Tissue engineering mucous membrane construction method based on degradable double-disc tracheal fistula patch and application

Abstract

The invention discloses a tissue engineering mucous membrane construction method based on a degradable double-disc tracheal fistula patch and application. Based on a degradable double-disc structure embedded tracheal fistula patch (CRP), an artificial mucous membrane is constructed on the surface of the CRP by utilizing a tissue engineering means to obtain an epithelialization patch (E-CRP); meanwhile, the E-CRP loaded with the tissue engineering mucosa is used for repairing a rabbit tracheal fistula model, the effect is investigated, and the result shows that the ordered regeneration and re-epithelization process of the defective part is accelerated while the tracheal fistula is effectively blocked.

Description

Technical Field

[0001] The invention belongs to the technical field of medical tissue repair materials, and relates to a tissue engineering mucosa construction and application thereof that can be used for tracheal fistula blocking. Background Art

[0002] Tracheal fistula (TF) refers to a pathological condition in which the trachea communicates with adjacent cavities or organs such as the pleural cavity, esophagus, and stomach, forming a fistula or fistula opening. This disease is complex and has a high mortality rate. It is a difficult point in thoracic surgery and requires timely treatment. According to literature reports, if patients with malignant TF are only given supportive treatment, their median survival period is only 1-6 weeks. If treatment is not timely, they may die in 6-12 weeks. 1 .

[0003] The current clinical treatment methods for TF mainly include surgical treatment and stent occlusion. Traditional surgical repair is not only cumbersome, but also has problems such as severe postoperative local infection, edema and fibrosis of the tissue around the fistula, and short-term repair effect. 2–4 Stent occlusion of TF can quickly restore the integrity of the airway, relieve dyspnea, and quickly relieve symptoms. This method is less invasive, simple to operate, and relatively safe in the short term, and has become an important treatment for TF.

[0004] The choice of stent is the key to the success of TF occlusion repair. Silicone-coated metal stents have become the first choice for tracheal stents in patients with malignant TF due to their excellent stability and flexibility. 5,6 In recent years, many new types of stents have been studied, such as biodegradable stents, drug-eluting stents, 3D printed stents, and tissue engineering stents. 7–11 A study at Ruijin Hospital used 3D printing technology to make tracheal Y-stents and customized tracheal stents for 6 patients, which effectively overcame the shortcomings of insufficient occlusion and easy displacement. However, it is only suitable for patients with small fistulas and may cause complex complications, making it difficult to promote. 12,13 Scholars at home and abroad have gained inspiration from the atrial appendage occluder in cardiology and tried to use the double-disc Amplatzer device to block tracheoesophageal fistula and tracheopleural fistula, which has overcome the shortcomings of traditional stents to a certain extent. 14,15 .

[0005] In addition, the current TF stent repair still has problems such as low occlusion rate, stent displacement and fracture, inadequate fistula occlusion, intraluminal neoplasia, and difficulty in re-epithelialization of the repaired area. 16–19 The double-disc metal stent currently used in clinical practice can only play a temporary filling role due to its non-degradability, and cannot achieve tissue regeneration and repair. It may even irritate or damage the nearby trachea and esophageal tissue, causing chronic inflammatory reactions and lifelong rejection problems. Even if the occlusion is successful, it is still possible that scar tissue and stenosis will form in the trachea due to difficulties in re-epithelialization.

[0006] In recent years, more and more studies have demonstrated the importance of re-epithelialization in tissue engineering tracheal reconstruction. 20–22 . Tracheal re-epithelialization refers to the process in which the epithelial cells that originally covered the trachea are damaged or shed after the trachea is damaged or inflamed, and the surrounding undifferentiated basal stem cells grow, migrate, and re-differentiate to repair the damaged area. Re-epithelialization can cover the damaged area, restore the structural integrity of the damaged mucosa, promote the regeneration and repair of mucosal cells, ensure the normal defense function of the airway, and promote the healing process of the injury, thereby reducing the occurrence of complications such as infection, bleeding, and scar tissue hyperplasia, improving the prognosis of the disease, and increasing the patient's survival rate and quality of life. 23,24 In tracheal defect repair, re-epithelialization is the key factor for successful repair. If the defect area cannot be successfully re-epithelialized, it may cause tracheal stenosis, occlusion and other problems. 25 .

