Functional decellularized tissue engineering tracheal graft compounded with latissimus juglandis flap lining
By using a combination technology of decellularized tracheal stent and composite latsm flap ribbon muscle in tracheal transplantation, the problems of insufficient support, high difficulty in repairing large-scale tracheal defects, and high difficulty in immune rejection and revascularization of tracheal transplantation are solved, and the safe and effective repair of tracheal transplantation is achieved.
Patent Information
- Application Number
- CN202411719141.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-03-04
AI Technical Summary
Existing tracheal replacements face problems such as insufficient support, high difficulty in immune rejection and revascularization when repairing large-scale tracheal defects, resulting in repair failure and serious complications.
The preparation method of decellularized tracheal scaffold for tracheal transplantation is adopted, including repeated freeze-thaw and decellularization steps, to form a decellularized tracheal scaffold with a relatively complete structure and matrix, and lined with a composite lats and lats and flap, covering the ribbon muscle to form a functional decellularized tissue-engineering tracheal graft.
The ideal decellularization effect of the tracheal transplant stent is achieved, the structure and matrix are retained, the mechanical performance requirements of the tissue engineering stent is met, and the revascularization and internal and external blood circulation traffic are quickly achieved through the lining of the flap and the outer covering of the ribbon muscle, avoiding the problems of immune rejection and insufficient support.
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Abstract
Description
Technical Field
[0001] The invention relates to a functional acellular tissue engineering tracheal graft lined with a composite platysma muscle flap, and belongs to the technical field of tissue engineering and regenerative medicine. Background Art
[0002] The trachea is a component of the respiratory system. It is tubular and composed of cartilage, muscle, connective tissue and mucosa. The upper part is connected to the larynx, and the lower part has two branches to the lungs. Trauma, congenital stenosis, tumor diseases and other causes can lead to severe tracheal damage. Small tracheal defects can be repaired by pulling and suturing. However, for large tracheal defects (tracheal resection length >50% or more than 6cm in adults, >30% in children), direct "end-to-end anastomosis" may fail due to excessive tension on the anastomosis. Therefore, there is an urgent need to use tracheal substitutes to achieve safe repair of large tracheal defects.
[0003] The biggest special feature of tracheal repair lies in its unique blood supply. The blood supply of the trachea is segmental, and there is no direct central nutrient artery and drainage vein connected to the recipient vascular system. This segmental distribution of blood supply not only limits the scope of circumferential tracheal resection, but also greatly increases the difficulty of tracheal graft revascularization. Therefore, when constructing a tracheal substitute for repairing a defective trachea, it is necessary to focus on its revascularization ability to effectively achieve revascularization after tracheal substitute transplantation.
[0004] At present, there are three types of tracheal substitutes used in clinical practice: autologous flap tissue trachea, allogeneic tracheal transplantation, and tissue-engineered trachea. Among them, the autologous flap tissue trachea is mainly composed of tubular autologous tissue with a vascular pedicle, with additional supports as support. This type of trachea is well vascularized and has good tissue compatibility, but because it does not have the structure and function of the original trachea, it has the risk of stenosis and collapse, and lacks the ability of cilia to clear mucus. The allogeneic trachea is a donor trachea that is transplanted in situ after several weeks of ectopic revascularization. Compared with the autologous flap tissue trachea, it can retain the structure and function of the original trachea, has sufficient biomechanical properties, and has a good degree of revascularization, but it requires a donor, and the revascularization time is longer, and immunosuppressants need to be used. Tissue engineering trachea is to implant recipient stem cells in a biodegradable scaffold or acellular donor trachea, and fill the graft through stem cell differentiation and recruitment of surrounding cells. Compared with autologous flap tissue trachea and allogeneic tracheal transplantation, it has sufficient biomechanical properties and growth potential, and does not require immunosuppressants, but it has high requirements for transplantation technology, and revascularization is difficult, making it difficult to achieve blood supply reconstruction. It can be seen that although these three tracheal substitutes can achieve certain clinical effects, they cannot simultaneously overcome the problems of insufficient support, immune rejection, and lack of blood supply to the graft. When they are used to repair defective trachea, they often fail due to serious complications such as local infection, tracheal stenosis, immune rejection, tracheal softening, and granulation tissue formation. Summary of the invention
[0005] In order to solve the above problems, the present invention provides a decellularized tracheal stent for tracheal transplantation, wherein the preparation method of the decellularized tracheal stent for tracheal transplantation comprises a repeated freezing and thawing step and a decellularization step;
[0006] The repeated freezing and thawing steps include: cooling the animal trachea to -90--70°C at a cooling rate of -1.5--0.5°C / min, and then freezing it for 3-5 hours at -90--70°C to obtain a frozen animal trachea; thawing and rewarming the frozen animal trachea at 36-38°C until the frozen animal trachea is rewarmed to 36-38°C to obtain a thawed and rewarmed animal trachea; one freezing operation and one thawing and rewarming operation are regarded as one freezing and thawing cycle, and the freezing and thawing are repeated 2-4 times to obtain a frozen and thawed animal trachea;
[0007] The decellularization step comprises: soaking the frozen and thawed animal trachea in a sodium deoxycholate aqueous solution with a concentration of 3 to 5 g / mL, and then shaking at a constant temperature of 36 to 38° C. and 150 to 500 r / min for 5 to 8 hours to obtain the animal trachea treated with sodium deoxycholate; soaking the animal trachea treated with sodium deoxycholate in a physiological saline containing 1500 to 3500 KU / L DNase-I, first incubating at 36 to 38° C. for 3 to 6 hours, and then shaking at a constant temperature of 36 to 38° C. and 300 r / min for 5 to 8 hours to obtain the animal trachea treated with DNase-I; one sodium deoxycholate treatment and one DNase-I treatment are regarded as one decellularization cycle, and the decellularization is repeated 5 to 7 times to obtain a decellularized tracheal stent for tracheal transplantation.
[0008] In one embodiment of the present invention, before the repeated freezing and thawing step, the method for preparing the decellularized tracheal stent for tracheal transplantation further comprises a pretreatment step, a sterilization step and a cryoprotection step;
[0009] The pretreatment step comprises: killing the mammal, taking the full length trachea of the mammal under sterile conditions, and stripping the loose connective tissue on the trachea to obtain the animal trachea;
[0010] The sterilization step comprises: firstly flushing the animal trachea in physiological saline, and then soaking the trachea in an aqueous solution containing 100-250 mg / L gentamicin and 150,000-250,000 U / L penicillin for 2-10 minutes to obtain a sterilized animal trachea;
[0011] The cryoprotection step comprises: soaking the sterilized animal trachea in a cryoprotection solution, and then leaving it to stand at 0 to 8° C. for 15 to 30 minutes to obtain the soaked animal trachea.
