Bi-crosslinking composite permeation hydrogel tracheal patch as well as preparation material and preparation method thereof
Through ultrasonic crosslinking technology using catechol grafted methacrylic anhydride gel sheet and fibrinogen composite solution, a porous structure of double crosslinked composite permeable hydrogel tracheal patch is formed, which solves the adhesion and blood supply difficulties of existing hydrogel materials in tracheal repair, and promotes tissue repair and chondrocyte proliferation of tracheal defects.
Patent Information
- Application Number
- CN202510418611.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-08-12
AI Technical Summary
In the repair of tracheal tubes, existing hydrogel materials have problems such as poor tissue adhesion, insufficient mechanical properties, poor tissue permeability, difficulty in blood supply or vascularization, and it is difficult to effectively repair tracheal defects.
A lyophilized gel sheet with catechol grafted methacrylic anhydride gelatin was used as a gel base patch, combined with a fibrinogen composite solution, and a double crosslinked composite permeable hydrogel tracheal patch with a porous structure was used to trigger crosslinking by ultrasonic to form a porous structure. Calcium-carrying liposomes were used to release calcium ions to activate transglutaminase, realize covalent crosslinking of fibrinogen, and form a hydrogel.
Good tissue adhesion and Piezo1 gene expression activation are achieved, which promotes chondrocyte proliferation and effectively repairs tracheal defects.
Smart Images
Figure CN120459373A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technology and relates to a tracheal patch, in particular to a double-crosslinked composite permeable hydrogel tracheal patch and a preparation material and a preparation method thereof. Background Art
[0002] Clinically, tracheobronchial injuries caused by trauma, tumors, and iatrogenic procedures can seriously endanger patients' lives and health. Because the trachea is a complex, multilayered structure, with ciliated epithelium covering the tracheal surface and connective tissue such as smooth muscle and blood vessels on the outer surface, direct longitudinal suturing can easily lead to iatrogenic tracheal stenosis and other complications. Furthermore, when tracheal lesions exceed their maximum extent, tracheal reconstruction is extremely difficult. Therefore, finding a suitable tracheal defect repair method is crucial for tracheal injury repair and reconstruction.
[0003] In recent decades, there have been many case reports of the use of developed biomaterials and prepared artificial implants to repair various damaged tissues. Various biomaterials made of natural or synthetic materials, including hyaluronic acid, gelatin, chitosan, sodium alginate, polycaprolactone (PCL), polylactic acid glycolic acid (PLGA), etc., have been reported to be used in tissue repair. Among them, hydrogels have been widely used in tissue engineering and organ regeneration due to their good biocompatibility, tissue elasticity, water retention and drug delivery properties. For example, methacrylated gelatin (GelMA) hydrogel has been widely studied in the field of tracheal repair due to its excellent water retention, biocompatibility and strong ability to encapsulate cells, which is beneficial to the migration and growth of cells during tissue regeneration and wound healing. However, GelMA has disadvantages such as poor tissue adhesion, insufficient mechanical properties, poor tissue permeability, difficulty in obtaining direct blood supply or vascularization after in situ transplantation, and poor tissue adhesion. Summary of the Invention
[0004] Based on the above problems, the present invention provides a double-cross-linked composite permeable hydrogel tracheal patch and its preparation materials and preparation methods, which can form a tracheal patch in situ at the tracheal defect. The prepared double-cross-linked composite permeable hydrogel tracheal patch has good tissue adhesion and can effectively activate Piezo1 gene expression under ultrasound triggering, effectively activate chondrocyte proliferation, and promote tissue repair of defective trachea.
[0005] The purpose of the present invention is achieved through the following technical solutions:
[0006] The present invention provides a double-crosslinked composite permeable hydrogel tracheal patch preparation material, comprising a gel base patch and a fibrinogen composite solution;
[0007] The gel base patch is a freeze-dried gel sheet of catechol-grafted methacrylic anhydride gelatin, which has a porous network structure, which is conducive to the infiltration and penetration of the fibrinogen composite solution;
[0008] The fibrinogen complex solution contains fibrinogen, calcium-loaded liposomes and transglutaminase, and has the ability to form a hydrogel through ultrasonic cross-linking. Ultrasonic waves can effectively induce the release of calcium ions in the calcium-loaded liposomes, thereby triggering the formation of the hydrogel.
