Collect the old culture medium and replace it with new culture medium every two days for a total of 10 days; (3-3) The collected old culture medium was subjected to gradient centrifugation at 300 × g for 10 minutes and the supernatant was collected; (3-4) Centrifuge at 2000 × g for 10 minutes and collect the supernatant; (3-5) Centrifuge at 10,000 × g for 30 minutes and collect the supernatant; (3-6) Centrifuge at 100,000 × g for 70 min at 4 °C, remove the supernatant, resuspend the remaining precipitate in PBS, and centrifuge again at 100,000 × g for 70 min; (3-7) The precipitate was resuspended in 200 μL PBS, filtered through a 0.22 μm membrane, and stored at −80°C.

[0049] Example 4. Comparison of 3D-EVs and extracellular vesicles obtained under 2D culture conditions.

[0050] (1) Preparation of extracellular vesicles obtained under 2D culture conditions: (1-1) Fifth generation human umbilical cord mesenchymal stem cells were routinely cultured in a 150 mm culture dish using serum-free medium, and the old medium was collected and replaced with new medium every two days for a total of 10 days; (1-2) The collected culture medium was subjected to gradient centrifugation at 300 × g for 10 minutes and the supernatant was collected; (1-3) Centrifuge at 2000×g for 10 minutes and collect the supernatant; (1-4) Centrifuge at 10,000 × g for 30 minutes and collect the supernatant; (1-5) 100,000 × g, centrifuge for 70 min at 4°C, remove the supernatant, resuspend the remaining precipitate in PBS, and centrifuge again at 100,000 × g for 70 min; (1-6) Resuspend the precipitate in 200 μL PBS, filter through a 0.22 μm membrane, and store at -80°C.

[0051] (2) Cell proliferation assay: (2-1) Experimental groups: negative control group (0D group), extracellular vesicles group obtained under 2D culture conditions (2D group), and 3D-EVs group (3D group).

[0052] (2-2) The fifth generation human epidermal cells were seeded at 3000 cells per well in a 96-well plate, with 5 replicates per group. After the cells adhered for 24 hours, 100 μL of basal culture medium was added to each well of the 0D group; 100 μL of basal culture medium containing 1 billion extracellular vesicles obtained under 2D culture conditions was added to each well of the 2D group; and 100 μL of basal culture medium containing 1 billion 3D-EVs was added to each well of the 3D group.

[0053] (2-3) After 24 hours, the cell proliferation was detected according to the detection method of the CCK-8 detection kit (Cell Counting Kit-8, CCK-8 kit: Biotime Biotechnology, product number: C0038).

[0054] Test results such as Figure 2 The cell activity of the 3D group was significantly higher than that of the 0D group and the 2D group, which proved that the 3D-EVs had a more obvious effect in promoting cell proliferation.

[0055] Example 5: Preparation and verification of repair hydrogel.

[0056] (1) Preparation method of the repair hydrogel: (1-1) adding the 3D-ECM prepared in Example 1, the 3D-EVs prepared in Example 3, and sodium hyaluronate powder to a NaOH solution containing a BDDE crosslinking agent, and stirring in a 40° C. water bath for 2 hours to perform a crosslinking reaction; (1-2) adding HCl dropwise to the reaction solution to adjust the pH to neutral to terminate the cross-linking reaction; (1-3) Wash the reaction product with sterile ultrapure water to obtain the repair hydrogel.

[0057] (2) Preparation of repair hydrogels with different raw material dosages: In order to study the effects of different raw material dosages on the repair efficacy of the repair hydrogel, repair hydrogels No. 1 to No. 3 were prepared according to the raw material dosages recorded in Table 1.

[0058]

[0059] (3) The prepared repair hydrogel No. 1, repair hydrogel No. 2 and repair hydrogel No. 3 were used in animal experiments to verify their cartilage regeneration and repair capabilities.

[0060] Experimental animals: 24 experimental rabbits aged 7 months were provided by Zhuhai Baishitong Biotechnology Co., Ltd. The animals had free access to pure water and standard maintenance feed.

[0061] Grouping: The mice were randomly divided into blank control group, no treatment group, commercial control group, repair hydrogel No. 1 group, repair hydrogel No. 2 group and repair hydrogel No. 3 group, with 4 mice in each group.