[0012] In one embodiment of the present invention, the method for preparing the decellularized tracheal scaffold for tracheal transplantation comprises the following steps:
[0013] Pretreatment steps: killing the mammal, taking the full length trachea of the mammal under sterile conditions, and stripping the loose connective tissue on the trachea to obtain the animal trachea;
[0014] Sterilization step: rinse the animal trachea in physiological saline first, and then soak it in an aqueous solution containing 200 mg / L gentamicin and 200000 U / L penicillin for 3 minutes to obtain the sterilized animal trachea;
[0015] Cryoprotection step: After the sterilized animal trachea is immersed in the cryoprotection solution, it is placed in a 4°C refrigerator for 20 minutes to obtain the immersed animal trachea;
[0016] Repeated freezing and thawing steps: firstly cooling the immersed animal trachea to -80°C at a cooling rate of -1°C / min, and then continuously freezing at -80°C for 4 hours to obtain a frozen animal trachea; thawing and rewarming the frozen animal trachea at 37°C until the frozen animal trachea is rewarmed to 37°C to obtain a thawed and rewarmed animal trachea; one freezing operation and one thawing and rewarming operation is regarded as one freezing and thawing cycle, and repeated freezing and thawing is performed 3 times to obtain a frozen and thawed animal trachea;
[0017] Decellularization step: soak the frozen and thawed animal trachea in a sodium deoxycholate aqueous solution at a concentration of 4g / mL, and then shake it at a constant temperature of 37°C and 300r / min for 6 hours to obtain the animal trachea treated with sodium deoxycholate; soak the animal trachea treated with sodium deoxycholate in physiological saline containing 3000KU / L DNase-I, first incubate it at 37°C for 4 hours, and then shake it at a constant temperature of 37°C and 300r / min for 6 hours to obtain the animal trachea treated with DNase-I; take one sodium deoxycholate treatment and one DNase-I treatment as one decellularization cycle, cycle decellularization 6 times, and obtain a decellularized tracheal scaffold for tracheal transplantation.
[0018] The present invention also provides a functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap, the functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap comprising a platysma muscle flap, a strap muscle and the above-mentioned decellularized tracheal stent for tracheal transplantation; the platysma muscle flap is lined with the decellularized tracheal stent for tracheal transplantation, and the subcutaneous tissue side of the platysma muscle flap is in contact with the inner wall of the decellularized tracheal stent for tracheal transplantation; the strap muscle is wrapped around the outside of the decellularized tracheal stent for tracheal transplantation. The flap is composed of skin with blood supply (blood supply) and its attached subcutaneous tissue. The strap muscle is a smooth muscle, which is mainly distributed in internal organs, blood vessels, respiratory tract, digestive tract, etc. It is not controlled by will and is also called involuntary muscle. The decellularized tracheal stent is a tracheal substitute obtained by decellularizing the animal trachea.
[0019] In one embodiment of the present invention, the decellularized tracheal stent for tracheal transplantation is provided with a plurality of through holes.
[0020] In one embodiment of the present invention, the through holes have a diameter of 1 to 2 mm and a hole spacing of 3 to 5 mm.
[0021] In one embodiment of the present invention, the strap muscles include anterior cervical strap muscles; the anterior cervical strap muscles include the sternohyoid muscle and / or the sternothyroid muscle.
[0022] In one embodiment of the present invention, the method for preparing the functional acellular tissue-engineered tracheal graft lined with the composite platysma flap comprises the following steps:
[0023] Punching step: taking the above-mentioned decellularized tracheal stent for tracheal transplantation, cutting it longitudinally along the tracheal membrane, and punching the decellularized tracheal stent for tracheal transplantation to obtain a decellularized tracheal stent with through holes;
[0024] Plastic steps: Take the platysma muscle flap of the animal, keep the blood supply on the left and right sides of the flap, and suture the upper and lower ends of the flap into a tube with both ends not closed, so as to obtain the platysma muscle flap tube of the animal with the subcutaneous tissue side facing outward and the skin side facing inward;
[0025] Assembly steps: opening the membrane portion of the decellularized tracheal stent with through holes to obtain an opened decellularized tracheal stent; taking an animal platysma flap tube, and annularly fixing the opened decellularized tracheal stent on the outside of the animal platysma flap tube, so that the subcutaneous tissue side of the platysma flap is in contact with the inner wall of the decellularized tracheal stent, to obtain a decellularized tracheal stent lined with the platysma flap;
[0026] Implantation steps: Cut off the distal end of the strap muscle downward, retain the blood supply of the proximal end, and suture it to the outside of the decellularized tracheal stent lined with the platysma muscle flap, so as to obtain a decellularized tracheal stent covered with strap muscle and lined with platysma muscle flap; implant the decellularized tracheal stent covered with strap muscle and lined with platysma muscle flap as a whole in the subcutaneous tissue, and remove it after the internal and external blood supply is connected, so as to obtain a functional decellularized tissue engineering tracheal graft lined with a composite platysma muscle flap. When removing the decellularized tracheal stent covered with strap muscle and lined with platysma muscle flap, cut off the blood supply of the platysma muscle flap, and the blood supply of the strap muscle is continuous). When using, trim the graft to the size required for the tracheal defect to further repair the tracheal defect.
[0027] In one embodiment of the present invention, the punching step is: take the above-mentioned decellularized tracheal stent for tracheal transplantation, cut it longitudinally along the tracheal membrane, and use a puncher to punch the decellularized tracheal stent for tracheal transplantation to obtain a decellularized tracheal stent with through holes.
[0028] In one embodiment of the present invention, the plastic step is: taking an animal platysma muscle flap, retaining blood supply on the left and right sides of the flap, placing a columnar support on the skin side of the animal platysma muscle flap, and then suturing the upper and lower ends of the flap around the columnar support into a tube with both ends not closed, to obtain an animal platysma muscle flap tube with the subcutaneous tissue side facing outward and the skin side facing inward.
