Bamboo fungus-based biological material, preparation method and application of bamboo fungus-based biological material in digestive tract perforation

Abstract

The invention discloses a bamboo fungus-based biological material, a preparation method and application of the bamboo fungus-based biological material in digestive tract perforation, and the bamboo fungus-based biological material is obtained by decellularizing bamboo fungus stipe and then drying, and keeps a natural three-dimensional porous morphology structure. The material has good compression elasticity, hemostatic property and biocompatibility, and animal experiments prove that the plugging device formed by curling the material has good plugging effect and repair promoting effect on gastrointestinal perforation.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, relates to natural source biomaterials, and specifically relates to a bamboo fungus-based biomaterial, a preparation method and use thereof in digestive tract perforation. Background Art

[0002] Digestive tract perforation is a serious gastrointestinal disease. Due to the full-thickness damage of the digestive tract wall tissue, gastrointestinal contents are prone to overflow into the abdominal cavity and cause infection, leading to serious consequences and high mortality. Traditional treatments for gastrointestinal perforation include open surgery and laparoscopic minimally invasive surgery closure, which may still cause leakage or surgical sutures to cause granulomas. At present, there have been some reports that materials are made into rods or dumbbells as gastric occluders, relying on the expansion of the material to achieve the closure of the perforation. Patent document CN116196046A discloses a gastric perforation repair device and a mold for processing a gastric perforation repair device, wherein the gastric perforation repair device has a first state and a second state, and the gastric perforation repair device includes a connector, a first sealing body and a second sealing body. The first sealing body and the second sealing body in the first state can converge in the direction close to the first axis to be placed in the surgical catheter, so that the gastric perforation repair device of the present application can be placed in the surgical catheter, thereby making it easy to be used in laparoscopic minimally invasive surgery. In the second state, the first sealing body is unfolded and sealed on the inner wall of the stomach to cover the gastric perforation, so that gastric juice cannot flow out through the gastric perforation. The second sealing body is unfolded and connected to the outer wall of the stomach so that the gastric perforation repair device can be stably fixed to the stomach wall. The materials used in the repair device have proteins and stem cell exosomes. However, this repair device is prefabricated with a mold and has a fixed size. It is impossible to flexibly adjust the diameter of the repair device and the sealing tension on the perforation wall according to the actual perforation situation. In addition, due to the presence of immunogenicity, the tissue compatibility of the protein material with the patient's digestive tract wall layer is also uncertain, which may lead to poor tissue repair at the perforation. Since the digestive tract perforation occluder has high requirements on the mechanical properties and biocompatibility of the material, new materials and perforation occluders are still under development. Summary of the invention

[0003] In view of this, one of the purposes of the present invention is to provide a bamboo fungus-based biomaterial having good mechanical properties that are more compatible with soft tissues, excellent biocompatibility, and good sealing properties for perforations.

[0004] The technical solution is as follows:

[0005] A bamboo fungus-based biomaterial is obtained by decellularizing the bamboo fungus stalk and then drying it, and maintains a natural three-dimensional porous morphology structure.

[0006] In one embodiment, the decellularization process of the above-mentioned bamboo fungus-based biomaterial is as follows: remove the cap and stipe from fresh red-stemmed bamboo fungus, retain the stipe, and rinse it clean; then immerse the stipe in a surfactant solution to remove cells; then remove the residual surfactant, wash and dry it to obtain the decellularized bamboo fungus stipe.

[0007] The second object of the present invention is to provide a method for preparing bamboo fungus-based biomaterials. The technical solution is:

[0008] A method for preparing the bamboo fungus-based biomaterial as described above, the key steps are:

[0009] S1, remove the cap and stipe of fresh red-stemmed bamboo fungus, keep the stipe, rinse and set aside;

[0010] S2, immersing the stipe in a surfactant solution and shaking to remove cells;

[0011] S3, taking out the stipes, washing them thoroughly, and obtaining the acellular bamboo fungus stipes;

[0012] S4, finally, freeze-drying the decellularized bamboo fungus stipes to obtain bamboo fungus-based biomaterials.

[0013] In one embodiment, in the above step S2, the surfactant solution is a sodium dodecyl sulfate solution.

[0014] In one embodiment, in the above step S3, the cells are fully washed with PBS solution or physiological saline.

[0015] The third object of the present invention is to provide a use of bamboo fungus-based biomaterial. The technical solution is:

[0016] The use of the bamboo fungus-based biomaterial as described above in preparing a hemostatic material for wounds of humans or mammals or a filling material for wounds in the digestive tract.

[0017] In one embodiment, the bamboo fungus-based biomaterial is used to prepare a digestive tract perforation occluder.

[0018] In one embodiment, the bamboo fungus-based biomaterial is used to prepare a gastrointestinal perforation occluder.

[0019] The fourth object of the present invention is to provide an occluder for treating digestive tract perforation. The technical solution is:

[0020] The invention discloses an occluder for treating digestive tract perforation, which comprises an occluding body formed by folding or winding the bamboo fungus-based biological material as described above after being moistened, and the occluding body forms the occluder after being dried and shaped.

[0021] In one embodiment, the blocking body is loaded with functional substances.

