Lung extracellular matrix, preparation method and 3D printing biodegradable airway stent

Abstract

The invention discloses a lung extracellular matrix, a preparation method and a 3D printing biodegradable airway stent. The preparation method of the lung extracellular matrix comprises the following steps: freezing and thawing lung tissues, sequentially decellularizing SDS and TritonX-100, air-drying, crushing and freezing to obtain the lung extracellular matrix, and carrying out basic identification on the lung extracellular matrix by using a DNA determination and HE dyeing method. The biological ink is prepared from the prepared lung extracellular matrix, and then the novel airway stent with high biocompatibility and good mechanical property is successfully developed through the 3D biological printing technology.

Description

TECHNICAL FIELD

[0001] The present application relates to biodegradable scaffolds, in particular to a lung extracellular matrix, a preparation method and a 3D printed biodegradable airway scaffold. BACKGROUND

[0002] Central airway obstruction (CAO) refers to the airway stenosis of trachea, left and right main bronchus and right intermediate segment bronchus caused by various causes. The most common causes are benign and malignant tumors, tuberculosis, and post-intubation scar stenosis. The clinical manifestations can be cough, sputum, dyspnea, and even asphyxia death in severe cases. The current common treatment methods mainly include drug therapy, surgical operation, bronchoscopic interventional therapy, etc. However, in the long-term clinical practice, it is found that drug therapy has poor curative effect, and surgical operation cannot solve the problems such as long stenosis segment, special stenosis site or airway stenosis caused by advanced malignant tumor, and postoperative restenosis risk may still occur after surgical operation. Therefore, bronchoscopic interventional therapy provides more choices for patients with benign and malignant airway stenosis. Bronchoscopic interventional therapy methods include balloon dilatation, high-frequency electric knife, laser, cryotherapy, airway stent and other techniques. Airway stent is often used for patients who cannot maintain airway patency after various treatments, or as a temporary emergency airway stent when the patient has life-threatening conditions for later treatment. The appearance of airway stent is similar to a hollow tubular structure of normal physiological airway, which provides support to the airway wall by the mechanical properties of the stent material itself, thereby realizing the function of opening the airway. In addition to having certain mechanical properties, it also needs to have certain biocompatibility to reduce foreign body reaction and avoid airway restenosis after granulation tissue formation. Although the current highest usage rate is self-expandable metal stent, for patients with benign airway central stenosis, the incidence of related complications is high with the extension of implantation time, such as stent fracture, airway perforation, and difficult removal. Therefore, the US Food and Drug Administration (FDA) issued a warning in 2005 against the use of SEMS (Self-Expandable Metal Stents) in benign airway diseases. Clinicians believe that silicone stents have good biocompatibility, high elasticity and radial support force, and the biggest advantage is that they can be removed and have a lower restenosis rate than bare metal stents, so they are mostly preferred in such diseases. However, in long-term clinical practice, it is found that there are still related complications, such as stent displacement, stent restenosis, sputum retention, and granulation proliferation. The reason is mostly due to the difference between the stent material and the airway tissue and the current non-individualized stent size. In recent years, the research of new airway stents mainly includes the following aspects: drug-eluting stents, biodegradable stents, and individualized airway stents, but most of them are in the preclinical research stage, and the biocompatibility and mechanical properties cannot be perfectly coordinated. Therefore, we expect the emergence of an 'ideal airway stent', a stent with high biocompatibility, self-degradation, excellent mechanical properties, and individualization, which can solve the above problems.

[0003] In recent years, the concept of personalized medicine has been increasingly accepted, and 3D printing technology has been developing rapidly. It can customize the unique implantable device of the patient through CT scan data. In theory, the use of 3D printing to customize the patient's personalized airway stent should be able to significantly reduce stent migration, granuloma proliferation and other related problems. Decellularized extracellular matrix is one of the current research hotspots of natural biological materials. It stands out among various materials with excellent biocompatibility. It not only has the advantages of natural biocompatibility and low immunogenicity, but more importantly, it has cell adhesion surface receptors. Previous studies have used it as a coating solution on the surface of synthetic biomaterials, which can not only reduce inflammation and foreign body reactions to synthetic implantable medical devices, but also enhance cell adhesion and achieve revascularization.

[0004] Currently, dECM (decellularized Extracellular Matrix) has been studied as one of the components of bio-ink, but most of them are for the construction of organoids and the establishment of in vitro disease models. For airway diseases, most of them focus on tissue-engineered tracheal replacement, and there is no related research in the field of airway stents. SUMMARY

[0005] The present application aims to provide a lung extracellular matrix, a preparation method and a 3D printed biodegradable airway stent, which has better anti-migration ability and biocompatibility.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is:

[0007] A preparation method of a lung extracellular matrix, comprising the following steps:

[0008] S1, freeze-thawing lung tissue;

[0009] S2, slicing the freeze-thawed lung tissue, sequentially decellularizing with SDS and TritonX-100, air-drying, crushing and freezing to obtain a lung extracellular matrix, wherein pressure is applied to the lung tissue during the sequential decellularization process with SDS and TritonX-100, and preferably, a pressure pot is used to apply pressure to the lung tissue.

[0010] The application uses a freeze-thaw + sodium dodecyl sulfate (SDS) + polyoxyethylene tert-octylphenyl ether (Triton X-100) sequential slice immersion method to prepare a porcine lung decellularized extracellular matrix (dECM), and uses a DNA determination method and an HE staining method to perform basic identification on the dECM.

