Application of lung extracellular matrix hydrogel in preparation of lung injury repair material

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of lung extracellular matrix hydrogel in preparation of a lung injury repair material. A methacrylic anhydride group is grafted on a lung tissue-derived extracellular matrix, and the lung extracellular matrix hydrogel capable of being quickly photo-crosslinked is provided, is high in stability, effectively promotes angiogenesis and prevents postoperative leakage. When the photo-crosslinking lung extracellular matrix hydrogel is applied to a lung injury animal model, the generation of blood vessels is remarkably promoted, the regeneration of pulmonary alveoli is promoted, and the fibrosis level is also remarkably reduced. Compared with clinically used sealants alpha-cyanoacrylate gel and fibrin glue, the sealant has an excellent lung injury repairing effect, provides a new strategy for trauma and iatrogenic lung injury repairing, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of lung extracellular matrix hydrogel in the preparation of lung injury repair materials. Background Technology

[0002] Lung injury poses a serious threat to life, especially in cases of acute trauma, such as traumatic lung injury from traffic accidents or construction accidents, and iatrogenic injury during lung surgery. This injury typically disrupts the integrity of the pleura and the alveolar-capillary membrane barrier, leading to air leakage and pneumothorax, which can result in respiratory failure if not treated promptly. Angiogenesis plays a crucial role in lung injury repair. Newly formed blood vessels maintain tissue oxygen and nutrient supply, help maintain the three-dimensional structure of lung tissue, and guide lung tissue remodeling, all of which are essential for the restoration and long-term stability of normal lung function.

[0003] Lung tissue-derived extracellular matrix (ECM) preserves the natural microenvironment of lung tissue, including a complex network of structural proteins, glycosaminoglycans (GAGs), signaling molecules, and biochemical and biomechanical cues, providing an ideal platform for guiding alveolar regeneration, regulating inflammatory responses, and tissue remodeling. Applying lung tissue-derived ECM to prepare hydrogels may be a potential method for sealing damaged areas to prevent postoperative air leakage. However, compared to lung extracellular matrix, extracellular matrix hydrogels exhibit significant differences in their three-dimensional chemical environment and mechanical properties, which will severely impact angiogenesis during lung injury repair. During hydrogel preparation, the retention of pro-angiogenic growth factors and their appropriate release mechanisms, as well as the porosity and connectivity of the hydrogel, are crucial for angiogenesis. In vivo, angiogenesis is a complex, multifactorial regulated process involving not only growth factors and the extracellular matrix but also being influenced by factors such as the three-dimensional chemical environment and mechanical properties.

[0004] To promote angiogenesis, it is usually necessary to add additional growth factors to the extracellular matrix hydrogel. Therefore, how to construct a lung injury repair material that does not require the addition of additional growth factors, prevents postoperative leakage, and effectively promotes angiogenesis is a problem faced in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the use of lung extracellular matrix hydrogel in the preparation of lung injury repair materials, with the aim of providing a lung injury repair material that does not require the addition of additional growth factors, prevents postoperative leakage, and effectively promotes angiogenesis.

[0006] This invention provides the use of a lung extracellular matrix hydrogel in the preparation of lung injury repair materials, wherein the lung extracellular matrix hydrogel comprises the following raw materials in parts by weight: pulmonary extracellular matrix grafted with methacrylic anhydride 1-1.5 parts, photoinitiator 0.1-1 part; The grafting rate of the pulmonary extracellular matrix grafted with methacrylic anhydride is 83%-87%.

[0007] Preferably, the pulmonary extracellular matrix hydrogel comprises the following raw materials in mass percentage: pulmonary extracellular matrix grafted with methacrylic anhydride 1-1.5%, photoinitiator 0.1-1%.

[0008] Preferably, the grafting rate of the pulmonary extracellular matrix grafted with methacrylic anhydride is 85%-87%.

[0009] Preferably, the pulmonary extracellular matrix grafted with methacrylic anhydride is prepared from raw materials comprising pulmonary extracellular matrix and methacrylic anhydride.