[0022] In one embodiment, the bamboo fungus-based biomaterial is cut and unfolded along the axial direction of its stipe to form a decellularized freeze-dried bamboo fungus stipe (DDRS) membrane, which is then moistened, flattened and rolled around its long axis from the outer side to form an onion-like device, thereby obtaining the occluding body. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The Fourier transform infrared (FTIR) absorption spectrum of decellularized freeze-dried bamboo fungus powder;

[0024] Figure 2 The morphological structure analysis of the bamboo fungus stipe includes: (A) Scanning electron microscope (SEM) images of the outer side, inner side, and cross-section of decellularized and fresh bamboo fungus samples; (B) Micro-computed tomography (Micro-CT) images of fresh bamboo fungus samples;

[0025] Figure 3 The figure is an analysis of the mathematical rules of pores in the decellular bamboo fungus stipe; where: (A) cross-sectional image of the decellular bamboo fungus stipe; (B) computer-simulated cross-sectional image of the decellular bamboo fungus stipe; (C) computer-simulated 3D model of the decellular bamboo fungus stipe; (D) a simulated bamboo fungus stipe model (d1) with a diameter of 2.5 cm and a thickness of 8 mm printed on bio-silicone and a concentric circle model of the same size (d2); (d3) stress-strain curves of the bamboo fungus model and (d4) concentric circle model under cyclic compression in a direction parallel to the cross section; (d5) stress-strain curves of the bamboo fungus model and (d6) concentric circle model under cyclic compression in a direction perpendicular to the cross section;

[0026] Figure 4 The images of fresh bamboo fungus / decellularized freeze-dried bamboo fungus stained sections are periodic acid-Schiff (PAS) staining, Giemsa (GIMESA) staining, and fluorescent whitening agent (CWS) staining respectively;

[0027] Figure 5 This is a transmission electron microscopy (TEM) image of decellularized freeze-dried bamboo fungus stalk (DDRS);

[0028] Figure 6 The water absorption rate and surface permeability of decellularized bamboo fungus stalks; (A, B) images and water absorption rate curves of decellularized bamboo fungus stalks before and after water absorption; (C, D, E) permeation test results of decellularized bamboo fungus stalks with different reagents;

[0029] Figure 7The results of the cyclic compression test of decellularized bamboo fungus stalk / gelatin sponge, including: (A) Schematic diagram of the compression test and experimental photos of the decellularized bamboo fungus stalk sample; (B) Photos of the water absorption-squeezing drainage-water absorption deformation recovery process of the decellularized bamboo fungus stalk sample; (C, E, G) Cyclic compression curves of gelatin sponge samples and (D, F, H) decellularized bamboo fungus stalk samples at different compression rates;

[0030] Figure 8 The results of the subcutaneous embedding experiment (tissue compatibility experiment) of the acellular bamboo fungus stalk occluder;

[0031] Fig. 9 These are the results of the cytotoxicity experiment of the acellular bamboo fungus stalk occluder.

[0032] Fig.10 The SEM images of the samples after the in vitro hemostasis experiment of the decellularized bamboo fungus stalk (DDRS) and gelatin sponge (GS) samples;

[0033] Fig.11 Results of animal experiments (endoscopic occlusion of porcine stomach perforation) using acellular bamboo fungus stalk occluders, including: (A) schematic diagram of the experimental process, (B) image of endoscopic puncture in the stomach, (C) image of endoscopic placement of the occluder, (D) image of the occluder swelled with water to block the perforation, (E) experimental capsule endoscopy, (F) images of the perforation site on days 7 and 14 after occluder placement, (G) complete healing of the perforation site, and (H) H&E staining of the occluded site and normal tissue. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with embodiments and drawings.

[0035] Example 1 Preparation and properties of decellularized bamboo fungus

[0036] 1.1 Preparation of decellularized freeze-dried bamboo fungus stalks (DDRS)

[0037] Experimental methods

[0038] (1) Remove the cap and stipe of fresh Dictyophora rubrovalvata M.Zang purchased from the market, keep the stipe, rinse and set aside. Prepare a 0.1% sodium dodecyl sulfate (SDS) solution, put it into a conical flask, put the Dictyophora rubrovalvata stipe into it, seal it with gauze, and place it in a spiral oscillator (100 rpm) at room temperature for 72 hours, changing the solution every 24 hours. The purpose of this step is to destroy the cell structure.

[0039] (2) Remove the bamboo fungus stalk, rinse it repeatedly with PBS solution, and then place it in an ultrasonic cleaner and clean it with PBS solution for 24 hours (change the solution every 4 hours).

[0040] (3) The washed bamboo fungus stalks were soaked in 75% alcohol for 4 h and then taken out to fully remove residual SDS. They were then soaked in PBS liquid and placed on a spiral oscillator for 4 h (100 rpm). The liquid was changed every 1 h to obtain decellularized bamboo fungus stalks.

[0041] (4) The decellularized bamboo fungus stalks were placed in a -80°C refrigerator for 24 hours to solidify, and then dried in a vacuum freeze dryer for 24 hours to obtain decellularized freeze-dried bamboo fungus stalks (DDRS), which were sealed and stored.

[0042] 1.2 Fourier transform infrared absorption spectroscopy (FTIR) analysis of decellularized freeze-dried bamboo fungus stalks

[0043] Experimental methods

[0044] (1) Place the DDRS sample in liquid nitrogen for 2 min, take it out and quickly grind it into powder for later use.

