Artificial fascia and its construction method and application

Abstract

The application discloses an artificial fascia which is a composite material with a five-layer structure and is composed of fat stem cell-loaded amniotic membrane, PRP gel and polyurethane film; the five-layer structure material is in sequence of fat stem cell-loaded amniotic membrane, PRP gel, polyurethane film, PRP gel and fat stem cell-loaded amniotic membrane; the fat stem cell-loaded amniotic membrane is acellular amniotic membrane paved with fat stem cells and is prepared by the following method: inoculating fat stem cells on the acellular amniotic membrane and growing for 7-14 days to obtain the fat stem cell-loaded amniotic membrane. The application further discloses a preparation method of the artificial fascia and application of the artificial fascia in preparation of a biological patch for treating hernia or in preparation of a mesh for treating pelvic organ prolapse. The artificial fascia has high tensile strength, good tissue compatibility, good immune function and degradability, is a biological composite material with excellent performance, can be used for treating hernia and pelvic organ prolapse and has great application potential.

Description

Technical Field

[0001] This invention relates to an artificial fascia, its construction method and application, belonging to the field of biocomposite materials technology. Background Technology

[0002] Hernia is a common clinical surgical condition in elderly men, and is usually treated with biological patches. However, existing biological patches have the drawback of low tensile strength, which often leads to rapid degradation and premature loss of their supporting function, resulting in hernia recurrence. Therefore, it is necessary to develop a biomaterial with high tensile strength.

[0003] Pelvic organ prolapse (POP) is a multifactorial disease caused by weakened support from the connective and muscular tissues of the pelvic floor, leading to the prolapse of pelvic organs into or outside the vagina. It is a symptom of pelvic floor dysfunction (PDD). POP severely impacts the quality of life and mental and physical health of middle-aged and elderly women. Treatment typically involves mesh implantation. However, while chemically synthesized meshes, such as polypropylene meshes, exhibit superior mechanical properties and success rates, their non-degradability and poor tissue compatibility can cause significant problems such as vaginal wall erosion, chronic pain, infection, and discomfort during intercourse. The incidence of secondary surgeries due to these complications is higher than that of autologous tissue repair surgeries. Therefore, it is necessary to develop a biomaterial with high tissue compatibility and good immunomodulatory properties. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides an artificial fascia, a method for constructing the fascia, and its application as a biological patch for treating hernia and as a mesh for treating pelvic organ prolapse.

[0005] This invention is achieved through the following technical solution:

[0006] An artificial fascia is a composite material with a five-layer structure, consisting of adipose-derived stem cell-loaded amniotic membrane, PRP gel, and a polyurethane membrane. The five layers are, in order: adipose-derived stem cell-loaded amniotic membrane, PRP gel, polyurethane membrane, PRP gel, and adipose-derived stem cell-loaded amniotic membrane; wherein the adipose-derived stem cell-loaded amniotic membrane is a decellularized amniotic membrane covered with adipose-derived stem cells.

[0007] Furthermore, the thickness of the PRP gel is 0.2–0.4 mm, preferably 0.3 mm.

[0008] Furthermore, the thickness of the polyurethane film is 0.2 to 0.4 mm, preferably 0.3 mm.

[0009] Furthermore, the adipose-derived stem cell-loaded amnion is prepared by the following method: adipose-derived stem cells are seeded onto decellularized amnion and grown for 7–14 days. Specifically, the decellularized amnion is laid flat on a culture plate, inoculated with a culture medium containing adipose-derived stem cells, then the decellularized amnion is flipped over, and the other side is inoculated with the same culture medium; after culturing for 7–14 days, the amnion is obtained.

[0010] Furthermore, the decellularized amnion can be prepared by the following method:

[0011] (1) Take a fresh amniotic membrane and soak it in a preliminary disinfection solution to disinfect the amniotic membrane and inhibit bacterial growth;

[0012] The preliminary disinfection solution is a PBS solution containing 50 μg / ml penicillin, 50 μg / ml streptomycin and 2.5 μg / ml amphotericin B.

[0013] (2) Remove the amnion, wash it with sterile water, then wash it with sodium hypochlorite solution, and then wash it with sterile water again. During the washing process, the amnion can be shaken to fully unfold it and achieve better washing results.

[0014] (3) Immerse the cleaned amnion in the cryopreservation solution, take it out and place it on filter paper, spread it out fully, cover it with another layer of filter paper, and repeat the freeze-thaw cycle twice.