[0011] According to the embodiments of the application, the application can be further optimized, and the following is a technical solution formed after optimization:

[0012] In one preferred embodiment, the sequential decellularization steps of SDS and Triton X-100 specifically include soaking in 1-3% SDS for 12-24 hours, washing with deionized water, soaking in 1-3% Triton X-100 for 12-24 hours, washing with deionized water and soaking for 12-24 hours.

[0013] In one preferred embodiment, after the freezing, sterilization and pepsin treatment are further included, and the pepsin treatment includes adding the lung extracellular matrix and pepsin in an acetic acid solution and soaking, preferably, the concentration of the acetic acid solution is 0.2-1.5 mol / L, the volume ratio of the dECM and pepsin is (4-6):1, and the soaking time is 2-4 days.

[0014] In one preferred embodiment, the freezing and thawing temperature is -90 to -70℃, and the freezing and thawing time is 12-36 hours.

[0015] In one preferred embodiment, the thickness of the lung tissue slice is 1-2 mm.

[0016] In one preferred embodiment, the air-drying temperature is 30-40℃, and the freezing temperature is -90 to -70℃.

[0017] The application further discloses a lung extracellular matrix prepared by the preparation method of the lung extracellular matrix.

[0018] Preferably, the lung tissue needs to be removed of residual impurities and blood on the surface of lung cells before freezing and thawing.

[0019] Based on the same inventive concept, the application further discloses a 3D printing biodegradable airway stent prepared from the lung extracellular matrix, the 3D printing biodegradable airway stent has a hardness of 50-80HA, a Young's modulus of 0.1-0.4MPa, and a modulus less than 1MPa, and is non-toxic to cells; the 3D printing biodegradable airway stent has a hardness of 55-70HA and a Young's modulus of 0.2-0.3MPa.

[0020] The airway stent prepared by the application has a hardness of 50-80HA, while the hardness of a silicone airway stent material is 60-80A, which is close to that of a silicone stent. The Young's modulus of the stent is 0.1-0.4MPa, which is easy to deform, and can be placed by a bronchoscope. The modulus of a human trachea reported in previous studies is in the range of 1-20MPa, and the modulus in the application is less than 1MPa, so the mechanical properties of the stent are not incompatible with the natural tissue.

[0021] In one preferred embodiment, the lung extracellular matrix and the hydrogel are mixed to prepare a biological ink, and the 3D printing biodegradable airway stent is prepared by printing the biological ink.

[0022] In one preferred embodiment, the hydrogel comprises GelMA and ALG, preferably, the mass concentration of the GelMA is 8-12%, the mass concentration of the ALG is 1-3%, and the concentration of the lung extracellular matrix is 3-4%.

[0023] The stent prepared in the ratio range has better viscoelasticity, hardness and mechanical properties.

[0024] The application develops a new type of stent, which is prepared from a mixed biological ink of lung acellular extracellular matrix, Gelatin Methacryloyl (GelMA) and Alginate (ALG) with excellent biocompatibility, and is formed by 3D biological printing technology. Since the mixed biological ink based on the lung acellular extracellular matrix is used as a new type of biological ink, the prepared 3D printing airway stent can have better anti-migration ability and biocompatibility, which provides a new idea for the development of a new type of airway stent.

[0025] In the early stage of the application, the lung acellular extracellular matrix is successfully prepared by using the perfusion method combined with SDS (Sodium dodecyl sulfate) + TritonX-100 (polyethylene glycol octylphenyl ether), which proves that the method has little effect on the key proteins of the lung extracellular matrix and the ultrastructure of the tissue.

[0026] Compared with the prior art, the application has the following beneficial effects:

[0027] 1、The prepared porcine lung acellular extracellular matrix does not have obvious nuclear structure, and DNA determination shows that the concentration of the acellular group is significantly reduced (P<0.01);

[0028] 2、The dECM / GelMA / ALG hydrogel of different concentrations is prepared, and as the concentration of dECM increases, the porosity decreases and the degradation time is prolonged;

[0029] 3、The airway stent is printed by using the 3D bioprinting method, and the cytotoxicity experiment does not have obvious cytotoxicity (P>0.05), and the mechanical properties are similar to those of the silicone stent. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a schematic diagram of a slice pressure kettle soaking method in an embodiment of the present application;

[0031] Figure 2 is a schematic diagram of general changes of the acellular lung tissue in an embodiment of the present application, Figure 2 Figure A is a lung without decellularization treatment; Figure B is after SDS soaking for 6 hours; Figure C is after SDS soaking for 12 hours; Figure D is after TritonX-100 soaking for 6 hours; and Figure E is after all reagents are soaked;

[0032] Figure 3 is a comparison diagram of HE staining and DNA content of the control group and the acellular group in an embodiment of the present application, Figure 3 Figure A is HE staining of the control group and the acellular group; and Figure B is a comparison of DNA content of the control group and the acellular group;

[0033] Figure 4 is a schematic diagram of GelMA / ALG mixed bio-ink gel in an embodiment of the present application, Figure 4-1 is 20% GelMA+4% ALG hydrogel (group 2 in Table 3-1); Figure 4-2 is 20% GelMA+2% ALG hydrogel (group 3 in Table 3-1); Figure 4-3 is 20% GelMA hydrogel (group 4 in Table 3-1); Figure 4-4 is 10% GelMA+4% ALG hydrogel (group 5 in Table 3-1);