[0045] (2) Use Fourier transform infrared spectrometer to detect the characteristic absorption peaks of DDRS samples.

[0046] Experimental Results

[0047] like Figure 1 As shown, the infrared characteristic absorption spectrum of DDRS is at 3312 cm -1 A broad peak appears at 2929cm, which is the stretching vibration peak of hydrogen bond OH, indicating the existence of intermolecular and intramolecular hydrogen bonds; -1 A characteristic peak appears at 1010-1100cm, which is caused by CH stretching vibration. The absorption peak in this area is the characteristic absorption peak of sugars. -1 There are two absorption peaks at 1036cm, which are characteristic absorption peaks of pyranoside. -1 There is a strong absorption peak at 1646cm -1 The relatively strong absorption peak is the characteristic absorption peak of C=O, 901cm -1 The presence of the absorption peak at indicates that the sample has β-glycosidic bonds. Therefore, it can be seen that DDRS is basically composed of polysaccharides, which is consistent with the prior art that bamboo fungus contains a large amount of bamboo fungus polysaccharides.

[0048] 1.3 Scanning electron microscopy (SEM) detection of fresh bamboo fungus and decellularized freeze-dried bamboo fungus

[0049] Experimental methods

[0050] (1) Fresh bamboo fungus stalks and DDRS were cut into several cubic samples with a cross-section of 0.5 cm × 0.5 cm. The fresh bamboo fungus samples were freeze-dried and set aside for use.

[0051] (2) Samples were prepared using conventional methods, and gold was sprayed on different surfaces of the material, such as the inner side, outer side, and cross section, and the sample microstructure was observed using SEM. The outer side refers to the surface in contact with the outside air under natural growth conditions, and the inner side is the opposite. The cross section is the cross section perpendicular to the long axis of the stipe.

[0052] Experimental Results

[0053] like Figure 2 As shown in A, the bamboo fungus cell is a hollow spherical structure (cell diameter is about 20-30 μm), the inner side is relatively smooth, and there are no obvious micropores on the surface. Internal macropores are observed in the cross section; after magnification, it can be seen that the membrane constituting the macropores is composed of multiple layers of spherical cells.

[0054] 1.4 Micro-CT detection of fresh bamboo fungus

[0055] Experimental methods

[0056] (1) Prepare several dried samples of fresh bamboo fungus according to the method in 1.3.

[0057] (2) The samples were scanned on a Micro-CT machine and the data were analyzed using CTVOX software to analyze the structures of the samples in different directions and at different levels.

[0058] Experimental Results

[0059] The Micro-CT image of the fresh bamboo fungus sample reflects the internal structure of the bamboo fungus stipe, which corresponds to the internal pore structure of the DDRS. Figure 2 B, DDRS is an annular structure with an inner hole in cross section, and the annular wall has a channel structure that is larger inside and smaller outside, forming a structure that is dense outside and loose inside, which is consistent with the structure shown in the SEM image.

[0060] The three-dimensional porous structure morphology of the cross section of the decellularized bamboo fungus stalk is as follows: from the center to the outside, the annular wall has a three-layer pore structure, and the pore diameter of each layer gradually decreases, the number of pores gradually increases, and the pores between adjacent layers have channels.

[0061] 1.5 Establishment of mathematical model of bamboo fungus pore structure and printing of 3D bionic model

[0062] The inventors found that after the decellularized bamboo fungus stalks were crushed and made into cylindrical samples through a mold, the cylindrical samples were difficult to recover their shape after being compressed and deformed in the radial and axial directions, while the moist decellularized bamboo fungus stalks had good recovery properties. Therefore, they speculated that the matrix structure of the bamboo fungus stalks itself gave it elasticity, and used the common elastic material bio-silicone to make different structural models to study the differences in their mechanical properties.

[0063] Experimental methods

[0064] (1) Take a few fresh bamboo fungus stalks, cut the middle section into several sections perpendicular to the long axis, and use a digital camera to take a cross-section image of the bamboo fungus. Then use ink to print the cross-section image on an A4 paper, place a ruler next to the paper, and scan and save it with a digital camera.

[0065] (2) Use ImageJ to measure and record the pore sizes of all pores on the cross section. All pores on the cross section are approximately regarded as circles. The radius of the hollow circle inside the stem of the bamboo fungus is defined as R. The average pore size of each layer of pores from the inside to the outside is D 1 , D 2 , D 3 (The average pore size is the average of the inner diameters of all polygons in each layer, using the arithmetic mean.) Record the number of pores in each layer on the cross section, marked as N from inside to outside. 1 、N 2 、N 3 .

[0066] (3) Use MATLAB software to analyze the correlation between the above parameters and construct a mathematical model.

[0067] (4) Using bio-silicone as the printing material, a porous bionic bamboo fungus model with a diameter of 2.5 cm and a solid concentric cylinder model were obtained by 3D printing.

[0068] (5) Cyclic compression tests were performed on the bionic bamboo fungus model and the concentric circle model in a direction parallel to the cross section. The compression was performed at a speed of 200 mm / min to 80% strain and repeated 10 times to compare the compression strengths of the two.

