Use of lung extracellular matrix hydrogel in the preparation of lung injury repair materials

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to the use of a lung extracellular matrix hydrogel in the preparation of a lung injury repair material. The application grafts methacrylic anhydride groups on a lung tissue-derived extracellular matrix, and provides a lung extracellular matrix hydrogel which can be rapidly photo-crosslinked, has high stability, effectively promotes angiogenesis, and prevents postoperative leakage. The photo-crosslinked lung extracellular matrix hydrogel of the application is applied to a lung injury animal model, significantly promotes angiogenesis, promotes alveolar regeneration, and also significantly reduces the fibrosis level. Compared with a sealing agent alpha-cyanopropyl acrylate gel and a fibrin glue which are used in clinical application, the photo-crosslinked lung extracellular matrix hydrogel has excellent effects on repairing lung injury, provides a new strategy for repairing traumatic and iatrogenic lung injury, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to the use of a lung extracellular matrix hydrogel in the preparation of a lung injury repair material. BACKGROUND

[0002] Lung injury poses a serious threat to life, especially in the case of acute trauma, such as traumatic lung injury caused by traffic accidents and construction accidents, and iatrogenic injury in lung surgery. Such injury often damages the pleural integrity and alveolar-capillary membrane barrier, leading to air leakage and pneumothorax, which may cause respiratory failure if not treated in time. In the process of lung injury repair, neovascularization is of great significance. The newly formed blood vessels can maintain the oxygen supply and nutrient supply of the tissue, help maintain the three-dimensional structure of the lung tissue, and guide the remodeling of the lung tissue, which is very important for the normal function recovery and long-term stability of the lung tissue.

[0003] Lung tissue-derived extracellular matrix (ECM) retains the natural microenvironment of lung tissue, including a complex network of structural proteins, glycosaminoglycans (GAGs), signaling molecules, biochemical and biomechanical cues, providing an ideal platform for guiding alveolar regeneration, regulating inflammatory response and tissue remodeling. Applying lung tissue-derived ECM to prepare hydrogel may be a potential method to seal the damaged area to prevent postoperative air leakage, but compared with lung extracellular matrix, the three-dimensional chemical environment and mechanical properties of extracellular matrix hydrogel have significant differences, which will seriously affect the angiogenesis in the process of lung injury repair; whether the pro-angiogenic growth factors are retained during the preparation of the hydrogel, and whether these factors can be released in a suitable manner, the porosity and connectivity of the hydrogel are also crucial for angiogenesis. In the in vivo environment, angiogenesis is a complex multi-factor regulation process, which not only involves growth factors and extracellular matrix, but also is influenced by many factors such as three-dimensional chemical environment and mechanical properties.

[0004] In order to promote the generation of blood vessels, it is usually necessary to add growth factors to the extracellular matrix hydrogel, therefore, how to construct a lung injury repair material that does not need to add growth factors, prevents postoperative leakage and effectively promotes angiogenesis is a problem faced by the field. SUMMARY

[0005] In view of the defects of the prior art, the present application provides the use of a lung extracellular matrix hydrogel in the preparation of a lung injury repair material, aiming to provide a lung injury repair material that does not need to add growth factors, prevents postoperative leakage and effectively promotes angiogenesis.

[0006] The present application provides the use of a lung extracellular matrix hydrogel in the preparation of a lung injury repair material, the lung extracellular matrix hydrogel comprising the following raw materials by weight:

[0007] 1-1.5 parts of lung extracellular matrix grafted with methacrylic anhydride groups.

[0008] Photoinitiator 0.1-1 part;

[0009] The grafting rate of the lung extracellular matrix grafted with methacrylic anhydride groups was 83% to 87%.

[0010] Preferably, the lung extracellular matrix hydrogel comprises the following raw materials in weight percentages:

[0011] Lung extracellular matrix grafted with methacrylic anhydride groups: 1-1.5%.

[0012] Photoinitiator 0.1-1%.

[0013] Preferably, the grafting rate of the lung extracellular matrix grafted with methacrylic anhydride groups is 85% to 87%.

[0014] Preferably, the lung extracellular matrix grafted with methacrylic anhydride groups is prepared by reacting raw materials including lung extracellular matrix and methacrylic anhydride.

[0015] Preferably, the molar ratio of free amino groups to methacrylic anhydride in the lung extracellular matrix is ​​1:10 to 1:40.

[0016] Preferably, the pH of the reaction is 9-10, the temperature of the reaction is 10-60 °C, and the reaction time is 12-48 hours.