[0029] In one embodiment of the present invention, the columnar support comprises expanded sponge, nanocotton and / or gelatin sponge.
[0030] In one embodiment of the present invention, the assembly steps are: opening the membrane portion of a decellularized tracheal stent with a through hole to obtain an opened decellularized tracheal stent; taking an animal platysma flap tube, wrapping the opened decellularized tracheal stent in a ring shape around the outside of the animal platysma flap tube, and fixing it with sutures so that the subcutaneous tissue side of the platysma flap is in contact with the inner wall of the decellularized tracheal stent, thereby obtaining a decellularized tracheal stent lined with a platysma flap.
[0031] In one embodiment of the present invention, during the implantation step, when the decellularized tracheal stent covered with strap muscles and lined with platysma muscle flap is removed, the vascular pedicle on one side of the strap muscles is cut off and the vascular pedicle on the other side of the strap muscles is retained, so that the blood supply to the strap muscles is continuous.
[0032] In one embodiment of the present invention, the length of the platysma flap tube is 2 to 3 cm; the length of the decellularized tracheal stent is 2 to 2.5 cm.
[0033] The present invention also provides a method for preparing the functional acellular tissue engineering tracheal graft lined with the composite platysma flap, the method comprising the following steps:
[0034] Punching step: taking the above-mentioned decellularized tracheal stent for tracheal transplantation, cutting it longitudinally along the tracheal membrane, and punching the decellularized tracheal stent for tracheal transplantation to obtain a decellularized tracheal stent with through holes;
[0035] Plastic steps: Take the platysma muscle flap of the animal, keep the blood supply on the left and right sides of the flap, and suture the upper and lower ends of the flap into a tube with both ends not closed, so as to obtain the platysma muscle flap tube of the animal with the subcutaneous tissue side facing outward and the skin side facing inward;
[0036] Assembly steps: opening the membrane portion of the decellularized tracheal stent with through holes to obtain an opened decellularized tracheal stent; taking an animal platysma flap tube, and annularly fixing the opened decellularized tracheal stent on the outside of the animal platysma flap tube, so that the subcutaneous tissue side of the platysma flap is in contact with the inner wall of the decellularized tracheal stent, to obtain a decellularized tracheal stent lined with the platysma flap;
[0037] Implantation steps: Cut off the distal end of the strap muscle downward, retain the blood supply of the proximal end, and suture it to the outside of the decellularized tracheal stent lined with the platysma muscle flap, so as to obtain a decellularized tracheal stent covered with strap muscle and lined with platysma muscle flap; implant the decellularized tracheal stent covered with strap muscle and lined with platysma muscle flap as a whole in the subcutaneous tissue, and remove it after the internal and external blood supply is connected, so as to obtain a functional decellularized tissue engineering tracheal graft lined with a composite platysma muscle flap. When removing the decellularized tracheal stent covered with strap muscle and lined with platysma muscle flap, cut off the blood supply of the platysma muscle flap, and the blood supply of the strap muscle is continuous). When using, trim the graft to the size required for the tracheal defect to further repair the tracheal defect.
[0038] In one embodiment of the present invention, the through holes have a diameter of 1 to 2 mm and a hole spacing of 3 to 5 mm.
[0039] In one embodiment of the present invention, the strap muscles include anterior cervical strap muscles; the anterior cervical strap muscles include the sternohyoid muscle and / or the sternothyroid muscle.
[0040] In one embodiment of the present invention, the punching step is: take the above-mentioned decellularized tracheal stent for tracheal transplantation, cut it longitudinally along the tracheal membrane, and use a puncher to punch the decellularized tracheal stent for tracheal transplantation to obtain a decellularized tracheal stent with through holes.
[0041] In one embodiment of the present invention, the plastic step is: taking an animal platysma muscle flap, retaining blood supply on the left and right sides of the flap, placing a columnar support on the skin side of the animal platysma muscle flap, and then suturing the upper and lower ends of the flap around the columnar support into a tube with both ends not closed, to obtain an animal platysma muscle flap tube with the subcutaneous tissue side facing outward and the skin side facing inward.
[0042] In one embodiment of the present invention, the columnar support comprises expanded sponge, nanocotton and / or gelatin sponge.
[0043] In one embodiment of the present invention, the assembly steps are: opening the membrane portion of a decellularized tracheal stent with a through hole to obtain an opened decellularized tracheal stent; taking an animal platysma flap tube, wrapping the opened decellularized tracheal stent in a ring shape around the outside of the animal platysma flap tube, and fixing it with sutures so that the subcutaneous tissue side of the platysma flap is in contact with the inner wall of the decellularized tracheal stent, thereby obtaining a decellularized tracheal stent lined with a platysma flap.
[0044] In one embodiment of the present invention, during the implantation step, when the decellularized tracheal stent covered with strap muscles and lined with platysma muscle flap is removed, the vascular pedicle on one side of the strap muscles is cut off and the vascular pedicle on the other side of the strap muscles is retained, so that the blood supply to the strap muscles is continuous.
[0045] In one embodiment of the present invention, the length of the platysma flap tube is 2 to 3 cm; the length of the decellularized tracheal stent is 2 to 2.5 cm.
[0046] The present invention also provides the functional acellular tissue engineering tracheal graft lined with the composite platysma flap or the use of the method in preparing a product for repairing tracheal defects.
[0047] The technical solution of the present invention has the following advantages:
[0048] 1. The present invention provides a decellularized tracheal stent for tracheal transplantation. The preparation method of the decellularized tracheal stent for tracheal transplantation comprises a repeated freezing and thawing step and a decellularization step; the repeated freezing and thawing step comprises: cooling the animal trachea to -90 to -70°C at a cooling rate of -1.5 to -0.5°C / min, and then freezing at -90 to -70°C for 3 to 5 hours to obtain a frozen animal trachea; thawing and rewarming the frozen animal trachea at 36 to 38°C until the frozen animal trachea is rewarmed to 36 to 38°C. The trachea of the animal treated with sodium deoxycholate is obtained by immersing the trachea of the animal treated with sodium deoxycholate at 36-38°C and 150-500 r / min for 5-8 hours, and the trachea of the animal treated with sodium deoxycholate is obtained by immersing the trachea of the animal treated with sodium deoxycholate in a sodium deoxycholate aqueous solution with a concentration of 3-5 g / mL. After being placed in physiological saline containing DNase-I, it is first incubated at 36-38°C for 3-6 hours, and then constantly shaken at 36-38°C and 300r / min for 5-8 hours to obtain the animal trachea treated with DNase-I; one sodium deoxycholate treatment and one DNase-I treatment are regarded as a decellularization cycle, and the decellularization is repeated 5-7 times to obtain a decellularized tracheal stent for tracheal transplantation. Experiments have confirmed that the decellularized tracheal stent for tracheal transplantation can retain a relatively complete structure and matrix to meet the requirements of mechanical properties of tissue engineering scaffolds while achieving a very ideal decellularization effect. Therefore, the decellularized tracheal stent for tracheal transplantation has great application prospects in the preparation of products for repairing tracheal defects.