[0009] In some specific embodiments, in the fibrinogen complex solution, the concentration of the fibrinogen is 15-30 mg / mL, for example, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL; ... In the original composite solution, the concentration of the calcium-loaded liposomes is 2-10 mg / mL, for example, 3 mg / mL, 4 mg / mL, 6 mg / mL, 5 mg / mL, 7 mg / mL, 8 mg / mL, and 9 mg / mL; the concentration of the transglutaminase is 50-150 nM, for example, 50 nM, 60 nM, 70 nM, 80 nM, 80 nM, 90 nM, 100 nM, 100 nM, 110 nM, 120 nM, 130 nM, and 140 nM.
[0010] Specifically, the preparation method of the gel base patch includes:
[0011] (1) obtaining methacrylic anhydride gelatin, and subjecting the obtained methacrylic anhydride gelatin to a carboxylation reaction to obtain carboxyl-modified methacrylic anhydride gelatin;
[0012] (2) Carboxyl activation reaction of carboxyl-modified methacrylic anhydride gelatin was carried out using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide to obtain catechol-grafted methacrylic anhydride gelatin gel, which was freeze-dried to obtain a gel base patch with a porous network structure.
[0013] Specifically, the preparation method of the fibrinogen complex solution is: dissolving fibrinogen with a NaCl solution, and then mixing the solution with calcium-loaded liposomes and transglutaminase to obtain the fibrinogen complex solution.
[0014] Specifically, the preparation method of the calcium-loaded liposomes is: lecithin, cholesterol and methoxypolyethylene glycol-phosphatidylethanolamine (mPEG-DSPE) are mixed, dissolved in an organic solvent, dried to form a film, and then mixed with a CaCl2 solution and ultrasonically hydrated to obtain the calcium-loaded liposomes.
[0015] In a specific embodiment, the organic solvent is chloroform, the added molar ratio of lecithin, cholesterol, and mPEG-DSPE is 52:43:5, and the concentration of CaCl2 is 20 mg / ml.
[0016] The present invention also provides a method for preparing a double-crosslinked composite permeable hydrogel tracheal patch, which uses the double-crosslinked composite permeable hydrogel tracheal patch to prepare a material, comprising the following steps:
[0017] Step 1, injecting the fibrinogen complex solution onto the surface of the gel base patch;
[0018] Step 2: triggering the cross-linking of the fibrinogen composite solution by ultrasound to form a double-cross-linked composite permeable hydrogel tracheal patch.
[0019] Specifically, in actual application, the gel base patch is attached to the surface of the tracheal defect tissue, and the fibrinogen composite solution is added to the surface of the gel base patch by injection. The fibrinogen composite solution penetrates into the interior of the gel base patch and the surface of the tracheal defect tissue, and then the fibrinogen composite solution is cross-linked by ultrasound triggering, thereby forming a double-cross-linked composite permeable hydrogel tracheal patch in situ at the tracheal defect.
[0020] In some specific embodiments, in step 1, the volume ratio of the injected volume of the fibrinogen complex solution to the gel base patch is 1-2:1, for example, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1.
[0021] In some specific embodiments, in step 2, the ultrasonic frequency is 1-3 MHz, for example, 2 MHz; the ultrasonic intensity is 1-3 W, for example, 2 W; and the ultrasonic time is 1-30 min, for example, 2 min, 5 min, 10 min, 15 min, 20 min, 25 min.
[0022] The present invention provides a double-crosslinked composite permeable hydrogel tracheal patch, which is prepared by using the above-mentioned double-crosslinked composite permeable hydrogel tracheal patch preparation material or according to the above-mentioned preparation method.
