Preparation method and application of double-disc structural tracheal fistula patch

Abstract

The invention discloses a preparation method and application of a double-disc structural tracheal fistula patch. The method comprises the following steps: constructing a silk fibroin (SF)-based double-disk structure embedded repair patch (CRP) with a physical and chemical double-crosslinking network; and by utilizing the ultra-light weight, good elasticity and water absorption, the double-sided disc-shaped structure can be embedded into a defect repair position, so that the effect of tight repair is achieved. The tracheal fistula can be stably repaired in situ, complications such as low success rate, stent displacement fracture, insufficient fistula orifice plugging and new biological hyperplasia in a lumen caused by a traditional stent are avoided, re-epithelization of a defect part is effectively promoted, the stent is expected to become an ideal stent for treating the tracheal fistula, and a new hope is brought to treatment of the tracheal fistula.

Description

Technical Field

[0001] The invention belongs to the technical field of medical tissue repair materials and relates to a preparation method and application of a double-disc structured tracheal fistula patch. 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. Currently, the main clinical methods for treating TF include surgical treatment and stent closure. Surgical treatment usually involves resection of tissues around the fistula opening, followed by suturing or reconstruction. For some patients with larger fistula tissue defects, in addition to directly suturing the fistula opening, it is often necessary to use autologous tissues such as pericardium, muscle tissue, pedicled skin flaps, etc. for repair. 1,2 In addition to conventional surgery, thoracoscopic and mediastinoscopy surgeries also have successful cases. The surgeon often needs to design a personalized surgical method based on the nature and size of the fistula. If necessary, partial tracheal resection and anastomosis, lobectomy, etc. are also required. Not only is the process cumbersome, but there are also certain limitations, such as severe local infection after surgery, edema and fibrosis of the tissue around the fistula, and the repair effect is not lasting. 2 Stent occlusion of the fistula can quickly restore the integrity of the digestive tract or respiratory tract and relieve symptoms quickly. This method is less invasive, simple to operate, and relatively safe in the short term. It has become an important treatment for TF. 3 .

[0003] The selection of stents is the key to successful occlusion repair. Commonly used tracheal stents include silicone stents, uncoated metal stents, hybrid stents (metal stents covered with silicone membranes, polypropylene membranes) and stents under development (biodegradable stents, drug-eluting stents, 3D printed stents), etc. 4–6 The coated metal stent has the characteristics of both metal stents and silicone stents, with an ideal inner diameter, self-expansion and good fit with the tracheal wall, small mobility, little inhibitory effect on mucociliary function, a variety of diameters to choose from, can be placed under bronchoscopy, and is easy to place. It has become the preferred tracheal stent for patients with malignant TF. 7 After the fistula is blocked, the airway can be quickly rebuilt, breathing difficulties can be relieved, and fluid intake can be restored, which can improve the quality of life and prolong life. However, the success rate of the above TF stent repair is 70% to 100%, and there are also problems such as stent displacement and fracture, inadequate fistula blocking, neoplasia in the lumen, and difficulty in re-epithelialization of the repaired area. 8–11In recent years, several teams have tried to use autologous fat pads and fill them into the fistula through bronchoscopes, achieving good clinical results. A study by Ruijin Hospital applied 3D printing technology to the production of tracheal Y-stents, customizing tracheal stents for 6 patients, effectively overcoming the shortcomings of insufficient closure and easy displacement of tracheal stents. However, it is only suitable for patients with smaller fistulas and is difficult to be widely promoted. 12 In addition, scholars at home and abroad have also tried to use the Amplatzer device for atrial septal defect closure to block tracheoesophageal fistula and tracheopleural fistula, and have achieved certain results. 13,14 In addition, the Over-the-scope clip (OTSC) anastomotic clip system has been gradually promoted at home and abroad in recent years. Due to its advantages such as large wingspan, strong bite force, and precise positioning, it can achieve a treatment effect close to surgical suture under endoscopy, and to a certain extent overcomes the shortcomings of traditional stents that are easy to shift and fall off. 15,16 .

[0004] As mentioned above, surgical repair has problems such as severe local infection after surgery, edema and fibrosis of tissue around the fistula, and short-lasting repair effect. Current stent repair has problems such as unstable success rate, stent displacement and breakage, inadequate closure of the fistula, proliferation of neoplasms in the lumen, and difficulty in re-epithelialization of the repair area.

[0005] In recent years, more and more studies have demonstrated the importance of tracheal re-epithelialization in tissue engineering tracheal reconstruction. 17–19 . 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. 20,21 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. 22 At present, there is no effective solution to the problem of re-epithelialization of the tracheal repair area. This is because the migration and regeneration capacity 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. 22,23 .