[0017] Preferably, the reaction time is 24-48 hours.

[0018] Preferably, the lung extracellular matrix is ​​obtained from lung tissue through decellularization, pulverization, and digestion.

[0019] Preferably, the decellularization process includes: treating lung tissue with a surfactant;

[0020] And / or, the pulverization process includes: grinding decellularized lung tissue into powder in a ball mill, and sieving 10... -200 Particles;

[0021] And / or, the digestion process includes: placing the pulverized lung tissue in a digestive enzyme solution for digestion.

[0022] Preferably, lung tissue is perfused with an anticoagulant before decellularization.

[0023] And / or, the specific process of the decellularization treatment comprises: the lung tissue is treated with an anionic surfactant with a volume fraction of 0.2-1.0 % for 1-3 hours, after water washing, the lung tissue is treated with a non-ionic surfactant with a concentration of 0.1 % to 1.0 % for 4-12 hours, the anionic surfactant is at least one selected from the group consisting of sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, sodium deoxycholate, sodium cholate, rhamnolipid, and sodium lauroyl glutamate, and the non-ionic surfactant is at least one selected from the group consisting of Triton X-100, Tween 20, Tween 80, Pluronic F127, and NP-40;

[0024] And / or, in the process of the pulverization, the particles with a size of 10 -75 are screened out;

[0025] And / or, the specific process of the digestion comprises: 5-20 mg / mL of the pulverized lung tissue is placed in a 0.5-3 mg / mL pepsin solution with a pH of 1-3, and is digested at 20-37 ℃ for 12-72 hours.

[0026] Preferably, the photoinitiator is selected from the group consisting of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, photoinitiator 907, photoinitiator IHT-PI659, and photoinitiator I2959.

[0027] The application grafts methacrylate anhydride groups on the lung tissue-derived extracellular matrix by screening process conditions and hydrogel formulations, and provides a lung extracellular matrix hydrogel which can be rapidly photo-crosslinked, has high stability, effectively promotes angiogenesis, and prevents postoperative leakage. When the photo-crosslinked lung extracellular matrix hydrogel of the application is applied to a lung injury animal model, it significantly promotes angiogenesis, promotes alveolar regeneration, and also significantly reduces the level of fibrosis. Compared with the sealant α-cyanoacrylate gel and fibrin glue used in clinical practice, the photo-crosslinked lung extracellular matrix hydrogel of the application has excellent effect on repairing lung injury, provides a new strategy for repairing traumatic and iatrogenic lung injury, and has good application prospect.

[0028] Obviously, according to the above content of the application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the application.

[0029] The above content of the application will be further described in detail through the specific embodiments in the form of examples. However, it should not be understood that the scope of the above subject matter of the application is limited to the following examples. Any technology realized based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1Figure 1 is a graph showing the results of characterization and cell compatibility of the acellular lung; wherein, Figure 1 Figure 2 is a graph showing the results of surface morphology and histological changes of the lung tissue before and after decellularization, Figure 1 Figure 3 is a graph showing the results of DNA content determination of the lung tissue before and after decellularization, **** indicates p <0.0001, Figure 1 Figure 4 is a graph showing the results of collagen content determination of the lung tissue before and after decellularization, there is no significant difference between the two groups of data, Figure 1 Figure 5 is a graph showing the results of live and dead staining experiment, cytoskeleton staining experiment and scanning electron microscopy of cells seeded in the acellular lung scaffold.

[0031] Figure 2 Figure 6 is a graph showing the results of grafting rate under different reaction times by o-benzaldehyde method, wherein there is no significant difference between the data groups of 24 hours-48 hours.

[0032] Figure 3 Figure 7 is a nuclear magnetic resonance spectrum of lung extracellular matrix (Lung-ma) grafted with methacrylate; wherein, Figure 3 Figure 8 is a 1H-NMR spectrum of Lung-ma, Figure 3 Figure 9 is Figure 3 Figure 10 is an enlarged view of the dashed box area in Figure 9.

[0033] Figure 4 Figure 11 is a general view of lung extracellular matrix hydrogel; wherein, Figure 4 Figure 12 is a photo of lung extracellular matrix hydrogel before gelation, Figure 4 Figure 13 is a photo of lung extracellular matrix hydrogel after gelation, Figure 4 Figure 14 is a photo of lung extracellular matrix hydrogel of different concentrations.