[0023] The present invention has the following beneficial effects:
[0024] The present invention discloses a double-crosslinked composite permeable hydrogel tracheal patch preparation material comprising a gel base patch and a fibrinogen composite solution. The gel base patch is a freeze-dried gel sheet of catechol-grafted methacrylic anhydride-treated gelatin with a porous network structure. The fibrinogen composite solution comprises fibrinogen, calcium-loaded liposomes, and transglutaminase, and is capable of ultrasonic crosslinking to form a hydrogel. By injecting the fibrinogen composite solution onto the surface of the gel base patch and then triggering crosslinking under ultrasound, the present invention can form a double-crosslinked composite permeable hydrogel tracheal patch in situ at the tracheal defect site. The double-crosslinked composite permeable hydrogel tracheal patch exhibits good tissue adhesion and, under ultrasound triggering, can effectively activate Piezo1 gene expression and chondrocyte proliferation, thereby promoting tissue repair of the defective trachea. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a photograph of the appearance of the double-crosslinked composite permeable hydrogel tracheal patch prepared in Example 1;
[0026] Figure 2 This is a cross-sectional SEM photograph of the gel-based patch prepared in Preparation Example 2;
[0027] Figure 3 This is a diagram showing the tissue adhesion test results of the double-crosslinked composite permeable hydrogel tracheal patch prepared by the present invention;
[0028] Figure 4 This is a graph showing the results of a chondrocyte activation test using the double-crosslinked composite permeable hydrogel tracheal patch of the present invention;
[0029] Figure 5 This is a graph showing the results of the test for activated Piezo1 gene expression of the double-crosslinked composite permeable hydrogel tracheal patch of the present invention;
[0030] Figure 6 This is a graph showing the test results of local tissue regeneration evaluation using a rabbit animal model using the double-cross-linked composite permeable hydrogel tracheal patch of the present invention. DETAILED DESCRIPTION
[0031] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in the art or in accordance with the product specifications are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.
[0032] The following embodiments of the present invention are intended to further illustrate the present invention. It should be noted that the following examples are only examples within suitable ranges, wherein specific process parameters are for reference only and are not intended to be limited to the specific numerical values exemplified below. Those skilled in the art can select parameters within suitable ranges according to actual needs based on the description herein.
[0033] Calcium-loaded liposomes (Lip-Ca 2+ ) can release calcium ions under ultrasound triggering, which can activate transglutaminase. The activated transglutaminase can catalyze transacylation, resulting in covalent cross-linking of lysine and glutamine side chain residues of the fibrinogen molecule, ultimately triggering the formation of a fibrinogen hydrogel. The present invention injects a fibrinogen composite solution containing fibrinogen, calcium-loaded liposomes, and transglutaminase onto the surface of the gel base patch. After complete penetration, cross-linking is triggered under ultrasound to form a double-cross-linked composite permeable hydrogel tracheal patch in situ at the tracheal defect. The double-cross-linked composite permeable hydrogel tracheal patch has good tissue adhesion and can effectively activate Piezo1 gene expression and chondrocyte proliferation under ultrasound triggering, thereby promoting tissue repair of the defective trachea.
[0034] Preparation Example 1 Preparation of Methacrylated Gelatin (GelMA)
[0035] Gelatin was dissolved in phosphate buffered saline (PBS, pH = 7.4) at 10% (w / v) for 3 hours at 55 ° C under magnetic stirring. Methacrylic anhydride (MA) (0.8 mL per 1 g of gelatin) was added dropwise with stirring (0.2 mL / min) and the pH was adjusted to 7.6. The resulting solution was stirred for 3.5 hours under the same conditions to replace the lysine and hydroxylysine groups of the gelatin with MA groups. The solution was quenched by adding four times preheated PBS (45 ° C), stirred for 35 minutes, and then dialyzed with warm water (42 ° C) using 14 kDa dialysis tubing (6 days) to remove unreacted MA. The transparent dialysate was filtered through a microporous membrane (0.45 μm) to remove insoluble debris, frozen horizontally in a Falcon tube at −78°C (2 days), and lyophilized for 3.5 days to obtain porous foamy methacrylic anhydride-modified gelatin (Gelatin Methacryloyl, GelMA), which was stored in a refrigerator until use.