[0007] At present, there is no effective solution to the problem of re-epithelialization of tracheal repair areas. This is because there are many difficulties in re-epithelialization of tracheal repair. First, the migration and regeneration ability of the tracheal mucosal epithelium is limited. Especially in the case of severe injury or chronic inflammation, it is difficult to re-epithelialize the tracheal injury area. 25,26 Secondly, when tracheal mucosal epithelial cells are extracted and cultured in vitro, usually only the airway basal cells of the basal layer can be separated. Basal cells are a type of multifunctional stem cells, but their proliferation and differentiation abilities are very limited, making it difficult to meet the needs of large-area tracheal repair. 23,27,28 Finally, during tracheal repair, the low transplant survival rate of epithelial cells greatly limits tracheal re-epithelialization.

[0008] Therefore, studying how to effectively block tracheal fistula and achieve re-epithelialization of tracheal defect is an important research direction in the field of tracheal repair. We need to develop a new method to construct tissue engineering mucosa on the surface of double-disc patch, so that while efficiently blocking tracheal fistula, re-epithelialization of tracheal defect can be effectively achieved, so as to improve the treatment effect of tracheal fistula and the quality of life of patients. Summary of the invention

[0009] In order to solve the problem of difficulty in re-epithelialization of the TF repair area, the present invention has developed a tissue engineering artificial mucosa construction method based on a degradable double-disc structure embedded patch, which is used to repair TF and promote re-epithelialization of the repair area. The traditional method of culturing tracheal epithelial cells is to immerse the cells in a culture medium for culture; however, the cells in this culture system cannot undergo mucociliary differentiation. In order to reproduce the layered mucociliary phenotype observed in vivo, tracheal epithelial cells must be cultured on an air-liquid interface system. The main feature of air-liquid interface culture is that the basal surface of the cell is in contact with the liquid culture medium, while its apical surface is exposed to the air. A common cell inoculation method is to inoculate the cells onto a permeable membrane of a cell culture insert. In the early stage of cell culture, both the apical and basal chambers need to be added with culture medium. When the number of cells is close to full, the culture medium in the apical chamber is blown dry and the culture medium in the basal chamber is retained. This culture system simulates the situation in the human airway and induces cells to differentiate into a mucociliary phenotype.

[0010] Based on this idea, the first aspect of the present invention is based on a degradable double-disc structure embedded TF patch (CRP), and uses tissue engineering methods to construct an artificial mucosa on the surface of CRP to obtain E-CRP; secondly, the rabbit TF model is repaired using E-CRP loaded with tissue engineering mucosa, and the effect is investigated. The results show that while effectively blocking TF, it accelerates the orderly regeneration and re-epithelialization process of the defect site.

[0011] To achieve the above object, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides a method for constructing a tissue engineering mucosa based on a degradable double-disc tracheal fistula patch, comprising the following steps:

[0013] A. Isolation and expansion of airway basal cells

[0014] The mammalian tracheal mucosa was isolated in vitro and the harvested tracheal mucosa was cut into 1 mm 2 The small pieces were washed thoroughly in PBS containing 1% antibiotics and digested with 0.15% collagenase at 37°C for 1 hour. The digestion solution was then filtered through a filter and resuspended and centrifuged to obtain airway basal cells. The isolated airway basal cells were isolated in PneumaCult TM -Ex medium, and when the cells reached 90% confluence, they were passaged and the second generation of airway basal cells were used to pre-construct E-CRP.

[0015] B. Pre-built E-CRP

[0016] After subculturing, cells were collected and treated with PneumaCult TM -Ex medium was resuspended to a concentration of 6*10 7cell / ml high concentration cell suspension was added dropwise to the surface of CRP, incubated at 37°C for 4 hours, and then PneumaCult TM -Ex culture medium was used to keep the liquid level below the level of the inoculated cells and cultured for 3 days.