[0006] Therefore, there is a need for a new type of tracheal repair stent that can effectively block tracheal fistula while promoting tracheal re-epithelialization and preventing complications during the re-epithelialization process, so as to improve the treatment effect of tracheal fistula and the quality of life of patients. Summary of the invention

[0007] The present invention is directed to the above-mentioned problems and provides a double-disc structured tracheal fistula patch and a preparation method thereof. The technical idea of ​​the present invention is as follows: construct a double-disc structured embedded repair patch (CRP) with a physical and chemical double cross-linked network based on silk fibroin (SF); utilizing its ultra-light weight, good elasticity and water absorption, the double-sided disc-shaped structure enables it to be embedded in the defect repair position to achieve a tight repair effect. A stable in situ repair of tracheal fistula is achieved, avoiding complications such as low success rate, stent displacement and fracture, insufficient fistula closure, and neoplasia in the lumen caused by traditional stents, and effectively promoting re-epithelialization of the defective part.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing a double-disc structured tracheal fistula patch, comprising the following steps:

[0010] (1) Mold preparation

[0011] The mold preparation method includes: making the mold by 3D printing or by stacking and assembling multiple layers of orifice plates.

[0012] For personalized molds, double-disc models are prepared through three-dimensional modeling, 3D printing, and the reverse molding method.

[0013] For molds for mass production, the mold is formed by assembling a multi-layer perforated plate. The multi-layer perforated plate has four layers, from the bottom to the top, the first layer is used as the bottom plate and no holes are set. The centers of the holes of the second to fourth layers are aligned, and the raw material liquid is poured into them to form a double-disc structured tracheal fistula patch of the corresponding shape according to the shape of the holes.

[0014] Since the tracheal fistula needs to be blocked, a disc larger than the fistula size needs to be set inside and outside the trachea at the same time, and a connecting part that can pass through the fistula hole is required to achieve fistula blocking. Therefore, the size of the holes made in the second and fourth layers is larger than the size of the holes made in the third layer.

[0015] In terms of mold material, a material with a smooth surface and strong resistance to high and low temperatures is selected. Preferably, the multi-layer perforated plate of the present invention is made of silicate glass, acrylic glass, polytetrafluoroethylene, stainless steel, etc.;

[0016] The shape can be designed according to the shape of the fistula, for example, the second and fourth layers of the plate can be made of circular, elliptical, oblong or other irregular shapes. The third layer of the plate has a cylindrical or special shape, which is slightly larger than the size of the fistula, so that it can be stuck in the fistula and block the hollow.

[0017] (2) Preparation of SF porous scaffolds

[0018] A 3-15% w / v silk fibroin aqueous solution is placed in an ice bath and stirred continuously to prevent the SF from undergoing a β-folding reaction due to changes in the surrounding temperature. The SF solution is then mixed with a cross-linker and a catalyst and placed in a mold. The epoxide groups at both ends of the cross-linker combine with the amino and hydroxyl groups on the silk fibroin to establish a chemical fiber cross-linking network to prepare a SF porous scaffold.

[0019] Preferably, the concentration of the silk fibroin aqueous solution is 5% w / v, the concentration of the crosslinking agent is 3 mmol / g, the concentration of the catalyst is 0.25 v / v%, and the stirring speed of the silk fibroin aqueous solution is 10-100 rpm.

[0020] The crosslinking agent is selected from any one of ethylene glycol diglycidyl ether (EGDE), glycerol diglycidyl ether (GDGE), butanediol diglycidyl ether (BUDGE), ethylene glycol diglycidyl ether (EDGE) and poly (ethylene glycol) diglycidyl ether (PEGDGE); the catalyst is selected from any one of N,N,N',N'-tetramethylethylenediamine, ammonium persulfate and ammonium sulfate.

[0021] (3) Freeze shaping

[0022] The prepared SF porous scaffold was placed in a -80°C refrigerator for rapid freezing and shaping for 1 hour, and then transferred to a low temperature of -20 to -10°C for continuous crosslinking for 24 to 48 hours. After the crosslinking was completed, it was placed at room temperature for thawing for 6 hours. After thawing, the porous scaffold after the crosslinking reaction was completed was soaked in deionized water to wash away the residual crosslinker and catalyst on the scaffold;

[0023] (4) Post-processing

[0024] After washing, precool it at -80°C in a refrigerator for 6 to 10 hours, and obtain the CRP scaffold after vacuum freeze-drying for 24 hours, which is then sterilized by irradiation for later use.

[0025] In a second aspect, the present invention provides a double-disc structured tracheal fistula patch prepared by the above method. The internal network of the CRP patch includes a β-folded physical structure and a chemical cross-linked structure formed by cross-linking with an epoxide cross-linking agent (EDGE), which form a physical / chemical double cross-linked network, giving the CRP ultra-light weight, good elasticity, water absorption and compression resistance.

[0026] In addition, the CRP scaffold has a multi-level microporous structure inside. This is because we placed the pre-crosslinked solution under freezing conditions, and the water solvent was frozen into an ice crystal template, so that the interconnected honeycomb structure of CRP has a high porosity, which is conducive to cell adhesion, growth and survival. Through SEM, it can be seen from the cross section that the CRP has an interconnected void structure with a pore diameter of approximately 72.24±17.02μm, while the morphology of CRP is observed from the front, and the surface has even finer micropores (10.19±2.00μm). The pore size inside and on the surface of CRP was further measured using ImageJ software. This surface pore structure leads to a higher initial contact angle during the dynamic contact angle test, but does not affect the excellent hydrophilicity and strong water absorption capacity exhibited by CRP.