[0049] 2. The present invention provides a functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap, wherein the functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap comprises a platysma muscle flap, strap muscles and a decellularized tracheal stent for tracheal transplantation; the platysma muscle flap is lined with the decellularized tracheal stent for tracheal transplantation, and the subcutaneous tissue side of the platysma muscle flap is in contact with the inner wall of the decellularized tracheal stent for tracheal transplantation; the strap muscles are wrapped around the outside of the decellularized tracheal stent for tracheal transplantation. The functional decellularized tissue-engineered tracheal graft lined with the composite platysma muscle flap uses a decellularized tracheal stent for tracheal transplantation with multiple holes, is lined with a platysma muscle flap, and is covered with strap muscles on the outside. It effectively combines the advantages of three tracheal substitutes: autologous flap tissue trachea, allogeneic tracheal transplantation, and tissue-engineered trachea. It can not only retain the structure and function of the original trachea, but also has good biomechanical properties and growth potential, does not require the use of immunosuppressants, and can quickly achieve revascularization and internal and external blood transportation. At the same time, the functional decellularized tissue-engineered tracheal graft lined with the composite platysma muscle flap has good compatibility with autologous flap tissue. In addition, the functional decellularized tissue-engineered tracheal graft lined with the composite platysma muscle flap can quickly achieve re-epithelialization, and the skin epithelium lining the platysma muscle flap metaplasia into ciliated epithelium has good mucus clearance ability. Therefore, the functional decellularized tissue-engineered tracheal graft lined with the composite platysma muscle flap has great application prospects in the preparation of products for repairing tracheal defects. BRIEF DESCRIPTION OF THE DRAWINGS
[0050] Figure 1 : HE staining results of decellularized tracheal scaffolds for tracheal transplantation.
[0051] Figure 2 : DAPI staining results of decellularized tracheal scaffolds for tracheal transplantation.
[0052] Figure 3 : Results of MHCⅠ and MHCⅡ staining of decellularized tracheal scaffolds for tracheal transplantation.
[0053] Figure 4 : Results of COLⅡ staining of decellularized tracheal scaffolds for tracheal transplantation.
[0054] Figure 5 : Results of DNA residual detection in decellularized tracheal scaffolds for tracheal transplantation.
[0055] Figure 6 : Elastic modulus test results of decellularized tracheal scaffolds for tracheal transplantation.
[0056] Figure 7 :Elastic modulus testing process of decellularized tracheal scaffolds for tracheal transplantation.
[0057] Figure 8 : Scanning electron microscopic observation of decellularized tracheal scaffolds for tracheal transplantation.
[0058] Fig. 9 : Masson staining results of decellularized tracheal scaffolds for tracheal transplantation.
[0059] Fig.10 : Safranin O staining results of decellularized tracheal scaffolds for tracheal transplantation.
[0060] Fig.11 : Comparison of fresh normal trachea and decellularized tracheal scaffolds for tracheal transplantation.
[0061] Fig.12 : A decellularized tracheal stent with through holes.
[0062] Fig.13 : Platysma flap incision.
[0063] Fig.14 : Animal platysma flap tube.
[0064] Fig.15 : Acellular tracheal scaffold lined with platysma muscle flap (through-hole side).
[0065] Fig.16 : Acellular tracheal scaffold lined with platysma flap (suture side).
[0066] Fig.17 : Acellular tracheal scaffold covered with strap muscles and lined with platysma muscle flap.
[0067] Fig.18 : Results of skin alignment suturing on both sides (observation window retained).
[0068] Fig.19 : Auricular cartilage.
[0069] Fig. 20 : Acellular cartilage scaffolds lining the platysma flap.
[0070] Fig.21 : Acellular cartilage scaffold covered with strap muscles and lined with platysma muscle flap. DETAILED DESCRIPTION
[0071] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.
[0072] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.
[0073] Example 1: A decellularized tracheal stent for tracheal transplantation and its preparation
[0074] This embodiment provides a decellularized tracheal stent for tracheal transplantation. The preparation method of the decellularized tracheal stent for tracheal transplantation comprises the following steps:
[0075] Pretreatment steps: the animal (the animal here refers to a mammal) is killed by air embolism through the marginal ear vein, the full-length trachea is removed under sterile conditions, and the loose connective tissue on the trachea is stripped to obtain the animal trachea;
[0076] Sterilization step: rinse the animal trachea in physiological saline first, and then soak it in an aqueous solution containing 200 mg / L gentamicin and 200000 U / L penicillin for 3 minutes to obtain the sterilized animal trachea;
[0077] Cryoprotection step: After the sterilized animal trachea is placed in a sterile biological bag, a cryoprotectant solution (purchased from OriGen) is first added to the sterile biological bag so that the sterilized animal trachea is immersed in the cryoprotectant solution, and then the sterile biological bag is placed in a 4°C refrigerator for 20 minutes to obtain the immersed animal trachea;
[0078] Repeated freezing and thawing steps: After the soaked animal trachea is taken out from the sterile biological bag, it is placed in a freezer of a program cooling instrument, first cooled to -80°C at a cooling rate of -1°C / min, and then continuously frozen at -80°C for 4 hours to obtain a frozen animal trachea; after the frozen animal trachea is taken out from the freezer, it is placed in a 37°C constant temperature water bath for water bathing until the frozen animal trachea is rewarmed to 37°C to obtain a thawed and rewarmed animal trachea; one freezing operation and one thawing and rewarming operation is a freezing and thawing cycle, and the freezing and thawing are repeated 3 times to obtain a frozen and thawed animal trachea;
[0079] Decellularization step: After the frozen-thawed animal trachea is immersed in a 4% (w / v, g / mL) sodium deoxycholate aqueous solution, it is shaken at a constant temperature of 37°C and 300 r / min for 6 hours to obtain the animal trachea treated with sodium deoxycholate; after the animal trachea treated with sodium deoxycholate is immersed in physiological saline containing 3000KU / LDNase-I (purchased from Invitrogen), it is first incubated at 37°C for 4 hours, and then shaken at a constant temperature of 37°C and 300 r / min for 6 hours to obtain the animal trachea treated with DNase-I; one sodium deoxycholate treatment and one DNase-I treatment are regarded as one decellularization cycle, and the decellularization is repeated 6 times to obtain a decellularized tracheal stent for tracheal transplantation.