[0034] Figure 5 is a scanning electron microscope and porosity of GelMA / ALG / dECM hydrogel of different concentrations, Figure 5 (left): Figure A is 10% G / 2% A hydrogel; Figure B is 3% d / 10% G / 2% A hydrogel; Figure C: 4% d / 10% G / 2% A hydrogel; and Figure D: 5% d / 10% G / 2% A hydrogel. Figure 5(Right): Column chart of porosity of each group;

[0035] Figure 6 Figure 6 is a schematic diagram of in vitro swelling and degradation curves of each group of gels;

[0036] Figure 7 Figure 7 is a schematic diagram of dECM / GelMA / ALG hydrogel airway scaffold extrusion experiment, Figures A-C are comparative diagrams of mixed hydrogel (10%G / 2%A) airway scaffold before and after straight blood vessel clamp extrusion; Figures D-F are comparative diagrams of mixed hydrogel (3%d / 10%G / 2%A) airway scaffold before and after straight blood vessel clamp extrusion;

[0037] Figure 8 Figure 8 is a mechanical property test of dECM / GelMA / ALG hydrogel airway scaffold, Figures A and B are schematic diagrams of hydrogel standard sample for tensile instrument test; Figure C is a stress-strain curve of 10%G / 2%A hydrogel. Figure D is a stress-strain curve of 3%d / 10%G / 2%A hydrogel;

[0038] Figure 9 Figure 9 is a schematic diagram of cell toxicity experiment of each group of scaffolds. DETAILED DESCRIPTION

[0039] The present application will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0040] Table 1 English abbreviation explanation

[0041]

[0042]

[0043] 1. Research materials

[0044] Pig lungs were purchased from the local market, and human normal bronchial epithelial cells (Beas-2B) were a cell line preserved in the Central Laboratory of Xiangya Third Hospital, Central South University.

[0045] 2. Experimental reagents and instruments

[0046] The main reagents used in the experiment are shown in Table 2-1; the main experimental instruments are shown in Table 2-2.

[0047] Table 2-1 Main experimental reagents

[0048]

[0049]

[0050] Table 2-2 Main materials and instruments

[0051]

[0052]

[0053] 3 Preparation and identification of lung acellular extracellular matrix

[0054] 3.1 Acellularization by sequential soaking

[0055] The pig lung acellular extracellular matrix of this example was prepared by sequential soaking of freeze-thawing, SDS + Triton X-100. The general experimental procedure is as follows:

[0056] 1) Washing: The entire trachea of the pig lung was perfused with tap water to remove surface impurities and as much residual blood as possible;

[0057] 2) Freezing: -80°C for 24 hours;

[0058] 3) Slicing: Remove the thicker tracheal part, and the remaining lung tissue is cut with a slicer, controlling the thickness to be 1-2 mm;

[0059] 4) Soaking: Sequential soaking at room temperature, the soaking order is: 1% SDS soaking for 12 hours - deionized water washing - 1% Triton X-100 soaking for 12 hours - deionized water washing and soaking for 12 hours;

[0060] 5) Air drying and crushing: After air drying at 37°C, place it in a crusher for crushing;

[0061] 6) Cryopreservation: -80°C freezing storage.

[0062] 3.2 Evaluation of pig lung acellular extracellular matrix

[0063] 3.2.1 Gross observation

[0064] 3.2.2 HE staining

[0065] Preparation of lung tissue paraffin sections

[0066] 1) Sampling: Take an appropriate size of fresh lung tissue and acellular lung tissue, soak them in 4% paraformaldehyde, fix them for more than 1 day, and then label them well, then place them in a dehydration box;

[0067] 2) Dehydration and wax immersion: Place the dehydration box in a basket, then place it in a dehydration machine for gradient alcohol dehydration. After dehydration, immerse it in paraffin for the next procedure;

[0068] 3) Embedding: First, pour the melted paraffin into the embedding mold, then gently pick up the tissue block after the wax immersion with a heated curved blunt forceps, mark the embedding surface in time, then bury it in the melted paraffin. Wait for a while, and after the surface is roughly solidified, place it in -20°C for accelerated solidification. After solidification, pick it up with forceps and carefully remove the excess paraffin around the tissue block with a blade and trim it into a regular square as much as possible;

[0069] 4) Sectioning: Place the trimmed wax block on the clamp table of the microtome, cut it into thin sections about 4 μm thick, and then store it at room temperature for later use;

[0070] 5) Mounting: Place the sections in a water bath at about 44°C, lay them flat on the liquid surface, gently pick them up with forceps and attach them to the appropriate position on the glass slide, and then bake the sections in a 60°C oven for 2 hours.

[0071] HE staining of lung tissue sections

[0072] 1) Paraffin section deparaffinization and hydration: Place the control and experimental slides in turn in xylene for 20 minutes, 3 times, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and distilled water for 5 minutes.

[0073] 2) Hematoxylin staining: Place the slides in hematoxylin reagent for about 5 minutes, rinse with tap water, then differentiate, quickly wash with water, and return to blue with PBS.

[0074] 3) Eosin staining: Place the slides in eosin staining solution for 2 minutes.

[0075] 4) Dehydration and mounting: Dehydrate the slides in gradient alcohol and xylene again, and mount them with neutral balsam;

[0076] 5) Microscopic observation.