[0027] Therefore, the third aspect of the present invention provides the use of the above-mentioned double-disc structure tracheal fistula patch in the regeneration and repair of tracheal fistula.

[0028] A fourth aspect of the present invention provides a minimally invasive fiberbronchoscope implantation regeneration and repair solution for tracheal fistula, including the double-disc structure tracheal fistula patch described above.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention constructs a tracheal fistula repair membrane CRP based on physical and chemical double cross-linking of silk fibroin, which has ultra-light weight, good elasticity and water absorption, and a double-sided disc-shaped structure that enables it to be embedded in the defect repair position to achieve a tight repair effect. The repair effect of CRP was tested on a rabbit tracheal fistula model, and the effectiveness of CRP in repairing TF was proved by imaging and histological methods. It can achieve in situ repair of tracheal fistula, promote orderly regeneration of tracheal defect sites, and accelerate the epithelialization process of defect sites. It is expected to become an ideal stent for the treatment of tracheal fistula, bringing new hope to the treatment of tracheal fistula.

[0031] Combining clinical CT three-dimensional imaging and endoscopic technology, customized CRP suitable for various types of fistulas is produced by preparing molds of corresponding geometric shapes. After constructing artificial epithelium, the fistula is sealed under endoscopy. This can achieve the purpose of minimally invasive and efficient repair, shorten the patient's treatment time, reduce the occurrence of complications, thereby improving the overall medical efficiency, and has important clinical transformation value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1The preparation and physical and chemical performance test of the double-disc structured tracheal fistula patch are shown: (A) Preparation process of CRP: four acrylic plates are assembled to form a CRP mold-----phase separation is induced under freezing conditions----the β-sheet of the silk protein molecular chain reacts with the epoxy-amino / hydroxyl group of the silk protein to form a physical and chemical double cross-linked network; (B) Physical display of CRP; (C) FTIR spectrum analysis of CRP; (D) SEM observation of the pores of CRP at different magnifications in the cross section and surface. The red arrows are internal macropores, and the blue arrows are surface micropores. Scale bar: 50μm, 10μm; (E) The pore size of the internal macropores and surface micropores of CRP; (F) The curve of the change of dynamic water contact angle with time; (G) The uniaxial compression curve of CRP in dry and wet states; (H) The elastic modulus of CRP in dry and wet states; (I) The general picture of the compression and rebound of CRP in wet state; (J) The cyclic compression curve of wet CRP.

[0033] Figure 2 The results of CRP repairing rabbit tracheal fistula in 3 weeks are shown: (A) Schematic diagram of two methods of repairing tracheal fistula, CRP and surgical suture. (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) CRP repair and surgical suture diagram; (D) (I) bronchoscopic image and (II) gross anatomical diagram of repair 3 weeks later; (E) histological staining results of tracheal axial section; (FL) Evaluation of the effect of CRP group and suture group after 3 weeks of repair.

[0034] Figure 3 The results of CRP repair of rabbit tracheal fistula after 6 weeks are shown: (A) X-ray and CT imaging results, the red arrow indicates the repair site of the tracheal fistula; (B) (I) bronchoscopic image and (II) gross anatomical diagram after 6 weeks of repair; (C) histological staining results of tracheal axial section; (DJ) evaluation of the effects of the CRP group and suture group after 3 weeks of repair.

[0035] Figure 4 The exploration of CRP in clinical application is shown: (A) CT image of a patient with tracheal fistula; (B) 3D reconstruction model of the patient's tracheal fistula; (C) Personalized CRP suitable for the shape of the fistula was developed based on the reconstruction model; (D) Personalized CRP preparation process; (E) General diagram of the customized human-sized CRP; (F) Finite element analysis of the equivalent stress and surface equivalent stress of E-CRP during exhalation; (G) The morphological recovery process of CRP, CRP can achieve compression loading and water-induced morphological recovery; (H) Schematic diagram of fiber bronchoscope-assisted in-situ non-invasive repair; (I) Simulation of bronchoscopic interventional non-invasive repair process on sheep trachea. DETAILED DESCRIPTION

[0036] 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.

[0037] In view of the key clinical problem of TF, this embodiment designs a chimeric repair membrane (CRP) based on physical and chemical double cross-linked silk protein, which can be used to repair the rabbit tracheal fistula model, can achieve immediate repair of airway defects, stably maintain airway patency, and effectively prevent tracheal stenosis, and accelerate the re-epithelialization process of the repair area. At the same time, it is further combined with clinical CT three-dimensional imaging and tracheal endoscopy technology to explore clinical applications.