[0010] Preferably, the molar ratio of free amino groups in the pulmonary extracellular matrix to methacrylic anhydride is 1:10-1:40.

[0011] Preferably, the pH of the reaction is 9-10, the temperature of the reaction is 10-60 ℃, and the time of the reaction is 12-48 hours.

[0012] Preferably, the time of the reaction is 24-48 hours.

[0013] Preferably, the pulmonary extracellular matrix is prepared from lung tissue through decellularization, crushing, and digestion.

[0014] Preferably, the process of decellularization comprises: treating the lung tissue with a surfactant; and / or, the process of crushing comprises: grinding the decellularized lung tissue into powder in a ball mill, and screening particles of 10 -200 ; and / or, the process of digestion comprises: digesting the crushed lung tissue in a digestive enzyme solution.

[0015] Preferably, the lung tissue is perfused with an anticoagulant before decellularization. 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; And / or, in the process of pulverization, the particles with a size of 10 -75 are screened out; And / or, the specific process of the digestion comprises: 5-20 mg / mL of the pulverized lung tissue is placed in a pepsin solution with a concentration of 0.5-3 mg / mL and a pH of 1-3, and is digested at 20-37 ℃ for 12-72 hours.

[0016] 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.

[0017] The application grafts methacrylate anhydride groups on a 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 has excellent effect on repairing lung injury, provides a new strategy for repairing traumatic and iatrogenic lung injury, and has good application prospect.

[0018] Obviously, according to the above content of the application, according to the ordinary technical knowledge and 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.

[0019] The above content of the application will be further described in detail through the following 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

[0020] Figure 1 are results of characterization of the decellularized lung and cell compatibility of the decellularized lung; wherein,Figure 1 A is the result chart of the surface morphology and histological changes of lung tissue before and after decellularization, Figure 1 B is the result chart of DNA content determination of lung tissue before and after decellularization, **** indicates p <0.0001, Figure 1 C is the result chart of collagen content determination of lung tissue before and after decellularization, there is no significant difference between the two groups of data, Figure 1 D is the result chart of live and dead staining experiment, cytoskeleton staining experiment and scanning electron microscope of cells inoculated in the decellularized lung scaffold.

[0021] Figure 2 is the result chart of grafting rate under different reaction times by o-benzaldehyde method, and there is no significant difference between the data groups of 24 hours-48 hours.

[0022] Figure 3 is the nuclear magnetic resonance spectrum of lung extracellular matrix (Lung-ma) grafted with methacrylate; wherein, Figure 3 A is the ¹H-NMR spectrum of Lung-ma, Figure 3 B is Figure 3 A is the enlarged view of the dashed box area.

[0023] Figure 4 is the general view of lung extracellular matrix hydrogel; wherein, Figure 4 A is the photo before the lung extracellular matrix hydrogel is formed, Figure 4 B is the photo after the lung extracellular matrix hydrogel is formed, Figure 4 C is the photo of lung extracellular matrix hydrogel with different concentrations.

[0024] Figure 5 is the characterization chart of the pro-angiogenic effect of lung extracellular matrix hydrogel with different concentrations.

[0025] Figure 6 is the result chart of quantitative statistics of the pro-angiogenic effect of lung extracellular matrix hydrogel with different concentrations, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0026] Figure 7 is the result chart of degradation experiment of lung extracellular matrix hydrogel with different concentrations.

[0027] Figure 8 is the result chart of the occlusion efficiency of lung wound occlusion of lung extracellular matrix hydrogel with different concentrations, wherein, **** indicates p <0.0001, ns indicates p≥ 0.05, no significant difference.

[0028] Figure 9 Figure showing the result of lung extracellular matrix hydrogel promoting alveolar regeneration; wherein, Figure 9 A is the result of immunohistochemical analysis of SFTPB, and the arrow indicates alveolar epithelial cell clusters, Figure 9 B is the result of quantitative statistics of the number of type II alveolar cells, * indicates p <0.01, **** indicates p <0.0001, ns indicates p ≥ 0.05, no significant difference.