[0077] 3.2.3 DNA content and DNA electrophoresis

[0078] According to the manufacturer's instructions, use the universal column type genomic DNA extraction kit to quantify the DNA content of the decellularized tissue. The specific process is as follows:

[0079] DNA extraction:

[0080] 1) Place 25 mg of control and experimental samples in a homogenizer, add 80 μL of Buffer GTL, grind thoroughly, then pour into a centrifuge tube, and add 100 μL of Buffer GTL;

[0081] 2) Add 20 μL Proteinase K. Then mix the sample thoroughly with vortex shaker. Place in 56°C water bath until the tissue is completely lysed, and invert or shake the centrifuge tube every few hours to disperse the sample;

[0082] 3) Add 200 μl Buffer GL, then mix the sample thoroughly with vortex shaker, place in 70°C water bath for 10 minutes, after a brief centrifugation, add 200 μl absolute ethanol to the centrifuge tube, and vortex shaker thoroughly;

[0083] 4) Briefly centrifuge, and pour the resulting solution into the Spin Columns DM that have been loaded into the collection tubes. Centrifuge at 12,000 rpm for 1 minute, and then discard the waste and reposition the Spin Columns;

[0084] 5) Add 500 μl Buffer GW1 to the Spin Columns, and centrifuge at 12,000 rpm for 1 minute, and then discard the waste and reposition the Spin Columns;

[0085] 6) Add 500 μl Buffer GW2 to the Spin Columns, and centrifuge at 12,000 rpm for 1 minute, and then discard the waste and reposition the Spin Columns;

[0086] 7) Then centrifuge at 12,000 rpm for 2 minutes, and discard the waste. Allow the Spin Columns to air dry;

[0087] 8) Place the Spin Columns in a new centrifuge tube, and add 50-200 μl sterile water to the middle of the Spin Columns. Allow the Spin Columns to stand at room temperature for 2-5 minutes, and then centrifuge at 12,000 rpm for 1 minute. The resulting DNA solution can be stored at -20°C.

[0088] DNA determination: Adjust the microspectrophotometer to zero with enzyme-free water, and place 2 μL of the DNA solution on the test platform. Measure the absorbance at 260 nm and 280 nm, respectively. The absorbance is ideal in the range of 0.2-1.2. Calculate the concentration.

[0089] 4 Preparation of biological ink

[0090] 4.1 Decellularized extracellular matrix pretreatment

[0091] 1) Sterilization: soak the dECM in hydrogen peroxide for 4-6 hours, and irradiate it with ultraviolet light for more than 6 hours;

[0092] 2) Pepsin treatment: place the dECM and pepsin (volume ratio of 5:1) in 0.5 mol / L acetic acid solution, and soak them at room temperature for 3 days until the dECM is completely dissolved;

[0093] 3) Neutralization: adjust the pH of the dECM solution to 7.3-7.4 with 1 mol / L NAOH solution;

[0094] 4) Osmotic pressure adjustment: mix the dECM solution and 10X PBS at a volume ratio of 10:1 to balance the osmotic pressure;

[0095] 5) Centrifugation: filter the undigested part;

[0096] 6) Freeze-drying: freeze-drying treatment;

[0097] 7) Storage: store at 4°C.

[0098] 4.2 GelMA and photoinitiator preparation

[0099] 4.2.1 Preparation of 0.25% (w / v) initiator standard solution

[0100] 1) Take 20 ml PBS and add it to a brown bottle containing initiator LAP (containing 0.05 g LAP);

[0101] 2) Heat to dissolve in a 40-50°C water bath for 15 minutes, with several oscillations during the process.

[0102] 4.2.2 Preparation of GelMA solution

[0103] 1) Take the required mass of GelMA and place it in a centrifuge tube;

[0104] 2) Take the appropriate amount of initiator standard solution and add it to the above centrifuge tube, oscillate to fully soak;

[0105] 3) Heat to dissolve in a 60-70°C water bath for 20-30 minutes, with several oscillations during the process. Standby.

[0106] 4.3 Preparation of alginate (ALG)

[0107] 1) Take the required mass of alginate and place it in a beaker;

[0108] 2) First, add alginate to deionized water in a certain proportion, heat in a 70°C constant temperature water bath, and stir while heating;

[0109] 3) Place and defoam for standby use.

[0110] 4.4 Bio-ink proportioning and hydrogel preparation, performance evaluation

[0111] The 20% / 10% GelMA solution (w / v, g / ml) and 4% / 2% alginate solution (w / v, g / ml) were mixed thoroughly at 1:1 (v:v), perfused into a cylindrical solid gel, and then gelled by irradiation at 405 nm for about 30 seconds. Subsequently, a weight of 50 g was applied for 30 seconds to observe whether the gel deformed or cracked after the pressure was removed. The optimal concentration ratio of GelMA to ALG was determined. Then, 3%, 4%, and 5% of dECM lyophilized powder (w / v, g / ml) was added to prepare three different dECM / GelMA / ALG bio-ink hydrogels A, B, and C, and their properties were tested.

[0112] 4.4.1 Observation of gel microstructure

[0113] 1) Place the sample in 2.5% glutaraldehyde for 6-12 hours;

[0114] 2) Discard the fixing solution and rinse the sample with 0.1 mol / L pH 7.0 phosphate buffer for three times, each time controlled within 15 minutes;

[0115] 3) Fix the sample with 1% osmium acid solution for 1-2 hours;

[0116] 4) Discard the previous liquid and rinse the sample again with 0.1 mol / L pH 7.0 phosphate buffer for three times, each time controlled within 15 minutes;

[0117] 5) Gradient alcohol dehydration treatment: 30% ethanol solution for 15 minutes, 50% ethanol solution for 15 minutes, 70% ethanol solution for 15 minutes, 80% ethanol solution for 15 minutes, 90% ethanol solution for 15 minutes, 95% ethanol solution for 15 minutes, 100% ethanol solution for 20 minutes and repeated twice;

[0118] 6) Treat the sample with a mixture of acetic acid and isoamyl acetate for 30 minutes;

[0119] 7) Discard the mixture from the previous step and treat the sample with pure isoamyl acetate for 1 hour, carbon dioxide critical point drying treatment;

[0120] 8) Coating, scanning electron microscope observation.