[0036] Preparation Example 2 Preparation of Gel Base Patch
[0037] Grafting of catechol onto the GelMA backbone using carboxylation and EDC / NHS chemical activation:
[0038] (1) GelMA (1 g) prepared in Preparation Example 1 was dissolved in PBS (20 mL) and stirred at 50°C; succinic anhydride (500 mg dissolved in 10 mL of dimethyl sulfoxide) and triethylamine (0.5 mL) were added to the GelMA solution; the resulting mixture was stirred at 52°C for 12 h, diluted with PBS (1.5 times) to terminate the reaction, dialyzed with warm water (6 days), and lyophilized to obtain carboxyl-modified methacrylic anhydride gelatin (GelMA-COOH).
[0039] (2) The obtained GelMA-COOH (1 g) was mixed with MES buffer (10 mL; pH = 5). N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) (300 mg), N-hydroxysuccinimide (NHS) (300 mg), and dopamine hydrochloride (200 mg) were added in sequence. The mixture was stirred for 15 hours, dialyzed with warm water under acidic conditions (pH, 4.2), and finally freeze-dried to obtain a freeze-dried gel sheet of catechol-grafted methacrylic anhydride gelatin (GelMA-C), i.e., a gel base patch.
[0040] Example 1 Preparation of double-crosslinked composite osmotic hydrogel
[0041] (1) Preparation of calcium-loaded liposomes: Weigh lecithin / cholesterol / mPEG-DSPE (52:43:5, mol / mol / mol), where the weight of mPEG-DSPE is 2 mg, dissolve in 1 ml of chloroform at 38°C, evacuate and dry using a rotary evaporator to form a lipid film. Next, add 1 ml of 20 mg / ml CaCl2 solution to the dried lipid film and treat it with an ultrasonic cell disruptor at 10% energy intensity for 10 minutes to hydrate the dried lipid film to obtain dispersed multilamellar liposomes. Impurities in the liposome solution are then removed by passing through 0.45 μm and 0.22 μm membrane filters (Milex, Ireland). Next, the calcium-loaded liposomes are dialyzed with isotonic buffer (0.6 M sodium chloride) to remove free calcium and stored at 4°C for use.
[0042] (2) Fibrinogen complex solution: Fibrinogen was dissolved in 750 μl of 0.9% NaCl to a final concentration of 22.42 mg / mL, and then mixed with 250 μl of the calcium-loaded liposome solution prepared above. A trace amount of transglutaminase with a final concentration of 8.69 mM was mixed with the above mixed solution to prepare 1 ml of fibrinogen complex solution.
[0043] (3) Preparation of double cross-linked composite osmotic hydrogel: 1 ml of the fibrinogen composite solution prepared above was added dropwise to a volume of 1 cm 3The surface of the GelMA-C freeze-dried gel base patch prepared in Preparation Example 2 was observed to observe the hydrogel penetration rate, situation and depth. After the fibrinogen composite solution penetrated into the interior of the gel base patch, it was sonicated for 100 seconds using an ultrasonic probe to trigger the crosslinking of the fibrinogen composite solution to obtain a double-crosslinked composite permeable hydrogel.
[0044] The researchers further explored the release of calcium ions from the fibrinogen complex solution and found that, under ultrasound, the calcium-loaded liposomes rapidly released 85% of the calcium ions within 120 seconds. The presence of the calcium-loaded liposomes promoted hydrogel formation to a certain extent. This suggests that ultrasound can significantly induce the release of calcium ions from the liposomes, thereby triggering hydrogel formation.
[0045] Figure 1 This is a photo of the surface morphology of the double-cross-linked composite permeable hydrogel prepared in Example 1. It can be seen that the fibrinogen composite solution forms effective penetration in the GelMA-C freeze-dried gel patch as the base. The fibrinogen composite solution can quickly penetrate into the GelMA-C gel base patch and form a hydrogel under ultrasound, forming an adhesive system.