[0080] The decellularized tracheal scaffolds for tracheal transplantation were stored in PBS buffer (0.01 M, pH 7.4) at 4°C until tested and used.
[0081] Experimental Example 1: Performance Verification of Decellularized Tracheal Scaffolds for Tracheal Transplantation
[0082] This experimental example verifies the performance of the decellularized tracheal stent for tracheal transplantation prepared in Example 1. The verification process is as follows:
[0083] New Zealand rabbits were used as experimental animals, and the decellularized tracheal scaffolds for tracheal transplantation were prepared using the full-length trachea of the New Zealand rabbits according to the method of Example 1. The decellularized tracheal scaffolds for tracheal transplantation prepared according to Example 1 were subjected to HE staining, and the staining results are shown in FIG. Figure 1 The decellularized tracheal scaffold for tracheal transplantation prepared by the method of Example 1 was stained with DAPI. The staining results are shown in Figure 2 The decellularized tracheal scaffold for tracheal transplantation prepared by the method of Example 1 was stained with MHCⅠ and MHCⅡ. The staining results are shown in Figure 3 The decellularized tracheal scaffold for tracheal transplantation prepared by the method of Example 1 was subjected to COLⅡ staining. The staining results are shown in Figure 4 The decellularized tracheal stent for tracheal transplantation prepared by the method of Example 1 was tested for residual DNA. The test results are shown in Figure 5 The elastic modulus of the decellularized tracheal stent for tracheal transplantation prepared by the method of Example 1 was tested using a tensile machine. The test results are shown in Figure 6 , the detection process is shown in Figure 7 The decellularized tracheal scaffold for tracheal transplantation prepared by the method of Example 1 was observed using a scanning electron microscope. The observation results are shown in Figure 8 (SEM × 5000 times). The decellularized tracheal scaffold for tracheal transplantation prepared by the method of Example 1 was subjected to Masson staining. The staining results are shown in Fig. 9The decellularized tracheal scaffold for tracheal transplantation prepared by the method of Example 1 was stained with Safranin O. The staining results are shown in Fig.10 The comparison of fresh normal trachea and the decellularized tracheal scaffold for tracheal transplantation prepared by the method of reference Example 1 is shown in Fig.11 .
[0084] Figure 1 to Figure 4 , Figure 6 , Figure 8 to Figure 10 In the table, NT refers to fresh normal trachea, FDT refers to animal trachea that has undergone one decellularization cycle, TDF refers to animal trachea that has undergone three decellularization cycles, and SDT refers to animal trachea that has undergone six decellularization cycles. Figure 5 In the table, Control refers to fresh normal trachea, grounp1 refers to trachea of animals that have undergone three decellularization cycles, and grounp2 refers to trachea of animals that have undergone six decellularization cycles. * indicates p < 0.05 compared with NT.
[0085] Depend on Figures 1 to 10 The results show that after the treatment of Example 1, the decellularized tracheal scaffold for tracheal transplantation can retain a relatively complete structure and matrix to meet the requirements of mechanical properties of tissue engineering scaffolds while achieving a very ideal decellularization effect. Among them, programmed cooling, sodium deoxycholate treatment and DNase-I treatment have a great influence on the decellularization effect and mechanical properties of the decellularized tracheal scaffold for tracheal transplantation, so the operation needs to be strictly controlled.
[0086] Example 2: A functional acellular tissue-engineered tracheal graft lined with a composite platysma muscle flap and its preparation
[0087] The present embodiment provides a functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap, the functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap comprises a platysma muscle flap, anterior cervical strap muscles and a decellularized tracheal stent for tracheal transplantation prepared in Example 1; the platysma muscle flap is lined with the decellularized tracheal stent for tracheal transplantation, and the subcutaneous tissue side of the platysma muscle flap is in contact with the inner wall of the decellularized tracheal stent for tracheal transplantation; the anterior cervical strap muscles are wrapped around the outside of the decellularized tracheal stent for tracheal transplantation; a plurality of through holes are provided on the decellularized tracheal stent for tracheal transplantation; the through holes have a diameter of 1.5 mm and a hole spacing of 3.5 mm.
[0088] The method for preparing the functional acellular tissue engineering tracheal graft lined with a composite platysma muscle flap comprises the following steps:
[0089] Punching step: taking a 2 cm long decellularized tracheal stent for tracheal transplantation, cutting it longitudinally along the tracheal membrane, and using a sterile puncher to punch the decellularized tracheal stent for tracheal transplantation to obtain a decellularized tracheal stent with through holes;
[0090] Plastic steps: Take an animal platysma muscle flap with a length of 3 cm (the platysma muscle flap here refers to a platysma muscle flap with a width of about 2 cm in the middle of the neck, and the animal here refers to a mammal), retain the blood supply on both sides of the flap, place an expansion sponge on the skin side of the animal platysma muscle flap, and suture the upper and lower ends of the flap around the expansion sponge into a tube with both ends not closed, so as to obtain an animal platysma muscle flap tube with the subcutaneous tissue side facing outward and the skin side facing inward;
[0091] Assembly steps: opening the membrane portion of the decellularized tracheal stent with a through hole to obtain an opened decellularized tracheal stent; taking an animal platysma flap tube, wrapping the opened decellularized tracheal stent in a ring shape on the outside of the animal platysma flap tube, and fixing it with absorbable sutures so that the subcutaneous tissue side of the platysma flap is in contact with the inner wall of the decellularized tracheal stent, thereby obtaining a decellularized tracheal stent lined with the platysma flap;
[0092] Implantation steps: cut off the distal end of the anterior cervical strap muscle of the animal (here the animal refers to a mammal), retain the blood supply of the proximal end, and suture it to the outside of the decellularized tracheal stent lined with the platysma muscle flap, so as to obtain a decellularized tracheal stent covered with the strap muscle and lined with the platysma muscle flap; implant the decellularized tracheal stent covered with the strap muscle and lined with the platysma muscle flap as a whole in the subcutaneous tissue, and take it out after the internal and external blood supply is connected, so as to obtain a functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap; when taking out the decellularized tracheal stent covered with the anterior cervical strap muscle and lined with the platysma muscle flap, cut off the blood supply of the platysma muscle flap, and cut off the vascular pedicle on one side of the anterior cervical strap muscle, retaining the vascular pedicle on the other side of the anterior cervical strap muscle, so as to ensure continuous blood supply to the anterior cervical strap muscle.