[0017] C. E-CRP culture

[0018] Replace PneumaCult TM -ALI culture medium, construct an air-liquid interface culture system ALIS, so that the basal surface of the cells is in contact with the culture medium and the apical surface is exposed to the air, further inducing the differentiation of airway basal cells on CRP and constructing E-CRP loaded with artificial mucosa; the culture medium is changed every two days, and after 4 weeks of in vitro induction, the tissue-engineered epithelialized E-CRP patch is harvested.

[0019] Preferably, the double-disc tracheal fistula patch of the present invention is prepared using silk fibroin.

[0020] The mammalian tracheal mucosa comes from humans, mice, rabbits, monkeys, sheep or pigs.

[0021] In a second aspect, the present invention provides a re-epithelialized tissue engineering mucosa, which is prepared by any of the methods described above.

[0022] The present invention systematically verified the constructed tissue engineering mucosa E-CRP from three aspects: structure, characteristic proteins and specific gene expression, and confirmed that the constructed E-CRP successfully achieved tissue engineering reconstruction of airway mucosal epithelium in both structural and functional dimensions in vitro. The results of the tracheal fistula animal model experiment showed that the effect of E-CRP after 6 weeks of repair was basically the same as that of natural trachea, and it fully helped the process of re-epithelialization and epithelial cilia in the defect area.

[0023] The third aspect of the present invention provides the use of the above-mentioned re-epithelialized tissue engineering mucosa in the regeneration and repair of tracheal fistula.

[0024] A fourth aspect of the present invention provides a tracheal fistula regeneration and repair component, comprising the above-mentioned re-epithelialized tissue engineering mucosa.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This study is based on a degradable double-disc embedded TF patch (CRP). Tissue engineering methods were used to construct an artificial mucosa on the surface of CRP to obtain E-CRP for blocking TF, while accelerating the orderly regeneration and re-epithelialization process of the defect site, thereby solving the problem of re-epithelialization of the tracheal fistula defect area.

[0027] From a technical perspective, E-CRP is based on degradable biomaterials, so it has high biocompatibility and can integrate well with the patient's autologous tissue, reducing the risk of rejection and infection. At the same time, because E-CRP is loaded with artificial mucosa, it can achieve rapid epithelialization in the tracheal cavity of the transplant, thereby exerting the physiological function of the airway, which helps to restore the patient's respiratory function and reduce the occurrence of complications. This technology is expected to become an important method for constructing active patches for tracheal fistulas, bringing new hope to the treatment of patients with tracheal fistulas.

[0028] From a socioeconomic perspective, E-CRP can save patients whose lives are threatened by tracheal diseases or injuries and provide them with hope for rebirth. It can effectively repair damaged trachea and enable patients to breathe normally, thus greatly improving their quality of life. Traditional tracheal treatments may require complex surgeries and long-term medical care, which imposes a heavy economic burden on patients and their families. As an emerging treatment method, E-CRP is expected to reduce the medical burden and reduce patients' medical costs by reducing the number of surgeries and shortening recovery time. With the continuous maturity of technology and the gradual expansion of the market, it is expected to bring huge economic benefits to related industries. In addition, the successful application of E-CRP not only provides new options for the treatment of tracheal diseases, but also promotes the development of tissue engineering and regenerative medicine in the medical field, and provides new ideas for the repair and regeneration of other organs and tissues.

[0029] In summary, E-CRP has significant advantages in terms of technical, social and economic effects. With the continuous development of technology and the gradual expansion of the market, it is expected to bring good news to more patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The overall design and repair effect of tissue engineering mucosa construction based on degradable double-disc tracheal fistula patch are shown.