[0038] 1. Preparation of double-disc structure tracheal fistula patch

[0039] The silk fibroin (SF) aqueous solution (5.0% w / v) was placed in an ice bath and stirred continuously to prevent the SF from undergoing a β-folding reaction due to changes in the surrounding temperature. The SF solution was then mixed with the cross-linking agent ethylene glycol diglycidyl ether (EGDE) (3mmol / g) and the catalyst N,N,N',N'-tetramethylethylenediamine (TEMED) (0.25v / v%) and placed in a mold. The epoxide groups at both ends of EDGE can combine with the amino and hydroxyl groups on the silk fibroin to establish a chemical fiber cross-linking network to prepare a SF porous scaffold.

[0040] The scaffold was first placed in a -80℃ refrigerator for rapid freezing and shaping for 1 hour, then transferred to -10℃ for continuous crosslinking for 24 hours. After crosslinking, it was placed at room temperature for thawing for 6 hours. The porous scaffold after the crosslinking reaction was soaked in deionized water to wash away the residual crosslinker and catalyst on the scaffold. After washing, it was precooled in the refrigerator for 8 hours, and the CRP scaffold was obtained after vacuum freeze drying for 24 hours, and irradiated for sterilization.

[0041] By designing molds of different shapes, various geometric shapes and ultra-light structures can be easily manufactured to match different tissue defects. This process is convenient, fast, low-cost, and has broad clinical translation value.

[0042] 2. Stent Appearance and Material Characterization

[0043] Figure 1 B shows a physical display of CRP, with two discs at both ends connected by a column in the middle.

[0044] The internal network of CRP includes a β-folded physical structure and a chemical cross-linked structure formed by cross-linking with an epoxide cross-linking agent (EDGE), which together form a physical / chemical double cross-linked network. EDGE reacts with the amine and hydroxyl groups on SF to generate secondary amines and ethers, respectively. Figure 1 C can be seen at 3276cm in the Fourier transform infrared spectrum. -1 The broad and strong absorption peak is the secondary amine absorption peak, 1230 cm -1 The characteristic peak of ether appeared at 1619 cm -1 、1512cm -1 The characteristic absorption peaks correspond to hydrophilic NO and CO. Using this basic construction strategy, many other protein biological components, such as collagen and acellular matrix materials, can be introduced into the double-crosslinked network system. In the future, a silk protein porous scaffold with bioactive microenvironment biomimetic can be established according to the characteristics of each protein component. In addition, we also conducted X-ray diffraction experiments, and the results showed that there was a typical peak at 28.4°, which is the characteristic peak of the β-folded crystal structure of the silk protein chain. This physical / chemical double-crosslinked network design gives CRP ultra-light weight, good elasticity and water absorption.

[0045] The CRP scaffold has a multi-level microporous structure. This is because we placed the pre-crosslinked solution under freezing conditions, and the water solvent was frozen into an ice crystal template, so that the interconnected honeycomb structure of CRP has a high porosity, which is conducive to cell adhesion, growth and survival. Through SEM, it can be seen from the cross section that the CRP has an interconnected void structure ( Figure 1 D), the pore diameter is about 72.24±17.02μm, while the surface of CRP has even smaller micropores (10.19±2.00μm) when observed from the front. ImageJ software was used to further measure the pore size inside and on the surface of CRP. The internal pore size is much larger than the surface micropores ( Figure 1 E). This surface pore structure results in a dynamic contact angle test ( Figure 1 F), but it does not affect the excellent hydrophilicity and strong water absorption capacity of CRP.

[0046] The double cross-linked network can significantly improve the compressive resistance of CRP to resist the pressure difference between inside and outside the trachea. We conducted uniaxial compression tests on CRP in dry and wet states ( Figure 1 G), and found that CRP exhibited extremely strong rigidity in a dry state, but its rigidity decreased significantly in a wet state, with the elastic modulus decreasing by nearly 40 times ( Figure 1 H). On the contrary, dry CRP has almost no elasticity, while wet CRP quickly becomes elastic due to the interaction of hydrogen bonds and the hydrophilicity of abundant amino and hydroxyl groups, and can withstand multiple deformations ( Figure 1I), the wet CRP exhibited extremely strong elastic properties in the cyclic compression test. During the 10 rebound compressions, the rebound curve remained stable ( Figure 1 J). By utilizing the elastic properties of this wet CRP, we can stuff it into defect areas smaller than its size and achieve a tight repair effect.

[0047] In general, the double-crosslinked SF network design enables CRP to have comprehensive synergistic effects, such as appropriate multi-level micropores, hydrophilicity, biocompatibility, and super elasticity in wet state, which can achieve tracheal defect repair of any shape. The mold assembly method makes the preparation process simpler and can also achieve mass production, which has broad clinical application and transformation value.

[0048] 3. Animal Model Experiment

[0049] To examine the actual effect of CRP in the repair of tracheal fistula, we preliminarily verified the feasibility of CRP in repairing tracheal fistula in a rabbit tracheal fistula model and evaluated the repair effect of CRP compared with traditional suture at two time points: 3 weeks and 6 weeks.