[0029] Figure 10 Figure showing the result of lung extracellular matrix hydrogel promoting angiogenesis; wherein, Figure 10 A is the result of CD31 immunohistochemical staining in the repair area, Figure 10 B is the result of quantitative statistics of the vascular density, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001, ns indicates p ≥ 0.05, no significant difference.

[0030] Figure 11 Figure showing the result of Masson trichrome staining and its quantitative results, wherein, Figure 11 A is the result of Masson trichrome staining, Figure 11 B is the result of collagen area statistics, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001. DETAILED DESCRIPTION

[0031] In the following examples and experimental examples, the reagents and materials not specifically specified are commercially available.

[0032] Example 1: A lung extracellular matrix hydrogel and a preparation method thereof The lung extracellular matrix hydrogel of the present example is prepared by grafting methacrylic anhydride groups on the lung extracellular matrix and then photo-crosslinking. The specific method is as follows: 1. Preparation of acellular lung 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 out. 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.

[0033] In other embodiments, the concentration of SDS during decellularization treatment can also be adjusted in the range of 0.2-1.0 %; the time of SDS treatment can also be adjusted in the range of 1-3 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 4-12 hours.

[0034] 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.

[0035] 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 5-20 mg / mL; the pH of the pepsin solution can also be adjusted in the range of 1-3; the concentration of the pepsin solution can also be adjusted in the range of 0.5-3 mg / mL; the temperature of the pepsin solution can also be adjusted in the range of 20-37 °C; and the time of digestion in the pepsin solution can also be adjusted in the range of 12 hours to 72 hours.

[0036] 2. Preparation of lung extracellular matrix hydrogel Acellular lung was added with NaOH to adjust pH to 9-10, and then methyl acrylate (MA) was added drop by drop in batches, with the molar ratio of free amino groups (from Acellular lung) to MA being 1:20 (in other embodiments, the molar ratio of free amino groups to MA can also be adjusted to be in the range of 1:10-1:40). NaOH was added to adjust pH to be in the range of 9-10, the reaction temperature was 25°C, the reaction time was 24 hours (in other embodiments, the reaction time can also be adjusted to be in the range of 5 minutes-48 hours, preferably 12-48 hours), and Lung-ma was synthesized. After the reaction was completed, the reaction was terminated by dilution with 5 times the volume of deionized water, and then deionized water was used for dialysis for 72 hours to remove unreacted acid anhydride and byproducts. Lung-ma was freeze-dried to obtain a white flocculent product, which was stored at 4°C after being sealed.

[0037] In 10 mg / mL Lung-ma (dissolved in physiological saline, and in other embodiments, PBS can also be used instead) was added a photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate LAP) and mixed uniformly, the concentration of the photoinitiator was 0.5% w / v, and the reaction was initiated under light irradiation to form a gel, and the wavelength of the light irradiation was 405 nm. In this embodiment, 1% lung extracellular matrix hydrogel (Lung-ma gel) was prepared.

[0038] In this embodiment, the calculation method of the amount of Acellular lung was as follows: the content of free amino groups in Acellular lung was determined by using o-benzaldehyde (OPA) method, and the actual amount of Acellular lung was calculated according to the content.

[0039] In other embodiments, the concentration of Lung-ma reacted with the photoinitiator can also be adjusted to be in the range of 5-40 mg / mL; the photoinitiator can also be selected from photoinitiator 907, photoinitiator IHT-PI659 or photoinitiator I2959; the concentration of the photoinitiator can also be adjusted to be in the range of 0.1-1% w / v; and the wavelength of the light irradiation can also be adjusted to be in the range of 365-405 nm.

[0040] 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 process of repairing lung injury, and its effect of repairing lung injury is significantly better than that of the sealant and fibrin glue commonly used in clinical practice.

[0041] Example 2: A lung extracellular matrix hydrogel and a preparation method thereof 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.