[0121] 4.4.2 Gel water absorption rate and in vitro degradation rate

[0122] 9) After the scaffold is dried with filter paper, weigh it (W0), then immerse it in complete culture medium, place it in a 37°C constant temperature water bath, take out the gel at different times, dry the excess water with filter paper, weigh it again (Wt), and calculate the gel swelling rate and in vitro degradation rate.

[0123]

[0124] 5 3D-printed airway stent

[0125] The stent was printed using the biomaker system of Beijing Shangpu Biotechnology Co., Ltd. The preset printing parameters were: nozzle temperature: 21°C, platform temperature: room temperature, printing speed: 5mm / s, starting layer speed: 5mm / s, empty running speed: 20mm / s, extrusion speed: 1mm / s, layer height: 0.2mm. The stent was placed in a 2% calcium chloride solution for ion crosslinking immediately after printing, and then washed with PBS for 3 times. 3 3 3 3

[0126] 5.1 General observation of airway stent

[0127] 5.2 Evaluation of mechanical properties of stent

[0128] Hardness test: The hardness of the material was tested using a pointer-type Shore hardness tester. The method was performed according to GB / T531.1-2008.

[0129] Tensile property test: Standard mechanical sample was prepared, and the method was performed according to GB / T13460-2008 using a universal material testing machine.

[0130] 5.3 Evaluation of biocompatibility of stent

[0131] 5.3.1 Cell culture

[0132] The culture medium was prepared by configuring 10% fetal bovine serum and 1% penicillin-streptomycin-amphotericin B in DMEM. Beas-2B cells were cultured at 37°C and 5% CO2. The culture medium was replaced every 2 days. When the cells covered 90% of the culture bottle, they were digested with 1ml of 0.25% trypsin, and then cultured in 3 new culture bottles.

[0133] 5.3.2 Cytotoxicity test

[0134] Each stent was weighed after drying, immersed in complete culture medium at a ratio of 0.2g / mL, and placed in a 37°C environment for 24 hours of extraction, then placed in a 4°C refrigerator for standby. Take Beas-2B cells, the cell density is 1X10 4 ​​​​ / mL, inoculated in 96-well plates, 100 μL of cell suspension per well, 100 μL of scaffold extraction solution was added after incubation for 24 hours after adhering to the complete culture medium, and CCK-8 detection was performed at the same time period after 24 hours and 48 hours; 10 μL of CCK-8 was added according to 10% (V / V) per well, and the cells were further incubated at 37°C for 1 hour, and the absorbance at 450 nm was measured by an enzyme-labeled instrument.

[0135]

[0136] 6Statistical methods

[0137] The measurement data were expressed as mean ± standard deviation (SD). Independent sample t-test was used to compare the data between two groups. P<0.05 indicated that there was a significant difference between groups. All statistical analyses were performed using GraphPad Prism 9.4.1 software.

[0138] 7Results

[0139] 7.1Preparation and evaluation of porcine lung acellular extracellular matrix

[0140] The sliced porcine lung tissue was decellularized by freeze-thawing + SDS + Triton X-100 sequential immersion method, and was spread as much as possible on the surface of the reagent solution, and a certain pressure was applied to it by pressing the pot to make it fully contact with the solution, as shown in Figure 1 .

[0141] 7.2Observation of lung acellular tissue gross specimen

[0142] Morphological changes: Fresh porcine lung tissue structure is tight and elastic, most of which is suspended on the surface of the immersion solution. During the decellularization process, the tissue thickness becomes thinner than before, the volume increases slightly, and part of the matrix can be seen in the trachea.

[0143] Color change: After the fresh porcine lung tissue was washed and stored at -80°C, the color became pale, the central part turned into ivory white after 6 hours of SDS immersion, and the edge part or thinner part became translucent, as shown in Figure 2 -B. The transparent part increased significantly after 12 hours, as shown in Figure 2 -C. After Triton X-100 immersion, it was basically transparent, and small bubbles could be seen inside, as shown in Figure 2 -D. After the completion of the immersion, the porcine lung acellular tissue was translucent, as shown in Figure 2 -E.

[0144] 7.3Comparison of HE staining and DNA content of porcine lung acellular tissue and fresh lung tissue

[0145] Control group: visible alveoli, airway and vascular structure, red-stained fibrous tissue mixed with capillaries, no obvious red blood cell residues were found in the capillaries, and a large number of blue-stained cell nuclei were clearly visible. Figure 3 -A.

[0146] After decellularization by soaking method, the DNA concentration of untreated fresh lung tissue and decellularized lung tissue measured by universal columnar genomic DNA extraction kit is as follows: untreated fresh lung tissue: 303.58 ± 29.44; dECM tissue DNA concentration: 35.21 ± 4.85 (unit: ng / mg dry weight tissue, n = 3). Compared with the DNA content of the two groups, it was found that the DNA concentration of the decellularized group was significantly lower than that of the untreated group, with statistical difference (P < 0.001), as shown in Figure 3 -B. The obtained dECM meets the standard: dsDNA < 50 ng / mg (dry weight), and no obvious cell nucleus is found by HE staining.