[0069] (6) Cyclic compression tests were performed on the bamboo fungus and the concentric circle model in a direction perpendicular to the cross section. The compression was performed at a speed of 80 mm / min until the strain was 80%. The cycles were repeated 10 times and the compression strengths of the two were compared.

[0070] Experimental Results

[0071] The inner pore radius R of the hollow section of the bamboo fungus stem and the pore diameter D of the pores on the hollow section (the average diameter D of each layer from the inside to the outside) 1 , D 2 , D3 ) is a significant composite quartic linear model. Through statistical analysis, the following formula is obtained:

[0072] ①D 1 =-3594.43+1818.29×R-343.56×R 2 +28.76×R 3 -0.90×R 4

[0073] ②D 2 =-283.03+139.84×R-25.99×R 2 +2.16×R 3 -0.07×R 4

[0074] ③D 3 =-512.63+241.18×R-42.40×R 2 +3.31×R 3 -0.10×R 4

[0075] This formula can predict the pore sizes of the three pore layers on the hollow section of the bamboo fungus stipe (D 1 , D 2 , D 3 ) as a function of the hollow radius R. The coefficient of determination of the mathematical model fitting is above 0.95, and the prediction ability is good. Therefore, the pore size of each pore layer determines the number of polygons inside the pore layer, and the three layers (N 1 、N 2 、N 3 ). Three binary complete polynomial regression models were constructed to accurately predict the number of holes in each layer:

[0076] ④N 1 =∑ n i＝0 ∑ n j＝0 β ij x 1 i y 1 j , where x 1 =R,y 1 =R+D 1 ,β is the model fitting coefficient;

[0077] ⑤N 2 =∑ n i＝0 ∑ n j＝0 γij x 2 i y 2 j , where x 2 =R+D 1 ,y 2 =R+D 1 +D 2 ,γ is the model fitting coefficient;

[0078] ⑥N 3 =∑ n i＝0 ∑ n j＝0 δ ij x 3 i y 3 j , where x 3 =R+D 1 +D 2, ,y 3 =R+D 1 +D 2 +D 3 ,δ is the model fitting coefficient.

[0079] Through software analysis, it was found that when the number of pore layers N from the inside to the outside of the bamboo fungus stem is 3, a better prediction effect can be achieved. From the two-dimensional model diagram showing the relationship between D, R and N, it can be seen that there is an excellent curve relationship between them. The prediction calibration diagram shows that the predicted value is very close to the actual value, and the determination coefficient of the model fitting reaches above 0.95.

[0080] The above formulas ① to ⑥ can be used to describe the mathematical characteristics of the three-dimensional pore morphology structure of the bamboo fungus stipe.

[0081] By setting the hollow radius R of bamboo fungus, the previous model is used to predict D 1 , D 2 , D 3 、N 1 、N 2 、N 3 , and simulate the cross-sectional pattern of DR by computer. For example, when we take the hollow radius R = 8.6, we can randomly set it through the previous model:

[0082] D 1 =2.85,D 2 =1.92,D 3 =1.22,N 1 =17,N 2 =33,N 3 =45.

[0083] like Figure 3 As shown in A and 3B, using the above seven parameters and MATLAB software, computer simulated images and annotation diagrams of DR cross sections can be obtained.

[0084] like Figure 3 As shown in Figures C and 3D, the three-dimensional data model of the DDRS 3D printing object was successfully obtained through computer modeling, and the bionic bamboo fungus model was successfully printed using bio-silicone. The obtained imitation bamboo fungus sample has elasticity and can be folded and compressed. The bionic bamboo fungus model with an outer diameter of 2.5cm and a thickness of 8mm and the concentric circle model of the same size printed with bio-silicone were subjected to cyclic compression experiments in the direction parallel or perpendicular to the cross section. Under the same strain and speed, the maximum compression strength of the bionic bamboo fungus model was significantly greater than that of the concentric circle model, regardless of whether it was in the parallel or perpendicular direction. This result shows that the mathematically regular pore structure inside the bamboo fungus stalk may give it natural good compression elasticity and strength.

[0085] 1.6 Staining of paraffin sections of fresh and decellularized lyophilized bamboo fungus

[0086] Experimental methods

[0087] (1) Fresh bamboo fungus and decellularized bamboo fungus samples were fixed in formalin-acetic acid-alcohol fixative (FAA fixative) and placed in a 4°C refrigerator for 24 h.

[0088] (2) The fixed samples were immersed in ethanol solutions of different concentrations to remove moisture, gradually increasing from low concentration to high concentration (50%, 70%, 80%, 90%, 100%), and immersed for 15 minutes, and repeated 3 times.

[0089] (3) Soak the tissue sample in molten paraffin to allow the tissue sample to penetrate and infiltrate the paraffin.

[0090] (4) The infiltrated tissue sample is placed in a pre-made paraffin box, and molten paraffin is added and solidified to form a paraffin block.

[0091] (5) Using a sliding microtome, cut the paraffin block into slices with a thickness of approximately 5 to 10 μm.

[0092] (6) Soak the sections in an organic solvent such as xylene for multiple times to remove the paraffin.

[0093] (7) Staining: The sections were stained with various stains (PAS, GIMESA, CWS).

[0094] (8) Sealing: Place the stained sections on a glass slide, add transparent glue and solidify to make a glass slide.