[0034] Figure 5 Figure 15 is a characterization graph of the pro-angiogenic effect of lung extracellular matrix hydrogel of different concentrations.

[0035] Figure 6 Figure 16 is a graph showing the quantitative statistical results of the pro-angiogenic effect of lung extracellular matrix hydrogel of different concentrations, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0036] Figure 7 Figure 17 is a graph showing the degradation experiment results of lung extracellular matrix hydrogel of different concentrations.

[0037] Figure 8 Figure 18 is a graph showing the sealing efficiency of lung wound sealing of lung extracellular matrix hydrogel of different concentrations, wherein, **** indicates p<0.0001, ns indicates p ≥0.05, no significant difference.

[0038] Figure 9 This image shows the results of how extracellular matrix hydrogels promote alveolar regeneration in lung cells; among them... Figure 9 Figure A shows the immunohistochemical analysis results of SFTPB, with arrows indicating alveolar epithelial cell clusters. Figure 9 B is a graph showing the quantitative statistical results of the number of type II alveolar cells, where ** indicates... p <0.01, **** indicates p <0.0001, ns indicates p ≥0.05, no significant difference.

[0039] Figure 10 The results of the effect of lung cell extracellular matrix hydrogel on promoting angiogenesis are shown in the figure; Figure 10 Image A shows the results of CD31 immunohistochemical staining in the repair area. Figure 10 B is a graph showing the quantitative statistical results of blood vessel density, where * indicates... p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001, ns indicates p ≥0.05, no significant difference.

[0040] Figure 11 This is a graph showing the results of the Marson trichrome staining experiment and its quantitative analysis. Figure 11 Image A shows the results of Masson's trichrome staining. Figure 11 B is a graph showing the statistical results of collagen area, * indicates... p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001. Detailed Implementation

[0041] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0042] Example 1: A lung cell extracellular matrix hydrogel and its preparation method

[0043] The lung extracellular matrix hydrogel of this embodiment was prepared by grafting methacrylic anhydride groups onto the lung extracellular matrix and then photocrosslinking it. The specific method is as follows:

[0044] 1. Preparation of acellular lung

[0045] Fresh porcine lung was isolated and the surface blood was washed with deionized water. A large volume of anticoagulant such as sodium citrate, oxalate, heparin or ethylenediaminetetraacetic acid was perfused from the trachea until no obvious blood flow was observed. The lung tissue was cut into thin slices with a thickness of 0.2 mm (in other embodiments, the thickness of the lung tissue can also be adjusted in the range of 0.2 mm to 5 mm), and then subjected to decellularization treatment. The decellularization process was as follows: 0.5% (v / v) sodium dodecyl sulfate (SDS) treatment for 3 hours, followed by physiological saline washing; and then 0.1% Triton X-100 treatment for 12 hours. After the treatment, the slices were washed with deionized water to remove residual reagents and cell debris, and then freeze-dried and stored at 4 °C.

[0046] In other embodiments, the concentration of SDS during decellularization treatment can also be adjusted in the range of 0.2% to 1.0%; the time of SDS treatment can also be adjusted in the range of 1 hour to 3 hours; the concentration of Triton X-100 can also be adjusted in the range of 0.1% to 1.0%; and the time of Triton X-100 treatment can also be adjusted in the range of 4 hours to 12 hours.

[0047] The freeze-dried acellular lung was ground into fine powder in a ball mill, and the powder was sieved to obtain particles with a size of 10 -75 The particles were suspended in a 1 mg / mL pepsin solution with a pH of 2 at a concentration of 10 mg / mL, and digested at 25 °C for 48 hours to form a uniform acellular lung.

[0048] In other embodiments, the size of the sieved particles can also be adjusted in the range of 10 -200 The concentration of the particles can also be adjusted in the range of 5 mg / mL to 20 mg / mL; the pH of the pepsin solution can also be adjusted in the range of 1 to 3; the concentration of the pepsin solution can also be adjusted in the range of 0.5 mg / mL to 3 mg / mL; the temperature of the pepsin solution during digestion can also be adjusted in the range of 20 °C to 37 °C; and the time of digestion can also be adjusted in the range of 12 hours to 72 hours.