[0031] Figure 2The reconstruction and in vitro characterization of tissue-engineered mucosa based on a degradable double-disc tracheal fistula patch are shown: (A) Construction process of the embedded patch (E-CRP) for tissue-engineered mucosa reconstruction; (B) Light microscopy images and specific immunofluorescence staining of in vitro expanded airway basal cells; (C) Panoramic and local images of E-CRP inoculated with airway basal cells after calcein staining, scale: 1 mm, 200 μm; (D) Fluorescence staining of live and dead cells 1 / 3 / 5 / 7 days after E-CRP inoculated with airway basal cells; (E) Statistical graph of the proportion of live cell fluorescence intensity at 1 / 3 / 5 / 7 days; (F) 3D structure of E-CRP after calcein staining after 21 days of culture in the air-liquid interface system (ALIS) Image; (G) SEM observation of the regenerated airway mucosa morphology in the cross-section and surface of E-CRP at different magnifications. The green arrow indicates the reconstructed airway mucosa layer. Scale bars: 50 μm, 10 μm; (H) H&E staining and immunofluorescence staining of epithelial markers, including cell nucleus DAPI (blue), ZO-1 (red), Muco5AC (yellow) and AC-Tubulin (green); (I) qPCR detection of the gene expression profile of airway basal cell marker genes (p63, ck-5); (J) qPCR detection of the gene expression profile of goblet cells (Muc5B) and ciliated cells (FoxJ1). The gene expression data have been normalized according to the housekeeping gene β-actin.

[0032] Figure 3 The results of mucosal reconstruction patch repair of rabbit tracheal fistula after 6 weeks: (A) Schematic diagram of E-CRP repair of tracheal fistula; (B) X-ray and CT imaging results: the red arrow indicates the repair site of tracheal fistula, L: left side; R: right side; H: head side; F: foot side; A: front; P: back; (C) Bronchoscopy image of E-CRP repair after 6 weeks; (D) Gross image of the inside and outside of the trachea after E-CRP repair after 6 weeks; (E) Histological staining results of tracheal axial section; (FK) Comparative evaluation of E-CRP repair and natural gas trachea after 6 weeks. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The key clinical problem is that the repaired area of ​​TF patch is difficult to re-epithelialize. Figure 1As shown, the present invention designs a method for tissue engineering airway mucosal epithelial reconstruction based on the double-disc structure embedded patch CRP, constructs a double-disc patch E-CRP loaded with artificial mucosa, which can effectively accelerate the tissue regeneration and re-epithelialization process in the repair area on the basis of achieving instant repair of airway defects and stably maintaining airway patency, thereby preventing tracheal stenosis.

[0035] The following describes the reconstruction method of tissue engineering mucosa and the application of tracheal fistula occlusion using a silk fibroin double-disc structured tracheal fistula patch as a carrier.

[0036] 1. Reconstruction and structural characterization of tissue-engineered mucosa

[0037] Artificial mucosal epithelium of CRP was reconstructed in vitro using tissue engineering methods, and a re-epithelialized embedded repair membrane (E-CRP) was constructed. A series of in vitro characterizations of E-CRP were performed.

[0038] The tracheal epithelium of the respiratory tract is a pseudostratified ciliated columnar epithelium, which is mainly composed of three types of cells: ciliated cells, secretory cells (mainly goblet cells that secrete mucus) and basal cells. When tracheal mucosal epithelial cells are extracted and cultured in vitro, usually only the basal layer of airway basal cells can be isolated. Basal cells are a type of multifunctional stem cell, but their proliferation and differentiation capabilities are very limited. Figure 2 As shown in A, the construction idea of ​​the present invention is to extract airway basal cells from the rabbit tracheal mucosa, culture them after in vitro amplification and inoculate them on the surface of CRP. Since the traditional two-dimensional (2D) immersion culture system only supports cells with basal cell phenotype and cannot reflect the real situation of airway epithelium, we use an air-liquid interface system (ALIS) to simulate the living environment of airway epithelium in vitro, induce the airway basal cells on CRP to differentiate into airway-specific pseudostratified ciliated columnar epithelium, thereby constructing artificial re-epithelialized CRP (E-CRP), as follows:

[0039] First, airway basal cells with repair and differentiation functions were isolated and expanded from the tracheal mucosa of New Zealand rabbits in vitro. The harvested mucosa was cut into 1 mm 2 The small pieces were washed thoroughly in PBS containing 1% antibiotics and digested with 0.15% collagenase at 37°C for 1 hour. Afterwards, the digestion solution was filtered through a filter and resuspended and centrifuged to obtain airway basal cells. The isolated airway basal cells were isolated in PneumaCult TM -Ex medium (STEMCELL, USA) was used for culture. When the cells reached 90% confluence, they were passaged and the second generation of airway basal cells were used for subsequent experiments.