[0015] The cryopreservation solution was prepared by mixing glycerol and DMEM culture medium in a 1:1 volume ratio;

[0016] The specific operation of repeated freeze-thaw cycles is as follows: freeze at -80°C for 1 hour, then thaw at 37°C for 0.5 hours, then freeze at -80°C for 1 hour, and then thaw at 37°C for 0.5 hours.

[0017] (4) Place the amnion after repeated freeze-thaw cycles in Triton X-100 solution and incubate on a shaker to remove cells initially; then wash the amnion with PBS solution by shaking.

[0018] (5) Place the amnion in a lipase solution and incubate on a shaker to remove epithelial cells and other impurities. Then wash the amnion with PBS solution by shaking.

[0019] The lipase used was TYPE II L8070 lipase purchased from Solarbio Biotechnology Co., Ltd.; the concentration of the lipase solution was 1000 U / L.

[0020] (6) Place the amnion in a genipin solution to crosslink, then rinse with distilled water;

[0021] Place the amnion in a saturated glycine solution, soak it, change the solution until the solution no longer changes color, and rinse repeatedly with distilled water;

[0022] The amnion was placed on aluminum foil and irradiated with 254 nm ultraviolet light.

[0023] Lay the amnion flat on the nitrocellulose membrane with the chorionic membrane side facing the nitrocellulose membrane, press it under the iron tray, and freeze it at -40℃ for 10 to 15 hours.

[0024] (7) Freeze-dry the frozen amnion until the moisture content is 5% to 7%;

[0025] (8) Cut the dried amnion into pieces, put them into plastic bags, vacuum seal them, and sterilize them by gamma irradiation.

[0026] Furthermore, the adipose-derived stem cells can be prepared by the following method:

[0027] (1) Isolation of adipose stem cells: Take the collected adipose tissue and wash it with physiological saline; add an appropriate amount of type I collagenase solution or trypsin solution for digestion; filter and centrifuge; discard the oil and enzyme solution on the upper layer of cell precipitate; resuspend the cell precipitate with physiological saline, blow it apart, filter it through a cell filter, centrifuge, and discard the supernatant to obtain stromalvascular fraction (SVF) cells.

[0028] (2) Culture and passage of adipose stem cells: The stromal vascular component cells obtained above are cultured in a routine manner. When the cell confluence reaches 70% to 80%, they can be passaged. Collect P2 or P3 generation cells and freeze them for later use.

[0029] Furthermore, the specific washing procedure is as follows: place the adipose tissue in a container (such as a T175 culture flask), first aspirate (using a pipette) the upper yellow oil layer and discard it, then aspirate the lower red liquid layer and discard it; add an appropriate amount of physiological saline to the remaining adipose tissue, shake vigorously for 3 minutes to thoroughly wash the adipose tissue, let it stand for 3-5 minutes, and aspirate the lower aqueous phase; repeat the washing of the adipose tissue three times until the lower aqueous phase is clear.

[0030] Furthermore, the specific operation method of the digestion is as follows: after adding the enzyme solution, seal the opening with a sealing film, shake vigorously for 5-10 seconds, place it in an air bath constant temperature shaker, and digest for 60 minutes at 37°C and 70 rpm, shaking vigorously for 5-10 seconds every 15 minutes during the digestion process.

[0031] Furthermore, the PRP gel is preferably an autologous PRP gel to avoid rejection reactions, allergic reactions, etc.

[0032] Furthermore, the PRP gel contains 50 ng / ml interleukin-10 and 30 ng / ml macrophage migration inhibitory factor.

[0033] The polyurethane membrane is a conventional product already available in the prior art and can be purchased on the market.

[0034] The method for constructing the artificial fascia includes the following steps:

[0035] (1) Place the amniotic membrane loaded with adipose-derived stem cells on the bottom of a petri dish, and then spread a layer of PRP gel on top of it;

[0036] (2) Lay the polyurethane film flat on the above-mentioned PRP gel;

[0037] (3) Lay a layer of PRP gel on the polyurethane membrane as described above;

[0038] (4) Place another piece of adipose stem cell-loaded amniotic membrane on the above PRP gel to obtain artificial fascia.

[0039] The artificial fascia is used as or in the preparation of a biological patch for treating hernias, and as or in the preparation of a mesh for treating pelvic organ prolapse.