[0049] 2. Preparation of lung extracellular matrix hydrogel

[0050] The Acellular lung is added with NaOH to adjust the pH to 9-10, and then methyl acrylate (MA) is added drop by drop in batches, and the molar ratio of free amino groups (from the Acellular lung) to MA is 1:20 (in other embodiments, the molar ratio of free amino groups to MA can also be adjusted in the range of 1:10-1:40). NaOH is added to adjust the pH to the range of 9-10, the reaction temperature is 25°C, the reaction time is 24 hours (in other embodiments, the reaction time can also be adjusted in the range of 5 minutes-48 hours, preferably 12-48 hours), and Lung-ma is synthesized. After the reaction is completed, the reaction is terminated by diluting with 5 times the volume of deionized water, and then deionized water is used for dialysis for 72 hours to remove unreacted anhydride and byproducts. Lung-ma is freeze-dried to obtain a white flocculent product, which is sealed and stored at 4°C.

[0051] In 10 mg / mL Lung-ma (dissolved in physiological saline, and in other embodiments, PBS can also be replaced) is added with a photo initiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate LAP) and uniformly mixed, the concentration of the photo initiator is 0.5% w / v, and the reaction is initiated under light irradiation to form a gel, and the wavelength of the light irradiation is 405 nm. In this embodiment, 1% lung extracellular matrix hydrogel (Lung-ma gel) is prepared.

[0052] In this embodiment, the calculation method of the amount of Acellular lung is as follows: the content of free amino groups in the Acellular lung is determined by the o-benzaldehyde (OPA) method, and the actual amount of the Acellular lung is calculated according to the content.

[0053] In other embodiments, the concentration of Lung-ma reacted with the photo initiator can also be adjusted in the range of 5-40 mg / mL; the photo initiator can also be selected from photo initiator 907, photo initiator IHT-PI659 or photo initiator I2959; the concentration of the photo initiator can also be adjusted in the range of 0.1-1% w / v; and the wavelength of the light irradiation can also be adjusted in the range of 365-405 nm.

[0054] In a lung injury animal model, the lung extracellular matrix hydrogel prepared in this embodiment has a significant pro-angiogenic effect, has excellent ability to promote lung tissue regeneration, and significantly reduces the level of fibrosis in the lung injury repair process, and its effect of repairing lung injury is significantly better than that of the commonly used sealant and fibrin glue in clinical practice.

[0055] Example 2: A lung extracellular matrix hydrogel and a preparation method thereof

[0056] A photo-crosslinked lung matrix hydrogel was prepared according to the method of Example 1, except that in Step 2, Lung-ma was reacted with the photo-initiator at a concentration of 15 mg / mL. A 1.5% lung extracellular matrix hydrogel was prepared in this example.

[0057] Comparative Example 1 A lung extracellular matrix hydrogel and a method for preparing the same

[0058] A photo-crosslinked lung matrix hydrogel was prepared according to the method of Example 1, except that in Step 2, Lung-ma was reacted with the photo-initiator at a concentration of 5 mg / mL. A 0.5% lung extracellular matrix hydrogel was prepared in this example.

[0059] Comparative Example 2 A lung extracellular matrix hydrogel and a method for preparing the same

[0060] A photo-crosslinked lung matrix hydrogel was prepared according to the method of Example 1, except that in Step 2, Lung-ma was reacted with the photo-initiator at a concentration of 20 mg / mL. A 2% lung extracellular matrix hydrogel was prepared in this example.

[0061] Comparative Example 3 A lung extracellular matrix hydrogel and a method for preparing the same

[0062] A photo-crosslinked lung matrix hydrogel was prepared according to the method of Example 1, except that in Step 2, Lung-ma was reacted with the photo-initiator at a concentration of 40 mg / mL. A 4% lung extracellular matrix hydrogel was prepared in this example.

[0063] The technical solutions of the present application are further described below through experiments. The samples acellular lung and Lung-ma used in the following experimental examples were prepared according to the method of Example 1. The 1% Lung-ma gel was prepared according to the method of Example 1, the 1% Lung-ma gel was prepared according to the method of Example 1, the 1.5% Lung-ma gel was prepared according to the method of Example 2, the 0.5% Lung-ma gel was prepared according to the method of Comparative Example 1, the 2% Lung-ma gel was prepared according to the method of Comparative Example 2, and the 4% Lung-ma gel was prepared according to the method of Comparative Example 3.

[0064] Experimental Example 1 Evaluation of the effect of acellular lung

[0065] I. Experimental method

[0066] 1. Histological changes of acellular lung tissue

[0067] The surface morphology of the acellular lung was observed by scanning electron microscopy. Hematoxylin-eosin staining, nuclear staining, Masson's trichrome staining, and Sirius red staining were used to observe the histological changes of the acellular lung. The collagen content in the lung tissue was quantitatively detected by hydroxyproline determination method. The calculation method of collagen content was the amount of collagen contained in each g of lung tissue dry weight ).