[0095] (9) Observe the morphological structure of each sample at different magnifications under a microscope and collect images.

[0096] Experimental Results

[0097] After decellularization and freeze-drying, the macroscopic and microscopic structures of bamboo fungus stalks did not change significantly compared with fresh bamboo fungus, indicating that decellularization did not damage the matrix morphology. In order to evaluate the decellularization effect, different staining methods were used to observe the paraffin sections (PAS, Gimesa, CWS) of bamboo fungus stalks before and after decellularization. Figure 4 It can be seen that under different staining methods, the cell morphology of the bamboo fungus stalk did not change significantly before and after decellularization, and no obvious nucleus or cytoplasm was stained.

[0098] 1.7 Transmission electron microscopy (TEM) observation of the microscopic morphology of decellularized bamboo fungus

[0099] In order to show the more subtle structure of DDRS, TEM detection was supplemented.

[0100] Experimental methods

[0101] (1) Sample preparation: 1 mm*2 mm slices of decellularized freeze-dried bamboo fungus were placed in a 2.5% glutaraldehyde solution and placed in a 4°C refrigerator for overnight solidification to prepare ultrathin slice samples.

[0102] (2) After attaching the sample to the carbon film, place it on the sample stage of the TEM and scan and take an image.

[0103] Experimental Results

[0104] like Figure 5 As shown, the distinct boundaries between the cell walls form a dark-light-dark system, which may facilitate the entry and exit of water molecules or other substances between cells.

[0105] 1.8 Water absorption rate experiment of decellularized freeze-dried bamboo fungus

[0106] Experimental methods

[0107] (1) Prepare DDRS samples of 2 cm × 2 cm in size and weigh them at room temperature, recorded as M1; the sample quantity is 3.

[0108] (2) Each group of freeze-dried samples was immersed in pure water, taken out at different time points, the visible water on the surface was removed, and the samples were weighed and recorded as M2.

[0109] The water absorption rate calculation formula is:

[0110] Water absorption (%) = (M2-M1 / M1) × 100

[0111] Experimental Results

[0112] like Figure 6 A and 6B, DDRS can absorb more than 25 times its weight in water. At the same time, due to the existence of multi-level pores in the material, the expansion rate after water absorption is not high.

[0113] 1.9 Surface permeability of the inner and outer surfaces of decellularized freeze-dried bamboo fungus

[0114] Experimental methods

[0115] (1) Prepare 10 mL of 1% Giemsa (GIMSA), sodium fluorescein and fuchsin solution and set aside. Take 6 mL of the volunteer's anticoagulated blood (sodium citrate) and set aside.

[0116] (2) Prepare several DDRS samples and gelatin sponge (GS) samples for future use.

[0117] (3) The decellularized bamboo fungus samples were placed on the table with the outer side or inner side facing upwards, and the gelatin sponge samples were placed on the table at the same time. 5 μL of anticoagulant blood was dripped on the surface, and the penetration process was recorded with a camera.

[0118] (4) Place the decellularized bamboo fungus samples with the outer and inner sides facing upwards, and drip 10 μL of Giemsa and 10 μL of sodium fluorescein on the surface respectively, and record the penetration process with a camera.

[0119] (5) Pour 50 mL of fuchsin solution into two 100 mL beakers, and gently place the decellularized bamboo fungus samples on the surface of the solution with the outer and inner sides facing upwards, respectively. Observe the penetration speed and area, and record the penetration process with a camera.

[0120] Experimental Results

[0121] like Figure 6 C-6E, when the DDRS material was placed in the diluted magenta reagent with different faces facing down, the outer surface had a faster penetration rate facing downward. When 10 μL of diluted GIMSA solution and sodium fluorescein solution were dropped on both sides of the DDRS material, it can also be seen that the penetration rate from the outside was faster than that from the inside. At the same time, when 5 μL of anticoagulant was also added to the surface of the material, the results were similar to the previous results and were faster than gelatin sponge (GS). This phenomenon may be due to the multilayer and hierarchical pore structure of DDRS, in which the mesh openings distributed on the outside of the bamboo fungus stalk enable the liquid to diffuse quickly after entering the macropores, and this structure is lacking on the inner side. In summary, DDRS exhibits obvious hydrophilicity, and the asymmetric pore distribution on the inside and outside makes it show different penetration effects when in contact with liquid.

[0122] 1.10 Compression performance test

[0123] The compressive elastic properties of DDRS samples were studied using a quantitative method.

[0124] Experimental methods

[0125] (1) Cut the DDRS and gelatin sponge samples into square samples of 1 cm × 1 cm respectively. The thickness of the DDRS and gelatin sponge samples is 4 to 6 mm and they are fully moistened with water.

[0126] (2) Each sample was placed in a universal tensile testing machine and compressed to 70%, 80%, and 90% strain at a speed of 40 mm / min. The cycle was repeated 10 times. The number of samples in each group was 3.

[0127] Experimental Results

[0128] like Figure 7 A and 7C-7H, after completely absorbing water, the DDRS samples completed 10 cycles of compression at a maximum strain of 90% and returned to a round shape. In addition, compared with gelatin sponge, DDRS also exhibited greater mechanical strength under cyclic compression. At the same compression rate and strain (40mm / min and 10 cycles), the maximum compressive strength of DDRS can be 10 times that of gelatin sponge, especially at 90% strain, the maximum compressive strength of DDRS can reach 200Kpa.