[0050] 3.1 CRP in situ repair of tracheal fistula for 3 weeks

[0051] like Figure 2 A. First, the rabbit was anesthetized and the skin was prepared. The midline of the neck was incised to fully expose the trachea. A 3 mm diameter defect was opened in front of the rabbit's trachea using a trephine drill to simulate clinical tracheal fistula. The experimental group used CRP for immediate repair in situ, while the control group underwent conventional surgical suture of the tracheal fistula. The actual surgical diagram is shown in Figure 2 C.

[0052] After 3 weeks of repair, the survival rates of the two groups were compared, showing that the CRP group was significantly better than the suture group. Five rabbits were repaired in each group. The 3-week survival rate of the CRP group was 100%, while that of the repair group was only 40%, and the rest died within 2-3 weeks after surgery. After the autopsy of the dead rabbits, it was found that all three rabbits died of severe suppurative secondary infection, and a large amount of yellow purulent accumulation appeared at the suture of the trachea. This is because the inside of the trachea has been in a state of communication with the external environment, and there is a lack of airway mucosal epithelial protection. If the tracheal defect is not sealed or sutured in time, it is easy to be attacked by pathogens in the ambient air, secondary chronic infection and inflammatory response, which will gradually spread to other parts of the body and the blood, causing systemic infection, and even leading to septic shock, and ultimately damaging multiple organs, leading to organ failure and death. This also explains why the rabbits in the suture group died within 2-3 weeks after surgery.

[0053] Three weeks after the repair, the surviving rabbits in both groups underwent in vivo X-ray and CT imaging to structurally evaluate the tracheal repair effect. Figure 2 B, Lateral X-ray of rabbits showed an obvious tracheal stenosis in the middle of the trachea in the suture group, while the trachea in the CRP group remained basically unobstructed. It was more obvious on CT three-dimensional imaging that there was an obvious lumen deformation and stenosis in the right front of the trachea in the suture group. This was due to the tension of the tracheal cartilage ring, which caused the direct suture to increase the tension of the tube wall, resulting in distortion, deformation and stenosis. We calculated the stenosis rate of the diameter of the repaired area of ​​the tracheal fistula on CT imaging ( Figure 2 F) and the percentage of the cross-sectional area to the normal tracheal cross-sectional area ( Figure 2 G), it can be seen that the CRP repair group was significantly better than the surgical suture control group.

[0054] Furthermore, we used CT 3D imaging to reconstruct the trachea of ​​the two groups in three dimensions. We can see that the defect in the front center of the trachea in the CRP group is still visible. This is because the CRP has not been completely degraded at this time, the surrounding tissue cells have not been fully grown, and there is a certain density difference between the repair area and the surrounding tissue. Although the defect in the control group appears to be sutured and repaired in the CT 3D reconstruction, it can be clearly seen that the wall of the tube is narrowing inward. Over time, the stenosis of the wall caused by this tension will gradually worsen, and even scar fibrosis will occur. If the tension is too great, it will cause the wound to crack again and secondary infection.

[0055] Intravital bronchoscopic imaging of the trachea of ​​two groups of rabbits ( Figure 2 DI) and gross anatomy ( Figure 2 D.II), it can be seen that at 3 weeks, CRP was still attached to the inner wall of the trachea, playing a temporary blocking role. The color and shape of the tissue in the repaired area after dissection were very close to the surrounding normal tissue. Although the repaired area in the suture group began to heal, due to the tension of the trachea itself, the lumen under bronchoscope had already shown obvious stenosis, and the repaired area showed slight white fibrosis tissue after dissection.

[0056] We then performed axial section staining on the tracheal samples from both groups at 3 weeks. Figure 2E, observe the internal tissue repair in the tracheal fistula area. H&E and Masson staining showed that tissue cells had begun to grow inside the entire CRP, mainly collagen and fibers. Although it showed a loose and disordered tissue structure, it could prove that CRP had excellent tissue compatibility, and the internal pore structure could promote the migration and growth of surrounding tissue cells. In the suture group, due to the incomplete absorption of the suture and the foreign body reaction of the rabbit itself, foreign body granulomas appeared locally, which is also one of the common postoperative complications in clinical practice. Over time, granulomas can still be gradually absorbed by the body. β-Tubulin and Muco5AC staining showed that a large number of fibroblasts and mucous cells crawled into and infiltrated the CRP along the internal pore structure of CRP during the repair process, thereby promoting tissue healing. The degree and number of fibroblast and mucous cell infiltration in the suture group were significantly lower than those in the repair group.

[0057] Regarding the re-epithelialization problem, we performed immunofluorescence staining of the airway epithelial marker total keratin PCK and the ciliated epithelial marker FoxJ1 protein. The results showed that at 3 weeks, epithelial cells that had migrated from the surrounding growth had appeared in the CRP repair area, forming a preliminary regenerated epithelium. However, the negative expression of FoxJ1 indicated that the epithelial cells at this time had not yet begun to ciliate and were still in the state of undifferentiated airway basal cells. Due to the tension of the trachea, the fistula in the suture group could not be well apposed, which affected the re-epithelialization of the trachea to a certain extent. In addition, we also performed immunofluorescence staining of the vascularization-specific indicators vWF & α-SMA. The degree of neovascularization in the CRP group was significantly higher than that in the suture group.