[0040] The artificial fascia of this invention uses a polyurethane membrane as its core and amniotic membrane, which has low immunogenicity, as its covering. PRP gel is used as an adhesive material to ensure a tight bond between the two. This invention adds two inflammatory inhibitory factors to the PRP gel. Due to the infiltration of immune cells into the implant, especially the various enzymes released by neutrophils, the degradation of the implant can be accelerated. The addition of inflammatory inhibitory factors delays the residence time of the artificial fascia in the body, allowing for the migration and colonization of the body's own cells. With the implantation of fibroblasts and the secretion of collagen, the body's own regenerative capacity is activated, gradually replacing the graft. The artificial fascia of this invention has high tensile strength, good tissue compatibility, excellent immune function, and is biodegradable. It is a high-performance biocomposite material with great potential for treating hernias and pelvic organ prolapse. Attached Figure Description

[0041] Figure 1 Schematic diagram of HE staining results of decellularized amnion.

[0042] Figure 2 : Schematic diagram of the morphology of adipose-derived stem cells (primary cells).

[0043] Figure 3 Schematic diagram of the morphology of adipose-derived stem cells (P2 generation cells).

[0044] Figure 4 Schematic diagram of HE staining results of amniotic membrane loaded with adipose stem cells.

[0045] Figure 5 Schematic diagram of HE staining results for artificial fascia, normal fascia, and decellularized amnion. Detailed Implementation

[0046] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0047] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0048] Example 1: Preparation of decellularized amnion

[0049] (1) Take a fresh human amnion and soak it in a preliminary disinfection solution for 20 minutes to disinfect the amnion and inhibit bacterial growth;

[0050] The initial disinfection solution was a PBS solution containing 50 μg / ml penicillin, 50 μg / ml streptomycin, and 2.5 μg / ml amphotericin B.

[0051] (2) Remove the amnion and rinse with sterile water (4 times, 10 minutes each time), then rinse with 0.05% sodium hypochlorite solution (sodium hypochlorite inactivates the virus and does not damage the amnion; 10 minutes), then rinse with sterile water (4 times, 10 minutes each time).

[0052] (3) Place the cleaned amnion in the cryopreservation solution, take it out and place it on filter paper, spread it out fully, cover it with another layer of filter paper, freeze it at -80℃ for 1 hour, then thaw it at 37℃ for 0.5 hours, freeze it at -80℃ for 1 hour, and then thaw it at 37℃ for 0.5 hours (i.e., repeat the freeze-thaw cycle twice).

[0053] The cryopreservation solution was prepared by mixing glycerol and DMEM culture medium in a 1:1 volume ratio;

[0054] (4) The amnion after repeated freeze-thaw cycles was placed in 1% Triton X-100 solution and incubated at 37°C on a shaker (80 rpm / min) for 24 hours to initially remove cells; then the amnion was washed with PBS solution by shaking (10 minutes).

[0055] (5) Place the amnion in a 1000 U / L lipase solution and incubate at 37°C on a shaker (80 rpm / min) for 15 hours to fully remove epithelial cells and other impurities, while ensuring the integrity of the amnion structure and preserving the growth factors in the amnion, which account for 75% of the total growth factors in the amnion; then wash the amnion with PBS solution by shaking (3 times, 10 minutes each time).

[0056] The lipase was TYPE II L8070 lipase purchased from Solarbio Biotechnology Co., Ltd.

[0057] (6) Crosslinking of decellularized amnion: The amnion was placed in a 0.5% genipin aqueous solution and crosslinked at 20°C for 24 h, then rinsed with distilled water;

[0058] Place the amnion in a saturated glycine solution, soak it, change the solution until the solution no longer changes color, and rinse repeatedly with distilled water;

[0059] Place the amnion on aluminum foil at a height of 6 inches from a 254 nm UV lamp and irradiate for 2 hours.

[0060] Lay the amnion flat on the nitrocellulose membrane (with the chorionic membrane side facing the nitrocellulose membrane), press it under an iron tray, and freeze it at -40°C for 12 hours;

[0061] (7) Freeze-dry the frozen amnion at -30°C and 0.2 Mbar until the water content is 6% (about 6 hours). At this time, the amnion reaches a better dehydration state and will not generate free radicals during subsequent γ-ray sterilization.