[0129] This experiment shows that DDRS not only has good water absorption but also has excellent compression elasticity.

[0130] 1.11 Shape memory test of decellularized freeze-dried bamboo fungus

[0131] Experimental methods

[0132] (1) Cut DDRS into 0.5 cm thick samples and soak them in pure water until they are completely absorbed.

[0133] (2) Squeeze out the water and fold it repeatedly, then place it in a culture dish filled with pure water and observe how the sample recovers its shape in water.

[0134] Experimental Results

[0135] Experiments have found that Figure 7 B, After squeezing out the water and folding several times, DDRS can restore its original shape in water.

[0136] 1.12 In vivo degradation experiment

[0137] Experimental methods

[0138] (1) Cut 0.5 cm thick DDRS into square samples of about 1 cm × 1 cm, control the quality to 5 mg for later use, and sterilize them under ultraviolet light in a biological safety cabinet for 1 hour.

[0139] (2) Nine male SD rats were anesthetized by intraperitoneal injection of sodium pentobarbital, and the back skin was shaved and the surgical area was disinfected with 75% alcohol.

[0140] (3) A 1-cm-long incision was made on the back of the rats, and the prepared samples were embedded subcutaneously and sutured. The rats were kept at a constant temperature of 25°C and fed with unlimited food and water for 2, 4, or 6 weeks.

[0141] (4) At the designed time point, the rats were killed by cervical vertebrae cutting, the skin and subcutaneous mass were completely removed, the blood stains on the surface were washed with saline, and the rats were fixed in 10% neutral formalin solution overnight.

[0142] (5) The subcutaneous mass was completely peeled off, the excess tissue fascia was removed, the liquid on the surface of the mass was wiped dry and weighed, and the in vivo degradation curve was drawn. At the same time, routine hematoxylin and eosin (H&E) staining was performed to observe the tissue inflammatory response.

[0143] Experimental Results

[0144] like Figure 8 The mass size of the mass was significantly reduced at week 4 compared with week 2, and the material was completely degraded after week 6. H&E results showed that the severity of the foreign body reaction decreased over time.

[0145] 1.13 Cell culture and cytotoxicity experiments

[0146] Cell culture

[0147] (1) Fibroblast (3T3) recovery

[0148] a. Take out the 3T3 cell cryopreservation tube from the liquid nitrogen tank and quickly place the tube in a 37℃ water bath until the cells are completely thawed.

[0149] b. Slowly add the thawed cell solution into the preheated DMEM culture medium and gently shake the culture bottle to evenly disperse the cells in the culture medium.

[0150] c. Place the cell culture flask in a 37°C, 5% CO 2 The cells were cultured in a constant temperature incubator.

[0151] (2) 3T3 cell culture

[0152] a. Wash 3T3 cells with PBS to evenly distribute the cells in the culture flask.

[0153] b. Use 0.25% trypsin to release the cells from the culture flask.

[0154] c. Add an equal volume of DMEM medium to stop digestion and collect the cell pellet by centrifugation.

[0155] d. Wash the cell pellet with PBS and add new DMEM medium to evenly disperse the cells in the medium.

[0156] e. Place the cell culture flask in a constant temperature incubator at 37°C and 5% CO2 for culture.

[0157] (3) 3T3 cell culture

[0158] a. Prepare DMEM medium and add 10% fetal bovine serum.

[0159] b. Wash the 3T3 cells with PBS to evenly distribute the cells in the culture flask.

[0160] c. Add pre-warmed DMEM culture medium and gently shake the culture flask to evenly disperse the cells in the culture medium.

[0161] d. Place the culture flask in a 37°C, 5% CO 2 The cells were cultured in a constant temperature incubator.

[0162] e. Regularly observe cell growth, replace with fresh culture medium, and perform cell passaging.

[0163] Cytotoxicity assay method

[0164] (1) Take 5 mg of DDRS sample and sterilize it under UV light in a biosafety cabinet for 1 hour before use.

[0165] (2) The samples were rinsed three times with sterile PBS and then rinsed three times with DMEM medium containing 10% fetal bovine serum (FBS).

[0166] (3) The sample was immersed in 5 mL of DMEM medium containing 10% fetal bovine serum and placed in a 4°C refrigerator for 24 hours.

[0167] (4) The soaking solution was aspirated and diluted with DMEM medium containing 10% fetal bovine serum to 3 / 4, 1 / 2, and 1 / 4 of the original solution, respectively, to obtain DDRS soaking solutions with concentrations of 1 mg / ml, 0.75 mg / ml, 0.5 mg / ml, and 0.25 mg / ml, respectively.

[0168] (5) Add the counted 3T3 cells to each group of immersion solution to prepare a 3T3 cell suspension (50 / ul). Add 100 ul of the cell suspension to a 96-well plate and incubate at 37°C and 5% CO. 2The cells were cultured in an incubator for 1, 2, and 3 days, and the corresponding fresh culture medium was replaced every two days (n=3). The control group used standard culture medium.