[0147] 7.4 Preparation of dECM lyophilized powder and determination of the optimal bio-ink ratio

[0148] 7.4.1 Preparation of dECM lyophilized powder The dECM after 72 hours of pepsin treatment is a mixed homogeneous white turbid liquid, in which flocculation can be occasionally seen. After freeze-drying treatment, it can be white powder, occasionally white flaky material.

[0149] 7.4.2 Determination of the ratio of GelMA / ALG hydrogel bio-ink

[0150] At present, most studies control the concentration of GelMA and ALG in the bio-ink combination in the range of 5%-10% and 2%-4% respectively. Referring to the relevant literature, we configured various concentrations of GelMA / ALG bio-ink (as shown in Table 3-1), and compared the elastic properties of each group of hydrogels after crosslinking under the pressure of 50g, as shown in Figure 4 Through the approximate evaluation of each group of gels, it was found that the appearance of the fourth group, i.e. the pure GelMA group, was transparent, and the hardness and elasticity were better, but the touch was the driest. In the case of the same concentration of GelMA, with the increase of the ALG concentration, the elasticity and hardness of the gel were obviously improved. Reducing the concentration of GelMA, the hardness and elasticity were obviously worse. Considering that the scaffold needs to have certain water absorption to keep it moist and good strength and elasticity, 20% GelMA and 4% ALG are selected to mix according to the volume ratio of 1:1, i.e. the final concentration of 10% GelMA / 2% ALG is selected as the final bio-ink ratio.

[0151] Table 3-1 GelMA / ALG different concentration ratio (table according to the order of ALG concentration from low to high)

[0152]

[0153] Table 3-1 GelMA / ALG different concentration ratio (continued)

[0154]

[0155] 7.5 Mixed hydrogel evaluation

[0156] 7.5.1 Macroscopic and microscopic structure

[0157] Referring to the above experimental results, we finally determined and configured the mixed final concentration of 10% GelMA / 2% ALG bio-ink, and added dECM freeze-dried powder according to 3%, 4%, 5% proportion (W / V, g / ml). After adding the freeze-dried powder, mix appropriately, and there is no obvious stratification of the liquid. Three kinds of dECM / GelMA / ALG hydrogel bio-ink with different concentrations were prepared. After 405 nm light irradiation, the control group and other groups were roughly the same in color, and the experimental group was roughly uniform in appearance. The appearance of the gel was opaque, and a small amount of flocculent material was visible inside. After photocuring, there was no flow, and there was a certain elasticity. As shown in Figure 5 , the electron microscope showed that the white fibrin monomer was attached to the three-dimensional network structure of the gel. The image j software was used to analyze the porosity of the electron microscope image. The porosity of 10% G / 2% A hydrogel was 21.64%, the porosity of 3% d / 10% G / 2% A hydrogel was 16.79%, the porosity of 4% d / 10% G / 2% A hydrogel was 9.85%, and the porosity of 5% d / 10% G / 2% A hydrogel was 8.85%. It shows that with the increase of dECM concentration, the gel porosity can be gradually reduced.

[0158] 7.5.2 Water absorption and in vitro degradation rate of mixed hydrogel

[0159] Considering that the mixed hydrogel needs to be placed in the body in the later stage, the material itself will interact in the body fluid environment in the body. In the ideal state, it can maintain its basic form for a certain period of time, and then it can degrade itself. Therefore, it is placed in complete culture medium, and the temperature is controlled at 37 degrees Celsius to simulate the in vivo environment and evaluate its in vitro degradation. As shown in Figure 6As shown: most of the polymer hydrogel materials have swelling properties, before 72 hours, with the increase of dEMC concentration, the swelling rate also gradually increased, after 72 hours, each group began to gradually degrade. Take 2 weeks as the observation period, it is found that the 4% dECM / 10% GelMA / 2% ALG group and the 3% dECM / 10% GelMA / 2% ALG group can slow down the degradation rate compared with the group without dECM. In addition, with the increase of dECM concentration, the degradation rate gradually slows down. But considering that the higher the dECM concentration, the higher the risk of pollution later, and the shorter the time of maintaining the mechanical properties of the scaffold, the final concentration of the scaffold bio-ink is determined as 3% d / 10% G / 2% A.

[0160] 7.6 Preparation and mechanical evaluation of 3D printed biodegradable airway scaffold of dECM / GelMA / ALG

[0161] 7.6.1 3D printed airway scaffold

[0162] The mixed bio-ink is printed by extrusion molding. The scaffold is 14mm in diameter, 1mm in wall thickness, and 7mm in height. The mixed hydrogel (10% G / 2% A) airway scaffold is white and translucent, while the scaffold with 3% is opaque and milky white. Both scaffolds have smooth and continuous surfaces and consistent thickness.

[0163] 7.6.2 Mechanical properties of 3D printed scaffold

[0164] 7.6.2.1 Dynamic changes of the scaffold

[0165] The airway scaffold is placed in the airway with the help of rigid bronchoscopy or flexible bronchoscopy. The present application applies radial pressure to the 3D printed biodegradable airway scaffold, as shown: Figure 7 After removing the pressure, both groups can recover to the original state, and the elasticity is good. Therefore, it is possible to place it through a hard bronchoscope.