[0068] 2. DNA content determination

[0069] The DNA detection refers to YYT 1876-2023 (Determination of DNA Residue in Tissue Engineering Medical Products Animal-derived Biomaterials: Fluorescent Staining Method. The calculation method of DNA content is the amount of DNA contained in each mg of lung tissue dry weight (ng).

[0070] 3. Biocompatibility

[0071] Primary airway epithelial cells were seeded on the acellular lung scaffold and co-cultured for 3 days. The cells seeded on the acellular lung scaffold were subjected to live and dead staining, cytoskeleton staining, and scanning electron microscopy characterization.

[0072] II. Experimental results

[0073] Scanning electron microscopy showed that the scaffold still maintained relatively intact alveolar and airway structures Figure 1 A). Hematoxylin-eosin staining and nuclear staining results showed that there was no obvious cell residue in the acellular lung scaffold, and the DNA content decreased from 409.50 ± 25.45 ng / mg before decellularization to 34.01 ± 3.52 ng / mg, which was lower than the internationally recognized standard (50 ng / mg) Figure 1 B). Masson's trichrome staining and Sirius red staining confirmed that the key ECM structural components were effectively preserved, and there was no statistically significant change in collagen content before and after decellularization Figure 1 A and 1C).

[0074] Live and dead staining experiments showed that after primary airway epithelial cells were seeded on the scaffold and co-cultured for 3 days, the cells remained highly active, with very few dead cells Figure 1 D). The seeded cells showed a more stretched morphology on the scaffold surface and could aggregate and proliferate in the pores of the acellular lung Figure 1 D). The acellular lung scaffold showed excellent biocompatibility and did not affect the growth and proliferation of cells.

[0075] Example 2 Determination of methacrylic anhydride grafting rate

[0076] I. Experimental methods

[0077] Lung-ma was prepared according to the method in Example 1, except that in step 3, the reaction times between acellular lung and MA were 5 minutes, 10 minutes, 20 minutes, 1 hour, 4 hours, 6 hours, 12 hours, 24 hours, 36 hours, and 48 hours, respectively. The residual amino groups in the acellular lung were determined using the o-benzaldehyde (OPA) method, and the grafting rate was calculated.

[0078] II. Experimental Results

[0079] like Figure 2 As shown, the grafting rate ranges from 67.5% to 86.6%. The grafting rate increases continuously with increasing reaction time. When the reaction time is 12 hours, the grafting rate reaches over 83%, and when the reaction time is increased to 24 hours, the grafting rate reaches its highest point at 86.6%. Further extending the reaction time does not significantly change the grafting rate. Therefore, the preferred reaction time in this invention is 24 hours.

[0080] Example 3 Characterization of grafted methacrylic anhydride

[0081] I. Experimental Methods

[0082] The success of grafting methacrylic anhydride onto Lung-ma was characterized by proton nuclear magnetic resonance spectroscopy.

[0083] II. Experimental Results

[0084] In the ¹H-NMR spectrum ( Figure 3 In the Lung-ma sample, characteristic peaks of methacryloyl groups can be observed: methylene and methyl protons (CH2=C(CH3)-CONH−) appear at 5.59 ppm and 5.36 ppm, respectively, the methyl peak (CH2=C(CH3)-CONH−) is located at 1.85 ppm, while the proton peak corresponding to the free amino group (-NH2) disappears at 2.93 ppm, indicating that methacrylic anhydride was successfully grafted onto Lung-ma.

[0085] Example 4: Macroscopic view of lung extracellular matrix hydrogel

[0086] I. Experimental Methods

[0087] Photocrosslinked lung extracellular matrix hydrogels were prepared according to the method in Example 1, with the difference that in step 2, the concentrations of Lung-ma reacting with the photoinitiator were 5 mg / mL, 10 mg / mL, 20 mg / mL, and 40 mg / mL, respectively, and the concentration of the photoinitiator LAP was 0.1% w / v. Lung extracellular matrix hydrogels of 0.5%, 1%, 2%, and 4% were prepared and photographed.

[0088] II. Experimental Results

[0089] Figure 4 Pictures of lung extracellular matrix hydrogels under different Lung-ma concentrations. It is proved that the method of the present application can successfully prepare lung extracellular matrix hydrogels.