[0093] Experimental Example 2: Performance verification of a functional acellular tissue-engineered tracheal graft lined with a composite platysma muscle flap
[0094] The performance verification experiment of the functional acellular tissue engineering tracheal graft lined with the composite platysma muscle flap was carried out with reference to Example 2. The experimental process is as follows:
[0095] The decellularized tracheal stent for tracheal transplantation prepared in Experimental Example 1 with a length of 2 cm was cut longitudinally along the tracheal membrane, and the decellularized tracheal stent for tracheal transplantation was punched with a sterile puncher to obtain a decellularized tracheal stent with through holes; the through holes had a diameter of 1.5 mm and a hole spacing of 3.5 mm (the decellularized tracheal stent with through holes is shown in FIG. Fig.12 );
[0096] New Zealand rabbits were used as experimental animals. Two parallel incisions with a length of 3 cm were made on the neck of the experimental animals. After placing an expansion sponge between the two parallel incisions, the platysma flap between the two parallel incisions was sutured around the expansion sponge to form a tube with both ends not closed, thus obtaining an animal platysma flap tube with the subcutaneous tissue side facing outward and the skin side facing inward. The spacing between the two parallel incisions was 3 cm and the incision length was 3 cm. At this time, the two ends of the animal platysma flap tube were still connected to the neck of the experimental animals and were not separated from the blood supply (see the incision of the platysma flap). Fig.13 , animal platysma flap tube Fig.14 );
[0097] The membrane portion of the decellularized tracheal stent with a through hole is cut open to obtain an opened decellularized tracheal stent; at the platysma flap tube of the experimental animal, the opened decellularized tracheal stent is wrapped around the outside of the platysma flap tube of the experimental animal and then fixed with absorbable sutures so that the subcutaneous tissue side of the platysma flap is in contact with the inner wall of the decellularized tracheal stent to obtain a decellularized tracheal stent lined with the platysma flap; at this time, the decellularized tracheal stent lined with the platysma flap is connected to the neck of the experimental animal through the inner platysma flap tube of the animal and is not disconnected from the blood supply (the decellularized tracheal stent lined with the platysma flap is shown in FIG. 1 ). Figure 15-16 );
[0098] The distal end of the anterior cervical strap muscle of the experimental animal was cut off, the blood supply of the proximal end was retained, and it was sutured to the outside of the decellularized tracheal stent lined with the platysma muscle flap, so as to obtain a decellularized tracheal stent covered with strap muscle and lined with platysma muscle flap; at this time, the decellularized tracheal stent lined with platysma muscle flap was connected to the neck of the experimental animal through the inner platysma muscle flap tube of the animal and the outer anterior cervical strap muscle at the same time, and was not separated from the blood supply (the decellularized tracheal stent covered with strap muscle and lined with platysma muscle flap is shown in FIG. Fig.17 );
[0099] The skin on both sides of the two parallel incisions were sutured in position, so that the decellularized tracheal stent covered with strap muscles and lined with platysma muscle flaps was implanted subcutaneously, and observation windows were made at both ends to observe the changes of the decellularized tracheal stent covered with strap muscles and lined with platysma muscle flaps every day (the results of the suture of the skin on both sides are shown in Fig.18 );
[0100] After 4 weeks of continuous observation through the observation window, it was found that the decellularized tracheal stent covered with strap muscle and lined with platysma muscle flap quickly achieved revascularization and internal and external blood flow. At this time, the decellularized tracheal stent covered with strap muscle and lined with platysma muscle flap buried under the skin was taken out and used as a functional decellularized tissue engineering tracheal graft lined with a composite platysma muscle flap for the repair of tracheal defects. When removing the decellularized tracheal stent covered with anterior cervical strap muscle and lined with platysma muscle flap, it is necessary to cut off the blood supply of platysma muscle flap, and cut off one side of the vascular pedicle of the anterior cervical strap muscle, retaining the other side of the vascular pedicle of the anterior cervical strap muscle, so that the blood supply of the anterior cervical strap muscle is continuous. Among them, when the decellularized tracheal stent for tracheal transplantation without perforation was used as a control to prepare a functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap, it was found that the implantation process could not quickly achieve revascularization, the internal and external blood circulation could not communicate, and the graft quickly necrotized and could not be used. Therefore, the decellularized tracheal stent for tracheal transplantation needed to be perforated.
[0101] When the functional decellularized tissue-engineered tracheal graft lined with the removed composite platysma muscle flap was transplanted into the trachea of a New Zealand rabbit, the blood supply to the tissue-engineered trachea was still guaranteed after the tissues at both ends of the muscle flap were cut off. This result further confirmed that the functional decellularized tissue-engineered tracheal graft lined with the composite platysma muscle flap that can be used for tracheal defect repair was successfully constructed. In actual use, the functional decellularized tissue-engineered tracheal graft lined with the composite platysma muscle flap can be trimmed to the size of the tracheal defect to repair the defect.
[0102] In summary, the functional decellularized tissue engineering tracheal graft lined with the composite platysma flap provided in Experimental Example 2 uses a decellularized tracheal stent for tracheal transplantation with multiple holes, lined with platysma flap, and covered with strap muscle on the outside, which effectively combines the advantages of three tracheal substitutes: autologous flap tissue trachea, allogeneic tracheal transplantation, and tissue engineering trachea. It can not only retain the structure and function of the original trachea, but also has good biomechanical properties and growth potential, does not require the use of immunosuppressants, and can quickly achieve revascularization and internal and external blood transportation. In addition, the platysma flap is simple to make, with materials collected nearby and good vascularization effect, which is convenient for further repair of defective trachea.