[0166] 7.6.2.2 Mechanical properties of the scaffold

[0167] For the two groups of samples, the universal material testing machine measures the elongation and draws the stress-strain curve to evaluate the Young's modulus and the breaking elongation. The pointer type Shore hardness tester measures the hardness of the 10% G / 2% A hydrogel airway scaffold, and the Shore hardness is 59HA, as shown in Figure 8The stress strain curve of C shows the tensile properties: peak stress: 3.0 MPa; strain at break: 1069.238%; elongation at break: 1064%. The calculated Young's modulus: 0.777 MPa. The Shore hardness of 3% d / 10% G / 2% A hydrogel airway stent: 56.5 HA; peak stress: 2.3 MPa; strain at break: 1042.51%; elongation at break: 1068%; Young's modulus: 0.298 MPa. The hardness of the silicone airway stent material is 60-80 HA

[30] , and the elastic modulus is about 0.3 MPa

[31] The hydrogel airway stent of the present application is similar to it, and in addition, its high elongation at break proves that this stent is very tough.

[0168] 7.6.3 Biocompatibility of the stent

[0169] In order to determine the biocompatibility of the new airway stent, the stent was extracted with complete culture medium as the extraction liquid at 37°C for 24 hours, and then the material extraction liquid was co-cultured with Beas-2B cells for 24 hours and 48 hours. As shown in Figure 9 we found that there was no significant difference in cell activity between the group without 3% dECM stent and the group with 3% dECM stent at the same time period (P>0.05).

[0170] 8 Discussion

[0171] Current research on lung dECM is mostly focused on the construction of disease models, organ chip research, cell culture, organoid construction, etc. As for the airway stent field, it is mainly to construct tissue engineering trachea by using cartilage dECM, collagen and other materials, or to directly decellularize the trachea as a substitute for tissue engineering trachea. However, there is still a lot of research space for lung tissue extracellular matrix, which accounts for 50% of the non-alveolar components of the entire lung tissue, and there is no relevant research in the airway stent field. This invention innovatively uses pig lungs, which are highly similar in anatomical structure to humans and have a wide source, as one of the materials for biological ink after decellularization, to realize a new type of airway stent with high biocompatibility and biodegradability by using 3D printing technology.

[0172] Nowadays, the decellularization technology is increasingly mature, and the current decellularization methods can be roughly divided into three categories: physical, chemical or biological. The choice of these three methods depends on the cell type, the density, thickness and lipid content of the tissue. If necessary, multiple methods can be combined to improve the efficiency of decellularization. The freeze-thaw method is used as the first step of decellularization in the present invention, but the effect of this method alone is not good enough, so the sequential decellularization method of freeze-thaw, SDS and TritonX-100 is adopted in the present invention. The order of TritonX-100 at the end can help remove the residual SDS in the tissue. Because different organs have different characteristics, the way of decellularization is also different, which generally includes perfusion method, pressure gradient method, supercritical fluid, soaking, stirring and other methods. At present, the perfusion method is mostly used for decellularization of large organs or organs with vascular systems. Although the present invention is also aimed at the lung, which is an organ with a vascular system, the ultimate goal is to make it one of the formulas of bio-ink, only the basic components of ECM need to be maintained, not the advanced structure. The slice soaking method can make the tissue have a larger surface area, and may have a faster and higher yield decellularization process. In addition, lung tissue is different from other organs, as a gas-containing tissue, it often floats on the surface of the liquid. The present invention innovatively uses a pressure kettle to ensure that the lung tissue is always inside the liquid by applying a certain pressure to it, which is conducive to further improving the efficiency of decellularization. In summary, the present invention adopts the slice immersion method combined with freeze-thaw and SDS+TritonX-100 decellularization. From the general appearance of the extracellular matrix, it can be clearly seen that after sequential soaking with reagents, the lung tissue changes from pale to translucent, and even the fine structure can be clearly seen. By HE staining and DNA determination, the present invention proves that most of the nucleated cells have been successfully removed, effectively avoiding the related immune system response caused by the xenogeneic biological material, and providing conditions for the subsequent preparation of bio-ink material. In addition, the present invention successfully realizes the high efficiency, convenience and yield of decellularized extracellular matrix production, and lays a certain foundation for future mass production.

[0173] The natural lung tissue-derived dECM cannot be directly used for bio-printing, and needs to be prepared into a pre-gel solution or powder for subsequent mixing with other materials after being digested by pepsin. Although the dECM powder after pepsin pretreatment of human dECM is convenient to mix, its mechanical properties will also be lost to a certain extent. Most studies have shown that the dECM gelation time is long and the crosslinking speed is slow. Therefore, it is difficult to construct specific shapes with single dECM in the printing process, and it is a good strategy to adopt multi-component bio-ink to balance the advantages and disadvantages of different materials, so as to configure a bio-ink that meets the requirements of printing, mechanics and other related requirements.