[0046] Example 2 Preparation of double-crosslinked composite osmotic hydrogel
[0047] (1) Preparation of calcium-loaded liposomes: Weigh lecithin: cholesterol: mPEG-DSPE (52:43:5, mol / mol / mol), where the weight of mPEG-DSPE is 4 mg, dissolve in 1 ml of chloroform at 38°C, evacuate and dry using a rotary evaporator to form a lipid film. Next, add 1 ml of 20 mg / ml CaCl2 solution to the dried lipid film and treat it with an ultrasonic cell disruptor at 10% energy intensity for 10 minutes to hydrate the dried lipid film to obtain dispersed multilamellar liposomes. Impurities in the liposome solution are then removed by passing through 0.45 μm and 0.22 μm membrane filters (Milex, Ireland). Next, the calcium-loaded liposomes are dialyzed with isotonic buffer (0.6 M sodium chloride) to remove free calcium and stored at 4°C for use.
[0048] (2) Preparation of fibrinogen complex solution: Fibrinogen was dissolved in 750 μl of 0.9% NaCl to a final concentration of 40.356 mg / mL, and then mixed with 250 μl of the calcium-loaded liposomes prepared above and a trace amount of transglutaminase with a final concentration of 8.69 mM in the mixed solution to prepare 1 ml of fibrinogen complex solution.
[0049] (3) Preparation of double cross-linked composite osmotic hydrogel: 1 ml of the fibrinogen composite solution prepared above was added dropwise to the 0.6 cm 2 solution prepared in Preparation Example 2 through a syringe. 3The surface of the base gel patch is covered with a film, and the fibrinogen composite solution is allowed to penetrate into the base gel patch. The film is ultrasonically treated for 600 seconds using an ultrasonic probe to trigger cross-linking of the fibrinogen composite solution to obtain a double-cross-linked composite permeable hydrogel.
[0050] <Performance Test>
[0051] (1) Scanning electron microscopy (SEM)
[0052] The cross-sectional morphology of the gel substrate patch prepared in Preparation Example 2 was measured using a field emission scanning electron microscope (SEM, ApreoSHiVoc). Specifically, the GelMA-C gel substrate patch prepared in Preparation Example 2 was immersed in deionized water for pre-freezing for 10 minutes, and then freeze-dried for 24 hours using a freeze dryer. During the SEM sample preparation process, the freeze-dried hydrogel fibers were immersed in liquid nitrogen for brittle fracture, and the cross-section was sputtered with gold for SEM photography. The test results are shown in Figure 2. Figure 2 shown.
[0053] Result analysis: From Figure 2 As shown in the SEM image, it can be found that the interior of the GelMA-C gel base patch has a porous network structure, which is conducive to the effective penetration of the liquid fibrinogen complex solution.
[0054] (2) Tissue adhesion test
[0055] The GelMA-C gel base patch prepared in Preparation Example 2 was pre-attached to the top of the pork tissue. After it softened and attached, 1 ml of the prepared fibrinogen composite solution was dripped onto the top of the GelMA-C gel base patch according to the preparation method of Example 1 to allow it to fully penetrate. After the penetration was completed, the ultrasonic activation system was used to observe the formation of the double-crosslinked composite permeable hydrogel to obtain the double-crosslinked composite permeable hydrogel. Next, the pig muscle tissue was rotated and squeezed to deform the double-crosslinked composite permeable hydrogel, and the adhesion of the double-crosslinked composite permeable hydrogel was observed. The test results are shown in the photo. Figure 3 shown.
[0056] Result analysis: It was found that after the fibrinogen composite solution was injected above the GelMA-C gel base patch, it could form effective penetration inside the GelMA-C gel base patch and Figure 3 It can be seen that the prepared double-crosslinked composite osmotic hydrogel still firmly adheres to the pork tissue after repeated folding, which proves that the double-crosslinked composite osmotic hydrogel prepared by the present invention has extremely strong tissue adhesion.