[0103] Comparative Example 1: A functional acellular tissue-engineered tracheal graft lined with a composite platysma muscle flap
[0104] The present comparative example provides a functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap, wherein the functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap is: on the basis of Example 2, the decellularized tracheal scaffold for tracheal transplantation in Example 1 is replaced with a decellularized cartilage scaffold for tracheal transplantation; wherein the preparation method of the decellularized cartilage scaffold for tracheal transplantation is: on the basis of Example 1, the full-length trachea of an adult New Zealand rabbit is replaced with auricular cartilage of an adult New Zealand rabbit.
[0105] Experimental Example 3: Performance verification of a functional acellular tissue-engineered tracheal graft lined with a composite platysma muscle flap
[0106] The performance verification experiment of the functional acellular tissue engineering tracheal graft lined with the composite platysma muscle flap prepared in Comparative Example 1 was conducted as follows:
[0107] On the basis of Experimental Example 2, the decellularized tracheal scaffold for tracheal transplantation prepared in Experimental Example 1 was replaced with a decellularized cartilage scaffold for tracheal transplantation; wherein, the preparation method of the decellularized cartilage scaffold for tracheal transplantation is as follows: on the basis of Experimental Example 1, the full-length trachea of a New Zealand rabbit was replaced with auricular cartilage of a New Zealand rabbit (auricular cartilage see Fig.19 , acellular cartilage scaffold lined with platysma flap Fig. 20 The results of skin suture on both sides are shown in Fig.21 ). When using acellular cartilage scaffolds for tracheal transplantation, the auricular cartilage is cut into strips and wrapped around the outside of the platysma flap tube of the animal without drilling.
[0108] After 4 weeks of continuous observation through the observation window, it was found that although the acellular cartilage scaffold covered with strap muscles and lined with platysma muscle flaps also achieved rapid revascularization and internal and external blood circulation, the strip auricle cartilage after cutting was insufficient to effectively replace blood vessels. Considering this point, the experiment was repeated using uncut auricle cartilage and uncut auricle cartilage with through holes, which also failed to achieve effective vascularization.
[0109] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.
Claims
1. A decellularized tracheal stent for tracheal transplantation, characterized in that: The method for preparing the decellularized tracheal stent for tracheal transplantation comprises a repeated freezing and thawing step and a decellularization step; The repeated freezing and thawing steps include: cooling the animal trachea to -90--70°C at a cooling rate of -1.5--0.5°C / min, and then freezing it for 3-5 hours at -90--70°C to obtain a frozen animal trachea; thawing and rewarming the frozen animal trachea at 36-38°C until the frozen animal trachea is rewarmed to 36-38°C to obtain a thawed and rewarmed animal trachea; one freezing operation and one thawing and rewarming operation are regarded as one freezing and thawing cycle, and the freezing and thawing are repeated 2-4 times to obtain a frozen and thawed animal trachea; The decellularization step comprises: soaking the frozen and thawed animal trachea in a sodium deoxycholate aqueous solution with a concentration of 3 to 5 g / mL, and then shaking at a constant temperature of 36 to 38° C. and 150 to 500 r / min for 5 to 8 hours to obtain the animal trachea treated with sodium deoxycholate; soaking the animal trachea treated with sodium deoxycholate in a physiological saline containing 1500 to 3500 KU / L DNase-I, first incubating at 36 to 38° C. for 3 to 6 hours, and then shaking at a constant temperature of 36 to 38° C. and 300 r / min for 5 to 8 hours to obtain the animal trachea treated with DNase-I; one sodium deoxycholate treatment and one DNase-I treatment are regarded as one decellularization cycle, and the decellularization is repeated 5 to 7 times to obtain a decellularized tracheal stent for tracheal transplantation.
2. The decellularized tracheal stent for tracheal transplantation according to claim 1, characterized in that: Before the repeated freezing and thawing step, the method for preparing the decellularized tracheal stent for tracheal transplantation further comprises a pretreatment step, a sterilization step and a cryoprotection step; The pretreatment step comprises: killing the mammal, taking the full length trachea of the mammal under sterile conditions, and stripping the loose connective tissue on the trachea to obtain the animal trachea; The sterilization step comprises: firstly flushing the animal trachea in physiological saline, and then soaking the trachea in an aqueous solution containing 100-250 mg / L gentamicin and 150,000-250,000 U / L penicillin for 2-10 minutes to obtain a sterilized animal trachea; The cryoprotection step comprises: soaking the sterilized animal trachea in a cryoprotection solution, and then leaving it to stand at 0 to 8° C. for 15 to 30 minutes to obtain the soaked animal trachea.
3. The decellularized tracheal stent for tracheal transplantation according to claim 1 or 2, characterized in that: The method for preparing the decellularized tracheal stent for tracheal transplantation comprises the following steps: Pretreatment steps: killing the mammal, taking the full length trachea of the mammal under sterile conditions, and stripping the loose connective tissue on the trachea to obtain the animal trachea; Sterilization step: rinse the animal trachea in physiological saline first, and then soak it in an aqueous solution containing 200 mg / L gentamicin and 200000 U / L penicillin for 3 minutes to obtain the sterilized animal trachea; Cryoprotection step: After the sterilized animal trachea is immersed in the cryoprotection solution, it is placed in a 4°C refrigerator for 20 minutes to obtain the immersed animal trachea; Repeated freezing and thawing steps: firstly cooling the immersed animal trachea to -80°C at a cooling rate of -1°C / min, and then continuously freezing at -80°C for 4 hours to obtain a frozen animal trachea; thawing and rewarming the frozen animal trachea at 37°C until the frozen animal trachea is rewarmed to 37°C to obtain a thawed and rewarmed animal trachea; one freezing operation and one thawing and rewarming operation is regarded as one freezing and thawing cycle, and repeated freezing and thawing is performed 3 times to obtain a frozen and thawed animal trachea; Decellularization step: soak the frozen and thawed animal trachea in a sodium deoxycholate aqueous solution at a concentration of 4g / mL, and then shake it at a constant temperature of 37°C and 300r / min for 6 hours to obtain the animal trachea treated with sodium deoxycholate; soak the animal trachea treated with sodium deoxycholate in physiological saline containing 3000KU / L DNase-I, first incubate it at 37°C for 4 hours, and then shake it at a constant temperature of 37°C and 300r / min for 6 hours to obtain the animal trachea treated with DNase-I; take one sodium deoxycholate treatment and one DNase-I treatment as one decellularization cycle, cycle decellularization 6 times, and obtain a decellularized tracheal scaffold for tracheal transplantation.