[0090] Experimental Example 5 In vitro pro-angiogenic effect of photo-crosslinked lung extracellular matrix hydrogels

[0091] I. Experimental Methods

[0092] Lung extracellular matrix hydrogels were prepared according to the method of Example 1, except that in step 2, when Lung-ma reacted with the photo-initiator, the concentration of Lung-ma was 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, and 40 mg / mL, respectively, and the concentration of the photo-initiator LAP was 0.1% w / v. Lung extracellular matrix hydrogels of 0.5%, 1%, 1.5%, 2%, and 4% were prepared, respectively.

[0093] The above lung extracellular matrix hydrogels (40 ) and the positive control Matrigel were evenly spread on the bottom of a 24-well plate, and human umbilical vein endothelial cells (HUVECs) were inoculated on the surface thereof at a density of 6 x 104 / well, and the tube formation of HUVECs was observed.

[0094] II. Experimental Results

[0095] From Figure 5 , 6 , it can be seen that lung extracellular matrix hydrogels of 0.5%, 1%, and 1.5% have obvious pro-tube formation effect, lung extracellular matrix hydrogel of 2% has significantly decreased pro-tube formation effect, and lung extracellular matrix hydrogel of 4% inhibits tube formation. Therefore, lung extracellular matrix hydrogels of 0.5% to 1.5% have excellent pro-angiogenic effect.

[0096] Experimental Example 6 Stability of lung extracellular matrix hydrogels

[0097] I. Experimental Methods

[0098] Lung extracellular matrix hydrogels were prepared according to the method of Example 1, except that in step 2, when Lung-ma reacted with the photo-initiator, the concentration of Lung-ma was 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, and 40 mg / mL, respectively, and the concentration of the photo-initiator LAP was 0.1% w / v. Lung extracellular matrix hydrogels of 0.5%, 1%, 1.5%, 2%, and 4% were prepared, respectively, and were freeze-dried.

[0099] The above lyophilized lung extracellular matrix hydrogel was weighed (W0) W 0 ), and placed in 10 mL PBS buffer (pH 7.4) at 37°C, 60 rpm constant temperature shaker, respectively, after 2, 6, 8, 10, 12, 14 days, rinse the surface with deionized water, freeze-dried and weighed (Wt) W t ), and 3 parallel groups were set up for each group (n = 3). The residual mass percentage was calculated. The degradation performance of the gel can be calculated by the formula:

[0100]

[0101] wherein Wt is the mass of the lyophilized hydrogel scaffold at time t, and W0 is the initial mass of the lyophilized hydrogel scaffold.

[0102] II. Experimental results

[0103] The degradation experiment results are shown in Figure 7 . The 0.5% gel degrades faster, with the maximum degradation curve slope and the maximum degradation rate in the first 6 days of degradation, and a 50% mass loss on the 6th day; subsequently, the degradation rate slows down and tends to be balanced. In contrast, the 1%, 2% and 4% gels exhibit similar degradation rates, with smaller degradation curve slopes and lower degradation rates, and the hydrogels lose about 10-18% of their mass on the 6th day of degradation. Therefore, under physiological conditions, the lung extracellular matrix hydrogel of 1-4% has better stability, which is conducive to its in vivo function.

[0104] Example 7 Leakage sealing efficiency of lung extracellular matrix hydrogel

[0105] I. Experimental method

[0106] 1. Experimental grouping

[0107] This experimental example is divided into seven groups, namely α-cyanacrylate gel group, fibrin glue group and 0.5%, 1%, 1.5%, 2%, 4% Lung-ma gel group.

[0108] 2. Animal experiment

[0109] Six Sprague-Dawley rats weighing 200-250 g were selected and anesthetized with isoflurane. A tracheal catheter was inserted through the mouth and connected to a small animal ventilator for mechanical ventilation: tidal volume 2.2 mL, respiratory rate 70 times / min, positive end-expiratory pressure 2.2 cm H2O, and the airway pressure F0 was collected as the lung basic air pressure, and the lung air tightness before and after sealing was evaluated by airway pressure measurement. Then a median thoracotomy was performed to expose the lungs; a 1 cm incision was made on the surface of the lungs with scissors to form an air leak. 100 The wound was repaired by sealing it with 0.5%, 1%, 1.5%, 2%, and 4% lung extracellular matrix hydrogel (prepared by the method in Example 1), and with α-cyanoacrylate gel (100g), a commonly used clinical lung sealant. ) and fibrin glue (100 As a control, postoperative airway pressure F1 was collected 5 minutes after stabilizing breathing. The sealing efficiency of the gel can be calculated using the formula:

[0110]

[0111] In the formula, F1 is the postoperative airway pressure and F0 is the initial airway pressure.