[0042] Comparative Example 1 A lung extracellular matrix hydrogel and a method for preparing the same 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.

[0043] Comparative Example 2 A lung extracellular matrix hydrogel and a method for preparing the same 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.

[0044] Comparative Example 3 A lung extracellular matrix hydrogel and a method for preparing the same 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.

[0045] 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.

[0046] Experimental Example 1 Evaluation of the effect of acellular lung I. Experimental method 1. Histological changes of acellular lung tissue The surface morphology of acellular lung tissue was observed using scanning electron microscopy, and histological changes were observed using hematoxylin-eosin staining, nuclear staining, Masson's trichrome staining, and Sirius red staining. The collagen content in the lung tissue was quantitatively determined using a hydroxyproline assay; the collagen content was calculated as the amount of collagen contained per gram of dry lung tissue. ).

[0047] 2. Determination of DNA content DNA testing is performed according to YYT 1876-2023 (Determination of DNA Residue in Animal-Derived Biomaterials for Tissue Engineering Medical Products: Fluorescent Staining Method). The DNA content is calculated as the amount of DNA (ng) contained in each mg of dry lung tissue.

[0048] 3. Biocompatibility Primary airway epithelial cells were seeded onto decellularized lung scaffolds and co-cultured for 3 days. Cells seeded onto the decellularized lung scaffolds were characterized by live / dead staining, cytoskeleton staining, and scanning electron microscopy.

[0049] II. Experimental Results Scanning electron microscopy showed that the stent still maintained relatively intact alveolar and airway structures. Figure 1 A). Hematoxylin-eosin staining and nuclear staining results showed no obvious cellular 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 is lower than the internationally recognized standard (50 ng / mg). Figure 1 B). Masson's trichrome staining and Sirius red staining confirmed that 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).

[0050] Live-dead staining experiments showed that after primary airway epithelial cells were seeded onto a scaffold and co-cultured for 3 days, the cells maintained high viability with very few dead cells. Figure 1 D). Seeded cells exhibit a more extended morphology on the scaffold surface and are able to aggregate and proliferate in the acellular lung pores. Figure 1 D). Acellular lung scaffolds exhibit excellent biocompatibility and do not affect cell growth and proliferation.

[0051] Experimental Example 2: Determination of Methacrylic Anhydride Grafting Rate I. Experimental Methods 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.

[0052] II. Experimental Results 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.

[0053] Example 3 Characterization of grafted methacrylic anhydride I. Experimental Methods The success of grafting methacrylic anhydride onto Lung-ma was characterized by proton nuclear magnetic resonance spectroscopy.

[0054] II. Experimental Results 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.

[0055] Example 4: Macroscopic view of lung extracellular matrix hydrogel I. Experimental Methods 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.

[0056] II. Experimental Results Figure 4Pictures of lung extracellular matrix hydrogels with different Lung-ma concentrations. It is proved that the method of the present application embodiment can successfully prepare lung extracellular matrix hydrogels.

[0057] Experimental Example 5 In vitro pro-angiogenic effect of photo-crosslinked lung extracellular matrix hydrogels I. Experimental method The 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.

[0058] 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 at a density of 6x104 / well, and the tube formation of HUVECs was observed.

[0059] II. Experimental results 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, while 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.

[0060] Experimental Example 6 Stability of lung extracellular matrix hydrogels I. Experimental method The 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.

[0061] The above freeze-dried lung extracellular matrix hydrogels were taken and weighed W 0Each sample was placed in 10 mL of PBS buffer (pH 7.4) and incubated in a shaker at 37 °C and 60 rpm for 2, 6, 8, 10, 12, and 14 days. Afterward, the samples were removed, rinsed with deionized water, freeze-dried, and weighed. W t Each gel group was tested in triplicate (n=3). The percentage of residual mass was calculated. The degradation performance of the gel can be expressed using the following formula: In the formula, Wt is the mass of the hydrogel freeze-dried scaffold at time t, and W0 is the initial mass of the hydrogel freeze-dried scaffold.