4. A functional acellular tissue-engineered tracheal graft lined with a composite platysma muscle flap, characterized in that: The functional decellularized tissue-engineered tracheal graft lined with the composite platysma flap comprises a platysma flap, strap muscles and the decellularized tracheal stent for tracheal transplantation according to any one of claims 1 to 3; the platysma flap is lined with the decellularized tracheal stent for tracheal transplantation, and the subcutaneous tissue side of the platysma flap is in contact with the inner wall of the decellularized tracheal stent for tracheal transplantation; the strap muscles are wrapped around the outside of the decellularized tracheal stent for tracheal transplantation.
5. The functional acellular tissue engineering tracheal graft lined with a composite platysma muscle flap as claimed in claim 4, characterized in that: The decellularized tracheal stent for tracheal transplantation is provided with a plurality of through holes; the diameter of the through holes is 1 to 2 mm, and the hole spacing is 3 to 5 mm.
6. The functional acellular tissue engineering tracheal graft lined with a composite platysma muscle flap as claimed in claim 4, characterized in that: The strap muscles include the anterior cervical strap muscles; the anterior cervical strap muscles include the sternohyoid muscle and / or the sternothyroid muscle.
7. The functional acellular tissue engineering tracheal graft lined with a composite platysma muscle flap according to any one of claims 4 to 6, characterized in that: The method for preparing the functional acellular tissue engineering tracheal graft lined with a composite platysma muscle flap comprises the following steps: Punching step: taking the decellularized tracheal stent for tracheal transplantation according to any one of claims 1 to 3, cutting it longitudinally along the tracheal membrane, and punching the decellularized tracheal stent for tracheal transplantation to obtain a decellularized tracheal stent with through holes; Plastic steps: Take the platysma muscle flap of the animal, keep the blood supply on the left and right sides of the flap, and suture the upper and lower ends of the flap into a tube with both ends not closed, so as to obtain the platysma muscle flap tube of the animal with the subcutaneous tissue side facing outward and the skin side facing inward; Assembly steps: opening the membrane portion of the decellularized tracheal stent with through holes to obtain an opened decellularized tracheal stent; taking an animal platysma flap tube, and annularly fixing the opened decellularized tracheal stent on the outside of the animal platysma flap tube, so that the subcutaneous tissue side of the platysma flap is in contact with the inner wall of the decellularized tracheal stent, to obtain a decellularized tracheal stent lined with the platysma flap; Implantation steps: cut off the strap muscle downward at the distal end, retain the blood supply to the proximal end, and suture it to the outside of the decellularized tracheal stent lined with the platysma muscle flap, so as to obtain a decellularized tracheal stent covered with the strap muscle and lined with the platysma muscle flap; implant the decellularized tracheal stent covered with the strap muscle and lined with the platysma muscle flap as a whole in the subcutaneous tissue, and take it out after the internal and external blood supply is connected, so as to obtain a functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap.
8. The functional acellular tissue engineering tracheal graft lined with a composite platysma muscle flap as claimed in claim 6, characterized in that: The punching step comprises: taking the decellularized tracheal stent for tracheal transplantation according to any one of claims 1 to 3, cutting it longitudinally along the tracheal membrane, and punching the decellularized tracheal stent for tracheal transplantation with a puncher to obtain a decellularized tracheal stent with through holes; The plastic step comprises: taking an animal platysma muscle flap, preserving blood supply on both sides of the flap, placing a columnar support on the skin side of the animal platysma muscle flap, and suturing the upper and lower ends of the flap around the columnar support into a tube shape with both ends not closed, so as to obtain an animal platysma muscle flap tube with the subcutaneous tissue side facing outward and the skin side facing inward; The assembly steps are as follows: opening the membrane portion of a decellularized tracheal stent with a through hole to obtain an opened decellularized tracheal stent; taking an animal platysma flap tube, wrapping the opened decellularized tracheal stent in a ring shape around the outside of the animal platysma flap tube, and fixing it with sutures so that the subcutaneous tissue side of the platysma flap is in contact with the inner wall of the decellularized tracheal stent, thereby obtaining a decellularized tracheal stent lined with a platysma flap.
9. A method for preparing a functional acellular tissue engineering tracheal graft lined with a composite platysma muscle flap as claimed in any one of claims 4 to 8, characterized in that: The method comprises the following steps: Punching step: taking the decellularized tracheal stent for tracheal transplantation according to any one of claims 1 to 3, cutting it longitudinally along the tracheal membrane, and punching the decellularized tracheal stent for tracheal transplantation to obtain a decellularized tracheal stent with through holes; Plastic steps: Take the platysma muscle flap of the animal, keep the blood supply on the left and right sides of the flap, and suture the upper and lower ends of the flap into a tube with both ends not closed, so as to obtain the platysma muscle flap tube of the animal with the subcutaneous tissue side facing outward and the skin side facing inward; Assembly steps: opening the membrane portion of the decellularized tracheal stent with through holes to obtain an opened decellularized tracheal stent; taking an animal platysma flap tube, and annularly fixing the opened decellularized tracheal stent on the outside of the animal platysma flap tube, so that the subcutaneous tissue side of the platysma flap is in contact with the inner wall of the decellularized tracheal stent, to obtain a decellularized tracheal stent lined with the platysma flap; Implantation steps: cut off the strap muscle downward at the distal end, retain the blood supply to the proximal end, and suture it to the outside of the decellularized tracheal stent lined with the platysma muscle flap, so as to obtain a decellularized tracheal stent covered with the strap muscle and lined with the platysma muscle flap; implant the decellularized tracheal stent covered with the strap muscle and lined with the platysma muscle flap as a whole in the subcutaneous tissue, and take it out after the internal and external blood supply is connected, so as to obtain a functional decellularized tissue-engineered tracheal graft lined with a composite platysma muscle flap.
10. Use of the decellularized tracheal stent for tracheal transplantation according to any one of claims 1 to 3, the functional decellularized tissue engineering tracheal graft lined with a composite platysma flap according to any one of claims 4 to 8, or the method according to claim 9 in preparing a product for repairing tracheal defects.