[0040] The airway basal cells were collected after passage and expanded, and then the cells were purified by PneumaCult TM-Ex medium was resuspended to a concentration of approximately 6×10 7 cell / ml high concentration cell suspension was added dropwise to the surface of CRP, incubated at 37°C for 4 hours, and then the culture medium was slowly added (the liquid level should not exceed the plane of the inoculated cells). TM -Ex expansion medium was replaced with PneumaCult TM -ALI (STEMCELL, USA) culture medium was used to construct an air-liquid interface culture system (ALIS) to further induce the differentiation of airway basal cells on CRP, thereby constructing E-CRP loaded with artificial mucosa. The culture medium was changed every two days, and after 4 weeks of in vitro induction, the tissue-engineered epithelialized E-CRP patch was harvested.

[0041] The morphological structure of the expanded airway basal cells was verified under a light microscope, and their characteristic proteins were verified by specific immunofluorescence staining of CK-5 and p63 ( Figure 2 B), proving that the airway basal cells extracted and amplified by the present invention have sufficient vitality and purity. Subsequently, the airway basal cells amplified in vitro were inoculated onto the surface of CRP, and the living cells were stained with calcein. Through full-field fluorescence imaging, it can be seen that the airway basal cells basically cover the entire surface of the patch ( Figure 2 C). Live and dead cell staining was performed on basal cells cultured on CRP for 1, 3, 5, and 7 days ( Figure 2 D) and the corresponding fluorescence intensity analysis ( Figure 2 E), we can see that airway basal cells can stably adhere and survive on the CRP surface, and as the culture time increases, the basal cells can stably proliferate. When the basal cells are expanded to completely cover the CRP surface, we transfer the pre-constructed E-CRP to ALIS for culture, prompting the airway basal cells to differentiate on the CRP surface. After 21 days of induction of differentiation, we stained the E-CRP surface with calcein again. Through 3D fluorescence imaging, we can see that some positive cells (red arrows) have cilia-like structures ( Figure 2 F), indicating that the airway basal cells on the E-CRP we constructed have undergone preliminary differentiation. Figure 2 G. By observing the cross section and surface of E-CRP through a scanning electron microscope, we can see that there is a layer of tightly laid tissue on the surface of E-CRP that is closely connected to CRP. The above results structurally prove the initial successful reconstruction of the artificial mucosal epithelial layer on E-CRP.

[0042] In order to prove the differentiation of basal cells on E-CRP from the perspective of protein function, we selected the tracheal epithelium as a protective barrier with three key characteristics: tight junction proteins maintain the integrity of the epithelium; mucins capture particulate matter and pathogens; and the movement of airway cilia transports these particulate matter and pathogens out of the airway. The E-CRP of the reconstructed epithelium was subjected to histological and immunofluorescence staining: the H&E results showed that the scaffold structure was intact, the airway epithelium basically only existed on a single side of the scaffold, and the cells were distributed continuously and tightly; the junction protein ZO-1 proved that the airway basal cells formed tight connections with each other; Muco5AC positivity proved that some airway basal cells had differentiated into mucus cells, which have the function of secreting mucin; AC-tubulin staining proved that the cells expressed abundant tubulin, suggesting that the cells initially differentiated into ciliated columnar epithelium ( Figure 2 H).

[0043] In order to verify the differentiation of airway basal cells on E-CRP at the gene expression level, we selected three groups of samples: airway basal cells before differentiation, airway cells not cultured on ALIS, and E-CRP cultured on ALIS. We performed qPCR detection on the three main cell marker genes of pseudostratified ciliated columnar epithelium: basal cell gene markers (ck5, TP63), differentiated goblet cell gene markers (muc5B), and ciliated cell gene markers (FoxJ1). Figure 2 From the mRNA gene expression profiles of I and 2J, we can see that the airway cells on E-CRP have undergone significant changes in gene expression after differentiation and culture on ALIS. The gene expression of genes representing basal cells is downregulated, and the gene expression of genes representing goblet cells and ciliated cells is significantly upregulated, while the gene expression of airway basal cells that have not been cultured with ALIS does not change significantly. These two results, on the one hand, prove that the ALIS culture we proposed is essential for the differentiation of airway basal cells on E-CRP, and on the other hand, prove that the airway basal cells we inoculated have differentiated in terms of gene expression.