[0057] (3) Activation of chondrocyte proliferation test
[0058] We co-cultured the double-crosslinked composite osmotic hydrogel sample prepared in Example 1 with chondrocytes and triggered them with ultrasound. After the cells produced contact inhibition, we used methylene blue staining to evaluate the state and morphology of the chondrocytes. We also used ImageJ to evaluate the cell number and compared it with the control group. The test results are as follows: Figure 4 shown.
[0059] Result analysis: The double-crosslinked composite osmotic hydrogel of the present invention can effectively activate the proliferation of chondrocytes under ultrasound triggering, and the chondrocytes form contact inhibition, and the color of the cells is significantly deepened after methylene blue staining.
[0060] (4) Activation of Piezo1 gene expression test
[0061] The cultured HUVEC cells and chondrocytes were fixed with 4% PFA for characterization. The double-crosslinked composite permeable hydrogel sample prepared in Example 1 was incubated with blocking solution (PBS containing 0.03% Triton-X100 and 3% normal goat serum) at RT for 30 minutes. Subsequently, the sections and cells were incubated overnight at 4°C in the primary antibody diluted in the blocking solution. The primary antibody used: Piezo1 (1:200). The secondary antibody is such as: goat anti-mouse IgG (1:1000. The sample was stained with DAPI (1:1000; ThermoFisherScientific) for identification of cell nuclei. For frozen sections, slides were mounted using VECTASHIELD mounting medium (Vectorlab). All images were analyzed using fluorescence (NikonTE2000-UJapan) and confocal microscopy. CD31+ capillaries and α-SMA+ blood vessels were quantified using ImageJ software. The test results are shown in Figure 2. Figure 5 shown.
[0062] Result analysis: The double-cross-linked composite osmotic hydrogel of the present invention can effectively activate Piezo1 gene expression under ultrasound triggering, and the immunofluorescence signal is significantly enhanced. The effective activation of Piezo1 gene expression can significantly promote the activation of the MMP signaling pathway, thereby effectively promoting the proliferation of osteoblasts and endothelial cells, and ultimately effectively promoting tracheal repair.
[0063] (5) Rabbit animal model to study local tissue regeneration evaluation
[0064] New Zealand white rabbits (3 months old) were used to evaluate the feasibility of local tracheal reconstruction using a double-crosslinked composite permeable hydrogel. Following the preparation method described in Example 1, a GelMA-C gel base patch was applied to the surface of the tracheal defect as the base of the composite material. Furthermore, 1 ml of a fibrinogen composite solution was added dropwise via syringe to the surface of the GelMA-C gel base patch. Hydrogel penetration was observed, and ultrasound was used to trigger hydrogel crosslinking. The rabbits' localized tracheal defects measured 0.4 x 0.2 cm, and the above materials were applied.
[0065] All rabbits were euthanized after 60 days of modeling, and the harvested trachea was fixed with 4% PFA. After processing the tissue, the fixed samples were embedded in paraffin. Then, 5 μm-sized sections were prepared using an HM340E microtome (ThermoFisherScientific). H&E staining was performed to confirm the degradation of the hydrogel. MT staining was performed to determine the fibrotic area. The paraffin sections of each group were dewaxed and fixed in Bouin solution at RT overnight. The fixed sections were stained with Weigert hematoxylin iron solution for 10 minutes at RT and with Biebrich scarlet acid fuchsin solution for 15 minutes. Finally, the sections were stained with aniline blue for 5 minutes. The sections were washed continuously between each staining step. The test results are shown in Figure 2. Figure 6 shown.
[0066] Result analysis: Figure 6 As shown, after the double-crosslinked composite osmotic hydrogel of the present invention was applied to the animal tracheal defect for 2 months, the defective tracheal tissue was effectively repaired, and new connective tissue and cartilage tissue were formed, and the tracheal repair effect was significant and effective.
[0067] In summary, the present invention injects a fibrinogen composite solution into the surface of the gel base patch and triggers its cross-linking under ultrasound, thereby forming a double-cross-linked composite permeable hydrogel tracheal patch in situ at the tracheal defect. The double-cross-linked composite permeable hydrogel tracheal patch has good tissue adhesion and can effectively activate Piezo1 gene expression and chondrocyte proliferation under ultrasound triggering, thereby promoting tissue repair of the defective trachea.