[0062] (8) Cut the dried amnion into pieces, put them into plastic bags, vacuum seal them, and sterilize them by γ-ray (cobalt-60) irradiation.

[0063] The decellularized amniotic membrane prepared above was stained with hematoxylin and eosin (using standard experimental methods). The staining results are as follows: Figure 1 As shown, by Figure 1 It is evident that the decellularized amnion is rich in collagen tissue and has no cells on its surface.

[0064] Example 2: Preparation of adipose-derived stem cells

[0065] (1) Isolation of adipose-derived stem cells

[0066] ① Adipose tissue collection: The donor area is locally anesthetized with tumescent solution. A 20 ml syringe equipped with a liposuction cannula with a diameter of 1.5-3.5 mm and a side hole of 3.0-5.0 mm is inserted into the donor area to create negative pressure. 150 ml of adipose tissue is manually and evenly aspirated in a "fan-shaped" manner. The tumescent solution is composed of the following components: 250 ml of physiological saline, 10-15 ml of 2% lidocaine solution, and 0.25 mg of adrenaline.

[0067] Before collection, a comprehensive health check is conducted on the donor of adipose tissue (including general information, medical history, and family history of genetic diseases), as well as screening for pathogenic microorganisms and investigating their stay in high-risk epidemic areas. Requirements: good physical condition, complete medical examination report, no family history of genetic diseases, no malignant tumors, no acute or chronic infectious diseases, and no hematological diseases; normal complete blood count and differential, and normal liver and kidney function; negative for specific human viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.) and negative for Treponema pallidum. In addition to providing adipose tissue, the donor must also provide at least 8 ml of peripheral blood for retesting, ABO blood typing, and HLA-I and HLA-II typing for traceability.

[0068] ② Collect the adipose tissue and wash it with physiological saline: Put 100 ml of extracted fat into a fat collection bottle, and aliquot 50 ml of fat into a T175 culture flask; use a 10 ml pipette (without the pipette tip) to first aspirate and discard the upper yellow oil layer of the T175 culture flask, and then aspirate and discard the lower red liquid layer; add 100 ml of physiological saline to the T175 culture flask, shake vigorously for 3 minutes to thoroughly wash the adipose tissue, let it stand for 5 minutes, and aspirate the lower aqueous phase; repeat the washing operation of adipose tissue three times until the lower aqueous phase is clear, and record the final volume of the remaining washed adipose tissue in the T175 culture flask.

[0069] Add an equal volume of type I collagenase solution (concentration 0.05%, mass percentage) and digest for 60 min; filter through a sterile transfusion set filter and centrifuge at 400 g for 10 min; discard the oil and enzyme solution on the upper layer of the cell pellet; resuspend the cell pellet with physiological saline, disperse the cells, pass the cells through a 100-mesh filter, centrifuge at 400 g for 10 min, discard the supernatant, and obtain stromal vascular component cells (SVF).

[0070] The specific operation method of digestion is as follows: after adding enzyme solution, seal with sealing film, shake vigorously for 5-10 seconds, place in air bath constant temperature shaker, digest for 60 minutes at 37℃ and 70 rpm, and shake vigorously for 5-10 seconds every 15 minutes during digestion.

[0071] (2) Culture and passage of adipose stem cells

[0072] ① Primary culture: Resuspend the prepared matrix vascular component cells in 10 ml of serum-free culture system. After the cells are dispersed, add 8 ml of serum-free culture system. Seed the cells evenly into 3 T-75 culture flasks, and add 4 ml of serum-free culture system to each flask. Place the culture flasks horizontally in an incubator at 37°C and 5% carbon dioxide for routine culture.

[0073] The serum-free culture system consists of serum-free culture medium (Lonza, 12-725F) and serum-free additive (PALL, 15950-017) in a volume ratio of 50:1.

[0074] ② Cell medium change: After primary culture for 24 h, the medium was completely changed; thereafter, the medium was completely changed every 3 days, and the cells were placed in a carbon dioxide constant temperature and humidity (37℃, 95% humidity) incubator for routine cell culture with 5% carbon dioxide.