[0058] Accordingly, we used ImageJ to perform statistical quantification on each group of immunofluorescence staining to evaluate the repair effect of CRP and surgical suture on tracheal fistula at 3 weeks: the fluorescence proportion of β-Tubulin (2H) showed that the infiltration degree of fibroblasts in the CRP group was significantly higher than that in the suture group; the fluorescence proportion of Muco5A (2I) showed that the infiltration degree of mucus cells in the CRP group was significantly higher than that in the suture group; the PCK positive coverage rate (2J) showed that the epithelialization degree in the CRP group was higher than that in the suture group; the FoxJ1 / PCK ratio (2K) showed that although the epithelialization degree in the CRP group was high, the epithelial ciliation degree was low, and the regenerated epithelium was mostly in an undifferentiated state; the fluorescence proportion of vWF&α-SMA (2L) showed that the neovascularization degree in the CRP group was significantly higher than that in the suture group.

[0059] It can be seen that the initial repair effect of CRP in situ repair of tracheal fistula has been revealed at 3 weeks, but there are still shortcomings such as insufficient CRP degradation, insufficient epithelialization in the repair area, and low differentiation of the regenerated epithelium. However, compared with the traditional surgical repair group, there have been significant improvements in the degree of tracheal stenosis, infection prevention, regenerated tissue infiltration, re-epithelialization and tissue vascularization.

[0060] 3.2 CRP in situ repair of tracheal fistula for 6 weeks

[0061] In response to the problems existing in CRP in situ repair at 3 weeks, we found that 6 weeks after the CRP group repaired the tracheal fistula, not only was the repair effect significantly better than that of the suture group, but the shortcomings of insufficient CRP degradation, low epithelialization in the repair area, and low differentiation of the regenerated epithelium that existed at 3 weeks were further optimized.

[0062] from Figure 3 The X-ray and CT images in A clearly show that the trachea repaired in the CRP group has a smoother wall without obvious stenosis and gaps, and the gap of the tracheal fistula after CT three-dimensional reconstruction disappears. This is due to the full degradation of CRP and the continuous infiltration of surrounding tissues after 6 weeks, which has formed relatively mature tissue inside the CRP. However, the suture group showed obvious tracheal stenosis in imaging 6 weeks after repair, which may be caused by fibrosis and scarring of the fistula after suture. Similarly, we calculated the narrowing rate of the diameter of the tracheal fistula repair area on CT imaging ( Figure 3 D) and the percentage of the cross-sectional area to the normal tracheal cross-sectional area ( Figure 3 E), it can be seen that the CRP repair group was still significantly better than the suture group at 6 weeks.

[0063] from Figure 3 In B, under the bronchoscope (I), it can be seen that the inner wall of the tube in the CRP group is smooth and continuous, and the mucosal coverage is complete. At the same time, the gross image after dissection (II) can also show that the repaired area in the CRP group is basically integrated with the surrounding normal tissue. In the suture group, both under the bronchoscope and the gross anatomical image, very obvious tube wall deformity and stenosis can be seen, which is caused by scar contracture after surgical suture.

[0064] To further compare the internal repair effects, we performed histological staining and immunofluorescence staining on the repair samples of the two groups at 6 weeks, as in the case of 3 weeks. Figure 3C. H&E and Masson staining showed that there was abundant tissue formation inside the CRP repair group, which was still mainly collagen and fiber. However, due to fibrosis and scarring after suturing, the fistula in the suture group contracted and protruded into the internal cavity to form tracheal stenosis. β-Tubulin and Muco5AC staining showed that a large number of fibroblasts and mucus cells fully infiltrated the CRP, and they were more orderly and dense than the tissue at 3 weeks; while the degree and number of fibroblasts and mucus cells in the suture group were significantly lower than those in the stent repair group, and they were all limited to the sutured area, failing to repair the original defect. Regarding the re-epithelialization and epithelial ciliation of the tracheal defect, the CRP group showed a better re-epithelialization effect and preliminary ciliation process at 6 weeks than at 3 weeks. From the results of airway epithelial-specific markers PCK and FoxJ1 staining, not only the regenerated epithelium crawling around the airway was distributed continuously, but the ciliated cell marker FoxJ1 also had a preliminary positive reaction, indicating that the airway basal cells crawling from the surrounding normal mucosa began to undergo preliminary differentiation in the CRP repair area, which was significantly better than the re-epithelialization and epithelial ciliation results of the right surgical suture group. In addition, in terms of vascularization-specific indicators vWF & α-SMA, the degree of neovascularization in the CRP group was also significantly higher than that in the suture group.