[0044] In summary, we have systematically verified E-CRP from three aspects: structure, characteristic proteins and specific gene expression, proving that the E-CRP we constructed successfully achieved tissue engineering reconstruction of airway mucosal epithelium in vitro in both structural and functional dimensions.

[0045] 2. Animal Model Experiments

[0046] In order to verify the effect of E-CRP on the re-epithelialization of tracheal fistula after tissue engineering mucosal epithelium reconstruction, E-CRP loaded with artificial mucosal epithelium was used to repair tracheal fistula in rabbits. After 6 weeks of repair, the re-epithelialization and epithelial cilia of the airway mucosa were compared with those of natural gastritis ( Figure 3 A).

[0047] from Figure 3 The X-ray and CT imaging results of B show that the tracheal fistula area repaired by E-CRP has a patency of the tracheal wall that is basically the same as the normal trachea, without obvious stenosis and gaps. In addition, no gaps can be seen in the trachea after CT reconstruction, indicating that, like the CRP at 6 weeks, the E-CRP has been degraded and mature tissue has been formed inside. Figure 3 C Under bronchoscope, it can be seen that the inner wall of the lumen in the patch area loaded with artificial mucosa is smooth and continuous, and the mucosa is fully covered. Figure 3 In the anatomical image of D, it can be seen that the inside and outside of the tracheal fistula repair area are basically integrated with the surrounding normal tissue. Similarly, we calculated the diameter (3F) and cross-sectional area (3G) of the tracheal fistula repair area on CT imaging and compared them with the diameter and cross-sectional area of ​​the natural gas tube. It can be seen that the effect of E-CRP after 6 weeks of repair is basically the same as that of the natural gas tube.

[0048] To further compare the internal repair effect, we also performed histological and immunofluorescence staining on the samples after 6 weeks of E-CRP repair, e.g. Figure 3 E. H&E and Masson staining showed that there was abundant tissue infiltration and collagen deposition inside the E-CRP repair area. β-Tubulin and Muco5AC staining showed that a large number of fibroblasts and mucus cells fully infiltrated the interior of the E-CRP. For the re-epithelialization and epithelial cilia of the trachea, E-CRP showed amazing epithelial repair ability. At 6 weeks, not only was the pck coverage rate 100%, but the fluorescence intensity ratio of the ciliated cell marker FoxJ1 to PCK exceeded 100%, indicating that the construction of artificial mucosa on E-CRP fully helped the process of re-epithelialization and epithelial cilia of the defect area.

[0049] We used ImageJ to statistically quantify the immunofluorescence staining results of the E-CRP repair area and the natural duct area to evaluate the repair effect of E-CRP at 6 weeks: the fluorescence ratio of β-Tubulin (3H) showed that the infiltration degree of fibroblasts in the E-CRP group was basically close to the natural duct at 6 weeks; the fluorescence ratio of Muco5A (3I) showed that the infiltration degree of mucus cells in the E-CRP group was no different from the natural duct; the PCK fluorescence density (3J) showed that the re-epithelialization of the artificial epithelium constructed by E-CRP was even better than that of the natural epithelium; the FoxJ1 / PCK ratio (3K) showed that the re-epithelialization degree of the artificial epithelium constructed by E-CRP was not only high, but also the degree of epithelial cilia exceeded that of the natural duct. So far, E-CRP has significantly promoted the re-epithelialization effect of the defective part of the tracheal fistula through the method of tissue engineering to regenerate the airway mucosa, and perfectly solved the problem of re-epithelialization in the process of tracheal fistula repair.

[0050] Conclusion

[0051] TF has always been a major clinical problem. Traditional surgery and stent occlusion methods have extremely high treatment risks and are difficult to overcome the problem of re-epithelialization. In recent years, with the rapid development of tissue engineering and regenerative medicine, the use of degradable biological scaffold patches and tissue engineering mucosal regeneration methods to repair TF has become a new treatment strategy.