[0075] ③ Cell Collection: On day 8 of culture, when the primary cultured cells reach 80%–90% confluence, the cells are digested and harvested: the serum-free culture system in the culture flask is collected into a 50 ml centrifuge tube and centrifuged for 10 min (room temperature, 400 g); the T-75 culture flask is rinsed once with 20 ml of physiological saline, the saline is discarded, and 2 ml of 0.25% trypsin preheated at 37℃ is added to the T-75 culture flask; under a microscope, the cells are observed until 80% of them become round and detach and float, at which point the digestion is terminated with the centrifuged serum-free culture system. The digestion process is carried out at 37℃ for 4 min.

[0076] Collect the terminated cell suspension into a 50 ml centrifuge tube, wash the T-75 culture flask once with 10-20 ml of physiological saline, pour the washing solution into the 50 ml centrifuge tube, centrifuge at 300 g for 10 min, discard the supernatant, blow away the centrifuged cell pellet, dilute with physiological saline to 40 ml, centrifuge at 300 g for 10 min to obtain the cell pellet.

[0077] We perform quality and performance tests on cells (including bacteria, mycoplasma, endogenous and exogenous viruses, cell viability, cell identification, growth characteristics, cell purity, homogeneity, and endotoxins).

[0078] The morphology of primary cells and passaged cells (P2) is as follows: Figure 2 , Figure 3 As shown.

[0079] ④ Cryopreservation: Dilute the cell pellet obtained in the above steps with 1.5 ml of serum-free culture system and mix well; slowly add 1.5 ml of cryopreservation solution pre-cooled at 4℃ for 30 min and mix well; add 3 ml of the mixed cell suspension to 3 cryopreservation tubes, 1 ml per tube; freeze the cells using a programmed freezing device (cool to -80℃, 90 min) and then store them in a liquid nitrogen tank (-196℃) for later use.

[0080] The cryopreservation solution consists of 20% (volume percentage) DMSO (Sigma, D5879) and 80% serum-free culture system.

[0081] Example 3: Preparation of amnion loaded with adipose-derived stem cells

[0082] (1) Spread the decellularized amnion in a six-well plate and adjust the adipose-derived stem cell culture medium (primary cells from Example 2) to a seeding density of 1×10⁻⁶. 4 / ml, inoculated onto decellularized amnion membrane, 0.5 ml / cm 2 Place it in an incubator and incubate at 37°C with 5% carbon dioxide for 1 hour.

[0083] (2) Using sterile forceps, turn the decellularized amnion over and inoculate the other side of the decellularized amnion with 1 ml / cm of adipose-derived stem cell culture medium. 2 Place it in an incubator and incubate at 37°C with 5% carbon dioxide for 1 hour.

[0084] (3) Culture in a CO2 incubator at 37℃ with 5% CO2 for 7 days to obtain adipose-derived stem cells loaded with amniotic membrane. Change the medium once during this period. Take a small piece of adipose-derived stem cell-loaded amniotic membrane and perform HE staining, as shown... Figure 4 As shown, by Figure 4 It is evident that the decellularized amnion lacks cellular components, and cells with blue nuclei are visible on its surface, indicating that adipose-derived stem cells grow on the surface of the decellularized amnion.

[0085] Example 4: Preparation of autologous PRP gel

[0086] (1) Collect 50 ml of peripheral blood strictly according to aseptic requirements. Use a special blood bag (containing anticoagulant) to collect the blood. During the collection process, gently shake the blood bag up and down while collecting the blood to prevent clotting. Before collection, the patient needs to provide an infectious disease test report, including hepatitis A, hepatitis B, hepatitis C, hepatitis E, HIV, and syphilis, to ensure the authenticity and accuracy of the patient's infectious disease test.

[0087] (2) In a biosafety cabinet, transfer peripheral blood to a centrifuge tube, centrifuge at 550 g for 10 min at RT, and collect the upper layer of platelet-rich plasma.

[0088] (3) Centrifuge again (2000 g, 15 min) to adjust platelet concentration to an appropriate level (2 × 10⁻⁶). 9 Add thrombin (Penglai Nuokang Pharmaceutical Co., Ltd., H20041419) and calcium gluconate (Shanghai Pujin Linzhou Pharmaceutical Co., Ltd., H41022648) (PRP / activator 9:1), and add interleukin 10 (Yiqiao Shenzhou, ILO-H5219) 50 ng / ml and macrophage migration inhibitor factor (Novus Biological, USA, F60103H011) 30 ng / ml. Gel forms within 3 min.