[0068] The above describes the embodiments of the present invention in conjunction with the accompanying drawings. However, the present invention is not limited to the above examples and various modifications can be made based on the inventive purpose of the present invention. Any changes, modifications, substitutions, combinations, or simplifications based on the methods and technical contents disclosed in the present invention shall be considered equivalent replacement methods and shall fall within the scope of the present invention as long as they are within the spirit and principles of the present invention.
Claims
1. A double-crosslinked composite permeable hydrogel tracheal patch preparation material, characterized in that: Includes a gel-based patch and a fibrinogen complex solution; Wherein, the gel base patch is a freeze-dried gel sheet of catechol grafted methacrylic anhydride gelatin; The fibrinogen complex solution comprises fibrinogen, calcium-loaded liposomes and transglutaminase, wherein the calcium-loaded liposomes release calcium ions under ultrasonic triggering.
2. The double-crosslinked composite permeable hydrogel tracheal patch preparation material according to claim 1, characterized in that: In the fibrinogen complex solution, the concentration of the fibrinogen is 15-30 mg / L, the concentration of the calcium-loaded liposome is 2-10 mg / mL, and the concentration of the transglutaminase is 50-150 nM.
3. The double-crosslinked composite permeable hydrogel tracheal patch preparation material according to claim 1, characterized in that: The preparation method of the gel base patch comprises: (1) obtaining methacrylic anhydride gelatin, and subjecting the obtained methacrylic anhydride gelatin to a carboxylation reaction to obtain carboxyl-modified methacrylic anhydride gelatin; (2) Carboxyl activation reaction of carboxyl-modified methacrylic anhydride gelatin was performed using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide to obtain catechol-grafted methacrylic anhydride gelatin hydrogel, which was freeze-dried to obtain the gel base patch.
4. The double-crosslinked composite permeable hydrogel tracheal patch preparation material according to claim 1, characterized in that: The preparation method of the fibrinogen composite solution is as follows: after dissolving fibrinogen with a NaCl solution, the solution is mixed with calcium-loaded liposomes and transglutaminase to prepare the fibrinogen composite solution.
5. The double-crosslinked composite permeable hydrogel tracheal patch preparation material according to claim 1, characterized in that: The preparation method of the calcium-loaded liposome is as follows: lecithin, cholesterol and mPEG-DSPE are mixed, dissolved in an organic solvent, dried to form a film, then mixed with a CaCl2 solution, and ultrasonically hydrated to obtain the calcium-loaded liposome.
6. The double-crosslinked composite permeable hydrogel tracheal patch preparation material according to claim 5, characterized in that: During the preparation of calcium-loaded liposomes, the organic solvent was chloroform, the added molar ratio of lecithin, cholesterol, and mPEG-DSPE was 52:43:5, and the concentration of the CaCl2 solution was 20 mg / ml.
7. A method for preparing a double-crosslinked composite permeable hydrogel tracheal patch, characterized in that: The double-crosslinked composite permeable hydrogel tracheal patch according to claim 1 is used to prepare the material, comprising the following steps: Step 1, adding the fibrinogen complex solution to the gel base patch; Step 2: triggering the cross-linking of the fibrinogen composite solution by ultrasound to form a double-cross-linked composite permeable hydrogel tracheal patch.
8. The method for preparing the double-crosslinked composite permeable hydrogel tracheal patch according to claim 7, characterized in that: In step 1, the volume ratio of the fibrinogen complex solution to the gel base patch is 1-2:
1.
9. The method for preparing the double-crosslinked composite permeable hydrogel tracheal patch according to claim 7, characterized in that: In step 2, the ultrasonic frequency is 1-3 MHz, the ultrasonic intensity is 1-3 W, and the ultrasonic time is 1-30 min.
10. A double-crosslinked composite permeable hydrogel tracheal patch, characterized in that: The double-cross-linked composite permeable hydrogel tracheal patch is prepared using the material for preparing the tracheal patch according to claim 1 or the preparation method according to claim 7.