[0065] Accordingly, we also used ImageJ to perform statistical quantification for each group of immunofluorescence staining to evaluate the repair effect of CRP and surgical suture on tracheal fistula at 6 weeks: the fluorescence proportion of β-Tubulin ( Figure 3 F) shows that the infiltration degree of fibroblasts in the CRP group was significantly higher than that in the suture group at 6 weeks; the fluorescence ratio of Muco5A ( Figure 3 G) showed that the degree of mucus cell infiltration in the CRP group was significantly higher than that in the suture group; PCK positive coverage rate ( Figure 3 H) showed that the degree of re-epithelialization in the CRP group was higher than that in the suture group; the ratio of FoxJ1 / PCK ( Figure 3 I) indicates that the CRP group not only improved the degree of re-epithelialization, but also greatly improved the degree of epithelial cilia; the fluorescence ratio of vWF & α-SMA ( Figure 3 J) showed that the degree of neovascularization in the CRP group was significantly higher than that in the suture group.

[0066] Thus, we have successfully constructed a double-disc structure embedded repair patch (CRP) based on SF with a physical and chemical double cross-linked network. This embedded patch can achieve stable in situ repair of tracheal fistula, avoiding complications such as low success rate, stent displacement and fracture, inadequate fistula closure, and neoplasia in the lumen caused by traditional stents, and effectively promote re-epithelialization of the defective part.

[0067] 4. Exploration of CRP application in clinical practice

[0068] A typical tracheoesophageal fistula patient was screened from the thoracic surgery database of Shanghai Ninth People's Hospital. The patient developed a tracheoesophageal fistula 5 weeks after esophageal cancer surgery. CT images showed that the fistula was located in the intrathoracic segment of the trachea, opened in the membranous part, connected to the esophagus, and had an irregular shape with a maximum diameter of about 1.5 cm ( Figure 4 A). Figure 4 B, Through clinical CT images, we performed a three-dimensional reconstruction of the patient's tracheal fistula and its surrounding tissue structure. Green represents the trachea, yellow represents the esophagus, and the red part in the middle is the tracheal fistula. We constructed a customized CRP model (4C) that is compatible with the special morphology and characteristics of the fistula.

[0069] In order to cope with the various fistulas of different sizes and shapes in clinical practice, we used a mold reaction method to prepare CRP with special shapes in a personalized way. Figure 4 D. We first used 3D printing technology to obtain a resin CRP that matched the actual modeling size, and then prepared a mold for silk fibroin reaction by silicone molding. In this mold, the silk fibroin was physically and chemically double cross-linked to obtain a CRP that perfectly matched the actual tracheal fistula. Figure 4 E is the personalized CRP display chart we prepared. This method can not only easily prepare CRP of different sizes and shapes, but also the whole process is safe, efficient and convenient, and has broad prospects for clinical transformation.

[0070] Before conducting TF repair experiments in large animals, we studied the mechanical behavior of CRP in TF repair by theoretical calculation and finite element analysis. During exhalation, there is positive pressure inside the trachea and negative pressure outside the trachea. This results in a force that acts mainly from the inside to the outside on the inner disk of CRP. Stress calculations on the inner disk surface show that the force is mainly concentrated on the edge of the disk, providing effective support ( Figure 4 F), and the same when inhaling. This analysis process allows us to simulate the behavior of the stent in the body, taking into account factors such as material elasticity and stress distribution. By optimizing the design of the CRP, we can ensure that the stent can withstand the necessary pressure and stress after implantation, maintain structural stability and integrity, and maximize the treatment effect, reduce complications such as infection and fistula recurrence, and promote faster healing of fistulas.

[0071] With the help of bronchoscope, we can accurately implant customized CRP into the location of fistula to achieve in situ minimally invasive repair. CRP has excellent elasticity and can be compressed into a large volume to be loaded into the working channel of bronchoscope. It also has the ability to quickly recover its deformation due to water responsiveness. After reaching the predetermined position, it can expand to its original shape to block the fistula. Figure 4G). Therefore, when the E-CRP stored in the working channel of the bronchoscope reaches the vicinity of the fistula, the doctor will use a guide wire to push the E-CRP out of the channel, and then restore it to its original form to block the fistula. Once the CRP is implanted, the doctor can also use the bronchoscope to observe its fit. If the position of the CRP needs to be adjusted, the doctor can operate through the bronchoscope to ensure the best fit of the E-CRP.

[0072] In order to verify the feasibility of the minimally invasive repair method of CRP, we simulated the repair process of this interventional surgery in a sheep tracheal fistula model. Figure 4 H, we first constructed a special tracheal fistula on the sheep trachea, and then used a delivery tube to simulate the working channel of the bronchoscope, loaded CRP into the end of the tube, and followed the tube to the location of the tracheal fistula. Then, we gently used the guide wire to push out the CRP stent to expand its outer disk, and slowly pulled back the delivery tube, so that the CRP outer disk remained outside the fistula, and the inner disk expanded in the trachea. The upper and lower disks fully covered the fistula to form a blockage, achieving in situ minimally invasive repair of sheep tracheal fistula ( Figure 4 I).

[0073] In the future, with the assistance of bronchoscopy technology, doctors can fully utilize CRP to achieve minimally invasive and efficient repair of tracheal fistula, which has important clinical translational value.