[0169] (6) Dilute the CCK-8 stock solution with incomplete DMEM medium to prepare a 10% CCK-8 working solution. At the designed time points (days 1, 2, and 3), discard the medium in each well, add 100 μl of CCK-8 working solution to each well, and place the wells in the incubator again in the dark for 30 minutes.

[0170] (7) Place the 96-well plate in an ELISA reader and measure the absorbance at a wavelength of 450 nm.

[0171] Experimental Results

[0172] like Fig. 9 The number of live cells was counted by CCK-8 test, and it was found that there was no significant difference between the culture solution of each concentration sample and the control group, indicating that DDRS had no obvious cytotoxicity (n=3). This shows that in addition to biodegradability, DDRS also exhibits excellent biocompatibility.

[0173] Example 2: In vitro hemostasis of decellularized bamboo fungus

[0174] Experimental methods

[0175] (1) Cut DDRS and GS into 0.5 cm pieces respectively 3 Several samples were placed in a biosafety cabinet and sterilized under ultraviolet light for 1 hour before use;

[0176] (2) Place the sample into each well of a 24-well microplate and mix with 100 μL of whole blood or platelet suspension;

[0177] (3) After exposure for different time periods, the samples were rinsed with PBS, fixed with 2.5% glutaraldehyde, and dehydrated with a series of gradient alcohol solutions. After drying, the samples were sprayed with gold using conventional methods and observed using a scanning electron microscope.

[0178] Experimental Results

[0179] like Fig.10, platelet aggregation and fibrin network formation occurred after DDRS and anticoagulant contact for 40 seconds; however, this phenomenon was not found in the control gelatin sponge group. For a more intuitive display, the SEM image was enlarged and pseudo-colored, with blue representing activated platelets, red representing red blood cells, and white representing fibrin network. Under the same reaction conditions between the two materials and platelets, the platelets on the surface of DDRS significantly aggregated and formed a fibrin network after 1 minute of reaction, while dispersed unactivated platelets and a small amount of aggregated platelets were found in the gelatin sponge group under SEM. This phenomenon shows that DDRS has stronger hemostatic properties than traditional hemostatic gelatin sponges, and can therefore be used as a wound hemostatic material.

[0180] Example 3: Use of decellularized bamboo fungus for blocking pig stomach perforation in vitro and in vivo

[0181] 3.1 In vitro pig gastric perforation occlusion experiment

[0182] Experimental methods

[0183] (1) The DDRS was cut open from the middle and flattened, cut into regular squares, moistened with pure water and flattened to form a DDRS membrane, which was then rolled along the long axis and rolled from the outside into an onion-shaped device, fixed and dried to form a bamboo fungus occluder with a diameter of about 3 mm. When rolled, the adjacent layers of the DDRS membrane fit tightly together.

[0184] (2) Place the porcine stomach serosa purchased from the market on a beaker with the surface facing upwards, cut a 5 mm long opening on the greater curvature of the stomach, seal the perforated part with a device, and pour simulated gastric fluid on the surface. Observe the blockage after 24 hours.

[0185] Experimental Results

[0186] After the incision of the pig stomach was filled with water, water could be seen leaking from the perforated part; after the perforated part was blocked with the bamboo fungus occluder, the middle section of the bamboo fungus occluder was stuck in the perforation, and the two ends expanded and expanded, and the gap no longer leaked. The pig stomach filled with the bamboo fungus occluder was placed on a beaker, and simulated gastric fluid was poured on the surface. No liquid leakage was found after 24 hours.

[0187] It is easy for a person skilled in the art to understand that the outer surface of the DDRS has an open porous structure, and liquid permeates faster. The flattened and curled DDRS membrane has a tendency to recover its deformation after absorbing water, so the volume expands and fills the perforation. In actual use, two or more layers of DDRS membranes can also be stacked and curled to obtain a bamboo fungus occluder with a larger diameter.

[0188] 3.2 In vivo pig stomach perforation occlusion experiment

[0189] To further demonstrate that the device can be delivered endoscopically to treat gastrointestinal perforation, a miniature pig gastric perforation model was used.

[0190] Experimental methods

[0191] (1) Prepare the bamboo fungus occluder according to section 3.1, dry it at room temperature for 24 hours, and sterilize it under ultraviolet light in a biological safety cabinet for 1 hour before use;

[0192] (2) The pigs were anesthetized by intramuscular injection of sodium pentobarbital and xylazine hydrochloride. A vertical perforation with a diameter of approximately 5 mm was made in the gastric wall using an electric scalpel under endoscopy, and excess tissue was removed using biopsy forceps.

[0193] (3) Clamp one end of the bamboo fungus occluder with a hemostatic forceps and connect it to the front of the endoscope to deliver the device to the wound site through the esophagus.

[0194] (4) Three minutes after surgery, water was sprayed on the device to expand and observe the perforation. At the same time, capsule endoscopy was performed on the 7th and 14th days after surgery to evaluate wound healing.

[0195] Tissue sectioning and H&E staining

[0196] (1) After obtaining the living tissue, it was fixed with 10% formalin solution, and then the fixed tissue was dehydrated with gradient alcohol xylene, embedded in paraffin, sliced ​​(7 microns), dried, and dewaxed with xylene alcohol.