[0062] II. Experimental Results Degradation experiment results are as follows Figure 7 As shown, the 0.5% gel degrades more rapidly, exhibiting the steepest degradation curve and the highest degradation rate during the first 6 days, with a 50% mass loss by day 6. Subsequently, the degradation rate slows and tends to reach equilibrium. In contrast, the 1%, 2%, and 4% gels show similar degradation rates, with steeper degradation curves and lower degradation rates; the hydrogels lose approximately 10-18% of their mass by day 6. Therefore, under physiological conditions, 1%–4% lung extracellular matrix hydrogels possess better stability and are more conducive to their function in vivo.

[0063] Experimental Example 7: Leakage Sealing Efficiency of Extracellular Matrix Hydrogel for Lung Cells I. Experimental Methods 1. Experimental Grouping This experiment was divided into seven groups: α-cyanoacrylate gel group, fibrin gel group, and 0.5%, 1%, 1.5%, 2%, and 4% Lung-ma gel groups.

[0064] 2. Animal experiments 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. Airway pressure F0 was collected as the baseline lung pressure, and lung airtightness before and after sealing was assessed by airway pressure measurement. Subsequently, a midline thoracotomy was 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 isoflurane was dripped into the lesion site. 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. After stable breathing for 5 minutes, the postoperative airway pressure F1 was collected. The sealing efficiency of the gel can be calculated by the formula: In the formula, F1 is the postoperative airway pressure, and F0 is the initial airway pressure.

[0065] II. Experimental results The results are shown in Figure 8 . The 0.5% lung extracellular matrix hydrogel is difficult to stably seal the wound during lung respiration, and the sealing efficiency is poor. The 1%, 1.5%, 2%, and 4% lung extracellular matrix hydrogels have excellent lung air leakage sealing effect, and there is no significant difference with the clinically commonly used α-cyan acrylate gel and fibrin glue. Therefore, the 1%~4% lung extracellular matrix hydrogel is expected to be used for lung injury sealing to prevent air leakage and pneumothorax.

[0066] Experimental Example 6: Lung extracellular matrix hydrogel for lung injury repair I. Experimental method 1. Experimental grouping This experimental example is divided into six groups, namely, the sham operation group, the operation group, the α-cyan acrylate gel group, the fibrin glue group, and the 1% Lung-ma gel group and the 2% Lung-ma gel group.

[0067] 2. Animal experiment 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 / minute, positive end-expiratory pressure 2.2 cm H2O. Then, the median thoracotomy was performed to expose the lungs; a 1 cm incision was made on the surface of the lung to form an air leak. 100 of 1% or 2% lung extracellular matrix hydrogel was added to the wound site for repair, and the clinically commonly used lung sealant α-cyan acrylate gel (100 ) and fibrin glue (100 ) were used as controls. For the sham operation group, only the lungs were exposed without making an incision. For the operation group, after making an incision on the surface of the lung, no other treatment was performed.

[0068] The samples were taken at 7 days and 14 days after the operation, and immunohistochemical staining was performed to detect SFTPB (a marker of type II alveolar cells) and CD31 (a marker of vascular endothelial cells) to evaluate lung regeneration.

[0069] II. Experimental results 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.

[0070] 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.

[0071] 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.

[0072] Based on the above experiments on angiogenesis, stability, wound closure during lung respiration, and animal experiments on lung injury, this invention preferably uses 1-1.5% lung extracellular matrix hydrogel, which does not require the addition of growth factors, effectively prevents postoperative leakage, and effectively promotes angiogenesis.

[0073] It can be seen from the above examples and experimental examples that the lung tissue-derived extracellular matrix is grafted with methacrylate anhydride groups by screening process conditions and hydrogel formula, a lung extracellular matrix hydrogel capable of being rapidly photo-crosslinked is provided, the lung extracellular matrix hydrogel 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 the generation of blood vessels, promotes the regeneration of alveoli, and also significantly reduces the level of fibrosis. 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: 1-1.5 parts 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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