[0074] V. Conclusion

[0075] 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 materials science and regenerative medicine, the use of biomaterials to repair TF has become a new treatment strategy. Silk fibroin is a research hotspot in the field of tissue engineering due to its good biocompatibility and degradability, as well as the advantages of easy processing and molding. In this study, a CRP based on a double cross-linked network SF was developed for repairing TF. Its excellent tracheal tissue repair effect was verified in a rabbit tracheal fistula model, effectively improving the problem of airway re-epithelialization. Combined with clinical CT reconstruction and tracheal endoscopy technology, we can achieve minimally invasive interventional repair of TF of different shapes, which has important clinical transformation and application value. In short, CRP has broad application prospects in tracheal fistula repair. Through further research and optimization, it is expected to provide safer and more effective treatment options for patients with tracheal fistula.

[0076] The references cited in the background technology of the present invention are as follows:

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[0100] 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 preparing a double-disc structured tracheal fistula patch, characterized in that: The steps include: (1) Use 3D printing to make personalized custom molds or use multi-layer plate stacking and assembly to make mass production molds; (2) The silk fibroin aqueous solution is placed in an ice bath and stirred continuously, and then the SF solution is mixed with a cross-linking agent and a catalyst and placed in a mold. The epoxide groups at both ends of the cross-linking agent combine with the amino and hydroxyl groups on the silk fibroin to establish a chemical cross-linking network to prepare a SF porous scaffold; (3) The prepared SF porous scaffold is placed in a -80°C refrigerator for rapid freezing and shaping, and then transferred to a low temperature of -20 to -10°C for continuous crosslinking. After the crosslinking is completed and the mold is removed, it is placed at room temperature for thawing. After thawing, the porous scaffold after the crosslinking reaction is completed is soaked in deionized water to wash away the residual crosslinking agent and catalyst on the scaffold; (4) After washing, precool in a refrigerator for 6 to 10 hours, and then freeze-dry in a vacuum for 24 hours to obtain the CRP scaffold, which is then sterilized by irradiation for later use.

2. The method for preparing the double-disc structured tracheal fistula patch according to claim 1, characterized in that: in, In step (1), the mold preparation method includes: preparing the mold by three-dimensional modeling, 3D printing a double-disc model, and a reverse molding method; forming the mold by assembling a plurality of perforated plates, from the bottom to the top, the first layer to the fourth layer, the first layer is a flat plate, the hole size of the second layer and the fourth layer is larger than the size of the hole of the third layer, the hole centers of the second to fourth layers are aligned, and the periphery of the four layers is clamped together to form an integrated mold.

3. The method for preparing the double-disc structured tracheal fistula patch according to claim 2, characterized in that: in, The mold material prepared by the 3D printing mold-turning method includes silica gel; the mold material prepared by the multi-layer plate assembly is selected from silicate glass, acrylic glass, polytetrafluoroethylene or stainless steel. The space left by the mold is the shape of the patch that will be formed later. The patch is a double-disc structure, including two larger upper and lower disks and a smaller connecting part in the middle. The cross-section of the large disk is circular, elliptical or other irregular shapes, and the thickness of the disk is 1 to 3 mm; the cross-section of the connecting part is circular or a special shape, and the thickness is 1 to 3 mm. The hole size of the second and fourth layers of boards is 3 to 8 mm, and the hole size of the third layer of boards is 1 to 3 mm.

4. The method for preparing the double-disc structured tracheal fistula patch according to claim 1, characterized in that: in, In step (2), the concentration of the silk fibroin aqueous solution is 3-15% w / v, the concentration of the crosslinking agent is 3 mmol / g, the concentration of the catalyst is 0.25 v / v%, and the stirring speed of the silk fibroin aqueous solution is 10-100 rpm; The cross-linking agent is selected from ethylene glycol diglycidyl ether EGDE, glycerol diglycidyl ether GDGE and butanediol diglycidyl ether BUDGE, or ethylene glycol diglycidyl ether EDGE and poly (ethylene glycol) diglycidyl ether (PEGDGE); The catalyst is selected from any one of N,N,N',N'-tetramethylethylenediamine, ammonium persulfate and ammonium sulfate.

5. The method for preparing the double-disc structured tracheal fistula patch according to claim 1, characterized in that: in, In step (3), the SF porous scaffold is rapidly frozen and shaped in a -80°C refrigerator for 1-3 hours, cross-linked at a low temperature of -20 to -10°C for 24 to 48 hours, and thawed for 3 to 6 hours.

6. The method for preparing the double-disc structured tracheal fistula patch according to claim 1, characterized in that: in, In step (4), the precooling temperature in the refrigerator is -80°C and the precooling time is 6 hours; The vacuum freeze-drying process steps are as follows: after the dryer is pre-cooled, the sample is placed in the dryer, the temperature is set at -80°C, the pressure is between 0.1 and 0.3 mbar, and freeze-dried for 48 hours.

7. A double-disc structured tracheal fistula patch, characterized in that: The method is prepared by any one of claims 1 to 6.

8. Use of the double-disc structured tracheal fistula patch according to claim 7 in the regeneration and repair of tracheal fistula.

9. A tracheal fistula regeneration and repair component, characterized in that: Including the double-disc structure tracheal fistula patch as described in claim 7.

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