[0052] In order to solve the problem of difficulty in re-epithelialization of the TF repair area, this study developed a tissue engineering artificial mucosa construction method based on a double-disc structure embedded patch to repair TF and promote re-epithelialization of the repair area. First, airway basal cells with repair and differentiation functions were isolated and expanded in vitro, and the airway basal cells were loaded onto the double-disc structure CRP surface and induced to differentiate in the air-liquid interface system (ALIS), thereby constructing E-CRP loaded with artificial mucosa. We used E-CRP to repair the rabbit tracheal fistula model, and the imaging and histological results proved that this method can perfectly solve the problem of re-epithelialization of the tracheal repair area.

[0053] This technology is expected to become an important method for constructing active patches for tracheal fistulas, thereby helping TF patients recover their respiratory function and reduce the occurrence of clinical complications. It has broad application prospects in tracheal fistula repair and brings new hope to the treatment of TF patients.

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[0083] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for constructing tissue engineering mucosa based on a degradable double-disc tracheal fistula patch, characterized in that: The steps include: A. Isolation and expansion of airway basal cells: Isolate and expand airway basal cells with repair and differentiation functions from mammalian tracheal mucosa in vitro; B. Pre-constructed E-CRP: After passage, cells were collected and treated with PneumaCult TM -Ex medium was used to prepare a high concentration cell suspension after resuspending, and then added to the surface of CRP. After incubation, PneumaCult TM -Ex medium, so that the liquid level does not exceed the plane of the inoculated cells, and culture for 3 days; C. E-CRP culture: Replace PneumaCult TM -ALI culture medium, construct an air-liquid interface culture system ALIS, so that the basal surface of the cells is in contact with the culture medium and the apical surface is exposed to the air, further inducing the differentiation of airway basal cells on CRP, constructing E-CRP loaded with artificial mucosa, and after a certain period of in vitro induction, a tissue-engineered epithelialized E-CRP patch is obtained.

2. The method for constructing tissue engineering mucosa based on a double-disc tracheal fistula patch according to claim 1, characterized in that: In step A, the airway basal cells are isolated and expanded as follows: the harvested tracheal mucosa is cut into 1 mm 2 The small pieces were washed thoroughly in PBS containing 1% antibiotics and digested with 0.15% collagenase at 37°C for 1 hour. The digestion solution was then filtered with a filter and resuspended and centrifuged to obtain airway basal cells. Isolation of airway basal cells in PneumaCult TM -Ex medium, and when the cells reached 90% confluence, they were passaged and the second generation of airway basal cells were used to pre-construct E-CRP.

3. The method for constructing tissue engineering mucosa based on a double-disc tracheal fistula patch according to claim 1, characterized in that: In step B, the second generation of airway basal cells was treated with PneumaCult TM -Ex medium was resuspended to a concentration of 6*10 7 Cells were suspended at a high concentration of 50 cells / ml and dripped onto the surface of the CRP. After incubation at 37°C for 4 hours, the culture medium was slowly added so that the liquid level did not exceed the plane of the inoculated cells.

4. The method for constructing tissue engineering mucosa based on a double-disc tracheal fistula patch according to claim 1, characterized in that: In step C, during the in vitro induction process, the culture medium was changed every two days, and after 4 weeks of in vitro induction, the tissue-engineered epithelialized E-CRP patch was harvested.

5. The method for constructing tissue engineering mucosa based on a double-disc tracheal fistula patch according to claim 1, characterized in that: The double-disc tracheal fistula patch is prepared by using biological materials such as silk fibroin, collagen, gelatin, and decellularized extracellular matrix.

6. The method for constructing tissue engineering mucosa based on a double-disc tracheal fistula patch according to claim 1, characterized in that: The mammalian tracheal mucosa is derived from humans, mice, rabbits, monkeys, sheep or pigs.

7. A re-epithelialized tissue engineering mucosa, characterized in that: The method is prepared by any one of claims 1 to 6.

8. Use of the re-epithelialized tissue engineering mucosa according to claim 7 in the regeneration and repair of tracheal fistula.

9. A tracheal fistula regeneration and repair component, characterized in that: Comprising the re-epithelialized tissue-engineered mucosa as described in claim 8.

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