[0112] II. Experimental Results

[0113] The results are as follows Figure 8 As shown, 0.5% lung extracellular matrix hydrogel is difficult to stably close wounds during lung respiration, resulting in poor sealing efficiency. 1%, 1.5%, 2%, and 4% lung extracellular matrix hydrogels exhibit excellent air leakage sealing effects and show no significant difference compared to clinically commonly used α-cyanoacrylate gel and fibrin glue. Therefore, 1%–4% lung extracellular matrix hydrogels hold promise for sealing lung injuries and preventing air leakage and pneumothorax.

[0114] Experimental Example 6: Application of Lung Extracellular Matrix Hydrogels in Lung Injury Repair

[0115] I. Experimental Methods

[0116] 1. Experimental Grouping

[0117] This experiment was divided into six groups: sham surgery group, surgery group, α-cyanoacrylate gel group, fibrin gel group, 1% Lung-ma gel group, and 2% Lung-ma gel group.

[0118] 2. Animal experiments

[0119] Six Sprague-Dawley rats, weighing 200–250 g, were anesthetized with isoflurane. An endotracheal tube was inserted through the mouth and the rats were connected to a small animal ventilator for mechanical ventilation: tidal volume 2.2 mL, respiratory rate 70 breaths / min, and positive end-expiratory pressure 2.2 cm H2O. A midline thoracotomy was then performed to expose both lungs; a 1 cm incision was made on the lung surface with scissors to create an air leak. 100 mg of [unspecified substance] was dripped into each lesion site. Repairing the wound involves sealing the wound with 1% or 2% lung extracellular matrix hydrogel, using clinically common lung sealant α-cyanoacrylate gel (100... ) and fibrin glue (100 As a control group, only the lungs were exposed without any incisions in the sham surgery group. In the surgical group, incisions were made on the surface of the lungs, and no other treatment was performed.

[0120] Stools were collected 7 and 14 days post-surgery for immunohistochemical staining to detect SFTPB (type II alveolar cell marker) and CD31 (vascular endothelial cell marker) to evaluate lung regeneration.

[0121] II. Experimental Results

[0122] Immunostaining with SFTPB and CD31 showed that the number of SFTPB-positive mature alveolar epithelial cells and CD31-positive vascular networks in the lung tissue of the 1% Lung-ma gel group was significantly higher than that in the fibrin glue group and the surgical group. Figure 9 , 10 The number of SFTPB-positive mature alveolar epithelial cells and CD31-positive vascular networks in the lung tissue of the 2% extracellular matrix hydrogel group was significantly lower than that of the 1% extracellular matrix hydrogel group, and there was no significant difference compared with the surgical group and the α-cyanoacrylate gel group. This indicates that the 1% extracellular matrix hydrogel of the present invention has the effect of promoting alveolar regeneration and angiogenesis, and has a better ability to promote lung tissue regeneration compared with other control groups of 2% extracellular matrix hydrogel.

[0123] Fourteen days post-surgery, Masson's trichrome staining results showed that 1% lung extracellular matrix hydrogel significantly reduced the degree of fibrosis. Figure 11 Quantitative analysis showed that severe fibrosis occurred in the surgical group, the α-cyanoacrylate gel group, and the 2% lung extracellular matrix hydrogel, with collagen area fractions of 19.18% ± 2.95, 25.87% ± 3.68, and 12.78% ± 0.61, respectively. While clinically used fibrin gel reduced collagen deposition by 51% (9.34% ± 1.30), obvious fibrous plaques were still visible. In contrast, the 1% lung extracellular matrix hydrogel of this invention reduced collagen levels to near normal (3.98% ± 1.54), a reduction of 79% compared to the surgical group, 85% compared to the α-cyanoacrylate gel, and 57% compared to the fibrin gel.

[0124] The above results indicate that the 1% lung extracellular matrix hydrogel prepared in this embodiment of the invention has the effect of promoting angiogenesis in vivo, has excellent ability to promote lung tissue regeneration, significantly reduces the level of fibrosis during lung injury repair, and its effect on repairing lung injury is significantly better than surgical and clinically commonly used sealants and fibrin glue.