[0062] Treatment: The blank control group was a group of experimental rabbits that were raised and cared for normally during the experiment. The remaining experimental rabbits were drilled with an animal modeling ball drill on the right hind limb articular cartilage surface with a diameter of 4 mm and a depth of 3 mm. The untreated group used 150 μL of 0.9% saline to inject the cartilage hole; the commercial control group injected 150 μL of commercial human extracellular matrix gel (Hunan Meibai Biomedicine Co., Ltd.; batch number: C003-231202) to fill the defect hole; the remaining three groups used 150 μL of the corresponding repair hydrogel to fill the defect hole. The experimental rabbits were killed 10 weeks after surgery, and the right hind limb modeling knee joint was sampled and fixed with 4% paraformaldehyde.

[0063] Experimental results: Take photos of the fixed samples and record them as follows: Figure 3 As shown, Figure 3 The middle (a) is the blank control group; Figure 3 The middle (b) is the untreated group, where obvious defects and less new tissue can be observed; Figure 3 The middle (c) is the control group of the product, where new tissue can be seen growing at the defect site, but the color of the tissue is obviously different from that of the surrounding natural cartilage tissue; Figure 3 The middle (d) is the repair hydrogel group 1, which has a lot of new tissue at the defect site and is very close in color to natural cartilage tissue.

[0064] Histopathological examination: The cartilage tissue of the sample was first decalcified and then stained with HE. Figure 4 shown. Figure 4 (a) in the middle is the staining image of normal articular cartilage tissue in the blank control group, where the complete cartilage cross section can be observed; Figure 4The middle (b) is the staining image of the untreated group, with less new tissue at the defect site; Figure 4 The middle (c) is a staining image of the control group of the product, where new tissue grows at the defect, but it is not completely full. Figure 4 (d), (e), and (f) are the staining images of repair hydrogel groups 1 to 3, respectively. It can be observed that the experimental groups that filled the defect holes all had a repair effect on the cartilage defects, among which the repair effect of repair hydrogel group 1 at (d) was the best, with the highest tissue integrity, which was very close to normal cartilage tissue, and the regenerated tissue integrity was high.

[0065] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A repair hydrogel, characterized in that: The raw materials include 3D-ECM, 3D-EVs and sodium hyaluronate. The 3D-ECM is an extracellular matrix extracted from human dermal fibroblasts through 3D culture in a hydrogel scaffold, and the 3D-EVs are extracellular vesicles extracted from human stem cells through 3D culture in a hydrogel scaffold.

2. The repair hydrogel according to claim 1, characterized in that: The preparation method of the 3D-ECM comprises the steps of: (1) preparing a methacryloylated hyaluronic acid solution containing human dermal fibroblasts as a printing ink; (2) using an extrusion 3D printing device to print the printing ink and then photocuring it; (3) The solidified hydrogel scaffold was immersed in culture medium, dextran sulfate was added to induce cells to secrete extracellular matrix, and cultured for 5 to 7 days; (4) removing the hydrogel scaffold, adding hyaluronidase to lyse it, and centrifuging to remove the lysate and collect the precipitate; (5) Add cell lysis buffer and collect the precipitate by centrifugation, which is 3D-ECM.

3. The repair hydrogel according to claim 2, characterized in that: The methacryloyl hyaluronic acid solution also contains an initiator.

4. The repair hydrogel according to claim 2, characterized in that: The concentration of the dextran sulfate was 20 μg / mL.

5. The repair hydrogel according to claim 2, characterized in that: The 3D-ECM is also freeze-dried and / or ground.

6. The repair hydrogel according to claim 1, characterized in that: The preparation method of 3D-EVs comprises the steps of: (a) preparing a gelatin solution and a sodium alginate solution, and adding human stem cells to prepare a printing ink; (b) using an extrusion 3D printing device to print and solidify the printing ink; (c) The solidified hydrogel scaffold is immersed in culture medium, and the old culture medium is collected and replaced with new culture medium every two days; (d) The collected old culture medium was subjected to gradient centrifugation, and the precipitate was resuspended and filtered through a 0.22 μm membrane to obtain the 3D-EVs.

7. The repair hydrogel according to claim 6, characterized in that: The printing ink in step (a) contains fibrin and / or transglutaminase.

8. The repair hydrogel according to any one of claims 1 to 7, characterized in that: The raw materials of the repair hydrogel include, by mass volume ratio: 1-5% of the 3D-ECM, 0.1-1% of the 3D-EVs and 5-10% of the sodium hyaluronate.

9. The method for preparing the repair hydrogel according to claim 8, characterized in that: After the 3D-ECM, the 3D-EVs and the sodium hyaluronate are mixed, a cross-linking agent is added and stirred in an alkaline environment until cross-linking is formed, and then the pH value of the reaction solution is adjusted to neutral to terminate the cross-linking.

10. Use of the repair hydrogel according to any one of claims 1 to 8 in the preparation of cartilage repair drugs.

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