[0174] The application successfully prepares a shaped hydrogel by a mold pouring method, light curing combined with ion crosslinking after a porcine lung extracellular matrix (dECM) is frozen, crushed, and pepsin-digested to convert it into a hydrogel precursor solution, and a GelMA / ALG / dECM bio-ink is configured. The optimal ratio of GelMA and ALG is determined through different concentration ratio experiments. It is found that the combination of 10% GelMA and 2% ALG has better viscoelasticity and hardness. The addition of lung dECM provides receptors for cell adhesion, and the application realizes the configuration of a biocompatible degradable bio-ink. Both GelMA and ALG are porous materials, and when the microstructure of the mixed hydrogel is observed, it is found that the pore size decreases with the increase of the dECM concentration, which is consistent with the findings of Agrawal P. This feature facilitates the determination of the dECM concentration in the later stage, enabling the scaffold to load cells and achieve functionalization. In addition, the materials used to prepare the scaffold in the application are biodegradable materials, and their degradation rate is generally related to the concentration of the bio-ink, the crosslinking mechanism, and the in-vivo and in-vitro environment. Through in-vitro degradation experiments of mixed hydrogels with different ratios, it is found that the mixed hydrogel prolongs the degradation time with the increase of the dECM concentration. The degradation product will eventually completely dissolve in the complete culture medium, and the gel remains soft during degradation, which also indicates that the degradation product will not cause damage to the airway wall. As for the degradation time, the 3% dECM and 4% dECM groups all have a longer degradation time compared with the group without adding dECM. However, the uncontrollable degradation rate and the change in degradation mechanics are difficult problems that need to be solved in the future. The mixed bio-ink in the application has high viscosity and shear thinning ability, and is especially suitable for extrusion type 3D printing, which is a simple and low-cost method. The bio-ink is extruded through a pressure system, and the structure we need is constructed by layer-by-layer deposition, while being supplemented by light curing. Although the precision needs to be improved, it is sufficient for the preliminary design of the scaffold in the application. The appearance of the scaffold printed in the application is similar to the silicone scaffold on the market, and the material leaching solution has been confirmed to have no toxicity to cells. Combined with related mechanical experiments, it is found that the new airway scaffold of 3% dECM / 10% GelMA / 2% ALG has a hardness of 56.5 HA, which is close to the hardness of the silicone airway scaffold material in the range of 60-80 A. The Young's modulus of this scaffold is 0.298 MPa, which is easy to deform and can be placed by a bronchoscope. The modulus of the human trachea reported in previous studies is in the range of 1-20 MPa, and the modulus in the application is less than 1 MPa, so it does not exhibit mechanical properties incompatible with natural tissues.The dECM concentration is now about 3-5% low concentration, because one of the main components of dECM, collagen, has a triple helix structure and intermolecular forces, which may change the cross-linking effect of the mixed hydrogel, so that too high dECM concentration will affect the mechanical properties of the mixed hydrogel.

[0175] After biological and mechanical performance evaluation, the airway stent prepared by the application has good elasticity and toughness, and no cytotoxicity, and can self-degrade within a certain period of time.

[0176] The above-mentioned examples should be understood as being only for more clearly illustrating the present application, but not for limiting the scope of the present application, and after reading the present application, the modifications of various equivalent forms of the present examples by those skilled in the art all fall within the scope defined by the appended claims of the present application.

Claims

1. A method of preparing a lung extracellular matrix, characterized by, The method comprises the following steps: S1, freezing and thawing lung tissue; S2, slicing, SDS and TritonX-100 sequential cell removal, air drying, crushing and freezing of the frozen and thawed lung tissue to obtain lung extracellular matrix, and pressure is applied to the lung tissue during the SDS and TritonX-100 sequential cell removal process, preferably, a pressure pot is used to apply pressure to the lung tissue.

2. The method of claim 1, wherein the lung extracellular matrix is prepared from a lung tissue selected from the group consisting of lung, lung cancer, and lung tumor tissue. The SDS and TritonX-100 sequential cell removal step specifically comprises soaking in 1-3% SDS for 12-24 hours, washing with deionized water, soaking in 1-3% TritonX-100 for 12-24 hours, and washing with deionized water and soaking for 12-24 hours. ​ 3. The method for preparing lung extracellular matrix according to claim 1, characterized in that, After freezing, sterilization and pepsin treatment are further included, the pepsin treatment comprises adding lung extracellular matrix and pepsin in an acetic acid solution and soaking, preferably, the concentration of the acetic acid solution is 0.2-1.5 mol / L, the volume ratio of the dECM and pepsin is (4-6):1, and the soaking time is 2-4 days.

4. The method of preparing pulmonary extracellular matrix of any one of claims 1-3, wherein, The freezing and thawing temperature is-90 to-70℃, and the freezing and thawing time is 12-36 hours.

5. The method for preparing lung extracellular matrix according to any one of claims 1-3, characterized in that, The thickness of the lung tissue slice is 1-2 mm.

6. The method of claim 1-3, wherein the lung extracellular matrix is prepared by, The air drying temperature is 30-40℃, and the freezing temperature is-90 to-70℃.

7. A lung extracellular matrix, characterized in that, The lung extracellular matrix is prepared by the method of any one of claims 1-6.

8. A 3D printed biodegradable airway scaffold made using the lung extracellular matrix of claim 7. The 3D printed biodegradable airway stent has a hardness of 50-80 HA, a Young's modulus of 0.1-0.4 MPa, and a modulus less than 1 MPa, and is non-toxic to cells; the 3D printed biodegradable airway stent has a hardness of 55-70 HA, a Young's modulus of 0.2-0.3 MPa.

9. The 3D printed biodegradable airway stent of claim 8, wherein, The lung extracellular matrix and hydrogel are mixed to prepare a biological ink, and the 3D printed biodegradable airway stent is printed using the biological ink.

10. The 3D printed biodegradable airway stent of claim 9, wherein, The hydrogel comprises GelMA and ALG, preferably, the mass concentration of the GelMA is 8-12%, the mass concentration of the ALG is 1-3%, and the concentration of the lung extracellular matrix is 3-4%.

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