[0125] Based on the above-mentioned pro-angiogenesis experiment, stability experiment, wound closure experiment in lung respiration process and lung injury animal experiment, the lung extracellular matrix hydrogel of 1-1.5% is preferred, which does not need to add growth factors additionally, effectively prevents postoperative leakage and effectively promotes angiogenesis.

[0126] It can be seen from the above examples and experimental examples that the lung extracellular matrix hydrogel provided by the application is quickly photo-crosslinked by screening process conditions and hydrogel formula, has high stability, effectively promotes angiogenesis and prevents postoperative leakage by grafting methacrylate anhydride groups on lung tissue-derived extracellular matrix. The photo-crosslinked lung extracellular matrix hydrogel of the application is applied to a lung injury animal model, significantly promotes the generation of blood vessels, promotes alveolar regeneration and also significantly reduces the fibrosis level. Compared with the sealant alpha-cyanocrylate gel and fibrin glue used in clinical use, the photo-crosslinked lung extracellular matrix hydrogel has excellent effect of repairing lung injury, provides a new strategy for repairing traumatic and iatrogenic lung injury and has good application prospect.

Claims

1. The use of lung extracellular matrix hydrogel in the preparation of lung injury repair materials, characterized in that, The lung extracellular matrix hydrogel comprises the following raw materials in parts by weight: One portion of lung extracellular matrix grafted with methacrylic anhydride groups. Photoinitiator 0.1-1 part; The grafting rate of the lung extracellular matrix grafted with methacrylic anhydride groups was 83% to 87%.

2. The use of the lung extracellular matrix hydrogel according to claim 1 in the preparation of lung injury repair materials, characterized in that: The grafting rate of the lung extracellular matrix grafted with methacrylic anhydride groups is 85%~87%.

3. The use of the lung extracellular matrix hydrogel according to claim 1 in the preparation of lung injury repair materials, characterized in that: The lung extracellular matrix grafted with methacrylic anhydride groups is prepared by reacting raw materials including lung extracellular matrix and methacrylic anhydride.

4. The use of the lung extracellular matrix hydrogel according to claim 3 in the preparation of lung injury repair materials, characterized in that: The molar ratio of free amino groups to methacrylic anhydride in the extracellular matrix of lung cells is 1:10 to 1:

40.

5. The use of the lung extracellular matrix hydrogel according to claim 3 in the preparation of lung injury repair materials, characterized in that: The reaction is carried out at a pH of 9-10, at a temperature of 10-60 °C, and for a duration of 12-48 hours.

6. The use of the lung extracellular matrix hydrogel according to claim 5 in the preparation of lung injury repair materials, characterized in that: The reaction time is 24-48 hours.

7. The use of the lung extracellular matrix hydrogel according to claim 1 in the preparation of lung injury repair materials, characterized in that: The lung extracellular matrix is ​​obtained from lung tissue through decellularization, pulverization, and digestion.

8. The use of the lung extracellular matrix hydrogel according to claim 7 in the preparation of lung injury repair materials, characterized in that, The decellularization process includes: treating lung tissue with a surfactant; And / or, the pulverization process includes: grinding decellularized lung tissue into powder in a ball mill, and sieving 10... -200 Particles; And / or, the digestion process includes: placing the pulverized lung tissue in a digestive enzyme solution for digestion.

9. The use of the lung extracellular matrix hydrogel according to claim 8 in the preparation of lung injury repair materials, characterized in that, Lung tissue was perfused with an anticoagulant before decellularization. And / or, the specific process of the decellularization treatment includes: treating lung tissue with an anionic surfactant at a volume fraction of 0.2-1.0% for 1-3 hours, washing with water, and then treating with a nonionic surfactant at a volume fraction of 0.1%-1.0% for 4-12 hours, wherein the anionic surfactant is selected from at least one of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium deoxycholate, sodium cholate, rhamnolipid, and sodium lauroyl glutamate, and the nonionic surfactant is selected from at least one of Triton X-100, Tween 20, Tween 80, Pluronic F127, and NP-40; And / or, during the crushing process, 10 are screened. -75 Particles; And / or, the specific digestion process includes: placing 5-20 mg / mL of pulverized lung tissue in a 0.5-3 mg / mL pepsin solution at pH 1-3, and digesting it at 20-37 °C for 12-72 hours.

10. The use of the lung extracellular matrix hydrogel according to claim 1 in the preparation of lung injury repair materials, characterized in that: The photoinitiator is selected from lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, photoinitiator 907, photoinitiator IHT-PI659 or photoinitiator I2959.

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