[0089] Example 5: Construction of Artificial Fascia

[0090] (1) Place the decellularized amniotic membrane (2 cm × 4 cm) loaded with adipose stem cells (the thickness of the amniotic membrane is about 0.02 to 0.5 mm) on the bottom of a petri dish, use a syringe to draw up PRP gel (before solidification), remove the needle, spread a layer of PRP gel (0.3 mm) on the decellularized amniotic membrane, and wait for 3 min to solidify.

[0091] (2) Lay the polyurethane film (approximately 0.3 mm thick, 2 cm × 4 cm) flat on the above PRP gel.

[0092] (3) Use a syringe to draw up PRP gel (before solidification), remove the needle, spread a layer of PRP gel (0.3 mm) on the polyurethane membrane, and wait 3 minutes for solidification.

[0093] (4) Lay another piece of adipose stem cell-loaded decellularized amnion (2 cm × 4 cm) on PRP gel to obtain artificial fascia, which consists of 5 layers of materials in the following order: adipose stem cell-loaded amnion, PRP gel, polyurethane membrane, PRP gel, adipose stem cell-loaded amnion.

[0094] Biomechanical testing: The artificial fascia prepared above was stretched using a biomaterials mechanical testing machine probe until equilibrium was reached; the specimen length at this point is the initial test height. The specimen was subjected to 10 consecutive stretches at a rate of 0.084 cm / min each time. The proportionality constant between longitudinal stress and longitudinal strain is the elastic modulus of the material. Decellularized amnion was used as a control. The results are shown in Table 1.

[0095] Table 1

[0096] Grouping Number of specimens Elastic modulus (MPa) Decellularized amnion 5 2.67±0.36 Artificial Fascia 5 1.29±0.28

[0097] As shown in Table 1, the decellularized amniotic membrane has a larger elastic modulus than the artificial fascia, indicating that due to the addition of cellular components, the artificial fascia is softer and tougher, and is closer to the fascia tissue in the body.

[0098] Animal Experiment: Twelve New Zealand white rabbits (2 months old, weighing 1.5–2 kg) were randomly divided into two groups of six each. Group 1 received acellular amniotic membrane, and Group 2 received artificial fascia. The rabbits were anesthetized, placed in a lateral prone position, and the skin was incised along the midline of the back. Hemostasis was achieved, and the dorsal fascia was fully exposed. A 10 mm × 10 mm piece of fascia was removed. Absorbable sutures were used to suture and fix the corresponding size of acellular amniotic membrane or artificial fascia to the defect site, ensuring complete closure with appropriate suture tension. Animals in each group were sacrificed using the air embolization method at 14 and 60 days post-operation. Tissue from the implantation site on the back was collected for histological examination. Figure 5 As shown.

[0099] Depend on Figure 5 It was observed that both groups showed significant inflammatory cell infiltration 14 days post-operation. At 60 days post-operation, the artificial fascia group showed reduced inflammatory cell infiltration compared to the decellularized amniotic membrane group. The fascia collagen fibers exhibited good morphology and structure, with numerous fibroblasts growing between the collagen fibers. Only a small amount of inflammatory cell infiltration was observed at the implant boundary.

[0100] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. An artificial fascia, characterized in that: It is a composite material with a 5-layer structure, consisting of adipose-derived stem cell-loaded amniotic membrane, PRP gel, and polyurethane membrane. The 5-layer structure consists of the following materials in sequence: adipose-derived stem cell-loaded amniotic membrane, PRP gel, polyurethane membrane, PRP gel, and adipose-derived stem cell-loaded amniotic membrane; wherein the adipose-derived stem cell-loaded amniotic membrane is a decellularized amniotic membrane covered with adipose-derived stem cells. The thickness of the PRP gel is 0.2–0.4 mm; the thickness of the polyurethane film is 0.2–0.4 mm. The PRP gel contains 50 ng / ml interleukin-10 and 30 ng / ml macrophage migration inhibitory factor. The amniotic membrane loaded with adipose-derived stem cells is prepared by the following method: the decellularized amniotic membrane is laid flat on a culture plate, inoculated with a culture medium containing adipose-derived stem cells, and then the decellularized amniotic membrane is flipped over and inoculated with a culture medium containing adipose-derived stem cells on the other side; after culturing for 7 to 14 days, the amniotic membrane is obtained.