[0197] (2) H&E staining: Place the sections in a hematoxylin solution for staining for 5-7 minutes, then treat with acid water and ammonia water for 5-10 seconds each, treat with running water for 1 hour, then decolorize with 70% and 90% alcohol for 10 minutes, and finally stain with eosin for 2-3 minutes, and dehydrate with pure alcohol and xylene.

[0198] (3) Use neutral resin to seal the slide.

[0199] (4) Scan the stained slides.

[0200] Experimental Results

[0201] like Fig.11 The experiment found that 3 minutes after the bamboo fungus occluder was placed in the perforation, it was found that it gradually expanded and fixed at the perforation position. Through the magnetic control capsule to observe the surgical site, it was found that on the 7th day, the bamboo fungus occluder was wrapped by the gastric mucosa, the wound gradually closed, and there was no fluid leakage around the wound; on the 14th day, the gastric mucosa at the surgical site was completely repaired. Two months after treatment, the gastric tissue was observed under endoscopy and it was found that the gastric mucosa at the surgical site was completely repaired.

[0202] H&E staining of the collected gastric tissue showed that the closed gastric perforation was completely bridged with the regenerated mucosa, and the gastric mucosa was fully repaired. It can be seen that the gastric mucosa at the repair site is thicker than the mucosa around the wound, which is a positive manifestation of the healing process of the gastric mucosa. The above results show that the bamboo fungus occluder can be delivered to the perforation site through an endoscope to release and occlude the perforation in vivo, can withstand the force of gastrointestinal peristalsis without leakage, and promote the closure and healing of gastrointestinal perforations.

[0203] It is easy for a person skilled in the art to understand that, during the preparation of the bamboo fungus occluder, functional substances such as antibacterial drugs and anti-inflammatory drugs can be loaded inside the material during the curling and shaping process of the DDRS, thereby obtaining an occluder carrying functional substances.

[0204] Through the above experimental research, we found that:

[0205] (1) The decellularized bamboo fungus stalk has excellent compressive elasticity, and this mechanical property is related to its natural three-dimensional porous structure;

[0206] (2) The acellular bamboo fungus stalk has good hydrophilicity and strong water absorption capacity, and the volume does not change much after absorbing water, and can return to its original shape after absorbing water;

[0207] (3) The decellularized bamboo fungus stalk has no obvious cytotoxicity, good biocompatibility, and can be naturally degraded in the body;

[0208] (4) The acellular bamboo fungus stalk has good coagulation performance, which is stronger than that of traditional gelatin sponge and can be used as a wound hemostatic material;

[0209] (5) The penetration capacity of the inner and outer surfaces of the decellularized bamboo fungus stalks is different. The outer surface of the sample cut longitudinally penetrates faster into the inner surface, while the inner surface penetrates slower.

[0210] (6) The decellularized bamboo fungus stalk has good sealing properties for gastrointestinal perforation, and because the overall expansion rate of DDRS is low after absorbing water, it does not exert much pressure on the tissue at the perforation, effectively sealing and promoting wound repair;

[0211] (7) The decellularized bamboo fungus stalk material is of natural origin, low cost, safe and controllable, and has the potential for clinical use.

[0212] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, ordinary technicians in this field can make various similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the scope of protection of the present invention.

Claims

1. A bamboo fungus-based biomaterial, characterized in that It is obtained by decellularizing and drying the stem of bamboo fungus, and retains a natural three-dimensional porous morphology structure.

2. The bamboo fungus-based biomaterial according to claim 1, characterized in that The decellularization process is as follows: remove the cap and stipe of fresh red-stemmed bamboo fungus, keep the stipe, and rinse it clean; then immerse the stipe in a surfactant solution to remove cells; then remove the residual surfactant, wash and dry it to obtain the decellularized bamboo fungus stipe.

3. The method for preparing bamboo fungus-based biomaterial according to claim 1, characterized in that The steps are: S1, remove the cap and stipe of fresh red-stemmed bamboo fungus, keep the stipe, rinse and set aside; S2, immersing the stipe in a surfactant solution and shaking to remove cells; S3, taking out the stipes, washing them thoroughly, and obtaining the acellular bamboo fungus stipes; S4, finally, freeze-drying the decellularized bamboo fungus stipes to obtain bamboo fungus-based biomaterials.

4. The method for preparing bamboo fungus-based biomaterial according to claim 3, characterized in that: In step S2, the surfactant solution is a sodium dodecyl sulfate solution.

5. The method for preparing bamboo fungus-based biomaterial according to claim 3, characterized in that: In step S3, the cells are thoroughly washed with a PBS solution or physiological saline.

6. Use of the bamboo fungus-based biomaterial according to claim 1 in preparing a hemostatic material for wounds of humans or mammals or a filling material for wounds of the digestive tract.

7. The use according to claim 6, characterized in that: The bamboo fungus-based biomaterial is used for preparing a digestive tract perforation occluder.

8. The use according to claim 6, characterized in that: The bamboo fungus-based biomaterial is used for preparing a gastric or intestinal perforation occluder.

9. An occluder for treating digestive tract perforation, characterized in that: The invention comprises a blocking body formed by folding or rolling the bamboo fungus-based biological material according to claim 1 after being moistened, and the blocking body forms an occluder after being dried and shaped.

10. The occluder according to claim 9, characterized in that: The blocking body is loaded with functional substances.

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