2. The artificial fascia according to claim 1, characterized in that, The amniotic membrane loaded with adipose-derived stem cells was prepared by the following method: decellularized amniotic membrane was laid flat in a six-well plate, and the concentration of the adipose-derived stem cell culture medium was adjusted to 1×10⁻⁶. 4 / ml, inoculated onto decellularized amnion membrane, 1 ml / cm 2 Place it in an incubator and incubate at 37°C with 5% carbon dioxide for 1 hour; (2) Using tweezers, flip the decellularized amnion over and inoculate the other side of the decellularized amnion with 1 ml / cm of adipose-derived stem cell culture medium. 2 Place it in an incubator and incubate at 37°C with 5% carbon dioxide for 1 hour; (3) After culturing at 37°C and 5% carbon dioxide for 7 days in an incubator, adipose-derived stem cells loaded with amniotic membrane are obtained.

3. The artificial fascia according to claim 1, characterized in that, The decellularized amnion was prepared by the following method: (1) Take a fresh amniotic membrane and soak it in a preliminary disinfection solution to disinfect the amniotic membrane and inhibit bacterial growth; (2) Remove the amnion, wash with sterile water, then wash with sodium hypochlorite solution, and then wash with sterile water again; (3) Immerse the cleaned amnion in the cryopreservation solution, take it out and place it on filter paper, spread it out fully, cover it with another layer of filter paper, and repeat the freeze-thaw cycle twice. (4) Place the amnion after repeated freeze-thaw cycles in Triton X-100 solution and incubate on a shaker to initially remove cells; Then the amnion membrane was washed with PBS solution by shaking. (5) Place the amnion in a lipase solution and incubate on a shaker to remove epithelial cells and other impurities. Then wash the amnion with PBS solution by shaking. (6) Place the amnion in a genipin solution to crosslink, then rinse with distilled water; Place the amnion in a saturated glycine solution, soak it, change the solution until the solution no longer changes color, and rinse repeatedly with distilled water; The amnion was placed on aluminum foil and irradiated with 254 nm ultraviolet light. Lay the amnion flat on the nitrocellulose membrane with the chorionic membrane side facing the nitrocellulose membrane, press it under the iron tray, and freeze it at -40℃ for 10 to 15 hours. (7) Freeze-dry the frozen amnion until the moisture content is 5% to 7%.

4. The artificial fascia according to claim 1, characterized in that, The adipose-derived stem cells were prepared by the following method: (1) Isolation of adipose stem cells: Take the collected adipose tissue, wash it with physiological saline; add an appropriate amount of type I collagenase solution or trypsin solution for digestion; filter and centrifuge; Discard the lipids and enzyme solutions on the upper layer of the cell pellet; resuspend the cell pellet in physiological saline, disperse it, filter it through a cell filter, centrifuge, and discard the supernatant to obtain the stromal vascular component cells; (2) Culture and passage of adipose stem cells: The stromal vascular component cells obtained above are cultured in a routine manner. When the cell confluence reaches 70% to 80%, they can be passaged. Collect P2 or P3 generation cells and freeze them for later use.

5. The artificial fascia according to claim 4, characterized in that, The specific washing procedure is as follows: Place the adipose tissue in a container, first remove the upper yellow oil and discard it, then remove the lower red liquid and discard it; add an appropriate amount of physiological saline to the remaining adipose tissue, shake vigorously for 3 minutes to thoroughly wash the adipose tissue, let it stand for 3-5 minutes, and remove the lower aqueous phase; repeat the washing procedure three times until the lower aqueous phase is clear. The specific operation method of digestion is as follows: after adding enzyme solution, seal with sealing film, shake vigorously for 5-10 seconds, place in air bath constant temperature shaker, digest for 60 minutes at 37℃ and 70 rpm, and shake vigorously for 5-10 seconds every 15 minutes during digestion.

6. The method for constructing the artificial fascia according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Place the amniotic membrane loaded with adipose-derived stem cells on the bottom of a petri dish, and then spread a layer of PRP gel on top of it; (2) Lay the polyurethane film flat on the above-mentioned PRP gel; (3) Lay a layer of PRP gel on the polyurethane membrane as described above; (4) Place another piece of adipose stem cell-loaded amniotic membrane on the above PRP gel to obtain artificial fascia.

7. The use of the artificial fascia according to any one of claims 1 to 5 in the preparation of a biological patch for treating hernia, or in the preparation of a mesh for treating pelvic organ prolapse.

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